key: cord- -hmzoxqu authors: alibrahim, omar s.; heard, christopher m.b. title: extracorporeal life support: four decades and counting date: - - journal: curr anesthesiol rep doi: . /s - - - sha: doc_id: cord_uid: hmzoxqu extracorporeal membrane oxygenation (ecmo) or extracorporeal life support (ecls) is a form of heart lung bypass that is used to support neonates, pediatrics, and adult patients with cardiorespiratory failure for days or weeks till organ recovery or transplantation. venoarterial (va) and venovenous (vv) ecls are the most common modes of support. ecls circuit components and monitoring have been evolving over the last years. the technology is safer, simpler, and more durable with fewer complications. the use of neonatal respiratory ecls use has been declining over the last two decades, while adult respiratory ecls is growing especially since the h n influenza pandemic in . this review provides an overview of ecls evolution over the last four decades, its use in neonatal, pediatric and adults, description of basic principles, circuit components, complications, and outcomes as well as a quick look into the future. ecmo or ecls has been around for more than four decades. ecls, which is the name better describes this technology, is a modified form of cardiopulmonary bypass that is used to support patients with cardiopulmonary failure unresponsive to conventional treatment. ecls is performed to drain blood from the venous system, remove carbon dioxide (co ), add oxygen (o ) through an artificial lung (the oxygenator), and return the blood using a pump to the body via an artery as in va ecmo or a vein as in vv ecmo [ ••, - ] . it is important to recognize that ecls is a support modality but not a cure. it provides time for other diagnostic and therapeutic measures to be pursued allowing injured organs to recover meanwhile abundance of oxygenation and optimum tissue perfusion is guaranteed. ecls is a complex, invasive, highrisk, and costly technology, and it should only be conducted in centers with sufficient experience, knowledge, and expertise in that field. the use of silicone rubber membrane in the 's [ , ] provided a strong framework until kolobow perfected the development of the silicone membrane oxygenator in the 's [ ] . this invention allowed the prolonged use of the heart lung bypass machine by clinicians outside the operating room. several groups were working on this concept in the 's and 's. dr. bartlett and bioengineer drinker successfully applied ecls in the laboratory for days using the newly developed membrane oxygenator with lower heparin dosage to minimize bleeding. dr. bartlett, who is considered by many as the father of ecmo, moved to the university of california at irvine in , where he continued to do his work in the laboratory to optimize cardiopulmonary bypass for prolonged use. in , dr. hill reported the first successful use of ecls in a young adult who suffered from severe hypoxic respiratory failure secondary to motorcycle accident in santa barbara, california [ ] . same year, dr. bartlett performed the first successful use of va ecmo on a baby who suffered from low cardiac output syndrome immediately after mustard atrial baffle operation for transposition of great vessels. the first neonatal respiratory survival was not reported until when drs bartlett and gazzaniga successfully used va ecmo for a full-term newborn who was suffering from severe hypoxic lung disease thought to be secondary to meconium aspiration (ma) [ ] . in , dr. bartlett and colleagues published their experience in the use of ecmo for newborn respiratory failure [ ] . he described the use of ecmo in moribund newborn infants; patients survived. all were deemed unresponsive to conventional therapy. the right atrium (ra) and the aortic arch (aa) were cannulated via the internal jugular vein (ijv) and the carotid artery (ca), respectively. primary diagnoses were hyaline membrane disease, sepsis, and persistent fetal circulation including congenital diaphragmatic hernia (cdh) and ma. this paper concluded that ecmo use has decreased mortality and morbidity in newborn respiratory failure, and that ecmo may be used effectively in older patients with respiratory failure if used before irreversible lung damage occurs. this report was a milestone in the evolution of ecmo around the world. many clinicians took notice and came to michigan to learn ecmo. they took this technology back to their hospitals which lead to the widespread use of ecls globally especially in neonatal respiratory failure. va and vv ecls are the most commonly used modes of support [ ••, , , , . va ecls provides cardiac and pulmonary support. therefore, this can be used in ards patients suffering from cardiac or circulatory failure as in severe septic shock. it is the ideal mode of support in cases of severe cardiogenic shock (e.g., cardiomyopathy, myocarditis). support can be partial or total depending on site of cannulation, size and position of the cannulae used, and the native cardiac function. oxygen delivery (do ) is the amount of oxygen delivered to the tissues each minute. it equals the oxygen content times the cardiac output. the more blood diverted to the ecls circuit (maximum cardiac output) with the presence of normal hemoglobin (maximizing oxygen content); do is maximized. va ecls cannulation can be done using one of three access points: first by using the transcervical approach, placing the venous cannula in the ra via the right ijv and the arterial cannula in the aa via the right ca. it is important to assure that the tip of the arterial cannula is away from the aortic valve as this can damage the valve and hinders the myocardial recovery. this approach may provide - % extracorporeal support as part of the blood volume still passes through the patient's native heart and lung. the second approach is the central or transthoracic approach with direct venous cannula placement in the right atrial appendage and arterial cannula placement in the aa. this approach is typically used in postoperative cardiac patients once they fail coming off cpb. this may provide total ecls support as the ecls circuit captures all blood volume and none passes through the native heart. it also has been described in patients with severe septic shock [ ] . the third approach is cannulating the femoral artery and the femoral vein. this approach has been described in adults and older children and has been used in cases of emergency cannulation or extracorporeal cardiopulmonary resuscitation (ecpr). va ecls provide circulatory support. in most cases, inotropes and vasopressors are weaned off soon after ecls initiation. coronary perfusion is provided by retrograde flow via the arterial cannula, which emphasizes the importance to have its tip accurately placed in the aortic arch away from the aortic valve. on the other hand, va ecls requires accessing and mostly ligating the carotid artery. there is an increased risk of systemic embolization with potential end organ damage mainly brain strokes. left ventricular afterload is increased, which may hinder myocardial recovery. vv ecls is ideal for pulmonary support in cases of severe respiratory failure. it does not provide cardiac nor circulatory support. blood is typically drained from the venae cavae and retained to the right atrium. that can be done either by multisite cannulation; usually by draining from a venous cannula in the inferior vena cava via the femoral vein and returning to the ra via the ijv; or single-site cannulation with the use of a double lumen catheter, draining the blood from the venae cavae and returning it to the ra. currently, most of venovenous access is achieved by cannulating the right ijv using a double lumen catheter, which minimizes the problem of recirculation that dominates the multisite access approach. other than sparing the carotid artery and minimizing the risk of systemic embolization especially embolic strokes [ ] , the benefits of vv ecls for pediatric respiratory ecls are evident. the pulmonary blood flow is maintained in vv ecls with well-oxygenated blood, which is considered a great vasodilator of the pulmonary circulation that leads to reduction of pulmonary vascular resistance (pvr). as a result, the afterload of the rv will decrease, augmenting its systolic and diastolic function. these effects continue through to the left side of the heart as the lv preload is optimized and coronary perfusion is maintained with highly oxygenated blood. there is no increase in lv afterload, and the risk of "myocardial stun" in minimized, with resulting improvement in myocardial oxygen delivery and myocardial performance [ , ] . other modes of ecls support are used. venovenoarterial or vva ecls has been increasingly used as a hybrid between va and vv ecls. this is typically used in older pediatric and adult patients with initial va ecls using femoral vein and artery. coronary and upper body perfusion may be inadequate. this can be overcome by adding another venous cannula in the ra via the ijv and connecting it via a y-connection to the returning "arterial" limb, so oxygenated blood is returned to the ra, passes through the native heart to perfuse the coronaries, the carotid arteries and the upper body, meanwhile providing circulatory support via the femoral arterial access. arteriovenous or av ecls is not commonly used in pediatrics. this method can be very effective for co removal utilizing only - % of the cardiac output. the femoral artery and vein are accessed; blood flows through a membrane lung (oxygenator) without a pump using the gradient difference between the arterial and venous pressures. this method, also known as the extracorporeal co removal (ecco r), is ideal for adults with chronic obstructive pulmonary disease (copd) with exacerbation and patients with near-fatal asthma where hypercapnia is the main problem [ , ] . the circuit is usually very simple which potentially makes it safer to maintain. vascular access and cannulae placement has evolved over the years. open surgical technique using the neck vessels and through the chest have been the main traditional methods especially for va support in neonates and young children with cardiorespiratory failure. percutaneous cannulation is becoming more popular and is currently considered the standard of care for vv ecls in pediatrics and adults using the current double lumen cannulae and for va support when accessing the femoral vein and artery. the open surgical approach provide the advantage of visualizing the vessel(s), estimating the appropriate-size cannula, and placement under vision. however, this approach is more time consuming, can lead to more bleeding at the site, and in many instances, requires ligation of the distal end of the vessel [ ••] . percutaneous techniques is faster, may decrease the risk of surgical site bleeding, no distal ligation of vessel is required, and provides a simpler way of decannulation without the need to explore the cannulation site or ligating the vessel. the semi-open technique or the percutaneous-assisted technique has been described especially for vascular access in neonates requiring vv support using double lumen catheter. a small transverse incision is made just above the right clavicle, the right ijv is visualized and then a percutaneous approach is used by accessing the vein cm distally using seldinger technique. transthoracic cannulation is used as an extension of cardiopulmonary bypass if the patient is not able to come off bypass in the operating room. the chest is open via median sternotomy; cannulae are placed directly in the ra and the aorta. decannulation is the procedure that is needed to terminate ecls. cannulae that are surgically placed have to be removed by exploring the surgical site, carefully pulling them out while maintaining homeostasis and ligating the vessels. cannulae placed percutaneously can be withdrawn at the bedside with pressure applied over the site for - min. there is no need for vessel ligation. however, percutaneously placed arterial cannulae may need surgical exploration and vessel repair. ecls circuit designs differ among institutions, although the main components and principles are similar. these components experienced significant evolution especially over the last decade [ •, •] . roller head (semi-occlusive) pumps have been traditionally used in ecls. they are similar to the pumps used in cpb. the blood is squeezed forward through the tubing "the raceway" against a plate at two pressure points in the pump housing while the roller head is rotating. this provides continuous forward motion of the blood towards the oxygenator and then back to the body. these pumps depend on gravity for the venous drainage into the pump (preload), so the patient has to be at a certain height ( - cmh o) from the pump and the bladder reservoir for it to work. whenever there is an interruption to that flow either secondary to hypovolemia, pleural or pericardial tamponade pathology, or kinking in the tubing; the pump will just slow down or stop till the venous return is reestablished or the cause of the problem is corrected. newer pumps have servo-regulation capabilities that allow the ecls specialists to set the alarms so the pump will slow down once certain negative venous (access) pressures are reached. this allows the specialist to troubleshoot and address the problem preventing many interruptions and stoppage of pump flow [ , •, fuhrman, ] . the centrifugal pumps are also used for ecls support. they are used in most pediatric and all adult patients ecls supported. many centers have transitioned from roller head to centrifugal pumps over the last years. centrifugal pumps are non-occlusive. earlier designs used spinning rotor with bearings and seals that lead to excessive head generation. those pumps needed to be replaced frequently, adding to the morbidity and mortality of these patients. with advancement in technology, the newer pumps utilize magnetic levitation to suspend and spin the impeller. their blood-handling qualities have also improved, minimizing heat generation that lead to reduction in circuit related hemolysis and air cavitation. the blood enters these pumps at the apex and gets expelled at the base towards the membrane oxygenator. other advantages include easy set up, small priming volume, ability to trap air and debris within the vortex, and lack of dependency on gravity for blood drainage. these pumps can be placed at any level relative to the patient, which make them suitable for inter and intrahospital transport. the membrane oxygenator, also known as the artificial lung, is responsible for gas exchange in ecls. the kolobow silicone membrane oxygenator was, for decades, the only available gas exchange device in the market. it was constructed of a flat, reinforced silicone membrane envelope that is wound in a spiral coil around a polycarbonate spool. there was a highly gas-permeable barrier separating blood and gas compartments, with no direct blood-gas interface. gas transfer occurs by molecular diffusion as it does in the human lung. the silicone membrane oxygenator was effective in gas exchange, but its compact design created long blood path and high resistance that made it harder to de-air and more challenging to prime. a separate blood warmers (heat exchanger) was needed for most of these devices. it was not unusual to replace these devices during ecls or to need more than one oxygenator to support older pediatric and adult patients. the silicone membrane oxygenators are not available in the market anymore. these hurtles lead to the development of newer generation of devices, the hollow fiber oxygenators. these devices consist of micro porous material where gas exchange takes place by bulk gas transfer via a direct gas to blood interface. these devices are easy to prime, have low resistance, and provide efficient gas exchange. but the longevity of these devices is limited, plasma leak into the gas phase would occur as early as few hours from ecls initiation, that lead to early failure of these devices and the need to be replaced urgently. in the early s, a newer design of these devices became available [ ] . these new devices incorporate the advantageous characteristics of the membrane oxygenator and the hollow fiber oxygenator together using polymethylpentene (pmp) and polyurethane fibers. the pmp is a micro porous material that is very efficient in gas exchange for extended period of time. these devices are durable and may attenuate the inflammatory response during ecls initiation. they have low resistance to blood flow, which makes them easy to prime, reducing the potential for thrombus formations and oxygenator failure. the rated flow is a measure that is used to describe the function of all gas exchange devices. the rated flow, which is the amount of normal venous blood that can be raised from to % oxyhemoglobin saturation in a given period of time, is high which allows many centers to use onesize device for all patients regardless of their size and weight [ , •] . neonatal ecls ( - days) neonates are still compromise the majority of patient population supported by ecls. as of july extracorporeal life support organization's (elso) report, a total of , neonates were supported by ecls, the majority ( , ) with severe respiratory failure with a survival rate of % [ ] . the most common diagnoses are meconium aspiration syndrome (mas), cdh, sepsis [ ] , persistent pulmonary hypertension of the newborn, and respiratory distress syndrome. mas used to be the most common diagnosis till recently. in the early 's, other treatment options such as high frequency ventilation (hfv), surfactant, and inhaled nitric oxide (ino) became more available [ ] [ ] [ ] . it is believed that the increasing use of these therapies has led to the significant reduction of ecls use in this patient population [ , , , ] especially in neonates with mas. there were around neonatal ecls runs reported every year for the last years compared to almost annual cases in the early s. the use of neonatal ecls peaked in with a total of neonates that year from approximately centers around the globe. va ecls is still the most common mode used in neonates, followed by vv ecls using the double lumen cannula [ ] . therapeutic hypothermia during neonatal ecls did not result in improved outcome up to years of age [ ] . in contrast to neonatal respiratory ecls; neonates requiring ecls for cardiac reasons have poorer survival rate of % [ ••] . mortality in neonates with congenital heart disease requiring ecls has not significantly changed over the last - years, despite the dramatic increase in its use for that purpose [ ] . in a recent study, low body weight, single ventricular physiology, lower ph before ecls, and longer time from intubation and mechanical ventilation to ecls deployment were associated with increased mortality in neonates requiring ecls for cardiac indications. mortality in this category is similar in surgical and nonsurgical patients. this data highlighted the importance of early initiation of ecls before acidosis and organ dysfunction occur [ ••, , ••, , ] . this concept was described previously; there is no welldefined criteria defining the optimal timing of ecls initiation in this population. cdh occurs in about : live births. the presence of pulmonary hypoplasia may result in pulmonary hypertension (phtn) with hypoxia, hypercapnia, and acidosis that could be evident soon after birth. variety of ventilation strategies and other treatment modalities (e.g., ino) may be needed in the first few days of life [ ••, ] . ecls have been used for more than three decades in cdh patients with phtn unresponsive to maximum conventional therapy. va and vv ecls have been used effectively; however, va ecls is more commonly used as it unloads the rv that may aid in restoring myocardial function. looking at elso data, cdh mortality did not change significantly over the last two decades. overall, the reported survival rates for cdh and ecls is about - % [ ] . as in other neonatal ecls, the oxygenation index (oi) has been used to determine need for ecls support. oi more than for more than h is considered an indication for ecls. pre-ecls factors (e.g., apgar score, pao , pco , ph) failed to predict outcome or prognosis [ ••] and clinicians should reevaluate selection criteria for not offering ecls to this selected group of patients. the timing of defect repair of ecls-managed patients is controversial. there are two main groups, on or off ecls. surgery performed during ecls can be either early in the ecls course or when phtn is resolved just before decannulation. a review of over cases from the cdh study group (cdhsg) registry [ ] over a -year period evaluated the outcome of surgery either during or after ecls. taking into account other outcome-associated variables such as duration of ecls run, type of surgical repair, and patient factors, patients repaired post ecls had a significantly better outcome. the odds of dying were . times greater if the repair was performed during ecls. this may be associated with a reduced bleeding risk as well as bias towards patients who have improved more quickly allowing ecls decannulation. the ability to wean off ecls within a two-week period may contribute positively to the outcome with respect to the timing of surgery [ ] . if the patient was weaned off ecmo within weeks, surgical correction post ecmo was associated with a significantly better outcome and a significantly reduced risk of bleeding when compared to patients at that institution who were repaired on ecls. the early repair on ecls has been suggested to offer the benefit of surgery before the anasarca becomes extensive and allows recovery from the physiologic insult while on ecls, hopefully without prolonging the ecls duration. a review of repair within days of ecls [ ] demonstrated that the risk of bleeding at surgical site was < %, the operative repair took less than h with a survival rate of %. other ecls-related bleeding complications were no different from those reported to the elso registry. anticoagulation management during ecls for cdh is pivotal especially if cdh repair is performed during ecls run. a review of atiii use in (target activity > %) cdh ecls patients [ ] when compared to the institutions historical control demonstrated that the use of atiii lead to significant reduction in the utilization of ffp, packed red cells, and platelets in the first days of ecls. the use of large volumes on blood products may adversely affect lung mechanics and delay recovery. amicar and tranexamic acid used peri-operatively could be helpful to minimize bleeding. pulmonary hypoplasia is an important component with respect to the need for ecls support related to hypoxia and hypercapnia. there have been case reports on the use of perfluorocarbons (pfc) to support alveolar maturation. a prospective randomized study of cdh patients on ecls [ ] with or without pfc use evaluated lung growth using l vertebral body size for comparison. there was about % increase in the left (affected) lung size during the pfc use. there were no noted side effects or complications from the pfc use. however, there was no comment on the lung growth in the non pfc group, and mortality was not significantly different (small n = ). viral and bacterial pneumonia causing acute respiratory failure and acute respiratory distress syndrome (ards) are common causes of morbidity and mortality in the picu. ecls has been successfully used as a rescue therapy for these patients unresponsive to conventional methods [ ] [ ] [ ] [ ] . as of july , a total of , pediatric patients received ecls, with patients supported for respiratory indications with a survival rate of % (elso) and in one report up to % [ ] . patients with respiratory syncytial virus infection, aspiration pneumonia, and near-fatal asthma [ ] has better chances to survive while those with ards related to sepsis, pertussis, fungal pneumonia, disseminated herpes simplex virus infection, immunodeficiency, multiorgan failure, and longer duration of mechanical ventilation (> days) before ecls deployment have higher odds of mortality [ , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . pre-ecls severe acidosis in addition to renal failure and need for continuous renal replacement therapy (crrt) have been related to lower survival, longer ecls duration, and higher complication rate [ , ] . ecls utilization in pediatric population has slightly increased over the last - years [ , ] . this increase has been steadier since with a total of cases reported that year, later peaked in with pediatric ecls in total. this increase is believed to be due to expanding the inclusion criteria for this patient population [ , ] , increase use of ecls in patients after congenital heart surgery, widespread of use of vv ecls, the advancement and the use of double lumen cannula in children, in addition to the expansion in extracorporeal cardiopulmonary resuscitation (ecpr) use [ , ] . conditions and comorbidities like immunosuppression, malignancy, and sepsis are considered acceptable indications for ecls these days, but would have been contraindications to ecls - years ago [ , , , [ ] [ ] [ ] [ ] [ ] [ ] . patients are more complex with more comorbidities. paden et al. [ ] reported an increase in pediatric ecls comorbidities from % in to % in . recent reports showed that pediatric ecls patients with malignancies and immunodeficiency could have a reasonable outcome with a survival rate - % [ , , ] . patients with bone marrow (bmt) and stem cell transplant present a particular challenge. gow et al. [ ] showed ecls survival rate of % based on the elso registry data in . of those four patients who survived ecls in that study, only one patient was able to leave the hospital. the development of renal failure and multiorgan dysfunction were considered risk factors for death. there are few case reports that described successful use of ecls in bmt and stem cell transplant patients secondary to different etiologies [ ] . the decision to offer ecls in this patient population should be on a case-by-case basis. providers should take into consideration the overall patient prognosis from the underlying illness, assess the presence of multiorgan dysfunction and understand the family wishes for their loved ones before considering ecls as an option. va support has been for many years the mode of choice for pediatrics respiratory ecls [ ] . it is still the predominant mode found in the elso registry. the utilization of vv ecls is gaining popularity in pediatrics [ , , , , ••, ] . in , vv ecls cases outnumbered va ecls cases in pediatric respiratory indications. now, in , vv ecls is considered the standard of care in pediatric patients with severe respiratory failure unresponsive to conventional therapies. pettignano et al. [ ] reported the early successful use of vv ecls for this patient population. eighty patients received ecls in his center over a period of years ( - ) . sixty-eight patients received vv ecls with a survival rate of % compared to va ecls patients with a survival rate of %. the cannulation techniques for vv ecmo in pediatrics have also evolved over the last years. multisite venovenous cannulation was the preferred method used in pediatric respiratory ecls. the continued advancement of vvdl, especially in the late 's when the bicaval wire-reinforced catheters was approved by the food and drug administration, single-site cannulation using these cannulas became a common practice [ ••, ] . in , vvdl cannulation for vv ecls represented % of the total cannulation. the use of vvdl cannulas have provided improved vv ecls pump performance with evidence suggesting reduction of the risk of recirculation traditionally related to the multisite approach. these cannulas can be inserted percutaneously, but need to be performed under imaging guidance to avoid the risk of atrial perforation or disruption to the hepatic vein [ , ] . using fluoroscopy, ultrasonography, or a combination of both is recommended. ards in adults is well described in the literature with high rates of morbidities and mortality that can exceed %. many conventional methods were studied to minimize this risk with mixed results [ ] . ecls use in adults with ards was first described in with dr. hill's experience. this was followed by the first multicenter randomized trial of the use of ecls in adults with ards conducted by zapol et al. [ ] at the national institute of health (nih) in . the results were disappointing as the mortality rate was > %. this put the brakes on using ecls for adult population for two decades. in , dr. bartlett and colleagues [ ] described their experience at the university of michigan in the largest retrospective study discussing the use of ecls in adult patients with ards between and with a survival rate of %. a protocol-driven algorithm was used in their institution since guiding the treatment of severe ards including the use of ecls. while on ecls, lung rest strategies, minimal anticoagulation, and optimization of oxygen delivery were the key factors. these results were encouraging, and ecls was perceived again as a viable and successful option for adults with ards not responsive to maximum conventional therapy. this regained confidence in adult respiratory ecls was boosted by the encouraging results of the cesar trial (efficacy and economics assessment of conventional ventilatory support versus extracorporeal membrane oxygenation for severe adult respiratory failure) by peek and colleagues in the uk [ ] . this study showed improved survival rate without disability at months after discharge from the hospital ( vs %; rr= . ; ci . - . , p = . ) in adult patients with ards transferred to ecls center. sixty-eight patients were supported by ecls of whom ( %) survived. as considered by many, the real rebirth of adult respiratory ecls occurred in . this milestone was driven by two major events: the influenza a (h n ) pandemic and by the availability of adult-size bicaval dual-lumen cannula. these cannulae are inserted percutaneously using seldinger technique under ultrasound or fluoroscopy in the right ijvand positioned to allow drainage of venous blood from the venae cavae and reinfusing to the ra. looking at the elso registry, there were less than reported cases of adult respiratory ecls per year, with then a substantial increase in with cases. this increase continued and peaked in with cases. more than % of the total runs in these reports are venovenous ecls mode (vv, vvdl, and vvdl-v) with overall survival rate of % [ ] . patients with viral or bacterial pneumonia, asthma, and trauma are more likely to survive [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . longer duration on mechanical ventilation before ecls, multiorgan failure, central nervous system (cns) events, fluid overload [ , , ] , non-pulmonary infections, higher peak inspiratory pressures, acidosis (ph < . ) and higher paco are associated with poor outcome. interestingly, the use of neuromuscular blockade and prone positioning before ecls was favorable. this initial experience of adult respiratory ecls with h n -induced ards came from australia and new zealand [ ] with survival rate up to % at the end of the study period. other reports from other countries came out describing their experience during the h n pandemic in adults with variable result, but mostly with survival rates - % [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the review of these reports revealed the importance to emphasize on the fact that ecls is a complex, high-risk, and costly technology, and it should only be conducted in centers with sufficient experience, knowledge, and expertise in managing ecls. as a result, a position paper [ ••] was published in july by an international group of physician and health care providers in order to provide physicians, care providers, hospital administrators, and policy makers a description of the optimal approach to organizing ecls program for adults with respiratory failure to ensure safety and proficiency. vv ecls using the bicaval dual-lumen cannula is the most common practice in adult respiratory indications [ , ] . ecls flow of - l/min would be ideal to provide adequate oxygenation and ventilation for an adult with severe ards. additional venous drainage cannula (usually in the femoral vein) may be warranted to achieve this goal. arterial oxygen saturation more than % would be acceptable given that clinical and laboratory evidence of adequate oxygen delivery to the tissues is achieved. ecco r is a modality of extracorporeal support that is increasingly utilized in adult respiratory population when hypercapnia is the main drive behind the need for excessive minute ventilation and ecls support. ecco r allows the use of low blood flow ( . - l/min) via a pumpless device to remove co efficiently. this typically is achieved by av support accessing the femoral artery on one side and the femoral vein on the other side. this approach is ideal for patients with copd exacerbation and hypercapnia. once on vv ecls or ecco r, patients can soon be placed on minimal ventilator settings "rest settings," continuous positive airway pressure (cpap), or even get extubated to protect their lungs from further injury [ ] [ ] [ ] . early tracheostomy provides patients with less discomfort and allows early mobilization that would be beneficial to facilitate recovery. early mobilization and rehabilitation is considered mandatory while patients on vv ecls as a bridge to lung transplantation [ , [ ] [ ] [ ] . there are few contraindications for ecls that include advanced age, severe disability (wheelchair bound), intracranial bleeding, uncontrolled coagulopathy, mechanical ventilation more than a week with high peak pressure and oxygen requirements [ ] . the use of ecls in pregnancy is uncommon; there are significant concerns regarding bleeding, fetal demise, and thrombotic complications. pregnant women with h n infection have higher risk of mortality and morbidity compared to nonpregnant women [ ] . over the last few years, especially during the h n influenza pandemic, there have been many reports of successful ecls use in pregnancy or postpartum period related to ards [ , , , •] . in the case series from australia and new zealand [ ] , they reported the use of ecls in pregnant thirty-one reports were found, reports of va ecls and of vv ecls with a total of patients. overall maternal survival rate was % while fetal survival was slightly lower than %. most common indications were severe ards, postpartum cardiogenic shock, and amniotic fluid embolism. in one case, delivery by cesarean section was performed during ecls; otherwise, delivery of the fetus was deferred. anticoagulation management was conservative maintaining lower therapeutic levels of activated clotting time (act) and activated partial thromboplastin time (aptt). the author recommends using act levels - s and aptt - s. there were few cases with mild to moderate amounts of postpartum vaginal bleeding with one case of catastrophic hemorrhage. the most common bleeding sites were around tracheostomy and ecls cannulation sites. the use of ecls in pregnancy is controversial. there are no guidelines for its use in this patient population yet, however recent reports over the last years showed that ecls has been successfully used in pregnancy and postpartum patients for cardiopulmonary failure with good maternal and fetal outcomes. ecls seems to be underutilized in this patient population. careful patient selection and cautious anticoagulation management can reasonably minimize bleeding risk. acute hypoxic respiratory failure and ards secondary to trauma is well recognized and is associated with high mortality and morbidity. pulmonary contusions occur in % in these patients. reid et al. [ •] reported their experience over years (april to april ) utilizing ecls for respiratory indications in trauma patients. fifty-two patients were included in that review with moderate to severe head injury in ( %) cases. all patients had multiple traumas and most of them ( %) had rib fractures with hemothorax and pneumothorax. thirty-one percent of these patients underwent surgical procedure while on ecls. vv ecls and ecco r were the main modes of support. their overall survival rate was %. out of the patients with intracranial bleeding, ended up with an external ventricular drain and three underwent craniotomies. multiorgan failure was the major cause of mortality; only one patient suffered from catastrophic bleeding and one with severe brain damage who eventually died. other reports [ , ] including more recent study by guirand et al. [ ] supported the use of ecls mostly by vv support in trauma ards patients with favorable outcomes. ecls is still considered the most common form of mechanical circulatory support in patients with cardiac failure unresponsive to conventional therapies. cardiac ecls has been consistently growing among different age groups over the last few years [ , , ••, , ] . as of july , a total of , cardiac ecls cases reported at the registry with an overall survival rate of . %, with % in neonates, % in pediatrics, and % in adults (elso). most of these patients (> %) is supported by va ecls either via cervical or central cannulation, the later being more common in the immediate post-operative period in neonates. pediatric patients with myocarditis have the best chances to survive (up to %). cardiac ecls is provided as a mean to organ recovery or as a bridge to transplant. newer devices like ventricular assist devices (vad) are more durable. its use for cardiac support is growing over the last years. vads are smaller and simpler devices compared to ecls, which allows early mobilization. ecpr is defined as applying ecls during cardiac arrest while performing cardiopulmonary resuscitation, or when repetitive arrest events occur without return of spontaneous circulation for > min [ ] . as of july , there are cases of ecpr reported to the elso registry with in neonates, in pediatrics, and in adults with overall survival rate of % ( % in neonates and pediatrics and % in adults) [ ] . the use of ecpr has increased over the years [ ] . pre ecpr acidosis, prematurity, and complication during ecls (crrt, intracranial bleeding, persistent metabolic acidosis) are associated with increase odds of death, while cardiac disease (e.g., arrhythmias, myocarditis) and neonatal respiratory illness are associated with improved outcome [ , , ••, , ] . recent reports showed the increasing use of ecpr in adult population with a trend towards decreased survival in recent years ( %). ecpr practice is institution dependant and it has been mainly limited to cardiac icus and for in-house arrest only. there have been few reports of ecpr use for out-of-hospital cardiac arrest with documented survival and intact neurological outcome [ , ] . the extracorporeal life support organization (elso) was founded in (https://www.elso.org). it is an international non-profit consortium of health care centers and individuals who are dedicated to the development, evaluation, and improvement of ecls. elso maintains the world's largest registry of data on ecls patients, with voluntary submission of this data. it provides elso centers with biannual reports on center-specific as well as worldwide number of cases, outcomes, and complications. other functions of elso include providing data for quality assurance and research, which has been growing. there are just fewer than , ecls publications listed in pubmed as of january . it also publishes the elso redbook, which is considered the definitive reference for ecls. elso has been partnering with its global chapters (euroelso, asia-pacific elso, latin american elso, and south and west asia elso) to advance ecls support internationally. there are published guidelines that are available for public describing useful and safe ecls practice; these guideline are revised regularly. bleeding is still a major complication during ecls. forty-six mechanical and patient related complications are recorded through the registry every year. oxygenator failure is the most common mechanical complication. table summarizes the most common events reported to elso as of july [ ] . ecls patient management is complex. it starts with patient selection and initiation of ecls. this is a large topic to cover in this context, so for that reason; we will briefly cover four major areas: ventilator management, anticoagulation, fluid and nutrition, and neurological management. understanding the role of ecls in respiratory indications is the key. it is important to mention that ecls is a support modality not a cure. it provides efficient gas exchange using an artificial lung, allows time for ecls providers to treat the underlying lung disease and prevent further iatrogenic lung injury. gentile ventilation is a very important principle in respiratory ecls. using high pressures trying to "open the lungs" is a dangerous maneuver that can lead to further lung injury and poor outcomes [ ] . applying lung "rest settings" on the ventilator should occur within the first few hours after initiation of ecls [ •] . this can be achieved in many formats. conventional ventilation using synchronized intermittent mandatory ventilation (simv) pressure control (pc) with pressure support (ps) is one way. minute ventilation will be brought down to a minimum. pc of cmh o above positive end expiratory pressure (peep) with a rate of , peep of - cmh o and ps of - is acceptable as long as the peak inspiratory pressure (pip) stays below and never exceeds cmh o. in addition to being protective of further iatrogenic lung injury, the use of this mode allows the providers to prevent any swings in pip that may occur using a volume control mode and enable them to objectively calculate the dynamic compliance of the lung as a measure of daily progress. hfov can also be used during ecls. it can be advantageous in cases of severe air leak syndrome, severe pulmonary edema, and pulmonary hemorrhage. it is important to minimize the minute ventilation and bring ventilation settings (amplitude and frequency) to a minimum. mean airway pressure should be kept below cmh o and never exceeds . it is uncommon to maintain patients on hfov during ecls course; its use could make it difficult to perform pulmonary toilet, assess tidal volumes, and patient might need heavy sedation. airway pressure release ventilation (aprv) or bi-level mode can be used emphasizing the same principles. p high can be set at - cmh o as long as the total pressures stay below cmh o and never exceeds . t high (time allowed for p high to be delivered) can be extended ( - s) to guarantee further recruitment as patient can breathe spontaneously during this. cpap with ps is a modality that is more acceptable now especially in awake patients with or without tracheostomy. patients, especially adults, can be extubated early in the ecls course. pulmonary toilet is pivotal in managing these patients, by frequent suctioning of mucous plugs and secretion. bronchoscopy should be used liberally for that purpose. saline installation in the endotracheal tube can facilitate that. other materials like pulmozyme and perfluorocarbon liquid have been used with variable results. the interaction between the blood and the biomaterials of the ecls circuit can lead to unwarranted effects by activating platelets and coagulation factors that promotes thrombosis and consumptive coagulopathy [ ] . as a result, using anticoagulation therapy is needed. the use of unfractionated heparin (ufh) has been the gold standard. it is cheap, available, has a short half-life, and can be reversed with protamine sulfate if needed. on the other hand, ufh is an indirect anticoagulant; it works by potentiating antithrombin (at) effect to inhibit "free" thrombin; it does not inhibit clot-bound or circuitbound thrombin. monitoring and managing anticoagulation therapy during ecls is challenging especially in neonates and young children. laboratory monitoring is typically done act has been, for decades, the most commonly used routine whole blood test [ ] . it measures how many seconds that takes a blood sample to form a clot. it is inexpensive, and can be performed quickly at bedside test, but it is not specific and provides a general idea about coagulation. for example, a prolonged act (> ) could indicate thrombocytopenia, platelet dysfunction, consumptive coagulopathy, excessive heparin, or a combination of these events. more detailed testing is needed to determine the next appropriate action [ ] . acceptable range is - s. that can be lower ( s) if there are concerns about bleeding. aptt is a plasma-based test that measures time to fibrin formation. it has been an acceptable mean to titrate anticoagulation therapy, and there is decent experience among providers, but it could show a lot of variability, and its use in critical conditions might be questionable [ ••] . acceptable range of . - . x patient baseline or - s is reasonable. anti-xa assay (heparin level, heparin assay) is a plasmabased test that measures the ufh effects based on its ability to catalyze at inhibition on factor xa. appropriate anti-xa levels of . - . unit/ml correlate well with ufh effects and showed to minimize blood sampling, blood products transfusions with less bleeding and clotting complications. thromboelastogram (teg) is another whole blood point of care test that examines the clot formation, strength, and fibrinolysis. it is not widely available; there is an element of subjectivity to the results interpretation, and there is limited data on improved outcomes with its use. direct antithrombin inhibitors' use has been documented in ecls in cases of heparin induced thrombocytopenia (hit) or heparin resistance. bivalirudin and argatroban have been used with some promising results [ , ] . for more details on this topic, please visit elso website-guidelines https:// www.elso.org/resources/guidelines.aspx. optimal sedation and analgesia during ecls remains poorly defined. many studies have demonstrated the need to escalate sedation requirement during ecls [ ] [ ] [ ] [ ] [ ] . fentanyl and morphine [ ] are the most commonly used opioids in ecls. escalation of these medications have been documented and attributed to an increase in the volume of distribution, increased sequestration in the circuit tubing and oxygenator, and decreased metabolism, at least in the case of morphine use [ ] , to its active metabolites. the use of benzodiazepine (e.g., midazolam and lorazepam) and dexmedetomidine [ , ] are common practices in different age groups. dexmedetomidine is an α- adrenergic receptor agonist that has analgosedative effects that may facilitate weaning heavy sedation. it is not uncommon for ecls patients to be heavily sedated during the first few days. the use of muscle relaxation is commonly used in cannulation, decannulation, and during procedures. it should not be a routine practice. nondepolarizing agents are commonly used in picu settings including ecls patients when necessary. these agents can be used as continuous infusion (e.g., cisatracurium, atracurium, vecuronium) or bolus dosing (rocuronium, vecuronium). cisatracurium is considered an appropriate agent to use in ecls patients because of its reasonable recovery time once turned off ( - min), safety profile, and ability to use in patients with multiorgan failure. cisatracurium is eliminated by ester hydrolysis and hofmann elimination. it is recommended to perform regular neurological examination on heavily sedated patients early in their course once or twice a day by lifting off the muscle relaxation and possibly reducing the sedation infusion. the goal of minimizing sedation while maintaining comfort is ideal. non-medical maneuvers including child life support, music, playing games, reading books, and family involvement play a major role in caring for these patients with less sedation, less withdrawal, and faster recovery. frequent neurological examination is critical during ecls. intracranial complications especially during va ecls are serious [ ] [ ] [ ] . clinicians should be vigilant, performing neurological assessments daily. a sudden unilateral change in the diameter of one pupil should prompt an aggressive investigation for an acute intracranial pathology especially bleeding. serial head ultrasound can be performed at the bedside for neonates and young infants receiving ecls to assess for intracranial abnormalities. adequate nutrition is pivotal for recovery in critical illness. enteral nutrition, even at trophic amounts, is preferred [ ] to maintain gut integrity, reduce risk of bacterial translocation and risk of tpn-related cholestasis, but total parenteral nutrition can be used if needed. maintaining strict fluid balance in ecls patients is crucial. fluid overload has been associated with increased mortality [ , ••] . it is not unusual to require large volume of fluids at the initiation of ecls, but clinician should be proactive instead of reactive in fluid management. the use of diuretics, concentrating medication infusion, and the judicious early initiation of ultrafiltration and crrt has proven to reduce ecls duration and length of stay [ , , , ] . ecls or ecmo is an acceptable mode of support in neonates, pediatrics, and adults with acute cardiorespiratory failure unresponsive to conventional therapies with an overall survival of %. the use of ecls is growing especially in adult respiratory indications, and will continue to grow. bleeding is still one of the most challenging complications. the development of new devices over the last few years resulted in a much simpler, safer, and prolonged ecls support. new styles of patient management including minimal sedation, spontaneous breathing, early tracheostomy, and early mobilization are becoming more common. the next generation of ecls devices will be easier to manage by caregivers, less thrombogenic, and more durable with less or no need for systemic anticoagulation. as per dr. bartlett, these are the highlights of the next era in ecls care "ecmo iii." conflict of interest omar al-ibrahim and christopher m.b. heard declare they have no conflict of interest. human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by any of the authors. wb saunders. neonatal ecmo has resulted in significant improvement the survival of neonates with cardiorespiratory failure unresponsive to conventional therapy 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oxygenation for influenza a (h n ) severe respiratory failure in japan the italian ecmo network experience during the influenza a (h n ) pandemic: preparation for severe respiratory emergency outbreaks extracorporeal membrane oxygenation (ecmo) in patients with h n influenza infection: a systematic review and meta-analysis including studies and patients receiving ecmo extracorporeal membrane oxygenation for pandemic influenza a (h n )-induced acute respiratory distress syndrome: a cohort study and propensity-matched analysis extracorporeal membrane oxygenation in pandemic flu: insufficient evidence or worth the effort? referral to an extracorporeal membrane oxygenation center and mortality among patients with severe influenza a (h n ) extracorporeal membrane oxygenation for influenza a(h n ) acute respiratory distress syndrome extracorporeal membrane oxygenation in adults with severe respiratory failure: a multi-center database the use of ecls in acute respiratory failure in adults is growing rapidly. ecls care is complex, high risk and costly modality. adult ecls should be conducted in centers with experience extracorporeal membrane oxygenation for respiratory failure in adults contemporary extracorporeal membrane oxygenation for adult respiratory failure: life support in the new era emerging indications for extracorporeal membrane oxygenation in adults with respiratory failure extracorporeal life support for adults with severe acute respiratory failure active rehabilitation and physical therapy during extracorporeal membrane oxygenation while awaiting lung transplantation: a practical approach concept of "awake venovenous extracorporeal membrane oxygenation" in pediatric patients awaiting lung transplantation extracorporeal membrane oxygenation in awake patients as bridge to lung transplantation. american journal of respiratory and critical care medicine h n influenza virus infection during pregnancy in the usa pandemic (h n ) influenza, pregnancy and extracorporeal membrane oxygenation extracorporeal membrane oxygenation for severe ards in pregnant and postpartum women during the h n pandemic modern use of extracorporeal life support in pregnancy and postpartum ecls use in well-selected pregnant and postpartum patients appears to be safe and associated with encouraging maternal and fetal survival rate with low complications ):r . the use of vv ecls and pumpless (av) in patients with acute respiratory failure and severe thoracic trauma represents an excellent support modality traumatic lung injury treated by extracorporeal membrane oxygenation (ecmo) application of ecmo in multitrauma patients with ards as rescue therapy venovenous extracorporeal life support improves survival in adult trauma patients with acute hypoxemic respiratory failure: a multicenter retrospective cohort study extracorporeal membrane oxygenation to support cardiopulmonary resuscitation in adults extracorporeal membrane oxygenation to aid cardiopulmonary resuscitation in infants and children ecls indications are expanding especially in children and adults with cardiorespiratory failure. adverse effects during ecls are common and underscores the need for skilled ecls management and trained ecls personnel and teams american heart association national registry of cpr investigators. outcomes among neonates, infants, and children after extracorporeal cardiopulmonary resuscitation for refractory inhospital pediatric cardiac arrest: a report from the national registry of cardiopulmonary resuscitation extracorporeal cardiopulmonary resuscitation outcomes in term and premature neonates extracorporeal cardiopulmonary resuscitation versus conventional cardiopulmonary resuscitation in adults with out-of-hospital cardiac arrest: a prospective observational study extracorporeal cardiopulmonary resuscitation for patients with out-of-hospital cardiac arrest of cardiac origin: a propensity-matched study and predictor analysis mechanical ventilation during extracorporeal life support (ecls): a systematic review extracorporeal membrane oxygenation in the adult: a review of anticoagulation monitoring and transfusion variability in anticoagulation management of patients on extracorporeal membrane oxygenation: an international survey. pediatric critical care medicine: a journal of the society of critical care medicine and the world federation of pediatric intensive and critical care societies evaluation of heparin assay for coagulation management in newborns undergoing ecmo anticoagulation management using unfractionated heparin in ecls is variable. bleeding and thrombosis are common in pediatric ecls and are associated with increased mortality the dosing and monitoring of argatroban for heparin-induced thrombocytopenia during extracorporeal membrane oxygenation: a word of caution bivalirudin versus heparin as an anticoagulant during extracorporeal membrane oxygenation: a case-control study increased sedation requirements in patients 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children: clinical article brain injury associated with neonatal extracorporeal membrane oxygenation in the netherlands: a nationwide evaluation spanning two decades positive fluid balance at day of adult ecls is an independent predictor of -day mortality. that may reflect disease severity continuous renal replacement therapy with an automated monitor is superior to a free-flow system during extracorporeal life support enhanced fluid management with continuous venovenous hemofiltration in pediatric respiratory failure patients receiving extracorporeal membrane oxygenation support key: cord- -f hks authors: chronopoulos, antonios; kalluri, raghu title: emerging role of bacterial extracellular vesicles in cancer date: - - journal: oncogene doi: . /s - - - sha: doc_id: cord_uid: f hks shedding of microbial extracellular vesicles constitutes a universal mechanism for inter-kingdom and intra-kingdom communication that is conserved among prokaryotic and eukaryotic microbes. in this review we delineate fundamental aspects of bacterial extracellular vesicles (bevs) including their biogenesis, cargo composition, and interactions with host cells. we critically examine the evidence that bevs from the host gut microbiome can enter the circulatory system to disseminate to distant organs and tissues. the potential involvement of bevs in carcinogenesis is evaluated and future research ideas explored. we further discuss the potential of bevs in microbiome-based liquid biopsies for cancer diagnostics and bioengineering strategies for cancer therapy. extracellular vesicles (evs) are released by all three domains of life-eukaryotes, bacteria, and archaea-and represent a universal, evolutionarily conserved mechanism for intercellular communication [ ] . our growing appreciation of the functional significance of the human microbiota in health and disease has triggered a marked interest in microbial-derived evs and their functional role in intrakingdom and inter-kingdom communication, transfer of nutrients within microbial communities, delivery of virulence factors and toxins, horizontal gene transfer, and modulation of host immunity [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in recent years, the advent of next generation sequencing technologies combined with large scale initiatives like the nih-funded human microbiome project and the eufunded metagenomics of the human intestinal tract consortium has expanded our understanding of the human microbiome and helped shape an evolving view of the human body as a "supraorganism" that harbors trillions of bacterial and human cells in~ : ratio [ , [ ] [ ] [ ] . these large consortia have revealed staggering microbial gene diversity in the human microbiome as well as tremendous inter-individual variability in the microbial species inhabiting the human body [ , ] . interestingly, while the human genome project has revealed a total number of , genes for the human genome [ ] , currently accepted estimations from analyzing the bacterial communities in the gut have yielded more than , bacterial genes vastly outnumbering the number of genes contributed by the human genome [ , ] . a healthy human host is inhabited by more than microbial species [ ] . most of those belong to a few dominant taxonomic groups, or phyla (actinobacteria, bacteroidetes, firmicutes, proteobacteria, cyanobacteria, fusobacteria) that gives some level of consistency across individuals at the higher taxonomic levels; however, the relative proportions of those phyla, as well as the individual microbial species and genes can vary dramatically across individuals, even in the case of monozygotic twins [ , ] . individual humans may be . % identical to one another in terms of their host genome (with . % of the variation being mostly snps and small indels) but completely different (up to %) in their microbiomethe collective genome of one's microbial communities [ ] . the relationship of these complex microbial communities (bacteria, fungi, viruses, and archaea) with their human host can be either symbiotic (mutually beneficial), commensal (neutral co-existence), or potentially pathogenic [ ] . accumulating evidence has revealed microbe-host interactions can critically influence host health by manipulating host immune responses, nutrient metabolism, maintenance of the intestinal barrier, and protection from pathogen colonization [ , [ ] [ ] [ ] . similarly, disruption of the gut microbiome (a term known as microbial dysbiosis) has been critically implicated in a range of human diseases including cancer, mental health, and cardiovascular and immune disorders [ ] [ ] [ ] . the intersectionality of microbiome research and evs owing to advancements in both fields has emerged as promising research endeavor. the increasing appreciation that microbiota-derived ev can enter the systemic circulation and be detected in human body fluids is likely to stimulate completely new areas of investigation in microbiome research, biomarkers and liquid biopsies, bev-based therapeutics, onco-immunology, as well as fundamental microbial ev biology. bacterial cells communicate with their host and other bacteria through direct contacts and secretion of soluble products, such as metabolites (e.g., short-chain fatty acids), lipoglycans, quorum sensing peptides, nucleic acids, proteins, and membrane vesicles, also known as bacterial extracellular vesicles (bevs) [ , ] . both pathogenic and commensal bacteria secrete bevsspherical membrane-enveloped particles ranging in size from - nm that disseminate part of the biological content of the parent bacterium into the extracellular milieu [ , ] . detailed proteomic and biochemical analyses have shown that bevs carry diverse cargo including membrane-bound and periplasmic proteins, enzymes (such as autolysins) and toxins, polysaccharides, nucleic acids (dna and rna), and peptidoglycan [ , ] . however, there does not appear to be a single mechanism modulating bev export, nor universal markers for bev cargo [ ] . several lines of evidence suggest that bevs are heterogeneous in their structure, size, density, and molecular cargo composition, and this heterogeneity stems from the different biogenesis routes, the unique membrane envelope structure of the parental bacterium they originate from as well as the genetic background of the producing strain and the growth conditions [ ] . mounting evidence shows bevs can follow different formation routes, which can lead to distinct bev subtypes with different molecular cargo and thus potentially different biological function [ , ] . gram-negative bacteria follow two main pathways for vesicle formation. the first formation route involves blebbing of the outer membrane of the bacterial envelope, generating outer-membrane vesicles (omvs); and the second pathway entails explosive cell lysis forming outer-inner membrane vesicles (oimvs) and explosive outer-membrane vesicles (eomvs) [ , ] . gram-positive bacteria produce cytoplasmic membrane vesicles (cmvs) through endolysin-triggered bubbling cell death [ , ] . generally, omvs are the archetypal bevs and most heavily studied. the membrane blebbing process giving rise to omvs generally occurs through a disruption of crosslinks between the outer membrane and the underlying peptidoglycan cell wall layer. regardless of their biogenesis route, we collectively refer to all those vesicle subtypes as bevs. furthermore, gram-negative and gram-positive bacteria have a distinctly different cell envelope architecture, which is reflected in the membrane composition of the bevs they produce [ , ] . the cell wall of gram-negative bacteria consists of a thin layer of peptidoglycan (a polymer-like mesh made of sugars and amino acids) in the periplasmic space between two membrane bilayers; the inner (or cytoplasmic) and outer membrane. the outer membrane contains lipopolysaccharides (lps; also known as endotoxin) on its outer leaflet and various membrane-bound proteins and channels such as porins that facilitate non-vesiclemediated transport. in contrast, gram-positive bacteria completely lack an outer membrane but have a much thicker peptidoglycan cell wall, which is linked to the underlying cytoplasmic membrane via lipoteichoic acids (lta) [ , ] . mirroring this envelope architecture, gram-negative bevs consist of an outer membrane with an interior leaflet of phospholipids and an exterior leaflet of lps, which is known to engage toll-like receptor (tlr ) [ , ] . gramnegative bevs are typically enriched in various outermembrane proteins, such as ompa and encapsulate periplasmic luminal components. however, the presence of cytoplasmic cargo (dna, rna, virulence factors) is debated and likely contingent on the specific biogenesis route of the three different gram-negative bev subtypes (omv, oimvs, and eomvs). previous reports that have found nucleic acids and cytoplasmic proteins in omvs might not have been bona fide omvs, formed through controlled blebbing of the outer membrane as this mechanism does not give direct access to cytoplasmic cargo. instead, it is the biogenesis mechanism of the double bilayered oimv and/or the single bilayered-eomv subtypes that enables direct access to and encapsulation of cytoplasmic cargo. indeed, endolysin-mediated degradation of the peptidoglycan cell wall (a key process in explosive cell lysis) results in cell envelope fragments that can recircularize and enclose released chromosomal and plasmid dna and other nucleic acids or cytoplasmic components. gram-positive bev, also known as cmvs as previously mentioned contain both membrane and cytoplasmic components and show lta on their surface that can engage the toll-like receptor (tlr ) [ , ] . a number of other environmental factors can also affect the rate of vesicle production and even bias toward certain biogenesis routes. some of these include media composition, temperature, growth phase, iron and oxygen availability, exposure to antibiotics, and genotoxic stress [ , ] . the localization of chromosomal dna in bevs from various gram-negative pathogenic bacteria (pseudomonas aeruoginosa, porphyromonas gingivalis, salmonella typhimurim) is mostly surface-associated (or extraluminal) with smaller amounts located in the intraluminal space [ ] . sequencing of the intraluminal bev dna has been found to be enriched in specific regions of the bacterial chromosome involved in virulence, stress response, antibiotic resistance, and metabolism. it remains speculative at this point, whether surface-associated versus intraluminal bev dna serves different functions. it is not unreasonable to assume a potential role for external dna in biofilm formation versus a role for internal bev dna in intercellular communication and horizontal gene transfer of antibiotic resistance or virulence genes [ ] . in addition to potentially modulating the innate immune response via or more cytosolic dna sensors, the possibility that pathogenic bev-derived dna can be transferred and detected in the nucleus of non-phagocytic cells (e.g. epithelial cells) [ ] , raises the intriguing possibility that bacterial genetic material could be transferred to human somatic cells and integrated into the host genome. integration of bacterial dna sequences has been in fact detected more frequently in human cancer cells versus normal cells, especially in gastrointestinal (gi)-related tumors with close proximity to the gut microbiome, suggesting a potential role of bacterial dna in carcinogenesis [ ] . bevs contain numerous microbe-associated or pathogenassociated molecular patterns (mamps/pamps) including lps, lipoproteins, peptidoglycan, and bacterial nucleic acids. the mamp content of bevs enables them to engage with host pattern recognition receptors (prr) in immune cells as well as nonimmune cells (e.g., epithelial cells at mucosal surfaces) to promote host pathology, immune tolerance, or confer protective immunity [ ] . the diverse immunomodulatory effects of bevs depend largely on the specific parental bacterium and its relationship with the host. for instance, bevs from pathogenic bacteria have the potential to exacerbate infection by dampening immune responses [ , ] , or trigger an overexaggerated immune reaction resulting in sepsis [ , ] . in contrast, bevs from symbiotic or commensal bacterial species in the gi tract promote maturation and immunological tolerance to confer protection from colitis or sepsis [ , ] . depending on the localization of prrs and the specific route of bev uptake, the recognition of bev-associated mamps/pamp can occur in various host cellular compartments, including the plasma membrane, endosomes, and cytoplasm [ ] (fig. ) . cell surface members of the tlr family, namely tlr and tlr , recognize extraluminal bev ligands such as lps and lta molecules, peptidoglycan, and lipoarabinomannan [ ] [ ] [ ] [ ] [ ] . the cytosolic receptors nucleotide-binding oligomerization domain-containing protein (nod ) and (nod ) are directly involved in sensing peptidoglycans (key component of bacterial cell wall) present in bevs secreted from pathogenic or commensal bacteria [ ] [ ] [ ] [ ] . nod / are key components of innate immunity and critical in host defense against bacterial infections and modulation of inflammatory responses. more recently nod / have been involved in maintaining intestinal homeostasis and microbiota balance [ , ] . intraluminal bev nucleic acids can be detected via dna-sensing and rna-sensing receptors. following endocytosis, bev rna cargo can be sensed through endosomal tlrs including tlr , tlr , tlr , and tlr . similarly, rnas delivered into the cytoplasm following fusion of bevs with the host cell plasma membrane can activate cytosolic rna sensors such as rig-i-like receptors [ ] . in a similar way, bev dna cargo can be sensed through the endosomal tlr or the cytosolic dnasensing cyclic gmp-amp synthase-stimulator of interferon genes pathway, although direct evidence for the latter is lacking [ , , ] . various routes of bev endocytosis have been described in host cells including macropinocytosis, lipid-raft-dependent and lipid-raft-independent endocytosis, as well as dynamin-, caveolin-, and clarithindependent entry [ ] . the utilization of different paths of uptake may reflect the size heterogeneity of bevs and the size selectivity of each route of endocytosis. in general, prr activation triggers the activation of kinases and transcription factors, that lead to the production of cytokines and chemokines resulting in the recruitment of immune cells and upregulation of co-stimulatory molecules commonly involved in adaptive immunity. tlrs for instance, signal via the adapter proteins myeloid differentiation primary response or tir-domain-containing adapter-inducing interferon-β, leading to downstream activation and nuclear translocation of transcription factors including nuclear factor kappa-lightchain-enhancer of activated b cells, interferon-regulatory factor, and activator protein- to induce production of proinflammatory cytokines and type i interferons [ , ] . there exists a growing consensus that bevs from the resident microbiota can enter the systemic circulation [ , [ ] [ ] [ ] [ ] [ ] [ ] . bevs released by bacteria in the gut lumen can cross the epithelial barrier to gain access into the underlying submucosa enabling them to interact with various resident immune cell populations (dendritic cells, neutrophils and macrophages) as well as potentially disseminate more widely around the body via the systemic or lymphatic circulation to reach distant tissues and organs or even the brain (fig. ) . the presence of systemic circulating bevs was recently reported by tulkens et al. in the plasma of patients with altered intestinal barrier function [ ] . of note, a number of communicable and noncommunicable diseases as well as lifestyle factors can trigger gut microbial dysbiosis thereby altering intestinal permeability, including obesity, diabetes, antibiotic use, diet and caloric restriction, or sleep deprivation. in the aforementioned study, patients with hiv, inflammatory bowel disease, or intestinal mucositis were found to have elevated circulating lps-positive bevs relative to healthy controls. the level of circulating bevs correlated positively, but modestly, with the plasma levels of zonulin-a biomarker of epithelial barrier integrity-that phosphorylates zo- proteins in epithelial and endothelial cells leading to tight junction disassembly. while these bevs are likely to be originating by gut bacteria, the authors did not fully prove it leaving the possibility of contributions from microbial niches in other body sites. simulating compromised tight junction integrity using an in vitro colitis model (caco- epithelial monolayer challenged with dss) also resulted in paracellular translocation of bevs. the same group by tulkens et al. most recently reported a detailed protocol for recovering bevs with high specificity from human body fluids, including blood plasma and stool through the sequential implementation of sizeexclusion chromatography and density-gradient ultracentrifugation [ ] . the underlying physical property that distinguishes bevs from host-derived eukaryotic evs (eevs) in blood plasma is their differential buoyant density. when run on a density gradient, eevs float typically at . - . g/ml while bevs are slightly heavier at . - . g/ml. this allows for label-free isolation of bevs from eevs, although subsequent biochemical characterization is typically needed. this was the first study that provided a preliminary characterization of bevs in human body fluids with ompa and lps being the markers for bev identification. however, a low yield of bev in blood plasma (~ bev/ml) was typically noted. interestingly, plasma contained bevs from both gram-negative (tlr reporter assay) and gram-positive bacteria (tlr reporter assay) and immunogold electron microscopy also identified the presence of both single-bilayered and double-bilayered bevs, the latter being indicative of oimvs, which typically contain cytoplasmic cargo and nucleic acids. the presence of chromosomal dna in circulating oimvs was not pursued further and s rrna sequencing data are lacking. implementation of rigorous experimental controls is imperative for samples with low microbial biomass that are inherently prone to misinterpretation due to contaminating molecules from commonly used laboratory reagents in dna extraction kits and library preparation [ , ] . while these studies leave the impression that entry of bevs in the circulatory system occurs only under certain conditions of compromised gut epithelial barrier integrity, this phenomenon may be common even in the steady state of healthy individuals. indeed, a recent study looked into the biodistribution of fluorescently labeled bevs (derived from the major human gut commensal bacteria bacteroides thetaiotaomicron) following oral administration in mice under normal healthy conditions [ ] . while the majority of the labeled bevs remained in the lumen of the gi tract, a small population could enter the circulatory or lymphatic systems via the gi tract. some of the bevs were found to accumulate in the liver, suggesting that they can transmigrate through the intestinal epithelium and enter the hepatobiliary system through the portal vein. a portion of the bevs could also be detected in the heart and lungs suggesting bevs can cross several host cellular barriers including epithelial barrier and the lymphatic-vascular endothelium to enter the bloodstream and disseminate systemically. even if the gut epithelial barrier is not compromised leading to increased paracellular transport, bevs could use other mechanisms to enter the circulatory system, such as active trans-cellular migration across the intestinal epithelium [ ] . in fact, active trans-cellular migration across epithelial monolayers has been observed for eukaryotic exosomes (e.g., endocytosis, mvb formation, and exocytosis across the other side of the layer) and has even been suggested as a possible mechanism for breaching the blood-brain barrier [ ] . luminal antigens and bevs may also be captured by dendritic cells (from the underlying lamina propria) and transported through the intestinal epithelium or via the assistance of mucus-secreting goblet cells [ , [ ] [ ] [ ] . in addition, intestinal m-cells (specialized epithelial cells of the mucosa-associated lymphoid tissue) could be involved in the translocation of luminal bevs to the submucosa and systemic circulation [ ] . macrophages infected with pathogenic bacteria are known to release exosomes with bev-associated components that can elicit pro-inflammatory responses. the uptake, intracellular trafficking, and processing of bev cargo by the intestinal epithelium needs to be further investigated to examine the possibility of de novo secretion of host epithelial-cellderived exosomes carrying luminal bev-associated cargo; this would represent a mechanism of transferring luminal antigens and bevs directly to antigen-presenting cells in the submucosa. circumstantial evidence for this exist from a study in which exosomes recovered from the basolateral compartment of bev-treated epithelial cells were found to encapsulate bev antigens as well as from a co-culture model in which exposure of dendritic cells to conditioned media from bev-stimulated epithelial cells resulted in polarization to a mixed t h -type and t h -type response [ , [ ] [ ] [ ] . a case for the existence of blood-derived bevs has also been reported in a transgenic mouse model of alzheimer's disease [ ] . metagenomic sequencing of the blood-derived bev dna revealed taxonomical diversity that reflected the diversity of the intestinal microbiota as well as a distinct bev-associated microbial landscape relative to wild-type controls. blood-derived bevs could represent an alternative to fecal sampling for profiling the gut microbiome and evaluating pathogenic variations in the intestinal microbiota (dysbiosis) in the context of neurodegenerative diseases. a few studies have also reported the presence of bacterial nucleic acids in the brain [ ] . in view of the presence of bevs in the bloodstream and their ability to cross boundary epithelial layers, it is interesting to speculate that a fraction of the circulating bevs might gain access to the brain through the blood-brain barrier or alternatively be produced by brain-resident bacteria. the gut microbiome is highly dynamic and impacted by a plethora of environmental factors such as diet, exercise, sleep habits, and medications. the presence of bevs in systemic circulation through their translocation from gi tract or other microbial niches has challenged our notion that blood is a sterile compartment and has put bevs in the spotlight as long-range "hormonal-like" mediators of interkingdom communication. future investigations are expected to delve deeper into the cross-talk between the resident microbiota and distant organs mediated by systemically circulating bevs, as this would further expand our understanding of how the human microbiome can regulate tissue and organ homeostasis in health and disease. microbial dysbiosis is a major contributing factor in oncogenesis and tumor progression for a number of gi-tractrelated malignancies, including gastric, colorectal, liver, and pancreatic cancer, and might even influence the treatment response to chemotherapy and immunotherapy [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the mechanisms by which bacteria affect carcinogenesis and tumor progression are largely unknown. some of the tumor-promoting mechanisms could be direct such as by inducing genomic instability or indirect by generating a proinflammatory tumor microenvironment (tme) and suppression of immunosurveillance [ , ] . although bevs have been implicated in the pathogenesis of inflammatory disease [ , ] , their role in cancer remains unknown. it is also uncertain whether the tumor-promoting or tumorinhibiting effects of the inter-kingdom cross-talk between the intestinal or intra-tumoral bacteria and the host cells in the tme is mediated through secreted microbial metabolites such as scfas or bevs. it is a tantalizing possibility that the gut microbiome may be implicated in all carcinomas including those that are ostensibly remote from the gi lumen (or its drainage) through the immunomodulatory action of systemically circulating bevs on recipient cells in distant organs, or even participating in the formation of pre-metastastic niches. some indirect preliminary evidence toward this direction came out from a seminal retrospective pan-cancer examination of whole-genome sequencing datasets in the tcga ( cancer types from , treatment-naïve patients totaling , samples) for microbial reads that found unique microbial signatures in tissue and blood that could discriminate between and within most major types of cancer, including tumors distally from the gi tract [ ] . the authors used various computational approaches, including independently trained machine learning models to filter, normalize, and classify microbial sequences. after stringent filtering approaches to remove potential contamination that discarded up to . % of total sequencing reads, a total of . % of the total reads could be mapped to sequences of microbial origin and resolved to a particular genus. the tcga-derived blood-borne microbial dna (mbdna) signatures could discriminate within and between most cancer types even in low-grade tumor stages and in cancers lacking genomic alterations. apart from the retrospective bioinformatic analysis, the group further validated the mbdna models while benchmarking against plasma-based cell-free tumor dna (ctdna) assays in a separate cohort, which included a group of healthy controls and a group of individuals with advanced-stage cancer, including prostate cancer, lung cancer, and melanoma. remarkably, plasmabased cell-free mbdna retained strong generalizable discrimination between healthy controls and grouped patients with cancer with specificity and sensitivity exceeding % while also retaining high discriminatory capacity in pairwise comparisons between the three individual types of cancer and their respective healthy controls, except for melanoma. this study raises the possibility of using microbial-based liquid biopsies for early detection of certain cancers and tumors lacking known mutational drivers. such a microbiome-based oncology diagnostic tool might provide distinct advantages over regular ctdna assays in predicting and stratifying patients based on differential survival and treatment response. this is because oncogenesis can proceed at variable rates in hosts with similar cancer mutational landscapes but with host microbial factors playing a much bigger role. in fact, pancreatic tumors from long-term survivors have a high quantity and quality of neoantigens [ ] , that exhibit homology to infectious disease-derived peptides, suggesting a neoantigen molecular mimicry with microbial epitopes, and demonstrating host microbial factors can be predictive of patient outcomes. this was corroborated with a recent study showing that the tumor microbiome in pdac patients is predictive of long versus short-term survival irrespective of the genomic composition of the tumor [ ] . it remains undetermined whether the observed microbial nucleic acids in the blood came from live or lysed bacteria or bevs. the presence of live bacteria in blood is improbable as it would likely cause some degree of bacteremia but conclusive data could be made available through culturomics, rather than metagenomics, to identify the existence of otherwise hard-to-culture live blood-borne bacteria. the most likely scenario, given the results obtained from the plasma-derived, cell-free dna validation cohort as well as the accumulating evidence arguing for the presence of dna-containing circulating bevs, that at least a significant portion of those nucleic acids is associated with bevs. more work will be needed to determine whether this is the case, and whether the dna-containing microbial evs are driving cancer or are merely passengers. to date there is no mechanistic study investigating how bevs can impact oncogenesis and tumor progression, and their role is likely to be nuanced and context-dependent. previous studies of bevs in infectious diseases could provide hints for their putative role in cancer. for instance, microbial dysbiosis in cancer could trigger the systemic release of microbiota-derived bevs that could act as tumorpromoting entities by invoking tolerogenic immune reprogramming of the tme. this could be achieved through different routes by bevs driving suppressive cellular monocytic differentiation in a tlr-dependent manner, to indirectly elicit t-cell anergy [ ] . the distal action of systemic gut-derived circulating bevs might also be the missing link between conditions associated with persistent disturbances in gut microbiota and metastatic dissemination of pre-established tumors. bev-associated mamps could interact with host cells in distant organs to initiate proinflammatory signaling and trigger alterations in the myeloid landscape to foster pre-metastatic niches for future colonization. from a therapeutic standpoint one could speculate that systemic administration of commensal healthy host-derived bevs directly to tumor-bearing hosts could represent a superior alternative to fecal microbiota transplantation that is currently being pursued in clinical trials [ ] . bevs possess several intrinsic properties that have made them appealing candidates for vaccine development against infectious pathogens. bevs exhibit high stability to a wide range of temperatures and treatments and are nonreplicative in nature and thus safe, and carry many of the same immunogenic surface-and membrane-associated components of their parental bacterium [ ] . depending on the originating species, bevs can stimulate both humoral and cell-mediated immunity and together with their nanoparticulate nature, provide them with their own adjuvanticity, as they are able to enhance t-cell response to antigens. for instance, the clinically approved omv-based cmenb vaccine containing three highly immunogenic proteins confers broad protective antibody responses against different neisseria meningitidis (n. menigitidis) serogroup b isolates [ ] . notably, the same omv-based vaccine that protects against n. meningitidis also confers a level of cross-protection against neisseria gonorrhoeae likely due to the genetic and antigenic similarity shared between the two pathogens [ , ] . the endotoxicity of bevs can be easily manipulated through genetic engineering techniques. one could speculate that bevs from certain commensal bacteria may have therapeutic value. in the future, we expect to see surging interest in the potential of bevs as cancer immunotherapeutic agents to elicit durable antitumor immune responses or alternatively as personalized or universal cancer vaccines. the potential of bevs in cancer immunotherapy was highlighted by a recent report showing that systemic intravenous administration of gram-negative bevs from the genetically modified escherichia coli (e. coli) msbb −/− strain (endotoxin-free) has a selective tropism for tumor tissue (potentially through the epr effect) and a remarkably capability of inducing long-term antitumor immune responses through the production of cytokines cxcl and interferon-γ that can fully eradicate established tumors without notable adverse effects [ ] . similar antitumor effects were also observed for the grampositive bevs derived from lactobacillus acidophilus (l. acidophilus) and staphylococcus aureus (s. aureus). there is also an enormous potential in using genetic engineering techniques to modify bacteria and subsequently purify recombinant bevs for use as cancer vaccines. bevbased cancer vaccines decorated with multiple heterologous tumor antigens on their surface and/or immunostimulatory bacterial dna (cpg motifs) for targeted delivery to antigen-presenting cells hold immense potential for eliciting a strong durable antitumor immune response potentially in conjunction to ctla- and anti-pd immunotherapies. conflict of interest md anderson cancer center and rk hold patents in the area of exosome biology and are licensed to codiak biosciences, inc. md anderson cancer center and rk are stock equity holders in codiak biosciences, inc. rk is a consultant and scientific adviser for codiak biosciences, inc. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. the biology, function, and biomedical applications of exosomes current understanding of the human microbiome membrane vesicle release in bacteria, eukaryotes, and archaea: a conserved yet underappreciated aspect 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title: viral proteins that enhance membrane permeability date: journal: viral membrane proteins: structure, function, and drug design doi: . / - - - _ sha: doc_id: cord_uid: dvxrwzqw nan during the infection of cells by animal viruses, membrane permeability is modified at two different steps of the virus life cycle (carrasco, ) (figure . ). initially, when the virion enters cells, a number of different-sized molecules are able to co-enter the cytoplasm with the virus particles (fernandez-puentes and otero and carrasco, ) . membrane potential is reversibly destroyed, being restored several minutes later. endosomes are involved in the co-entry process, since inhibitors of the proton atpase block early permeabilization even with viruses that do not require endosomal function. a chemiosmotic model has been advanced to explain the molecular basis of early membrane modification by virus particles (carrasco, ) . the viral molecules involved are components of virions: glycoproteins when enveloped particles are analyzed or, still unidentified, domains of the structural proteins in the case of naked viruses. attachment of the particle to the cell surface receptor does not alter membrane permeability by itself. inhibitors that hamper virus decapsidation, still allowing virus attachment to the cell surface, block early membrane permeabilization (almela et al., ) . at late times of infection, when there is active translation of late viral mrnas, the plasma membrane becomes permeable to small molecules and ions (carrasco, ) ( figure . ). different viral molecules may be responsible for this late enhancement of membrane permeability, including viroporins (gonzalez and carrasco, ) , glycoproteins, and even proteases (chang et al., ; blanco et al., ) . this chapter is devoted to reviewing some characteristics of membrane permeabilization by viral proteins. in addition, the methodology used to assay enhanced permeability in animal cells is described. finally, the design of selective viral inhibitors based on the modification of cellular membranes during virus entry or at late times of infection is also discussed. a number of hydrophilic molecules, including some antibiotics, poorly permeate through cellular membranes (contreras et al., ; lacal et al., ) . this is the case of hygromycin b, anthelmycin, blasticidin s, destomycin a, gougerotin, and edein complex. the aminoglycoside antibiotic hygromycin b (mw ) is produced by streptomyces hygroscopicus. this hydrophilic molecule is an efficient inhibitor of protein synthesis in cell-free systems but interferes very poorly with translation in intact cells. however, the modification of the plasma membrane by viruses or by other means leads to a rapid blockade of translation (carrasco, ) . concentrations of the antibiotic ranging from about . - mm are added to the culture medium and protein synthesis is estimated by incubation with radioactive methionine for hr (see figure . ) (gonzalez and carrasco, ) . in addition to its simplicity, the hygromycin b test has a number of advantages for assaying changes in membrane permeability. one is its great sensitivity, and another is that this test measures membrane modifications only in cells that are metabolically active. moreover, in cultures where some cells are uninfected, hygromycin b would only enter virus-infected cells that are synthesizing proteins. the hygromycin b test has been applied with success to prokaryotic (lama and carrasco, ) and eukaryotic cells, including yeast carrasco, , ) and mammalian cells (gatti et al., ; gonzalez and carrasco, ) . alpha-sarcin is unable to pass through these pores. alpha-sarcin is a protein of amino acid residues, which is produced by aspergillus giganteus (oka et al., ) . this protein inhibits translation by modifying ribosomes in an enzymatic manner. thus, a molecule of alpha-sarcin is able to inactivate a great number of ribosomes by hydrolysis of the a -g phosphodiester bond in the s rrna (chan et al., ) . this toxin does not enter mammalian cells because it does not attach to the cell surface and is therefore unable to cross the plasma membrane. however, alpha-sarcin efficiently interferes with protein synthesis in cell-free systems or in cells where the permeability barrier has been destroyed (fernandez-puentes and . alpha-sarcin co-enters cells in conjunction with virus particles, and is liberated to the cytoplasm (otero and carrasco, ; liprandi et al., ) . in this manner, this toxin irreversibly blocks translation several minutes after virus entry. the molecular basis of the co-entry of macromolecules with virus particles has been analyzed in detail elsewhere (carrasco, (carrasco, , . apart from alpha-sarcin, a number of proteins that interfere with translation, many of them of plant origin, have been described (fernandez-puentes and carrasco, ; lee et al., ) . the release of all these toxins into cells is enhanced by virus particles. not only proteins, but also other macromolecules, including nucleic acids efficiently co-enter with virus particles (cotten et al., ) . however, none of these macromolecules passes into cells at late times of infection. apart from the use of translation inhibitors that do not easily permeate into intact cells, a number of assays can be employed to assess modifications in membrane permeability. amongst these assays, we can list the following. cells are preloaded with radioactive uridine and the exit of nucleotides can be monitored after induction of viroporin expression (gonzalez and carrasco, ) . unlike with most amino acids, the pool of uridine nucleotides is abundant in the cell interior, thus providing a convenient and sensitive assay for monitoring the exit of molecules from cells. other tests use radioactive glucose derivatives (e.g., -deoxyglucose), which cannot be metabolized and accumulate in cells. the analysis of the release of radioactive compounds that are not actively transported into cells leads to the failure to measure enhanced membrane permeability. entry of o-nitrophenyl-␤-d-galactopyranoside (onpg) into the bacterial cells can be determined very simply. this ␤-galactosidase substrate can be incubated with bacterial cells and production of the resulting compound can be followed by determining the absorbance at nm (lama and carrasco, ) . there are a number of non-vital dyes employed to characterize cell mortality. it should be noted that the entry of these compounds, in fact, determines a modification in cell membranes, which in some cases does not directly correlate with cell death. trypan blue is a dye widely used for monitoring enhanced membrane permeability. however, this assay is not very sensitive. in addition, trypan blue staining does not discriminate between metabolically active or dead cells, as the hygromycin b tests does. another assay employs the polyamine neurobiotin that needs specific connexin channels to enter mammalian cells (elfgang et al., ) . permeabilization of cell membrane increases uptake of this cationic molecule. internalized neurobiotin can be detected in paraformaldehyde-fixed cells, by fluorescence microscopy, using fluorescein isothiocyanate-conjugated streptavidin (gonzalez and carrasco, ) . propidium iodide (pi) is a dna-intercalating compound that does not enter intact cells. however, those cells that exhibit increased membrane permeability are able to take up pi, which can be assayed by cell fluorometric analysis (arroyo et al., ) . the appearance in the culture medium of cellular enzymes is a clear indicator of cell mortality. this is the case for lactic dehydrogenase and bacterial ␤-galactosidase, present outside the cells (sanderson et al., ) . commercial kits to measure this enzymatic activity are available. the release of cellular proteins to the medium occurs at very late times of viral infection, when cells have already died. this alteration takes place at much later times after hygromycin b entry can be detected (blanco et al., ) . viroporins are small proteins encoded by viruses that contain a stretch of hydrophobic amino acids (gonzalez and carrasco, ) . typically, viroporins are comprised of some - amino acids. the hydrophobic domain is able to form an amphipathic ␣-helix. the insertion of these proteins into membranes followed by their oligomerization creates a hydrophilic pore. the architecture of this channel is such that the hydrophobic amino acid residues face the phospholipid bilayer while the hydrophilic residues form part of the pore. in addition to this domain, there are other features of viroporin structure, including a second hydrophobic region in some viroporins that also interacts with membranes. this second interaction may further disturb the organization of the lipid bilayer. these proteins may also contain a stretch of basic amino acids that acts in a detergent-like fashion. all these structural features contribute to membrane destabilization. more recently, another domain has been described in some glycoproteins and also viroporins that has the capacity to interact with membranes. this domain is rich in aromatic amino acids and is usually inserted at the interface of the phospholipid bilayer (suarez et al., ; sanz et al., ) . this type of interaction also leads to membrane destabilization, further enhancing membrane permeability. a number of viroporins from different viruses that infect eukaryotic cells have been reported. this group of proteins includes picornavirus b and a, alphavirus k, retrovirus vpu, paramyxovirus sh, orthomyxovirus m , reovirus p , flavivirus p , phycodnavirus kcv, coronavirus e, and rhabdovirus alpha p. a recent review devoted to viroporins discusses the structure and function of a number of proteins of this group (gonzalez and carrasco, ) , and so the details of each particular viroporin will not be reviewed in this chapter. the main activity of viroporins is to create pores at biological membranes to permit the passage of ions and small molecules. the cloning and individual expression of viroporin genes has allowed their effects in bacterial and animal cells to be analyzed. thus, the expression of this type of viral gene enhances the permeation of ions and several hydrophilic molecules in or out of cells (carrasco, ) . in addition, the purified viroporin molecules open pores in model membranes, providing a system that is amenable to biophysical analysis (fischer and sansom, ) . the pore size created by viroporins allows the diffusion of different molecules with a molecular weight below about , da. the main step affected in animal viruses containing a deleted viroporin gene is the assembly and exit of virions from the infected cells (klimkait et al., ; liljestrom et al., ; loewy et al., ; betakova et al., ; watanabe et al., ; kuo and masters, ) . these genes are not essential for virus replication in culture cells, but the plaque size is much smaller in viroporin-defective viruses. notably, virus entry and gene expression in viroporin-deleted viruses occur as in their wild-type counterparts. an aspect of viroporin function at the molecular level that is still not understood is the link between pore activity and virus budding. in addition to small hydrophobic viral proteins, there are other virus products that promote membrane permeabilization. this occurs with a number of virus glycoproteins (gp) that are known to increase cell membrane permeability, such as the human immunodeficiency virus gp (chernomordik et al., ; arroyo et al., ) , the ebola virus gp (yang et al., ) , the cytomegalovirus us protein (maidji et al., ) , the vaccinia virus a l protein (sanderson et al., ) , rotavirus vp and ns proteins (charpilienne et al., ; newton et al., ) , the hepatitis c virus e protein (ciccaglione et al., ) , and the alphavirus e protein (nyfeler et al., ; wengler et al., ) . the architecture of some viral glycoproteins is such that upon oligomerization, the transmembrane (tm) domains may form a physical pore. in principle, two different regions of a viral fusion glycoprotein could form pores. one such region contains the fusion peptide that would create a pore in the cell membranes upon insertion (skehel and wiley, ) , while the tm domain would form a pore in the virion membrane (wild et al., ) . moreover, sequences adjacent to the tm region could have motifs designed to destabilize membrane structure (suarez et al., ) . entry of enveloped animal viruses leads to early membrane permeabilization, which is mediated by the formation of the two pores (fusion and tm) formed by viral fusion glycoproteins. this early permeabilization induced during the entry of virions requires conformational changes of the fusion glycoproteins. by contrast, after virus replication, newly synthesized glycoproteins may affect membrane permeability when they reach the plasma membrane (figure . ). this modification is achieved only by the tm domain, while the fusion peptide does not participate in this late modification. in viruses that lack the typical viroporin, its function could be replaced by these pore-forming glycoproteins, while for other viruses viroporin activity may be redundant (bour and strebel, ) . in the latter case, pore formation may be generated by viral glycoproteins and viroporins (figure . ). we would like to propose the possibility that pore-forming glycoproteins play a key role mainly during virus entry and, in some cases, also during virus budding, while viroporins come into action when viruses need to exit the cell. early membrane permeabilization is always carried out by a virion component. in the case of enveloped viruses, this early event is executed by a structural glycoprotein, which is coupled to the fusion process. an understanding of fusion at the molecular level also requires an explanation of the phenomenon of early membrane permeabilization. we have advanced the idea that viral glycoproteins involved in membrane fusion participate in the dissipation of the chemiosmotic gradient, thus providing the energy to push the nucleocapsid and neighboring macromolecules to the cell interior (carrasco, ; irurzun et al., ) . fusion glycoproteins do not simply serve to bridge the cellular and the viral membrane, but instead are designed to open pores in both membranes. this pore-opening activity may be necessary to lower membrane potential and to dissipate ionic gradients. several chapters of this book are devoted to the detailed description of the structure and function of these glycoproteins, so we will focus our attention on viral glycoproteins that permeabilize membranes when individually expressed in cells. these membrane active proteins may exhibit this activity later on in the virus life cycle. rotavirus infection provokes a number of alterations in cellular membranes during infection . amongst these alterations, there is an increase in the concentration of cytoplasmic calcium . several rotavirus proteins exhibit membrane-destabilizing activity. the enterotoxin nsp induces alterations in membrane permeability (tian et al., ) . the individual expression of the non-structural glycoprotein nsp has the ability to increase the concentration of cytoplasmic calcium. this increase may be mediated by activation of phospholipase c activity (dong et al., ) . rotavirus particles induce the co-entry of protein toxins into cells (cuadras et al., ) . at least two structural components possess the ability to permeabilize cells, including vp protein and vp glycoprotein (charpilienne et al., ; irurzun et al., ) . infection of lymphocytic human cells by hiv- enhances membrane permeability to ions and several compounds (voss et al., ; gatti et al., ) . there are at least three different hiv-encoded proteins responsible for these alterations: vpu protein, the retroviral protease, and the fusion glycoprotein gp . apart from the fusion peptide, there are two regions of gp that exhibit membrane permeability; one is located at the carboxy terminus (arroyo et al., ; comardelle et al., ) and another corresponds to the membranespanning domain (arroyo et al., ) . the c-terminus of gp includes two - residues, which may form cationic amphipathic ␣-helices, designated as lentivirus lytic peptides and (llp- and llp- ). synthetic llp- peptide forms pores in planar phospholipid bilayers late early figure . . participation of pore formation by viral glycoproteins and viroporins in membrane permeability. early membrane permeabilization is coupled to the fusion activity of the corresponding viral glycoprotein. this fusion glycoprotein may create two pores. one is located at the target cell membrane and the other is formed by the tm domain. late membrane permeabilization may be carried out by viroporins or by the tm domains of viral glycoproteins. (chernomordik et al., ) , permeabilizes hiv- virions to deoxyribonucleoside triphosphates (zhang et al., ) , and induces alterations in ion permeability of xenopus oocytes (comardelle et al., ) . inducible expression of the hepatitis c virus e glycoprotein increases membrane permeability in bacterial cells. the ability of e to modify membrane permeability has been mapped to the carboxy terminus of the protein (ciccaglione et al., (ciccaglione et al., , . similar permeabilization was found with escherichia coli cells that synthesize semliki forest virus e glycoprotein after exposure to low ph (nyfeler et al., ) . finally, overexpression of vaccinia virus a l glycoprotein produces changes in the morphology, permeability, and adhesion of mammalian cells. the potential capacity of a l protein to form pores at the plasma membrane promotes the entry of calcium ions and pi and the release of lactic dehydrogenase into the culture medium (sanderson et al., ) . different approaches have been envisaged for the design of compounds that interfere with virus replication based on modifications in membrane permeability. one such approach makes use of inhibitors of cellular or viral functions that do not permeate easily into intact animal cells. notably, these agents selectively enter into virus-infected cells (carrasco, ; benedetto et al., ) . most of the inhibitors used thus far interfere with protein synthesis, although compounds that affect other functions could also be employed. entry of these agents leads to the inhibition of translation specifically in virus-infected cells, leading to a profound inhibition of virus growth (contreras et al., ; carrasco, ; gatti et al., ) . although this approach discriminates well between uninfected or virus-infected cells in culture, the high toxicity of the agents thus far assayed has hampered its use in whole animals. perhaps future searches for less toxic compounds would make this approach amenable to application in therapy. in fact, some of the plant toxins that co-enter with virus particles have been described as being antiviral agents (lee et al., ) . even compounds such as hygromycin b, which has been used in the veterinary field as an antibacterial agent, could also be used as an antiviral compound for rotavirus infections (liprandi et al., ) . the paradigm of an inhibitor of a viral ion channel is amantadine (hay, ) (figure . ) . this compound has been used as an anti-influenza agent in humans (de clercq, ) . the target of amantadine is the influenza-encoded protein m . residues , , , and of m determine the amantadine sensitivity of this ion channel. a drawback of amantadine is the high doses necessary to affect influenza. the search for more effective compounds may provide a more efficacious treatment for this illness. compounds that interfere with the functioning of other viroporins have also been described. this is the case of amiloride derivatives that block hiv- vpu activity (ewart et al., ) . in this manner, the production of infectious hiv- is reduced in the presence of this agent. recently, long alkyl-chain iminosugar derivatives have been found to interfere with the function of the hepatitis c virus p protein as an ion channel (griffin et al., ; pavlovic et al., ) . these compounds exhibited antiviral activity with bovine viral diarrhea virus, which is closely related to the hepatitis c virus (durantel et al., ) . much effort has been concentrated recently on the development of antiviral agents that inhibit the fusion step of hiv. binding of hiv gp to the cd receptor is followed by further interaction of this viral glycoprotein with the coreceptor molecules cxcr and ccr . after this initial interaction, the conformation of the ectodomain of the tm glycoprotein gp is profoundly modified. exposure of the fusion peptide at the amino terminus of gp triggers its insertion into the target cellular membrane, leading to the fusion of the viral and the cellular plasma membranes. all these steps have been used as targets for anti-hiv therapy (cooley and lewin, ) . as regards the fusion step, a variety of peptide mimetic inhibitors have been developed. the pioneering work on peptide t has demonstrated that this compound is a potent inhibitor of gp -induced membrane fusion. t exhibits antiviral activity in hiv-infected patients. the detailed mechanism of action of t at the molecular level is known. this peptide is homologous with amino acids within the c-terminal heptad repeat region (hr ) of hiv- gp . t competitively binds to hr and interferes with the formation of the six helix hr -hr bundle complex necessary for membrane fusion. at present there are a great number of peptides that interfere with binding of gp or with gp -induced membrane fusion and that have been tested for their anti-hiv activity and clinical efficacy. in this regard, t is one of the second generation of hr- peptide mimetic inhibitors that consists of amino acids. pro is a soluble cd receptor (cd -igg ) that binds to and neutralizes gp before virus binding occurs. sch-c is an oxime-piperidine compound that is a coreceptor antagonist. this small molecule acts as an inhibitor of ccr . md is a non-peptidic, low molecular weight bicyclam compound that prevents interactions between cxcr and gp , blocking signal transduction from cxcr . future research in this field will provide us with additional antiviral compounds to add to the anti-hiv armory. inhibitors of poliovirus uncoating efficiently block the early membrane permeabilization induced by virus particles membrane permeabilization by different regions of the human immunodeficiency virus type transmembrane glycoprotein gp a human virus protein, poliovirus protein bc, induces membrane proliferation and blocks the exocytic pathway in the yeast saccharomyces cerevisiae identification of regions of poliovirus bc protein that are involved in cytotoxicity inhibition of animal virus production by means of translation inhibitors unable to penetrate normal cells the vaccinia virus a . l gene encodes a hydrophobic -amino-acid virion membrane protein that enhances virulence in mice and is conserved among vertebrate poxviruses cell killing by hiv- protease the human immunodeficiency virus (hiv) type envelope protein is a functional complement to hiv type vpu that enhances particle release of heterologous retroviruses membrane leakiness after viral infection and a new approach to the development of antiviral agents entry of animal viruses and macromolecules into cells modification of membrane permeability by animal viruses the sequence of the nucleotides at the alpha-sarcin cleavage site in rat s ribosomal ribonucleic acid membrane permeabilization by small hydrophobic nonstructural proteins of japanese encephalitis virus solubilized and cleaved vp , the outer glycoprotein of rotavirus, induces permeabilization of cell membrane vesicles an amphipathic peptide from the c-terminal region of the human immunodeficiency virus envelope glycoprotein causes pore formation in membranes mutagenesis of hepatitis c virus e protein affects its membrane-permeabilizing activity hepatitis c virus e protein induces modification of membrane permeability in e. coli cells a synthetic peptide corresponding to the carboxy terminus of human immunodeficiency virus type transmembrane glycoprotein induces alterations in the ionic permeability of xenopus laevis oocytes inhibition, by selected antibiotics, of protein synthesis in cells growing in tissue cultures hiv- cell entry and advances in viral entry inhibitor therapy high-efficiency receptormediated delivery of small and large ( kilobase) gene constructs using the endosome-disruption activity of defective or chemically inactivated adenovirus particles rotaviruses induce an early membrane permeabilization of ma cells and do not require a low intracellular ca ϩ concentration to initiate their replication cycle antiviral drugs: current state of the art rotavirus infection alters na ϩ and k ϩ homeostasis in ma- cells the rotavirus enterotoxin nsp mobilizes intracellular calcium in human intestinal cells by stimulating phospholipase c-mediated inositol , , -trisphosphate production study of the mechanism of antiviral action of iminosugar derivatives against bovine viral diarrhea virus specific permeability and selective formation of gap junction channels in connexin-transfected hela cells amiloride derivatives block ion channel activity and enhancement of virus-like particle budding caused by hiv- protein vpu viral infection permeabilizes mammalian cells to protein toxins viral ion channels: sructure and function inhibition of hiv type production by hygromycin b the human immunodeficiency virus type vpu protein enhances membrane permeability human immunodeficiency virus type vpu protein affects sindbis virus glycoprotein processing and enhances membrane permeabilization the p protein of hepatitis c virus forms an ion channel that is blocked by the antiviral drug the action of adamantanamines against influenza a viruses: inhibition of the m ion channel protein entry of semliki forest virus into cells: effects of concanamycin a and nigericin on viral membrane fusion and infection the human immunodeficiency virus type -specific protein vpu is required for efficient virus maturation and release the small envelope protein e is not essential for murine coronavirus replication antibiotics that specifically block translation in virus-infected cells expression of poliovirus nonstructural proteins in escherichia coli cells. modification of membrane permeability induced by b and a poliovirus-mediated entry of pokeweed antiviral protein in vitro mutagenesis of a full-length cdna clone of semliki forest virus: the small , -molecular-weight membrane protein modulates virus release productive penetration of rotavirus in cultured cells induces co-entry of the translation inhibitor alpha-sarcin the -kilodalton membrane protein of semliki forest virus is involved in the budding process accessory human cytomegalovirus glycoprotein us in the unique short component of the viral genome promotes cell-to-cell transmission of virus in polarized epithelial cells effect of rotavirus infection on intracellular calcium homeostasis in cultured cells rotavirus nonstructural glycoprotein nsp alters plasma membrane permeability in mammalian cells expression of semliki forest virus e protein in escherichia coli. low ph-induced pore formation complete nucleotide sequence of cdna for the cytotoxin alpha sarcin proteins are cointernalized with virion particles during early infection the hepatitis c virus p protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives overexpression of the vaccinia virus a l integral membrane protein promotes ca ϩ influx into infected cells interfacial domains in sindbis virus k protein. detection and functional characterization coiled coils in both intracellular vesicle and viral membrane fusion membrane interface-interacting sequences within the ectodomain of the human immunodeficiency virus type envelope glycoprotein: putative role during viral fusion the nonstructural glycoprotein of rotavirus affects intracellular calcium levels alteration of intracellular potassium and sodium concentrations correlates with induction of cytopathic effects by human immunodeficiency virus influenza a virus can undergo multiple cycles of replication without m ion channel activity entry of alphaviruses at the plasma membrane converts the viral surface proteins into an ion-permeable pore that can be detected by electrophysiological analyses of whole-cell membrane currents peptides corresponding to a predictive alpha-helical domain of human immunodeficiency virus type gp are potent inhibitors of virus infection identification of the ebola virus glycoprotein as the main viral determinant of vascular cell cytotoxicity and injury amphipathic domains in the c terminus of the tm protein (gp ) permeabilize hiv- virions: a molecular mechanism underlying natural endogenous reverse transcription this work was supported by grants from the fundación para la investigación y prevención del sida en españa ( ), instituto de salud carlos iii ( / ), and the dgicyt pm - . the authors also acknowledge the fundación ramón areces for an institutional grant awarded to the centro de biología molecular "severo ochoa." key: cord- -nrzjedhi authors: dasgupta, s; auth, t; gompper, g title: nano- and microparticles at fluid and biological interfaces date: - - journal: j phys condens matter doi: . / - x/aa sha: doc_id: cord_uid: nrzjedhi systems with interfaces are abundant in both technological applications and biology. while a fluid interface separates two fluids, membranes separate the inside of vesicles from the outside, the interior of biological cells from the environment, and compartmentalize cells into organelles. the physical properties of interfaces are characterized by interface tension, those of membranes are characterized by bending and stretching elasticity. amphiphilic molecules like surfactants that are added to a system with two immiscible fluids decrease the interface tension and induce a bending rigidity. lipid bilayer membranes of vesicles can be stretched or compressed by osmotic pressure; in biological cells, also the presence of a cytoskeleton can induce membrane tension. if the thickness of the interface or the membrane is small compared with its lateral extension, both can be described using two-dimensional mathematical surfaces embedded in three-dimensional space. we review recent work on the interaction of particles with interfaces and membranes. this can be micrometer-sized particles at interfaces that stabilise emulsions or form colloidosomes, as well as typically nanometer-sized particles at membranes, such as viruses, parasites, and engineered drug delivery systems. in both cases, we first discuss the interaction of single particles with interfaces and membranes, e.g. particles in external fields, non-spherical particles, and particles at curved interfaces, followed by interface-mediated interaction between two particles, many-particle interactions, interface and membrane curvature-induced phenomena, and applications. systems with interfaces are abundant in both technological applications and biology. while a fluid interface separates two fluids, membranes separate the inside of vesicles from the outside, the interior of biological cells from the environment, and compartmentalize cells into organelles. the physical properties of interfaces are characterized by interface tension, those of membranes are characterized by bending and stretching elasticity. amphiphilic molecules like surfactants that are added to a system with two immiscible fluids decrease the interface tension and induce a bending rigidity. lipid bilayer membranes of vesicles can be stretched or compressed by osmotic pressure; in biological cells, also the presence of a cytoskeleton can induce membrane tension. if the thickness of the interface or the membrane is small compared with its lateral extension, both can be described using two-dimensional mathematical surfaces embedded in three-dimensional space. we review recent work on the interaction of particles with interfaces and membranes. this can be micrometer-sized particles at interfaces that stabilise emulsions or form colloidosomes, as well as typically nanometer-sized particles at membranes, such as viruses, parasites, and engineered drug delivery systems. in both cases, we first discuss the interaction of single particles with interfaces and membranes, e.g. particles in external fields, non-spherical particles, and particles at curved interfaces, followed by interface-mediated interaction between two particles, many-particle interactions, interface and membrane curvature-induced phenomena, and applications. keywords: membranes, nanoparticles, capillary interactions, lipid bilayers, emulsions, viruses, interfaces (some figures may appear in colour only in the online journal) original content from this work may be used under the terms of the creative commons attribution . licence. any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and doi. also compartmentalize cells and thereby define organelles. transmembrane transport is essential for the communication both inside a cell as well as of cells with their environment [ , ] . the interaction of particles and pathogens with biological membranes-and therefore also their cellular uptake and intracellular transport-crucially depends on the particle size, shape, softness, and surface functionalization. nowadays a whole zoo of micro-and nanoparticles can be fabricated from various materials, with engineered shapes and surface functionalizations. the particles can be used for applications in food science [ ] [ ] [ ] , cosmetics [ , , ] , as antimicrobials [ , ] , and in nanomedicine [ ] [ ] [ ] ; therefore systematic studies and a careful consideration of potentially toxic effects are required [ ] [ ] [ ] [ ] [ ] . figure shows examples for oblate, bullet-shaped, pill-shaped, and dumbbell-shaped microparticles that are made from a polymeric material. figure shows cube-like, rod-like, irregularly-shaped, and spindle-like metal and metal-oxide nanoparticles. all these particles can also be considered as model systems for 'particles' found in nature. for example, the malaria parasite is micrometer-sized and has an egg-like shape [ , ] . milk contains casein micelles with sizes below nm that stabilize fat globules [ ] . viruses resemble roundish, filamentlike, and bullet shapes with sizes below nm [ ] [ ] [ ] ; in particular, the filamentous ebola and marburg viruses are of much interest due to their enhanced virulence that leads to high mortality rates [ , ] . fluid interfaces are rough on the molecular scale and can be analytically well described by a hyperbolic tangent-shaped density profile [ , ] , see figure . however, the typical interface width is much smaller than the sizes of the particles that we consider. in order to study the interaction of particles with interfaces, the interface can therefore be thought of as mathematical surface with its physical properties characterized by an interface tension. lipid-bilayer membranes often consist of many components, e.g. different lipids and cholesterol, and biological membranes usually also contain membrane proteins. this can lead to phase separation within the membrane, as shown in figure . the typical thickness of a lipid bilayer membrane is ≈ d nm and cannot be neglected for small nanoparticles with sizes of few nanometers. for nanoparticles with radii of nm and above, the membrane can be described as mathematical surface with curvature-elastic properties. the possiblity to model both fluid interfaces and biological interfaces (membranes) using mathematical surfaces is our motivation for discussing particles at interfaces and at membranes in a single review article. for biological interfaces, we will focus on sufficiently large particles for which a continuum model is not only feasible, but also more appropriate than an atomistic model. the deformation energy of the surface can then be calculated using the helfrich hamiltonian [ ] , here, the interface conformation is characterized by the two principal curvatures at each point of the interface, c and c , that enter the hamiltonian via the mean curvature = + h c c ( )/ and the gaussian curvature = k c c . the total deformation energy is obtained by integration over the entire interface area s. tension γ, bending rigidity κ, spontaneous curvature c , and gaussian saddle splay modulus κ describe the mechanical and elastic properties of the interface. equation ( ) applies to biological interfaces if the bending energy contribution dominates, and to fluid interfaces if the energy is given only by the tension term. for spherical particles at planar fluid interfaces, the particle size, the interface tensions between the particles and the two phases, as well as the interface tension between both phases characterize the system. if the interface tensions between the particles and both phases are identical, the particles attach to the interface because their presence reduces direct contact between both phases without any additional costs for the contact of the particles with the fluids. for high-tension interfaces and micrometer-sized particles, such as for silica particles at oil-water interfaces, the attachment energy gain can be as high as k t b [ ] . therefore, such particles are irreversibly adsorbed to the interface. if the interface tensions of the particles with both fluids differ, the attachment energies of the particles can be strongly reduced. for particles attached to membranes, the membrane elastic properties in equation ( ) and the adhesion energy between particles and membranes characterize the system. here, s ad is the membrane area adhered to the particle, and the adhesion strength w for the contact interaction can be mediated by van der waals forces, by electrostatic interactions, and by specific adhesion (receptor-ligand bonds). for small spherical particles attached to nearly planar membranes and weak adhesion strengths, wrapping may not be energetically favourable, while for spherical particles with a radius of nm and for a high adhesion strength = − w k t . nm b the energy gain through wrapping can be as high as k t b . particle shape can strongly alter the attachment energy. table provides an overview of typical attachment energies of spherical particles to interfaces and membranes. for particles at interfaces, we assume that they have equal interface tensions with both fluid phases and that they are therefore half immersed in each phase. for particles at membranes, we assume that they are attached to a membrane with half of their surface area and that there is no deformation energy cost for the membrane surrounding the particles . interface deformations induced by particles lead to interface-mediated interactions and self-assembly; many-particle systems minimize the deformation energies also with respect to the particle positions. for example, capillary forces between micrometer-sized ellipsoidal particles increase with increasing aspect ratio and the energy gain at particle contact can be as high as k t b already for ellipsoid aspect ratios of - [ , ] . membrane-mediated binding energies of few k t b have been calculated for spherical particles at membranes [ , , ] . table provides an overview of interface-mediated and membrane-mediated bond energies for particles at contact. structures on the micrometer scale can readily be observed using light microscopy. in order to access the nanometer scale, more sophisticated techniques, such as electron microscopy or super-resolution microscopy, have to be employed [ ] [ ] [ ] [ ] [ ] [ ] . whereas ramsden and pickering have reported on particle-stabilised emulsions already in the early th century [ , ] , images of nanoparticles, such as bacteriophages, have only been reported in s, soon after electron microscopy has become available [ ] . since the very early studies of particles at fluid and biological interfaces, observation techniques and abilities to engineer particles have continuously advanced. besides these experimental developments, also mesoscopic and atomistic modeling and computer simulation techniques and speed have rapidly developed. both allows the characterization of particles at interfaces with increasing accuracy; this review article provides an overview of recent achievements. for both fluid and biological interfaces, we will discuss similar aspects: attachment to the interface, mechanisms for deforming the interface, particle orientation at the interface for nonspherical particles, long-range and short-range interactions, many-particle interactions, and applications. many concepts can easily be transferred between systems with fluid and with biological interfaces. we stress this parallelity by the analogous structure of the sections for both systems. section is devoted to particles at fluid interfaces, section to particles at biological membranes. the overview of the experimental studies of selfassembly of microparticles at fluid interfaces may not only benefit scientists investigating such systems, but also those interested in nanoparticles at biological membranes, where systems are less accessible experimentally and fewer studies are therefore available. particles adsorb at fluid interfaces because they reduce the interface area and thereby also the total interface energy. here, we have to account for the interface energies between the three phases, liquid , vapor (or a second liquid) v, and solid s. the system can be characterized by the three interface tensions γ sv , γ v , and γ s , see figure . the force balance at the contact line, where the liquid-vapor interface is located at the particle, is given by the young-dupré equation, which defines the contact angle θ c , see figure . the energy difference to bring a spherical particle with radius a from the bulk phase to the interface is the trapping energy [ ] [ ] . copyright © american chemical society. where τ is an effective line tension and l c is the length of the contact line. for micrometer-sized particles, the contrib ution of the line tension is negligible; the trapping energy is proportional to the liquid-vapor interface tension γ v and decreases with decreasing contact angle, see figure . for small particles with sizes below one micrometer line tension becomes relevant [ ] , for large particles with sizes of several micrometers gravity has to be taken into account [ ] [ ] [ ] . the eötvös or bond number compares the contributions of interface tension and gravity to the total energy, where ρ ∆ is the density difference of the two phases, g is the gravitational acceleration, and l is a characteristic length of the particle. similarly, the energies due to line tension and surface tension are compared using the dimensionless number ( ) . (a) a vertical slice of width σ . and height σ is shown with the interface region highlighted. (b) corresponding mean density profile z ( ) . reproduced with permission from [ ] . © epla. all rights reserved. for micrometer-sized particles and typical density differences between, for example, silica and water, ≈ − bo [ ] ; for line tensions and the corresponding surface tensions measured for polystyrene and poly(methyl methacrylate) particles at oil-water interfaces, = li . - . [ ] . in the following sections, we mainly discuss systems with bo and li , where both gravity as well as line tension can be neglected. the estimate for li indicates that line tension may not always be entirely negligible for small micrometer-sized and large nanometer-sized particles. line tension may qualitatively alter the behaviour of non-spherical particles at interfaces: for example, ellipsoidal and cylinderical particles may undergo orientational changes in order to minimise the length of the contact line. in [ ] , contact angles have been measured for spherical particles and for prolate ellipsoidal particles with various aspect ratios obtained by deforming initially spherical particles. here, an apparent decrease of the contact angle with increasing aspect ratio of the particles has been measured. this can be attributed to a line tension and a contact angle that is independent of particle shape [ ] . the experiment predicts an effective line tension τ ≈ nn that also includes experimentally-observed heterogeneities in the contact line. other values for line tensions reported in the literature are in the pn to µn range [ ] . one of the key reasons for the stability of colloidal assemblies at interfaces and of pickering emulsions are the very high trapping energies for the particles [ , ] , see table . calculations for non-spherical particles show that the trapping energies depend only weakly on particle shape and increase for both oblate and prolate deformations of spherical particles, see figure . at curved interfaces, the trapping energies of spherical particles have been shown to depend on the interface curvature and therefore on the laplace pressure difference at the interface [ ] . for spherical particles and a contact angle θ = c , the trapping energy decreases with increasing interface curvature. for contact angles θ ≠ c , the trapping energy either increases or decreases depending on the sign of the contact angle and on whether the interface is curved towards or away from the particles. the white arrow points to a cholesterol oriented in between the monolayer leaflets. reprinted with permission from [ ] . copyright © national academy of sciences. [ , ] . based on [ ] and [ ] . to satisfy the young-dupré equation locally at every point on the three-phase contact line, see figure , the contact lines and therefore also the interfaces are often not planar for non-spherical particles. also particle-surface inhomogeneities, either due to roughness or chemical surface patterning, e.g. for janus particles, may induce contact-line undulations. furthermore, imposed fields, such as gravity for large mm-sized particles or electromagnetic fields for electrically-charged or for magnetic particles, may lead to interface deformations. the capillary forces due to the system's tendency to reduce the total interface area between liquid and vapor is the origin of the capillary forces between particles, see table ; very strong bond energies of k t b for micrometer-sized particles correspond to interface height perturbations of only nm [ ] . the importance of hydrodynamic interactions for the dynamics of particles at interfaces is characterized by the capillary number with fluid viscosity η and typical particle velocity v. for typical velocities of µ − m s for particles with sizes of up to few micrometers in water [ ] , we find < − ca . therefore, for the systems discussed in this review article ca , such that hydrodynamic interactions can be neglected. particle velocities can therefore be directly related to the forces acting on the particles and the friction by the fluid. self-assembly of colloidal particles at fluid interfaces is governed by both direct interactions, such as van der waals and electrostatic interactions, and indirect interactions, such as forces due to overlap of interface distortions, popularly coined as capillary interactions [ , ] . in , pieranski reported a two-dimensional colloidal crystal of spherical polystyrene colloids with a triangular lattice structure at an air-water interface induced by an asymmetric charge distribution [ ] , forced into two dimensions by the interface. a large number of more recent studies on colloids at interfaces that assemble due to interface-mediated and direct interactions show a rich variety of two-dimensional structures. an overview of both single-particle and many-particle systems at interfaces is provided in the remainder of this section. in sections . - . , we focus on various aspects of the interaction of single particles with interfaces, while in sections . and . , we discuss two-particle and many-particle interactions, respectively. section . focuses on the special case of particles at interfaces that are half immersed into an ordered fluid. we finally discuss applications in section . . an undulating contact line at a particle distorts a surrounding planar fluid interface. for a cylindrically-symmetric system, such as a spherical particle at an interface, the young-laplace equation that describes the interface deformation is best expressed in cylindrical coordinates [ ] , the height profile φ h r, ( ) of the interface can then be expressed as product of a function of the radial coordinate r and a function of the angular coordinate φ around the sphere, the general solutions, are characterized by the contact line at the particle, described by Φ m, , φ m, , and r m, . the interface deformation decays faster with increasing distance from the particle the larger m, i.e. the larger the number of undulations along a circle around the particle. the contact-line undulations can be expressed as multipole expansion, where the contact radius r c is often similar to the particle radius, h m are the expansion coefficients, and φ m, the phase spherical particles at a fluid-fluid or fluid-gas interface in a system with a solid spherical particle (s), a fluid (l), and a gas/ second fluid (g). the contact angle θ c for the green particle is , while the contact angle for the red particle is smaller than . the three interface tensions γ sv , γ s , γ v are sketched for each particle using arrows. they indicate the force balance given by the young-dupré equation that holds for all points along the three-phase contact lines. angles. the first two terms of the expansion vanish, because without additional external forces the particles are at their optimal height and orientation at the interface. the lowest multipole contribution and the most important contribution for long-ranged interactions is the quadrupolar deformation with = m [ ] , where h characterizes the undulation amplitude of the contact line. the long-ranged interactions between two particles in the far field therefore decay with − d , where d is the interparticle distance. experimentally, imprinted contact-line shapes have to be distinguished from mere surface roughness. whereas surface roughness often leads to temporary and random pinning of the contact line, engineered contact-line deformations are stable and well controlled. for instance, undulating plates allow the engineering of the interface deformation and the tuning of the capillary interaction between particles from attractive to repulsive [ , ] . however, also nanometric roughness on microspheres or disc-like particles leads to strong capillary interactions due to contact-line pinning [ , [ ] [ ] [ ] . the temporary nature of contact-line pinning can be observed for instance for metastable orientations of dumbbell-shaped particles at interfaces; the distribution of particle orientations relaxes towards the globally stable particle orientation when the system ages [ ] . in , lucassen first suggested that particles with complex shapes can induce interface distortions in absence of gravity [ ] . he systematically calculated the interaction between sinusoidal interface deformations. however, the most common interface distortion due to non-spherical particles is the quadrupolar deformation, which is found for all elongated particles with homogeneous surface functionalization at planar interfaces in the far field [ , , ] . for ellipsoidal particles, quadrupolar interface deformations can also be found in the near field [ , , ] . for contact angles θ < c , the interface is pulled down at the tips and pulled up near the long sides of the particle-and reverse for the inverse-wetting condition with θ > c , see figure (a). for ellipsoidal particles and θ = c -and for spherical particles-a planar interface remains undeformed. strength and nature of the interface distortions can be characterized using different quantifications: contour maps of the interface distortion, see figures (a) and (b), shapes of contact lines, see figure (c), differential heights ∆u of the interface between the crest and trough along the contact lines, see figure (d), or height profiles along cross-sections, see figure (e). for well-defined particle shapes, in particular for ellipsoidal particles and quadrupolar interface deformations, the characterization of the interface deformation fields is uniquely determined by ∆ = − u z z max m in , where z max is for both particles. the spherical particle approaches the ellipsoidal particle at the side. reprinted with permission from [ ] . copyright ( ) american chemical society. (d) maximal height difference ∆u of the contact line for ellipsoidal particles with several aspect ratios b a ⩽ / ⩽ . the numerical data are normalized by the half the length of the minor axis a and plotted as function of the contact angle θ c . the maximum value of ∆u, depicted by ×, shifts to smaller contact angles with increasing particle aspect ratio, as indicated by the grey lines that serve as guides to the eye. reprinted with permission from [ ] . copyright ( ) american chemical society. the maximum and z min the minimum height along the contact line. the height difference ∆u for ellipsoidal particles vanishes for θ = c and for θ = c and assumes a maximal value ∆u max for θ < < c for ellipsoids with aspect ratios b a . ⩽ / ⩽ [ ] , see figure (d). the position of the peak shifts from larger to smaller contact angles with increasing aspect ratios of the particles [ ] , while the height of the peak increases from ∆ ≈ u a . . studies are available for ellipsoidal particles [ , , ] , for cylindrical [ ] , for cuboidal particles [ ] , and for rounded box-like particles [ ] . the height difference along the contact line of elongated particles can be thought of as a measure for their capillary interaction strength. systematic theoretical and experimental calculations thereby provide routes to tailor capillary assembly of multiple particles. the experimental determination of contact angles using light microcopy is difficult, because the same interface deformation can be derived for two different contact angles. this 'contact-angle mystery' arises because the differential interface height distortion, i.e. ∆u a / , does not show a monotonic increase with the contact angle θ c , see figure (d). due to the non-monotonic profile for ellipsoidal particles, for each value of ∆u a / there are two possible values for θ c , see figures (a) and (b). however, the 'mystery' can be resolved by measuring a second quantity, such as the excess area s s / plotted in figure (c), i.e. the ratio of the projected area by the contact line and the projected area at θ = c [ , ] . in figure , results from theoretical contact-angle calculations for ellipsoidal particles with various aspect ratios are plotted together with corresponding experimental measurements of ∆u a / and with s s / as function of the contact angle. the lower branch for smaller θ c has already been reported in [ ] , the upper branch more recently in [ ] . only with the knowledge of both, experimental values for ∆u a / and for s s / , the correct branch of contact angle solutions can be singled out. a new technique using electron microscopy provides means to measure contact angles more directly than optical microscopy. this so-called freeze-fracture shadow-casting cryo-sem (fresca) has been used to measure contact angles for wetting of spherical and ellipsoidal micro-and nanoparticles at liquid-liquid interfaces [ ] . for more complex particle shapes, the connection between interface deformation and particle aspect ratio can be different from the case of 'simple' elongated particles. for example, figures (a)-(e) shows deformations around rounded box-like particles, where rises and dips depend on local particle shape rather than aspect ratio. cuboidal particles can produce octupolar distortion fields with eight lobes (rises and dips), see figure (f). for rounded cylinders, there can even be multiple branches of possible contact angles due to the multiple peaks in the ∆u a / variation for certain aspect ratios (unpublished results). table provides an overview of the dominant multipole contributions for interface deformations and corresponding systems, several of them are discussed in more detail in the following. we discuss here gravity, buyoancy, and thin films in part and non-planar interfaces in part . . . . gravity, buyoancy, and thin films. the presence of graviational forces leads to floatation forces that cannot be neglected in the regime of large absolute values of the bond number [ , [ ] [ ] [ ] [ ] [ ] [ ] ] . particles either sink into the interface / , for ellipsoidal particles with aspect ratios in the range b a . ⩽ / ⩽ . the data from [ ] is plotted together with numerical data taken from [ ] . for a spherical particle, s s / varies as θ sin c , as shown by the solid line above. for all contact angles between and , s s / attains higher values for ellipsoidal particles in comparison to the analytical estimate obtained for a spherical particle. adapted with permission from [ ] . copyright ( ) american chemical society. if they are heavier than the fluid or float up if they are lighter than the fluid (buyoancy). gravitational forces therefore induce dominant monopole interface deformations, so-called 'capillary charges', see equation ( ) [ ] . some fascinating outcomes of the interplay of capillarity and gravity are water striders [ ] , meniscus-climbing insects [ , ] , and the cheerios effect in cereal bowls [ ] . monopole interface deformations are obtained as well for particles in thin films, where instead of gravitation the small thickness of the film compared with the particle size induces interface deformations [ , ] , see figure . the ideal interface positions on the particle are incompatible with the film thickness, which leads to immersion forces. for a thin film of fluid at a solid interface, the horizontal projection of the immersion force is [ ] πγ with the radius r c of the three-phase contact lines at the particles, the mean meniscus slope angle Ψ c at the contact line, and the distance d cc between the particles. this expression holds ( /( )) / , where ρ ∆ is the density difference between the fluid and the gas. figure compares the floatation forces between particles with immersion forces for polystyrene latex particles in a water film on a glass substrate; in this example, the floatation forces decrease faster with increasing interparticle distance than the immersion forces. as an instructive example, we discuss froth floatation as an application in more detail [ ] . here, grains of one solid are carried away with the froth, while grains of a different solid sink to the bottom of the floatation system. the selective adsorption of a collector chemical onto a mineral in a flotation plant determines-among others-the attachment of the mineral to bubbles used for separation. the stability of the froth with the particles depends mainly on the properties and the amounts of particles [ ] . the so-called capillary pressure due to the liquid that drains from the film because of gravity measures the pressure when the film ruptures. this critical pressure increases if particles stabilise the film, e.g. spherical particles with contact angles far below , and decreases if particles destabilise the film, e.g. particles with sharp edges. / . the heights are scaled by the length scale r. reprinted with permission from [ ] copyright of the royal society of chemistry (f) deformation profiles of an interface around a particle with hauser's cube shape at contact angle θ = . whether a particle stabilizes or destabilizes a film furthermore depends on the orientation that the particle assumes at the interface [ ] , see section . . figure shows how a thin film ruptures due to the presence of a non-spherical sharpedged particle. particles at curved interfaces experience forces not only due to the presence of the interface, but also because of the laplace pressure. curvature gradients of the interface lead to lateral forces, and curved interfaces modify interface-mediated interactions between particles [ , , , , , ] , see figure ; vice versa, particles at high densities can induce spontaneous interface curvature [ ] . mean curvature h and deviatoric curvature δ = − c c c determine the trapping energy [ ] where e f is the trapping energy at the flat interface, see equation ( ). for a spherical colloid of radius a on a spherical drop- , which is the interface curvature-induced contribution. this term is evaluated by considering contributions due to both the interface energies and the work done against the laplace pressure. a similar estimate for the trapping energies attempted previously in [ ] without including the work done by the laplace pressure resulted in the correction term to be . this result is consistent with equation ( ) when the contribution due to the pressure term, , is added to it [ ] . for a minimal surface with mean curvature = h , such as for a catenoidal interface shape, the correction term compared with the flat interface depends only on the deviatoric curvatue δc. the trapping energy of a colloidal particle on a catenoidal minimal surface is thus . on an interface with varying curvature, the lateral force on the trapped particle is given by [ thus, spherical particles on interfaces with curvature gradients experience forces depending on both the absolute values and the gradients of the mean and deviatoric curvature to move towards regions of high mean and deviatoric curvatures (and therefore also high gaussian curvatures because . recent experiments of microspheres on interfaces with different curvatures have evaluated these forces to be of the order = − ± [ ] . theoretical estimates of capillarity at curved interfaceswhich neglect pinning-suggest that capillary forces for particles that are smaller than the capillary length where ρ is the density of the fluid, are proportional to both a and the gradient of the gaussian curvature, ∇k [ ] . this has been verified in experiments where large microposts induce a curvature gradient and cause smaller particles to migrate along the deformed interface to assemble in regions of high gaussian curvature, see figure . experimental evidence further suggests that capillary migration on curved interfaces can be enhanced by contact-line pinning. contact-line undulations, which lead to finite local interface curvatures, induce capillary forces of the order ⋅ -⋅ k t b , which are much larger compared with the weak forces due to capillarity for perfectly smooth spherical particles. the experiments demonstrate that rough microspheres, microdisks, and rod-like particles move along deterministic trajectories to regions of maximum deviatoric curvature [ , , ] . non-spherical particles do not only migrate figure . comparison between immersion and floatation capillary forces between two spherical latex particles in a water film on a glass substrate: dependence of the capillary interaction energy, ∆w, on the particle radius, r. the distance between the particles is = l s , the contact angle θ α = c , and the liquid-vapor interface tension γ γ = v . see figure for a sketch of the system. reprinted with permission from [ ] . copyright ( ) american chemical society. translationally by sensing the background interface curvature, but also orient themselves to align their long axes, such that the excess area is minimized. for example, cylindrical particles reorient either parallel or perpendicular to the groove as the interface curvature changes from concave to convex [ ] . particle orientations for non-spherical particles, the orientation of the particles at interfaces, as well as particle shape, size, and surface properties have to be taken into account to determine adsorption energies and interface deformations. cube-like particles can be oriented in a corner-top or face-top orientation depending on the contact angle. elongated particles in a magnetic field or elongated janus particles in their stable orientations can be oriented with their long axes tilted with respect to the interface. one way to estimate the adsorption energy of a particle at an interface is to calculate the area of the fluid interface that is 'cut out'. this estimate can be exact if the interface tensions between particle and both phases are equal. for a particle that is symmetric with the interface acting as a mirror plane, the surrounding interface remains planar. however, in general particle-induced deformations of the surrounding interface can change adsorption energies and stable particle orientations even qualitatively. figure (a) sketches the various angles by which a cube-like particle can be rotated. figure for θ = c the flatinterface approximation predicts the edge-top orientation to be globally stable, while accounting for the deformation of the surrounding interface the corner-top orientation is found to be globally stable. figure (c) shows specific orientations for a cube-like particle at an interface. for a contact angle θ = . c , the globally stable state is the face-top orientation, while for θ = c the globally stable state is the corner-top orientation. figure (d) shows the predicted interface deformations for both cases using the same scale: hexapolar for the corner-top orientation and almost planar for the face-top orientation. capillary interactions between the cubes are therefore expected to be much stronger in the corner-top orientation compared with the facetop orientation. for particles with complex shapes, such as cube-like particles, a multitude of kinetically stabilised orientations and corresponding multipolar interface deformations is expected to be observed in experiments. a simple non-spherical particle with a stable tilted state is a janus dumbbell, which consists of two spherical particles made from different materials, see figures and . these particles do not deform planar interfaces, because the contact line around each of the spherical particles is a circle. figure (a) shows the experimentally observed orientations for dumbbell particles that consist of an hydrophobic spherical particle of radius r a and a hydrophilic spherical particle of radius r p . in their lowest-energy orientation, the spherical particles intersect the interface at different heights. the tilt angle of the dumbbell in the lowest energy state is [ ] see figure (b). here θ p is the contact angle at the polar particle, θ a is the contact angle at the apolar particle, α = r r a p / is the relative size of the two lobes, and with decreasing aspect ratio, the tilt angle of the janus dumbbell increases. for aspect ratio = b a / , only one of the spherical particles is in contact with the interface. however, also for cases where the tilted orientation of the dumbbell is predicted to have the lowest energy, a substantial fraction of particles are found experimentally in one of the kinetically stable orientations, where only one of the spherical particles intersects the interface [ ] . the lowest-energy orientation, where both spheres are at the interface, coexists with two kinetically stable orientations where only one of the spheres is at the interface. because the energy for an orientation where only one spherical particle is in contact with an interface does not depend on the tilt angle, an arrest in such a state could be caused by surface roughness. in these trapped states, the particles can therefore reorient to the tilted orientation only by diffusion. unlike janus dumbbell particles, janus ellipsoidal particles that are half hydrophobic and half hydrophilic do not show a continuous transition of their the orientation between a tilted long axis and a perpendicular long axis to the interface, see figures and . energy landscapes for various orientations of an ellipsoidal and a dumbbell particle are shown in figure . the lowest energy states for different aspect ratios are indicated in the figure using green symbols; the discontinuous transition in the particle orientation is clearly visible. the tilted orientation is only stable above a threshold aspect ratio and the orientation of the particle orientation jumps to the perpendicular orientation below this aspect ratio [ ] . these energies for various particle orientations have been calculated numerically, under the assumption of a flat interface surrounding the particles. whereas for ellipsoidal particles with homogeneous surface properties the deformations of the surrounding interface are quadrupolar with a dip at the tips and a rise at the long sides for contact angles below , see section . , the deformations of the interface around janus ellipsoids can show both a dip and a rise along their long sides [ ] , see figure . similar interface deformations have been observed for double-hydrophilic janus cylinders with aspect ratios . , . , and reprinted with permission from [ ] . copyright ( ) by the american physical society. . [ ] , see figure . the cylinders are found in kinetically trapped end-on orientations as well as globally stable tilted orientations. the cylinders have asymmetric hydrophilicity unlike most studies where the janus particles have amphiphilic properties [ ] . in their tilted orientations, they induce hexapolar interface deformations that lead to capillary interaction. complex particle assemblies have been observed due to the multi-lobed deformations and the non-determininistic assembly. fields. an external magnetic field can be used to tune the orientation of prolate ellipsoidal magnetic particles at fluid interfaces [ , , ] , see figure . the field tends to align the magnetic dipole with the direction of the applied magnetic field, where µ and h are the dipole and the field, respectively. the angle φ indicates the particle orientation and µ = | || | b h represents the field-dipole strength. the total free energy of the particle at the interface, neglecting particle-induced interface deformations, can therefore be written as where s is the area of the interface 'cut out' by the particle, and a sv is the area of the particle in contact with the vapor/the second liquid. if the field is oriented normal to the interface and if the direction of the permanent magnetic dipole coincides with the major axis of the ellipsoid, the analytical estimation of the tilt energy predicts that at a critical field strength the particle 'jumps' from a tilted orientation to a vertical orientation. here, φ = and φ = correspond to vertical and horizontal particle orientations, respectively. in figure , the energies of magnetic prolate ellipsoidal particles are shown as function of their orientation angle for various aspect ratios and field strengths. for particles with aspect ratio = b a . / and length of the minor axis a = nm , the energy barrier between vertical and tilted states can be several hundred k t b , such that the particles are trapped in one of both orientations. lattice-boltzmann simulations have been used to investigate the orientations of magnetic ellipsoidal particles in external fields including the deformations of the surrounding interface [ ] . the simulations show that the interface deformations around the particles significantly affect the tilt angles for a given dipole strengths, altering the properties of the reorientation transition. figure shows a simulation snapshot of the deformed interface and a plot of the tilt angle for various field strengths together with the analytical approximation discussed above. the simulation snapshot shows that the interface deformations remove more interface than in the planar-interface approximation, which lowers the free energy. deviations from the approximate result are found mainly for high aspect ratios. the discontinuous transitions allows the switching of the particle orientations using external magnetic fields, which could find applications for instance using its dynamically-tunable optical properties for electronic readers [ ] . furthermore, also the dipolar interface deformations can be switched on and off and thus lead to switchable capillary interactions between particles adsorbed at fluid interfaces. overlaps of interface deformations around particles lead to interface-mediated interactions, also called (lateral) capillary forces. capillary interaction energies between two particles, see figure , can be calculated based on the difference between the changes of the fluid-vapor interface area for placing two particles at given center-of-mass separation d cc and for placing two isolated particles at a planar interface. the corresponding capillary interaction energy is [ ] where ∆s ab is the change of the fluid-vapor interface area around the interacting particles, and ∆s a and ∆s b are the changes of the interface areas around the single particles. interface-mediated interactions follow power laws in the far field and are thus long-ranged. in the near field, the interactions strongly depend on the particle shapes. because the dynamics occurs in the stokes regime for small capillary numbers, figure . amphiphilic dumbbell particles at an oil-water interface. (a) in experiments, three dumbbell orientations are found: only the hydrophilic sperical particle intersects the interface, only the hydrophobic spherical particle intersects the interface, and both spherical particles intersect the interface. the circles and arrows indicate the size of the particle crossection at the interface and the shadow length obtained by freeze-fracture, shadow-casting (fresca) cryo-scanning electron microscopy, that can be used to measure the contact angle. (b) schematic diagram highlighting the geometry of the dumbbells at a liquid-liquid interface. reprinted with permission from [ ] . copyright ( ) american chemical society. see equation ( ), the particle velocities are proportional to the interaction forces and can therefore be extracted for example from videos. analytical solutions for the interaction forces are available in the far field, while numerical calculations have to be used in the near field. for large distances between particles, the deformation field of particle a can be assumed to be small at the position of particle b and vice versa. the capillary interaction energy can then be calculated using the superposition of the interface deformations around the single particles [ , , ] where h a and h b are the interface height deformation fields due to particle a and b, respectively. the lowest multipole contribution for a given system, see table , dominates the interaction. for sufficiently large particles for that gravity or buyoancy have to be considered, the capillary forces act between two monopole interface deformations [ ] πγ with the capillary length defined in equation ( ), the mass density ρ ∆ between the upper and the lower phase, and the capillary charges the angle ψ i is the slope of the interface at radius r i , the radius of the contact line. for )/( ), particle mass density ρ i , fluid mass density ρ , and upper fluid/gas mass density ρ g . stamou et al first presented an analytical result for the pair potential between two quadrupolar deformation fields in polar coordinates [ ] . in this quadrupolar approximation, the energy between two ellipsoidal particles is figure . janus ellipsoid and janus dumbbell at an interface with corresponding energy landscapes for particle orientations. while the dumbbell smoothly transitions to the perpendicular orientation with increasing aspect ratio, the ellipsoid jumps from a small tilt angle to the perpendicular orientation beyond a critical aspect ratio. reprinted with permission from [ ] . copyright ( ) american chemical society. interestingly, the magnitudes of the interaction potentials are equal in s-s, t-t, and t-s orientation for equal center-of-mass distances. kralchevsky et al extended the multipole approach to multipoles of arbitrary orders [ ] . whereas for weak deviations from circular contact lines a polar multipole expansion is most appropriate, for ellipsoidal particles with higher aspect ratios the appropriate choice are elliptical multipoles [ ] . ( )/ ( / ) , are observed for both cylindrical and ellipsoidal particles [ , ] . deviations are observed for small interparticle distances, which is discussed in section . . . elongated particles that touch each other have lower bond energies than predicted by the quadrupolar approximation in side-by-side orientation and higher bond energies in end-to-end orientation, see figure . capillary self-assembly is determined by the contact interactions between the particles, therefore near-field interactions are important for all many-particle systems. uncharged ellipsoidal particles at low bond numbers preferentially align in side-byside orientation [ ] . the higher stability of the side-by-side orientation over the tip-to-tip orientation is obvious from equation ( ) and is qualitatively unchanged by the deviation from the ideal quadrupolar approximation in the near field. using triangulated surfaces and energy minimisation, numerical calculations of deformation energies have been applied for various particle shapes, orientations, and contact angles. figure shows snapshots and interaction potentials for pairs of identical ellipsoidal particles with aspect ratios = b a / and = b a / [ ] . the bond energies increase quadratically as function of the bond angle φ | | that quantifies deviations from the side-by-side orientation with φ = . figure shows the bond-bending energies for two ellipsoids with shows a linear elastic regime with φ = t g , where γ = g a . [ ] ; the torques may reach values up to k t b . a bending modulus for the polymer-like assemblies of particles at the interface can be extracted from many-particle calculations. if two ellipsoids with different aspect ratios interact, the stable assembly is an arrow [ ] , see figure . unlike ellipsoidal particles, cylindrical particles preferentially assemble in end-to-end orientation, see figure . because of the flat ends of cylindrical particles, the contact figure . configurations of double-hydrophilic janus cylinders at the air-water interface. the first and second columns are sem images of janus cylinders embedded in pdms slabs prepared by the gel trapping method. arrows indicate the location of the wettability separation line. the schematic representations show the side view of particle configurations at the air-water interface where four colors represent four different particle-fluid surfaces: weakly polar surface in air (black), weakly polar in water (red), strongly polar in air (blue, rarely shown), and strongly polar in water (cyan). the third column shows the corresponding optical microscopy images. the scales bars are µm . reprinted with permission from [ ] . copyright ( ) american chemical society. figure . sketch of an ellipsoidal particle adsorbed at a liquidliquid interface in (a) vertical, (b) horizontal, and (c) tilted orientation. the variables defining the geometry and orientation of the particle are discussed in the text. the direction of the magnetic field is indicated by b. reprinted from [ ] . © iop publishing ltd. all rights reserved. line can adjust its height rather freely if the height of the surrounding interface changes because the contact angle remains unchanged. when two cylindrical particles approach each other, the energy gain for wetting the surfaces overcomes the costs for the deformation of the surrounding interface, thus the interface forms a capillary bridge between the two ends. the bond-bending energies for cylindrical particles around their preferred tip-to-tip state are very high, with restoring torques up to = t k t b [ ] . therefore, such chains are usually not distorted. two cylindrical particles may switch to the metastable side-by-side orientation only for bond angles larger than φ ≈ c - , which can be achieved for particulate monolayers under compression or shear stress. the height of the capillary bridge that forms between two cylindrical particles decreases when the distance between them is increased, compare snapshots for = d a . / with = d a . / in figure ; it changes also if the particles are tilted with respect to each other, compare snapshots for = d a . / and φ = , and for = d a . / and φ = . for cylindrical particles, bond bending is not elastic as for ellipsoidal particles, but shows non-elastic hinging behaviour. therefore, both bond energies and bond-bending energies are significantly affected by deformations of the surrounding interface and by changes of the wetting of the planar faces of the particles. this importance of wetting energy is a qualitative difference for the capillary interactions between ellipsoidal and between cylindrical particles. the crossover between elastic bond-bending and nonelastic hinging can be further explored using superegg-shaped particles with variable edge curvatures, as discussed in [ ] . . reprinted from [ ] . © iop publishing ltd. all rights reserved. / . (b) comparison of the tilt angle of the particle obtained from the analytical, planarinterface approximation in [ ] and the numerical data in [ ] . although the numerical data qualitatively confirms the prediction of a discontinuos transition, there are quantitative deviations in particular at higher aspect ratio. reprinted from [ ] with permission from the royal society of chemistry. and ω indicate the orientation of the particle or , respectively, with respect to the vector joining the centers of the two particles. the center-to-center distance is given by d cc for a particle of aspect ratio b a / . reprinted with permission from [ ] . copyright ( ) american chemical society. in general, the presence of one particle changes the contactline position on the other particle. figure (c) shows the deformation of the initially circular contact line around a spherical particle by a nearby ellipsoidal particle. figure shows that although the spherical particle at a planar interface by itself does not deform the interface and does not induce interfacemediated attraction, an ellipsoidal and a spherical particle mutually attract each other. the attractive interaction between spherical and ellipsoidal particles is larger for side-on configurations of the spherical particles than for tip-on configurations. in both configurations, however, the attraction between spherical and ellipsoidal particles is significantly weaker than the attraction between two ellipsoidal particles with similar sizes that have equal aspect ratios and surface properties. attractive energies for capillary bonds and energy barriers between locally stable configurations are often high, therefore computer simulations show self-assembly in kinetically-trapped dendritic and raft-like structures [ ] . numerical calculations for two-particle, three-particle, and four-particle interactions predict a variety of stable and metastable configurations [ , ] . many-particle studies of particles at interfaces can also be extended to polydisperse particle mixtures [ ] . , and ellipsoidal particles with aspect ratio = b a / that have been obtained by deformation of spherical particles with µ = a m [ ] . for prolate ellipsoidal particles mostly the side-by-side orientation is observed in experiments, hexagonal networks can be stable if an additional repulsive electrostatic repulsion contributes to the particle-particle interaction. figure (a) shows self-assembly of slightly charged micrometer-sized ellipsoidal particles. the structure initially shows many tip-to-tip contacts; after slow relaxation it compactifies slightly and shows less triangular and more chain-like aggregates in side-by-side orientation after h [ ] , see figure (b). the chains of particles in side-by-side orientation can be thought of as 'colloidal polymers': worm-like chains or rings with bending elasticity. for non-spherical particles at planar interfaces, particleparticle interactions may affect the orientations of particles and vice versa. for example, cube-like particles at lower interfacial densities assemble on hexagonal and honeycomb lattices in corner-top orientation [ ] , and at higher interfacial densities on a square lattice in face-top orientation [ ] . figure shows how particle orientations affect interparticle interactions: chains form for prolate ellipsoidal particles that are oriented with their long axes parallel to the interface, while particles disperse homogeneously if they are oriented with their long axes perpendicular to the interface. particle orientation can be controlled using an external magnetic field [ ] , see section . . . a similar tunable (dipolar) interface deformation and therefore tunable capillary interaction can be achieved using magnetic spherical janus particles [ ] . interface curvature alters particle-induced interface deformations and therefore many-particle self-assembly, see section . . . in order to systematically understand the interaction of many particles at non-planar interfaces, it is important to study model systems with controlled curvatures [ , [ ] [ ] [ ] [ ] . for example, spherical particles at interfaces with saddle-like shapes induce quadrupolar interface deformations and therefore at low densities assemble on a square lattice [ ] , see figure (a); at high densities, the optimal packing on a hexagonal lattice is observed. the local lattice structure can be investigated using bond-orientational order parameters, where n j indicates the number of neighbors of a particle. the angle θ j is the angle between the bond with a neighboring particle j and an arbitrary reference axis. figure (b) shows that the lattice order switches continuously from square to hexagonal with increasing particle density. particles embedded within a nematic liquid crystal (lc) interact with the nematic directors in the vicinity of the colloid and therefore impose boundary conditions on the nematic order parameter at the colloid-lc boundary. this leads to longranged anisotropic elastic interactions for spherical and ellipsoidal particles dispersed in nematogenic fluids [ , ] . due to varying nature of nematic defects at the colloid-lc boundary, typically elastic multipoles, such as dipoles and quadrupoles, are observed. large pair interaction strengths render the self-assembled aggregates insensitive to thermal or hydrodynamic fluctuations [ ] . capillary interactions dominate the elastic energies [ ] . for microspheres in nematic films, strength and nature of the elastocapillary interactions depend on film thickness and particle size [ ] . for small thicknesses of films, giant elastic dipoles occur due to interface distortions. defects appear around particles, elastic dipoles and nematic elasticity counterbalance the strongly attractive capillary interactions and stabilize particles pairs at finite distance. figure shows how cylindrical nanoparticles first assemble at a nematic interface in their energetically favourable tip-to-tip orientation and then align with the nematic director [ ] . the weak elastic interactions are able to manifest themselves at flat nematic interfaces, but under curvature gradients again capillarity dominates [ ] . particle anisotropy shall also play a significant role for defect formation at colloid-lc boundaries and shall thus also control nature and strength of pair-particle interactions. future experiments and theoretical investigations to elucidate these elusive interplay between capillarity and elasticity are warranted. applications for (engineered) particles at interfaces range from emulsion stabilisation and colloidosomes to suppression of the coffee-ring effect and froth floatation. in addition to capillary forces, also for example marangoni flows, surfactants, electric charges, or gravity have to be taken into account. a classical application is the stabilisation of emulsions. here, the interactions of particles at interfaces on the mesoscale can be expected to determine macroscopic rheologial properties. in general, the strong adsorption of particles to interfaces leads to thermodynamically stable droplets of oil (water) suspended in water (oil) [ , , [ ] [ ] [ ] . at low particle concentrations, the mechanism of limited coalescence generates narrow and reproducible droplet-size distributions [ ] . phase inversion of droplet emulsions is obtained by increasing the volume fraction of oil (water). for mixtures for small hydrophilic and hydrophobic particles also sponge-like, bicontinuous phases and non-spherical colloidosomes have been observed [ , ] . particles with anisotropic surface functionalization, such as janus particles, can be used as amphiphilic colloidal surfactants [ , ] . particle-stabilised emulsions are used for example in food and cosmetic industries [ ] [ ] [ ] [ ] . colloidosomes are solid capsules engineered with controlled permeability and mechanical strength that can have sizes from sub-micrometers to millimeters [ ] . the capsules are prepared by self-assembly of colloidal particles to emulsion droplets that are then locked together for example using sintering or electrostatic binding of an oppositely-charged polyelectrolyte. this ensures that the shells remain intact when they are transferred to a different fluid. with the help of centrifugation, the particles are typically transferred into a solvent that is identical to the internal phase. colloidosomes can be used for encapsulation of drugs, proteins, vitamins, flavors, gas bubbles, and even living cells. particles that are homogeneously dispersed over an entire drop often form ring-like deposits when the drop evaporates, so-called 'coffee rings' [ ] . addition of ellipsoidal particles with aspect ratio . , obtained by deforming spherical particles with µ = a . m , to droplets that contain the spherical particles has been found to lead to more homogeneous deposition [ ] . for droplets that contain ellipsoidal particles only, a strong suppression of the coffee ring effect is already observed for very moderate aspect ratios ≈ b a . / . numerical calculations predict capillary attraction between spherical and ellipsoidal particles [ ] ; the uniform deposition may therefore be caused by capillarity-induced cluster formation. applications for that a more uniform deposition of the material instead of 'coffee rings' may be desired are inkjet printing, fabrication of microand nanostructures, and coating. froth floatation is used to separate minerals from gangue. the separation efficiency depends on both the particle-bubble attachment and the froth stability [ , ] . while the particle-bubble attachment can be adjusted using collector chemicals that modify the particle's surface properties, the film stability is affected by particle shape. one complication that decreases froth stability can be particles with sharp edges [ ] . therefore, a systematic understanding of the interaction of non-spherical particles with thin films and potentially also of immersion forces may help to improve froth floatation [ , , , ] . particles adsorb at biological interfaces, often called membranes, because of an adhesion strength w between the particles and the membranes. in addition to the adhesion strength, the minimal ingredients required to study spherical particles are the bending ridigity κ and the tension γ of the membranes, and the radius a of the particles. for small particles, molecular interactions are important. coarse-grained or even atomistic computer simulations are used to investigate theoretically translocation through and incorporation within membranes [ ] [ ] [ ] [ ] . experimentally, the interaction of small particles with membranes can be investigated using a combination of microfluidic devices, fluorescence microscopy, and electrophysiological measurements [ ] , by scattering techniques [ ] , and by quartz crystal microbalance and afm [ ] . for larger particles with radii > a nm , wrapping is the dominant mechanism of the interaction between particles and membranes, as will be discussed in detail in this section. particles that interact with membranes can be engineered and are found in biological systems with a wide variety of sizes, shapes, and surface functionalizations, see figure . comparing the different energetic contributions, large spherical nanoparticle adhesion and wrapping is determined by two characteristic crossover radii, the radius κγ * a that compares the bending energy with the membrane tension characterizes the crossover between the bending-dominated regime for particles with < κγ * a a and the tension-dominated regime for larger particles. while for nanoparticles bending energy is thus the main player, for micrometer-sized particles tension plays the dominant role. within the bending-dominated regime, small particles with < κ * a a w remain unwrapped, while larger particles get wrapped, determined by the subtle balance of bending and adhesion energy [ , [ ] [ ] [ ] . for given particle radius, the threshold adhesion strength κ = * w a / therefore marks the transition between the unwrapped and the complete-wrapped regime. adhesion may be mediated by van der waals interaction [ ] , hydrophobic interaction [ ] , electrostatic interaction [ ] , and specific adhesion via receptor-ligand bonds [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in biological media, nanoparticles can be surrounded with a corona of proteins that effectively changes their surface properties [ ] [ ] [ ] [ ] [ ] [ ] . throughout this section, we assume that the adhesion can be modeled by a continuous and mostly homogeneous adhesion strength, which is appropriate for many systems, but not always sufficient for / . an overview of both single-particle and many-particle systems at membranes is provided in this section. in sections . - . we focus on various aspects of the interaction of single particles with membranes, while in sections . and . we discuss two-particle and many-particle interactions, respectively. section . focuses on particles at membranes with spontaneous curvature. we finally discuss applications in section . . for a spherical particle and an infinitely large tensionless membrane, the membrane surrounding the particle assumes a catenoidal shape, see figure (a). this shape is found around all cylindrically-symmetric, 'conical' membrane inclusions, figure . interaction of a spherical particle with an ellipsoidal particle of aspect ratio = b a / . the contact angle θ = . c has been used for both particles. the energies are plotted for spherical particles with radii = r a and = r a that approach the ellipsoidal particle (a) at the side (b) and at the tip, here shown for a spherical particle with = r a . (c) interaction energies γ ∆e a / as function of d a cc / between the centers of mass of the particles. inset: doublelogarithmic plot and the fit of the numerical data to a power-law decay. reprinted with permission from [ ] . copyright ( ) american chemical society. which includes curved proteins [ , , ] and attached polymers [ ] [ ] [ ] [ ] . a catenoid is a minimal surface with vanishing mean curvature at every point and therefore with vanishing bending energy. the deformation energy cost for the membrane adhered to the particle and the adhesion energy gain for contact between particle and membrane can therefore be directly compared with each other in order to predict whether a particle gets wrapped or not. the wrapping transition is fully characterized using the critical particle radius κ * a w for wrapping in equation ( ) . however, in general the deformation energy of the membrane surrounding a particle will contribute to the total deformation energy of the membrane. a convenient way to characterize wrapping states of particles for various parameters are wrapping diagrams based on energy minimisation, analogous to phase diagrams in thermodynamics [ , , ] , see figure (b). spherical particles remain unwrapped below a threshold adhesion strength and get partialwrapped or complete-wrapped for higher adhesion strengths. particles at membranes with finite tension can be found in stable partial-wrapped states for adhesion strengths just above those for the binding transitions, see figure (b). the stable partial-wrapped states are separated by continuous transitions from the unwrapped states and by discontinuous transitions from the complete-wrapped states. the latter trans itions with energy barriers are associated with two spinodals that indicate those values for the adhesion strength beyond which particles transition spontaneously from partial-wrapped to completewrapped states and below which particles transition spontaneously from complete-wrapped to unwrapped states. membrane deformations induced by non-spherical particles depend on both particle shape, discussed in this section, and particle orientation, discussed in section . . in general, inhomogeneous surface curvature of particles corresponds to energy barriers for wrapping [ , ] . for non-spherical particles, membrane tension is therefore not required to stabilize partial-wrapped states; such particles are ideal membrane markers for imaging. thus not only size and aspect ratio, but also local particle surface curvature matters for wrapping of non-spherical particles [ ] . the importance of local particle surface curvature can be demonstrated well for hauser's cube-shaped particles. the phase diagram in figure shows that no membrane deformation energy costs arise if cube-like particles attach with a flat face to a membrane. the particles therefore adhere already for very small adhesion strengths and are found in shallow-wrapped states. the first energy barriers are encountered between shallow-wrapped and deep-wrapped states when the membrane has to bend around the highly-curved edges to increase wrapping from one face to five faces. the energy barriers between deepwrapped and complete-wrapped states correspond to the last four edges to be wrapped. both energy barriers shift to higher adhesion strengths for higher values of the membrane tension. we discuss here particles within the hydrophobic tail region of lipid bilayers in part and particles at curved membranes in part . particles with sizes of few nanometers may incorporate themselves in membranes and locally distort both lipid order and membrane thickness [ , , ] , see figure (a). while lipid order can only be investigated on the molecular scale, thickness variations can also be investigated using continuum models. here the headgroup positions of the monolayers can be modeled by mathematical surfaces with bending rigidity and monolayer tension, and a confinement potential which maintains the lipid bilayer thickness. analogous to integral membrane proteins with hydrophobic mismatch [ ] [ ] [ ] [ ] , particles that locally induce membrane-thickness deformations experience short-ranged deformation-mediated interactions. while the interactions may be repulsive at long distances as predicted by the continuum models, both continuum models and molecular dynamics simulations predict short-ranged attractive interactions and therefore clustering of nanoparticles within membranes. for high membrane tensions, the energy barrier for a third particle approaching a pair of particles is considerably decreased, such that particle aggregation may occur spontaneously [ ] , see figures (b) and (c). not only particle shapes, also membrane curvature prior to wrapping has to be taken into account for the interaction of particles with membranes. this can be demonstrated by calculating entry of particles into and exit of particles out of vesicles with different curvatures, see figure . the vesicles are assumed to freely adjust volume but keep a fixed membrane area. vesicles without particles thus adopt an overall spherical shape that is then locally distorted by binding a nanoparticle. the adhesion strength w and / , for vesicles with radius r v , are the relevant parameters to characterize this system. they are a subset of the full parameter space, determined in addition by membrane spontaneous curvatures and by osmotic pressures [ ] . for particles that enter vesicles from the outside, > c r , wrapping is hindered by an energy barrier. for particles that exit vesicles from the inside, < c r , upon increasing the adhesion strength the particles continuously transit from the free state via stable partial-wrapped states with increasing wrapping fractions to the complete-wrapped state. partial-wrapped particles can be used as probes for the local membrane curvature [ ] . a direct and barrierless transition between the free and the complete-wrapped state is only present for particles at infinitely large planar membranes. with decreasing absolute relative curvature c r , both the energy barriers for particle entry as well as the stability of partially-wrapped states for particle exit decrease. the adhesion strengths for the wrapping trans itions can be well approximated by the instability relations [ , ] for non-spherical particles, the orientation of particles at membranes-in addition to particle shapes, sizes, and surface properties-has to be taken into account to determine / and membrane tensions γ γ κ = a / . (a) the membrane deforms in a cylindrically symmetric way around the symmetry axis and the shape can be described by a radial deformation profile. for an infinitely large tensionless membrane, the shape of the free membrane is catenoidal. (b) the wrapping phase diagram shows that the nanoparticles are unwrapped below a critical adhesion strength = w , and completely enveloped for adhesion strengths beyond the thick solid line. the thin dotted line is the envelopment transition calculated neglecting the deformation energy of the free membrane. the thin dashed lines are the spinodals for spontaneous unwrapping from the complete-wrapped to the free/non-wrapped state and for spontaneous envelopment from the partially wrapped to the fully wrapped state. adapted with permission from [ ] . © epla. all rights reserved. wrapping states and membrane deformations. elongated particles can be oriented in rocket and in submarine orientation, with their long axes perpendicular and parallel to the membrane, respectively. janus particles preferably bind with their most adhesive side to the membrane. changing the orientation of particles in magnetic fields can be used to probe the elastic properties of membranes and cells. the orientations of elongated particles at membranes crucially depend on particle shapes, membrane elastic properties, and membrane-particle interactions, see figure . table summarizes wrapping states and transitions for various particles shapes and membrane elastic parameters based on the results of [ , , , ] . for prolate ellipsoidal particles, the particles bind to membranes with their points of lowest curvature at the sides of the particles. in these shallow-wrapped states, they are in the so-called 'submarine orientation'. for fast wrapping, if reorientation does not occur, the particles remain in submarine orientation until they reach the complete-wrapped state [ ] . for for two corresponding states at the w phase boundary: (a) a shallowwrapped state with approximately % of particle wrapped, and (b) a deep-wrapped state with approximately % of the particle wrapped. (c) phase diagram for wrapping of hauser's cube for reduced membrane tension γ γ κ = a / and reduced adhesion strength πκ = w wa /( ), where a is the particle surface area. we find a shallow-wrapped (sw), a deep-wrapped (dw), and a completewrapped (cw) state, separated by two discontinuous wrapping transitions, labeled as w and w . adapted with permission from [ ] . copyright ( ) american chemical society. morphology diagram of stable states of a particle adhering to a vesicle. the wrapping state is plotted for various values of rescaled adhesion energies κ = u wa / and relative curvatures c r of the vesicle membrane and the particle surface. the three black lines divide the diagram into three regions in which the particle is either partially wrapped, unwrapped, or fully wrapped. in the grey shaded region, the transitions between the unwrapped state and the wrapped state of a particle outside the vesicle require the crossing of an energy barrier. the red dashed lines are analytical instability lines derived from the stability relations in [ ] . reproduced from [ ] .-published by the royal society of chemistry. oa cc by . . slow wrapping, the particles are able to reorient to their minimal-energy states at every time, they transit from submarine orientation to the so-called 'rocket orientation' at deep wrapping. the rocket state is energetically preferable at deep wrapping, because only one of the pointed tips needs to be wrapped. from a stable deep-wrapped state, ellipsoidal particles then continuously transition to the complete-wrapped state [ ] . for rod-like particles, energy minimisation predicts that the particles readily bind with their blunt tips in rocket orientation at very small adhesion strengths. if the edges of the particles are sharp and their aspect ratios are small, the particles remain in rocket orientation until they reach complete wrapping; the transitions between shallow-wrapped and deep-wrapped, as well as between deep-wrapped and complete-wrapped states are discontinuous. for high aspect ratios or round edges of the particles, rod-like particles bind with their blunt tips in rocket orientation only with a very small fraction of their surface area. they then rotate to submarine orientation in the shallow-wrapped state, and back to rocket orientation in the deep-wrapped state [ , ] , see figure . in particular, the theoretical prediction for the rotation from submarine orientation to rocket orientation is in agreement with experimental observations for budding of filamentous viruses [ , ] . modes of entry for nanoparticle uptake by membrane wrapping: (i) submarine mode with the long axis of the particles oriented parallel to the membrane, (ii) rocket mode with the long axis oriented perpendicular to the membrane, and (iii) competition between submarine and rocket mode as observed for rod-like particles with high aspect ratios. the complete-wrapped particle is connected by an infinitely small catenoidal neck to the membrane; the particle orientation in this state is irrelevant. reprinted with permission from [ ] . copyright ( ) american chemical society. table . shape dependence of particle wrapping, based on the results of [ , , , ] . the membrane can be characterized by bending rigidity only, 'κ', or by bending rigidity and membrane tension, 'κ and γ'; the binding transition can occur at finite or vanishing adhesion strength w; the particle can be in submarine or rocket orientation; transitions can be continuous (cont.) or discontinuous (discont.) and may involve reorientation (reorient.). the binding transition for ellipsoids is independent of the membrane tension and is given in [ , ] . binding transition a fast wrapping at high adhesion strength, such that a bound ellipsoid cannot reorient to rocket orientation. b rocket mode for supereggs with blunt tips and small aspect ratio (e.g. = n and = b a . / ). reprinted with permission from [ ] . copyright ( ) american chemical society. the dynamics of wrapping of spherical nanoparticles is determined by the typical time scales for the relevant processes, such as membrane deformation [ ] , receptor or protein diffusion that may be hindered by a cortical cytoskeleton [ ] [ ] [ ] [ ] [ ] , and potential metabolic remodeling of the cytoskeleton [ ] [ ] [ ] . after initial contact between nanoparticles and membranes, the deformed area of the membrane surrounding the particles increases until half wrapping and then again decreases. while the membrane deformation is catenoid-like for small and for large wrapping fractions of particles, for finite membrane size the highest deformation energy costs are expected for about half-wrapped particles. formation of a defect in the neck towards the end of the wrapping process can induce the separation of bent and flat membrane and completes wrapping [ ] . the reorientation dynamics for elongated particles can be calculated using molecular dynamics simulations for nanoparticles that interact with initially flat lipid bilayer membranes [ , ] . also local free-energy analysis and incremental changes of the nanoparticle orientation in the direction of lowest energy at each time step allow to predict the wrapping pathway [ ] . in figure , the corresponding curvature-energy landscapes are displayed. a spherocylindrical nanoparticle that is initially oriented in the unfavourable rocket orientation first reorients towards submarine orientation. however, although energetically most favourable until half wrapping the particle may never actually reach submarine orientation. beyond half wrapping, the particle turns back to the then favourable rocket orientation, in agreement with the energetics discussed at the beginning of this section. we are not aware of systematic experimental studies for the interaction of lipid-bilayer membranes with nanoparticles that have anisotropic surface functionalization. however, micrometer-sized spherical particles that are half coated with ligands preferably orientate with the ligandcoated side towards the membrane during phagocytosis [ ] . furthermore, viruses have been modeled as partially adhesive particles [ ] . the reorientation of the malaria parasite in tipfirst orientation at the beginning of the invasion process may also be due to an adhesive gradient on the parasite surface [ ] . analytical calculations for partial-wrapped particles, e.g. janus particles, at non-spherical vesicles show that membrane curvature-induced forces pull the particles to regions with preferred membrane curvature [ ] . in analogy to magnetic particles with switchable orientation at fluid interfaces, micrometer-sized magnetic particles can also be attached to cell membranes with an underlying cytoskeleton [ ] . such microrheological measurements reveal glassy behaviour for various cell types [ ] [ ] [ ] [ ] . for red blood cells with their cortical spectrin cytoskeleton responsible for the shear elasticity of the complex cell membrane [ ] , the values of the elastic parameters obtained using magnetic particle microrheology agree well with those used for computer simulations for cell stretching and blood flow [ ] . membrane deformations induced by partial-wrapped particles lead to membrane-mediated interactions that minimise the sum of both membrane deformation energy and particlemembrane attachment energy [ ] . several recent studies are discussed in this section. however, much more is knownand this knowledge may partially be transferred to nanoparticles-about the related systems of lipid bilayer-mediated interactions between curved inclusions, both for membranedeformation mediated interactions [ , , , , ] , as well as for membrane-fluctuation mediated casimir interactions [ ] [ ] . membrane-mediated interaction between two parallel and long cylindrical particles at distance d attached to the same side of membranes under lateral tension is repulsive [ ] , / predicted by local energetics. the nanoparticles take a general laying-down-thenstanding-up sequence during endocytosis. the heat maps show the curvature energy for various particle orientation angles and wrapping fractions. nanoparticle wrapping at the turning points (i-v) along the endocytic pathways predicted by local free-energy analysis is schematically shown on the right: green-shaded areas are wrapped, while yellow-shaded areas are naked. reprinted with permission from [ ] . copyright ( ) american chemical society. where ξ γ κ = / is a characteristic reciprocal length, and a cyl is the cylinder radius. the interaction between two cylindrical particles attached to opposite sides of the membrane is attractive, here, the length of the cylinders is contained in the param eters for the bending rigidity, adhesion strength, and membrane tension for these effectively one-dimensional calculations. results for stronger membrane deformations can be found in [ ] . only few studies are available for far-field interactions between spherical nanoparticles that are partially attached to lipid bilayer membranes. figure shows both numerical calcul ations and experimental data. membrane-mediated particle attraction is found for distances below a , with an attractive energy well of κ ≈ − e . a for an interparticle distance of about a . [ ] . for typical lipid bilayer bending rigidities κ < < k t k t b b , this corresponds to binding energies the figure shows larger attraction strengths for membrane tensions γ < nn m / compared with membrane tensions γ µ > n m / . many computer simulation studies for membrane-mediated interactions between partial-wrapped nanoparticles, as shown in figure , rely on finite-element calculations. here, the bending energy is discretized on triangulated surfaces [ ] [ ] [ ] [ ] . large systems that require at the same time fine discretization in high-curvature regions around particles are computationally expensive, therefore membrane-mediated interactions between the particles have so far mostly been studied in the near field. however, long-ranged membrane-mediated interactions between partial-wrapped nanoparticles might be similar to long-ranged interactions between curved inclusions. for example, for two spherical-cap inclusions attached to the same side of planar membranes, the repulsive membranemediated interaction is [ ] where α and α are polar angles that determine the sizes of the spherical caps, a is their radius, and d is the distance between the centers of the caps. strongly-curved inclusions at very close distances experience an attractive interaction [ ] . interactions between two (spherical) nanoparticles have been shown to be attractive, see e.g. figure . this is surprising on the first view, because catenoid-like membrane deformations around partial-wrapped nanoparticles at large distances require only very small deformation-energy costs. if two spherical-cap inclusions or two particles approach each other on a planar membrane the ideal catenoid-like deformations cannot form any more because of the boundary conditions. therefore, the membrane-mediated interactions because of deformation-energy are expected to be repulsive [ , ] . however, for spherical particles the attractive membrane-mediated interactions because of membrane-particle adhesion energy dominate and lead to an overall attraction [ ] . for two partial-wrapped particles in the near field, not only the interparticle distance, but also the orientation of the pair of particles with respect to the membrane has to be taken into account. for example, in [ ] the connecting line between two particles adsorbed to a vesicle is parallel to the membrane at large distances and reorients to perpendicular orientation at small distances. the attractive interaction is significantly higher for this particularly stable tubular arrangement [ ] . many-particle interactions are important for membrane-mediated interactions, which has been demonstrated in several cases for integral membrane proteins with hydrophobic mismatch and for spherical-cap inclusions [ , , , ] . it is therefore essential to not only consider pair interactions between particles. we start our discussion with the 'inclusion case', i.e. with membrane deformation-mediated interactions only, where the membrane area attached to each particle is fixed. while two weakly-curved spherical-cap inclusions that are attached to the same side of the membrane repell each other as discussed in section . . , many inclusions have been observed to aggregate and to induce bud formation [ , , [ ] [ ] [ ] . a membrane that is curved prior to adhesion of inclusions or that gets curved by cooperative budding screens the repulsive interaction and can therefore lead to effective attraction. figure highlights catenoidal membrane deformations around inclusions on vesicles; the size of the catenoidal 'halo' shrinks with increasing background curvature, thereby screening the repulsive interaction between the inclusions. these catenoidal patches reduce the total bending energies of the vesicles, which vanish at optimal inclusion density. in comparison, on planar membranes curved inclusions always increase the deformation energy. a particle dimer switches from a linear aggregate on the membrane to a tubular aggregate for increasing wrapping fraction [ , ] , compare also section . for the orientation of elongated particles. on a vesicle, the wrapping fraction can be tuned by adjusting the reduced volume of the vesicle, where v is its actual volume and v sph is the volume of a spherical vesicle with the same membrane area. a small reduced volume also allows more than two particles to join the tube. the energy gain of such tubular assemblies compared with single, complete-wrapped particles strongly depends on the ratio of the range ρ of the particle-membrane interaction potential and the particle radius a. the finite potential range leads to a higher adhesion energy gain for tubular arrangements compared with single-particle buds, because some fraction of the tubular necks between particles are inside the interaction range; for example the energy gain per particle is about κ for ρ = a . / [ ] . simulation snapshots and energies for several configurations of three particles attached to membranes are shown in figure . if the particles are all located in the plane of the membrane, a third particle that attaches to form a linear aggregate gains membrane-mediated binding energy of few k t b and attaches without an energy barrier. in contrast, a particle that attaches from the side to the existing particle aggregate experiences an energy barrier and has a few k t b higher energy in the (metastable) bound state compared with the unbound state. bending energy has been shown to favour compact aggregation, . here, φ = corresponds to a linear arrangement of the particles. the four snapshots depict minimum-energy conformations at the angles φ = , , , and . reprinted with permission from [ ] . copyright ( ) by the american physical society. adhesion energy linear aggregation [ ] . also for tubular aggregates, the linear-tube configuration is preferred over the more compact, triangular configuration [ ] , see figure (b). linear aggregates in the plane of the membrane have been experimentally observed for colloidal particles bound to giant unilamellar vesicles (guvs) [ ] , tubular aggregates for the interaction of viruses with cells and guvs [ ] . figure (a) shows a phase diagram for many-particle systems in terms of bending rigidity κ and particle-membrane binding energy d [ ] . in the limit of small bending rigidities, partial-wrapped particles form a hexagonal cluster phase where the membrane penetrates in-between the particles. in the limit of high bending rigidities, the particles are barely attached to the membrane. they deform the membrane only weakly, therefore they also interact only weakly and are found in loose, mostly hexagonal aggregates. linear aggregates are observed inbetween both limites for biologically relevant bending rigidities κ < < k t k t b b . figure (b) shows a phase diagram in terms of particle diameter and particlemembrane binding energy. free unbound nanoparticles are found at low adhesion strengths and for small particle radii, then linear aggregates, tubular aggregates, and single-particle buds are observed with increasing adhesion strength [ ] . this sequence of configurations is consistent with the reorientation reported for elongated nanoparticles in [ ] , see section . . biological membranes are often not symmetric, the two monolayers usually consist of different lipids. this asymmetry can be modeled with the help of a spontaneous curvature c of the membrane. if the directions of spontaneous curvature and the curvature of the particle surface coincide, the spontaneous curvature facilitates complete wrapping compared with a symmetric membrane [ ] . at the same time, the direct transition between free and complete-wrapped states is replaced by a discontinuous transition. for a spontaneous curvature opposite to the curvature of the particle surface, the complete-wrapped state is shifted to higher adhesion strengths. furthermore, a new regime with partial-wrapped states is found. with increasing spontaneous curvature, also the regime of adhesion strengths where partial-wrapped states are stable increases. for fixed adhesion strengths, finite spontaneous curvatures lead to size selectivity for wrapping of nanoparticles, see figure . for vanishing or small spontaneous curvatures, all particles with radii beyond a threshold radius get figure . membrane-mediated interactions between many particles. (a) phase diagram for nanoparticle self-assembly in terms of membrane bending rigidity κ and particle-membrane binding energy d , which replaces the adhesion strength w in the simulations. the snapshots show typical aggregates in the h , the l, and the h phase (top to bottom). adapted with permission from [ ] . copyright ( ) by the american physical society. (b) phase diagram in terms of nanoparticle diameter a and d . with increasing d , the gaseous phase g with non-attached particles is followed by the linear aggregation phase l, the tubeformation phase t, and the single-particle bud phase b. the radius of the vesicle is σ = r v and the particle surface fraction is . . adapted with permission from [ ] . copyright ( ) by the american physical society. wrapped, see equation ( ) . with decreasing spontaneous curvatures, in addition to the lower threshold radius for complete wrapping, also an upper threshold radius beyond the that particles remain unwrapped is found [ ] . the regime with stable complete-wrapped states thus narrows to a small window in the particle radius for high negative values of the spontaneous curvature. such a preferred radius for particle wrapping has been observed in experiments [ , ] . size selectivity by spontaneous membrane curvature is therefore an alternative to receptor-based models for cellular uptake that are usually used to motivate an upper limit for the particle radius to achieve complete wrapping [ ] . on the one hand, particle-membrane systems can be used for applications in therapeutics and diagnostics. on the other hand, for numerous other applications in industry, potential toxic effects have to be considered. furthermore, nanostructured surfaces can be rationalised as nanoparticles bound to a substrate, which opens an entire new field for applications. for basic research, particle-membrane systems can serve as model systems to understand biological processes, such as viral budding, malaria invasion, and phagocytosis. . . . drug delivery. nanoparticles are potential drug-delivery vectors and tools for diagnosis [ ] [ ] [ ] [ ] [ ] . for diagnosis, in particular non-spherical nanoparticles with enhanced stability of partial-wrapped states can serve as membrane markers for imaging [ ] . for drug delivery, rough hydrophobic particles have been proposed to have a high drug-carrying capacity and high loading efficiency [ ] . such particles can prolong the time of release of a drug payload, which can be therapeutically advantageous. nanoparticles may even cross the blood-brain barrier via transcytosis and may therefore be applied for drug delivery in the brain [ , ] . recently hollow shell-shell nanocontainers with stimuli-responsive properties that could allow for controlled drug release have been suggested for drug delivery [ ] . for studies of potential toxic effects, not only single-particle properties, such as composition and size, but also particle concentrations play an important role [ ] . toxicity can be quantified by ec values that refer to the concentration of particles that induce a response of cells or organisms halfway between the base line and the maximum after h exposure time to nanoparticles. while agno leads to growth inhibition for algae already at concentrations of − − mg l , ag nanoparticles lead to growth inhibition only at concentrations between − . mg l and − mg l , see figure . most other organisms are less sensitive to nanoparticles and growth inhibition occurs only at higher particle concentrations compared with algae. in general, the ec values increase significantly with particle size, which means that the toxic effect is reduced. for mammalian cells they range from . . . nanostructured surfaces. multi-electrode arrays allow to electrically couple excitable cells to electronic devices, see figure . the challenge is to achieve an optimal coupling, which requires to minimize the cleft between the cell and the electrode and to achieve tight attachment of the cell to the substrate in general. while thin electrodes pierce cell membranes [ ] , micro-electrodes get wrapped [ , ] . a systematic study of different micro-electrode shapes reveals that mushroom-shaped pillars are engulfed more than cylindrical pillars without caps [ ] . furthermore, cells have been found to preferably engulf pillars in their center compared with their edge. quantitative evaluation of focused ion-beam cuts allows to extract normal cytoskeletal stresses between few pa for cylinders with caps and several hundred pa for cylinders without caps. sis. passive endocytosis of particles with a homogeneous adhesion strength between particles and membranes is often modified by specific adhesion and active processes for biological systems. figure shows viruses with different shapes and a malaria parasite that have similar sizes as the particles discussed earlier. one specific example for active wrapping is the invasion of the malaria parasite into an erythrocyte, see figure . reorientation of the parasite, possibly because of a gradient of adhesive molecules, is followed by invasion. during invasion, a tight junction forms and reseales the membrane, which completes the formation of the parasitophorous vacuole. for the egg-like shape of the malaria parasite, line tension at the tight junction, and anisotropic adhesion of the parasite leads to a complex phase diagram for passive endocytosis with stable non-wrapped, shallow-wrapped, deep-wrapped, and complete-wrapped states [ ] , see figure . as indicated in figure , secretion of unstructured membrane from the parasite and favourable spontaneous curvature of the erythrocyte membrane may help the parasite to overcome energy barriers for wrapping and to finally invade the cell. in addition, also motor forces have been proposed to assist invasion [ ] . another active biological 'wrapping' process is phagocytotic uptake, see figure . here, the growth of the phagocytotic cup with its actin cytoskeleton is the major dynamic process [ ] . quantification using microscopy and image analysis shows fast uptake at the beginning, followed by a plateau with weakly increasing wrapping fraction, and again fast uptake of the particle towards the end of the process [ ] . various mechanisms for phagocytotic uptake have been suggested, among them receptor diffusion and directed motion [ ] , a zipper-like mechanism based on membrane fluctuations and membrane adhesion to the particle [ ] , and hindered uptake by increase and relaxation of membrane tension [ ] . multi-phase fluid systems and cellular biological systems abound with interfaces. nano-and microparticles naturally collect at such interfaces, because their localization at the interfaces lowers their interaction energy with the environment. we have considered here two types of interfaces: fluid-fluid interfaces governed by interfacial tensions, and biological interfaces controlled by curvature elasticity. particles at both types of interfaces show many common behaviours. the interface attraction depends on particle size, shape, and surface properties. particles can orient in different ways at interfaces, they deform the interfaces around them, and these interface deformations lead to interface-mediated interactions and collective behaviour. although systems and concepts for particles at fluid and biological interfaces are very similar, and for instance sds stabilisation of droplets can drive the system from fluid interfaces towards membranes [ ] , there is a major difference. in many cases, particle sizes at fluid interfaces are in the micrometer range, while particle sizes at lipid bilayer membranes are in the nanometer range. this implies that different experimental techniques are required in both cases, where the nanometer scale makes particles at membranes more difficult to observe and more difficult to manipulate. therefore, we have a better knowledge about particles at fluid interfaces, and there is an urgent need for systematic and well-controlled experiments for particles at membranes. nevertheless, a unified numerical and theoretical description of both systems is possible-at least for particles above a threshold diameter of about − nm -on the basis of continuum models for two-dimensional surfaces and their deformations embedded in three-dimensional space. from the technological point of view, micrometer-sized particles at fluid interfaces can be used in many ways to control and tailor interface properties. such applications range for both the upper and lower lobes, after engulfment begins at ≈ t s there is an initial slow stage (light gray) followed by a much quicker second stage (dark gray). engulfment is complete by ≈ t s . reprinted from [ ] , copyright ( ), with permission from elsevier. from emulsion stabilisation through an effective reduction of the interface tension to the design of solid shells and surfaces with controlled optical properties. from a biological point of view, nanoparticles are interesting in nanomedicine as biomarkes and drug carriers, but their effect on cells also has to be assessed because of their potential nanotoxicity. in addition, there is a large range of biological particles, such as viruses and parasites, the interaction of which with cells is also highly desirable to be controlled. the interaction of single hard particles with interfaces is by now reasonably well understood. therefore, we believe that future research should move towards particle-mediated interactions and collective properties for many particles at both fluid and biological interfaces, as well as towards soft particles. for example, size and concentration effects for the interaction or nanoparticles with liposomes and polymersomes have been studied using electron microscopy and scattering [ ] [ ] [ ] . using force measurements, nanoparticlemediated adhesion between elastic gels has been measured [ ] . the hard particles can serve as 'glue' between soft interfaces. soft particles, e.g. microgel and polymeric particles, add further complexity by allowing the particle shape to adjust in response to the interaction with a fluid or biological interface [ , [ ] [ ] [ ] [ ] [ ] . finally, dynamical behaviour of particles at fluid and biological interfaces awaits further characterization. for instance, dynamics of wrapping of non-spherical particles by biological interfaces has been discussed in section . . , but calculations that include brownian motion are missing. colloids adsorbed to stabilised oil-water interfaces show different diffusion properties than colloids in bulk [ ] , systematic studies could be used to measure the viscosity of lipid bilayers. furthermore, nanoparticles in combination with superresolution microscopy can be used as probes to study dynamics in biological cells [ ] [ ] [ ] . to conclude, particles are already widely used for applications, but a systematic understanding of the interactions of engineered nano-and microparticles with soft and biological matter is often lacking. furthermore, our understanding of basic biological processes on the cellular scale, such as phagocytosis and blood-stage malaria, will benefit from a detailed understanding of particles at biological interfaces. we expect that in the future more systematic studies for particles at fluid and biological interfaces will allow 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nanoparticles in living endothelial cells protein-based fluorescent nanoparticles for super-resolution sted imaging of live cells super resolution imaging of nanoparticles cellular uptake and trafficking our research on the interaction of particles with biological membranes has been supported by the eu fp nmp collaborative project prenanotox ( ). key: cord- -zq kqf h authors: shen, hsin-hui; lithgow, trevor; martin, lisandra l. title: reconstitution of membrane proteins into model membranes: seeking better ways to retain protein activities date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: zq kqf h the function of any given biological membrane is determined largely by the specific set of integral membrane proteins embedded in it, and the peripheral membrane proteins attached to the membrane surface. the activity of these proteins, in turn, can be modulated by the phospholipid composition of the membrane. the reconstitution of membrane proteins into a model membrane allows investigation of individual features and activities of a given cell membrane component. however, the activity of membrane proteins is often difficult to sustain following reconstitution, since the composition of the model phospholipid bilayer differs from that of the native cell membrane. this review will discuss the reconstitution of membrane protein activities in four different types of model membrane—monolayers, supported lipid bilayers, liposomes and nanodiscs, comparing their advantages in membrane protein reconstitution. variation in the surrounding model environments for these four different types of membrane layer can affect the three-dimensional structure of reconstituted proteins and may possibly lead to loss of the proteins activity. we also discuss examples where the same membrane proteins have been successfully reconstituted into two or more model membrane systems with comparison of the observed activity in each system. understanding of the behavioral changes for proteins in model membrane systems after membrane reconstitution is often a prerequisite to protein research. it is essential to find better solutions for retaining membrane protein activities for measurement and characterization in vitro. the cell membrane separates intracellular components from the outside environment and is constituted by various phospholipids, cholesterol, glycolipids and proteins. integral membrane proteins have at least one polypeptide segment spanning the membrane bilayer whereas peripheral membrane proteins are temporarily attached to the lipid bilayer or to integral membrane proteins by various interactions such as hydrophobic, electrostatic and other types of non-covalent interactions. membrane proteins work as a selective filter to regulate molecules entering cells and also serve in communicating with the surrounding environment. thus, membrane proteins play an essential role in the physiological functions needed for cell survival. the functional activities of membrane proteins are modulated by the structure of the surrounding lipids molecules in the membrane [ , ] ; thus the composition of the lipid bilayer can affect the inter-or intra-molecular interactions between the lipid bilayer and membrane proteins [ ] . investigating membrane proteins in vivo is difficult because the membrane proteins are associated with a complex mixture of other proteins, and are prone to aggregation in solution [ ] . it is still a major challenge at this stage to extract information needed in vivo to address specific questions in the function of the cell membrane. to simplify cell membrane systems, model membranes such as monolayers, bilayers, liposomes and nanodiscs have been developed, enabling detailed investigation of membrane protein structure in lipid membranes. model membrane environments more closely resemble the natural lipid bilayer than alternatives such as detergents. however, many features of phospholipid structure need to be considered and optimized in the creation of a suitable model membrane. for example, the hydrophobicity of the lipid chain defined by the lengths of the fatty acid chains, is an important parameter for retaining protein activity. other factors affecting the reconstituted membrane protein activity are the chemical properties of the lipid head groups which control membrane hydrophilicity. both parameters are crucial in stabilizing membrane protein structure. there are a number of approaches used to create a model membrane in order to mimic properties of the native cell membrane, and we will review these various approaches for reconstituting membrane proteins into different types of model membrane-monolayers [ ] , supported planar lipid bilayer [ ] and liposomes [ ] as shown in figure a -c. we will also discuss the emerging technology of nanodiscs [ ] ( figure d ). nanodiscs are a new class of model membrane, with attractive properties that address shortcomings of other approaches in the study of membrane proteins. the first section gives a brief summary of each method and a comparison of their strengths and weaknesses. in the following section, we describe four case studies and will compare the protein activity changes when the membrane proteins are reconstituted into different model membranes. in these case studies, we demonstrate how protein activities are modulated by the lipid environment and discuss how this environment helps to retain protein activities. and black in b represent water and a substrate respectively. nanodiscs contain membrane scaffold proteins, shown in green. one of the most common approaches to study the membrane protein structure and activity uses a langmuir monolayer at the air-water interface. this method has been extensively used for more than a century [ , ] . reconstitution of membrane proteins helps obtain further information on their organization and structure in the langmuir membrane [ , ] . it is a simple method to create a phospholipid monolayer at an air-water interface. basically, a desired amount of lipid or lipid mixtures are dissolved in organic solvents such as chloroform or chloroform/ethanol mixtures, followed by spreading the lipid/solvent mixtures on the water surface. by evaporating out the solvent, the phospholipid molecules self-assemble vertically as a monolayer film at the air-water interface, with their hydrophilic head groups immersed in the water and their hydrophobic tail pointed to the air as shown in figure a [ ] . a major advantage of using the langmuir monolayer system is that parameters such as thickness, surface pressure, molecular area and subphase thickness can be well controlled [ ] . more advanced characterization techniques, such as Π-a isotherm uv-vis adsorption, x-ray reflectivity, ellipsometry and rheology, have been developed to gain detailed information on the binding of proteins onto the phospholipid monolayer and to monitor enzyme activities when binding to the monolayer [ ] . however, a limitation of langmuir monolayers is that the lack of a layer comparing to the natural cell structure (bilayer) and the high surface tension of water that can cause protein denaturation. despite this limitation, there are several successful studies using this approach. two types of membrane proteins in monolayer model membrane system will be briefly described below: rhodopsin [ , ] , bacteriorhodopsin [ , ] and the aliphatic peptide gramicidin [ , ] have been successfully reconstituted and studied in monolayers at the air-water interface. in order to obtain information on the secondary structure and orientation, the protein layer can be investigated in situ at the air-water interface by either polarization modulation infrared reflection absorption spectroscopy (pm-irras) or x-ray reflectivity in combination with surface pressure-area isotherms [ ] . the study of gramicidin is an example of such an approach, and while gramicidin is unfolded at high molecular area (low pressure), it is refolded upon compression and retains its precise structure and orientation. likewise, for both rhodopsin and bacteriorhodopsin, the secondary structures measured in monolayers are indistinguishable from that in native membranes when appropriate conditions are used. while some experiments have suggested that spreading of rhodopsin in certain conditions (> m/n) leads to denaturation [ ] , bacteriorhodopsin, in contrast, is very stable in most testing conditions (compression and temperature change). the different properties of the protein are probably due to the ability of baceriorhodopsin to form a stable two-dimensional crystalline structure at the air-water interface [ ] . phospholipid monolayers are simple model membrane systems that are perfectly suited to study the binding of peripheral proteins onto a membrane surface. peripheral membrane proteins spontaneously bind onto phospholipid monolayers at the air-water interface by injecting themselves into the subphase underneath the lipid monolayer. in most cases, useful information can be obtained by measuring the binding of peripheral proteins onto the monolayer. for example, the kinetics and dynamics of adsorption of myristoylated and nonmyristoylated recoverin onto phospholipid monolayers have been investigated using surface pressure isotherm described in figure [ ] . the curve can be fitted with stretched exponential which can convert into the rate of adsorption of myristoylated and nonmyristoylated which is . s − and . s − , respectively. this indicates that the adsorption of myristoylated recoverin is six times faster than nonmyristoylated recoverin. reconstituting enzymes into the langmuir monolayers at the air-water interface has been found to be a very useful approach to understand the hydrolysis of membrane phospholipids. for example, the interfacial recognition and adsorption of phospholipases a (pla ) and phospholipases c (plc) to the phospholipid membrane interface are poorly understood. by using this approach, it appears that both pla and plc are active at the monolayer model membrane, indicating that the kinetics of phospholipid hydrolysis at the air-water interface can be monitored by biophysical characterization techniques in situ such as pm-irras and infrared reflection adsorption spectroscopy [ ] . moreover, it has been found that in the presence of calcium, phospholipid hydrolysis by pla resulted in the production of calcium-palmitate complexes. this suggests that calcium is necessary for pla secretion. the formation of a supported lipid bilayer on a solid substrate was reported by tamm and mcconnell in as a new model membrane system to study the physical properties of biological membranes and their constituent lipid and protein molecules [ , ] . supported planar lipid bilayers are prepared by several methods [ , ] . vesicle fusion is the simplest method for supported bilayer formation and the fusion mechanism on a hydrophilic support is well understood [ , ] . essentially, the bilayer is prepared by the fusion of small unilamellar vesicles on solid supports such as sio , glass and modified gold surface by van der waals, electrostatic, hydration and steric forces. the supported lipid bilayer has polar hydrophilic headgroups facing the aqueous surroundings and two hydrophobic tails that face the interior of the membrane which more closely resembles biological membranes than the langmuir monolayer. the supported lipid bilayer can confer many key functions to biological membranes. however, one side of the hydrophilic head group is still tightly attached to the solid support and this may, in some cases, affect the fluidity of the model membrane. this matters, since integral membrane proteins may not diffuse in the plane of the membrane. furthermore the orientation of membrane proteins cannot be controlled in the supported planer lipid bilayer. to alleviate some of these problems, a new tethered polymer-supported planar lipid bilayer system was developed to investigate the reconstitution of integral membrane proteins in a laterally mobile form into the supported lipid bilayer [ ] . wagner and tamm [ ] have successfully designed a supported lipid bilayer on a polyethyleneglycol cushion shown in figure . the polymer cushion minimizes the interactions of the proteins with the substrate and the polymer. it also provides a soft support and, for increased stability, covalent linkage of the membranes to the supporting quartz or glass substrates. in low polyethyleneglycol concentration regimes, the bilayers were assembled with high lateral lipid diffusion coefficients ( . - . × − cm /s). cytochrome b and annexin v were used to test the polyethyleneglycol cushion system. two populations of laterally mobile proteins were observed in the polyethyleneglycol cushion-supported bilayers. approximately a quarter of cytochrome b diffused with a diffusion coefficient of . - . × − cm /s, and more than half of the cytochrome b diffused with a diffusion coefficient of ~ × − cm /s. similar results were found in the annexin v system. annexin v diffused with two populations with diffusion coefficients of × − cm /s and × − cm /s. the new polymer-supported lipid bilayer system has increased the mobile fraction and retained the full lateral mobility of both cytochromes b and annexin when integrated or bound to the supported lipid. although polymer cushions allow for successful integration of small membrane proteins into bilayers, further challenges stem from studies with large transmembrane proteins. polymer cushions cannot provide large transmembrane proteins with good solvent accessibility, or enough space for the motion; required for the activity. while several types of polymer cushions have been developed, including polymethyl methacrylate diblock polymer cushions [ ] , poly(ethylene imine) [ , ] cushions and poly(ethylene glycol) tethered lipopolymers [ ] , these cushions are mostly limited to a thickness of up to nm. a recent development of a maleic anhydride copolymer thin film has film thickness up to nm [ ] . the hydrophilic polymer-cushioned supported lipid bilayers provide a higher mobility and homogeneous distribution of the incorporated beta-amyloid precursor protein cleaving enzyme (bace) on the bilayer surface, and enhances the enzymatic activity of bace (increased from % to %). even so, the activity of the incorporated bace remains significantly lower ( %) than that of the native enzyme ( %). another important classic category of membrane proteins are the transporters of ions and small molecules. studies of how ion channels regulate the transport of substrates [ ] are important for fundamental biology. however, it is challenging to incorporate ion channels in supported lipid bilayers due to leakage or instability issues. detailed studies of ion channel conduction or gating require considerable period of time (possibly > h), and it is difficult to set up a stable and electrically quiet environment for the ion channel in planar lipid bilayer. a better alternative has proven to be reconstitution of ion channels into proteoliposomes. lipid vesicles, also known as liposomes, consist of a self-closed lipid bilayer. they have been widely used for more than years to reconstitute the membrane proteins in unilamellar phospholipid vesicles. liposomes are relatively easy to construct by procedures such as extrusion method or ultrasonication, with reverse-phase evaporation. furthermore, giant vesicles of unilamellar or multilamellar nature can be "micro-manipulated" under an optical microscope. reconstitution of membrane proteins in liposomes usually requires detergents wherein purified membrane proteins are solubilized in detergent, then mixed with the desired phospholipid vesicles forming an isotropic solution of mixed phospholipid-protein-detergent micelles. the detergent can then be removed slowly by dialysis, gel filtration or biobead adsorption. when the detergent concentration reaches a critical level, the protein will spontaneously associate with the phospholipid membrane to form biologically active liposomes, called proteoliposomes. however, it has been a hard feat to control the final orientation of protein in the proteoliposomes [ ] , as well as the amount of protein inserted due to the limited area available. in many cases, disorientation of the protein causes aggregation. despite these difficulties, there have been many successful cases of membrane proteins reconstituted in the proteoliposomes, and we describe two examples below. several integral membrane proteins have been successfully reconstituted into proteoliposomes such as rhodopsin [ ] , g proteins [ , ] , proapoptotic bcl- proteins and t-bid [ ] , phosphocholine cytidylyltransferase (ct) [ ] and p protein kinase c (pkc) [ ] . however, these studies also found that the resulting protein activities are sensitive to the membrane curvature of the liposomes. this indicates that different phospholipids can cause considerable curvature stress changes in the liposomes [ ] . specifically, the curvature stress has been suggested to modulate the free energy and folding of the integral membrane proteins [ ] . sometimes the activity of different enzymes is modulated by the same driving force of the membrane curvature, but there may also be variation of activity through different mechanisms. for example, the activities of both ct [ ] and pkc [ ] are enhanced by increasing the negative curvature strain of the membrane. the activity of ct appears to be directly coupled with the membrane curvature, in contrast, the activity of pkc does not have a direct relationship with the curvature strain and enzymatic activity [ ] . the activity of pkc is instead modulated by nonlamellar-forming lipids via a less direct mechanism. liposomes have been commonly used for reconstituting different types of transporters to allow for the free diffusion of solution or catalysis of obligatory co-transporters. a large number of functional membrane proteins have been successfully reconstituted into liposomes but only a few examples will be discussed here. the reconstitution of colicin ia and e in either soybean phospholipids or e. coli phospholipids show that there is channel formation in the liposomes but there are unspecific channels allowing passage of ions, such as rubidium, sodium, chlorine, potassium or phosphate but not of sugars [ , ] . an example of the reconstitution of selective transport comes from the d-glucose transporter, purified from human erythrocytes and extracted from detergents followed by incorporation into proteoliposomes. with incorporation of the d-glucose transporter, the proteoliposomes become permeable to d-glucose but not to l-glucose. the transport was inhibited by cytochalasin b which is a potent inhibitor of d-glucose transporter [ , ] . several types of atp-dependent ion transporters such as ca + /mg + -atpase, na + /k + -atpase, and h + /k + -atpase have been reconstituted into proteoliposomes [ ] . upon addition of atp, ions are observed to be transported inwards and can form a complex. the single-channel property of channels incorporated into proteoliposomes can be investigated using the well-known patch-clamp method [ ] . channel activity is monitored following excision of the patch from the proteoliposomes. ion-channel reconstitution makes possible the investigation of the influence of membrane lipid composition on channel function. the kinetic investigation of these channels under physiological conditions has been discussed elsewhere [ ] . another up-to-date method is using organic solvent or oil mixed with water that creates water-in-oil (w/o) microdroplets coated by phospholipid. the hydrophilic head group immerses in the water and the hydrophobic tail locates in the oil/organic solvent phase. the application of the water-in-oil system could cover a wide range of applications from monolayer, planer lipid bilayer and liposomes. funakoshi et al. [ ] and maglia et al. [ ] used a planer lipid bilayer formed by two microdroplets driven to come in contact to reconstitute ion channels in the bilayer. this method is extremely simple and reproducible. recently, the water-in-oil microdroplets are extended to form liposomes by using droplet-transfer method invented by yoshikawa [ ] . by using this approach, it is possible to modulate the lipid compositions of outer and inner leaflets and furthermore to orient a reconstituted membrane protein in liposomes [ ] . nanodiscs offer a solution to some of the challenges described in the previous sections. the first attempt to reconstitute membrane proteins in the phospholipid bilayers using nanodisc technology was initiated by sligar's group a decade ago [ ] . the nanodisc is a self-assembly of phospholipids and a membrane scaffold protein derived from human serum apolipoprotein a . the detergent, cholate, can be used to solubilize phospholipids and membrane scaffold proteins into a micelle mixture. following detergent removal with dialysis or bio-beads adsorbent, a nanodisc self-assembles. the phospholipid associates as a bilayer domain while the membrane scaffold protein wraps around the edges of the discoidal structure in a belt-like configuration ( figure d ). it is possible to modify the diameter of the bilayer disc by genetically engineering the apolipoprotein a by changing the number of amphipathic helices. by this approach, the diameter of nanodiscs can be made anywhere from . to nm, and therefore accommodate a range of membrane proteins. the ratio of phospholipid: membrane scaffold protein is precisely defined which helps engineer the different size of membrane proteins in the nanodiscs. detailed formation of different types of nanodiscs has been described elsewhere [ , ] . the great advantage of using nanodiscs is keeping the membrane proteins in aqueous solution, in native-like phospholipid bilayer environment that is soluble, stable, monodisperse and detergent-free. most important, it isolates proteins or complexes as individual particles in monomeric or oligomeric states for analysis by techniques that range from activity assays to electron microscopy. since , there have been more than membrane proteins reconstituted into nanodiscs [ ] , ranging from signaling receptors to transport machines. we will discuss the applications separately below. nanodiscs have been used to analyze the influence of binding substrate on monodisperse receptors which are isolated from the cell-surface membrane. those receptors include g protein-coupled receptors (gpcr) [ , ] , cholera toxin receptor ganglioside g m , bacterial chemoreceptor [ ] and epidermal growth factor receptor. introduced into nanodiscs, the receptors stay in monodispersed, controllable, predefined oligomeric states in which it is possible to characterize the oligomeric status. for example, two different gpcr proteins, the beta-adrenergic receptor (β ar) and rhodopsin [ ] have been extensively studied using nanodiscs. β ar was one of the first receptors assembled into nanodiscs which was found to be functionally active ( % of starting activity recovered) and shown coupling to its g-protein. rhodopsin is a light-activated gpcr present in the photoreceptor cells of the retina and transducin is an important g-protein naturally expressed in retina rods and cones. assembly of functional rhodopsin into nanodiscs was found to activate transducin with high efficiency and to isolate the high affinity of transducin-metharhodopsin ii complex. this provides strong evidence that the monomeric state of rhodopsin can activate and interact with the transducin. a dimeric rhodopsin nanodisc was separated for monomeric forms using sucrose density gradients. even with two rhodopsins in the nanodiscs, interaction with a single transducin molecule was observed and found to activate the transductin with high efficiency [ ] . numerous membrane associated enzymes have been incorporated into nanodiscs. cytochrome p (cyp) enzymes have been extensively studied, including cyp b [ ] , cyp b [ ] , cyp a [ ] and cyp [ , ] . this system has provided a means for studying the extensive collection of membrane bound cytochromes p with the same biochemical and biophysical tools that have been previously limited to use with the soluble p s. for example, the cytochrome p a (cyp a ) is a membrane-bound protein which is a human hepatic drug-metabolizing enzyme. most studies of the ligand binding by cyp a are carried out in the presence of detergents below their critical micelle concentrations [ , ] but are compared by the propensity of cyp a to aggregate. even in studies attempting to use liposomes, cyp a is unlikely to exist in its native state because the detergent concentrations are much higher than the phospholipid concentrations. as a result, the understanding of the structure and composition of cyp a in the lipid phase was limited and the membrane effect on cyp a ligand binding behavior is unclear. nanodiscs have been utilized to study cyp a which displays monophasic reduction kinetics. with a high lipid-protein ratio, cyp a is captured as a monomer. however, at lower lipid ratio, cy a self-associates and heterogeneous behaviors are induced. the nanodiscs prohibit self-association in this case as there is only one cyp a per nanodisc and show significant improvement in homogeneity and stability. this opens up new possibilities for detailed analysis of equilibrium and steady-state kinetic characteristics of catalytic mechanisms of cyp enzymes [ ] . the sec translocon is a membrane-embedded protein assembly that drives protein translocation into or across membranes. the core translocon is formed from a trimeric arrangement of secy, sece and secg [ ] . the secyeg promoter has transmembrane helices sitting in the phospholipid membrane. the oligomerization of secyeg has been proposed to be necessary to proper function. researchers were successful in reconstituting sec into membrane vesicles in and have had great success in characterizing several partial reactions of secyeg functions [ ] . reconstituting a single secyeg into a nanodisc with different types of lipids [ ] suggests that the acidic lipids can stabilize the secyeg channel in the nanodisc bilayer and trigger dissociation of the seca dimer. a model has been proposed by alami et al. [ ] , suggesting that the dissociation of the seca dimer provoked by the secyeg complex is followed by activation of the seca atpase. furthermore, dalal et al. [ ] , using the nanodisc technology, have also shown that only the secy dimer together with acidic lipids supports the activation of the seca translocation atpase. recently, a high resolution single-particle cryo-em structure of single secyeg complexes in nanodiscs, bound to translating ribosomes was first solved at subnanometer resolution [ ] . it allows the secyeg complex to be investigated in a natural lipid bilayer environment and identifies the ribosome-lipid interactions. wu et al. [ ] also used surface plasmon resonance to investigate the competitive binding of ribosomes and seca. the data suggest that both ribosomes and seca can interact simultaneously with secyeg complex during membrane protein insertion, but seca competes with ribosome when it binds to the secyeg complex. in the previous section, we have shown that membrane proteins can be assembled into four different types of model membrane and the activities of some of the membrane proteins can be retained, allowing their physicochemical properties to be studied. but is there a model membrane system that is the best for membrane-protein reconstitution? the reconstitution of the same membrane protein into different model membranes has been compared and, in this section, we list four membrane proteins with varying activities in different model membranes. ganglioside g m is a naturally occurring native receptor that binds to cholera toxin via hydrogen bonds [ ] . it is an excellent receptor for studying lipid-receptor interaction. several different approaches to reconstituting the glycolipid receptor g m in model membranes have enabled the measurement of binding of its interaction partner cholera toxin. in liposomes and supported lipid bilayer systems, the ganglioside g m is free to diffuse across long distances and exhibits a non-uniform lateral distribution, i.e., self-aggregation, even at low incorporation ratios. therefore the binding activity of ganglioside g m with cholera toxin b is restricted [ ] . investigations of ganglioside g m incorporated into nanodiscs found reduced protein aggregation. bricarello et al. [ ] found that the reconstitution of a low concentration of ganglioside g m in nanodiscs, shows binding of cholera toxin with a significantly higher affinity than in liposomes or supported lipid bilayers. this is due to the interaction of ganglioside g m with the headgroup region of the disc which reduces the oligomerization, thereby causing a potential effect on the affinity of toxin binding. thus, nanodisc technology restricts the ganglioside g m oligomerization by controlling the number of ganglioside g m monomer isolated by each nanodisc. borch et al. [ ] have also used sensor chip-based surface plasmon resonance (spr) technology to measure the detailed kinetic binding of the interaction between soluble molecules and membrane receptors inserted in the bilayer of nanodiscs. the corresponding spr sensorgrams are displayed in figure . overall, the change of the sensorgram indicates that the spr sensorchip is immobilized with histidine-modified nanodisc or the cholera toxin b bound to the nanodiscs. the sensorgrams in both figure a ,b shows the binding of nanodiscs ( ru) on the antibody immobilization surface on the sensor chip. by injecting the cholera toxin b over two flow cells presented in figure a and b, the spr sensorgrams can detect the interaction of the cholera toxin b with nanodisc with or without the existence of g m . it has been revealed that the captured % g m -nanodiscs bound ru of the cholera toxin b without binding to the capturing nanodiscs without g m ( figure b ). the measured kinetic values of the interaction are in agreement with those reported by previous studies on the interaction of the cholera toxin with the g m receptor embedded in different membrane systems. this, therefore, serves as a proof of concept that nanodiscs can be employed in kinetic spr studies. the nucleus envelope is composed of two bilayers (the outer nuclear membrane and inner nuclear membrane) and contains abundant ion channels, through which ions and small molecules diffuse between the cytoplasm, nucleoplasm and perinuclear (i.e., intermembrane) space. the nuclear ionic channels represent a ubiquitous structure in the nuclei in a wide range of cells, although little is known about its functional properties. to characterize nuclear ionic channels, guihard et al. [ ] attempted to reconstitute nuclear envelope vesicles derived from the canine liver nuclei into a planar lipid bilayer and giant proteoliposomes. they found that the success rate of nuclear envelope fusion into planar lipid bilayers was extremely low although cardiac nuclear ionic channels were successfully incorporated into planar lipid bilayers. the detection of the nuclear ionic channels activity was not possible. such a low efficiency can be explained by the clustering of nuclear envelope vesicles, and the low density of single vesicles, as well as the presence of residual chromatin and/or nuclear proteins (histones or lamins) which would prevent fusion events with the bilayer. another approach is reconstituting nuclear envelope vesicles into giant proteolipsosmes and detecting the single ion channel by the patch-clamp technique [ ] . large conductance, voltage-gated, k + and cl − selective nuclear ionic channels are characterized and plotted as a current-voltage relationship presented in figure a ,b respectively. it has been found that under asymmetrical / mm kcl conditions, the zero current potential for unitary currents is at mv ( figure a ). calculated from the goldman-hodgkin-katz (ghk) flux equation, a p k +/p cl − ratio is . . this value indicates the k + selectivity for this channel. in figure b , the cl − selective nuclear ionic channel yields a positive zero current potential of + . mv, with a p cl −/p k + ratio of , indicative of a high cl − selectivity over k + . this suggests super fusion of the channel under asymmetrical ( / mm) kcl conditions. the current-voltage relationship curves indicate that the nuclear ion channels can be functionally characterized by incorporating the proteins into the giant proteoliposomes where it is possible to retain their channel activity. furthermore, the measured activities are consistent with those described for native nuclear ion channels. p-glycoprotein, the most extensively studied atp-binding cassette transporter, has been implicated in the phenomenon of multidrug-resistance in tumor cells and has been suggested to play a significant role in drug absorption and deposition. how p-glycoprotein interacts with its substrates is still unknown. functional studies are limited because of the difficulty of obtaining large quantities of stable p-glycoprotein. besides that, no atpase activity of p-glycoprotein solubilized in detergent could be detected. when p-glycoprotein is reconstituted into proteolipsomes, it has detectable atpase activity; however, the whole complex is very unstable. heikal et al. [ ] have further found that p-glycoprotein reconstituted in the proteoliposomes has a half-life of less than one day. in , ritchie et al. [ ] performed a detailed study of drug-stimulated atpase kinase activity of p-glycoprotein using the nanodisc technology. the p-glycoprotein protein was reconstituted into both msp e d disc and liposomes in order to compare its atpase kinase activities. the results described in figure demonstrate that p-glycoprotein is functionally active when reconstituted into the nanodiscs (close squares). comparing to the atpase kinase activity of p-glycoprotein reconstitution in lipsosomes (close circles), there is a twofold increase in the maximum atpase activity in the nanodiscs. this could be due to the uniform orientations of p-glycoprotein in the nanodiscs while there are two possible orientations in liposomes. these data not only show that p-glycoprotein is functionally active when reconstituted into the nanodiscs, but that it also exhibits higher specific activity than the current standard reconstitution system. figure . comparsions of the atpase activity of p-glycoprotein in nanodiscs (square) and proteoliposomes (circle). open symbols: basal activity in the absence of drug; filled-in symbols: activity in the presence of nicardipine [ ] . atp-binding cassette transporters utilize the energy of atp hydrolysis to transport a wide range of substrates across cellular membranes and for non-transport-related processes such as translation of rna and dna repair [ ] . a member of the atp-binding cassette super family, the maltose transporter malfgk from e. coli, together with the substrate-binding protein male, is one of the best-characterized atp-binding cassette binding cassette transporters suitable for various reconstitution techniques. bao and fuong have reported the reconstitution of the maltose transporter in nanodiscs, in detergent and in proteoliposomes. the atpase activity of the malfgk complex in various environments is shown in figure . the data presented in the first column of figure show that the basal atpase activity for assembly in the nanodiscs and detergent (~ nmol/min/mg) is -fold higher than in proteoliposomes because of the decrease in the activation energy barrier of the transporter [ ] in detergent micelles and nanodiscs. however, in the presence of male, the rate of atp hydrolysis increases in all assembly conditions. this is because male captures maltose and delivers the sugar to the transporter. note that the basal atpase activity assembly in the nanodiscs dramatically increases from to nmol/min/mg. the maltose alone has no effect on the basal atpase activity in the nanodiscs and detergent. however, in nanodisc and detergent, an inhibition of the atpase activity was observed in the presence of both maltose and male in the nanodiscs. this is because that maltose reduces the binding affinity of the male-malfgk complex, which therefore has reduced the atpase activity. in proteoliposomes, the atpase activity (~ nmol/min/mg) shows a further -fold increase in the presence of both maltose and mele in the figure. the author used another type of male mutant which binds maltose with higher affinity. this male mutant, in contrast, shows a reduction of the atpase activity in proteoliposomes which has the same effect as the nanodiscs and detergents. overall, proteoliposomes have shown a low basal atpase activity because the lipid stabilized the transporter. however, the nanodiscs have been shown to be a better medium than proteoliposomes for studying the atp hydrolysis ability of atp-binding cassette transporters. this review summarizes and compares the most up-to-date methods for reconstituting membrane proteins into model membranes. there is no superior method for reconstituting membrane proteins in the model membrane; instead two or more model membranes should be considered, depending on the particular needs of the system and the proteins of interest. in general, systems based on lipid bilayers supported on a solid substrate are still the most favored and well-developed of the methods to study membrane proteins in the bilayer. this approach allows detailed study of the fundamental properties of biological membranes and is practical to reproduce the bilayer system. on the other hand, the proteoliposome is more suitable for ion channel reconstitution in the bilayer, as well as for combination with the patch-clamp method to detect the ionic selectivity of the channel. finally the self-assembled nanodiscs system provides a robust and common means for rendering these targets soluble in aqueous media while providing a native-like bilayer environment that maintains functional activities. nanodisc technology offers another way to prepare monodisperse samples of membrane proteins in the bilayer environment, and it is emerging as the favored approach in studies concerning membrane protein complexes. biochemical and functional characterization of the membrane association and membrane permeabilizing activity of the severe acute respiratory syndrome coronavirus envelope protein lipid-protein interactions in human erythrocyte-membrane acetylcholinesterase. modulation of enzyme activity by lipids correlation between the effect of the anti-neoplastic ether lipid -o-octadecyl- -o-methyl-glycero- -phosphocholine on the membrane and the activity of protein kinase calpha weak dependence of mobility of membrane protein aggregates on aggregate size supports a viscous model of retardation of diffusion structure and phase transitions in langmuir monolayers allogeneic stimulation of cytotoxic t cells by supported planar membranes effect of colicins ia and e on ion permeability of liposomes direct solubilization of heterologously expressed membrane proteins by incorporation into nanoscale lipid bilayers surface enhanced raman scattering of a lipid langmuir monolayer at the air-water interface lipid monolayers: why use half a membrane to characterize protein-membrane interactions? langmuir monolayer of artificial pulmonary surfactant mixtures with an amphiphilic peptide at the air/water interface: comparison of new preparations with surfactant polymyxin b-lipid interactions in langmuir-blodgett monolayers of escherichia coli lipids: a thermodynamic and atomic force microscopy study langmuir balance investigation of superoxide dismutase interactions with mixed-lipid monolayers modern physicochemical research on langmuir monolayers structure of rhodopsin in monolayers at the air-water interface: a pm-irras and x-ray reflectivity study formation, structure, and spectrophotometry of air-water interface films containing rhodopsin proton transport by bacteriorhodopsin in planar membranes assembled from air-water interface films structural and spectroscopic characteristics of bacteriorhodopsin in air-water interface films spectroscopic and structural properties of valine gramicidin a in monolayers at the air-water interface effects of gramicidin-a on the adsorption of phospholipids to the air-water interface organization, structure and activity of proteins in monolayers monitoring of phospholipid monolayer hydrolysis by phospholipase a by use of polarization-modulated fourier transform infrared spectroscopy supported planar membranes in studies of cell-cell recognition in the immune system supported phospholipid bilayers formation of high-resistance supported lipid bilayer on the surface of a silicon substrate with microelectrodes supported lipid bilayer self-spreading on a nanostructured silicon surface simulations of temperature dependence of the formation of a supported lipid bilayer via vesicle adsorption simulations of lipid vesicle rupture induced by an adjacent supported lipid bilayer patch membrane lateral mobility obstructed by polymer-tethered lipids studied at the single molecule level tethered polymer-supported planar lipid bilayers for reconstitution of integral membrane proteins: silane-polyethyleneglycol-lipid as a cushion and covalent linker reversible activation of diblock copolymer monolayers at the interface by ph modulation, : lateral chain density and conformation polymer-cushioned bilayers. i. a structural study of various preparation methods using neutron reflectometry polymer-cushioned bilayers. ii. an investigation of interaction forces and fusion using the surface forces apparatus controlled enhancement of transmembrane enzyme activity in polymer cushioned supported bilayer membranes orientation and reactivity of nadh kinase in proteoliposomes modulation of rhodopsin function by properties of the membrane bilayer role of lipid polymorphism in g protein-membrane interactions: nonlamellar-prone phospholipids and peripheral protein binding to membranes influence of the membrane lipid structure on signal processing via g protein-coupled receptors the apoptotic protein tbid promotes leakage by altering membrane curvature modulation of ctp:phosphocholine cytidylyltransferase by membrane curvature elastic stress the role of membrane biophysical properties in the regulation of protein kinase c activity membrane lipid polymorphism: relationship to bilayer properties and protein function elastic coupling of integral membrane protein stability to lipid bilayer forces reconstitution of colicin e into dimyristoylphosphatidylcholine membrane vesicles the permeability of bilayer lipid membranes on the incorporation of erythrocyte membrane extracts and the identification of the monosaccharide transport proteins binding of cytochalasin b to human erythrocyte glucose transporter conformational dynamics of na+/k+-and h+/k+-atpase probed by voltage clamp fluorometry the extracellular patch clamp: a method for resolving currents through individual open channels in biological membranes lipid bilayer formation by contacting monolayers in a microfluidic device for membrane protein analysis droplet networks with incorporated protein diodes show collective properties cell-sized liposomes and droplets: real-world modeling of living cells oriented reconstitution of a membrane protein in a giant unilamellar vesicle: experimental verification with the potassium channel kcsa phospholipid phase transitions in homogeneous nanometer scale bilayer discs membrane protein assembly into nanodiscs functional reconstitution of beta -adrenergic receptors utilizing self-assembling nanodisc technology transducin activation by nanoscale lipid bilayers containing one and two rhodopsins using nanodiscs to create water-soluble transmembrane chemoreceptors inserted in lipid bilayers atomic-force microscopy: rhodopsin dimers in native disc membranes single-molecule height measurements on microsomal cytochrome p in nanometer-scale phospholipid bilayer disks co-incorporation of heterologously expressed arabidopsis cytochrome p and p reductase into soluble nanoscale lipid bilayers the critical iron-oxygen intermediate in human aromatase the ferrous-oxy complex of human aromatase kinetics of dithionite-dependent reduction of cytochrome p a : heterogeneity of the enzyme caused by its oligomerization ligand binding to cytochrome p a in phospholipid bilayer nanodiscs the effect of model membranes the atpase activity of seca is regulated by acidic phospholipids, secy, and the leader and mature domains of precursor proteins nanodiscs unravel the interaction between the secyeg channel and its cytosolic partner seca two copies of the secy channel and acidic lipids are necessary to activate the seca translocation atpase cryo-em structure of the ribosome-secye complex in the membrane environment competitive binding of the seca atpase and ribosomes to the secyeg translocon crystal structure of cholera toxin b-pentamer bound to receptor gm pentasaccharide self-aggregation-an intrinsic property of g(m ) in lipid bilayers ganglioside embedded in reconstituted lipoprotein binds cholera toxin with elevated affinity nanodiscs for immobilization of lipid bilayers and membrane receptors: kinetic analysis of cholera toxin binding to a glycolipid receptor patch-clamp study of liver nuclear ionic channels reconstituted into giant proteoliposomes the stabilisation of purified, reconstituted p-glycoprotein by freeze drying with disaccharides chapter -reconstitution of membrane proteins in phospholipid bilayer nanodiscs abc transporters, mechanisms and biology: an overview discovery of an auto-regulation mechanism for the maltose abc transporter malfgk the authors gratefully acknowledge financial support from the australian research council (arc). hhs is an arc super science fellow and tl is an arc federation fellow. tl and lm were awarded the arc super science fellowships and grant (fs ). we thank victoria hewitt for her critical reading of the manuscript. key: cord- - caevjvh authors: falanga, annarita; galdiero, massimiliano; morelli, giancarlo; galdiero, stefania title: membranotropic peptides mediating viral entry date: - - journal: pept sci (hoboken) doi: . /pep . sha: doc_id: cord_uid: caevjvh the means used by enveloped viruses to bypass cellular membranes are well characterized; however, the mechanisms used by non‐enveloped viruses to deliver their genome inside the cell remain unresolved and poorly defined. the discovery of short, membrane interacting, amphipathic or hydrophobic sequences (known as membranotropic peptides) in both enveloped and non‐enveloped viruses suggests that these small peptides are strongly involved in breaching the host membrane and in the delivery of the viral genome into the host cell. thus, in spite of noticeable differences in entry, this short stretches of membranotropic peptides are probably associated with similar entry‐related events. this review will uncover the intrinsic features of viral membranotropic peptides involved in viral entry of both naked viruses and the ones encircled with a biological membrane with the objective to better elucidate their different functional properties and possible applications in the biomedical field. significant improvements have been achieved in recent years in the understanding of the multiple alternative ways of virus entry into susceptible cells. [ , ] the strategies employed by viruses to enter cells are different according to the presence or absence of a lipid bilayer surrounding the virus. enveloped viruses present a membrane bilayer while non-enveloped viruses lack this membrane and present on their surface only capsid proteins. the mechanism of cell invasion by the two groups of viruses is rather diverse and the foremost difference is the direct consequence of their distinctive physicochemical state at the interface that occurs at the time of the encounter between the virus and the cell membrane; in particular, two lipid membranes confronting each other in the case of enveloped viruses as opposed to a layer consisting only of proteins that face a lipid layer in the entry of naked viruses. enveloped viruses entry exploits direct fusion with a cellular membrane through the involvement of specialized viral fusion proteins, present on the viral membrane and the consequent transfer of the nucleocapsid into the cytoplasm. [ ] on the other hand, the entry of non-enveloped viruses, which lacking the outer viral membrane are unable to take advantage of the cellular mechanism of membrane fusion, involves the activation of viral lytic factors that induce cell membrane rupture. [ ] the major features in membrane interaction by enveloped and non-enveloped viruses are reported in figure . notwithstanding the different mechanisms of penetration, the key step is the entry process and the modification of the cell membrane which allows the viral genome to penetrate into the host cell and start the replication cycle in the appropriate cellular compartment. [ , ] the most critical barriers for viral penetration and replication are the plasma membrane, the cytoplasm, and any other membrane that needs to be crossed in order to have access to the sites where viral replication or assembly take place. the overall picture of the mechanism of viral entry is becoming increasingly complete; in fact, depending on their dimension and structure, they have acquired different strategies to penetrate and take control of cell functions. [ , ] the molecular details of the interactions at the interface of virus and cell surfaces are quite complex and highly variable, but there is a common idea that only a limited number of pathways allowing viruses to reach the sites of penetration exist, with enveloped and non-enveloped viruses presenting different and unrelated processes, but with general principles driving all fusion events. the main difference between fusion peptides of enveloped viruses and lytic factors of non-enveloped viruses is the fact that fusion peptides promote membrane fusion while lytic factors promote membrane disruption. in this review, we summarize current knowledge on the mechanism of membrane fusion of both enveloped and non-enveloped viruses with a focus on common principles. fusion peptide derived from enveloped viruses and lytic peptides from non-enveloped viruses are described with an effort to find undisclosed differences and similarities. we conclude reporting ground breaking applications of membranotropic peptides (both fusion and lytic peptides) which open a new exiting field of research. enveloped viruses depend on membrane fusion for cell penetration. [ ] the two main routes used by enveloped viruses to enter the cell are the endocytic and non-endocytic pathways. [ ] most enveloped viruses undergo endocytosis while only few are able to fuse directly to the plasma membrane. [ ] viruses that use the endocytic route for cellular internalization are able to escape into the cytosol avoiding lysosomal degradation. therefore, penetration invariably involves membrane fusion mediated by specific viral glycoproteins (catalysts) with the main difference being that viruses using endocytic pathways fuse their envelope with the endosomal membrane from the luminal side. [ ] thus, membrane fusion constitutes the essential and ubiquitous mechanism of entry of enveloped viruses, irrespective of their route of entry. [ , , ] viral fusion proceeds through a hemifusion stalk with merging of proximal leaflets, which culminates in the opening and expansion of a pore connecting the two sides of the membrane. [ ] the overall process is supported by a catalyst responsible of the lowering of the transition barriers and fusion proteins constitute the catalytic agents fulfilling this function. [ ] the entry involves several other critical steps which include cellular receptor or co-receptor binding, internalization, uncoating, and release of viral nucleic acids at the proper site of replication. [ , , ] various factors can mediate an efficient interaction between cells and viruses; for example, cholesterol rich domains are platforms for the entry of many enveloped viruses such as the influenza virus [ , ] and many viral membranes contain much more cholesterol than the mammalian plasma membranes from which they are derived. [ , ] viral fusion proteins undergo significant rearrangements from the pre-fusion to the post-fusion conformations which are triggered by either receptor binding, proteolytic cleavage or low endosomal ph, and eventually determine the exposure of previously sequestered hydrophobic peptides, loops, or patches, able to interact with and destabilize one or both the opposing membranes. [ , ] crystallographic data on pre-and post-fusion structures of viral fusion proteins has resulted in the identification of at least three distinct classes based on their three-dimensional organization and mechanism of fusion ( figure ). [ , ] class i fusion proteins includes many of the most studied human pathogens such as influenza virus, human immunodeficiency virus (hiv), and ebola virus; the key features is the requirement of a proteolytic cleavage to initiate fusion, [ ] activating these proteins which form an extended intermediate on the surface of the virion with the fusion peptide at the n-terminus of the protein engaged in the interaction with the target membrane and forming a link between the two fusing bilayers. the pre-hairpin structure is a unique state in which the fusion protein is simultaneously connecting two distinct membranes, the target membrane through the fusion peptide or patches and its own viral membrane through its transmembrane domain (tm). further conformational changes produce trimers of hairpins with a central a-helical coiled-coil structure. [ , ] the -helix formation is related to the opening of the fusion pore and provides the major driving force for the process. thus, peptides able to prevent -helix formation, interfering with refolding of the fusion glycoproteins act as potent viral entry inhibitors. [ , ] class ii fusion proteins, which include flaviviruses, alphaviruses, and bunyaviruses, are consistently different from those of class i and are mainly composed of b structures. [ ] these fusion proteins are often heterodimers or homodimers lying almost flat on the virion surface. [ ] they are organized in three globular domains: domain i is char- class iii fusion proteins have a mixed secondary structure with the central a-helical trimeric core similar to class i and two fusion loops located at the tip of an elongated b-sheet similar to class ii fusion proteins; the main representatives of class iii fusion proteins are protein g of vesicular stomatitis virus (vsv), [ , ] gb protein of herpes simplex virus (hsv), [ ] and of epstein-barr virus (ebv), [ ] and gp from insect-cell baculovirus. [ ] the post fusion structure is characterized by the presence of an internal fusion peptide in domain i organized in two hydrophobic fusion loops flanked by a b-sheet domain with a pleckstrin-like fold (domain ii). domain ii is nested with the largely a-helical domain iii, which is composed of trimers that give rise to an elongated, rod-like shape molecule. the a-helical domain is inserted in domain iv which is made of b-sheets and a very long c-terminal extension (domain v). interestingly, the multicomponent herpesvirus fusion machinery requires the presence of gb, gh/gl, and gd and multiple cellular receptors are engaged in the entry pathway in a cascade of molecular interactions. [ ] gh/gl and gb are part of the core fusion machinery and need to cooperate in order to trigger the initial lipid destabilization which culminates in the fusion of the two bilayers. [ ] [ ] [ ] despite the fact that it is still debated whether gh is merely a fusion regulator or it plays a more direct role in the fusion process, studies leave little doubt that also the gh/gl complex undergoes dynamic rearrangements during the fusion process. [ ] the crystallographic post-fusion structure of gb shows that it is a canonical class iii fusion protein; [ ] and several synthetic peptides derived from gb induce the fusion of large unilamellar vesicles and inhibit herpes virus infection. [ , , ] irrespective of their structural differences, the three classes of fusion proteins seem to induce membrane fusion by essentially the same generic mechanism and a common refolding pathway is highly suggestive for the conservation of several phases of the process. the non-enveloped viruses exploit a different mechanism for entry because they lack a membrane which surrounds the protein capsid; as a consequence they require capsid-dependent mechanisms for penetrating the cell membrane or for exiting from the endosome. [ , ] the entry process is much less known and involves a series of triggers producing conformational and structural rearrangements which end in the exposure and/or release of lytic factors. low ph, receptor interactions, protease cleavage, chaperone-assisted morphological changes, divalent cation chelation, or any combination of these factors which take place at the appropriate site of membrane penetration are essential triggers to produce the activated viral intermediate. [ ] the mechanism of membrane disruption involves proteolytic cleavage of the coat protein and programmed exposure/release of small membrane-lytic peptides. in this scenario, viral penetration is mediated by short, membrane interacting, amphipathic and/or hydrophobic sequences present in proteins undergoing a conformational modification which allows the exposure of these domains and their interactions with membranes. [ ] similarly to enveloped viruses, capsid proteins seem to be trapped in a metastable state waiting for a trigger to expose their membrane active peptides and the membrane penetrating ability of these sequences is fundamental for entry but at the same time their premature exposure has to be avoided before host cells provide the triggers. flock house virus (fhv) is one of the simplest and most studied non-enveloped viruses. the infection starts with the binding to one or more host cell receptors leading to a receptor-mediated endocytosis mechanism. [ ] the receptor binding induces a conformational change that initiates uncoating or viral disassembly once the particle is exposed to low ph within the endocytic pathway blocking this event often inhibits infection. the immature provirion fhv is initially assembled from copies of the coat protein alpha and, subsequently, it undergoes autoproteolytic cleavage to generate the mature infectious virion which contains a large n-terminal fragment, b ( amino acids), and a small c-terminal fragment, g ( amino acids). [ ] the capsid shell is constituted by the central region of b forms, while the g peptides are the amphipathic helices non-covalently associated with the capsid interior. the g peptides are responsible of the interaction with the membrane and its local disruption which ends in viral entry. these peptides are composed of an n-terminal amphipathic helix separated by a proline-glycine-proline turn from a hydrophobic c-terminal region. the amphipathic g helices are located in the interior of the capsid and when fhv enters cells through receptor mediated endocytosis, the g peptides are exposed and determine the disruption of the membrane with consequent release of the viral genome in the cytosol. [ ] this is a dynamic process with g peptides continuously, transiently, and reversibly exposed to the exterior of the capsid; g peptides may not only be exposed but also released from the virus particle which may represent a common paradigm of non-enveloped virus entry. in this dynamic process g peptides may continuously "sample" the environment until they encounter the appropriate cellular trigger; at this point the virus undergoes an irreversible conformational change in which the g amphipathic helices insert into the target membrane, allowing the viral rna to enter the cytoplasm. [ ] | m em b ra n otrop i c p ep ti d es many studies are devoted to the comprehension of the mechanism of insertion of fusion peptides, loops, or patches into monolayers inducing nipple formation and curvature in the target cellular membrane through many synergic interactions. the insertion in one leaflet of a closed bilayer will cause the increase of the surface area of the leaflet and the formation of a spontaneous curvature, which is one of the driving forces to reduce the energetic barrier needed for the achievement of fusion. [ ] fusion and/or membranotropic domains in viral fusion proteins contain aromatic residues which together with alanines and glycines [ ] contribute to the interaction with just one bilayer leaflet. many biophysical and structural techniques using synthetic analogues and model membranes have been used to determine physiologically relevant states during membrane partitioning. [ , ] the two structural motif widely found in fusion proteins and able to produce membrane curvature are the amphipathic a-helix and tilted peptides. [ , ] the segregation of hydrophobic and polar residues on the two opposite sides of the amphipathic a-helix is responsible of the superficial insertion into the upper monolayer which modifies the pack- similarly, also tilted peptides are characterized by an asymmetric distribution of hydrophobic residues, which again produces a modification of the organization of the membrane into which they insert. [ ] the membrane bound conformation of the influenza virus fusion peptide is characterized by the presence of a hairpin of two tightly packed, antiparallel a-helices; [ , ] the asymmetric insertion is determined by the fact that hydrophobic residues insert into the proximal membrane leaflet and polar residues project outward. the precise angle of the kink between the two arms depends on the sequence, the ph, and the lipid environment and determines the functional features necessary for activity. as a matter of fact, the compact hairpin structure drives favorable insertion, while its expanded structures promote subsequent membrane destabilization. [ ] canonical class ii and class iii fusion peptides correspond to loops which do not undergo conformational changes upon insertion into the target membrane; the interaction clearly involves few hydrophobic residues, and the insertion into the outer leaflet of the membrane is superficial and probably inadequate to destabilize membranes. thus, a cooperative effect attained upon fusion activation is the only explanation for activity. [ ] the idea of a single fusion peptide being exclusively responsible for the membrane perturbing activity has been overwhelmed by evidences supporting the concerted action of different membranotropic peptides, [ ] which together with the canonical fusion peptide are involved in the modification of membrane curvature. [ ] [ ] [ ] [ ] [ ] [ ] many viral fusion proteins present an additional hydrophobic membrane proximal region at the intersection between the ectodomain and the transmembrane anchor (tm), the so-called mper (or pre-tm); [ ] the unusual clustering of aromatic amino acid in these regions prompted the idea of their strong involvement in the fusion process. indeed, peptides corresponding to the pre-tm region partition into membrane interfaces and likely cooperate with fusion peptides and tm domains during apposition of membranes, enhancing the overall hydrophobicity of the environment and contributing to the distortion of the lipid membranes required for fusion. [ ] the pre-tm of hsv- gh interacts strongly with membranes; [ ] the pre-tm of gp of foamy virus induces fusion of model membranes; [ ] the aromatic domain of the glycoprotein s of severe acute respiratory syndrome virus (sars) partitions into lipid membranes and perturbs their integrity; [ ] the pre-tm region of the gp protein from ebola promotes perturbations of membranes when in a helical structure. [ ] hsv fusion involves both gb and gh and several membranotropic sequences are present in both glycoproteins, [ , ] although the precise role played by each of these regions remains to be elucidated. the two fusion loops at the tip of the domain ii of gb constitute a structural subdomain with the hydrophobic amino acids forming a crest lined on both sides by charged residues; [ ] the concerted use of the two peptides produced a significant distortion of the target membrane bilayer, while when they were used separately they presented a lower membrane penetration. [ ] the two charged residues represent a novel feature of fusion peptides with the presence of hydrophilic residues on either side favoring insertion. [ ] fusion of inner and outer monolayers is clearly involved but not formation of pores, indicating that bilayer perturbation not complemented by leakage is a typical feature of viral fusion peptides which violate the host membrane without compromising its integrity. [ ] several gh peptides are able to interact with membranes and play a role in the process. among these, gh , represents a key achievement in grasping the role of hydrophobic viral peptides. [ ] [ ] [ ] [ ] the peptide contains residues critical for interaction such as aromatic residues (tryptophan and tyrosines) which are known for their preferred location at the membrane interface and for their ability to facilitate oligomerization [ ] together with numerous hydrophobic residues (glycines, leucines, alanines) which are critical for membrane insertion; [ ] at the c-terminus there is an arginine residue which is key for establishing peptide-lipid interactions. gh penetrates into membranes from its n-terminal side, and assumes an amphipathic helical conformation. [ ] the n-terminal histidine acts as a switch for triggering viral fusion and strongly enhances the fusion activity; [ ] the role of the histidine has been reported also for paramyxoviruses [ ] and togaviruses. [ ] yao et al. [ ] in order to investigate how the fusion peptide (fp) and lipids also play a key role in this process, generating membrane curvature thanks to their physico-chemical properties. cholesterol is key as it selectively intercalates into the leaflet of the bilayer favoring its distortion without producing unfavorable hydrophobic/hydrophilic interactions. [ ] the secondary structure of the fusion peptide of hiv changes according to the cholesterol content in the membrane, being a-helical in the absence of cholesterol, but shifting to a b conformation with the increase of cholesterol. [ ] both the fully a-helical and the fully b-structured peptides are able to insert deeply inside the membrane, while the mixed secondary structures, present at intermediate cholesterol concentrations, are more superficially inserted into lipid bilayers and less effective in inducing membrane fusion. [ ] it is likely that different secondary structures and domains with different content of cholesterol might be involved in different stages of fusion. [ ] probably, lipid phase discontinuities between liquid ordered and disordered domains containing cholesterol produce membrane defects with exposed hydrophobic surfaces favoring a deeper insertion of fusion peptides, and promoting membrane fusion. [ ] in conclusion, the clear view is that membrane fusion is a very complex process involving several domains of the fusion proteins which interact directly or indirectly with biological membranes, and contribute to the merging of the viral envelope and cell membrane. some peptides (as the lytic peptides of nodaviruses, picornaviruses, and reoviruses) can be generated by an autocatalytic cleavage step of a precursor, whereas others can be generated from the proteolytic activity of cellular enzymes. [ , ] essentially we can classify lytic peptides in amphipathic a-helices and myristoyl groups; or we can classify them according to their mechanism of membrane action in those causing transient modification of the cellular membrane, pore formation, and total disruption of the limiting membrane. [ ] although being clearly different among themselves and with fusion peptides of enveloped viruses, they present notable similarities. incubation of hela cells with various peptides corresponding to the c terminus of the l protein of papillomavirus, determines the entrance of propidium iodide into cells. [ ] the incubation of the adenovirus internal protein vi with liposomes loaded with a fluorophore, caused the release of the entrapped fluorophore. [ ] similarly, incubation of vp * of rotavirus with liposomes entrapping a fluorophore caused its release, suggesting that vp * is sufficient to perforate the lipid vesicles. [ , ] the membrane disrupting g peptide of fhv also triggers the release of the fluorophore. [ , ] as for enveloped virus fusion peptides, the amphipathic a-helix seems to be a key structural motif also for non-enveloped viruses. the n-terminal amino acids of the fhv g peptide form an amphipathic a-helix which is commonly referred to as g , [ ] while the g peptide comprises also a c-terminal region which is commonly considered to play a supporting role for the correct positioning of g . the g peptide assumes a random coil conformation in solution, while it adopts a kinked helical conformation in model membranes; similarly to other membranotropic peptides also viral lytic factors seem to be able to adopt a membrane-active conformation when interacting with the lipid bilayer. [ , ] the g peptide is able to spontaneously partition into lipid bilayers and increases the membrane permeability of liposomes; [ , [ ] [ ] [ ] ] in particular, it mediates liposome lysis through the insertion only into the outer leaflet of the lipid bilayer, and locating parallel to the membrane surface, with the hydrophobic face of the helix packed against the membrane surface. [ , ] a concentration dependent membrane leakage process [ ] similar to other non-enveloped viruses such as poliovirus vp [ ] and reovirus l n [ ] is observed. similarly to the influenza [ ] and hiv gp [ ] fusion peptides from enveloped viruses, the amphipathic region of g peptide presents a kinked helical structure in solution. the rigid, boomerang like structure assumed by the influenza fusion peptide in lipid environment is required to promote membrane fusion; [ ] in fact, abolishing the kink in the structure or making it flexible eliminates membrane fusion [ ] probably for the failure of the fusion peptide to insert deep into the lipid bilayer and pack against the hydrocarbon moieties. at neutral ph the g peptide is able to cause membrane disruption; while at low ph it is only able to alter its location relative to the capsid, but does not increase its membrane interacting ability. [ ] surprisingly, thanks to a kinked structure and a tight alignment of the hydrophobic residues on one side of the peptide at low ph similar to influenza virus, fhv g peptide shows localized perturbation of lipid arrangements with no proof of pore formation. [ , ] while membrane destabilization requires the simple insertion of the fusion peptide into the outer leaflet of the lipid bilayer, leakage needs both a deeper insertion and an interaction between peptides inside the membrane. the amphipathic region of g peptide oligomerizes in bilayers [ ] with a low content of cholesterol, which being more fluid would promote association between peptides. [ ] the presence of the g c-terminus is absolutely necessary for virus entry; [ ] as a matter of fact, truncations, or point mutations in the c-terminal region of g determine a disordering of the pentameric bundle formed by the n-terminal amphipathic helices of g in fhv particles and hamper in vitro and in vivo membrane lysis. the pentameric bundles constitute the viral "membrane attack module," and an essential function of the g c-terminal region is to maintain this module in its correct conformation [ ] (figure ). the g and g peptides cause a similar localized disorder of the target bilayer and the presence of the cterminal hydrophobic helical region in full-length g make the peptide effective at concentrations achievable in the context of viral infections. the kinked helical structure seems to be a common trait of enveloped and non-enveloped viruses and differences in length and angularity of the helices as well as differences in the amino acid content may cause variations in the mechanism of interaction with the bilayer; the presence of this motif in viral proteins may signal a membrane associated role for this component during a certain step of the viral life cycle which adds to eventual other roles. adenovirus requires acidic ph for exposure of the amphipathic helix contained in its protein vi and disrupts endosomal membranes to release its nucleocapsid. [ ] mutations in the amphipathic helix reduce infection and endosome escape, supporting the view that both the hydrophobic character and a-helical structure are key to allow maximal membrane disruption. the n-terminal amphipathic helix of protein vi, as fhv g peptides, lies parallel to model membranes with the hydrophilic face interacting with the phospholipid head groups, and probably causes disruption of the bilayer by introducing positive curvature in figure schematic representation of fhv capsid (a). an expanded view of the crystallographic structure (pdb: ftb) of one subunit (a protein) showing the location of the amphipathic region of g peptide in yellow (b). schematic representation of a protein, which undergoes auto cleavage during maturation producing b and g (c) with relative sequence of g peptide membranes. [ ] mutations in the amphipathic region of protein vi, which prevent insertion into the membranes, severely affects membrane penetration and cellular entry. [ ] proteins vp and vp play a fundamental role in membrane penetration by poliovirus [ ] with the exposure of amphipathic a-helical nterminal approximately amino acid region of vp being necessary for liposome binding; [ ] while the n-terminus of poliovirus vp contains a hydrophobic myristoyl (c acyl) group for insertion into membranes. [ ] following receptor binding, the amphipathic a-helix within the n-terminal portion of vp , is exposed and probably forms pores to transfer the genome to the cytoplasm. interestingly, vp also performs a role in membrane penetration and the hypothesis is that vp primarily function is to secure the particle to the limiting membrane, while vp participates directly in pore formation. mammalian orthoreovirus protein l contains a small hydrophobic peptide (l n) with a myristoyl group at its n-terminus. [ ] disruption of cellular membranes, requires the complete dissociation of l n peptides from the particle; auto-cleavage of l during reovirus entry generates l n peptides that are linked to an n-terminal myristoyl group. after lipid association, the l n peptide changes conformation from an extended to a b-strand rich secondary structure. [ ] l n is able to generate size-selective pores in erythrocyte or liposomal membranes. it is likely that the b-hairpins in l n associate with the membranes forming a b-barrel pore. [ ] probably, membrane disruption caused by l n is similar to that of b-barrel toxins. [ ] it is highly probable that a future more detailed knowledge of the mechanism used by lytic peptides of non-enveloped viruses will lead to the discovery of more common features between enveloped and nonenveloped membranotropic peptides. membranotropic peptides thanks to their adaptness to interact with the membrane, are opening the way for numerous applications. [ , ] their ability to bind lipid membranes is correlated to their simultaneous hydrophobic and amphipathic nature, while their insertion into the bilayer is due to their capability to change conformation according to the environment; moreover, they are able to penetrate deep into the hydrophobic core but do not span the bilayer in a pore-like manner; on the contrary, they tend to self-associate at the interface between the membrane and the aqueous compartments. below are reported main applications. one of the main applications of fusion peptides of enveloped viruses is as inhibitors of viral penetration. their ability to directly interact with the hydrophobic surfaces present on cell membranes and/or fusion proteins allows them to interfere with virus entry. [ , ] the inhibition mechanism is still unclear but it is likely that inhibition of infectivity is correlated to inactive aggregates formed between the fusogenic stretches present in the viral protein and in the peptides. in particular, the formation of aggregates is related to their ability to oligomerize or to mimic the mode of binding of their original domains in their partner protein; thus, stabilizing a pre-fusion intermediate and preventing merging of the bilayers. [ , [ ] [ ] [ ] self-oligomerization of fusion peptides has been proposed to be responsible of inhibition by several groups. [ , ] hiv fusion peptides form structurally defined oligomeric complexes which have been considered responsible of inhibition; [ , ] moreover, mutants of the native sequence with a lower helical content and tendency to self-associate into b-sheets are able to inhibit membrane fusion with different magnitude and at various stages. [ ] virip is a peptide designed to target gp fusion peptide and thus block hiv- infection; it has undergone clinical studies and was demonstrated to be as active as peptides targeting the coiled-coil. [ , ] its clinical evaluation represents the proof of concept that membranotropic sequences could inhibit viral replication in infected individuals and may have potential clinical effectiveness. moreover, the knowledge that several domains are implicated in the fusion mechanism and may interfere with the intramolecular interactions between the several domains, clearly demonstrates that they all represent potential targets for the design of entry inhibitors. [ ] membranotropic peptides are emerging as delivery vectors. [ ] [ ] [ ] until now, the most widely used delivery vectors are cationic cell penetrating peptides (cpps), which enter essentially by endocytosis causing the entrapment of the cargo into endosomes with only minor quantities of the cargo able to reach the target where to exert the biological function. on the contrary, membranotropic peptides are internalized by direct penetration of the membrane and thus determine immediate bioavailability of the delivered molecule. fusion membranotropic peptides are particularly noteworthy because they can physically interfere with the membrane hydrophobic interior forming bulges that protrude from the membrane and ease contacts between fusing bilayers; in particular, they are able to translocate molecules through the plasma membrane directly into the cell, promoting lipid-membrane reorganizing processes, and causing local and temporary membrane destabilization with subsequent reorganization, circumventing the endosomal entrapment by favoring the escape from the endosome. [ , ] the internalization mechanism is also related to the toxicity of the internalized drug and the development of resistance. the gh is able to directly translocate across the membrane bilayer and to transport several cargos such as quantum dots, [ ] liposomes, [ ] dendrimers, [ , ] nanoparticles; [ ] it is also able to cross the bbb in vivo. [ , ] mpg is an amphipathic peptide composed of the fusion peptide of hiv- associated to an hydrophilic domain with positively charged residues derived from the nuclear localization sequence (nls) of simian virus (sv ) large t antigen (pkkkrkv), through a spacer (wsq). [ , ] in vitro mpg is able to deliver both sirna and dna after just h. [ ] the principal internalization mechanism was shown to be independent of the endosomal pathway and to involve the study of the internalization of g peptide derived from fhv [ ] revealed that this is mediated by relatively high cell surface adsorption leading to enhanced macropinocytic uptake and cytosolic distribution and also revealed a higher efficiency of internalization compared with tat. [ ] influenza virus fusion peptide has been used for increasing transfection efficiency, its ph-dependent fusogenic and endosomolytic activities are able to enhance lysosomal degradation before the contents of the endosomes are delivered to lysosomes. [ ] the development of industrial applications in drug delivery is probably one of the most exciting and fastest growing fields, with the possibility of these peptides to pass through the bbb and become an important player in the fight against all pathologies correlated to neurosciences. scientists envisage also a possible use of viral membranotropic peptides as an alternative to classical antibiotics in order to combat the antibiotic resistance problem. [ ] antimicrobial peptides (amps) are widely exploited and represent attractive candidates for the development of anti-infective agents; [ ] [ ] [ ] recently, attention has been devoted also to the exploitation of membranotropic peptides derived from viral fusion proteins as antibacterial drugs. in fact, some amps with helical structure seem to share high sequence (preference for alanines and glycines) and structure (amphipathic a-helix) similarity with fusion peptides and suggest a convergent evolution correlated to their ability to disturb lipid bilayers. [ ] the fusion domain of influenza virus was evaluated for its antibacterial activity; analysis showed that the amidation of the c-terminus is a key factor to render the fusion peptide an antibacterial peptide and optimization of the amphiphilic balance can improve efficacy. [ , ] the antibacterial activity of viral membranotropic peptides is not yet widely evaluated and much work is still open in this field; in particular, their mechanism of perturbation of membrane bilayers may allow the design of novel sequences with the ability to denature the membrane bilayer of bacteria which will add to their many roles. development of effective vaccines against viruses is another worldwide concern. a potent vaccine needs to be able to induce both [ ] in vivo prime-boost immunization enhanced humoral and cellular immune responses, suggesting the promising application of membranotropic peptides as vaccine candidates in future (table ) . the membrane entry of enveloped and non-enveloped viruses employs fundamentally different mechanisms, although common themes have emerged in the entry process. this similarity is essentially represented by the presence/exposure of small membranotropic peptides which cause membrane disruption and/or promote membrane fusion. entry involves membrane fusion versus perforation, but cellular triggering factors and structural intermediates appear to share some similarities. interestingly there is also some similarity with the mechanism used by bacterial toxins to cross biological membranes in order to reach the cytosol; in fact, many toxins, undergo conformational changes which allow them to initiate the translocation process. [ , ] how exactly both enveloped and non-enveloped viruses overcome host cell membrane barriers to deliver their genomes remains an intriguing problem. comprehensive structural and biochemical studies on enveloped viruses have brought to the conclusion that a unifying mechanism for host cell entry exists; where a membranotropic fusion loop, peptide, or patches catalyze fusion of the two membranes. in contrast, interaction of non-enveloped viruses with host cells during entry is less defined; while membrane active peptides have been discovered as necessary elements for entry in several well-studied non-enveloped virus capsids. in conclusion, it is now evident that the success of membranotropic peptides further stimulates challenging research on the unraveling of the many roles and applications that could be developed for both enveloped virus fusion peptides and small lytic peptides in nonenveloped viruses; membranotropic peptides are attracting increasing attention from the scientific community and their future will be dictated by the progresses in their industrial applications. http://orcid.org/ - - - entry of enveloped viruses into host cells: membrane fusion biophysical modulation of peptide-membrane interactions author biographies annarita falanga received the degree in food science and technology at the university of naples he earned his phd in virology from the university of cambridge (uk) in . he was appointed lecturer in microbiology in at the department of animal health of the faculty of veterinary medicine of the university of naples 'federico ii'. he moved to the faculty of medicine of the second university of naples where he was appointed associate professor and then full professor of microbiology giancarlo morelli is a full professor of chemistry at the university of naples 'federico ii she carried out research activities at the columbia university of new york in - . in she earned the position of assistant professor of inorganic chemistry at the university of naples and since she is associate professor. since , she is adjunct professor at loyola university chicago. research activities focus on antimicrobial peptides and drug delivery mechanisms. how to cite this article membranotropic peptides mediating viral entry key: cord- -ub p ngr authors: mollenhauer, hilton h.; james morré, d.; rowe, loyd d. title: alteration of intracellular traffic by monensin; mechanism, specificity and relationship to toxicity date: - - journal: nan doi: . / - ( ) -z sha: doc_id: cord_uid: ub p ngr monensin, a monovalent ion-selective ionophore, facilitates the transmembrane exchange of principally sodium ions for protons. the outer surface of the ionophore-ion comples is composed largely of nonpolar hydrocarbon, which imparts a high solubility to the complexes in nonpolar solvents. in biological systems, these complexes are freely soluble in the lipid components of membranes and, presumably, diffuse or shuttle through the membranes from one aqueous membrane interface to the other. the net effect for monensin is a trans-membrane exchange of sodium ions for protons. however, the interaction of an ionophore with biological membranes, and its ionophoric expression, is highly dependent on the biochemical configuration of the membrane itself. one apparent consequence of this exchange is the neutralization of acidic intracellular compartments such as the trans golgi apparatus cisternae and associated elements, lysosomes, and certain endosomes. this is accompanied by a disruption of trans golgi apparatus cisternae and of lysosome and acidic endosome function. at the same time, golgi apparatus cisternae appear to swell, presumably due to osmotic uptake of water resulting from the inward movement of ions. monensin effects on golgi apparatus are observed in cells from a wide range of plant and animal species. the action of monensin is most often exerted on the trans half of the stacked cisternae, often near the point of exit of secretory vesicles at the trans face of the stacked cisternae, or, especially at low monensin concentrations or short exposure times, near the middle of the stacked cisternae. the effects of monensin are quite rapid in both animal and plant cells; i.e., changes in golgi apparatus may be observed after only – min of exposure. it is implicit in these observations that the uptake of osmotically active cations is accompanied by a concomitant efflux of h(+) and that a net influx of protons would be required to sustain the ionic exchange long enough to account for the swelling of cisternae observed in electron micrographs. in the golgi apparatus, late processing events such as terminal glycosylation and proteolytic cleavages are most susceptible to inhibition by monensin. yet, many incompletely processed molecules may still be secreted via yet poorly understood mechanisms that appear to bypass the golgi apparatus. in endocytosis, monensin does not prevent internalization. however, intracellular degradation of internalized ligands may be prevented. it is becoming clear that endocytosis involves both acidic and non-acidic compartments and that monensin inhibits those processes that normally occur in acidic compartments. thus, monensin, which is capable of collapsing na(+) and h(+) gradients, has gained wide-spread acceptance as a tool for studying golgi apparatus function and for localizing and identifying the molecular pathways of subcellular vesicular traffic involving acid compartments. among its advantages are the low concentrations at which inhibitions are produced ( . – . μm), a minimum of troublesome side effects (e.g., little or no change of protein synthesis or atp levels) and a reversible action. because the affinity of monensin for na(+) is ten times that for k(+), its nearest competitor, monensin mediates primarily a na(+)-h(+) exchange. monensin has little tendency to bind calcium. not only is monensin of importance as an experimental tool, it is of great commercial value as a coccidiostat for poultry and to promote more efficient utilization of feed in cattle. the mechanisms by which monensin interact with coccidia and rumen microflora to achieved these benefits are reasonably well documented. however, the interactions between monensin and the tissues of the host animal are not well understood although the severe toxicological manifestations of monensin poisoning are well known. equine species are particularly susceptible to monensin poisoning, and a common effect of monensin poisoning is vacuolization and/or swelling of mitochondria in striated muscle. other pathological injuries to striated muscle, spleen, lung, liver and kidney also have been noted. a consistent observation is cardiac myocyte degeneration as well as vacuolization. differences in cellular response resulting from exposure to monensin (i.e., golgi apparatus swelling in cultured cells, isolated tissues, and plants vs.mitochondrial swelling in animals fed monensin) suggest that myocardial damage is due either to a monensin metabolite or is a secondary response to some other derivation. however, as pointed out by bergen and bates [ ], the underlying mode of action of ionophores is on transmembrane ion fluxes which dissipate cation and proton gradients. consequently, some or all of the observed monensin effects in vivo in animals could be secondary phenomena caused by disruption of normal membrane physiology resulting from altered ion fluxes. monensm, a na ÷ ionophore capable of collapsing na ÷ and h + gradients, has gamed wide-spread acceptance as a biochemical and biologacal investigative tool to study golgi apparatus function and to localize and identify the molecular pathways of subcellular vesicular traffic. among its advantages are the low concentrations at which lntubltions are produced ( . - . #m), a minimum of troublesome side effects (e.g., little or no change of protein synthesis or atp levels), and a reversible action [ ] . the purpose of this review is to examine the mechanism of action and specificity of monensln in na+/h ÷ exchange and to attempt to reconcile tlus to the large body of structural and biochemical information on monensin toxicity derived from animal studies in , pressman and co-workers [ ] reported a class of anubiotics that induced alkali ion permeability in mitochondna and other membranous systems. these antibiotics functioned as lonophores (ion-carriers) to carry ions across hpid barriers as complexes soluble in the hpid phase of the membranes. the potential use of tonophores as probes of biological function, or as potential therapeutic agents, was recognized very early [ ] [ ] [ ] [ ] , but major economic importance was not forthcoming until the discovery of monensin in and the recognlnon of tts potential in the poultry mdustrty as a coccxdtostat [ ] subsequently, ~t was discovered that tonophores also could improve feed converston in rumtnants such as cattle [ , ] , thus adding further to their commercial value of the more than lonophores that have been reported [ ] , three, monensln, lasalocld and sahnomysin, have widespread commercial use of those licensed, monensin is probably used most widely. several monensins have been tdentified [ , ] _ monenstn a, and specfftcally the sodium salt of monensin a (hereafter simply designated as monensm) ts derived from streptomvces cmnamonensts [ ] , and a crude mycehal preparation (rumensm') containing about _ % monensin ts used commercially. the on spectfictty of monensm xs ag > na >> k > rb > cs > li > ca [ , ] with approximately a -fold selectlvry for sodtum over potassium [ ] and little tendency to bind calcmm [ ] i! . one of the original interests in ionophores was their percewed potential for directly modifying intracellular iomc gradients, pamcularly ca +, winch would lead, hopefully, to the development of useful pharmacologic agents or, alternatively, provide a tool for studying cellular functtons medmted by changes m ca + [ , ] . of parttcular interest were divalent xonophores such as x- a (normally considered a ca + ionophore -note, however, x- a complexes na ÷ and k + almost as well as ca +) winch have been shown to mduce contractaon of aortic strips and increase the rate of contractility of tsolated perfused rabbit heart [ , ] , and release ca + from energy-loaded vesicles derived from the sarcoplasrmc renculum of muscle [ ] [ ] [ ] x- a may also mcrease blood flow through coronary artenes and mcrease cardiac output [ ] . the emphasis on heart physiology stemmed from this organ's strong dependence on calcmm for proper functioning [ ] . indeed, subsequent studies of x- a used as a feed additive for poultry and cattle has shown that the heart ~s a primary target for lonophore toxicity [ ] . x- a affects many other cellular functions such as release of b~olog~cally active agents and the induction of sperm acrosome reactions of several species [ , ] . it was soon realized, however, that many of the ionotroplc effects of the divalent ionophores could be duplicated with even greater efficiency by monovalent ionophores such as monensm which complexes na + but almost no ca ÷ tins response apparently occurs because the movement of na ÷ into a cellular compartment by monensin fac~htates the entry of ca + by a na+-out/ca +-ln exchange [ , , , ] . thus, a ca ÷ shaft ts stall the pnmary factor medmtlng cellular responses although other factors may also play significant roles m monensm physiology for example, many tono-phores, either directly or as a result ol the lomc imbalance. may transport, promote uptake, or release effector substances such as serotonm, lustamme, prostaglandm and catacholamlne which, m turn. have profound effects on cellular function [ . ] slmdarly. monensm. through alteration of the ph of mtracellular compartments may lnhlbr the release and/or transport of numerous agents and. in so doing, perturbate cellular function in cardiac tissues, both posmve and negative motroplsm has been observed sequentially (the variable factor being time) m tissues following exposure to monensin [ ] concentrahon of monensln may. also. cause similar posttlve/neganve ,notroptc responses [ , . finally. some monensln mgested b}¢ an animal is metabolized to other tonophores, the properties of which are largely unknown thus, ionophores, in spite of many common characteristics, differ indtvtdually in their effects on cell~ moreover. cells may respond to both direct tonophore interaction as well as secondary effects that develop from the initial ionophore reactions. the latter is partlcularly likely in the whole antmal where metabolites with unknown properties are produced from lonophore breakdown and where changes m the products of one organ can affect the function of other organs_ monensin is an open chain molecule that is capable of ton complexation through a cyclic form stabilized by hydrogen bonding between the carboxyl and hydroxyl groups charge transfer bonding wltinn the cavity formed is responsible for on binding (fig ) _ because the affinity of monensln for na + is -ttmes that for k+. tts nearest competitor in biological systems. bergan and bates [ ] ) the aniomc form of the lonophore is stabilized by the polar environment characteristic to the surface of a membrane_ the ionophore is capable of ion pamng with a metal cation either at the terrmnal carboxyhc acid moiety or at other internal sites the binding of a cation initiates the formation of a hpophahc, cyclic catlon-lonophore complex that can diffuse through the interior of the blmolecular membrane structure_ after traversing the membrane, the complex is again subjected to a polar environment where the electrostatic forces that had stablhzed the complex are no longer greater than the unfavorable gibbs free energy change of cyclzzation the ionophore then releases its enclosed cation and reverts to the low energy acyclic conformation monensin, like other carboxyhc lonophores, binds metal ions through liganding s~tes such that the ions become centrally oriented ( fig. ) and masked from the extraceuular environment [ , , ] . the outer surface of the ionophore-lon complex is composed largely of nonpolar hydrocarbon, which imparts a high solubility to the complexes m nonpolar solvents [ , , ] in biological systems, these complexes are freely soluble in the lipid components of membranes and, presumably, diffuse through the membranes from one aqueous membrane interface to the other [ , ] . once the ion traverses the membrane as a monensin-lon complex, the ion is released, and the monensin molecule picks up a proton to form an undissociated molecule which then retraverses the membrane to release the proton to the outside of the cell, vesicle, organelle, or other subcellular compartment [ ] (fig. ) . thus, the net effect for monensin is a trans-membrane exchange of monovalent ions for protons. the on transfer rates may be very high and can approach, or even exceed, normal enzyme diffusion rates [ ] , although the actual rate may be markedly altered depending on factors such as the concentration of k ÷ m the external medium and the type and concentration of permeant ion that accompariles the accumulated k + [ ] . however, effects of lonophores on cation transport and their distribution among different membrane-bounded compartments within the cell, will vary depending upon the physical and chemical properties of the different membranes. membrane fluidity, thickness, curvature, charge and orientation of polar head groups of phosphohpids, cholesterol content, and protein content, all influence solubihty, penetration, and expression of the lonophore [ ] . moreover, in asymmetric membranes (i.e., biological membranes), ionophores generally exhibit asymmetric transport properties [ ] . for example, kovac et al [ ] showed that both vahnomycm and nigericin (an lonophore similar to monensin) crossed the plasma membrane of saccharomyces cereotslae at a rather low rate but then were preferentially located, and active as ~onophores, m the tuner mltochondrlal membrane. thus, the physiological effects of monensln will depend on the membrane composition and functional characteristics of the different compartments involved. although mechanisms for on transfer through a blmolecular leaflet (membrane) have been proposed, questions still remain as to how this action is related to known effects of monensin on cell function and what relationships these may have, m turn, on blochermcal mechanisms leading to ammal toxaclty. while any consideration of ~onophore action must focus on the mechanism of lonophore interaction with biological membranes [ ] , the complexity of the process and the multiplicity of potentml pathways involved suggests that a single causal mechanism cannot explain both the cellular responses and the clinical expressions of toxic-]ty in animals monensin is cost-effective in increasing the yield of meat from both fowl and ruminants [ ] . in fowl, this increase m productivity is derived almost directly through the control of coccid]a that, if present, would adversely affect animal health [ ] in ruminants, increased product]vity appears to result from several factors, the most obvious being an increase in the effectiveness of feed utlhzation [ , , ] . these whole-animal effects (xe, systems effects) are well documented [ , [ ] [ ] [ ] and will be paraphrased only briefly here. the beneficial effects of monensm in cattle accrue, in part, through shifts m rumen rmcroflora population. for example, gram-positive bacteria (that are primarily acetate, butyrate, h , and formate producers) are inhibited by monensin; whereas, gram-negauve bacteria (many of which produce succinate) are less sensmve to monensm [ ] . the outer layer of the multflayered well of the gram-negatxve bacterium may contribute to this resistance by acting as a barrier to the penetration of [able i adapted from ledger and tanzer [ ] in ~e~retlon reduced secretion procollagen [ ] [ ] [ ] [ ] , fibronectm [ ] [ ] [ ] ] , proteoglycans [ , , ] , prolactln [ ] , alburmn [ ] , transfernn [ ] , promsuhn polypepudes [ ] , larmmn [ ] , a-amylase isoenzymes [ , ] , newly synthesized proteins [ ] , secretory proteins [ ] , proteins for fast axonal transport [ , ] , thyroxine-binding globuhn [ ] , acetylchohnesterase [ , ] , chononlc gonadotropin [ ] , phytohemagglutmm [ ] , very-low-density hpoprotem [ ] , maize rootcap polysaccharldes [ ] (see however, sticher and jones [ ] for lack of monensm effect), vesicular stomatltxs virus glycoprotein [ ] , extracellular matrix [ ] , type ii collagen [ , ] , reviews [ , , ] increased secreuon catecholarmne [ ] , catheps'n d [ ] _ defective processing pro-albumin to serum albumin [ ] , receptors for msuhn and somatatomedin c [ ] , pro-oplomelanocortm [ ] incomplete processing of ohgosacchandes (n-hnked and/or o-linked) myeloperoxldase [ ] , prenv glycoprotexn [ ] ; fibronectm [ ] , hcg subumts [ ] , blocked formation of complex ohgo-~acchandes [ ] , herpes simplex glycoprotelns [ ] , hla-dr-as-soclated mvanant chain [ ] , coronavlrus glycoprotem [ ] , review [ ] undersulphauon proteoglycans [ ] , glycosammoglycan chains [ ] , / -d-xyloside glycosammoglycans [ , ] in endocytosts and endosome actdzflcatton intubitlon of mternahzatlon arylsulfatase [ ] , lmmunoglobulln [ ] , a- -macroglobuhn [ ] , semlikl forest varus [ ] , horseradish peroxldase [ ] inhibition of dlssocmtion of mternahzed hgand asmloglycoprotems [ ] , asmlo-orosomucojd [ ] lnhlbxtlon of hgand transfer smb~s virus nuclear capsids to cytoplasm [ ] , epidermal growth factor, / -hexasamlmdase, low-density hpoprotein, lmmunoglobulin, and proteoglycans to lysosomes [ , , ] inhibition of acldfflcauon endocytlc vesicles [ ] [ ] [ ] , lysosomal and prelysosomal compartments [ ] , interference with semllka forest virus genome penetrauon [ ] , expression of diphtheria toxin [ ] ; recycling of ldl receptors [ ] , release of diphtheria toxin from endocyuc vesicles [ , ] lnhlbltton of mtracellular degradation proteoglycans [ ] , insuhn [ ] , lysosomal (methylarmne-sensxtlve) protem degradation [ ] inhibition of contraction of contractile vacuoles paramecmm aureha [ surface formation and growth altered secretion of cell surface molecules proteoglycan [ , , ] , type if collagen and/or procollagen [ , , ] ; fibronectin [ , , ] , lamlmn [ ] , mcorporauon of sulfaudes into myelin [ ] , incorporation of po protein and myehn basic proteins into myelin [ ] inhibition of scale morphogenesis scales of the green alga pyrarmmonas mconstans [ ] inhibition of cell spreading-cultured fibroblasts [ ] ; mesoderm ceils [ ] stimulation of receptor capping mouse t-lymphoma cells [ ] inhtbltlon of growth rye seedhngs [ ] , pelhu ] transport of molecule.~ recognition of independent secretory pathways acetylchohne receptor and acetylchohnesterase [ ] , membrane glycoprotems/ assembly of uukunlerm virus [ ] , ca +-dependent and ca +-mdependent secretion of a-amylase [ ] , proteoglycan and hyaluronate [ ] , prolactln [ ] ; galactosyl receptor [ ] maturation and/or transport of viral coat proteins vesicular stomatltis virus [ , ] , herpes sxmplex varus [ , ] , semhkj forest wrus [ ] , uukumerm wrus [ ] , alphavtrus [ ] , coronawrus glycoprotean [ ] , bovine herpes virus type glycoprotelns [ ] st~mulatlon of sugar and sugar nucleoude transport avian erythrocytes, isolated rat and mouse dmphragm muscle, and red cells [ , , ] ; mouse thymocytes [ ] redirection of secretory product plasmalemma to tonoplast [ ] inhibition of mtracellular transport: protein to rod outer segments [ ] , myeloperoxtdase [ ] , hcg subumts [ ] , accumulation of lammln [ ] ; gp glycoprotein [ ] , procollagen [ ] , fibronectm [ ] interactions wtth other toxm~ enhancement of toxaclty tlamuhn m swine [ ] , dlsulfide-hnked methotrexate-anti-transferrln receptor conjugate [ ] , specific cytotoxlcxty of a breast cancer-associated antigen lmmunotoxan m humans [ ] reduction of toxicity selenmm and vitarmn e [ ] monensm; although, a more direct influence revolving differenes in membrane energetlcs also has been implicated [ ] monensln also may decrease the degradation of dietary protein in the rumen and, thus, increase the amount of protein avaalable for dlgesuon and uptake in the small intestine [ ] . both a reduction in overall cell numbers m the rumen and a direct effect of monensln on bacterial protelnase and dearmnase activity have been suggested as contributing to this effect [ , ] . one of the first subcellular effects observed in relation to the topical apphcatlon of monensin was vacuolatlon of golgi apparatus clsternae [ ] . subsequently, xn vitro studies clearly demonstrated that monensin altered or inhibited numerous membrane-located phenomena (table i) . among these were the transfer of a -macroglobuhn from coated pits to receptosomes [ ] , recycling of low-density hpoprotem receptors [ ] , pinocytosis [ ] , transfer of product from endoplasmac reticulum to golgl apparatus [ ] , maturation and/or transport of viral coat proteins [ , , , , , , , ] , inhibition of transport of membrane proteins to rod outer segments [ ] , xnhibltion of cholesterol transport from the golgl apparatus to the mltochondnal site of steroidogenesis [ ] , blockage of phytohemagglutinln transport out of golgl apparatus and into protein bodies [ ] , inhibition of procollagen and fibronectm secretion from cultured human fibroblasts [ ] , inhibition of carbohydrate processing in cultured human flbroblasts [ ] , and inhibition of processing and cellular secretion [ , , , [ ] [ ] [ ] . additionally, cellular effects of monensm vary markedly depending upon the organism and the route of adrmnlstratlon. cultured cells, cells of tissue shces or explants, and plant organs that have received a topical exposure to monensin sufficient to inhibit growth or some cellular processes, usually show deviations in golgl apparatus structure and function. in contrast, ceils from animals poisoned by ingested monensin often exhibit gross mltochondrlal lesions without the corresponding golgl apparatus modifications. the reasons for these fundamental differences in cellular responses to monensin provide one focus for the present review and illustrate the many complexities surrounding the use of monensln as a probe specific to a single metabolic process. the primary functional unit of the golgi apparatus is a stack of membranous compartments (i e., the clsternae) each of which differs chemically, structurally, and functionally from the others [ ] [ ] [ ] [ ] . the number of cisternae per stack varies widely; although, in most animal cells and higher plant cells, there are about - cisternae per stack. each stack is polarized in the sense that product and membrane maturation appear to occur sequentially from a cls (fornung) face on one side to a trans (matunng) face on the other side [ ] . for simplicity, the stack may be divided into umts (measured from cis to trans faces) each of which represents a known set of functions. currently, there appear to be some - such units that make up each stack [ , ] . in reahty, however, it seems more likely that these changes in function occur gradually across the stack of clsternae rather than in discrete steps. there is, also, one or more membraneous structures (e.g., the trans golgl network [ ] or tgn and the partially coated retlculum [ ] or pcr) that he just off the trans poles of the stacks in plant cells, these structures appear to be derived from sloughed trans clsternae [ ] . tgn and pcr participate in the separation (i.e., sorting) of both secretion and endocytic products [ , [ ] [ ] [ ] [ ] and regulate the release of endocytosed substances through a ph-sensltlve mechamsm [ ] . the functions of these post-golgi apparatus structures are rapidly affected by monensin. several mechanisms for the movement of membrane and product through the golgi apparatus have been postulated. for example, movement may occur by sequential maturation of golgi apparatus elements (i.e., formation, maturation and loss of cxsternae) through the golgl apparatus stack [ ] . this would require the formation of new clsternae on one face of a golgl apparatus sack and commensurate loss of clsternae from the opposite face of the stack the source of these new clsternae is a special region of endoplasmic retlculum which gives rise to transition vesicles that move and condense on the forming (cls) face of the stack where they fuse together to form the new osternae [ ] [ ] [ ] product movement may also occur by shuttle vesicles at the peripheries of the clsternae that move proteins from one cisterna to the next [ ] . however, both direct (i e., nonvesicular) movement of substances into golgi apparatus cisternae and an endoplasmlc reticulum-mediated movement of product through the penpheral tubules of the clsternae must also be considered as viable options for the delivery and transfer of substances in and out of the golgl apparatus [ ] . the post-golgl apparatus structures appear to move membrane and product almost entirely via shuttle vesicles, many of which are coated [ ] [ ] [ ] monensin exerts its most profound effects on the trans cxsternae of the golgi apparatus stacks in those regions of the apparatus primarily associated with the final stages of secretory vesicle maturation and in post-golgl apparatus structures primarily associated with endocytosis and membrane/product sorting. because of its relative specificity, biologists have used monensln extensively as an inhibitor of trans golgl apparatus function. incorporation of radiolabeled [ s]methlonine into secreted lmmunoglobulin m molecules in monensintreated cells was reduced as was slalylatlon of immunoglobulin m and lymphoid cell surface glycoprotelns [ ] . these latter findings showed that the intracellular processing of n-asparagine-hnked oligosacchandes is altered in the presence of monensln with an effect primarily on those sugars (e.g., slalic acid, galactose, fucose) added late in the processing continuum [ ] flbronectin, secreted in human flbroblasts, was incompletely processed in the presence of monensln and exhibited a greater incorporation of mannose than did control protein molecules [ ] inhibition of fibronectln secretion in human melanoma also has been reported [ ] . similarly, when treated with monensln, rat astrocytes in primary culture accumulated lamimn, another matrix glycoprotem involved in cell adhesion [ ] . not only do the monovalent lonophores block transport and surface expression of several secretory glycoproteins in normal cell functioning [ , , ] and the transport of membrane glycoproteins or enveloped viruses [ , , , , , , , ] , they inhibit formation of cell surfaces including assembly of peripheral myelin [ , ] . in mouse thymocytes, monensin leads to ( stimulated incorporation of labeled sialyl-, galactosyl-, and n-acetyl glycosaminyl residues [ ] . this enhanced accumulation was not due to a &rect effect of monensin on glycosyltransferase activities but, rather, as a consequence of a greater entry and accumulation of labeled sugar nucleotides in the swollen clsternae_ galactosyl transferase itself was translocated through the golga apparatus at a slower rate with monensin_ however, the sialylatlon of the o- nked ohgosaccharides of the enzyme was unaffected by monensln treatment [ ] effects of monensin on glycosyltransferases also may be indirect. monensin has been reported to decrease galactosyltransferase activity m golgl apparatus of rat embryo fibroblasts [ ] although it had no effect on this activity in baby hamster kidney cells [ ] monensln is an especially useful inhibitor, since it blocks lntracellular transport of protein at the level of the golgl apparatus without directly affecting protein synthesis [ , ] the effect of monensin is considered to be on transport rather than on processing per se [ , ] one argument is that oligosaccharide processing of those glycoprotelns that reach the appropriate site occurs normally even xn monensin-treated cells [ , , , ] . however, these observations could be explained as well if processing of ollgosacchande chains of different secreted glycoprotelns occurred at different sites, only some of which were sensitive to monensln [ , ] . the ablhty of monensln to effectively 'freeze' processing of molecules at a particular stage had lead to its use in identifying transitory synthetic intermediates. examples include the insulin receptor where several polypeptlde precursors have been described [ ] , the intracellular accumulation of non-cleaved precursors of pituitary hormones that occur in the presence of monensin [ ] , and dissection of the pathway for secretion of gonadotropin by cultured human trophoblastic cells [ ] . in some instances, the effect of monensin may be to redirect, rather than block, the movement of golgl apparatus-derived product. for example, under normal conditions, proteins of developing seeds accumulate in a central vacuole which then partitions into smaller units of storage protein (i.e, the protein bodies). however, when treated with monensin, the golgi apparatus-derived transport of the protein vlclhn in pea cotyledon was redirected from the vacuole to the plasmalemma and the newly synthesized viclhn was released from the cotyledon cells to accumulate between the plasmalemma and cell wall [ ] monensin lnhibmon of golgi apparatus function ~s sufficiently well established [ , , , ] that the phenomenon is used widely as one criterion for verifying the passage of a biochemical entity through the golgi apparatus. thus, based on partial monensm inhibition, hammerschlag and co-workers [ , ] concluded that passage through the golgl apparatus was an obllgator~ step m the lntracellular routing of materials m ta,~t axonal transport_ bartalena and robblns [ ] showed that rnonensin ~mpeded the exit of thyroxin-binding globulin from the golgj apparatus w~thout affecting the terminal glycosylatlon of the protein yanagashito and hascail [ ] reported that monensin reduced and delayed transport of both secretory and membrane-associated forms of proteoglycans, suggesting passage through the golgl apparatus of rat ovarian granulosa cells m culture similarly, an involvement of the golgi apparatus m the transport of sulfatldes to myelin [ ] and phytohemaghitlnln to protein bodies m bean cotyledons [ ] were deduced from monensin mhlbmon fhckinger and co-workers [ ] using [ h]leucine, showed that all, or nearly all, of the protein secretory product of mouse epididymis principal cells pass through the golg~ apparatus in times approximately eqmvalent to those reported m other tissues. this transfer of product from golgi apparatus to the cell surface was largely blocked by monensin swelling of golgi apparatus cisternae observed in the electron microscope following fixation with glutaraldehyde is, perhaps, the most consistent visual in vitro demonstration of a monensin-mduced effect on a membranous cell compartment [ , , , , [ ] [ ] [ ] the swollen clsternae usually appear devoid of contents by electron microscopy (figs. and ) but an electron-dense substance may be precipitated through the osmium tetroxlde-zmc iodide reaction (unpubhshed data) although all clsternae of the golgi apparatus may swell in response to monensin ( fig. and a) , the major effect appears to be associated with the mature, or trans, parts of the golgi apparatus stacks (figs and b) [ , , ]_ gnffiths and co-workers [ ] showed that monensm inhibited the transport of viral membrane proteins from medml to trans golg~ apparatus cisternae, thus indicating a monensln block between medial and trans cisternae monensm also blocked tlamrmng of the high mannose bound to the viral membrane proteins and their conversion to complex ohgosacchartdes similarly, niemann and co-workers [ ] found that monensm blocked glycosylation of e glycoproteln of corona virus in infected mouse cells. srinlvas and co-workers [ ] reported failure to process simple endo-h-sensitive to complex endo-h-reslstant ohgosacchandes and reduced efficiency of cleavage of the prenv glycoprotein precursor to gp for evehne mouse cells infected with friend munne leukemia virus these findings indicate a block prior to entry into the golgl apparatus also, m cultured hepatoma cells, transport of vesicular stomatltis wrus (vsv) g protein was arrested prior to acquisition of endo-h resistance, suggesting a block early in the processing pathway [ ] . strous and co-workers [ ] showed that monensin affects primarily the galactosyltransferase-containlng c~sternae of the golgi apparatus based on studies of the metabohsm, localization, and biosynthesis of n-and o-linked oligosaccharides of galactosyltransferase in helz cells. the accumulation of incompletely processed glycoproteins indicates either an up-stream accumulation of secretory materials behind a golgl apparatus blockage by monensm [ ] or a monensin block near the exit site from endoplasmlc reticulum [ ] . monensin effects on golgi apparatus have been observed in a wide range of plant and animal species and appear to be a universal response to the topxcal applicatton of monensln. as pointed out above, monensln action is exerted on the trans half of the stacked clsternae (fig ) , often near the point of exit of secretory vesicles [ , , , , , , ] or, especially at low monensm concentrations or short exposure times, sometimes m the rmdreglon of the stacked cisternae [ , ] . intracellular transport may be blocked [ , , , , , ] , often wlthxn rmnutes after exposure to monensln [ , , , , ] . swollen units usually accumulate near the golgi apparatus [ , ] a monensm effect is quite rapid in both animal and plant cells; e, changes in golgi apparatus have been observed after only - rmn of treatment [ , , , ] . these early effects have been documented particularly well is suspension cultures of carrot (daucus carota l ) [ ] . when carrot cells were exposed to monensln at - m (which is approxamately the minimum concentration that will elicit a strong monensm response m plants), production of secretory vesicles ceased and, almost immediately, an increased number of clsternae in the dlctyosome stacks was observed. an average of one additional clsterna per stack was formed within the first - mln of monensin treatment and, in some experiments, a second clsterna was formed within about man. these effects occurred without significant swelling of cisternae. thereafter, vacuoles, representing intact swollen clsternae, began to accumulate in the cytoplasm at a rate of about one every - min (fig. ) . the mechanism postulated for this momentary increase of d~ctyosome clsternae was that monensln, acting on the trans pole of the dictyosome, blocked normal formation [ ] for details of procedure) the two samples differed onl~¢ m that the one dlustrated m {a) was adjacent to a 'natural' ( e_ uncut) surface of the hver lobe, whereat (b) was from a cell near the 'cut' surface of the tissue slice in both instances, come golgx apparatus (ga) clsternae were sv~ollen, however, m (a) swelling was progresse~e from cls to trans pole (direction of arrow) whereas m (b) swelling was t.onfined to the trans c~sterna note that m~tochondrm (m) were condensed of secretory vesicles but did not block the formation of new clsternae at the cis face of the apparatus however, as the trans clsternae began to swell, the swollen clsternae were eventually released as intact units that neither fragmented nor integrated (fused) with other cellular constituents (e.g., plasma membrane). with the scale producing green alga pyramimonas lnconstans, exposures of to several hours to monensln resulted m disorientation of the golg~ apparatus and disruption of scale morphogenesls [ ] . these effects were reversible more recent studies indicate that a similar pattern of swelling and accumulation of clsternae in the cytoplasm occurs in cultured animal cells [ ] . when h- hepatoma cells were treated for varying times with ~ to m monensin, one swollen clsterna per stack of clsternae was produced after - rain of treatment during tlus time, approximately one additional clsterna per stack was formed (fig. , inset) . as the clsternae veslculated, vacuoles began to appear m the cytoplasm these large swollen vacuoles were formed at the rate of one sin concentrations, the vacuoles were larger and appeared more rapidly than at low concentrations of monensln but the kinetics of vacuole formation were qualitatively similar. however, by h following treatment with - m momensin, all clsternae of the golgi apparatus appeared as vacuoles. the swollen trans compartments that accumulate in the golgl apparatus region with monensln inhibition may contain regmns that are clathrin coated; e.g., condensing secretory material (prolnsuhn) in pancreatic cells [ ] . the response of golgi apparatus of hver slices to monensin was qualitatively similar to that with hepatoma cells in culture [ ] . with liver shces, a fraction enriched in vacuoles was isolated and demonstrated to contain the trans golgl apparatus markers, galactosyltransferase, and thiarmne pyrophosphatase, in ratios sirmlar to those of golgl apparatus proper [ ] . in barley aleurone layers, the a-amylase and acid phosphatase activities that accumulated within aleurone cells following treatment with monensin, were localized in cellular components with buoyant densities intermediate between endoplasrmc reticulum and mitochondria and cosedimented with latent inoslne diphos-phatase activity, a putative golgi apparatus marker in plants [ ] . heupke and robinson [ ] reported a shift to higher density of golgi apparatus membranes from monensin-treated barley cells, a response no obvious from work with mammalian cells. the golgi apparatus clsternae that accumulated behind a monensin block in semlikl forest virus-infected bhk cells bound viral nucleocapsids, and the resulting increase in density permltted their separation by gradient centnfugatlon from other golgi apparatus elements [ ] . the effects of monensin on golgl apparatus, at least up to several hours of exposure, appear to be fully reversible [ , , , ] in carrot cells, normal secretory activity was resumed within nun after transfer of cells to a monensln-free medium; although, in these cells, the vacuoles formed during the monensln block, remained in the vicinity of the golgi apparatus for several hours or more, even after apparently normal secretory activity had resumed however, with the longer treatment times of several hours [ ] or days [ ] monensin apparently causes swelhng of golgi apparatus cisternae through a na+-in/h+-out exchange across the membranes leading to a net uptake of na ++ ci and entry of water [ , ] evidence in support of this concept was provided by studies with isolated chromaffin granules which lysed readily after brief exposure to monensln in na +-or k+-contalnlng isotomc media for swelling to occur, the membrane must normally be lmpermeant to cations as is known for the chromaffin granule_ the chromaffln granule membrane contains a h+-atpase which is electrogenexc and, in the presence of a permeant anion, acidifies the granule interior to ph _ thus, the operation of this pump in the presence of monensin drives net salt uptake [ ] . to test whether net salt uptake driven by the presence of a proton gradient also would explain the monensininduced swelling of golgi apparatus cisternae, wild-carrot cells in suspension culture were treated with drugs and inhlbltors known to interfere with proton gradients [ ] monensin-induced swelling of golgl apparatus in sjtu could be inhibited by the protonophore, carbonylcyanlde-p-trlfluoromethoxyphenylhydazone (fccp), but was only little affected by the inhibitor of lysosomal acidification, quercetln, or by the lysosomotropic amines, chloroquine, and ammonia. cyanide also dramatically decreased swelling, and arsenate (with prolonged treatments) reduced the number of swollen cisternae organic acids, by providing a readily permeable counterlon, promoted monensln-lnduced swelhng these data imply that the monensin-induced swelling of golgi apparatus cisternae involves a proton gradmnt at or near the mature poles of the golgi apparatus because monensin induces a " na+/h + exchange, and since the van't hoft factors for h + and na + are practically the same [ ] , the osmolanty of the cell content should not increase to cause swelhng without a net proton influx one explanation would be that the ph of golgi apparatus vesicles is highly regulated proton translocatlng atp hydrolyzing enzymes (h +-atpases) are associated with several components of cells that develop acidic intermrs such as endosomes, coated vesicles, lysosomes, and trans golgl apparatus clsternae [ ] . ewdence for the presence of an h +-atpase has been the demonstration of atp-dependent vesicle acxdlflatlon in golgl apparatus isolated from rodent liver [ ] [ ] [ ] , corn coleoptlles [ ] , and sycamore cells [ ] acldificatmn was demonstrated both by [lac]methylamlne uptake and by spectrophotometric assays of amd-quenched dye fluorescence (acridine orange, neutral red, or quinacrme) several lines of evidence confine the golgi apparatus h +-atpase to the trans cisternae. as emphasized in the preceding section, the early in sltu effects of monensm are frequently localized to the trans faces of the golgl apparatus the resultant swelling, whmh is proposed io be due to the accumulation of osmotically active ions in exchange for protons [ . ] , occurs predominantly m trans clsternae additionally, a basic congener o] dmltrophenol ( -( , -dlnitroanlhn o)- '-amlno-nmethyldlpropylamine, damp), which concentrates m acidic compartments as shown in fibroblasts by lmmunocytochemlstry is present onl) clsternae and vesicles associated with the trans faces of the golgl apparatus [ ] moreover, damp rapidly leaves these compartments when cells are incubated with monensm, thus further indicating that accumulation of damp is due to the acid ph some involvement of a low ph compartment is evidenced by the observatmn that some monensln effects on processing, ke, proteolytlc conversion of proalbumln [ ] , are mimicked by amines however, in promyelocytlc leukemia cells, processing of myeloperoxldase, while blocked by both monensln and chloroqulne, was not affected by nh~ cations, thus indicating that processmg is not necessarily influenced by ph-dependent mechanisms [ ] these results were interpreted as indicative of processing in golgl apparatus based on inhibition of transport by monensin and chloroquine rather than processing m lysosomes and other late, acidic compartments involving a ph-dependent mechanism [ confirmatmn of a trans location of the h ~-atpase has come from free-flow electrophoresis separations of golgl apparatus yielding cls, medial, and trans compartments m fractions of diffenng electrophoretic mobility [ , ] _ in these separatmns, proton pumping activity was found exclusively in the most electronegative fractions coming from the trans-most goigl apparatus re-gion_ gnfflng and ray [ ] have offered the suggestion that acldificatmn of clsternal lumlna may be part of an osmotic mechanism to compress and flatten the cisternae the latter, for example, might aid in the transfer of content into secretory vesmles. the inward pumping of protons would tend to favor the exit of na + and k + out of the clsternae furthermore, as the ph falls, those monovalent cations remaining would tend to combine with acidm groups of the golgx apparatus membranes, further reducing the osmolanty of the clsternal content relative to that of the external cytoplasm. thus, water would be driven osmotically out of the clsternae to both compress the clsternae, as seen along a pronounced cls to trans gradient for plant golgl apparatus [ ] , and, perhaps, to account for the condensation of secretory materials m condensing vacuoles and other trans golgi apparatus compartments (e.g, ref ). other cell compartments, including endocytic vesicles [ , ] , lysosomes [ , ] , multivesicular bodies [ ] , and coated vesicles [ ] [ ] [ ] have h+-atpases. all use h+-atpases to acidify their interiors and the enzymes responsible have been solubdized from lyso-somes and reconstituted into liposomes [ ] . vacuole (tonoplast) membranes [ ] , and possibly also plasma membranes of fungi [ ] , contain h÷-atpase. similarly, a gradient of acidification within the endocyuc pathway has been indicated from immuno-electron microscopy with protein a-colloidal gold and monospecific antibodies to the weak base pnmaquine [ ] . however, not all compartments with h+-atpases (e.g., cells, vacuoles, lysosomes, coated vesicles) swell in response to monensin. cell and/or vacuole swelhng may be hmlted due to the very large internal volumes revolved. the contracnon of contractile vacuoles of paramectum was inhibited reversibly by monensin and in a manner dependent upon the presence of na + [ ] but marked swelling was not observed. little is known about the swelhng response, ff any, of lysosomes, coated vesicles and other endocync compartments m response to monensin lntubition. their functions, however, are inhabited by monensin as will be emphasized in the section that follows. carboxyhc lonophores strongly inhibit proton uptake by photosynthenc preparations [ ] . in chloroplasts, swelling of thylakolds (inner membrane compartments) but not of the space between tuner and outer plastld membranes has been observed to result from monensln treatment [ ] . thylakold swelling, in contrast to swellmg of mitochondrial cristae and of golgl apparatus clsternae, was reduced upon mcubanon in darkness, again suggesting a relationship between swelling in the presence of monensln and the hght-driven proton gradient used for photophosphorylatlon [ ] mltochondrla have an outwardly directed energy-hnked proton pump and do not swell with monensln (rather, they tend to condense, see figs. , a and b) while the light-driven proton pump of chloroplasts and chromatophores, and that of the golgi apparatus pump, are directed reward causing the vesicles to swell. evidence for a secretory pathway bypassing the golga apparatus in the monensln-blocked cells is provided by kubo and pigeon [ ] who lnvesngated the effects of monensin on the synthesis and expression of membrane igm of a human lymphoblastold hne. they found altered processing of both /l, k chains and incomplete terminal glycosylations. yet, transport of the altered molecules was observed. that the aberrant processing did not influence markedly the membrane expression of the igm is consistent with a secretory pathway bypassing the golgl apparatus m monensin-blocked cells. slmdarly, a dual secretory pathway, only one part of which was suscepnble to monensin, was deduced from studies of a-amylase secretion m rice seed scutellum [ ] . in zea majze roots, monensln lnhib~ted secretion of aamylase but not polysaccharide slime [ ] . the blocked secretion that results m the intracellular accumulation of secretory products frequently is not absolute. some portion of the material synthesized is released from the monensm-mhabited cells and this material frequently exhibits an abnormal type of posttranslational modification. for example, those proteoglycans from chicken embryo chondrocytes secreted m the presence of monensln are vastly undersulfated [ , , , ] . thus, membrane and secreted molecules leaving the cell following a monensin block appear to have been denied the full range of processing enzymes they would normally encounter during transit through the cell however, whether the incompletely processed molecules bypass one or more parncular lntracelhilar compartments (eg, the golgi apparatus), or whether they pass through functionally incomplete compartments, remains to be determined. during maturation of uukunieml virus m baby hamster kidney cells, monensin appeared to mhibit a terminal step of virus assembly, but not the expression of virus membrane glycoproteins g and g at the cell surface. these findings suggest that both g and g could enter a functional transport pathway in the presence of monensm that bypasses the trans golgi apparatus compartment to become expressed at the cell surface [ ] . evidence for a golgi apparatus bypass has been presented m liver where secretory hpoproteins may move directly from endoplasnuc reticulum to the cell surface without direct golgl apparatus involvement [ , ] . at concentrations of - m or higher, monensin inhibited secretion of albumin, transferrln, and vsv proteins g and x destined for delivery to the cell surface to the same extent m rat hepatoma cells [ ] . this was taken as evidence that the same vesicles were used by all four proteins m their movement from golgl apparatus to the plasma membrane however, the time required to move from er to the golgi apparatus, based on sensitivity to endoglycosidase h, differed for secretory and membrane proteins. an even more striking observation was that following the monensm block, secretory proteins accumulated in an endo-h-sensitive form, whereas, membrane proteins were already endo-h-resistant. this strongly implies that membrane and secretory proteins are not m the same compartment initially and would support the concept of peripheral input of secretory proteins into the secretory vesicles of the golgi apparatus, at least in hver [ , , ] alonso and compans [ ] provided evidence for two distract pathways of glycoprotein transport in madin-darby canine kidney (mdck) cells only one of which was blocked by monensm however, in baby hamster kidney (bhk ) cells, both influenza virus and vsv maturation were sensitive to monensin. the vsv particles were synthesized m both mdck and bhk cells, but transport to the cell surface was blocked only in the mdck cells thus, there appear to be two distinct pathways of transport of glycoproteins to the plasma membrane in mdck cells, only one of which is blocked by monensln there is no information on the nature of the alternative transport vesicle that carries mfhienza virus to the cell surface of mdck cells if, in fact, a vesicle is involved. melroy and jones [ ] reported accumulation of normally secreted a-amylase within barley aleurone layers after monensln treatment. however, only isozyme was secreted normally whereas isozymes , and were not secreted also, in the perfused rat hver, monensin treatment has less of an effect on blliary secretion than on secretion of plasma proteins [ ] . sn-nllarly, in the transport of hla-dr a-and / chains [ ] , processing of n-linked carbohydrate chains to full endo-h resistance occurs however, with the associated i-chain, processing of both o-and n-linked carbohydrate chains ~s inhibited, and carbohydrate chains remain predornlnantly endo-h susceptible. here, the processing of membrane-associated proteins that occurs despite a monensln block may reside in intercalary clsternae that constitute the golgi apparatus region recently termed medial [ a ] there is now considerable evidence for monenslnsusceptible compartments in the endocytotic pathway. transfer of product to secondary lysosomes [ ] as well as virus penetration into cultured cells [ ] are impaired by monensm. stein and co-workers [ ] have shown that monensin blocks transfernn recycling by causing internahzed hgand to accumulate in the perinuclear regmn, primarily in multivesicular bodies, of the k cells used in the study. based on studies of hrp uptake in rat fibroblasts, wilcox and co-workers [ ] suggested that inhibition of endocytic events may be the consequence of an inhibition of membrane recycling within the cell rather than a direct effect of monensin at the cell surface. maxfleld [ ] reported that ~tm monensln resulted m an mcrease in internal ph of endocytic vesicles of cultured mouse fibroblasts from to above to account for its effects on receptor-mediated endocytosis similarly, marsh and co-workers [ ] concluded that the lnhlbmon of semhki forest virus penetration into cultured cells was the result of this increase in ph of endocyuc vacuoles and lysosomes above ph , the threshold for fusion activity of viral membranes monensln has also been shown to inhibit lysosomal degradation of protein by affecting lysosomal ph [ ] and to abolish aslaloglycoprotein degradation in cultures of rat hepatocytes through a ph shift in prelysosomal endocytlc vesicles [ ] . using digttal image analysis, tyco and co-workers [ ] showed that monensin raised the ph of endocytic vesicles in cultured human hepatoma cells and caused a hgand-lndependent loss o! receptors in other studies, monensln did not prevent lnternahzatlon of s-labeled proteoglycans by rat ovarian granulosa cells although their lntracellular degradation was completely inhibited [ ] . yet, degradation pathways involving proteolysls of both dermatin and heparln sulfate and limited endoglycosldlc cleavage of heparln sulfate continued_ these findings, while consistent with an involvement of both acidic and nonacldlc compartments, show that monensm inhibition is primarily on those processes that normally occur in acidic compartments such as endosomes or lysosomes by raising their ph. sirmlarly, with isolated hepatocytes, whittaker et al [ ] found no effect of monensin on insulin internalization but, rather, an impairment of ~ts degradation once lnternahzed. rustan and co-workers [ ] suggested that monensm inhibits both endo-and exo-cytosls by a similar mechanism, namely, disruption of proton gradients their conclusions were based on studies of rat hepatocytes in which monensm inhibited both secretion of very-lowdensity hpoprotelns, and binding and degradation of aslalofetuin both secretion and receptor binding were markedly decreased after only rmn of monensin treatment although no effect on protein synthesis was observed. however, secretion was more sensitive [o monensln than endocytosis, suggesting that monensin independently intubits endocytlc and secretory functions although the mechanisms may be similar. marnell et al [ ] explained the monensin block of the cytotoxic effect of &ptheria toxin on a sirmlar basis following endocytosls of the toxin, the toxin was assumed to penetrate the membrane of the endosome and enter the cytoplasm in response to an acid environment by neutralizing the ability of endosomes to acidify their interiors, monensin, like the lysosomotroplc amines, was able to block the low ph-dependent dissociation of receptor-hgand complexes and subsequent release of ligands either to the cytoplasm (viruses and toxins) or to lysosomes (endocytosed proteins such as ldl). this in turn would prevent recycling of receptors and membrane and eventually bring endocytosls to a halt due, not necessardy to an inhibition of the uptake processes per se, but perhaps, to blockage of an internal step very similar to that believed to be blocked at the goln apparatus. also consistent with sirmlar modes of monensin mhibition in processing both endocytlc and exocytic vesicles are findings that a single mutatmn in chinese hamster ovary cells impaired both golg¢ apparatus and endosomal functions in parallel included were the monensin sensitive steps of virus and toxin penetration from endosomes into the cytoplasm and of golgl apparatus-associated maturation of smbis virus [ ] . the alterations correlated with losses of atp-dependent vacuole acidification as if the atpase of endosome and golg~ apparatus shared a common genetically regulated subunit. ono et al. [ ] studied a monensln-reslstant mutant of mouse balb/ t cells which also proved to be a poor host for either vesicular stomatltis virus or semllkl forest virus multiplication. the mutant cells resistant to monensln, bound virus normally and contained acidic compartments. however, movement of virus from the cell surface to the endosome and lysosome compartments was extremely slow. thus, the ablhty of monensln to block processing of endocytic vesicles by making prelysosomal compartments less acidic, suggests a mechanism for perturbation of endocytosls based on its ionophorlc properties the mechanism could be slmxlar to the monensm-medlated exchange of monovalent alkah tons for protons that induces, by osmotic means, the observed swelhng of golgl apparatus clsternae. clearly, monensm does not interfere with the uptake and binding of particles at the cell surface. monensm is ineffective against activities that occur at the cell surface. hedm and thyberg [ ] showed that uptake of igg prebound to the cell surface was unaffected by monensln similar findings have been made in studies of receptor-medmted endocytosls of various other ligands [ , , ] . however, monensm may secondarily affect mternahzation through depletion of monensln-sens~tive receptor sites at the cell surface. this would occur if the cell surface receptors are recycled back into the cell and then blocked in the post-golgi region by monensin so that they could not return to the cell surface [ , , ] . thus, monensin inhibition of endocytlc events seems to be at the site of transfer from endocytlc vesicles to lysosomes [ , , , ] or, in monensin-sensltive endosomes, inhibition of the dissociation of ligand-receptor complexes [ ] most animal cells show a dose-related response to monensm that falls off rapidly at monensm concentrations less than - m consequently, most studies of monensln effects use monensin concentrations of - m or higher however, a few reports indicate a cellular response at monensm concentrations less than - m for example, receptor capping by lymphocytes is stimulated by low concentrations of monensin in the range - to - m and inhibited by monensm concentrations above - m [ ] cultured adrenal chromaffin [ ] and heart [ ] cells also are stimulated by low concentrations of monensln. though these effects occur at concentrations below the threshold effects for most golgl apparatus responses, they still presumably result from increased levels of cytoplasmic sodium due primarily to the ~onophore insertion at the plasma membrane fig (a) outer cap cells from a control (nontreated) maize root txp preserved by freeze-subsututlon [ ] . the form of dlctyosome (d) was normal and slrmlar to that following glutaraldehyde/osmlum tetroxade fixation (b) same except that root tip was treated for h with -s m monen~m before being preserved by freeze-substitution. the trans clsternae and/or secretory vesicles (arrowheads) were swollen, and mltochondna (m) were condensed (compare with mltochondna of a) w, cell wall, v, central vacuole swelling of golgl apparatus clsternae occurs in a wide range of plant and animal cells (see subsection iii-b ) and may be a universal response to monensln poisoning. however, in plants, and to a lesser extent animals, swelhng is influenced by the fixative used to preserve the cells specifically, morphological evidence of swelhng is less (in animal golgl apparatus) or nonexistent (in plant golgl apparatus) when the tissues are fixed in potassium permanganate as compared to fixation in ghitaraldehyde/osmium tetroxade [ ] . these effects could be due either to fixation artifacts or to differences between plant and animal golgl apparatus (e.g, ref_ ) this problem was evaluated by comparing the images of golgl apparatus preserved by various chemical fixatives as well as preservation by freezing and low temperature substitution in acetone and osmium tetroxade. presumably, the image following freeze sub-stitutlon would reflect the true ultrastructure more closely than the image following chemical fixation. the results of freeze substitution in both animal cells [ ] and maize root (figs a and b) , show swollen golgi apparatus c~sternae following monensln exposure in a pattern similar to that observed after glutaraldehyde/ osmium tetroxide fixation [ ] . however, using videoenhanced light microscopy and cultured bovine mammary epithelial cells, a marked swelling response to monensln was observed only after the addition of glutaraldehyde fixative to the monensln-incubated cells (morr , d j., mollenhauer, h h., spring, h., trendlenberg, m, morr , d m. and kartenbeck, j, unpubhshed data)_ thus, whether monensin-lnduced swelhng occurs m vlvo or is in response to aldehyde fixations remains an important questiion. no swelling was observed in golgl apparatus of protoplasts of carrot cells freshly prepared by digestion of cell walls when exposed to monensin even though such a response was obtained in the same cultures prior to wall dissolution [ ] . similarly, in thin slices of hver incubated in monensin, the golgl apparatus adjacent to cut edges of tissue slices showed a different swelling response than golgl apparatus adjacent to the uncut natural surfaces of the lobe (unpublished data) adjacent to a cut edge, fewer cisternae swelled and those that swelled were only in the most trans positions the basis for such differences is unknown but might, for example, indicate changes in cisternal proton pumping ability, or monensln uptake, in response to changes in the physiological state of the golgi apparatus brought about by the tissue excision the mechanism by which monensin interacts with coccldla and rumen rmcroflora is well documented [ , ] however, the interaction between monensm and the tissue of the host animal is less well understood even though the chnlcal manifestations of monensln poisoning are well known_ most striking are differences between the in wtro monensln effect ohserved in cultured plant and animal cells, and plants, and the in vwo effects observed m animals when used at recommended levels, either as a coccidlostat for poultry [ , ] or for cattle [ , , ] , monensin seldom causes poisoning nonetheless, misuse of the product, usually from improperly mixed or improperly distributed feed, may cause toxlcosis and death [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . horses, ponies and other equine species are particularly sensitive to monensln poisoning [ , . ] . the median lethal dosage (ldso) for the horse js - mg monensin per kg body weight compared to - mg per kg body weight for cattle and mg per kg body weight for poultry [ , , ] in mammals, the physical signs of monensln toxlcosls commonly include anorexia, diarrhea, depression, sweating, ataxm, palpitations of the heart, and sudden death following exercise [ , , , , [ ] [ ] [ ] [ ] stiffness of hindquarters and swollen gluteus muscles [ , ] , elevated pulse rate [ , , ] , and ecg abnormalities [ , , , ] also have been reported in fowl, the outstanding signs of monensln toxlcosls are drowsiness, excessive thirst, anorexia, depression and paralysis [ , , ] marked congestion in a variety of organs also has been noted [ ] severely poisoned birds may die in sternal recombency [ ] . routine clinical tests on serum from horses poisoned by monensm may show abnormally high values for blood urea nitrogen, total billrubin, creatlne kinase, lactate dehydrogenase and aspartate armnotransferase [ , , ] however, chnlcal manifestations are often variable makang interpretation and diagnosis difficult. moreover, serum levels of sodium, potassium, chlorine, calcium, phosphorus, and urea may remain at near-normal levels following monensin treatment [ ] _ in poisoned mammals and fowl, generalized congestion, hemorrhage, and macroscopic injury to striated muscle [ , , , ] , spleen [ , ] , lung [ ] , liver [ ] , and kidney [ , , , , ] have been noted. the most consistent macroscopic observation in ponies, cattle, pigs and fowl, has been cardiac myocyte degeneration and vacuollzatlon [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] in animals poisoned with relatively high doses of monensin, an initial condensation of heart mltochondria is often seen (fig ) however, with longer exposure times, some mitochondria swell and vacuolate with an almost total loss of matnx substance (fig ) _ intracnstae spaces generally remain unchanged with swelling being restricted to the mitochondrial matrix this is followed by loss or dilution of matnx components and a reduction in size of cnstae so that the rmtochondna appear as empty vacuoles with residual cristae typically, only some mitochondrla in a particular fiber become swollen and appear as vacuoles, and fig section of left ventricle from a rat treated wath a single mtraperitoneal mjecaon of mg monensm per kg body weight most rmtochondna (m) were either condensed or swollen in swollen rmtochondna, cnstae (arrowheads) were greatly reduced in extent but, otherwise, were of approxamately normal ttuckness thas dosage level (e_g, mg monensln per kg body wexght) is quite tugh for rats and often resulted m significant generahzed damage to the muscle fiber as dlustrated in the lower part of the rmcrograph however, rmtochondnal swelhng (but not condensauon) may occur as well at lower monensln dosages, even when no fiber damage can be identified ultrastructurally swelling and vacuohzatlon of mltochondna are progresslve with time whereas rmtochondnal condensation was dose-dependent and often occurred rapidly , a few were normal (arrowhead), but most were rmldly condensed swollen tmtochondna always appeared randomly distributed through a fiber although significant differences m rmtochondnal swelling were often noted between fibers (e g, see fig_ ) these are randomly distributed throughout the fiber (fig. ) . we have identified early stages of mitochondrial vacuolation and swelling in both the rat and pony (unpublished data), but these mitochondria were seldom plentiful, suggesting that transition to the vacuolated state, once started, was relatively rapid non-swollen mitochondria may appear condensed, especially after exposure to high levels of monensin ( fig. and ). granulation of mitochondna ( fig. ) was observed only occasionally and those granules that were present appeared similar to the tncalclum phosphate granules often associated with normal mitochondria [ , ] . however, whether monensln-lnduced granules contain ca ~ has not been determined. granules like these have been observed in lschermc and reperfused hearts and are considered indicative of calcium overload [ ] [ ] [ ] [ ] _ ca + overload may be a potential cause of cell death or cellular dysfunction in ischerma [ , [ ] [ ] [ ] [ ] [ ] [ ] although the effects are reversible if the cell is not too severely damaged [ , ] exaggerated matochondrial swelling has also been observed immediately following reperfuslon of hearts rendered lschemlc by occluding blood flow for a few minutes [ , ] whether excess mitochondrial ca + occurs as a result of monensin is not clear under appropriate condiuons, either monensin or na + in excess can block ca + accumulation and promote its release from both mitochondrla and sarcoplasrmc reticulum over a broad range of monensin and na + concentrations [ , , ] it is probable that the release of ca + by monensln can occur as the result of an increase in cytoplasmac na + from a monensin shuttle although monensin (at relatwely high concentrations) has been shown to release ca + directly m a na+-lndependent manner from cardiac sarcoplasmic retlculum [ ] in all probability, however, monensin is not present in myocytes at high concentrations since swelling of golgl apparatus cxsternae (a characteristic response to monensin) ~s never observed in these cells. the picture is further comphcated by the fact that ca + release patterns may vary according to type of cell (e g., white vs red muscle cells [ ] ) as well as the availability of extracellular ca + [ ] . although not related to this report, it may be of some interest to note that both mitochondrlal condensation and granulation are much more intense in x- a-treated animals (and cultured cells as well) than in comparable animals (or cells) treated with monensin [ ] _ the percentage of affected mltochondrla vaned markedly between muscle types and species of animal. analyses of striated muscle m ponies showed that there was a much greater ( -times) hkehhood of finding altered mltochondrla in heart tissues than m diaphragm or appendicular muscle [ ] a similar relationship existed in rats except that most swollen mitochondria were m the &aphragm [ ] . some antibioucs (e g, tiamuhn and avoparcm) may act synergistically with monensln to induce shifts in the distribution of swollen mltochondria [ ] and other cellular damage. differences in distribution patterns of swollen mltochondrla also were observed between red and white muscle fibers of the rat diaphragm [ ] . red and white fibers were dffferentmted structurally by size, mltochondrlal content, and z-band configuration [ , [ ] [ ] [ ] swollen matochondrm were present in all fiber types when the number of affected rmtochondna was small. however, when large numbers of swollen matochondrm were present, the dxsmbutlon pattern was heavily skewed toward the white muscle fibers (fig_ ). a differential effect of monensln was also noted by van vleet and co-workers [ ] who observed in swine severe damage m the diaphragm, vastus lateralls, sem~tendlnosus, triceps and intercostal muscles: moderate damage m longissimus lumborum muscle, and little (or minimal) damage in tongue. damage was greatest m muscles containing a high proportion of type fibers these distribution patterns, coupled with the characteristic form of the degenerating rmtochondrla, are not common observations and, therefore, may be strong indtcatots of monensin poisoning in animals_ swollen rmtochondrla have not been observed in any of the nonmuscle cells of the heart, diaphragm, or appendicular tissues or in hver, adrenal, or kidney cells thus, monensm adrmnistered to mammals in vlvo tends to induce rmtochondrial changes only m selected tissues and/or types of muscle fibers the mechanisms for these rmtochondrlal changes and reasons for the specificity are not known. these problems are compounded by the fact that matochondnal condensation, but not subsequent swelling and degeneration, occurs in cells exposed topically to monensin. whale mitochondnal aberrations are the primary morpholog~c indicators of monensin poisomng in striated muscle, other aspects of muscle ultrastructure may show deterioration [ ] or may remain relatively normal even with gross mltochondnal damage [ ] . generalized fiber and cell degeneration may occur following monensin poisoning [ , , ] . the extent of monensin-induced injuries appears to be time and dosage dependent. if death occurs shortly after monensin exposure, there may be httle or no recognizable evidence of pathologtcal change, at least in liver, kidney, and striated muscle. generalized necrosis appears to occur most often when monensln is administered over long periods of time. even with single doses of monensin, structural aberrations in striated muscle develop progressively over several days and then regress if the animal survives the imtial insult (unpublished data). we have not observed permanent injury in either striated muscle or liver of monensin-treated rats although such effects have been noted in other animals [ ] . many of the effects on striated muscle attributed to monensxn occur also with other lonophores irrespective of their ion specxficlties [ , ] this implies, again, that na + is not the direct cause of muscle perturbation but, rather, that the iomc imbalance resulting from the intracellular influx of na + triggers other cellular responses that lead to the observed perturbations. monensin is known to both inhibit and promote ca + accumulation in myocytes depending on the absence or availability, respectively, of external ca + stores [ , ] ; alter na + gradient-dependent ca + transfer through the basolateral plasma membranes of rat small intestine [ ] ; increase myocardial calcium activity [ ] , inhibit ca + accumulation by cardiac macrosomes or cause release of accumulated ca + stores [ ] ; and release ca + from macrosomes [ , ] . digitalis and other cardiac glycosides (which increase myocardial contractility) appear to act by altering intracellular na + concentration through inhibition of membrane-bound na +-, k+-actlvated adenosine triphosphatase which secondarily results in an increase in mtracellular ca + [see ] . calcium ionophores such as lasalocld and a have been suggested as potential probes for studying the effects of calcium imbalance on myocardial function [ ] . in retrospect, monensin affects the myocardlum in much the same way as do the calcium lonophores and, in some instances, to an even greater extent [ ] . these observations suggest that the xonotropic effects of monensln may be partially indirect; e g., through release of histamine and endogenous amines [ ] , or stimulation of synthesis and/or release of prostaglandln [ , , ] . thus, na + balance plays an indirect but critical role in modulating myocardial function however, a calcium-independent catecholamine depleting action of monensin in cultured rat pheochromocytoma cells [ ] and m bovine adrenal medullary cells and chromaffln granules [ ] suggests that monensln may also play a direct role in altering cellular function. similarly, sutko and co-wor~kers [ ] showed that both monensxn and mgencin produce their effects in guinea pig atria by direct action as well as by releasing catecholamines from tissue stores. differences in subcellular responses to monensin, between the whole ammal and isolated cells or organs, have been noted by us as well as by others [ ] . thus, swelling of golgl apparatus clsternae observed an cultured cells, tissue shces, and plant roots and stems, is not an aberration characteristic of the cells of animals poisoned by monensln. lack of golga apparatus welhng in animals infers that cells from monensin-poisoned ammals are bathed in body fluids containing less than - m monensln which is approximately the minimum effective dose of monensin that will cause sweulng of golgt apparatus clsternae in cultured animal cells. alternatively, lack of vacuolated and/or swollen mitochondna in cultured mammahan cells and plants generally imphes that the vacuolated and/or wollen rnttochondria observed in striated muscle from monensinpoisoned animals are secondary effects of monensin poisoning, perhaps caused by a metabohte of monensin [ , ] . alternatively, monensln could affect the synthesis and/or transport of humoral agents (e.g., catacholanunes) which, in turn, would alter muscle homeostasis and lead to the rmtochondnal aberrations observed in striated muscle. for plants, at concentrations of -s m, monensin was shown to inhibit gernunatlon and growth of ryegrass seedlings [ ] . the effects were primarily associated with poor root development and significant reduction of root mass as compared to controls leaf emergence and leaf mass was only slightly affected. at - m monensln, roots often did not emerge from the seed during germination and root mass of seedlings was often near zero. under these same conditions, shoot mass was reduced about % as compared to controls. monensin, a monovalent ion-selective ionophore, facilitates the transmembrane exchange of prinopally sodium ions for protons. the outer surface of the lonophore-ion complex as composed largely of nonpolar hydrocarbon, which tmparts a high solubility to the complexes in nonpolar solvents. in biological systems, these complexes are freely soluble in the lipid components of membranes and, presumably, diffuse or shuttle through the membranes from one aqueous membrane interface to the other. the net effect for monensin is a trans-membrane exchange of sodium ions for protons. however, the interaction of an ionophore with biologl-cal membranes, and its ionophorlc expression, is highly dependent on the blochemcial configuration of the membrane itself one apparent consequence of this exchange is the neutralization of acidic lntracellular compartments such as the trans golgi apparatus clsternae and associated elements, lysosomes, and certain endosomes. this is accompanied by a disruption of trans golgi apparatus clsternae and of lysosome and acidic endosome function. at the same time, golgl apparatus clsternae appear to swell, presumably due to osmotic uptake of water resulting from the inward movement of ions monensin effects on golga apparatus are observed in cells from a wide range of plant and animal species the action of monensin is most often exerted on the trans half of the stacked clsternae, often near the point of exit of secretory vesicles at the trans face of the stacked cisternae, or, especially at low monensln concentrations or short exposure times, near the rmddle of the stacked cisternae. the effects of monensln are quite rapid in both animal and plant cells; l.e, changes in golgl apparatus may be observed after only - min of exposure it is implicit in these observations that the uptake of osmotically active cations is accompanied by a concornltant efflux of h + and that a net influx of protons would be required to sustain the ionic exchange long enough to account for the swelling of clsternae observed in electron rmcrographs. in the golgi apparatus, late processing events such as terrmnal glycosylation and proteolytlc cleavages are most susceptible to inhibition by monensln. yet, many incompletely processed molecules may still be secreted via yet poorly understood mechanisms that appear to bypass the golgi apparatus in endocytosls, monensln does not prevent internalization however, intracellular degradation of internalized ligands may be prevented. it is becormng clear that endocytosls involves both acidic and non-acidic compartments and that monensln inhibits those processes that normally occur in acidic compartments. thus, monensln, which is capable of collapsing na + and h + gradients, has gamed wide-spread acceptance as a tool for studying golgi apparatus function and for locahzlng and identifying the molecular pathways of subcellular vesicular traffic involving acid compartments. among its advantages are the low concentrations at which inhibitions are produced ( - . /~m), a minimum of troublesome side effects (e g., little or no change of protein synthesis or atp levels) and a reversible action. because the affinity of monensin for na + is ten times that for k +, its nearest competitor, monensxn mediates primarily a na+-h + exchange monensin has little tendency to bind calcium. not only is monensin of importance as an experimental tool, it is of great commercial value as a coccidiostat for poultry and to promote more efficient utilization of feed m cattle the mechanisms by which monensln interact with cocctdla and rumen rmcroflora to achieve these benefits are reasonably well documented. however, the interactions between monensm and the tissues of the host animal are not well understood although the severe toxicological manifestations of monensln poisoning are well known equine species are particularly susceptible to monensm poisoning, and a common effect of monensln poisoning is vacuolizatton and/or swelling of rmtochondna m striated muscle other pathological injuries to striated muscle, spleen, lung, hver and kidney also have been noted a con-sistent observation is cardiac myocyte degeneration as well as vacuohzation differences m cellular response resulting from exposure to monensln (t e, golgi apparatus swelling in cultured cells, isolated tissues, and plants vs. mltochondrial swelling m animals fed monensln) suggest that myocardial damage is due either to a monensln metabohte or is a secondary response to some other derivation however, as pointed out by bergen and bates [ ] , the underlying mode of action of lonophores is on transmembrane ton fluxes which dissipate cation and proton gradients consequently, some or all of the observed monensin effects m vlvo m animals could be secondary phenomena caused by disruption of normal membrane physiology resulting from altered ion fluxes. the role of membranes in metabohc regulation polyether antibiotics -naturally occurnng acid ionophores polyether antibiotics -naturally occurnng acid ionopbores polyether antlbloucs -naturally occurnng acid ionophores polyether antibiotics -naturally occumng acid ionophores (west-icy polyether antibiotics -naturally occurring acid ionophores the role of membanes m metabohc regulalaon polyether antlbtotlcs -naturally occurnng acid ionophores comp blochem phys- o protoplasma clba found symp ongm and continuity of cell organelles (relnert endocytosls (paston, i and wdhngham proc. natl acad th ann proc equine medacane and surgery current vetennary therapy food ammal practice (howard an atlas of fine structure calcium antagomsts and cardiovascular disease key: cord- - gzc authors: wang, jiajun; lapinski, nicole; zhang, xiaohui; jagota, anand title: adhesive contact between cylindrical (ebola) and spherical (sars-cov- ) viral particles and a cell membrane date: - - journal: mech soft mater doi: . /s - - - sha: doc_id: cord_uid: gzc a critical event during the process of cell infection by a viral particle is attachment, which is driven by adhesive interactions and resisted by bending and tension. the biophysics of this process has been studied extensively, but the additional role of externally applied force or displacement has generally been neglected. in this work, we study the adhesive force-displacement response of viral particles against a cell membrane. we have built two models: one in which the viral particle is cylindrical (say, representative of a filamentous virus such as ebola) and another in which it is spherical (such as sars-cov- and zika). our interest is in initial adhesion, in which case deformations are small, and the mathematical model for the system can be simplified considerably. the parameters that characterize the process combine into two dimensionless groups that represent normalized membrane bending stiffness and tension. in the limit where bending dominates, for sufficiently large values of normalized bending stiffness, there is no adhesion between viral particles and the cell membrane without applied force. (the zero external force contact width and pull-off force are both zero.) for large values of normalized membrane tension, the adhesion between virus and cell membrane is weak but stable. (the contact width at zero external force has a small value.) our results for pull-off force and zero force contact width help to quantify conditions that could aid the development of therapies based on denying the virus entry into the cell by blocking its initial adhesion. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. viral infection is one of the major public health issues in the world. from one of the most lethal viruses, ebola virus, to the novel coronavirus (sars-cov- ) causing the present pandemic, countless people have died and been sickened from virus infection and complications. moreover, this is an ongoing concern because there will always be new viruses or mutated ones. it is therefore important to develop an understanding of how virus particles infect our body's cells. one of the vital moments during infection is the internalization of the virus particle, often by hijacking the normal physiological adhesive function of the receptors on the surface of the cell membrane. uptake of most virus particles into a host cell is mediated by the receptor-dependent adhesive interaction between cell-surface and virus-surface molecules [ ] [ ] [ ] . this initiates endocytosis, which comprises physiochemical interactions between the virus particle and cell membrane and several kinetic processes. during this process, the adhesive forces tend to bend and pull against tension to wrap around the viral particle [ , ] . because blocking this adhesion is a prime therapeutic target, it is important to electronic supplementary material the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users. understand the mechanics of initial virus adhesion to the cell. in particular, we aim to connect adhesion properties (measured, for example, by single-molecule force spectroscopy) and the behavior of the whole virus-cell membrane interaction. the mechanics and biophysics of this problem have been approached in a number of ways ranging from all-atom molecular dynamics simulations that contain atomistic structural detail to continuum models that contain only a few parameters that characterize virus, membrane, and adhesive properties [ , ] . the former provides tremendous descriptive and predictive power but are, by construction, specific to a particular system. the latter allow investigation of more general principles at the cost of specificity. there is a significant literature on continuum models of adhesion between particles (biological or inorganic) and membranes. (see reference [ ] for a review.) shanahan developed a model for compliant particle adhesion onto a rigid surface [ ] . long et al. modeled aspects of the fusion of synaptic vesicles with the plasma membrane, which is related to the problem of soft vesicles attaching onto a rigid surface [ ] . a similar model about kinetics of virus binding and fusion was developed by chou [ ] . zhang et al. studied nanoparticle cellular endocytosis by considering parameters such as ligand density [ ] . an important subclass of models, to which our work belongs, shares the feature that the total free energy is written as the sum of three contributions: from bending, tension, and adhesion. several of the models are based on the theory that the marginal reduction of the free energy during the adhesion process balances the marginal cost of bending the membrane [ ] [ ] [ ] . several studies consider the process of virus particle wrapping and engulfment by the cell membrane [ , ] . seifert and lipowsky ( ) studied the adhesion of vesicles and found that there is a transition between a bound and free state based on competition between bending and adhesion energies [ , ] . other studies have considered the combined effects of bending, tension, and adhesion, as the particle is wrapped partially to fully, sometimes representing the membrane as a continuum plate [ , , ] . although this problem of adhesion has been well-developed, much less attention has been paid to the contact mechanics of viruses onto a flexible membrane. this is of interest because such analyses can be used to interpret in detail atomic force microscopy (afm) force-spectroscopy measurements of force-deflection response used to characterize the adhesion process [ ] , particularly to interpret the force-spectroscopy studies on virus-host cell interactions [ ] [ ] [ ] . it is also of interest because virus attachment often occurs under external load, which is accounted for in a contact mechanics model but not in most of the literature cited above. in this work, we create a continuum model for the small-deflection adhesive contact mechanics of virus particle attachment onto the host cell membrane in terms of the principal biophysical properties of the virus, membrane, and their interaction. we seek equilibrium states by minimizing the total free energy of the virus-membrane system. in particular, we seek to describe the force-deflection and contact area-deflection relationships. these results also help to retrieve conditions for lack of adhesion, pull-off force, and contact area between the virus particle and cell membrane. the common physical basis for the model is to assume that, for given applied deflection, equilibrium states are achieved by a balance of adhesion energy of cell surface receptors, which drives adhesion, against the cost of the associated deformation, represented by bending and tension energies of the cell membrane [ , ] . we show that the solution is governed by two dimensionless material parameters that can be identified as a normalized bending stiffness and tension. (these parameters have previously been identified) [ , ] . in particular, quantities of interest such as the equilibrium contact size (if any) or force required to remove the virus from the membrane depend (in dimensionless form) only on these two parameters. the analysis is carried out for d cylindrical and d axisymmetric virus models representing, for example, a filamentous virus like ebola or a nominally spherical virus like sars-cov- . in the following sections, we first outline how the problem is posed for both cylindrical and axisymmetric models. in later sections, we present the results of the model. the supporting information contains details of derivations. we now describe in outline the continuum models for adhesive contact between the virus and cell membrane, driven by adhesion and external displacement or force, and resisted by tension and elastic bending. figure sketches the geometry of both cylindrical (i.e., d) and spherical (i.e., axisymmetric) models. aspects of the d model have been previously studied by mkrtchyan et al. [ ] , but without external force or displacement. for both the d and axisymmetric cases, we consider the case where the viral particles are stiff compared to the cell membrane to which they attach. all the parameters and non-dimensional parameters used for the models are summarized in table . the cell membrane, represented in fig. by the blue line, can be split into two parts, one that contacts the virus particle and a second one that is out-of-contact. the virus particles in the d and axisymmetric models are assumed to be cylindrical and spherical, respectively. because the virus is represented by a rigid cylinder, the region of the membrane in contact has a circular cross-section in d, ignoring end effects because the radius of the virus is quite small compared to its length [ ] . for example, the ebola virus has diameter of about nm, whereas its length can reach - μm. in the axisymmetric model, the virus particle is assumed to be spherical, and its region of contact with the membrane is a spherical cap. for example, the novel coronavirus (sars-cov- ) is nominally spherical with a diameter of around nm. since the diameters of both types of viruses are much smaller in size than the host cell but large compared to the thickness of the membrane (~ nm), we assume that the membrane of the host cell is originally flat and deforms when in contact with the virus (blue line, fig. ). the width of contact section in the d model is a, and the host cell membrane is supported some distance l = a + b away from the center of the virus attachment region. for nominally spherical viruses (such as hiv, zika virus and sars-cov- ), the contact region is circular with radius, a. the membrane of the host cell is supported some radial distance l away from the axis of symmetry. as in the d model, the membrane is assumed to be flat in its stress-free state; it deforms when in contact with the virus particle. for both d and axisymmetric models, the interaction between the viral particle and the cell membrane is driven by adhesive interactions and externally applied force or displacement. attachment of the viral particle to the cell membrane is resisted by energy required to bend the cell membrane and by the tension it is under. we presume that the tension is set by some effect such as osmotic pressure and is not constitutively linked to the deformation, i.e., it holds a constant isotropic value. in our models, the parameters that govern the adhesive contact mechanics are (more in table ) bending rigid κ, tension σ, adhesion free energy per receptor β, binding receptor density ρ, and the radius of the virus, r. these parameters are associated with contributions to the energy of the system: bending, tension, and adhesion (u bending , u tension , and u adhesion , respectively [ , ] . there is additionally the potential of the external force or an applied external displacement. in the work presented here, we carry out the contact mechanics calculation under displacement control, so it enters as a parameter. thus, total energy is the bending and tension energies are each the sums of contributions from two regions, region i where the membrane is in contact with the virus and region ii where it is free of applied lateral loads. we consider special limiting cases in which either tension or bending dominates over the other. calculating the energies in eq. ( ) requires knowledge of the contact region and the shape of the membrane outside the contact region. this is governed by the helfrich hamiltonian [ , ] which generally results in nonlinear euler-lagrange governing equations [ , , , ] . however, since in this work we are interested primarily in conditions close to the no-adhesion case, we can take advantage of a major simplification in the shape equation for small deformations. we can additionally neglect the fig. the geometry of the two models. (upper left) sketch of a stiff cylindrical (i.e., d) virus particle attaching onto a flexible membrane; (upper right) sketch of a stiff spherical virus particle attaching onto a flexible axisymmetric membrane; (lower left) mechanical model of d virus particle attachment driven by adhesion and external force or displacement and resisted by membrane bending and tension; (lower right) mechanical model of spherical virus particle attachment driven by adhesion and external force or displacement and resisted by membrane bending and tension coefficient of gaussian curvature in the helfrich hamiltonian because the processes we study involve no change in topology. with these two simplifications, the governing equation simplifies to a linear differential equation [ ] : typical values are tension σ~μn/m and bending rigidity κ~ - k b t [ , , ] , where k b is boltzmann's constant. in the following, we will use specific forms of eq. ( a) for plane and axisymmetric problems. the ratio ffiffiffiffiffiffiffiffi κ=σ p represents a length scale. if the size of the virus is large compared to this length scale, then tension dominates. conversely, if the virus is small compared to this length, then bending does. for the viral particles we wish to consider, using typical values given in table , we estimate this ratio to be about nm, which means that both tension and bending energies can be important. a normalized version of eq. ( a) is where α ¼ κ ρβr is a normalized bending stiffness, γ ¼ σ ρβ is normalized tension, and ρβ, as the product of number density of surface ligand-receptor pairs and the adhesion energy of one such pair, is the specific (per unit area) adhesion energy of the interface. thus, in normalized form, this problem is governed by two dimensionless material parameters, α and γ. versions of these two parameters are well known in the literature [ , , ] . in the d model, the membrane in region i conforms to the cylindrical virus particle so that the normalized deflection w(x) in region i is governed by the circular shape of the virus cross-section. the deflection w(x) in region ii is governed by the d version of eq. ( a) [ ] κ normalizing eq. ( ) as defined in table , we get bending stiffness normalized by adhesion α ¼ κ to better understand the behavior, we focus most of our attention on the two limits in which either tension or bending dominate. (we can ignore bending if α γ ≪ ; we can ignore tension if α γ ≫ .) in the axisymmetric model, the deflection w(r) in region i is constrained by contact of the membrane to the surface of the viral particle. the problem is axisymmetric, and so, deflection w(r) is a function of radial position only. in region ii, it is governed by the differential equation [ ] : where r is the radial distance from the lower pole. again, we focus on the two limits in which either tension or bending dominate. also, for the majority of the discussion in the remainder of this manuscript when we refer, for example, to tension, we mean normalized tension, since the discussion is almost exclusively of solutions to the normalized dimensionless governing equations and parameters. in normalized variables, the state of the system is described by five dimensionless variables (under displacement control): δ; a; l; α; γ, i.e., the normalized displacement of the virus, contact width/radius, size of the membrane patch, normalized bending stiffness, and tension. we find that the effect of l is weak, and we hold it fixed in this work at l ¼ , thus reducing to four the number of dimensionless variables. in region i, the deflection is specified by the fixed shape of the virus, and δ; a. in region ii, the deflection of the membrane is given by solving eqs. ( ) or ( ) subject to clamped boundary conditions where the membrane is held fixed and continuity and smoothness of deflection at the intersection between the two regions. for example, in the axisymmetric spherical virus case where bending dominates, the deflection in region ii is given by the solution of eq. ( ) and is where the constants c , c , c , c are obtained by the boundary and continuity conditions (see si for details). with the solution for deflection known, the various contributions to energy can be computed in normalized form. again, for the same case, where the first term represents adhesion energy (negative in value), the second term the bending energy inside the contact region, and the third term the bending energy in region ii. in general, the total energy associated with a particular configuration of the system can be written as we now impose two equilibrium conditions. the first enforces the condition that for other fixed parameters, the contact radius adjusts to satisfy the condition that when this condition can be satisfied, it provides a solution a * δ; α; γ À Á , eliminating the explicit dependence of total energy on contact width so that u total ¼ u total δ; α; γ À Á . (this condition can sometimes be satisfied exactly; on other occasions, it is computed numerically.) next, we apply the condition that the force is conjugate to the applied deflection so that in this way, for all the different conditions studied, we obtain force-deflection f δ; α; γ À Á and contact width-deflection a δ; α; γ À Á relations. a special point in the force-deflection trace is the maximum or pull-off force, f max α; γ ð Þ. similarly, an important point in the contact width-deflection trace is contact width corresponding to zero force, a α; γ ð Þ. both these quantities depend only on the two materials parameters. for the sake of brevity, those results are presented in si. the discussion just presented to obtain the solution in each of the cases studied is captured by the algorithm described below: we first present results for the cylindrical virus (fig. ). figure a and b show results in the bending-dominated limit and fig. c , d the tension-dominated limit. the tension-dominated limit can also be solved exactly without the small-deflection assumption. for consistency, in the main text, we present results for small deflections only. the exact result for this specific case is provided in supporting information s . . positive values of force represent tension. in the bending-dominated limit, the force-deflection relation is approximately (but not exactly) linear. its most interesting feature is a maximum in the force (for α < ), which corresponds (fig. b) to a reduction in contact width down to . with increasing bending stiffness α, the peak force reduces. at the critical value of α = , indicated by the dashed vertical lines, the pull-off force reduces down to . that is, there is no adhesion between the virus and cell membrane in the absence of external force. this result is consistent with conclusions obtained by models based only on the balance of bending and adhesion. for higher values of bending stiffness, the contact reduces to while still under compressive load. subsequently, the force-deflection behavior follows that of a cylinder in line contact with the membrane. of course, in reality, true line contact is not possible due to the finite thickness of the membrane. figure c shows the force-displacement results in the tension-dominated limit for a range of γ. the force-deflection response is nearly linear again, with slope that increases with increasing tension. interestingly, the force for all values of γ is identical at a positive (tensile) value for zero indentation. as a consequence, indentation depth at zero force is negative and increases with decreasing γ. the contact radius as a function of indentation depth is shown in fig. d . unlike the bending-dominated case, even for large γ, the contact width is quite substantial for considerable tensile extension. this is unlike the bending-dominated case in which there is no force-free adhesion if bending stiffness exceeds a critical value. this means that for the tension-dominated limit of the d model, there is not a critical value at which adhesion is blocked-see also fig. s in supporting information. figure shows results for the spherical virus adhesion. figures a and b show the force-deflection and contact radiusdeflection results in the bending-dominated limit. like in the d model, for α > , the contact reduces to while deflection is still negative. for this range of bending stiffness, contact radius at zero force is as is the pull-off force (see si). figure c shows force-deflection results for the tension-dominated limit. the results here are strikingly different from the d model in that the force jumps to , corresponding to a jump to in the normalized contact radius, fig. d . the pull-off force reduces with increasing tension, but only mildly so. we developed and studied adhesive contact between a stiff viral particle and a cell membrane. our model is based on a continuum description: minimization of energy that is composed of contributions from bending, tension, adhesion, and external force or deflection. we considered cylindrical (filamentous) and spherical viruses, representative of two typical viral shapes such as ebola and sars-cov- . the principal results of the analyses are normalized force-deflection and contact width/radius versus deflection as a function of two dimensionless materials parameters, α and γ, denoting normalized bending stiffness and tension, respectively. we paid attention to limits in which bending or tension dominate the other. in both the bending-dominated cases, a striking result is that for sufficiently stiff membranes, there is zero pull-off force and contact radius at zero force also vanishes. for larger stiffness, contact vanishes even under compressive indentation. the tensiondominated case is qualitatively different for the d and axisymmetric shapes. in the tension-dominated limit of the cylindrical model, the contact width a decreases very slowly with increasing deflection. in the axisymmetric geometry, in contrast, there is a welldefined pull-off force that corresponds to contact shrinking to . in our model, force is applied for two reasons, (a) because under physiological conditions, viral particles often adhere to endothelial and epithelial cells where they are subjected to fluid forces. although these forces are generally in shear with respect to the contact, for simplicity, we chose to apply normal forces as that preserves the symmetry of the problem. (b) one of the applications of our model is to help interpret nanoindentation experiments in which a small functionalized bead is pressed into contact with a cell surface and retracted. we believe that the force-displacement results can be related directly to the atomic force microscopy (afm) force-displacement measurements [ ] [ ] . our continuum model is a simple representation, attempting to capture many important details in only a few physical parameters. however, our model is suitable for application to interpretation of nanoindentation and force-spectroscopy experiments. it also advances understanding of the biomechanics of filamentous and spherical virus-adhesive contact mechanics to cell membranes. the biological context is, of course, more complicated. for example, in both the sars-cov- and ebola cases, adhesive receptors are elongated structures: the so-called spike [ ] and tim [ ] receptors, respectively. the continuum approach we have used represents these by an adhesion energy density. in so doing, important features, such as their discrete nature, can no longer be addressed [ , ] . also, receptors have often to protrude through a glycocalyx layer in order to overcome electrostatic repulsion to adhere, and all these details are lumped into the continuum parameters of our model. in our model, we chose to support the membrane at some distance from the contact axis. this represents a characteristic distance between points where the cytoskeleton attaches to the membrane. another way to account for the cytoskeleton would be to represent it by a foundation. a simple way would be to use a winkler foundation in which case the governing equation for the membrane shape (eq. ) would change to : ð a; bÞ this introduces a new dimensionless parameter, ϕ, with associated characteristic length: very approximately, the distance in our work can be thought of as representing this length. adhesion and fusion efficiencies of human immunodeficiency virus type (hiv- ) surface proteins multiple receptors involved in human rhinovirus attachment to live cells influenza virus binds its host cell using multiple dynamic interactions how does a virus bud? physical principles of nanoparticle cellular endocytosis the computational route from bilayer membranes to vesicle fusion fluid lipid membranes: from differential geometry to curvature stresses wrapping of nanoparticles by membranes adhesion of a liquid-filled spherical membrane adhesion energy can regulate vesicle fusion and stabilize partially fused states stochastic entry of enveloped viruses: fusion versus endocytosis adhesion of vesicles adhesion of vesicles and membranes elastic deformation of a fluid membrane upon colloid binding adhesion and wrapping in colloid− vesicle complexes kinetics of particle wrapping by a vesicle theory of plates and shells life at the nanoscale -atomic force microscopy of live cells biomechanical characterization of tim protein-mediated ebola virus-host cell adhesion entropy-driven tension and bending elasticity in condensed-fluid membranes adhesion of cylindrical colloids to the surface of a membrane biomechanical characterization of tim protein-mediated ebola virus-host cell adhesion elastic properties of lipid bilayers: theory and possible experiments shape transformations of vesicles: phase diagram for spontaneous-curvature and bilayer-coupling models frequency spectrum of the flicker phenomenon in erythrocytes cell surface area regulation and membrane tension membrane tension and membrane fusion sars-cov- infects t lymphocytes through its spike protein-mediated membrane fusion characterizing functional domains for tim-mediated enveloped virus entry detailed mechanics of membrane-membrane adhesion and separation. i. continuum of molecular cross-bridges detailed mechanics of membrane-membrane adhesion and separation. ii. discrete kinetically trapped molecular cross-bridges acknowledgments the authors would like to thank the reviewers for their helpful comments.funding this work was supported by the nih grant r ai - a and by nsf grant . key: cord- -tk v hoj authors: nan title: environmental and safety issues with nanoparticles date: - - journal: nanoparticle technology handbook doi: . /b - - - - . - sha: doc_id: cord_uid: tk v hoj nan since nanoparticles have superior surface activity and can be applied to the production of particles with various functions, they are extremely important for the future development of sophisticated material technologies. on the other hand, this superior activity of nanoparticles is a cause of trouble from the perspective of safety, and does not always have a positive influence on the environment. attention must also be paid to impact on health. nevertheless, all technologies have negative aspects, and overcoming these kinds of problems, we will be able to utilize the superior characteristics of nanoparticles for practical purposes. to achieve this goal, it is necessary to fully understand the influence of nanoparticles on the environment and the relevant safety issues. this chapter evaluates the relationship between nanoparticles and the environment, and also describes the trouble caused by nanoparticles as well as the safety issues. the relationship between nanoparticles and the environment will be clarified from the viewpoint of what kind of influence nanoparticles generated either artificially or naturally have on the environment. the influence on the indoor environment, where nanoparticles are produced, will also be clarified. the safety of nanoparticles will be clearly described from the perspective of the trouble caused by the superior surface activity of nanoparticles; the effect of the compositional characteristics of nanoparticles, and also the influence on health. a method for assessing the influence of nanoparticles using quantum dots is also explained. in the final section, methods for removing nanoparticles from gas and liquid are described as technology to control the influence of nanoparticles on the environment. in our atmospheric environment, particles ranging from several nanometers to several tenth micron orders are suspended. they are emitted into the atmosphere at the rate of . billion tons every year. emission sources are classified as either natural or artificial. natural particles occupy % of total particles, consisting mainly of salt particles (ϳ billion ton) from the sea and soil particles (ϳ . billion ton) from the land. on the other hand, the latter particles are brought about by human activities. although occupying only % of the total emitted particles, their size is mostly of submicron order and because they contain hazardous chemical components such as nitrates, sulfates, hydrocarbons, heavy metals, etc. in high concentration, their effects on the ecosystem are serious. fig. . . shows an overview of the size and concentration ranges of various aerosol particles. as it can be seen, the number concentration of atmospheric aerosol which we inhale every day ranges from several thousand particles per cm in clean area to several hundred thousands in dusty areas, and the size range lies between nm and several tens of micrometer. fig. . . shows mass-based size distribution of atmospheric aerosol particles. since the size distribution in the nanosize range appears only when the sources of particle generation exist, the size distribution is usually bimodal with peaks in the size range of a few to micron and submicron. the former peak consists of naturally generated coarse particles such as soil dust, sea salt spray, and so on. on the contrary, the latter contains plenty of artificially generated particles, some of which grow from molecules (in most cases vapor state) exhausted by human activities through chemical reaction, condensation, and coagulation. particle growth rarely leads to particles larger than m unless high concentration of vapors or particulate matters which cause the above-mentioned growth mechanisms exist in the atmosphere. as it can be seen from the differences in the particle generation process, fine particles generated from molecules or nanoparticles are much more complicated in their chemical component than the coarse particles, and sometimes have serious adverse health effects. such fine particles are called pm . , which is defined for particles less than . m including nanosized particles. recent epidemiologic investigation reports that the concentration of pm . showed a positive correlation to the mortality due to pulmonary diseases [ ] . various research techniques are used in order to understand the process of particle growth and to trace back to the source of pollution. an example is shown in fig. . . where a characteristic function of sulfur dioxide is shown taking into account all possible factors related to particle growth. where f is the characteristic function that expresses particle size, particle concentration, particle composition, and so on [ , ] . particulate materials in water are present in the form of colloids. these colloid particles are classified into inorganic colloids. examples of the former are oxides of aluminum, silicon and other substances, and typical examples of the latter are substances such as humic acid and fulvic acid. while the structure and molecular weight of particles vary depending on the area of water, it is known that what are usually present in water are comparatively small colloids (particles smaller than nm). the number concentration of colloid particles in ground water, or a typical water area environment, ranges from to (number / m ) and varies significantly depending on the geochemical conditions of the aquifer. it is known that in moving water, colloid particles sometimes act as a medium in conjunction with water and in some cases move faster than water. homogeneous porous layer such as a sand layer, and most of the colloid particles are trapped. the mechanism of partical trap in this layer is explained by the sand filtration theory. c and d are a gravel layer and a rock bed, respectively, and both have high water permeability with large gaps and cracks. particles can also pass through easily. safety and movement characteristics of colloid particles have a significant influence on the movement of materials such as ionized molecules in aquatic environments. since fine particles such as nanoparticles in particular are highly stable as colloid particles, it will be very important in the future to understand their influence. at the same time, these characteristics are considered to have a high potential to be developed for further application of nanoparticles. in most cases, nanoparticles in exhaust gases are studied from the viewpoint of the influence of total particulate matters on the environment. the term "nanoparticles" is used only in a few cases, "fine particles" is usually used for investigation. since nanoparticles are part of fine particles, this section will be described from this perspective. major sources of combustion exhaust gases are stationary large-scale combustors and diesel engines for stationary and portable use. for stationary combustors, fuels such as coal, oil, and gas are used. lighter fuels have a lower rate of particulate emission, but have a higher fine particle content including nanoparticles. fig. . . [ ] shows the frequency distribution in combustion of coal and heavy oil. fig. . . a and b are the distributions on a number and mass basis, respectively. as these figures clearly show, the total weight of particles of a size of m or smaller is extremely low, while their total number is, on the contrary, very large. it is clear that, while the total quantity of particulate material is far larger in coal combustion than in oil combustion, the difference is less when it comes to particles m or less in diameter, including nanoparticles. most of the particles contained in pulverized coal combustion exhaust gases are considered to be formed as particulate materials directly from ash content, which is originally contained in coal and also includes some unburned carbon. particularly, almost all large-size particles are considered to be this type of particle. on the other hand, fine particles include two types. one type is formed in the process by which low boiling point metal contained in coal ash is evaporated and vaporized in a high-temperature combustion field and then becomes particles in the exhaust gas cooling process. the other includes carbon particles formed in the gas phase, or so-called soot, which is generated due to the delay in oxygen supply for combustion of evaporated volatile matter in the initial stage. fig. . . [ ] shows the relationship between the trace metal content in coal ash and the particle diameter. aluminum with a high boiling point has a constant concentration regardless of the particle diameter. however it is obvious that in the case of metals with a lower boiling point, the smaller the particle diameter, the larger the content. with regard to particles with sizes m or smaller in the nano domain, it has been clarified that the generated amount is increased rapidly by reducing combustion air supply or by weakening the oxidation atmosphere in the volatile matter combustion area, for example, when air supply from a burner is reduced in twostage combustion. this also demonstrates the significant contribution of carbon particles formed in the gas phase. also in the case of ash from heavy oil combustion, there are large particles of a carbon residue type generated from sprayed liquid particles and particles formed in the gas phase as well. as in the case of coal, trace metal contained in heavy oil with a low boiling point is concentrated into fine particles and discharged. also in the case of the combustion of liquefied natural gas, carbon particles formed in the gas phase are generated, albeit in trace amounts. in contrast, only in the case of diesel engines, fuel is injected into the high-temperature and highpressure atmosphere produced by compressing only air to induce spontaneous ignition, and combustion continues with a heterogeneous mixture of fuel and air in the combustion chamber. therefore, particulate materials mainly consisting of unburnt carbon are generated due to incomplete combustion. fig. . . [ ] shows changes in the diameter of particles according to changes in the diesel engine load. it is obvious that the overall concentration of particles increases with the increase in the load rate of the engine. according to observations using sem, fine particles in diesel engine exhaust gases have also been found to comprise fine primary particles of a size several tens of nanometers, and coarse particles with carbon hydride condensed on the surface of secondary aggregates of primary particles. influence of particle diameter on trace element contents. the volume of industrial and domestic wastewater is increasing significantly year by year with the change in the lifestyle based on mass consumption and mass disposal brought about by the dramatic development of the economy and industry. effective advanced wastewater treatment is required because wastewater contains a variety of constituents such as particles, organic materials, and emulsion depending on the resource. inorganic nanoparticles are not generally stabilized in the liquid because they form aggregates of some sort more or less. for wastewater reclamation and reuse, these nanoparticles can be removed from the liquid by the advanced treatment processes such as membrane filtration following biological treatment processes. organic materials such as macromolecules are regarded as soft nanoparticles judging from their sizes, in contrast with hard inorganic particles. chemical mechanical polishing (cmp) is one of the fastest growing processes in semiconductor industry, and it has become an integral part of the state-of-the-art fabrication line for the multilayer wiring board of large-scale integrated circuit (lsi). besides the semiconductor devices, cmp is widely applied to the magnetic head, the magnetic memory, and the imaging devices. the process is primarily used for polishing the device side of a semiconductor wafer through the mechanical downforce of slurry abrasive in combination with chemical oxidation of wafer surface. in general, colloidal silica is used as abrasive slurry to planarize the oxide wafer surface. particles in slurry are highly charged to avoid aggregations between particles or between particles and wafer surfaces. during the process, large volumes of ultrapure water are consumed to clean the surface of the wafer, which generates large quantity of cmp wastewater typically having high solid content resulting from slurry abrasive particles of sio , al o , or ceo , depending on the nature of the cmp application. the quantity of cmp wastewater generated is expected to increase proportionally with the growing needs of the cmp processes. as a result, the treatment and reuse of cmp wastewater has become increasingly necessary. the cmp wastewater has been generally treated with the conventional chemical coagulation-sedimentation process, producing large quantity of sludge. currently, a membrane filtration process coupled with chemical pretreatment is used to separate the nanoscale particles from the cmp wastewater to reclaim the water [ , ] . wastewater of nanosized metal colloid, which is hard to be removed by coagulation-sedimentation process, is discharged in such diverse fields as metalworking factory, electronic components factory, and pigment-manufacturing factory [ ] . it is reported that various trace elements of heavy metal are contained in wastewater discharged from a pulp production plant [ ] . a spent emulsion, which contains nanosized copper colloid, is discharged from plants manufacturing copper cables for electrical industry, and the treatment for purification of effluents is examined by the integrated membrane system based on ultrafiltration (uf) and nanofiltration (nf) [ ] . a glass company generates the wastewater containing fine clay and glass particles from the grinding process of glass surfaces during production of crt glass used for tvs and monitors. separation of fine clay and glass particles by microfiltration (mf)/uf is examined in order to treat glass industry wastewater for reuse in the manufacturing process [ ] . the colored substances are free from regulatory constraint of water quality so far because they are not considered hazardous substances. however, water color is being recently used as a standard for the judgment of the purity in water because the removal of color becomes important for wastewater reclamation and reuse. dye works are scattered across the country as the industry with local tradition. the dyehouse effluent is discharged in large quantity, and it has extremely complex composition because it contains not only dye but also dyeing aid and finishing agent. in general, dye cannot be removed by standard biological treatment because of its low environmental biodegradability. dye wastewater is treated by coagulation-sedimentation and activated sludge processes, and nanoparticles produced in the course of the treatment are released into the environment [ ] . the color is often imparted by organic substances, predominantly humic substances. aquatic humic substances including humic and fulvic acids are a term referring to a broad class of naturally occurring mixture of organic compounds, ubiquitous in surface waters, ground waters, and soil pore waters. they are a complex mixture of heterogeneous organic materials in terms of elemental composition, chemical functionality, and molecular size distribution since humic substances can be derived from any organic materials, including plant and animal debris, microfauna, biowaste, pesticide, and others. the molecular weights of humic acids range from several thousands to several tens of thousands daltons, and those of fulvic acids range from several tens of thousands to several hundred thousands daltons. because of this versatility, humic substances are known to significantly affect the behavior of some pollutants in natural environments, such as trace metal speciation and toxicity, solubilization and adsorption of hydrophobic organic compounds, and disinfection by-product formation [ ] . melanoidins are natural polymeric compounds of dark brown color, and they are closely related to humic substances. they are produced by a set of consecutive and parallel nonenzymatic reactions taking place between amino compounds and carbohydrates during a maillard reaction [ ] . they are contained in the molasses wastewater from alcohol distillery, sugar processing and refinery industry, and glutamate processing industry. such wastewater containing melanoidins has frequently caused a coloration problem of water environment, and thus the suitable decolorization treatment is required in many fermentation and sugar industries using molasses. treatments by flocculation, ozonation, and electrolysis are promising in color removal [ ] . food-processing wastewater usually contains a variety of organic materials in varying degree of concentration. in cheese-making in the dairy products industry, only ϳ % of the initial milk volume becomes product, cheese, and the other % becomes by-product, liquid cheese whey. since cheese whey is a protein-and lactose-rich by-product of cheese production, its cost-effective utilization is becoming increasingly important. recent developments in membrane technology have provided exciting new opportunities for large-scale protein and lactose fractionation in whey treatment [ ] . in textile industry, typically it takes over l of water to process just kg of textile material. not only the washing water must be treated to recover important by-products such as lanolin, but bleaching and dyeing chemicals must also be removed before discharge back to the rivers [ ] . surfactants are a primary constituent of the detergent used in the household routinely, and also they are widely used in industry and agriculture because they have several functions such as washing, emulsification, and dispersion. the surfactants are usually present in the solution in the form of the micelle, and large amounts of surfactant wastewater are discharged in the rivers [ ] . pesticides whose molecular weight ranged from to da (ϳ nm) have been used in great quantities not only for agricultural use but also in golf links and resort. therefore, the wastewater and effluent treatments have become an important issue, and pesticide separation by nf membranes is found to be very efficient [ ] . the potential reclamation of high-quality water produced by the advanced treatment of the secondary effluent of the municipal sewage has come a long way in recent years. the sewage contains various components such as virus [ ] , pharmaceutical substances [ ] , and endocrine disrupting compounds derived from zoonotic excretory substances [ ] . the advanced treatment of such chemical contaminants at low level becomes increasingly important. as mentioned above, the removal of nanoparticles contained in wastewater is stringently required to recycle the reclaimed wastewater in a wide variety of industries such as chemical industry, textile industry, pulp and papermaking industry, food-processing industry, dairy products industry, and pharmaceutical industry. also for domestic wastewater, the reuse of the reclaimed wastewater for nonpotable purposes is becoming more and more important, and this is expected to raise awareness of the behaviors of nanoparticles contained in wastewater in order to upgrade the water treatment processes. in recent urbanized lifestyles people tend to spend more time in enclosed buildings or residences than outdoors. therefore, it is of great importance to characterize indoor particles and correlate between indoor and outdoor ones from the viewpoint of evaluating the influence of indoor air quality (iaq) on human health. as shown in table . . , indoor nanoparticles originate from the several sources such as products of chemical reactions, nonvolatile residues (nvrs) of liquid droplets, printers/photocopiers, combustion, bioaerosols, and infiltration of outdoor air. . . . secondary particle formation by gas phase ozonolysis for particle formation resulting from chemical reaction via ozone, the reaction of terpenes is very common in indoor environments as well as atmospheric ones. terpenes are emitted from fragrance-containing vegetable oils such as pine oil and citrus oil, and wooden materials including woody furniture [ ] . meanwhile, sources of emission of ozone are air cleaners, air-conditioners, laser printers using corona discharge, and infiltration of outdoor air. terpenes are generic terms of unsaturated organic compounds that are composed of isoprene as unit (e.g. -pinene and limonene). these compounds used for household applications readily react with ozone because they have one or more double bonds. it has been proposed that, as shown in fig. . . , the reaction mainly proceeds to form less volatile pinonic acid via pinonaldehyde of intermediate [ ] . furthermore, the acid-catalyzed reaction allows the products to convert into higher molecular weight compounds by the polymerization via carbonyl groups in the aldehydes and the aldol condensation [ ] . it has been reported that the resultant generated particles have a size distribution with a peak diameter of about nm, and that the products by terpenes ozonolysis irritate human airways [ ] . nanoparticles are also generated from air humidifiers or negative air-ion generators in which water is atomized. in general, humidifiers are mainly categorized into vaporization type and atomization type [ ] . the former does not entrain impurities in water when it is fed into indoor spaces of interest. meanwhile, the latter has the drawback that nvrs are suspended in spaces to be humidified by feeding water via spraying and sonication. the nvrs in tap water include colloidal particles and soluble fraction such as silicates, sulfates, carbonates, and chlorides. the size of nvr particles, d p _ r can be estimated from the following equation: where dp_ m is the droplet size, c the mass fraction of nvr in the droplets, m the droplet density, and r the nvr particle density, respectively. assuming that m-sized droplets ( m ϭ , kg/m , r ϭ , kg/m ) are formed by an ultrasonic nebulizer, and the mass fraction of nvr in city water is ppm ( Ϫ ), the nvr particle size, d p_r is estimated to be nm. recently, a wide variety of negative ion generators using the lenard effect, corona discharge, uv/photoelectron emission and electrospray have been commercialized and attention has been focused on features such as air purification and physiological activation [ ] . among them there are the ion generators that atomize water based on the lenard effect and electrospray form the nvr particles as by-product in addition to ion products if the supplied water contains nonvolatile impurities. fig. . . shows an example of electrical mobility distribution for ions generated by the electrospray method (positive in this case) [ ] . this method atomizes liquid fed to a tip of a capillary electrode to form fine droplets with large amounts of charge by applying high voltage between the tip and the downstream counter electrode. when water in the generated droplets evaporates and their surface charge density attains the charge limit called "rayleigh limit", this phenomenon induces their self-fragmentation followed by the formation of a high concentration of cluster ions. in this figure, the high-mobility peak on the right side corresponds to the cluster ions. these ions are nm in size assuming that they are singly charged. meanwhile, another peak on the figure results from nvr in water and then its height increases with the increase in the fraction of tap water in the fed liquid. the electrical-mobility-equivalent size of nvr particles measured by differential mobility analyzer (dma) -condensation nucleus counter (cnc) method ranges from to nm and their concentration is on the order of particles/cm . comparing the forementioned electrical mobility distribution with the particle size one, the nvr nanoparticles are estimated to hold about charges. accompanying the recent proliferation of computers, the use of inkjet printers and electrophotographic machines such as laser printer and photocopier is becoming common in homes as well as offices. it has been reported that these devices emit various sorts of pollutants. the eco-friendlinessoriented standards such as blue angle standard [ ] regulate the maximum permissible limits of benzene, styrene, total volatile organic compounds (tvoc), ozone and particles. as the regulation of particles is based on the emitted mass per hour, mainly the relatively coarser particles such as toner and dust adhering to paper have been targeted. however, some reports have revealed that nanoparticles are emitted from inkjet printers or laser printers [ ] . fig. . . depicts the size distribution of particles emitted from a laser printer measured by a scanning mobility particle sizer (smps). as seen in the figure, nanoparticles with a peak diameter of around nm are generated in printing mode, whereas the emission in the case of feeding paper without printing is about one third of the normal printing mode. furthermore, these particles were dried by passing them through a diffusion dryer because they are thought to originate from the nucleation of water vapor emitted from papers in the fixation process. as a result, it was found that most particles formed in the paper feed mode evaporated and then vanished, while particles in the printing mode contained nonvolatile components as well as water. from these results it is anticipated that the particles are derived from styrene remaining in the toner even though their composition has still not been identified. meanwhile, it is thought that during ink discharge ink-jet printers emit not only the main ink droplets but also their satellites (about m) to result in nanometer-sized nvrs during printing. one of the most significant source of indoor nanoparticles relevant to combustion is cooking such as frying and sautéing [ ] . some reports said that over % of the particles by number were in the ultrafine fraction range during cooking with bimodal peaks at and nm, attaining the number concentration on the order of particles / m and the emission rate of particles / h. owing to lifestyles in asian countries, cigarette smoke, incense, and mosquito coils also contribute to indoor nanoparticle levels [ ] . it was reported that especially in the indian subcontinent the combustion of biofuels such as straw and dried cattle manure used for cooking could have a significant impact on climate change in the south asian region [ ] . sidestream cigarette smoke also contains nanoparticles, having a concentration distribution with the main peak between . and . m [ ] . in addition, it was found that nanoparticles a peak size of nm formed by the nucleation of vapor fraction in filtered sidestream smoke immediately after burning when the dilution of smoke by air was insufficient [ ] . attention should be paid to air cleaners when a high concentration of cigarette smoke has to be treated by the cleaners using a single unit air filter. airborne virus particles or virions are typically in the - nm size range, and are a good example of nanoparticle bioaerosols. smaller viruses typically contain one subunit, which consists of an outer protein capsid, internal nucleic acid (e.g. dna and rna), and other internal proteins. corona virus that causes sars and influenza are good examples of them. the viruses most often are transmitted through direct contact with an infected person, such as by shaking hands, hugging or kissing, while sometimes it is spread by nasal droplets. however, it is still unknown how these virus particles behave in the case of airborne infection. recently, the studies that attempt to elucidate the behavior have been progressing [ ] . this section describes the sources of nanoparticle generation in industrial processes by categorizing them into specific processes where a cleanroom is used and other general ones. the sources of emission of unwanted nanoparticles in general workplaces are categorized as fumes from hot processes (e.g., smelting, refining, and welding) and from (incomplete) combustion processes. favorable conditions required for the generation of nanoparticles are found in workplaces where there is ( ) presence of vaporizable material, ( ) sufficiently high temperature to produce enough vapor, followed by condensation to form an independent aerosol, and ( ) rapid cooling and a large temperature gradient. there have been so many studies on occupational exposure to fine particles in the field of public health. in general, high spikes of nanoparticle concentration are observed during active operations, followed by a gradual decay after the operation, primarily because of coagulation, evaporation, dilution, and/or deposition. the fraction of the total number of nanoparticles generally decreases, whereas that of the number of submicrometer particles increases with time and distance from the point of emission. in order to accurately estimate exposure, the effects of spatial and temporal changes will need to be evaluated. therefore, it is important to identify the time required for the concentration to decline to the normal or background levels. as an example of reports on grinding processes, fig. . . shows the case where a steel substrate was ground upon using a high-speed grinder [ ] . from the figure the distribution of concentration of generated particles has a distinct bimodality, one with the finer peak at around nm and the coarser one at around m. the former results from within the grinder motor and the volatilization or combustion of amenable ground substrate and/or grinding materials, the latter from the mechanical abrasion and attrition. however, the resultant total concentration on the order of particles/cm is not so high. . . . industrial processes with cleanrooms cleanrooms and associated controlled environments (e.g., in the case of an iso class cleanroom, the maximum permissible airborne particle concentration is less than particles/m for particles with the size of . m or larger, while the airborne particle concentration in ordinary indoor environments is on the order of particles/m or higher) are usually adopted to avoid particle contamination in industrial processes where precision products such as engineered nanoparticles, semiconductors, and other electronic or optical devices are fabricated because the deposition of particles onto product surfaces causes their yield reduction and quality deterioration. the emission sources in cleanroom environments are tabulated in table . . . since some of the listed emission sources emit trace amounts of nanoparticles, these nanoparticles are not regarded as particulate contaminant but as chemical or molecular one. in this section, these nanometer-sized solid substances formed on solid surfaces by chemical reaction are also included. size distribution of nanoparticles generated when a steel plate was ground with a high-speed grinder. ( ) air exhaled by humans emissions from human bodies are a minor contribution in ordinary indoor situations because airborne particle concentration in such places is quite high, whereas the emission cannot be seen as negligible in cleanroom environments. the major human emissions are thought to be atmospheric dust deposited on clothes and skin fragments, and most of these particles are submicrometer in size. meanwhile, particles in exhaled air are composed of fine liquid droplets from spittle ( . % of water), and then evaporate to form nanoparticles of nvr. in fig. . . an example of size distributions of particles in exhaled air before and after smoking is shown [ ] . when measuring particles in exhaled air, the air was introduced into a measuring device after drying them by passing them through a diffusion dryer. the size distribution of particles in exhaled air before smoking (n db ϭ ) has a bimodality, one with the peak size of . m, and the other of nm or smaller. the former peak comes from atmospheric aerosols as it decreases with the increase in number of deep breaths in a clean booth (n db ). the latter originates from nvr particles of spittle droplets. incidentally, since smoking induces the rapid increase in number concentration of particles . m or larger by times or more and for nanoparticles by about double, special attention should be paid to the management of personnel's clothes such as face mask when they enter a cleanroom after smoking. ( ) emission from ionizers ionizers are commonly used in cleanrooms to eliminate electrostatic charge on substrates for precision electronic devices. the most popular ionizer is a corona-discharge type. corona-discharge type ionizers are categorized into the following three groups; ac, dc and pulsed-dc types. the issues of emission of contaminants such as ozone, no x and particles have been pointed out [ ] . these issues are also applicable to air cleaners using a corona discharger. among these problems is that the particle emission has a potential for particle contamination onto product surfaces and eventually decline in product yield. the particle emission, which has been studied since the s, is caused by foreign particle deposition onto electrodes, electrode erosion, and gas-to-particle conversion. the issue of electrode erosion can be solved by the improvement of electrode materials, whereas for the issue of gas-to-particle conversion, the airborne molecular contamination (amc) control to be ionized has to be made. it was reported that fundamentals ch. environmental and safety issues with nanoparticles corona-discharge ionizer (e.g. gas-to-particle conversion of low-molecular-weight cyclosiloxane) boron-containing particles from borosilicate glass fibers of hepa filter haze by chemical reaction on solid surfaces (precipitation of ammonium salt and silica) watermark on wafer surfaces at drying leakage from thin film and nanoparticles processing equipment silicon-containing compounds that precipitated on the electrodes result from the gas-to-particle conversion of low-molecular-weight cyclosiloxane (lmcs) from silicone sealant via corona discharge [ ] . ( ) boron-containing particles from hepa filter with borosilicate glass fibers the use of hepa and ulpa filters made of borosilicate glass fibers prevails in the most cleanrooms. it has been known that owing to chemical reaction in equation ( . . ) bf vapor is formed from glass fiber filters by passing hf gas leaking from wet cleaning equipment through the filters. boron, which is a dopant element for semiconductors, has been thought to be a contaminant that might cause failure in semiconductor devices if it comes from the surroundings. in addition, it has been revealed that trace amounts of boron in the form of boric acid (h bo ) are also formed from the fibers via the reaction with moisture in the surrounding air (equation ( . . )). fig. . . depicts the change in volatilized boron mass from various filters in terms of airborne boron concentration. especially, at the initial stage just after the initiation of ventilation, the volatilized boron mass increases with increase in relative humidity [ ] . boric acid, which is solid at room temperature, is surmised to form in the particulate form. however, its existence was identified only by chemical analysis because it is present only in trace amounts. haze might form on glass surfaces of lenses and mirrors for optical instruments if they are exposed for a long time to cleanroom environments where amcs are not controlled properly. the haze is more likely to bring about the insufficient light delivery onto a surface to be exposed in photolithographic processes. one of the reasons is that ammonium sulfate ((nh ) so ) is formed, which then precipitates on the glass surfaces via chemical reaction of sulfur dioxide (so ) with ammonia or amines. for another reason, hexamethyldisilazane (hmds) used as additive in resist coating or lmcs from silicone sealant is adsorbed, and then decomposed to form silica precipitates on glass surfaces by photochemical reaction during laser irradiation, followed by the unwanted decline in laser penetration [ ] . as another example a report said that tiny projections, which are also known as "haze", with a size of . m or smaller were formed on silicon wafer surfaces owing to the adsorption of organosilicate compounds in thin film formation processes with cvd. it is similarly caused by the precipitation of sio [ ] . ( ) watermarks on solid surfaces during drying when a silicon wafer surface is cleaned with deionized water and then dried in air, a watermark is formed on it via the mechanisms demonstrated in fig. . . . oxygen in air is dissolved and diffused into water droplets or adsorbed water on a wafer surface, followed by the formation of silicate compounds via silanol reaction. the watermark on a wafer surface is detected in the form of nanometer-sized particles by an electron microscope [ ] . ( ) leakage from nanoparticle production processes in regard to the risks to processing equipments by nanoparticle leakage from production processes, the vdi report in germany [ ] has been described in detail. the production of engineered nanoparticles can be generally categorized into two approaches. one is a "top-down" approach that is initiated with a bulk material and then breaks it into finer pieces using some form of energy such as etching, ball milling, sputtering, and laser ablation. the other approach is to synthesize materials from the atomic or molecular level by growth and assembly to form the desired nanoparticles. processes included in this "bottom-up" category are sol-gel, chemical vapor deposition, flame synthesis laser pyrolysis, and so on. most of these processes are performed in a closed reaction chamber installed in a cleanroom or associated controlled environment. human exposure to these engineered particles does not take place during synthesis unless there is an unexpected system failure (e.g. rupture of a seal). human exposure is more likely to occur after the manufacturing when opening the reaction chamber, drying the products, or in the post-process handling of the products. the release of nanoparticles during production chamber cleaning operations is another critical point. cleaning typically involves using water or some solvent. brushes, sponges, or tissues used in the cleaning will carry nanoparticles into the waste stream. disposal of the waste and wastewater may become a source of nanoparticle release into the environment. further, conditioning of nanoparticles such as compression, coating, and composition to form final products may also result in the release to the environment and resultant exposure although very few studies have been carried out on this subject. recent studies [ ] to evaluate the aerosol discharge during the handling of carbon nanotubes showed that the generation of nanoparticles occurred under vacuum to remove spilled nanotube materials or vigorous mechanical agitation. however, they reported that the concentrations were very low. in addition, measurements of nanoparticle levels during final packaging of carbon black, which is a typical engineered nanoparticle material, showed that there was no increase in nearby air [ ] . study on the safety of nanoparticles has started only recently, and no sufficiently systemized results have been obtained. what should be noted in particular is that the possibility of radial troubles caused by particulate matters are considered to increase by the decrease of particle diameter in nanoparticles. one typical example is the problem of dust explosion, caused by the high surface reactivity of fine particles. in other words, since nanoparticles are extremely fine particles, dust explosion is more likely to occur. explosion is more likely to occur because fine particles are different in their composition, in that low boiling point metal can be easily condensed, as described in section . . however, of particular note here is that, since all particles do not necessarily exist independently in the form of a single particle, the possibility of dust explosion does not simply increase as particles become finer. fine particles with sizes of m or smaller such as nanoparticles have an extremely high agglomeration propensity and secondary particles can be easily generated. therefore, in some cases they conversely behave like large particles. these are the points to be taken into consideration when studying the problems caused by nanoparticles. as shown in fig. . . [ ] , the effect of the particle diameter on dust explosion tends to be that the smaller the particle diameter of the dust, the lower the minimum explosion concentration. in other words, explosion can be induced under conditions of lower concentration of particles in air as the particle diameter becomes smaller. due to the difficulty of conducting experiments to suspend particles with the same size in a uniform concentration, this result was obtained from particles far larger than nanoparticles; however, it has been clarified qualitatively that the smaller the particle diameter, the higher the possibility of dust explosion. from the perspective of composition, the possibility of explosion increase if materials that react easily with oxygen at low temperature are condensed into particles of small diameter. therefore, with regard to the effect of particle diameter on dust explosion, more careful attention needs to be paid in the case of combined materials than in the case of uniform materials, as assumed in fig. . . . as described before, however, since nanoparticles are considered to exist often as agglomerates, it is necessary from the perspective of the particle diameter to take into consideration the diameter of not only primary particles but also of particles after agglomeration. to address the safety of nanoparticles, it will be important in the future to elucidate their behavior in detail including these factors. the terms 'nanoparticles' and 'nanomaterial' have been used for particles of which one representative dimension, for example, diameter of particles on cross-sectional diameter of fibers has at least nm or less. some people hold that the majority of such fine particles are exhaled without depositing in the respirator tract, and that therefore the particles may not cause pulmonary diseases. however, the properties of nanoparticles are known to be different from the bulk material they are derived from. in cases where the biological effects of bulk materials have been reported, nanosized particles of these materials may be expected to have stronger dose response for the health effects. every effort must be made to clarify the uncertainty on the risks of these nanomaterials [ ] . at the present time, there is no regulation or standard for assessing the biological effects of nanomaterials, and therefore there is a paucity of toxicological data concerning nanomaterials. much more systematic and strategic studies are needed to enable risk assessments for human health [ ] [ ] [ ] [ ] [ ] . as regards risk assessment and risk management of nanomaterials, the characterization and identification of anticipated risks should be first determined for chemical substances or foods. conventional assessment methods are applicable for water-soluble particles. for insoluble nanoparticles, the assessment of potential health hazards should be made based on their properties or toxicity and dose-response relationship. the risk is a product of hazard and exposure; even if a nanoparticle has a hazard, the risk is lower when the possibility of exposure to the nanoparticle is small [ ] . influence of particle diameter on lower limit of particle concentration for explosion. ( ) exposure routes for nanoparticles nanoparticles can either be deliberately introduced into the body for medical purposes (drug delivery systems) or absorbed involuntarily from the environment (inhalation of nanoparticle-containing dust in the air). a distinction should also be drawn between nanoparticles manufactured for industrial application and those unintentionally generated and released in the environment, such as welding fumes or diesel exhaust particles (dep). in the fields of environmental science and toxicology, numerous studies on the potential health hazards caused by ultrafine particles have been conducted. practically, there are several definitions of nanoparticles or ultrafine particles, however, findings regarding biological effects of the ultrafine particles are useful as a starting point for estimating the effects of nanoparticles on human health. human and animals contact with nanoparticles through various routes: nanoparticles can be inhaled in the air, swallowed in the water, ingested in food, and absorbed via the skin in cosmetics. for successful risk assessment, it is important to determine how nanomaterials or nanoparticles are used, such as composites, surface coating, or powders. coatings or powders have the potential to release a part of their nanomaterials into the environment. workers who come into contact with nanomaterials have the possibility of exposure to nanoparticles at the workplace. consumers of products using nanotechnology can also be exposed to them. attention needs to be paid to the environments and ecosystems in which nanoparticles and nanomaterials are released. nanoparticles in the products may change their size, quantity, and composition during their life-cycle of manufacturing, use, transportation, and disposal. ( ) respiratory uptake of nanoparticles inhalation is the main route of exposure to nanoparticles. particles inhaled with the air through the mouth and nose pass through the throat (nasopharynx and oropharynx) and tracheobronchial tree before reaching the alveolar region where oxygen moves from the alveoli to the blood and carbon dioxide moves from the blood to the alveoli. how deeply particles can penetrate and where they become deposited on each respiratory airway such as the nasal cavity, tracheobronchial tree, and the alveoli depend on their size under the various deposition mechanisms: inertial impaction, gravitational sedimentation and diffusion, etc. the respiratory airway includes the anterior nasal passage, posterior nasal passage, pharynx, larynx, trachea, main bronchi, bronchi, bronchioles, terminal bronchioles, alveolar duct, and alveoli, as shown in fig. . . . in the human lungs, the trachea divides asymmetrically into the right main bronchus that enters the right lung where it divides into three lobes, that is, an upper, a middle, and a lower, and the left main bronchus that enters the left lung where it divides into two lobes, that is, an upper and a lower. the trachea divides into two branches, dividing progressively to the terminal alveolus. to quantitatively assess the pulmonary particle deposition needs human lung morphology models, respiratory physiology based models of the entire lung airway system and aerosol deposition models based on many experimental findings. in , the icrp task group on lung dynamic (icrp: the international commission of radiological protection) published their revised lung model [ ] . the deposition, clearance, and translocation of particles in each of the compartments were described. while the model has been widely used in the nuclear field, it is applicable to conventional aerosols as well as radioactive aerosol. in the nuclear field, aerosols including radon progeny that used to be nanoparticles have been studied. fig. . . shows the deposition fractions of inhaled particles per adult nasal respiration of . m /h in each region re-calculated for the nasopharynx, tracheobronchial, and the pulmonary (alveolar) region based on the model. inhaled aerosol particles deposit on different regions depending on their size; for example, nanoparticles larger than nm deposit mostly in the alveoli and those less than nm deposit in the nasal cavity. how deeply particles penetrate into the lung depends on their size. nanoparticles can reach pulmonary region in the lung and deposit more intensively and this, therefore, has become one of the reasons for concern about the effects of nanoparticles on human health. however, deposition of nanoparticle chain-like agglomeration and fibrous particles such as carbon nanotubes cannot be estimated by this model. most inhaled nanoparticles are deposited on the surface of the respiratory tract. generally, if insoluble particles are deposited in the ciliated airspaces which are lined with a mucous layer, they are transported to the digestive tract with the mucous flow by mucociliary movements. particles deposited on nonciliated bronchioles and alveoli are phagocytosed by macrophages, which is a kind of white blood cell. as a result, their residence time is longer, however, they are usually transported to the ciliated upper part of the respiratory tract. removal of deposited particles described above is called clearance. when the amount of deposited particles is below a certain value, no health effect is produced. in relation to a macrophage response to particles, crystalline silica is hazardous, whereas titanium dioxide is not. pneumoconiosis is a well-known lung disease that is caused by exposure to dust particles of several micrometers in diameter. silicosis is a typical form of pneumoconiosis resulting from exposure to crystalline silica dust and characterized by a progressive fibrosis of the lungs. the macrophage-mediated clearance (phagocytes) was effective for micron and submicron particles. it has been reported that only % of deposited nanoparticles were removed by the clearance mechanism [ ] . it has been suggested that the remaining nanoparticles may pass through the alveolar walls, penetrate into the blood or lymphatic circulation, and be transported to other organs. many studies have shown that the smaller the particle size the greater the mobility or they pass easily through the alveolar wall and enter into the bloodstream. it is further presumed that the mechanism of health effects of nanoparticles on cardiovascular system other than the respiratory tract is similar to that in the airborne ultrafine particles from dep. in japan, two reference values, the 'administrative control levels' (acl) and the 'occupational exposure limits' (oels), are used for the regulation of hazardous chemicals as well as dust (particle matters). oels are recommended and revised every year by the japan society for occupational health. oel (oel-mean) for mean concentration of a chemical substance is defined as the reference value to the mean exposure concentration at or below which adverse health effects caused by the substance do not appear in most workers working for h a day, h a week under a moderate workload [ ] . the 'threshold limit value' (tlv) has the same definition (but may not be the same values as oel of the same substance) provided by the american conference of governmental industrial hygienists (acgih). acl is an index to determine the control class to judge the propriety of the working environment control based on the results for working environment measurement, which have been implemented for the unit work area in accordance with the working environment measurement standard. the results of working environment measurement are evaluated by classifying the working environments concerned into three control classes (control class i, ii, and iii). these classes are used as the standards to classify the level of the working environment concerned. among those subject to working environment measurement, these standards apply to workplace where dust, lead, organic solvent, and specified chemical substances are used. article of the industrial safety and health law stipulates that certain workplaces in which harmful substances are involved or harmful work operations are performed shall be the subject to working environment measurement. a % cut-off size of the dust particle is set at m for both standards and is far larger than nanosized particles. oel or acl [ ] for particulate matters are usually based on mass concentration, that is, milligram per cubic meter. therefore, if the particulate matters have broad size distribution, the contribution of nanoparticles is not large to in terms of mass of particles. evidence from a number of toxicological studies on insoluble particles indicates that the primary determinant of the health effect of particles depends on the surface area of particles deposited [ , ] . on the basis of the results from a number of in vitro studies of insoluble nanoparticles, a hypothetical cellular interaction has been proposed [ ] : inflammation and oxidative stress can be mediated by several primary pathways: ( ) the particle surface causes oxidative stress resulting in increased intracellular calcium and gene activation; ( ) transition metals released from particles result in oxidative stress, increased intracellular calcium, and gene activation; ( ) cell surface receptors are activated by transition metals released from particles, resulting in subsequent gene activation; or ( ) intracellular distribution of insoluble nanoparticles to mitochondria generates oxidative stress. in the workplace, the concentration of nanoparticles may be at a high level and most of the nanoparticles become agglomerates, while nanoparticles will form single nanoparticle at low levels in the general environment. it is a matter of debate whether agglomerates of nanoparticles react as a larger particle or a single nanoparticle in the lung or other organs. if insoluble particles are retained in the lung for a longer time without enough clearance mechanisms, they can cause pulmonary inflammation or pneumoconiosis. it is of interest that nanoparticles deposited in the lung can move into the blood vessel through alveolar epithelium and they can damage vessels or produce blood clots [ , ] . in a recent study, nanoparticles deposited in the nose may move directly to the brain via the olfactory bulb [ ] . ( ) biological effects of fullerene the biological effects of fullerene have being investigated intensively. in rats dosed orally with radioisotope-labeled c fullerenes, most were excreted in the feces and some were found in the urine. a small amount of them can be absorbed via the gastrointestinal tract. in contrast, in the same study, % of the same labeled fullerenes administered intravenously were retained after week, with most found in the liver [ ] . ld s (acute toxicity) by intraperitoneal injection in mice and rats were . and . g/kg, respectively. the dose of . g/kg orally in rats did not result in death. the reproductive translocation of fullerenes was also observed in mice. fullerenes have shown mutagenic activity in ames tests. fullerenes have shown no skin irritation or allergic reactions [ ] . on the other hand, fullerenes are being tested for possible medical use. fullerenes are basically hydrophobic but water-soluble derivatives have been synthesized to be used as drugs or its carrier. the derivative can be anticipated as drugs, for example, anti-aids drug. it has been stated that the toxicity of fullerenes changes due to slight structural changes including chemical modification [ ] . ( ) biological effects of carbon nanotubes carbon nanotubes are chemically stable and are similar in form and size to asbestos; these characteristics have given rise to concern that carbon nanotubes may have the potential to cause pulmonary diseases such as lung cancer and mesothelioma similar to asbestos. a few data are available concerning the biological effects of carbon nanotubes. the biological effects of carbon nanotubes are being researched. epithelioid granulomas and interstitial inflammation are induced in mice and rats following exposure to single-walled carbon nanotubes [ ] [ ] [ ] . untreated carbon nanotubes contain the nanoparticles of transition metals such as iron and nickel, which are used as catalysts in forming carbon nanotubes. these nickel-containing carbon nanotubes have been reported to be toxic [ ] . the concentration of airborne asbestos fibers is expressed as a number concentration, that is, fibres per cubic centimeter or fiber per liter. when fiber concentrations are determined by phase contrast light microscopy, the fibers with a diameter of less than m, a length longer than m, and a lengthto-diameter ratio (aspect ratio) greater than are counted [ ] . asbestos fibers having nanosized diameter were often observed in analyses of environmental samples using electron microscopy. international agency for research on cancer (iarc) rated asbestos as a known human carcinogen (group ) [ ] and the concentration of chrysotile asbestos is expressed as a risk level of . fiber/cm [ ] . health effects of vitreous fibers and other asbestos substitutes have been assessed to determine their oels or their carcinogenicity in humans. the health effects of carbon nanotubes are being intensively investigated now. the oel for carbon black respirable dust is mg/m and these for activated charcoal and graphite are . mg/m in each [ ] . in the ref. [ ] , while rats and mice inhaled carbon black with a particle diameter of ϳ nm at a concentration of - mg/m did not produce any specific changes, particles (agglomerate of small particles) of ϳ nm at a concentration of - mg/m produced early pulmonary changes. micronsized titanium dioxide particles are thought to have almost no toxicity and often used as a negative control substance. the oel for titanium dioxide is mg/m for respirable fraction [ ] . however, the results of a series of studies by oberdörster et al. [ , , , ] on submicron-and nanosized titanium dioxide suggested that as size decreases, inflammatory effects are intensified, and normally nontoxic substances may assume hazardous characteristics. fig. . . shows a part of the results by oberdörster et al. in which rats and mice were exposed to anatase titanium dioxide particles [ ] . their results have been frequently cited in the discussion of whether the health effects of fine particles should be based on its mass or its surface area. in fig. . . , percentages of neutrophils in lung lavage of rats are shown as indicators of inflammation after intratracheal instillation of different mass doses of and nm tio particles. the steeper dose response of nanosized tio particles is observed than for submicron tio particles when the dose is expressed as mass (fig. . . a ). if the same dose-response relationship as in fig. . . a is indicated as particle surface area (fig. . . b), the particle surface area seems to be a more appropriate dosimetric for comparing effects of different-sized particles of the same chemical structure. zinc oxide is a white powder and used in pigments. the oel is mg/m for its respirable fraction [ ] , and the value for zinc oxide fume which causes metal fume fever is under consideration. nanoparticles of transition metals and rare earth elements and their oxides will be used widely. since many of these metals and their oxides have biological effects, particular attention should be given to them. nickel compounds are rated as a human carcinogen (group ) by iarc. in particular, nickel oxide is particularly insoluble among the nickel compounds and remains longer in the lung. nanosized nickel oxide particles have greater toxicity to the lung than larger particles [ , ] . pulmonary inflammatory responses induced by nanosized cobalt particles have been reported [ ] [ ] [ ] . biological effects of nanosized particles of other transient metals such as iron and manganese have received attention [ ] . rare earth elements are a general term of chemical elements consisting of scandium (sc), yttrium (y), and a lanthanide series of elements from lanthanum (la) to lutetium (lu). these elements have been used in magnetic alloy, fluorescent and hydrogen storage alloy. particularly cerium oxide nanoparticles are frequently used as a fuel additive and are incorporated in cosmetics formulation. the potential biological and environmental effects of these elements have not sufficiently been investigated. it has been demonstrated that ld s for these elements in oral and intravenous administration are in a range from several dozens to several thousands milligrams per kilograms, indicating that none of these elements has high toxicity. in the results of studies in which the biopersistence and the distribution of rare earth compounds in the body were investigated, for example, the compounds deposited in bones and teeth, and organs including lung, liver, spleen and kidney following intratracheal, oral, intravenous, and intraperitoneal administration. although the compounds deposited predominantly in the liver other than bones, it has also been reported that the distribution of the compounds in the lung and spleen increased when the dose was increased [ ] . the demand for indium compounds has been sharply increasing. the compounds have been used in the materials for transparent electrodes for flat panel displays. in japan, the cases of pulmonary interstitial pneumonia and pulmonary fibrosis have been reported in workers engaged in cutting and grinding of sintered indium-tin oxide (ito) and potentially having inhaled the dusts released from ito [ , ] . the biological effects of indium arsenic compounds and indium phosphorus compounds also have been investigated. the acgih has proposed a value of . mg / m for their tlv, and the value has been applied tentatively in japan. we have been experiencing amazing progress of the technology on the processing for the nanometer-sized materials. the applications cover even biomedical engineering in addition to information technology, material, environmental science, and energy production [ ] [ ] [ ] [ ] [ ] . as the result, many kinds of new materials have been designed, fabricated, and discarded. from now on, this movement will be accelerated and even more new functional materials will be distributed in the world. here, we should not forget the safety of those materials in the process of the production, usage, and discard. without this safety assessment, we will go into the same problems as that of asbestos just we are facing now. first of all, we have to conduct experiments to reveal the minimal concentration for emergence of the toxicity. in other words, we have to fix the standard value for the threshold concentration for each material first of all. if we do not fix it, we should not use the material at any concentration, which means any engineering process could not be carried out. the applications in the various fields have started all over the world, and the safety assessment is urgently needed. in this article, we introduce the methods of the safety assessment of the semiconductor nanoparticles and describe the safety and the threshold depending on the surface treatment. the production process and the surface treatment play one of the most important roles for the safety of the nanoparticles. cd and se semiconductor nanoparticles coated with zns are one of the most widely used for the strong intensity of the fluorescent activity. cd oxide and se compounds once dispersed in the tri-octhil-phosphine oxide (topo) heated up to Њc generate nanoparticles by self-assembly. then zns enhances the stability of the structure and raises up the fluorescent intensity than without the coated one. nanoparticles, thus manufactured, as materials for a novel memory in the field of intelligence technology (it), and as super-micro devices for laser in the field of optics, have been developed all over the world [ ] [ ] [ ] [ ] [ ] . these nanoparticles cannot be dissolved into water, but dissolved into organic solvents like toluene. therefore, for biological and medical applications, various technologies for surface-conjugations to make them hydrophilic [ , ] have been developed. for example, nanoparticles covered with topo are hydrophobic because an alkyl group on them is hydrophobic. therefore, a technology for replacing this alkyl group with hydrophilic carbonic acid (making the whole particles soluble in water) has been developed [ ] . nanoparticles, thus surface-treated, can be dissolved into water, like sodium salt or potassium salt. with this method, various kinds of materials have been surface-conjugated. the mtt assay method is a way to evaluate the hazard assessment of nanoparticles, in which the activation metabolism in a mitochondrium in a cell is measured and the influence of nanoparticles on the prolification of the cell is qualitified. the mtt is a kind of tetrazolium, whose molecular formula is c h brn s. taken into a cell, it is decomposed by a dehydrogenase enzyme in a mitochondrium into a pigment called 'hormazan'. the measurement of the fluorescence intensity of the pigment shows the prolification of the cell [ ] [ ] [ ] . fig. . . . shows the hazard assessment of vero cells and kidney cells of the african green monkey against cdse/zns nanoparticles. the horizontal axis shows the concentrations of the nanoparticles and the vertical axis the fluorescence intensity of the hormazan at nm, that is, the metabolism of the cells. the figure indicates that cyto-toxicity is not observed for cells when the concentration is less than . m. this result means that this concentration is the threshold of the cell toxicity. likewise, cyto-toxicity is not observed for the hela cells and for the human primary cells. further, in order to find out how the sizes of nanoparticles influence the cyto-toxicity, the cyto-toxicities were evaluated with three kinds of quantum dots; one whose fluorescence wavelength is nm, red, one with nm, yellow, and the other with nm, green. the following results were obtained. the largest quantum dots whose fluorescence wavelength is nm show a tendency to give cyto-toxicity. cyto-toxicity is observed at concentrations more than m [ ] . another method to evaluate cyto-toxicity is the flow cytometry [ ] . the mtt assay alone cannot tell whether the toxicity observed is lethal to the cells or just restrains the prolification of them. in the flow cytometry, the nuclei of dead cells are dyed with propidium iodide (pi) after the nanoparticles are taken in and the ratios of the dead cells are measured. fig. . . shows the lethal cyto-toxicity of mua conjugated nanoparticles ( nm, green) against vero cells. the vertical axis indicates the numbers of the cells, and the horizontal axes show the fluorescence intensities and the cyto-toxicities. these experiments also show that dead cells cannot be observed at concentrations less than . m even though nanoparticles are taken in, as was shown in the mtt assay. however, at concentrations more than m, the nanoparticles taken in cause damage to more than the half of the cells. that is, the cyto-toxicity of mua quantum dots against cells is lethal [ ] . nanoparticles have been surface-conjugated for applications for various uses. some surface-conjugations cause more grave cyto-toxicity than others. therefore, relations between surface-conjugations and their safety for cells have to be considered. in order to find out the relations, the safety evaluations of nanoparticles surface-conjugated with two materials were made; one is with mua (quantum dots-cooh) and the other is with glycerol (quantum dots-oh), and their purified and unpurified particles. fig. . . shows that the purification reduces the cyto-toxicity for the quantum dots-oh, and that the toxicity remains the same after the purification for the quantum dots-cooh. mua itself, a material with which particles are conjugated, has cyto-toxicity. this experiment shows that toxicity against cells is connected not only with particles themselves but also with kinds of surfaceconjugations and degrees of purification. it is shown in the above experiments that the toxicity of nanoparticles is lethal to cells. next, we found out by the comet assay method whether the toxicity is derived from damaged dna. the method is a way to evaluate quantitative damage of dna by electrophoresis. the fragmented dna seeps out of their cells by treating cells, whose dna has been fragmented, with agarose-gel to break their cell membrane, and then electrophoresing them. it looks like the "tales of comets". cells, whose dna has not been fragmented, have their nuclei keeping their spherical shape after electrophoresis, and the tales of comets cannot be observed [ ] [ ] [ ] [ ] . fig. . . shows the results of the experiments with quantum dots conjugated with cooh (both purified and unpurified) to wtk- cells, a human lymphoblast mutation strain [ ] . the vertical axis shows the lengths of the tails, and the horizontal axis the concentrations of the quantum dots. the concentration of the nanoparticles is m. the results are as follows: the unpurified quantum dots with cooh caused damage to dna. on the other hand, the electrophoresis with the purified quantum dots does not show dna damage. this is probably because the dna damage has been repaired during the longer cultivation time. conducted with quantum dots conjugated with mua, and quantum dots-nh with an amino group. dna damage was not observed in either case. the results indicate that as far as particles themselves do not break down, the cyto-toxicity of quantum dots is derived from the chemical properties of the materials covering the quantum dots. the safety evaluation of nanoparticles has not been conducted sufficiently. as indicated above, the procedure for surface-conjugation could apply not only to cd/se nanoparticles but also to other nanoparticles. today, various kinds of techniques for surfaceconjugations have been available in academic papers, proceedings, and on the internet. some of them are widely known and others are patented. those techniques are all shared among the human race. even at this moment, the human beings are making breakthroughs in various fields and developing different kinds of technologies. sharing these technologies will lead to still more speedy developments of yet more advanced technologies. to achieve it, these technologies should be so structured that different fields, for example, bioimaging and biotechnology, structured on their own, can be linked to each other. such structured knowledge will play an essential part in merging different fields. in order to prevent nanoparticles release from a system so as to maintain environmental safety, the removal technique of nanoparticles must be established. in this section, separation techniques of particles from exhausted or suspended gas and liquid are described focusing on particles less than nm. generally, as shown in fig. . . , all particle separators for a dispersed system employ either one of three basic forms of particle separation. on the left hand side of the figure lie the separation methods in which particles are collected only by force field (electrostatic force, centrifugal force, gravity force, etc.), and the representative separator is electrostatic precipitator (esp). if some obstacles (collectors) are placed into the particle laden stream, particle separation is facilitated because particles are collected on obstacles with a smaller deviation from the fluid flow by the force exerting on the particles compared to the case without obstacles. typical collectors of this form are air filter, deep bed filter, etc. on the right hand side of the figure lie separators that collect particles utilizing only sieving effect of obstacles without any force field. in this case, geometrical size of channel between the obstacles must be smaller than that of particles. membrane filter, fabric filter, etc. belong to this group. when we apply the above collection forms to nanoparticles, the major collection mechanisms are brownian diffusion and electrostatic force for particles in gas, while sieving effect and interception/adhesion forces for those in liquid. as mentioned above, most airborne particles are collected by separators utilizing various kinds of forces such as gravity, centrifugal force, electrostatic forces, inertia, brownian diffusion force, and so on. therefore, the migration velocities or displacement of a particle per second due to the individual forces gives the basis for the comparison of removal efficiencies due to each force. in fig. . . , migration velocities of particles due to various forces are depicted against particle diameter at normal temperature and pressure for particle density of g/cm [ ] . as seen from the figure, the velocities due to gravity, centrifugal force, and inertia monotonically decrease with decreasing particle diameter, suggesting that the removal of nanoparticles with these forces is difficult. on the contrary, the velocities due to brownian diffusion and electrostatic forces increase with decreasing particle diameter for particles less than nm. this suggests that brownian diffusion and electrostatic forces are most effective in collecting nanoparticles. fig. . . summarizes typical conventional dust collectors. among them, the effective collectors for nanoparticles are esp and fabric/air filter. however, for the case of esp, which relies on only electrostatic force, nanoparticles (Ͻ nm) fail to carry even one electron resulting in low collection efficiency. in this case electrically charged filters are effective because we can expect the combined effects of electrostatic forces and brownian diffusion. among charged filters, so-called electret filter, which consists of permanently polarized fibers, is the most favorable filter because of its charge stability. particle penetration data of electret filter are plotted against particle diameter in fig. . . and compared with that of uncharged filter with the same structure. for the three combinations of charged states of fiber and particle there exist the most penetrating particle diameters. for the uncharged fiber, collection efficiency of uncharged particle has a minimum at nm and increases with decreasing particle diameter, showing that nm is the most penetrating particle size. for the charged fiber, particle collection efficiency is very high even for uncharged particle, and the efficiency for charged nanoparticles is extremely high because of strong coulombic force between fiber and particle. the experimental data plotted in fig. . . are qualitatively in good agreement with the theoretical prediction following the particle size dependency on particle migration velocity (shown in fig. . . ). however, as particle size becomes smaller and comparable with the size of a molecule, particles may rebound on a collector surface, and the adhesion probability of particles drops, resulting in a decrease in collection efficiency. fig. . . is an example of experimental data that confirm the particle rebound [ ] . the figure shows the penetration of nanoparticles through a grounded circular tube. the solid curve is the theoretical line derived by assuming that particles are deposited from a laminar flow in a tube by brownian diffusion. it is evident that experimental penetration deviates from the theoretical line for particles less than nm. this means that molecular behavior begins to appear when the particle size becomes as small as nm, and as a result, the collection efficiency is reduced. it should be noted that considerable amounts of nanosized particles are contained in diesel exhaust particles (dep), possibly penetrating through the honeycomb type (tubular channel) diesel particulate filters (dpf). there are two types of methods that differ in the way the nanoparticles in liquid are collected. the first group, called membrane filtration, constrains the particles by a membrane, and the liquid is allowed to flow freely through the membrane. in the second group of ultracentrifugation, the liquid is constrained in a rotating vessel, and the particles move freely within the liquid by an external field of acceleration caused by ultracentrifugal field. these methods have been quite extensively used in separation of macromolecules and molecules from liquid, and they are recently becoming important also in separation of nanoparticles from liquid. in pressure-driven membrane filtration processes, the pressure gradient across the membrane would force solvent and smaller species through the pores of the membrane, while the larger molecules/particles would be retained. membrane filtration processes are usually classified into three general categories according to the size of separating components, as shown in fig. . . . microfiltration (mf) is designed to retain suspended particles in the range of nm- m. ultrafiltration (uf), on the other hand, retains macromolecules or nanoparticles in the range of - nm (nominal molecular weight cut-off (nmwco) ranging from , to , , da). nanofiltration (nf) is a relatively new process that uses charged membranes, and it covers molecular sizes ranging from . to nm (nmwco ranging from to , ). it is useful in that it can separate dissociated forms of a compound from the undissociated form. one of the major factors limiting the use of membrane filtration is membrane fouling, resulting in a dramatic decline in flux with time of operation. to account for the membrane fouling, the resistancein-series model is frequently employed. the resistance model becomes ( . . ) where u is the permeate flux, v the filtrate volume per unit membrane area, the filtration time, p the applied transmembrane pressure, the viscosity of the permeate, r t the total resistance, r bm the resistance of the membrane per se plus the clogging of the membrane pores, r c the resistance of the filter cake, and r cp the resistance of the concentration polarization layer. significance of each resistance in membrane filtration is as follows. the membrane, even in the absence of any suspended particle, has a natural flow resistance. during membrane filtration, particles become attached to the pore channel of the membrane thereby reducing the flow channel dimension, or pores become blocked off completely. the last two effects lead to resistances that are due to adsorption and pore blocking. the blocking filtration model introduced by hermans and bredée [ ] , and grace [ ] is most commonly used as an interpretation of such phenomena. the clogging of the membrane pores is strongly influenced by such solution environment as ph and the ionic strength. the permeate flux of bovine serum albumin (bsa) (pi . , molecular weight , , stokes-einstein diameter . nm) solution by permeable mf membrane (nominal pore size . m) is lowest at around the isoelectric point [ ] . as the bsa molecule carries no net charge at the isoelectric point, the molecule is in its most compact state at that point. the bsa molecules deposit themselves rather densely onto the pore walls of the membrane to form a compact configuration with a smaller lateral electrical interaction between the molecules. as a result of this, the flow resistance increases markedly at around the isoelectric point. in dead-end membrane filtration, which has a feed and permeate stream, each with the same mass flow rate, the resistance of the filter cake plays a major part in the filtration resistance. therefore, the cake filtration theory can be applied, and thus the permeate flux u is described by ( . . ) where m is the ratio of wet to dry cake masses, s the mass fraction of solids in the solution, av the average specific filtration resistance, the density of the permeate, and v m the fictitious filtrate volume per unit membrane area, equivalent to the flow resistance of the membrane [ ] . for fine particle suspensions, colloidal forces which arise from interaction between the suspended particles control the nature of the filter cake. the average specific filtration resistance av and the average porosity av of the filter cake are strongly affected by the solution properties, including ph and electrolyte strength. for instance, in mf of suspensions of the titanium dioxide (pi . , the original mean specific surface area size nm), av goes through a minimum, and av is much larger near the isoelectric point [ ] , as shown in fig. . . . the titanium dioxide particles are destabilized around the isoelectric point where the van der waals attraction is more dominant. consequently, the particle tends to come together, that is, to flocculate, and the very porous flocs are then formed. thus, it is speculated that the filter cake formed from such porous flocs has often loose and wet structures. on the other hand, the filter cake becomes compact and dry when the particle carries the charge. since the most loose filter cake forms around the isoelectric ph, the filter cake is most permeable. it is interesting to note that the results in protein uf had a distinctly different behavior. in protein uf of bsa solution, the filter cake is in its most compact state around the isoelectric point [ ] , as shown in fig. . . . since the bsa molecules are hydrophilic colloids, their stability in the solution would appear to be influenced not only by the presence of a surface charge on the protein but also by hydration of the surface layers of the protein. the bsa molecules, because of hydrated layers surrounding them, are not destabilized by such consideration as depression of the electrical double layer. thus, the bsa molecules have water bound to them even around the isoelectric point. the hydrophilic bsa molecules maintain a dispersed state in the solution due to hydration of the surface layers of the protein even around the isoelectric point. when a bsa molecule acquires a charge, the filter cake becomes loose and wet due to electrostatic repulsion between the charged bsa molecules. this contrasts to the compact filter cake around the isoelectric point. the average specific filtration resistance av has a definite maximum around the isoelectric point since a compact filter cake provides a large hydraulic flow resistance. most membrane filtration processes are operated in the cross-flow mode, in which the feed is moved tangentially to the membrane surface so that the filter cake is continuously sheared off. during membrane filtration, particles in the feed are brought to the upstream surface of the membrane by convective transport, and this results in a higher local concentration of the rejected particles at the membrane surface as compared to the bulk solution which is referred to as concentration polarization [ ] . the particle concentration in the solution adjacent to the membrane varies from the value at the membrane surface, c m , to that in the bulk feed solution, c b , over a distance equal to the concentration boundary layer thickness . the resulting concentration gradient causes the particles to be transported back into the bulk solution due to diffusional effects. at steady state, the rate of convective transport of particle toward the membrane is balanced by the rate of particle transport through the membrane plus the rate of the diffusive back transport of particle. thus, the permeate flux u is given by ( . . ) where cp is the particle concentration in the permeate, k (ϭd/ ) the mass transfer coefficient, and d the diffusion coefficient. the osmotic pressure model assumes that the deviation from pure water flux occurs solely due to the osmotic pressure difference across the membrane, and thus the permeate flux u is given by ( . . ) where is the osmotic pressure, which is a function of the concentration. equation ( . . ) means that the effective driving force across the clean membrane is pϪ{ (c m )Ϫ (c p )}. replacing pϪ{ (c m )Ϫ (c p )} by the hydraulic pressure at the membrane surface, p m , equation ( . . ) reduces to the cake filtration equation. to minimize the effects of cake buildup and concentration polarization, membrane filtration is usually conducted using the cross-flow geometry in which the feed flow is parallel to the membrane and perpendicular to the filtrate flow [ ] . however, especially in mf the energy requirements associated with pumping the feed (plus any recirculation flow) along the membrane surface are typically very high. thus, there have been some innovations in recent years with cakeless filtration. the rotating disk module in which the membrane disk is stationary is suited for large-scale operation [ ] . it is possible to enhance the permeate flux by using the vibrating modules [ , ] . in the rotating cylinder device with the membrane on the inner rotating cylinder, counter-rotating taylor vortices within the annular gap are available [ , ] . dean vortices that twist and spiral in the direction of flow inside a highly curved channel, similar to vortices in rotary modules can result in enhanced flux [ ] . these vortices, or flow instabilities, induce turbulence into the system. periodic removal of the formed filter cake is also effective for enhancing the permeate flux. recently, several methods have been investigated: back washing using the filtrate or air pressurization [ ] , periodic rotation of the cylindrical membrane [ ] , pulsatile flow [ ] , high-frequency transmembrane pressure pulsing with a frequency around . - hz [ ] . dead-end upward filtration, where the filtrate flow is in the opposite direction to gravity, and dead-end inclined filtration, where the membrane is inclined, can reduce the cake formation onto the membrane in uf of nanoparticulate suspension and protein solutions. in upward uf of silica sol (mean diameter . nm) and bsa solution, a sustained permeate flux is achieved, as shown in fig. . . [ , ] , because the filter cake overlying the membrane is exfoliated continuously under the gravitational force acting on the particles comprising the filter cake. another approach for enhancing the permeate flux is to employ external force fields. electrofiltration, in which an applied electric field is used to drive charged particles away from the membrane surface, has been developed. in electrofiltration, the accumulation of the particles on the membrane surface is limited by the imposed electrophoretic force. in addition, the permeate flux through the filter cake is dramatically enhanced due to electroosmosis as a secondary electrokinetic phenomenon. this method can be applied to a broad combination ranging from mf of particulate suspension such as bentonite [ ] to uf of protein solution. fig. . . shows the reciprocal permeate flux (d /dv) versus the permeate volume per unit membrane area, v, for various values of the strength of the dc electric field, e [ ] . the steady permeate flux increases noticeably with the magnitude of the imposed field strength. also, a higher electric field strength causes the permeate flux to equilibrate more rapidly. a method has been developed for removing humic substances by hybrid uf combined with both flocculation and adsorption treatments, as shown in fig. . . [ ] . flocculation by use of polyaluminum chloride (pacl) is particularly effective for the removal of humic acids, which constitute the relatively high molecular weight fractions of humic substances, whereas adsorption by use of powdered activated carbon (pac) is able to remove fulvic acids of relatively low molecular weight effectively, which cannot be fully flocculated by pacl. hybrid uf in combination with flocculation and adsorption treatments exhibits high permeate quality because the flocs and pac are easily retained by the uf membrane. in ultracentrifugal sedimentation, ultracentrifugal force field of several tens of thousands of revolutions per minute is applied to a rotor. in recent years, ultracentrifugal sedimentation is employed for concentrating dilute protein solutions and for separating proteins and other large biological molecules from low-molecular-weight solutes or from much larger particles. fig. . . shows the results for ultracentrifugal sedimentation of an aqueous solution of the mixtures of bsa and egg white lysozyme (pi . , mw , ) measured using schlieren optics in an analytical ultracentrifuge [ ] . the angular acceleration of the rotor is , rad/s. the symbol r i and r i in the figure represent the distances from the center of rotation to the sedimentation boundary at time and , respectively. the electrical nature of macromolecules plays a significant role in determining the sedimentation behavior in ultracentrifugation of binary protein mixtures. in the ph range where both protein molecules were electropositive, the molecules sediment independently due to the electrostatic repulsive force acting between bsa and lysozyme molecules. denpun kagaku filtr. sep institute for quality assurance and certification: basic criteria for the award of the environmental label (printer ral-uz ) proc. air cleaning contam proc. air cleaning contam proc. air cleaning contam chemical contamination in semiconductor processing environments and its countermeasures the japan society of industrial machinery manufactures: report on behavior control of individual sort of contaminants - report on introduction of advanced technologies to environmental equipment industry industrial application of nanomaterials -chance and risks. future technologies, division of vdi technologiezentrum nanoscience and nanotechnologies: isbn recommendation of occupational exposure limits who: environmental health series . world health organization summaries & evaluations -nickel and nickel compounds rapid colorimetric assay for cellular growth and survival ouyou earozorugaku crossflow filtration: theory and practice encyclopedia of fluid mechanics: slurry flow technology filtr. sep key: cord- -ihh q f authors: nan title: posters p - p date: - - journal: eur biophys j doi: . /s - - -x sha: doc_id: cord_uid: ihh q f nan in order to understand mrna stability, we proceed to the studie of life time model system composed of the regb ribonuclease and the ribosomal protein s . the t bacteriophage life cycle is modulated by regb which mediate speci c mrna degradations. regb cleaved the mrna at the middle of the translation initiation region (shine-dalgarno sd). studies had shown that regb activity is enhanced with addition of s which is normaly required for the translation of mrna in case of unusual sd regions. s is considered as a key factor of translation's inititiation. composed of six similar domains (f -f ), it interacts with the ribosome by his f -f domains and with the mrna by f -f . we had shown that the f fragments got the same properties than the whole protein. many global architectures had been published (linear or globular conformation). then we try to understand what is the catalysis way of s in the regb activation and in rna recognition. we study the global conformation of f fragment. for that, we use nmr to determinate the domains interfaces. at rst we made the nmr backbone assignement of n/ c/ h labeled fragments (f -f ). then we analysed hsqc overlapping of each fragments in order to identify interfaces residues. the residues identi cation on s homologous model allowed us to determinate interaction region between domains. currently we study of s -rna interactions to determinate if the ligand conformation could change the interaction region and involve conformationals changes. translocation of amino acids from the membrane interface to the interior: theory and experimiment c. aisenbrey , e. goormaghtigh , j.-m. ruysshaert , b. bechinger ulp/cnrs, chemistry, rue blaise pascal, strasbourg, université libre, campus plaine cp / , brussels the interactions of a series of histidine-containing peptides with biological model membranes have been investigated by attenuated total re ection fourier transform infra red (atr-ftir) and oriented solid-state nmr spectroscopies. related peptides have previously been shown to exhibit antibiotic and dna transfection activities. the -residue lah x and lah x peptides were designed in such a manner to form amphipathic helical structures in membrane environments. four histidines and four/six variable amino acids x constitute one face of the helix whereas leucines and alanines characterize the opposite hydrophobic surface. the dichroic ratio of atr-ftir spectra, or the orientation-dependent n solid-state nmr chemical shift have been used to follow the ph-dependent transition from in-plane to transmembrane alignments upon increase in ph. a theoretical model of the topological modulations is presented and the experimental transition curves analysed in order to reveal the gibbs free energy of transition. the novel concept provides access to the free energy changes associated with the amino acids x incorporated into an extended -helix and in the context of phospholipid bilayers. for the peptides of the lah x series the gibbs free energies associated with the transition from the membrane interface to the bilayer interior follow the sequence of amino acids: l < a i < s f < t g < v w << y. here we present a novel solid-state nmr approach which allows for the accurate determination of the tilt and rotational pitch angles of peptides reconstituted into uniaxially oriented membranes. the method works with transmembrane or in-plane oriented peptides that have been labelled with , , - h -alanine and n-leucine at two selected sites. proton-decoupled n and h solid-state nmr spectroscopy at sample orientations of the membrane normal parallel to the magnetic eld direction have been used to characterize the tilt and rotational pitch angle of these peptides in considerable detail. the same samples when inserted into the magnetic eld at degrees tilted alignments provide valuable information on the rotational diffusion constants in membranes and thereby of the association and size of peptide complexes within the membrane environments. whereas monomeric transmembrane peptides exhibit spectral averaging and well-de ned resonances, larger complexes are characterized by broad spectral line shapes. in particular the deuterium line shape is sensitive to association of a few transmembrane helices. in contrast, the formation of much larger complexes affects the n chemical shift spectrum. aisenbrey ch. & bechinger, b. biochemistry , - ( ) a meccano set approach of joining trpzip a water soluble -hairpin peptide with a didehydrophenylalanine containing hydrophobic helical peptide p. chetal, v. chauhan, d. sahal international centre for genetic engineering & biotechnology,new delhi ,india a residues long, water soluble, monomeric -hairpin peptide "trpzip" [cochran et al ( ) pnas , - ], stabilized by tryptophan zipper has been linked via a tetraglycyl linker to a hydrophobic didehydrophenylalnine ( f) containing helical octapeptide. circular dichroism studies of this residues long peptide, "trpzipalpha" (ac-gewtwddatktwtwte-gggg-fal fal fa-nh ) in water have revealed the presence of both the -hairpin and the helical conformations. this is the rst instance where a f containing peptide has been found to display a helical fold in water. the uorescence emission wavelengths of tryptophan in ac-g-w-g-nh , trpzip and trpzipalpha were . , . and . nm respectively. the uorescence quantum yield of trpzip was . fold higher than trpzipalpha suggesting that proximal interactions between the -hairpin and the helix caused the quenching of tryptophan uorescence in the former by the fs in the latter. the molar ellipticity of the far uv couplet characteristic of trpzip was reduced in trpzipalpha and the cd based thermal melting temperatures at nm were c (trpzip) and c (trpzipalpha). a concentration dependent variable temperature cd study in water showed that in trpzipalpha, increasing temperature is detrimental to the -hairpin, but it augments the helical motif by intermolecular oligomerization. our results show that in water, trpzipalpha exhibits long-range interactions between two different secondary structures. structural-based differential stability in the yoeb-yefm toxin-antitoxin module i. cherny, e. gazit department of molecular microbiology and biotechnology, faculty of life sciences, tel aviv university, tel aviv , israel the speci c physiological role of natively unfolded proteins is only recently beginning to be explored. a notable case in which natively unfolded state appears to have physiological signi cance is the e. coli yoeb-yefm toxin-antitoxin (ta) module. a crucial element in proper functioning of ta systems requires physiological instability of the antitoxin in contrast to the stable pro le of its toxin partner. we have shown that yefm antitoxin is a natively unfolded protein, lacking secondary structure even at low temperatures. in contrast, its toxin partner has a well-folded conformation at physiological temperatures. we suggest that the structural-based differential thermodynamic stability between the two components is the cause for their differential physiological stability, since structural instability of the antitoxin exposes it to cellular quality-control machinery. we further revealed that yefm and yoeb interact and form tight complex and determined it stoichiometry. a potential use of ta systems is as novel antibacterial targets. indeed, we identi ed homologous yefm-yoeb systems in a large number of bacteria including major pathogens. we aim to design peptides capable of interfering with the yefm-yoeb interaction, thus releasing the toxin to execute its detrimental function. for this purpose, we identi ed a short linear determinant within yefm that is involved in yoeb interaction. this peptide motif will be optimized for development of antibacterial lead molecules. a. chatterjee, a. prabhu, a. ghosh-roy, r. v. hosur tata institute of fundamental research, mumbai, india- dynein light chain (ddlc ), a member of the cytoplasmic motor assembly exists as a monomer or a dimer (functional form) under different experimental conditions. here we report the unfolding characteristics of the monomeric ddlc at ph , due to urea and guanidine hydrochloride, by various biophysical techniques. it is observed that the unfolding pathways due to the two denaturants have many differences. urea unfolding seems to be two state, while guanidine unfolding is more complex. the nmr experiments carried out at low denaturant concentrations have enabled detailed characterization of the structure and dynamics of the near native excited states of the protein. these are similar to the native state in structure, except for the small extensions of the helices in the nterminal half of the protein. however, the local stabilities of the and -strands are perturbed and this occurs differently in the two denaturants. in the guanidine case the entire multi-stranded -sheet in the c-terminal half is destabilized. in either case the motional characteristics, seem to suggest the presence of a nite population of the dimer in the excited state ensemble. these states are suggested to be likely intermediates in the momoner-dimer transition, and their characterization here thus provides clues to the molecular mechanism of the transition. it is also envisaged that the near native excited states could play regulatory roles in the functioning of the protein. kinetic bottlenecks identi cation in different folding models f. cecconi , c. guardiani , r. livi infm -istituto di sistemi complessi -cnr, italy, centro interdipartimentale per lo studio delle dinamich complesse, università di firenze, italy, dipartimento di sica, università di firenze, italy the ww domains are a family of fast folding, compact, modular domains featuring a triple-stranded, antiparallel beta-sheet. the ww domain of the pin protein, due to the availability of a complete picture of the residues involved in thermodynamic stability and in the formation of the transition state, in particular, represents an excellent benchmark to test computational methods. the objective of the present work is to identify the kinetic bottlenecks in the folding process through md unfolding simulations at increasing temperatures. the kinetic bottlenecks are related to the establishment of contacts requiring the overcoming of a large entropy barrier and acting as a nucleus for the creation of further contacts. the key sites are therefore those involved in contacts showing a dramatic decrease in fractional occupation near the speci c heat peak. the technique was applied to the go model and to a model based on the knowledge of secondary structure, providing in both cases a picture of the folding process consistent with the experimental data. evidence is also shown that while the go model allows a more accurate prediction of the native structure, the folding pathway is better described by the other model. the protein talin plays a key role in coupling the integrin cell adhesion molecules to the actin cytoskeleton and in integrin activation. the globular head of talin, which binds -integrins, is linked to a rod containing an actin-binding site and binding sites for the protein vinculin, which regulates the dynamic properties of cell-matrix junctions. we have determined the structure of three domains which contain vinculin binding sites (vbss) and shown that each of these are made up of helical bundles. the structures of complexes between vinculin and peptides corresponding to the vbss show that the residues which interact with vinculin are buried in the hydrophobic core of the helical bundles of the talin domains. nmr studies of the interaction of one of these domains with vinculin shows that it involves a major structural change in the talin fragment, including unfolding of one of its four helices, to make the vbs accessible. while the observation of folding of unstructured regions of a protein on interaction with a 'partner' is quite common, this kind of major unfolding to permit a protein-protein interaction is much less common. ways in which it may be regulated will be discussed. conformational changes of eye lens proteins studied by combined saxs and high pressure s. finet , f. skouri-panet , a. tardieu european synchrotron radiation facility, rue horowitz, bp , grenoble france, institut de minéralogie et de physique des milieux condensés, rue de lourmel, paris france, protéines: biochimie structurale et fonctionnelle, case , quai st-bernard, paris france -, -and -crystallins are the main components of vertebrate eye lenses, with exceptional structural and associative properties. the crystallins are known to be exceptionally stable in vivo since they have to last the lifetime of the organism. they therefore represent an extreme case of stability versus unfolding and aggregation. these proteins are mainly beta strands. -crystallins are kda monomers (from to % sequence identity), and -crystallins are large hetero-oligomers of about kda. -crystallins are molecular chaperone; they belong to the ubiquitous superfamily of small heat shock proteins, shsps. here, the conformation and the stability of -and -crystallins were investigated by small angle x-ray scattering (saxs) and high pressure, depending upon temperature and ph. at room temperature, -crystallins have shown a partially reversible change in size from to kb, and this effect was enhanced by the combination of temperature and pressure. in the case of -crystallins and in the pressure range up to kb, the pressure was combined with temperature and ph. the results depend upon the different itself. structural studies on pore-forming peptides s. m. ennaceur , d. bown , j. m. sanderson chemistry department, university of durham, biology department, university of durham the resistance of pathological microorganisms to conventional antibiotic drugs has created a need for new antibacterial agents. biologically active antimicrobial peptides that act as primary defense agents in a large variety of species are thought to have the potential as precursors for a new range of drugs from antibiotics to cancer treatments. this study has attempted to analyse the structural properties of membrane peptides and proteins through the use of model systems that have been designed to mimic their natural counterparts: we have successfully synthesised model membrane peptides with a beta-sheet structural motif and have used a wide range of techniques to analyse their interactions with phospholipid (pc) membranes. the synthetic peptides were very hydrophobic and only soluble in uorinated alcohols such as hfip and to a lesser extent tfe. we found hfip to have a very strong af nity for pc membranes and carried out a series of experiments to investigate this af nity. binding of hfip to pc membranes was found to be reversible and we exploited this property in d crystal trials of our synthetic peptides. we over expressed the c-terminal domain of brka, a gram negative autotransporter protein, which forms a beta-barrel channel in the outer membrane (omp), for comparison with our model peptides. we performed d crystal trials on the omp and imaged the resulting protein arrays by stem and afm. a protein spontaneously folds into a unique native structure in physiological conditions. this process accompanies a huge loss of the conformational entropy (ce). our major concern is to specify the factor that can compete with the ce loss. the previous discussions concerning protein folding have been focused on contributions to the free energy of folding from the interaction potentials in a system. a view lacking in earlier studies is that the folding is critically in uenced by the translational movement of water molecules. when solute particles contact each other in a solvent, the excluded volumes for the solvent molecules overlap, and the total volume available to their translational movement increases. this leads to a gain in the translational entropy (te) of the solvent. this type of te effect should be much stronger in protein folding where the tight packing of the side chains occurs. an elaborate statistical-mechanical theory is employed to analyze the te of water in which a peptide or a protein molecule is immersed. it is shown that the te gain upon folding is large enough to compete with the ce loss. when water is replaced by another solvent whose molecular size is larger, the te gain decreases to a remarkable extent. we suggest that the entropic loss accompanying the self-assembly and the formation of ordered structures in a living system is compensated mostly by the te gain of water, highlighting an aspect of the crucial importance of water in sustaining life. domain ii of ribosome recycling factor is required for disassembly of the post-termination complex p. guo, l. zhang, y. feng, g. jing institute of biophysics, chinese academy of sciences, national laboratory of biomacromolecules, beijing, china ribosome recycling factor (rrf) consists of two domains and, in concert with elongation factor ef-g, triggers dissociation of the post-termination ribosomal complex. however, the exact function of the individual domains of rrf remains unclear. to clarify this, two rrf chimeras, ecodi/ttedii and ttedi/ecodii, were created by exchanging the rrf domains between the proteins from escherichia coli and thermoanaerobacter tengcongensis. the ribosome recycling activity of the rrf chimeras was compared with their wild-type rrfs by using in vivo and in vitro activity assays. the experiments show that like wild-type tterrf, the ecodi/ttedii chimera fails to complement the rrf ts phenotype of e.coli lj (frr ts ) strain and has no polysome breakdown activity. however, under the same conditions, the ttedi/ecodii chimera complements the rrf ts phenotype and has polysome breakdown activity equivalent to that of wild-type ecorrf. the results indicate that domain ii of rrf is the functional domain that is mainly responsible for the disassembly of the post-termination ribosomal complex, and the speci c interaction between rrf and ef-g on the ribosomes mainly depends on the interaction between domain ii of rrf and ef-g; while domain i of rrf is the main contributor for binding ribosomes and maintaining the stability of the rrf molecule. this study provides direct genetic and biochemical evidence for the assignment of individual functions of rrf domains. self-assembly of natural somatostatin into liquid crystalline nano brils w the natural neuropeptide somatostatin- is a cyclic tetradecapeptide hormone, with broad inhibitory effects on both endocrine and exocrine secretions. we report the self-assembly of somatostatin in solution, into stable liquid crystalline nano brils, based on the neuropeptide bioactive backbone conformation. the system was studied as a function of peptide concentration, milieu composition and time, using optical and electron microscopy, x-ray scattering, vibrational spectroscopy and sec/rp-hplc. in pure water, the formation of twisted nano brils (around nm wide and a few microns long) was characterized. their structure relies on the native somatostatin -hairpin and on intermolecular antiparallel -sheets networks. the nano brils were observed to laterally associate further with increased concentration and time, as well as to generate hexagonal phases. increase in ionic strength (sodium chloride, phosphate) was found to signi cantly favor the self-association process. the soft conditions of formation of the somatostatin nano brils support biological relevance, for instance to the biological mechanism of storage of the neuropeptide hormone. unraveling the physical origin of the structure of fully denatured ubiquitin s. golic grdadolnik, f. avbelj national institute of chemistry, hajdrihova , ljubljana, slovenia the structure and dynamics characterization of non-native states of proteins is crucial for understanding the mechanism of protein folding. recently many experimental studies have shown variations of conformational propensity and exibility along the backbone chain of fully denatured proteins. it has been supposed that areas of residual structure may serve as initiation sites of protein folding. however, the physical origin of these variations is still unclear. we analyze the structure of fully urea-denatured ubiquitin. the experimental veri cation of conformational propensities of protein backbone is obtained through structurally dependent nmr parameters. although the secondary structure of ubiquitin under strong denatured conditions is not detectable and no correlation with the native overall topology is found, the variations of nmr parameters along the backbone follow the secondary structure elements of its native state. we show that these variations are in accord with the recently developed electrostatic screening model of denatured proteins ( ). in this model, the backbone conformations of residues in unfolded protein are determined by local backbone electrostatic interactions and their screening by backbone solvation. many virulence factors from gram-negative bacteria are autotransporter proteins. the nal step of autotransporter secretion is c nterminal threading through the outer membrane (om), followed by folding. this process requires neither atp hydrolysis nor a proton gradient. pertactin, an autotransporter from bordetella pertussis and the largest -helix structure solved to date, folds much more slowly than expected based on size and native state topology, yet folding intermediates are not aggregation-prone. equilibrium denaturation results in the formation of a partially folded structure, a stable core comprising the c-terminal half of the protein. examination of the pertactin crystal structure does not reveal the origin of the enhanced c-terminal stability. yet sequence analysis reveals that, despite size and sequence diversity, all autotransporters are predicted to fold into parallel -helices, suggesting this structure may be important for secretion. for example, slow folding in vivo could prevent premature folding of in the periplasm prior to the assembly of the om porin. moreover, extra stability in the c-terminal rungs of the -helix may serve as a template for the formation of the native protein during secretion, and formation of the growing template may contribute to the energy-independent translocation mechanism. coupled with the sequence analysis, these results suggest a general mechanism for autotransporter secretion. thermal and functional properties of e.coli outer membrane protein-receptor fhua fhua is e.coli outer membrane protein, which transports iron into the bacterial cell and also serves as receptor for several phages. in order to get more deep information about structural properties of fhua, we've studied its thermal properties by means of calorimetry. we've also investigated the interaction between t , ira phages and directly fhua by means of viscometry. the calorimetric result of heat denaturation of membrane protein fhua and next deconvolution of the recorded calorimetric curve with two transitions has shown that in a chosen conditions the structure of fhua consists of domains. though both t and ira phages grow on the common bacterial strain (e.coli k ho ), expressing fhua the results of viscometric investigation show that under direct interaction of phages with fhua the receptor activity of protein revealed only for t phage. therefore, we conclude that other than fhua protein serves as receptor for ira phage. it should be mentioned that the phage dna ejection process induced by receptor was observed for the rst time by us in an incessant regime. electron spray ionization mass spectroscopy (esi-ms) is a powerful tool for the investigation of the protein folding or proteins non-covalent interactions in solution since charge state distributions (csds) in esi-ms are affected by the conformational state and mass relates on the association state. we used this tecnique to inquire at different ph and different conditions the dimerization process of the porcine and bovine -lactoglobulins that share a high sequences similarity and close d structures. dimerization oflactoglobulins is reversible and involves both electrostatic and hydrophobic interactions. it was possible to detect simultaneously both the monomeric and dimeric form of the proteins in solution, pointing out the different dimerization behaviour of the two isoforms. we assessed the maximal stability of the dimeric structure at ph for the porcine protein and ph for the bovine one. moreover we showed that bovine lactoglobulin has a stronger dissociation costant than the porcine protein. further we showed that it is possible to modulate the dimerization equilibrium of the bovine isoform at ph both increasing temperature and adding methanol without inducing denaturation of the protein. a possible novel method of protein structure prediction; a. ikehata division of structural bioinformatics science of biological spramolecular systems graduate school of integrated sciences mechanism of protein folding has been mysterious since an nsen's dogma was sugested.here i would like to propose a possible novel method of protein structure prediction; origami method. the method comes from a protein backbone property of uctuation and the residue hydrophobicity. l. marsagishvili , m. shpagina , z. podlubnaya institute of theoretical and experimental biophysics ras, pushchino state university amyloid brils are formed by proteins or their peptides in the result of a conformational transition from alpha helix into beta-sheet structure. despite the different nature of proteins-precursors their amyloids have common properties: beta-pleated sheet structure with individual beta-sheets oriented parallel to the main axis; insolubility in vivo; speci c binding to congo red and thyo avin-t. amyloid deposits are observed in different diseases such as myositis, myocarditis, cardiomyopathies and others. we showed that sarcomeric cytoskeletal proteins of titin family (x-, c-, h-proteins) of rabbit skeletal muscles are capable to form amyloid brils in vitro. these proteins already contain % of beta-sheet structure necessary for formation of amyloids. the amyloid nature of their brils was conrmed by electron, polarization and uorescence microscopy. as x-, c-, h-proteins form amyloid brils easily in vitro, there is a danger of fast growth their amyloid deposits in vivo. taking into account common properties of amyloids formed by different proteins, our results clear the ways for conducting by amyloidogenesis in human organs and tissues. work is supported by rfbr grants - - , "universities of russia" . . and program of the presidium ras "fundamental sciences for medicine". probing the folding capacity and residual structures in -and -residue fragments of staphylococcal nuclease d. liu, x. wang, y. feng, l. shan, j. wang national laboratory of biomacromolecules, institute of biophysics, chinese academy of sciences n-terminal fragments of staphylococcal nuclease (snase) with different chain lengths were used as a model system in the folding study. the detailed characterization of conformational states of - and - residues snase fragments (snase and snase ) and their v w and g w mutants can provide valuable information on the development of conformations in the folding of snase fragments of increasing chain lengths in vitro. in this study, the presence of retained capacity for folding and residual structures in snase and snase is detected by cd, uorescence, ftir, and nmr spectroscopy. snase is represented as an ensemble of interconverting conformations. the uctuating nascent helix-and âsheet-like structures, localized in regions of a -a and t -v , respectively are transiently populated in snase . the native-like tertiary conformations are obtained for g w and v w and for snase in the presence of . m tmao. analysis of the results of such studies indicate that folding of snase fragments is dominated by developing the local and non-local nucleation sites from native-like secondary structures and by intensifying the longrange interactions of residues at nucleation sites with residues further removed in sequence. thermal disruption of a spanning network of hydration water and conformational changes of elastin a. krukau , i. brovchenko , a. oleinikova , a. geiger international max-planck research school in chemical biology, otto-hahn str. , d- , germany, physical chemistry, university of dortmund, otto-hahn str. , d- , germany hydration water strongly in uences the structural and dynamical properties of biomolecules. the existence of a spanning hydrogenbonded network formed by the hydration water enables the function of biomolecules at low hydration levels. we can expect that the formation/disruption of the spanning network formed by the hydration water in solution also affects crucially the protein properties. we present the rst computer simulation study of the thermal disruption of a spanning network formed by the hydration water of a biomolecule (elastin-like peptide). this process obeys the laws of d percolation transition, similarly to the formation of a spanning water network with increasing hydration level [ ]. the spanning water network transforms into an ensemble of small water clusters with increasing temperature: it is still permanent at k and exists with probability % at k. in the same temperature interval, the conformation of the peptide changes noticeably: its radius of gyration increases sharply (by %) at about k. these two phenomena may be related to the "inverse temperature transition" at about k, where an elastin solution separates into two phases. in our simulations, the displacement of hydration water by the addition of a denaturant (urea) or by other peptide molecules causes an even stronger increase of the radius of gyration (up to % lipidic cubic phases formed by distinct water and lipid volumes provide bicontinuous d bilayer matrices that have speci c and controllable water channel sizes and large surface areas. these systems have proven to be also valuable as membrane mimetic structures, as promising matrices for controlled-release and delivery of proteins, vitamins and small drugs in pharmacological applications, and they offer a d lipid matrix for successful crystallization of membrane proteins which do not easily crystallize in bulk solution. the present study is directed towards a better understanding of the interplay between curved cubic lipid phases and the protein entrapped within their aqueous channel structures. as model systems, we have chosen a cubic ia d phase formed by an uncharged lipid, monoolein, and incorporated different proteins, such as cytochrome c, -chymotrypsin and insulin, in its narrow water channels. we show that the protein secondary structure and unfolding behaviour may be in uenced by the con nement and, vice versa, the topology of the lipid matrix may change as a function of protein size and concentration. in fact, even new cubic lipid structures may be formed that are not known in pure lipid systems. furthermore, we compare the aggregation scenario of insulin in bulk solution and in the narrow water channels of the cubic lipid matrix and discuss the differences found in terms of the geometrical limitations imposed by the con nement. after endocytosis, i.e. at acidic ph, the t domain inserts in the membrane of the target cell and helps the translocation of the catalytic domain into the cytoplasm. therefore, the t domain has a key role in the strategy of internalization of the toxin. the study of the interaction of the t domain with membranes and its ph dependence is important for a better understanding of the diphtheria toxin translocation mechanism. at least, two steps can be distinguished during the membrane insertion of the t domain. the rst step involves hydrophobic interactions with the membrane and is related to the ph-induced stabilization in a molten-globule state. in the second step, electrostatic interactions are preponderant and the ph-sensitivity comes from changes of the balance between repulsive and attractive electrostatic interactions. the role of the n-terminal part of the t domain in the second step has been investigated by studying peptides corresponding to the amphiphilic helices found in this part of the domain. the results are correlated with those obtained with a single trp mutant probing the n-terminal region of the whole domain. the translocation mechanism will be discussed in view of the physico-chemical properties of the peptides. in complex systems with many degrees of freedom such as peptides and proteins there exist a huge number of local-minimumenergy state. one way to overcome this multiple-minima problem is to perform a simulation in a generalized ensemble where each state is weighted by a non-boltzmann probability weight factor so that a random walk in potential energy space may be realized (for reviews, see refs. [ ] [ ] [ ] xas spectroscopy results show that there are two different structures of the metal binding site in the a peptide according to whether they are complexed with cu or zn ions. while the geometry around copper is suggestive of an intra-peptide binding with three histidine residues bound to the metal, the zinc site geometry is compatible with an inter-peptide aggregation mode. this result reinforces the hypothesis that assigns opposite physiological roles to the two metals, with zinc favouring and copper blocking peptides aggregation and consequent plaque formation. effect of pressure on the conformation of proteins. a molecular dynamics simulation of lysozyme a. n. mccarthy, r. grigera instituto de física de líquidos y sistemas biológicos (iflysib), conicet-unlp-cic, university of la plata, argentina the effect of pressure on the structure and dynamics of lisozyme was studied by md computer simulation at bar ( pa) and kbar using gromacs package. all-atoms (ff gmx) force eld were used for the minimization process and for all the md simulation and kept all protein bond lengths constrained (lincs algorithm). water molecules (spc/e model) were constrained using the settle algorithm. for the electrostatic forces we applied the reaction field method. lennard-jones interactions were calculated within a cut-off radius of . nm. the results have good agreement with the available experimental data, allowing the analysis of other features of the effect of pressure on the protein solution. the studies of mobility show that although the general mobility is restricted under pressure this is not true for some particular residues. from the analysis of secondary structures along the trajectories it is observed that the conformation under pressure is more stable, suggesting that pressure acts as a 'conformer selector' on the protein. the difference in solvent accessed surface (sas) with pressure shows a clear inversion of the hydrophilic/hydrophobic sas ratio, which consequently shows that the hydrophobic interaction is considerably weaker under high hydrostatic pressure conditions. direct observation of mini-protein folding using uorescence correlation spectroscopy h. neuweiler, s. doose, m. sauer applied laser physics & laser spectroscopy, university of bielefeld, bielefeld, germany the "trp-cage" motif represents the smallest and one of the fastest folding mini-proteins known to date. the globular fold is characterized by a hydrophobic core burying a single tryptophan (trp) residue. here, we report on the direct observation of trp-cage folding kinetics using uorescence correlation spectroscopy (fcs). our method is based on the selective uorescence quenching of oxazine dyes by trp which becomes ef cient only upon contact formation between the dye and the indole moiety of trp. by sitespeci cally labeling the dye to trp-cage, temporal uorescence uctuations of the dye-peptide conjugate, caused by intramolecular contact formation between dye and trp, directly report on conformational dynamics and folding transitions of the peptide chain. in order to measure uorescence uctuations directly in solution we used fcs on a confocal uorescence microscope setup. fcs allows us to reveal conformational dynamics with nanosecond timeresolution, under thermodynamic equilibrium conditions, and in highly dilute solutions (i.e. at nano-molar sample concentrations). our method con rms microsecond folding kinetics of the trp-cage motif, previously estimated with non-equilibrium temperature-jump techniques. we further investigated stability and folding rates under denaturing conditions and at various temperatures, giving further insight into structural transitions during the folding process. identi cation and mutagenesis of a region of tnt required for the stability of tnt-tni coiled-coil evolutionarily conserved heptad hydrophobic repeat (hr) domains present in troponin subunits tnt and tni are involved in alpha helical coiled-coil formation. using recombinant peptides from fast-skeletal tnt and tni, we examined the contributions of amino acid residues within these hr domains as well as anking these domains, to the stability of the coiled-coil interaction. a series of tnt fragments were tested for their ability to form coiledcoil with tni hr domain. we show that the tnt region - , although remains outside of the coiled-coil domain, is absolutely required for the stability of the coiled-coil. interestingly, the region tnt - contains few absolutely conserved residues that are potential candidate for ionic interaction, as predicted by molecular modeling. using single point mutants we show that among all the conserved residues, residue lysine is most important in stabilizing the coiled-coil interaction, whereas others play accessory role. we propose that the lysine initiates the stabilization of the coiled-coil interaction and then the other residues acts in a zipper like fashion. magnesium promotes conformational switching of ca sensor s. mukherjee, k. v. r. chary tata institute of fundamental research the importance of mg , one of the most abundant metal ion in the cell cytoplasm, relating to the calcium sensor mechanism is demonstrated. in this study it has been shown that the amino acid long calcium binding protein from entamoeba histolytica (ehcabp) can exist in three different forms namely, the calcium-free (apo) form, the magnesium bound (apo-mg) form and the calcium bound (holo) form. these three forms have been characterized using chromatographic, calorimetric as well as various spectroscopic techniques. there is a radical difference in the stability between the calcium free and ca bound forms. the calcium free form has molten globule like characteristics. mg stabilizes the closed conformation of the apo form, where the hydrophobic core remains buried. the presence of mg signi cantly alters the calcium binding cooperativity thereby increasing the cooperativity of the conformational switching between the open and closed conformation which is an important aspect of such regulatory proteins. a structural model for the molten-globular form of apo-ehcabp and its equilibrium folding towards completely folded holo state in presence and absence of mg will be presented. intermediate states of formin binding protein ww domain: explored by replica-exchange simulation y. mu school of biological science, nanyang technological university, singapore ww domain of formin-binding protein (fbp) is a model system for beta strand folding study. although it is small, only amino residues in total, the folding kinetics of fbp ww domain proved to be biphasic. an extensive molecular dynamics-monte carlo hybrid method, called replica-exchange simulation strategy is employed to study the folding/unfolding of the fbp ww domain in explicit water model. begin with randomly chosen conformations from high temperature unfolding trajectory distributed in replicas ( different temperatures covering from k to k), the simulation lasts nanoseconds in each replica. in the end an interesting distribution of conformations shapes up. we nd that there are three distinctive subgroups, one being the unfolded conformations with rmsd averaging around Å , one being native conformations with rmsd . Å, more interestingly, the third group having rmsd Å, an intermediate folding ensemble. by checking the intermediate ensemble in detail, we nd that it is quite heterogeneous and the heterogeneity mainly comes from the exibility of the c-terminal loop region. our ndings provide a microscopic picture of folding kinetics of the ww domain: the stable intermediate states with mis-registered hydrogen bonds on the c-terminal beta strand make this peptide folding as a three-state folding model rather than a usual two-state model. h. rezaei, f. eghiaian, j. perez, y. quenet, y. choiset, t. haertle, j. grosclaude institut national de la recherche agronomique, france in pathologies due to protein misassembly, low oligomeric states of the misfolded proteins rather than large aggregates play an important biological role. to get better insight into the molecular mechanisms of prpc/prpsc conversion, we studied the kinetic pathway of heat induced amyloidogenesis of the full length recombinant ovine prp (arq) at ph . . according to the size exclusion chromatography experiments, three sets of oligomers were generated from the partial unfolding of the monomer. the effect of concentration on the oligomerization kinetics was different for the three species obsreved suggesting that they are generated from distinct kinetic pathways. limited proteolysis and peptide analysis of the two best separated peaks showed a difference in the accessibility of the c-terminal domain of these two oligomers, and allowed the identi cation of regions undergoing a structural change during the conversion process. the analysis of correlations between oligomer populations as well as numerical kinetic modeling led us to propose a multi-step kinetic pathway describing the evolution of each species as a function of time. the existence of at least three distinct oligomerization pathways on one hand and the differences in the accessibility of the two puri ed oligomers on the other hand re ect the structural plasticity of the prp protein. studying the mechanism of retention of ovine prion protein in soils will tackle the environmental aspect of potential dissemination of scrapie infectious agent. the conformational transition from the monomeric cellular prion protein prp c in -helical structure into the aggregated -sheet-rich multimer prp sc is supposed to be responsible for the so-called prion diseases. it is commonly admitted that the recombinant prp could serve as model of conversion of the normal prion protein prp c . fundamentally, the interaction of proteins with surfaces either uid or solid involves both protein binding and unfolding. our goal in studying protein adsorption is to determine the nature and the amplitude of the structural changes occurring during non-speci c adsorption. the protein-clay interaction depends on several parameters such as protein hydration, net charge, charge distribution on the protein surface. ftir spectroscopy is well-suited to probe structural changes of proteins at a molecular level at aqueous/solid interfaces. the conformational states of the full-length ovine prp adsorbed on the electronegative clay surface are compared to its solvated state in deuterated buffer in the pd range . - , using ftir spectroscopy. during the intoxication of a cell, the diphtheria toxin binds to a cell surface receptor, is internalized and reaches the endosome. the translocation (t) domain from the toxin interacts with the membrane of the endosome in response to the acidic ph found in this compartment. this process drives then the passage of the catalytic domain of the toxin through the membrane into the cytoplasm. the interaction of the t domain with the membrane involves at least two steps. in the rst step occurring at ph , t adopts a molten globule conformation, which is able to bind super cially to the membrane through hydrophobic interactions by the c-terminal region (helices th and th ). in a second step occurring between ph and , penetration into the membrane involves electrostatic interactions. this step leads to a functional inserted state. trp was mutated to phe in order to use trp located in helix th as a probe of its behavior during the interaction with the membrane as a function of ph. we found that the second step is correlated with the reorganization of the n terminal region in the membrane and is controlled by electrostatic interactions. peptide conformational search using generalized simulated annealing method p. g. pascutti , f. p. agostini , c. osthoff , k. c. mundim , m. a. moret ibccf -ufrj -brazil, lncc -brazil, iq-unb -brazil, ceppev -fundação visconde de cairú / df-uefs -brazil the three-dimensional structure of proteins is mainly determined by the sequence of amino acids, making possible the development of ab initio methods for peptides and protein structure prediction. we proposed a stochastic method based on a classical force field and the generalized simulated annealing (gsa), which utilizes tsallis generalization of boltzmann-gibbs statistics. we have applied this method for peptide conformational search and as a complement for comparative modeling of proteins, searching the conformation for loops and mismatched sequence alignments. the gsa ef ciency depends on the right choice of the parameters involved in the conformational calculation: the qv parameter which de nes a function for visiting the molecular energy surface; and the qa parameter de ning an acceptance probability, both according with tsallis statistics. to avoid the conformational trapping in local energy minima we introduced a new parameter in this work, qt, to control the temperature decreasing. to investigate the qv, qa and qt best parameters set, we used the -alanine and -alanine peptides, which have a known alpha-helical structure in low dielectric environment. the global minimum energy occurs for the alpha-helix folded structure, and was found for qv ranging from . to . , qa from . to . , and qt from . to . . we observed also that convergence values for qv decrease while for qa and qt increase for folded structures. p. s. santiago, É. v. de almeida, m. tabak instituto de quimica de são carlos-usp cp são carlos sp brasil extracellular hbgp is a giant hemoglobin, similar to other annelid hemoglobins, having a molecular weigth of . mda. the effect of ctac in the oligomeric protein structure was assessed by optical absorption and emission spectroscopies. optical absorption spectra of hbgp . mg/ml as a function of ctac concentration from up to . mm evidentiate changes both in soret band at nm, and at nm associated to light scattering which appears at low ctac concentration. below mm of surfactant extensive light scattering occurs together with signi cant shifts of max of soret band from nm to around nm, which is probably due to oxidation of the original oxyhbgp. light scattering reaches a maximum value disappearing for higher ctac concentrations. fluorescence spectra show a signi cant increase in intensity ( fold) upon titration with ctac. this is consistent with the dissociation of the oligomer with signi cant reduction of intrinsic quenching of tryptophan uorescence due to the heme groups. similar data were obtained at protein concentration of mg/ml. in this case a signi cant increase of light scattering is observed with protein precipitation at a narrow ctac range followed by re-disolution at higher ctac concentration. differently from anionic sds surfactant, cationic one induces protein aggregation. support: cnpq and fapesp. in situ formation of silk protein nano-particles studied by small angle x-ray scattering m. w. roessle , c. riekel , d. i. svergun european molecular biology laboratory; outstation hamburg/germany, european synchrotron radiation facility; grenoble/france silk threads from the mulberry silkworm bombyx mori are used for the production of textiles since centuries. in modern applications silk proteins are chosen because of their good tensile strength, their high biocompatibility as well as of the resorbability for the human body. however, the processing of silk by the silkworm is barely understood. the silk proteins are stored as a solution in the glands of the silkworm and processed during the spinning into a co-block polymer like ber. in a rst step the random coil silk proteins are transformed into molecules with beta-sheet subdomains, which provide a protein-protein interface for the ber assembly. this transformation can be mimicked by a rheometer applying a shear force to the silk protein solution. applying combined small/wide angle x-ray scattering (saxs/waxs) transient formed silk nanoparticles upon increasing shear force were found. further details of these macromolecules were derived by xing the transient state with chemicals such as polyethylene oxide (peo). the resulting data can be analyzed in detail by saxs data evaluation software and low resolution models of the found nanoparticles were derived. moreover, the internal structure of the particles was explored as well as suggestions for the silk processing of the silkworm could be made. on the three-dimensional information of a protein sequence v. a. risso, l. g. gebhard, r. g. ferreyra, j. santos, m. noguera, m. r. ermacora universidad nacional de quilmes conicet in this work, lactamase of b. licheniformis (es l) was used as an experimental model to (a) study the conversion of sequential information into d structure and (b) to investigate the distribution of conformational information in the polypeptide chain. by a novel approach, over thirty connectivity variants of the polypeptide chain were prepared; witch were also nterminally truncated to a variable degree. the variants produced in e. clil were puri ed to homogeneity, refolded, and its structure content analyzed by circular dichroism, hydrodynamic behaviour and aggregation state. several variants were dimeric in solution, suggesting a possible general inespeci c stabilization mechanism. most variants were compact and had different degrees of secondary and tertiary structure. a strikingly large number of variants showed native like spectroscopic signatures and signi cant enzymatic activity, which means that the very elaborate active site of beta lactamase is formed, at least in fractions of the molecules, despite the absence of long stretches of sequence. these ndings are discussed in the light of the current knowledge of the protein folding process. var and lgg contributed equally to this work. on the three-dimensional information of a protein sequence v. a. risso, l. g. gebhard, r. g. ferreyra, j. santos, m. e. noguera, m. r. ermacora universidad nacional de quilmes conicet in this work, lactamase of b. licheniformis (es l) was used as an experimental model to (a) study the conversion of sequential information into d structure and (b) to investigate the distribution of conformational information in the polypeptide chain. by a novel approach, over thirty connectivity variants of the polypeptide chain were prepared; witch were also nterminally truncated to a variable degree. the variants produced in e. clil were puri ed to homogeneity, refolded, and its structure content analyzed by circular dichroism, hydrodynamic behaviour and aggregation state. several variants were dimeric in solution, suggesting a possible general inespeci c stabilization mechanism. most variants were compact and had different degrees of secondary and tertiary structure. a strikingly large number of variants showed native like spectroscopic signatures and signi cant enzymatic activity, which means that the very elaborate active site of beta lactamase is formed, at least in fractions of the molecules, despite the absence of long stretches of sequence. these ndings are discussed in the light of the current knowledge of the protein folding process. var and lgg contributed equally to this work. unfolding of the extrinsic proteins of photosystem ii ( kda, kda and kda) induced by pressure unfolding of the three extrinsic proteins of spinach photosystem ii induced by pressure has been systematically investigated. thermodynamic equilibrium studies indicated that these proteins are very sensitive to pressure. at o c all the proteins show a reversible unfolding transition by about mpa for kda, mpa for kda and mpa for kda. the ph and temperature dependence of pressure unfolding of these proteins were explored. the stabilization effect of reagents sucrose etc on the proteins was found to associate not only with the increase in the unfolding free energy, but also with the reduction of the absolute value of v u . pressure-jump studies of unfolding of kda protein revealed a negative activation volume for unfolding and a positive activation volume for refolding, indicating that in terms of system volume, the transition state lies between the folded and unfolded states. comparison of temperature dependence of v u # , v f # and v u indicated that the thermal expansivities of the transition state and the unfolded state are similar and larger than that of the folded state. aggregation-prone intermediate protein structures on the refolding pathway l. smeller , j. fidy , k. heremans semmelweis university dept. biophysics and radiation biology, budapest, hungary, department of chemistry, katholieke universiteit leuven, belgium the folding of the polypeptide chain into a native conformation can be studied by experimental systems, where the environmental parameters causing the denatured state can be easily and fast eliminated. one of such parameters is the high hydrostatic pressure. refolding of the unfolded protein can be studied after decompression. the refolding pathway can contain several intermediate states. on the other hand, the destabilization of the native proteins can populate conformations, where the polypeptide chain is not completely folded. these metastable conformations can easily aggregate. deposition of insoluble protein aggregates plays a crucial role in the conformational diseases (parkinson, alzheimer's disease, amyloidozis, etc.) stability and conformation of the above mentioned metastable conformations were investigated in case of myoglobin, apo-horseradish peroxidase, lipoxygenase. fluorescence and infrared spectroscopy and light scattering experiments were used to explore not only the structure but also the aggregation af nity of the intermediates in case of all the above mentioned proteins a well de ned temperature range could be determined, where the metastable not completely folded structures were populated considerably during the refolding process. these intermediate conformations were significantly more aggregation prone, than the native conformers existing in the same temperature range. aggregation processes in beta-amyloid peptides: effects of molecular chaperons a. sgarbossa, d. buselli, f. lenci cnr istituto bio sica, pisa, italy several neurodegenerative pathologies, like parkinson's, hungtington's and alzheimer's diseases, are related to the formation of small peptides aggregates, which amyloid brils originate from. understanding the molecular mechanisms responsible for these processes can, therefore, contribute to clarify the origin and, hopefully, to control the development of the afore mentioned diseases. here we report the results of an in vitro study aiming to affect the aggregation kinetic of - and - beta-amyloid peptides by means of an endogenous chaperone-like protein (alpha-crystallin) and an exogenous polycyclic aromatic pigment (hypericin) that can perturb the aggregation process through stacking interactions with the peptides aromatic residues. because of the well known problems in getting reproducible and reliable results, particular attention has been devoted to carefully check the preparation procedures of the samples. the effects of both alpha-crystallin and hypericin on the self-assembly process have been examined at different times of the aggregation kinetics. the results are discussed in relation with the involvement of different molecular structures in the amyloid brillation phenomenon. remodelling the folding of thioredoxin by removal of the c-terminal helix j. santos, j. m. del no iquifib and departamento de química biológica, facultad de farmacia y bioquímica, uba, junín , c aad buenos aires, argentina e. coli thioredoxin (trx) is a monomeric / protein of amino acids with a fold characterized by a central beta sheet surrounded by alpha helices. two subdomains are topologically noticeable, but it is unclear whether their folding occurs in a concerted fashion. subdomain trx - has been extensively studied as a model of a partially folded, with no tertiary or persistent secondary structure. this work describes the expression and characterization -by circular dichroism (cd), uorescence emission, size exclusion chromatography, chemical cross-linking and light scattering-of a novel engineered fragment (trx - ) lacking the last stretch of amino acids. after refolding from inclusion bodies, trx - shows a strong propensity to form soluble oligomers endowed with distinctive optical properties unlike those observed for the full protein. although trx - also shows signi cant changes in secondary structure, trp residues appear to occupy rigid and apolar environments. these ndings support the existence of an alternatively folded form for trx - . in addition, the secondary structure content of chemically synthesized peptide trx - and its ability to complement fragment trx - upon refolding were also evaluated by cd. taken together, the data herein presented shed light upon issues such as the distribution of information content relevant for folding along the polypeptide chain in regard to conformational stability. with grants from anpcyt, ubacyt and conicet. thermal aggregation of two "beta-protein" models at different ph values v. vetri, f. librizzi, v. militello, m. leone physical and astronomical sciences, univ. of palermo, italy & infm the structural stability of proteins strongly depends on the environment and the lost of this stability may trigger a partial unfolding, leading in turn to the formation of aggregates. such processes have been extensively studied also in view of their biotechnological and medical implications. in fact a large number of diseases is associated with protein misfolding and aggregation. conformational changes play a keyrole in the aggregation processes and have their onset under particular external conditions. the aggregation pathways and the topology of the obtained supramolecular structures sizeably depend on the details of the involved conformational changes, which are determined by the details of the external conditions. here we present an experimental study on thermal aggregation processes of two model proteins mainly composed from structures: -lactoglobulin and concanavalin a, at different ph values. the conformational changes of the proteins (whose association state depends by ph) and the aggregation pathways were monitored by intrinsic and suitable external dyes uorescence. at the same time, the growth of supramolecular structures was followed by measuring the rayleigh scattering of the excitation light. secondary structure changes were followed by circular dichroism measurements. the results show that at different ph values the aggregation processes of both proteins follow different pathways determined by the variations in the native structure and by the details of the involved conformational changes. a. verma , t. herges , w. wenzel iwr, forschungszentrum karlsruhe, po box , , karlsruhe, germany, int, forschungszentrum karlsruhe, po box , , karlsruhe, germany ab-initio protein structure prediction and the elucidation of the mechanism of the folding process are among the most important problems of biophysical chemistry. investigations of the protein landscape may offer insights into the folding funnel and help elucidate folding mechanism and kinetics. we investigate the landscape of the internal free-energy of the amino acid villin headpiece with a modi ed basin hopping method in the all atom force eld pff , which was previously used to fold several helical proteins with atomic resolution. we identify near native conformations of the protein as the global optimum of the force eld. more than half of the twenty best simulations started from random initial conditions converge to the folding funnel of the native conformation, but several competing low-energy metastable conformations were observed. from , independently generated conformations we derived a decoy tree which illustrates the topological structure of the entire low-energy part of the free energy landscape and characterizes the ensemble of metastable conformations. these emerge as similar in secondary structure content, but differ in the tertiary arrangement. exploring the free energy landscape of a folded protein by means of afm stretching experiments m. vassalli , b. tiribilli , l. casetti , a. torcini , a. pacini , a. toscano isc -cnr, florence -italy, csdc, univ. of florence -italy, anatomy dept, univ. florence -italy the aim of our work is to study the mechanically induced folding and refolding of single proteins by means of an atomic force microscope (afm). the resulting data will be analyzed with theoretical methods, both to determine the folding pathway and to gain information on the energy landscape of real systems. recently, experiments employing atomic force microscopy have shown that mechanical and thermal unfolding share several common features. we are using an afm to perform mechanical stretching experiments on single biomolecules. the experiments that can be performed are particularly well-suited to reconstruct the folding-unfolding pathways as well as the free energy landscape of the examined protein. in particular we are interested in the free energy pro le associated to titin and elastin, by considering a periodic loading of the afm cantilever, instead of the usual linear ramp, and measuring the force as a function of displacement. these experiments will be complemented by theoretical and numerical studies. molecular dynamics simulation of simple models but including the experimental geometry, will allow to examine in detail the effect of different experimental procedures (periodic loading versus linear ramp) proposed to reproduce equilibrium energy landscapes. moreover, we will investigate the limit of applicability of the jarzynski's equality which has been claimed to be able to be used to extract equilibrium results from non equilibrium measurements. association of subunits is a prerequisite for formation of the native structure of the dimeric ipmdh a. varga, É. gráczer, i. hajdú, p. závodszky, m. vas institute of enzymology, biological research center, hungarian academy of sciences, budapest, hungary to answer the question whether subunits are autonomous folding units, or their association at an early stage of folding is required for formation of the native protein structure, denaturation-renaturation experiments were carried out with the dimeric isopropylmalatedehydrogenase (ipmdh). denaturation was induced by guanidine hydrochloride, renaturation was initiated by dilution and followed by activity measurements and uorimetry. reactivation is a complex process with an initial lag phase, indicating the presence of an inactive intermediate. the kinetics of the process is independent of protein concentration, suggesting that association of the two polypeptide chains takes place much faster than the rate limiting rst order isomerisation step(s). restoration of protein uorescence during renaturation is also protein concentration independent, biphasic process, however the initial lag phase is replaced by an even faster burst increase of uorescence. the rst step leads to formation of an intermediate with a native-like uorescence spectrum. based on our experiments the following mechanism is proposed for refolding of ipmdh: d+d i i n , where d is denaturated monomer, i and i are inactive dimer intermediates, n is native dimer, that means initial association of the polypeptide chains during refolding is a prerequisite for formation of the native dimensional structure of ipmdh. we describe a new beamline for optical biophysic in construction on the synchrotron soleil. the high briliance of the synchrotron beam, associated with its tunability on a broad part of the electromagnetic spectrum make it an excitation source of choice for several biophysical optical techniques. the disco beamline we present will consist of three endstations : . the circular dichroism (cd) endstation will bene t from the inclusion of the energies accessible in the vuv wavelength range ( - nm) and from the natural polarization of the synchrotron beam. cd spectra of proteins covering a broad range of wavelenghts will enable better and ner structural analysis. moreover, new biological chromophores such as sugars which absorb in the deep uv will be accessible in cd. . the mass spectrometry endstation,will bene t from an ionisation beam with even greater energies (down to nm) comprising nebulisation at atmospheric pressure. photoionisation of macromolecular bio-structures without any solvent restriction will produce perfect analytes for mass spectrometry, . the multiparametric imaging endstation, build on a confocal microscope, will use the great tunability of the synchrotron radiation ( - nm) to excite samples at many wavelenghts simultaneously. the temporal component of the beam will allow natural lifetime imaging by phase modulation -demodulation. predictive all-atom protein folding with stochastic optimization methods the prediction of protein tertiary structure, in particular based on sequence information alone, remains one of the outstanding problems in biophysical chemistry. according to the thermodynamic hypothesis, the native conformation of a protein can be predicted as the global optimum of its free energy surface with stochastic optimization methods orders of magnitude faster than by direct simulation of the folding process. we have recently developed an all-atom free energy force eld (pff ) which implements a minimal thermodynamic model based on physical interactions and an implict solvent model. we demonstrated that pff stabilizes the native conformation of several helical proteins as the global optimum of its free energy surface. in addition we were able to reproducibly fold several helical proteins (the amino acid (aa) trp-cage protein, the villin headpiece ( aa), the conserved headpiece of the hiv accessory protein ( aa), the headpiece of protein a ( aa) and the -helix bacterial ribolsomal protein l ( aa), as well as several beta-sheet peptides. we used several stochastic optimization methods: the stochastic tunneling method, an adapted version of parallel tempering, basin hopping techniques and distributed evolutionary optimization strategies. we discuss advantages and limitations of this approach to de-novo allatom protein structure prediction. the independent thermal unfolding simulations of gb have been performed. physical property parameters of protein structure were chosen to construct a -dimension physical property space. then the -dimension property space was reduced to dimensions principle component property space. under the property space, the unfolding pathway ensemble of gb was obtained. the pathway ensemble likes a funnel that was gradually emanative from the native state ensemble to the unfolded state ensemble. the unfolding trajectories have the similar variable trend during the native state and the transition state ensemble. during the unfolded state, the unfolding trajectohries were divided into two types that one includes only one trajectory and the other include trajectories. the rst type of unfolded state was a discontinuity step distribution model, which is not random distribution. the second type of unfolded state was a near ellipsoid distribution model and a near random. there were substantial overlaps of unfolded state, indicating that thermal unfolded state consists of a con ned set of property values that makes the number of unfolded state of protein to be much smaller than that was believed before. the protein circular dichroism data bank (pcddb): a bioinformatics and spectroscopic resource b. a. wallace , l. whitmore , r. w. janes dept. of crystallography, birkbeck college, university of london, school of biological sciences, queen mary, university of london we describe the development and creation of the protein circular dichroism data bank (pcddb), a deposition data bank for validated circular dichroism spectra of biomacromolecules. its aim is to provide a resource for the biological and bioinformatics communities, by providing open access and archiving facilities for circular dichroism spectra. it is named by parallel with the protein data bank (pdb), a long-existing valuable reference data bank for protein crystal structures. it will permits spectral deposition via userfriendly web forms and will include automatic reading of a range of data formats and data mining from le headers to facilitate the process. it will be linked, in the case of proteins whose crystal structures and sequences are known, to the appropriate pdb and sequence data band les, respectively. a series of validation tools that will provide reports on data quality are included (and are accessible as stand-alone software). it is anticipated that this data bank will provide readily-accessible biophysical catalogue of information on folded proteins that may be of value in structural genomics programs, for quality assurance and archiving in industrial and academic labs, as a resource for programs developing spectroscopic structural analysis methods, and in bioinformatics studies. the relation of n-terminal residues and structural stability of l-chain apoferritin k. yoshizawa , k. iwahori , y. mishima , i. yamashita crest/jst, atrl-matsushita, nara institute of science and technology the denaturation of apoferritin by acidic solution was studied. ferritin, the ubiquitous iron storage protein, represents a well known polymeric assembly that is highly resistant to chemical and physical denaturants. it is a cage-shaped protein which is composed of subunits. natural vertebrate ferritins are copolymers of two different subunits, l-and h-chains. in the recombinant h-chain apoferritin (rhf), the structural stability is decreased by deletion of the n-terminal residues. we studied the effect of n-terminal residues of recombinant l-chain apoferritin (rlf) on the acidic ph denaturation and re-assembly. we constructed rlf and mutant rlfs which are lack of (fer ) or (fer ) amino acid residues from the n-terminus and investigated their stability by cd spectra. among three, fer has the least endurance against ph decrease. in the case of fer , the re-assembly of subunits into apoferritin can be performed by increasing solution ph without causing the by-product while huge aggregations are caused in fer and fer . the structural comparison of three mutants indicates that the hydrogen bonds of inter-and intra-subunits decrease by the loss of the n-terminal residues. therefore, it is elucidated that the hydrogen bonds of inter-and intra-subunits from n-terminal residues affect the molecule stability and re-assembly of l-chain apoferritin. pressure perturbation calorimetic studies of solvation, unfolding and aggregation of proteins r. winter university of dortmund, physical chemistry, otto-hahn-str. , d- dortmund pressure perturbation calorimetry (ppc) was used to study the solvation and volumetric properties of various proteins in their native and unfolded state. in ppc, the coef cient of thermal expansion of the partial speci c volume of the protein is deduced from the heat consumed or produced after small isothermal pressure jumps, which strongly depends on the interaction of the protein with the solvent or cosolvent at the protein-solvent interface. the effects of ph and various chaotropic and kosmotropic cosolvents (glycerol, sucrose, urea, guhcl, salts, etc.) on the solvation and unfolding behavior of the proteins was also investigated, and the observed volume and expansivity changes are correlated with further thermodynamic and spectroscopic properties of the systems. depending on the type of cosolvent and its concentration, speci c differences are found for the solvation properties of the proteins, and the volume change upon unfolding may even change sign. taken together, the data obtained lead to a deeper understanding of the solvation process of proteins in different cosolvents in their native and unfolded states. in addition, the effects of con nement and crowding on the solvational properties of the proteins were studied. finally, the use of ppc for studying intermolecular interactions and aggregation (amyloidogenesis) phenomena of proteins (e.g., insulin, prp) will be discussed. the in uence of semisynthetic derivatives of phenolic lipids on activity of yeast abc pumps phenolic lipids are the natural amphiphilic long-chain homologues of orcinol ( , -dihydroksy- -methylbenzene). they occur in numerous plants and microorganisms. resorcinolic lipids exhibit high af nity for lipid bilayer and biological membranes and are able to modify the activity of membrane enzymes (e.g. pla , ache). the in uence of semisynthetic derivatives of phenolic lipids on yeast pdr protein activity was studied by spectro uorimetric method using the potentiometric uorescence probe dis-c ( ). the probe is expelled from s. cerevisiae by abc pumps and can conveniently be used for studying their performance. two pump-competent s. cerevisiae strains and different pump-free mutant strains were used to experiment to check the effect of the semisynthetic derivatives of phenolic lipids on activity of abc transporters. two of these derivatives, named . and . , seem to affect the activity of pdr pumps. their in uence on activity of yeast plasma membrane multidrug resistance abc pumps is concentration-dependent. in uence of lipid membrane composition on pglycoprotein activity k. bucher, s. d. krämer, h. wunderli-allenspach department chemistry and applied biosciencies, eth zurich, switzerland p-glycoprotein (p-gp), a membrane atpase expelling many structurally unrelated compounds, is one of the major contributors to multidrug resistance. it is proposed that substrates bind to it within the membrane and are exported from there out of the cell. p-gp substrates are generally hydrophobic and their binding to the transporter is governed by their ability to partition into the membrane. the intimate association of both p-gp and its substrates with the membrane suggests that p-gp function may be regulated by the composition of the lipid bilayer. as detergents in uence the membrane properties and have been shown to affect p-gp atpase activity, we developed virtually detergent-free proteoliposomes to investigate the in uence of the membrane environment on the atpase activity of p-gp. the basal and substrate induced atpase activity was dependent on the cholesterol level of egg phosphatidylcholine (pc) membranes. the compound concentration at half maximal activation of p-gp (k m ) in proteoliposomes correlated with the af nity of the respective compound to liposomes consisting of the same lipids as the proteoliposomes tested. in conclusion, the basal and drug-induced atpase activity of p-gp is strongly dependent on the cholesterol content in detergent-free p-gp/egg pc/cholesterol proteoliposomes. m. berchel , j. jeftic , t. benvegnu , j.-y. thepot , d. plusquellec enscr umr cnrs , campus de beaulieu, rennes, université de rennes , umr cnrs , rennes bipolar lipids found in archaebacterial membranes, generally termed bolaamphiphiles, induce increased stability in membranes exposed to environments such as acidic conditions, high temperatures, high salt concentrations and/or absence of oxygen. we have synthesized a spin labeled unsymmetrical bolaamphiphile that selforganises in water solutions in multilamellar vesicles and shows slow ip-op phenomenon in comparison to conventional liposomes. generally, the ip-op from the exovesicular to the endovesicular membrane surface is a relatively slow process, which is due to the high energy barrier in transferring the polar amphiphilic heads through the lipophilic membrane. it can be involved in membrane transport mechanisms and in facilitating the transport, cells have evolved to use various supramolecular strategies. the half-life of the ip-op is estimated to more than twelve hours. we are now modulating the ip-op rate by incorporating chemical modi cations such as addition of cyclopentanes, double or triple bonds into the bridging chain of the molecule, in order to control the membrane transport via the ip-op mechanism. transport activity of the monocarboxylate transporter is increased by carbonic anhydrase h. m. becker, j. w. deitmer abt. allgem. zoologie, tu kaiserslautern, kaiserslautern the enzyme carbonic anhydrase (ca), which catalyses the conversion of co and h o to bicarbonate and protons, is present in nearly all animal cells, and is highly expressed in astrocytes. it is known that ca can bind to several membrane transporters, forming a transport metabolon and thereby enhancing the transport activity of the protein. in this project we have studied the functional interaction of the enzyme with the monocarboxylate transporter (mct ), which transports lactate and other monocarboxylates together with protons and is believed to play a pivotal role in the metabolite shuttling between astrocytes and neurons. therefore we expressed mct and then injected ca into xenopus oocytes. our results indicate a direct binding of ca to the mct , leading to a ca-induced increase in acid/base ux mediated by the transporter. interestingly, the effect was insensitive to the ca inhibitor ethoxyzolamide and to the nominal absence of co /hco , but disappeared when binding of ca to the mct was hindered. it seems, that ca, bound to the mct , mediates local buffer capacity by removing protons transported into the cell via the mct . this helps to stabilise the proton gradient close to the cell membrane, and thereby enhances the transport activity of the mct . these ndings suggest that ca can enhance metabolite-acid/base transport, by forming a transport metabolon with the mct . melibiose permease of e.coli (mel b) is a membrane bound ioncoupled sugar symporter that uses the favorable na , li , or h electrochemical potential gradient to drive cell accumulation ofor -galactosides. cysteine scanning mutagenesis, electrophysiological (ssm -solid supported membranes) and uorometric measurements, were used in order to better understand the role of speci c parts of the protein in the function of this symporter. the ssm technique combines a rapid solution exchange with the high sensitivity of planar lipid membranes. it employs a solid supported membrane as a capacitive electrode and allows the time resolved investigation of charge translocation during the catalytic cycle of such transporters as na /solute symporters. in order to obtain some more precise information about the function of mel b symporter, starting from the c-less melb, the mutant g c was constructed and from electrical, spectro uorimetric and fret measurements carried out on this mutant, in the absence and in the presence of speci c inhibitors, conclusions were drawn about the possible role of the helix iv in the function of the symporter. two-dimensional crystallization of co-reconstituted ca +-atpase, phospholamban and sarcolipin the sarcoplasmic reticulum ca -atpase and its regulators phospholamban (plb) and sarcolipin (sln) form a primary control mechanism in the recovery of resting state calcium levels in the myocardium. defects in the regulation of ca -atpase by plb and sln are central determinants in cardiac contractility and disease states such as cardiomyopathy. given the signi cance of these proteins, the structural details of their regulatory mechanisms remain an important future goal for the clinical improvement of heart disease. using co-reconstitution into proteoliposomes at low lipidto-protein ratios, we have examined the effects of mutation on the functional properties of plb and sln, revealing novel insights into calcium pump regulation. in addition, these same co-reconstituted proteoliposomes have been used for structural studies by electron cryo-microscopy. in an attempt to better de ne the structural interactions between plb, sln and ca -atpase, we have sought methods for the production of large two-dimensional crystals suitable for high resolution electron crystallography. we previously utilized the co-reconstituted proteoliposomes to produce long, tubular crystals suitable for helical reconstruction. our new procedure comprises three steps -co-reconstitution, membrane fusion, and crystallization -producing large two-dimensional crystals suitable for high resolution structural studies. herein, we will present our latest results characterizing the structural interaction between plb, sln and ca -atpase. t. v. demina , n. s. melik -nubarov , h. frey , e. e. pohl state university, chemistry department, moscow, russia, institute of organic chemistry, johannes-gutenberg-university, mainz, germany, humboldt university, charite, institute of celland neurobiology, berlin, germany multidrug resistance (mdr) of tumours is associated with overexpression of the p-glycoprotein responsible for an active drug ef ux from cells. block copolymers of ethylene oxide and propylene oxide (pluronics) are known to cause a pronounced chemosensitization of tumour cells. the effect may be due either to speci c polymer -protein interactions or to unspeci c lipid bilayer disturbance. we have shown recently that amphiphilic copolymers with various hydrophilic and hydrophobic blocks can disturb lipid bilayers. importantly, that block copolymers of propylene oxide and glycerol (ppo-pg) with hyperbranched "corona" induced larger effects then pluronics with linear polyethylene oxide chains. in the present work we have shown that ppo-pg copolymers increase dox cytotoxicity towards human erythroleukemia (k is , k /dox) and breast carcinoma (mcf /dox) resistant cell lines. using confocal and two-photon microscopy, we demonstrated that these copolymers accelerated dox penetration into resistant cells, inhibited efux and caused drug redistribution into nuclei. a clear correlation between the ability of the polymers to disturb lipid bilayers and favour drug accumulation in mdr cells was disclosed. this nding points to an unspeci c mode of the copolymers' chemosensitizing activity. voltage dependence of processes related to electrogenic membrane transporters electrogenic membrane transporters, such as the sodiumbicarbonate cotransporter (nbce ), may induce dependence on membrane potential upon processes which are innately voltageindependent. we tested this hypothesis by heterologously coexpressing electrogenic nbce from human kidney in oocytes of the frog xenopus laevis with the electroneutral rat monocarboxylate transporter mct . the apparent intracellular buffer capacity was increased by nbce expression and became voltage-dependent by mm/ mv membrane depolarisation. lactate transport via the mct not only became enhanced after co-expression with nbce , but also dependent upon membrane potential. injection of carbonic anhydrase caii from bovine erythrocytes into oocytes enhanced the ef cacy of nbce activity, identifying an additional, ca-sensitive, membrane current via nbce . our results show that nbce adds voltage-dependent buffer capacity to the cytosol; this is suggested to be the prime cause for enhancing acid/base-coupled transport and conferring membrane potential dependence on transporters which are stoichiometrically electroneutral. these interactions may have functional consequences for cells and tissues, where electrogenic and electroneutral processes interact, such as in brain, heart and muscle. . by using carboxy-snarf- as ph-sensitive uoroprobe and microspectro uorimetry, we now show that nhe activation is due to jun kinase (jnk) activation, resulting from reactive oxygen species (ros) produced during metabolism of b(a)p and might involve lipid raft. when analysing b(a)p-induced apoptosis, we have found that cariporide signi cantly reduces both nuclear fragmentation and caspase- like activity. we further show that nhe activation and/or alkalinization affects the mitochondrial ros production detected during the apoptotic cascade, likely via an effect on the complex iii of the electron transport chain. altogether, our results suggest that apoptotic xenobiotics, such as benzo[a]pyrene, induce an early activation of nhe that might play a signi cant role in the subsequent mitochondria-dependent apoptosis. conformational dynamics of a lactose proton symporter j. c. holyoake, m. s. sansom biochemistry department, university of oxford, south parks road, oxford, ox qu, u.k. transporter proteins are an essential class of proteins, catalysing the transfer of molecules across membranes. increasing numbers of transporter structures are becoming available, opening the way to study their dynamic properties using computational techniques. we present a study on the conformational dynamics of the lactose proton symporter lactose permease using molecular dynamics simulations. these simulations exhibit large scale conformational changes from the initial intracellularly open conformation to a more closed conformation that may be signi cant to the transport mechanism. the conformational change is analysed to identify the contributing motions. effects of nortriptyline and chlorpromazine on anthroylouabain-labeled na,k-atpase e. a. guevara , m. l. barriviera , a. hassón-voloch , s. r. louro departamento de física, puc-rio, rio de janeiro, brazil, instituto de biofísica carlos chagas filho, ufrj, rio de janeiro, brazil the effects of nortriptyline and chlorpromazine (cpz) on the uorescence properties of anthroylouabain (ao)-treated na ,k -atpase of electrocyte membranes from e. electricus are studied. na ,k -atpase oscillates between two major conformations e and e during ion transport cycle. the cardiotonic steroid ouabain speci cally inhibits this enzyme binding to the e conformation. the uorescent label ao presents increased uorescence when binding to the ouabain site of na ,k -atpase. tricyclic drugs such as the antipsychotic cpz and the antidepressant nortriptyline inhibit na ,k -atpase activity in the micromolar range. for the e enzyme, but not e , nortriptyline was found to increase the uorescence in a concentration dependent manner, suggesting a further stabilization of e . for both conformations, cpz induces negligible uorescence change up to µm. the uorescence of atpasebound ao, however, strongly increases upon ultraviolet exposure after cpz treatment at concentrations around µm. fluorescent products of cpz-photodegradation were studied in pure buffer and in the presence of membranes. the results suggest that cpz binds to na ,k -atpase and photolabels amino-acid residues near the ouabain binding site. how important is protein exibility for transport through ion channels? a. a. gray-weale university of sydney certain ion channels are selective for k+ over other ions, but the geometry of the pore does not explain selectivity because thermal uctuations are too large. i extend the usual treatment of ion channels with molecular dynamics simulation by calculating the static and dynamic pair correlations between monovalent ions and ion channels (gramicidin-a and kcsa), and also between certain small, complex cations and the gramicidin channel. this means not only the radial-distribution functions or the density pro les, but also correlations between the ion and the mass and charge densities of various regions of the protein. the advantage of this approach is that it systematically identi es the elements of the protein and modes of motion that contribute to selectivity, and illustrates the decay of correlations. recently, noskov et al. [ ] showed that thermal uctuations protect selectivity. my results on the interaction of ions with carbonyl groups agree with theirs, but take the analysis further to higher correlations. the key new element is the study of the time-correlation functions that describe the motion of the ions through the channel, borrowing methods originally developed for the study of dense or even supercooled liquids. the surface-enhanced infrared absorption spectroscopy (seiras) is used for the investigation of two membrane proteins, the cytochrome c oxidase (cco) and the bacteriorhodopsin (br). of central interest are the transport-mechanisms of electrons (cco) and ions (br). the main parts of the setup are an infrared light source, a hemispherical si-crystal, in which the beam is internal re ected and a plexiglas cell with buffer solution (see schematic scetch). the beam is totally re ected on the inner at surface of the crystal, but the evanescent wave excites surface plasmons in a chemically adsorbed gold-layer on the crystal (attenuated total re ection spectroscopy-atr). the protein to be analysed is attached at the gold surface and can absorb certain wavelengths. the gold is need for the surfaceenhancing effect. cco plays a major role in the respiratory chain, the retinal protein br is a photosynthetic protein. the cco is immobilized on the gold surface via the af nity of its histidine-tag to a nickel-chelating nitrilo-triacetic acid (nta) surface. for the br we incorporate the protein in a lipid membrane, which is attached on the gold surface by the sulfuric bindings of , -di-o-phytanyl-sn-glycerol- -tetraethylene glycol-d,l-lipoic acid ester lipid (dptl). the sensitivity of this method is further enhanced by modulation of an external parameter, like the electric potential. effects of copper ions on the escherichia coli growth and proton-potassium exchange copper ions are required for the function of many important enzymes in escherichia coli but can cause a number of toxic cellular effects also. it's interesting to reveal the in uence of copper ions on the growth of bacteria and proton-coupled membrane systems. upon transition of e. coli mc wild-type culture to stationary growth phase a decrease in redox potential (e h ) from the positive values ( + mv) to the negative ones (of - to - mv), resulting h production by formate hydrogenlyase (fhl) have been studied. copper increased a latent growth phase duration as well as delayed a logarithmic growth phase in concentration-dependent manner. during the anaerobic growth, the production of h was strongly inhibited in the presence of cucl ( mm). . mm cucl was inhibited h production under experimental conditions with glucose. the inhibitory effect of copper ions ( . mm) on n',n'dicyclohexylcarbodiimide (dcc)-sensitive h /k exchange was also observed: k uptake was decreased and the stoichiometry of dcc-inhibited ion uxes varied. interestingly, these effects on h and k uxes were absent for the mutant hd (hyc-operon for hydrogenase was deleted). we suggest that copper ions, inhibiting the activity of fhl, have an effect on h /k -exchanging mechanism which is the proton f f -atpase associated with k uptake trk system. this effect may be due to the relationship of fhl with the ion-exchanging mechanism above under fermentation at alkaline ph. lateral diffusion in tethered bilayer membranes m. jung, v. atanasov, w. knoll, i. koeper max planck institute for polymer research, mainz, germany tethered bilayer membranes (t'blm) provide a useful platform for the investigation of bilayer membranes as well as embedded membrane proteins. we have developed a modular system, which is suitable for surfaces providing gold and oxide surface coatings. these systems serve as a quasi natural environment for the study of membrane proteins, being functionally incorporated into a lipid bilayer, which is covalently bound (tethered) to the substrates. functionality could be shown using electrochemical methods. here we present a study of these systems, basically the membrane itself, using uorescence recovery after photobleaching (frap) studies in order to investigate lateral motion in the lipid bilayers. lateral mobility is essential for successful incorporation of large membrane protein complexes. we will present rst results of experiments that try to differentiate motions hindered due to the tethering from diffusion in free oating or suspended bilayers. the information gained in this study will serve for improvements in the chemical structure of the tethered molecules. we will develop the system as a basis for bio sensing applications, where embedded proteins will serve as actual sensing elements. a. d. ivetac, j. campbell, m. s. p. sansom biochemistry department, university of oxford, south parks road, oxford, ox qu, u.k. atp-binding cassette (abc) transporters form an important superfamily of membrane proteins which couple atp hydrolysis to the active transport of diverse compounds across the cell membrane. their biomedical relevance is highlighted in examples such as multidrug resistance to antibacterial and anticancer agents, and cystic brosis. the availability of crystal structures of three complete bacterial abc transporters provides an opportunity to study structurefunction relationships at the atomic level. in this work, we carry out multi-nanosecond molecular dynamics simulations of the vitamin b importer from e. coli (btucd), with both the complete multimeric transporter embedded in a phospholipid bilayer and the soluble subunits in a membrane-free environment, in an attempt to elucidate some of the conformational changes which arise during the transport event. atp-bound and atp-free structures are used to investigate the effect of nucleotide on the system. a range of analytical techniques have been applied to assess the dynamic behaviour of the protein during the simulations, which includes measurements of: conformational drift, residue exibility, transmembrane domain (tmd) movement, concerted protein motions, nucleotide-binding and translocation pathway changes. an in-vitro method was designed to measure transmembrane transport rates. liposomes were prepared by extrusion with dipalmitoyl phosphatidylcholine (dppc) and optionally cholesterol, and loaded with a peptide (zinc-insulin tagged with a uorescent group or bsa). after removal of the non-encapsulated peptide from the liposome solution by gel ltration, the release of the peptide from the liposomes was monitored by uorescence as a function of time at various temperatures. the transport was greatly accelerated by the presence of a speci c proprietary excipient molecule (cyclopentadecanolide -cpe ™), which effectively triggered the release of the peptide. a mathematical model was developed to quantify these results. a semi-empirical nonlinear equation involving four parameters ts the protein release pro les. then a neural network predictions model was used to correlate the different release condition parameters and the four semi-empirical tting parameters based on the experimental data sets. most release data t well with the mathematical model, further supporting our theory of a two step release mechanism. phenyltin the well known group of organotin compounds that exhibit toxic properties in relation to the biological systems are phenyltins. no studies have been performed as yet to establish directly whether organotins such as diphenyltin dichloride ( dpht) and triphenyltin chloride (tpht) cross the lipid bilayer. we have performed experiments that showed transfer of those compounds across the lipid bilayer using the stopped-ow technique and desorption of those compounds from a monolayer using the langmuire technique. obtained results demonstrate that dpht and tpht rst adsorb onto the lipid bilayer surface, in diffusion controlled manner, within a very short time ( . s), whereas the membrane passing was observed in a minute's time range. the long time kinetics show a complex dependence on the kind of compound, its concentration and the presence of cholesterol in the membrane. the desorption of both compounds from the monolayer to water subphase occurs in a minute's time range. these observations may explain the known fact, that the in uence of organic, amphiphilic tin (and also lead) compounds is more toxic than that of inorganic ones.the phenyltins much easier (compared with tin or lead ions ) penetrate e.g. blood -brain barrier. [ under the resting conditions ca concentration in agonistsensitive ca stores re ects a balance between active uptake mediated by a ca -atpase (serca) and passive ef ux of ca . this ca leak appears to be a common property of ca -storing organelles, but the nature of the leak in submandibular acinar cells remains unclear. we have studied the ca leak pathways in the endoplasmic reticulum (er) of acinar cells of rat submandibular salivary gland by directly measuring concentration ca in the er ([ca ] er ) in mag-fura /am preloaded cells while [ca ] i was clamped at a resting level with a egta/ca mixture. we have shown that thapsigargin (tg) or ca -free buffer treatment completely blocked ca uptake by serca after the rst minute of superfusion and caused a ca leak represented by continuous decline in [ca ] er . this ca leak from the er was not sensitive to tg, heparin and ruthenium red and therefore appears to be independent of the serca, the insp receptor and the ryanodine receptors. however, treatment with puromycin ( . - mm) to remove nascent polypeptides from er-ribosome translocon pores increased ca leak from the er by a mechanism independent of the serca, insp or ryanodine receptors. thus we conclude that basal ca leak from the er of submandibular acinar cells occurs through translocon pores in the er membrane. r. b. kishore, j. reiner, e. edgu-fry, a. jofre, k. helmerson physics laboratory, national institute of standards and technology we have developed a procedure to make lipid and polymer nanotubes of up to one cm long and nm in diameter, from the surface of giant liposomes and polymersomes, using micro uidics and optical tweezers. the liposomes and polymersomes were formed, using elcetroformation method, from phospholipids and amphiphilic diblock copolymers, respectively. the polymer tubes were made extremely robust by cross-linking them using chemical reactions. we are currently studying the transport of molecules in the crosslinked nanotubes for use in nano uidic networks. p-glycoprotein (p-gp) is an active membrane transporter capable of expelling out of the cell a large number of potentially cytotoxic amphiphilic molecules with unrelated chemical structures. as a consequence, p-gp may be responsible for multidrug resistance of tumors against chemotherapy (mdr); it also plays a key role in absorption, biodisposition and elimination of many pharmaceuticals. to understand the molecular mechanisms of the transmembrane drug transport mediated by pgp, it is highly desirable to design a convenient assay for measuring both p-gp atpase activity and p-gp transport function. to do so, we used inside-out native membrane vesicles, prepared from mdr cells and containing high amounts of p-gp. we took advantage of the speci c property of a uorescent dye, the carbocyanin jc- , known to be expelled out of mdr cells: above a critical concentration (the "cjc"), this dye forms j-aggregates which emit a uorescence at a wavelength very different from that emitted by the monomer. in the presence of mgatp, the p-gp-containing vesicles accumulated jc- , which exceeded locally the cjc and thus formed intraluminal j-aggregates; these aggregates allowed accumulated jc- both to be sequestered inside the vesicles, by dramatically slowing down its passive backdiffusion, and to be speci cally detected. kinetic characterization of this transport suggests that jc- is rst translocated to the exoplasmic lea et of the vesicle membrane before its internalization into the aqueous phase of the vesicle lumen. interaction between the energy metabolism and externally applied electric elds in yeast cells electric elds are often used for biophysical or biomedical treatment of biological cells, e.g. cell fusion or killing of cells. however, only a few data about the possible mechanisms of electrosensitivity of biological cells are available. since electrostimulation always induces depolarization of biomembranes, an impact of the energy metabolism is obvious due to the regeneration of electrochemical gradients by the expenditure of cellular energy. our aim is to investigate the interactions between externally applied electric elds and the aerobic/anaerobic energy metabolism of yeast cells. for this, we have constructed a new electrical interface for local stimulation of biological cells with variable duration and amplitude. when applying short lasting electrical pulses to yeast cells, we nd a direct response of the energy metabolism (measured by nadh-uorescence) to these pulses. a sudden and fast decrease in nadh is followed by a slower recovery of the uorescence signal. these nadh-signals are abolished in the presence of antimycin a or kcn, demonstrating the importance of mitochondrial energy production for this phenomenon. we attribute these changes to the immediate break down of atp as a consequence of the regeneration of the membrane potential (atpases) and the slower regeneration of atp by mitochondrial respiration. new insights of hypericin blood transport and its incorporation into the plasma membrane b. m. macri , g. stoian , m. l. flonta department of animal physiology and biophysics, faculty of biology, university of bucharest, department of biochemistry, faculty of biology, university of bucharest, bucharest, romania hypericin (hyp) is one of the active compounds from hypericum perforatum (an herb usually prescribed as antidepressant). the bioavailability of this hydrophobic molecule is a very important issue for medical applications. the goal of our work was the study of hyp blood transport mechanisms. techniques of absorbtion spectroscopy, electrophoresis and uorescence microscopy were used in order to de ne the properties of hyp-albumin and hyp-lipoproteins complexes and to explain the action of hyp at the plasma membrane level. hyp bind to several electrophoretic (sds-page) bands evidenced by mice plasma migration. both albumin and lipoproteins bind to hyp, forming complexes, during the blood transport process. different types of lipoproteins from males and females plasma mice were evidenced by gradient electrophoresis to bind hyp. hyp-albumin complex was also identi ed by absorbtion spectra, and the ratio a /a has a ph-dependence. hyp interaction with plasma membranes was also examined on cell culture by uorescence microscopy, and hyp plasma incorporation is a dose-and incubating time-dependent process. our results partially elucidate the plasma fractions that bind hyp, contributing to its blood transport. this study proposes a new mechanism of hyp cellular insertion, discussing its plasmatic membrane penetration due to its high hydrophobicity. the overexpression of p-glyoprotein (p-gp) is one of the major causes of multidrug resistance (mdr) in cancer chemotherapies. many p-gp inhibitors have been designed to reverse the mdr effect, but the structure-activity relationships of the p-gp inhibitors and substrates still remain largely unknown. until now, it is still very challenging to obtain the high-resolution p-gp structures and currently only low-resolution electron microscopy structure is available. this has caused the structure-based design of "p-gp-ignoring" therapeutic agents a remote goal. however, the recent determination of x-ray crystal structures of bacterial lipid a transporter, msba, has provided eligible structure templates for homology modeling of p-gp. we have therefore conducted explicit solvent molecular dynamics simulations of the fulllength ef ux pump, human p-gp, in an excessively hydrated popc bilayer to re ne the homology model. both free and atp-bound forms of p-gp have been simulated. the entire system consists of more than , atoms. our molecular dynamics simulations have shown that the overall architecture of p-gp remained very stable for tens of nanoseconds, while the observed membrane undulation was rather large. the simulation results have allowed us to investigate the conformational changes of p-gp upon atp binding in the ef ux process and to predict the possible binding site of various known substrates and inhibitors. the re ned structure models of p-gp by our simulations could be used as the basis for further drug design. electroporation (ep) is a phenomenon where increased permeability of cells exposed to an external electric eld is observed. the induced transmembrane voltage presumably leads to the formation of aqueous pores in the phospholipid bilayer, which increases permeability of the membrane for molecules and ions. ep is currently used in many biomedical applications including transfer of genes and electrochemotherapy of tumors. still, the molecular mechanisms of the process are not fully explained. recently it was proposed that ep could be monitored in real-time by measuring electric conductivity of tissue. so far the studies focused mostly on a single pulse, however in biomedical applications usually several pulses are used. in our study we used a train of electric pulses to analyse the relationship between electric conductivity and cell permeabilization. current-voltage measurements during and after pulse application were performed in dense suspension of cells. conductivity changes were analysed numerically using nite elements method and compared with the percentage of permeabilized cells. we obtained a transient increase in conductivity above a certain voltage with complete relaxation in < s. substantial changes in conductivity are also due to the diffusion of ions through membrane pores and osmotic swelling. we further show that relation between conductivity and permeabilization level is indirect. interaction of quinolones with bacterial porin ompf: uorescence quenching studies quinolones are widely used antibiotics witch develop their antibacterial action by inhibition of important bacterial enzymes. consequence of the internal location of their target of action, the translocation of this drugs trough the outer membrane is an essential step for their antibacterial action. in vivo studies have been showing that ompf is important for the entrance of some of these antibiotics, but the exact degree of involvement of this protein in the transport of the different members of this group of antibiotics, remains unknown. in this study, the quenching of the intrinsic tryptophan uorescence of ompf, in presence and in the absence of the drugs and by two distinct quenchers, was used as a rst approach, to elucidate ligandinduced structural changes and consequently prove the differential involvement of ompf in the entrance of these antibiotics in the bacterial cell. the results obtained reveal that the degree of interaction with the protein is related with the hydrophobicity of the different antibiotics. this kind of evidence suggests that the entry by the porin channel is not the only path used by these antibiotics and that it is more important for the latest generations of this group because of their increased hydrophilic characteristics. inhibition of multidrug resistance-associated protein mrp and kv channels by natural polyphenols k. michalak, a. teisseyre, b. ania-pietrzak department of biophysics, wroclaw medical university, poland resistance to cytotoxic agents remains a major obstacle to successful chemotherapy in cancer. best-characterized form of drug resistance is caused by the overexpression of genes encoding membrane drug pumps, like p-gp or mrp . in present study, activity of several plant polyphenols ( avonoids and stilbene) against mrp has been studied using functional assay based on ef ux of mrp uorescent substrate. very recently, a role of kv . potassium channels in proliferation of various cancer cells was suggested. in our study the effect of the plant polyphenols on voltage-gated potassium channels kv . was investigated by patch-clamp electrophysiological method. some of studied compounds were found to be active inhibitors of multidrug resistance-associated protein mrp and voltage-gated potassium channels, and their properties are promising for further research in the eld of anticancer activity of natural products. the transport mechanism of melibiose permease: a study using electrical measurements and uorescence techniques k. meyer-lipp , c. ganea , t. pourcher , g. leblanc , k. fendler max planck institut für biophysik, frankfurt, germany, c. davila medical university, bucharest, romania, cea, université de nice so a antipolis, nice, france the melibiose permease (melb) of escherichia coli is a membrane bound carrier that uses the favorable na+, li+, or h+ electrochemical potential gradient to drive cell accumulation of alfa-galactosides (melibiose, raf nose) or beta-galactosides (methyl- -thio-beta-dgalactopyranoside). electrophysiological techniques have proved to be extremely useful tools to investigate the mechanism of ion transfer across the membrane by ion-coupled transporters. using a solid supported membrane (ssm) as a capacitive electrode a rapid solution exchange can be combined with the high sensitivity of planar lipid membranes and allows time resolved investigation of the charge translocation during the catalytic cycle of na+/solute symporters. this technique has been combined with uorescence measurements, which report on structural changes during the substrate transport process of the carrier. we have used time resolved tryptophane uorescence, uorescence energy transfer with a uorescent sugar substrate and site speci c uorescence of a label attached to a cysteine residue on the protein. this allowed us to identify conformational transitions during the reaction cycle of the melibiose permease. we could assess their electrogenicity and determine rate constants. a kinetic model for na+ and melibiose binding and transport is presented. electrophysiological characterization of the vast number of annotated channel and transport proteins in the postgenomic era would be greatly facilitated by the introduction of rapid and robust methods for the functional incorporation of membrane proteins into dened lipid bilayers. we present an automated method for reconstitution of membrane proteins into lipid bilayer membranes, that substantially reduces both the reconstitution time and the amount of protein required. we have applied this well-de ned system to the characterization of a novel mitochondrial uncoupling protein, ucp and demonstrated that ucp exhibits protonophoric function exclusively in the presence of fatty acids, similar to that previously shown for its homologue ucp . the membrane conductance was proportional to the concentration of the reconstituted ucp in presence of oleic acid or eicosatrienoic acid, and was inhibited by atp. amphipols are amphipathic polymers designed to replace or supplement detergents in membrane protein solution studies. for the study of the ca -atpase from sarcoplasmic reticulum, previous experiments have revealed both advantages and disadvantages to the use of a polyacrylate-based amphipol, a - . these issues have been reinvestigated using four different amphipols. size exclusion chromatography showed that, although a - aggregates in the presence of millimolar concentrations of calcium -an effect that probably accounts for most of the aggregation of atpase/a - complexes observed in our previous work-, aggregation can be avoided by resorting to a sulfonated version of a - . we also found that all amphipols tested slowed down the rate of calcium dissociation from its binding sites and reduced atpase activity, while protecting the solubilized protein against denaturation. this suggests that association with the polymer may damp the protein's dynamics, perhaps due to the multipoint attachment of the polymer to its hydrophobic transmembrane surface. such a "gulliver" effect could contribute both to the protection of membrane proteins against denaturation and to the reversible inhibition of serca a. clc proteins are found from prokaryotes to mammals. they function as plasma membrane chloride channels or provide neutralizing anion currents for v-type h -atpases that acidify compartments of the endosomal/lysosomal pathway. vesicular clcs have been thought to be cl -channels, in particular because clc- and clc- mediate plasma membrane cl -currents upon heterologous expression. we have shown, however, that these two mainly endosomal clc proteins rather function as electrogenic cl /h exchangers, resembling the transport activity of the bacterial clc-e that has been crystallized. neutralization of a critical glutamate residue not only abolished the steep voltage-dependence of transport, but also eliminated the coupling of anion ux to proton counter-transport. clc- and clc- may still compensate the charge accumulation by endosomal proton pumps, but are expected to tightly couple vesicular ph-gradients to cl -gradients. calorimetry and mechanics of ca transporting systems in rat myocardial bigeminies a series of new n-oxides of tertiary amines (nta) was checked for its biological activity. individual compounds differed in the length of substituted alkyl chain. the primary goal was to nd if they can be used as effective antioxidants and, to what degree they modify used model (liposomes) and biological (erythrocytes, algae and cucumber) membranes. various methods were used in order to do that. a mechanism of the interaction between nta and membranes was studied by measuring their potency to hemolyse erythrocytes, to in uence a phase transition temperature in dppc liposomes, to change a membrane potential of algae cells. the measure of the interaction of nta with cucumber cells were potassium leakage, chlorophyll content and inhibition of growth of hypocotyls. antioxidative abilities of nta were determined by measuring their ef ciency to protect erythrocytes against membrane lipid oxidation induced by uv irradiation and by comparising their antioxidative ef ciencies with that of trolox (vitamin e analogue) in chromogen experiments. the mostly widely employed mechanism of drug extrusion in bacteria is via membrane transport proteins called ef ux pumps. in gram-negative bacteria, multidrug resistance is conferred by tripartite complexes, rather than by a single transport protein. through these systems, a wide range of substrates is expelled from the cytoplasm, through the periplasmic region, to the exterior of the cell. among these complexes, the acrab/tolc system in escherichia coli is formed by an inner membrane ef ux pump, acrb, an outer membrane protein, tolc, and a periplasmic protein known as an adaptor, acra. the components of this complex are studied, in order to provide insights into drug transport in bacteria. here we present a dynamics study on mexa, homologue of acra from pseudomonas aeruginosa. the protein has been studied by molecular dynamics simulations in bulk water. a structural adjustment by the periplasmic protein is required in order to engage both the bottom part of the om protein and the top region of im protein. the dynamics on mexa reveals a exible behaviour of the protein in water. the major concerted motions observed are the hinge-bending of the two domains, and the rotation of the -barrel domain. these can be related to the adaptation of mexa (and acra) to the om and im proteins during the process of assembly in forming the complex, and during the opening of the channels. electrophysiologic study of ap in chara corallina -indication of its biochemical nature the speci c conductance's of aqueous solution of electrolytes (viz.naf, nacl, nano , na so , kf, kcl, kno , k so , mgcl , cacl , fecl , mncl ,crcl ,cucl , cocl , )have been measured across peritoneum at temperatures between( - ) c.conductance attains a maximum limiting value at higher concentrations for each electrolyte due to a progressive accumulation of ionic species within the transmembrane region. the membrane becomes more and more conductive to incoming ions and attaining a limiting value due to the fact that an electrically neutral pore, which is speci c for a particular ion, is unlikely to contain more than one type of ion. consequently, at high electrolyte concentration, the pore saturates and the conductance's approaches a limiting value. the values of speci c conductance measured follow the sequence for anions; so > cl > no > f . whereas for the cations the sequence is k > na ; ca > mn > co > cu > mg ; cr > fe . the energy of activation for the cations as well as for the anions follows the sequence (for cations): the low temperature ( k) chlorophyll uorescence, photochemical activity, oxygen ash yield and oxygen burst decay of thylakoid membranes with different organization of the light-harvesting chlorophyll a/b complex of photosysytem ii (lhcii) were investigated after freeze-thaw cycle in criotoxic and cryoprotective medium. the increase of lhcii oligomerization, which is associate with signi cant reduction of the surface charge density of the thylakoid membrane, correlates with lower extent of freezing damage of the photosynthetic apparatus, when the procedure is carried out in cryotoxic medium (nacl). in the presence of the cryoprotective compound (sucrose) freezing damage is less pronounced and is not affected by the degree of the lhcii oligomerization. the mechanisms of damage and protection of photosynthetic apparatus in the process of freeze-thaw treatment are discussed. spectral and redox characterization of the novel heme ci in the cytochrome b f complex . this is an indication of different spin delocalization in the primary donor, for the mutant being typical of a monomeric oxidized bchl. considering the fact that the properties of both isolated and membrane-associated mutant rcs were similar, we conclude that missing bchl molecule from the mutant rc was the result of the introduced mutation but not of the protein puri cation procedure. authors acknowledge the support by the russian brf. we created two site-directed mutants, a s and l i in the d protein of photosystem ii in thermosynechococcus elongatus. both mutations are within the binding pocket of the primary quinone acceptor (q a ). we investigated the effects of the mutations in vivo and in isolated psii. while the l i mutant exhibits characteristics similar to the wild type, the a s mutation effects q a charge recombination measured by thermoluminescence and uorescencedecay. these results strongly indicate that the a s mutation induce a shift in the redox potential of q a . the a s accelerates the rate of photoinhibition, an effect consistent with the negative shift in the redox potential. epr was used to measure the temperature dependence of the electron transfer from q a to q b in the a s mutant. it was found to be indistinguishable from the wild type despite the difference in the midpoint potential of q a . this is taken as an indication as a gating mechanism on the acceptor side of psii similar to that in bacterial reaction centers. protochlorophyllide oxidoreductase takes an abnormal reaction pathway below the glass transition g. durin , d. j. heyes , c. n. hunter , d. bourgeois ibs and esrf, grenoble, france, krebs institute and r hill institute for photosynthesis, shef eld university, shef eld, uk motions through the energy landscape of proteins lead to biological function. at temperatures below a dynamical transition ( - k), the activity of some proteins cease. in this work, we describe an enzyme that, instead, engages into a non-productive pathway below k. protochlorophyllide oxidoreductase (por) catalyzes the reduction of protochlorophyllide (pchlide) into chlorophyllide (chlide), a key step in chlorophyll biosynthesis. por is one of the two enzymes known to require light for catalysis. when illuminated with gentle light at k, the complex of t. elongatus por with pchlide and nadph transforms into a nonuorescent intermediate. upon warming, several uorescent intermediates develop, and at k chlide is released. when illuminated at temperatures below k, por behaves differently. if gentle light is used, the reaction can not start. instead, if a blue laser source is used, the initial complex disappears, like at k. however, upon warming, a new intermediate develops that uoresces at nm and leads to a dead-end product. by using uorescence microspectrophotometry, we have measured the solvent glass transition temperature of the system to be k. the solvent glass transition, possibly controlling a por dynamical transition, may be the determinant that switches the enzyme reaction pathway from a non productive to a productive one. the nonproductive pathway results from a two-photons absorption mechanism, whereas the productive pathway is a one-photon mechanism. sensory rhodopsin ii from n. pharaonis (npsrii) forms a complex with its cognate transducer nphtrii in a : stoichiometry . light activation of npsrii leads to a movement of helix f which triggers a rotation of tm in nphtrii . the mechanism of signal transduction through the hamp region to the cytoplasmic domain of the transducer is still unknown. structural information exists for the transmembrane and cytoplasmic regions, however the hamp domain is not yet characterized. in order to obtain structural information on this domain, twenty-four residues in the membrane adjacent region ( - ), and six residues in the following region were spin labeled and investigated by cw and pulsed x-band epr. to analyze the overall architecture of the complex, doubly spin labeled variants between the transducer and the receptor were also engineered. depending on their function, the absorption spectra of rhodopsins can be tuned by the protein over a wide range. a major determinant for spectral shifts between different rhodopsins are electrostatic interactions between the chromophore retinal and the protein. we compute and compare the classical electrostatic potential at the retinal of three archaeal rhodopsins: bacteriorhodopsin (br), halorhodopsin (hr), and sensory rhodopsin ii (srii). these proteins are an excellent test case for understanding the spectral tuning of retinal. the absorption maxima of br and hr are very similar, while the spectrum of srii is considerably blue shifted. we nd that the electrostatic potential is similar in br and hr, but differs signi cantly in srii. a quantum mechanical model of a particle in a box with a step potential can qualitatively relate the differences between the electrostatic potentials of the proteins to the relative shifts of their absorption maxima. by decomposing the electrostatic potential into contributions of individual residues, we could identify six residues that are responsible for the differences in electrostatic potential between the proteins. three of these residues are close to the retinal, while the other three residues are more then angstroem away from the retinal. the counterion of the schiff base, which is frequently discussed to be involved in the spectral tuning, does not contribute to the dissimilarities between the electrostatic potentials. effect of uv-a radiation on thylakoid membranes with different organization p. ivanova, a. dobrikova, t. markova, s. taneva, e. apostolova institute of biophysics, bulgarian academy of sciences, so a, bulgaria the effect of uv-a ( - nm) radiation on the energy transfer and the photosynthetic oxygen evolution of thylakoid membranes from pea mutants was investigated. the membranes have different pigment composition, stoichiometry and organization of pigment-protein complexes. the aim of our work was to nd out whether uv-a induced damage is affected by the altered content and/or oligomerization of the main light-harvesting chlorophyllprotein complex (lhcii) in thylakoid membranes. the data for the effect of uv-a radiation on the oxygen evolution demonstrate that: (i) the inhibition of photosystem ii (psii)-mediated electron transport and ash-induced oxygen yields strongly depend on the amount of lhcii; (ii) the increase of the s o populations of psii centers in darkness is more pronounced in thylakoid membranes with smaller amount of lhcii; (iii) the inhibition of the oxygen evolution is related to the reduced number of the functionally active psii centres; (iv) the degree of impairing of active psii centres depend on the amount and oligomerization of lhcii. the results also show that the altered content and organization of lhcii in uence the uv-a light-induced changes in the energy transfer between psii and psi and within the supramolecular lhcii-psii complex. the effects of uv-a radiation on leaves and isolated thylakoid membranes are compared. sudden polarisation -a large change in the electric dipole moment between the excited and the ground state -is a well-known phenomenon for retinal chromophore. some early models of the energy transduction mechanism in bacteriorhodopsin (br) even attribute a primary functional role of that. however, it was apparently unrecognized that the maxwell theory intuitively predicts the appearance of an ultrafast transient electromagnetic radiation due to this dipole moment change. here we show that the existence of this type of radiation can be derived from semiclassical quantum electrodynamics as a second order phenomenon. in optical terms it corresponds to the previously unstudied resonant case of optical recti cation. recently we experimentally observed a major component in the fs coherent infrared emission of oriented purple membranes of br corresponding well to this effect (groma et. al, proc. natl. acad. sci. , , ). our theory predicts that such a signal holds detailed information on the dynamics of excited state polarization, opening a new branch of impulsive spectroscopy on asymmetric systems. beyond optical recti cation we found a complex phase a coherent oscillation living for a few ps, i.e. much longer than the excited state of br. fitting analysis resulted in at least seven vibrating modes in the - cm region, while windowed fourier transform indicated time-dependent frequency distribution. a. ghignoli, g. cercignani, s. lucia, g. colombetti istituto di bio sica, cnr -pisa, dipartimento di fisiologia e biochimica, università di pisa, italy the life cycle of ophryoglena ava, a histophagous ciliate dwelling in fresh waters, reportedly includes several stages that feature morphology changes and different phototactic responses. previous studies on the phototactic responses in o. ava during its phase of maximal positive phototaxis led to an action spectrum with two main peaks at and nm, and a minor peak at nm. starting from those results, we analyzed the phototactic response at various cell ages, using three broad-band interferential lters (fwhm = nm) centred respectively at , and nm, and constructed dose-effect curves for each band. a higher photosensitivity at nm, and lower photosensitivies with the other two lters ( and nm) have been observed at any cell age. however, the photosensitivities in the blue and orange regions show a different time course vs. cell age with respect to the photosensitivity in the green region. measures were also carried out on cells whose feeding cycle was altered by a -day starvation (a double time with respect to the standard protocol) before being fed at t = . the maximal photoresponse values reached by starved cells are lower than the highest values reached with standard cultures; in other words, a general reduction of the phototactic response is observed. these results suggest that, while feeding optimally induces cell division, it does not generally reset all cellular functions. a. quaranta , f. lachaud , y. pellegrin , p. dorlet , m.-f. charlot , s. un , a. aukauloo , w. leibl service de bioénergétique, cea-saclay, bât. , gif-sur-yvette cedex (france), laboratoire de chimie inorganique, bât. , université paris-sud, orsay (france) coordination complexes based on a photoactive rutheniumpolypyridyl moiety linked to simple, rigid ligands with binding sites for transition metals, are developed to mimic the light induced charge separation and water oxidation processes taking place in the photosynthetic apparatus. inspired by the structure around the donor side of photosystem ii a family of phenanthroline based ligands holding an imidazole, a phenol or an indole unit simulating the amino acids histidine, tyrosine and tryptophan in the oxygen evolving complex, were developed as models for proton-coupled electron transfer. in some of the molecules investigated the hydrogen bonding interaction present in the natural system is reproduced. combined data from photophysical, spectroelectrochemical studies and dft calculations evidenced the photogeneration of a phenoxyl or a tryptophan radical upon excitation of the chromophore in presence of an external electron acceptor, therefore mimicking the electron trade between p and tyrz-hist . finite element model to predict the electric potential distribution in ps i containing vesicles c. p. a. pennisi , e. chemineau , e. greenbaum , k. yoshida center for sensory motor interaction, aalborg university, denmark, chemical sciences division, oak ridge national laboratories, usa, facultad de ingeniería, uner, argentina photosynthetic reaction centers (rc) are integral membrane proteins and molecular photovoltaic structures. recently, it was suggested their use as triggers of voltage-gated ion channels in excitable cells, where a certain voltage threshold has to be reached to evoke a response (kuritz et al., ieee trans. nanobiosci. in press ). experimental studies with rc's reconstituted in lipid vesicles have shown different values of transmembrane voltage, depending on parameters like light intensity, rc concentration and membrane passive properties. ultimately, the purpose of this work is to have a tool to estimate the proximity, number and density of rc's required near a voltage-gated channel to activate an excitable cell. as a starting point, we aim to predict the spatial distribution of the membrane potential in vesicles. a nite element model was realized using a commercial package (femlab, comsol a/s). the three-dimensional distribution of the electrical potential near a single rc in the surface of a spherical vesicle was calculated. in terms of density, in conditions of saturating light, a minimum of , e rcs/cm is needed to develop a potential of mv, capable to activate voltage-gated sodium channels. microsecond time-resolved x-ray diffraction study of purple membrane t. oka , k. inoue , m. kataoka , n. yagi faculty of science and technology, keio university, japan, japan synchrotron radiation research institute (jasri), japan, nara institute of science and technology, japan the structural changes in the photoreaction cycle of bacteriorhodopsin, a light-driven proton pump, was investigated at a resolution of Å by time-resolved x-ray diffraction experiment utilizing synchrotron x-rays from an undulator of spring- . the xray diffraction measurement system, used in coupling with a pulsed yag laser, enabled to record diffraction pattern from purple membrane lm at a time-resolution of µsec over the time domain of µsec to msec. the low temperature ( k) chlorophyll uorescence, photochemical activity, oxygen ash yield and oxygen burst decay of thylakoid membranes with different organization of the light-harvesting chlorophyll a/b complex of photosystem ii (lhcii) were investigated after freeze-thaw cycle in cryotoxic and cryoprotective medium. the increase of lhcii oligomerization, which is associate with signi cant reduction of the surface charge density of the thylakoid membrane, correlates with lower extent of freezing damage of the photosynthetic apparatus, when the procedure is carried out in a cryotoxic medium (nacl). in the presence of a cryoprotective compound (sucrose) freezing damage is less pronounced and independent of the degree of the lhcii oligomerization. the mechanisms of damage and protection of photosynthetic apparatus during the freeze-thaw process are discussed. we have studied the effect of a cytokinin meta-topolin (mt, m) on senescence-induced changes in the photosynthetic apparatus of detached primary leaves of wheat (triticum aestivum l. cv. hereward). the senescing leaves were kept under continuous light conditions. mt signi cantly slowed down the senescenceinduced decrease in chlorophyll content and markedly stimulated violaxanthin zeaxanthin (z) conversion. the high z content was maintained even after an hour in darkness. mt treatment caused also the appearance of an emission band f peaking at - nm. this emission band is attributed to aggregates of lightharvesting chlorophyll a/b-binding proteins (lhc), the production of which is associated with a higher z content. the presence of lhc aggregates in mt treated leaves was documented also by electron microscopy imagines. besides the lhc aggregation, mt induced also a decrease in photosystem i content which was documented by electrophoresis and k-uorescent spectra. supported by grants frvs / and msm . recently high resolution images of bacterial photosynthetic membranes have revealed the organization of membrane proteins in these native membranes. the organization revealed is remarkable, and all the more so when we realize that these specialized, protein rich, membranes differentiate from the cytoplasmic membrane which has a more complex composition and is richer in lipids. analysis of the protein organization in these specialized membranes from several different bacteria suggest that the organization results from a phase separation of several different contiguous phases. in order to better understand our observations we have undertaken an examination of the different phase behaviors that are possible for membrane proteins considered as a two dimensional colloid. monte-carlo modeling of the phase diagram of this system shows the importance of interaction distance in the determination of system behavior. transcription of our observations on the model systems to the photosynthetic membranes suggests that electrostatic and elastic forces in the membrane are of particular importance in determining the high level order of membrane proteins. the recent crystal structure of photosystem i (psi) from synechococcus elongatus shows two quasi-symmetric branches of potential electron transfer cofactors including primary donor (dimer of chlorophylls p ), monomeric chlorophylls a and a and quinone a , bound to the psaa/psab heterodimer. so far, it is not clear if both potential electron transfer pathways are active in this process or only one of them. to solve this issue, we studied a set of mutants with methionine coordinating the primary electron acceptor, a , replaced by histidine, leucine, or serine in either of two branches. our results obtained with a technique of femtosecond transient absorption spectroscopy show that both branches are equally active in electron transfer. mutation in either branch slows the forward electron transfer between a and a from ps in wilde type psi to - ns in all these mutants. this strong effect is explained by signi cant change in the redox midpoint potential and change in the position of a by the mutations. i. s. zaharieva department of biophysics and radiobiology, faculty of biology, university of so a an approach to the investigation of structural and functional properties of the photosystem ii supramolecular complex in native photosynthesizing objects based on the registration of delayed chlorophyll a uorescence is developed. using a disc phosphoroscope, we register simultaneously: i) changes of the intensity of millisecond delayed uorescence (decayed in . - . ms time range) during the transition of the photosynthetic apparatus from dark to lightadapted state; ii) changes of the intensity of delayed uorescence decaying in different subintervals of the investigated time range; iii) dark relaxation curves at different moments of the transition; iv) changes of the intensity of prompt chlorophyll a uorescence. the analysis of these data allows the correlation of the delayed uorescence characteristics to particular processes occurring in the photosystem ii complex -proton or electrical gradient accumulation, changes in the redox state of quinone acceptors, changes in the pigment-protein complexes caused by different stress factors, for example temperature. three-dimensional structure of major light-harvesting antenna of photosystem ii from cucumber h. yan , z. liu , k. wang , t. kuang , j. zhang , l. gui , x. an , w. chang national lab of biomacromolecules, institute of biophysics, chinese academy of sciences(cas), datun road, chaoyang distr., beijing , china, lab of photosynthesis and environmental molecular physiology, institute of botany, cas, nanxincun, xiangshan, beijing , china the major light-harvesting antenna complex of photosystem ii (lhc-ii), the most abundant integral membrane protein, functions in light capture, energy transfer/distribution and photoprotection. lhc-ii from different species or conditions shows different spectral properties and variation in polypeptide and pigment components. this indicates some speci c function-related alterations in the organization of lhc-ii. here we report a . -Å crystal structure of cucumber homo-trimeric lhc-ii, organized in a perfect virus-like icosahedral particle. the electron-density map shows the reasonable existence of a chlorophyll (chl) a/b mixed binding site in the complex. the occurrence and locus of lactucaxanthin (lac) was seen directly for the rst time. based on the credible structure information, a mechanism of the energy transfer, regulation and excess excited energy dissipation under high light condition was proposed. coherent anti-stokes raman scattering microscopy (cars) is a new approach for chemical imaging of molecular systems within cells and tissues, with high sensitivity, high spatial resolution, and three dimensional sectioning capabilities, without using uorophores that are prone to photobleaching. this technique permits to map selectively molecular species, by using vibrational properties of their chemical bounds. the epi detected (e-cars) and forward detected (f-cars) intensities depends on the shape, the size of the sample, as well as the index of the solvent. in this presentation, after introducing the cars microscopy technique, we show the rst cars studies of the refractive effect of the sample, comparing the e-cars and f-cars signals for different diameters of polystyrene beads, in different refractive index solvents. we present several simulations, comparing forward-detected and backward-detected signals in different sized polystyrene beads, embedded in different index solvents, and we show that, the backwardre ected f-cars dominates the experimentally epi-detected signals. furthermore, we demonstrate experimentally and theoretically that the maxima of forward and epi-detected signals are generated at different positions along the z axis in the sample. we nally discuss how index mismatch in cells can alter cars images. the effects of static magnetic elds on humans have been the subject of continuous investigations. since one of the major static magnetic eld sources is nuclear magnetic resonance imaging (mri), the present study aimed to investigate the effects of . t magnetic eld that is produced by mri on humans. the study is carried out with voluntary and healthy young men from to years of age. the subjects informed about the purpose of the study at the beginning. the subjects exposed to minutes of . t static magnetic eld by means of putting the subjects into the magnetic resonance unit. ml blood was taken from each subject one minute before and one minute after exposure. t and t relaxation times and trace elements were measured in of pre and post exposure plasma of the subjects. the obtained post exposure values were compared with pre-exposure values of the subjects. pre and post exposure results were analyzed by means of student t-test. evaluation of tumor response of breast cancer patients by diffusion weighted mri k. a. danishad , v. seenu , u. sharma , p. k. julka , g. k. rath , n. r. jagannathan department of nmr, department of surgery, department of radiotherapy, all india institute of medical sciences, new delhi, india diffusion weighted mr imaging (dwi) measures the diffusion of water molecules in tissues and is quanti ed by apparent diffusion coef cient (adc). dwi can be used to differentiate tumors from normal tissue and also can be used to monitor the response of tumor to chemotherapy. thirteen healthy volunteers and twelve patients were recruited for the study. dw images were obtained prior to therapy (n= ) and after three cycles of therapy (n= ). the mean adc value of tumors ( . x . mm /s) was signi cantly less (p < . ) compared to the normal tissue ( . x . mm /s). decrease in adc in tumor is due to an increase in the cellularity which restricts the diffusion of water molecules. in patients receiving neo-adjuvant chemotherapy, the adc values were higher ( . x . mm /s) and were closer to that of the normal tissue (p < . ), indicating response of the tumor to chemotherapy. the post-therapy increase in adc is due to the cell damage caused by the therapeutic agents which increases the fractional volume of the interstitial space, causing an increase in the mobility of water. the study showed that dwi can be used non-invasively to assess the response of breast cancer patients to neo-adjuvant chemotherapy. quanti cation by optical imaging of gene electrotransfer in mouse muscle and knee optical imaging was evaluated for monitoring and quanti cation of the mouse knee joint and tibial cranial muscle electrotransfer (et) of a luciferase encoding plasmid. the substrate of luciferase (luciferin) was injected i.p or locally in the muscle or the knee joint. luminescence resulting from the luciferase-luciferin reaction was measured with a cooled ccd camera. luminescence of the knee joint and muscle were higher after local than after i.p injection of luciferin, but both measurements were highly correlated. local injection procedure was adopted. a signi cant correlation was observed between measurements in vivo and in vitro on the same muscle. reproducibility of individual luminescence measurements was also veri ed, and the luminescence levels were clearly dependant of the amount of plasmid injected. in vivo luciferase in the electrotransfered knee joint was detected for two weeks. intramuscular electrotransfer of . or µg of plasmid led to stable luciferase expression for days, whereas injecting µg plasmid resulted in a luminescence fall two weeks after electrotransfer. these decreases were, at least partly, related to the production of antibodies against luciferase. thus, optical imaging was shown to be a relevant technique to quantify variations of luciferase activity in vivo in one given tissue. furthermore, evaluating the effective amount of luciferase in tissues from in vivo luminescence levels requires calibration since it relies on conditions of the enzymatic reaction and light absorption. acute effect of corticosterone on nmda receptormediated ca + elevation in mouse hippocampal slices m. saito , s. sato , h. osanai , a. hirano , y. komatsuzaki , s. kawato department of physics and applied physics, college of humanities and sciences, nihon university, department of biophysics and life sciences, graduate school of arts and sciences, university of tokyo corticosterone (cort) is a principal glucocorticoid synthesized in the rodent adrenal cortex and secreted in response to stress. we examined the rapid effects of cort on n-methyl-d-aspartate (nmda) receptor-mediated ca signals in adult mouse hippocampal slices by using ca imaging technique. application of nmda caused a transient elevation of intracellular ca concentration followed by a decay to a plateau within sec. the min preincubation of cort induced a signi cant decrease of the peak amplitude of nmda-induced ca elevation in the ca region. the rapid effect of cort was induced at a stress-induced level ( . - µm). because the membrane non-permeable bovine serum albuminconjugated cort also induced a similar rapid effect, the rapid effect of cort might be induced via putative surface cort receptors. in contrast, cort induced no signi cant effects on nmdainduced ca elevation in the dentate gyrus. in the ca region, cort effects were not evaluated, because the marked elevation of nmda-induced ca signals was not observed there. in vivo subcellular structures recognized with phase k. nagayama , r. danev , n. usuda , y. kaneko , k. nitta , a. nakazawa , k. atsuzawa , m. tanaka , m. setou okazaki institute for integrative bioscience, okazaki, japan, fujita health university, school of medicine, toyoake, aichi, japan, saitama university, saitama, japan, tokyo metropolitan institute of gerontology, itabashi-ku, tokyo, japan phase contrast transmission electron microscopy has been developed to enable a high contrast and a high resolution observation for unstained ice-embedded samples. to enhance the image contrast, two methodologies have already been developed; i) scattering contrast for stained samples with small aperture diaphrams and ii) defocus contrast for unstained or stained samples with deep defocusing. the former prevails in histochemical sciences and the latter is popular in electron crystallography. both methods, however, have a common drawback that the contrast is only improved by impairing the image quality. this drawback can be removed with use of the phase contrast method using phase plates, which has traditionally been used in visible light microscopy. due to the severe obstacle of the charging of phase plates, however, the idea has not yet been materialized. we have solved the phase-plate charging problem. an experiment kv with tem for a whole cell from cyanobacterium unstained and ice-embed ful lled the expectation. only weak and vague contrast was obtained for the conventional image of the cell even with a very deep defocus. contralily a high-contrasted image has appeared for phase contrast images, where various ne structures are clearly recognized. this may be a rst example to observe nanometer scale structures in details in the intact cell. other examples including intact state intravesicular structures will be shown. j. lichtenberger, p. fromherz max-planck-institute for biochemistry we cultured bovine chromaf n cells on an array of electrolyteoxide-silicon eld-effect transistors (eos fet) and monitored granule secretion. by stimulation with barium chloride, vesicles are released into the narrow sheet of electrolyte between the chip surface and the plasma membrane. the interaction of released protons with the silicon dioxide surface of the chip alters the threshold voltage of the transistor and gives rise to a measurable signal. simultaneously performed measurements with a carbon bre showed a correlation of the transistor signals and amperometric current traces. we conclude that the transistors are able to monitor exocytosis on a single vesicle level. to elucidate the role of protons, we destroyed the proton gradient across the vesicle membrane by nigericin and valinomycin. as a result a massive reduction of the transistor signals was induced, whereas there was only little change of the amperometric records. we conclude that released protons are responsible for the detection of vesicles with transistors. the individual transistor records of vesicle exocytosis can be explained by combining the dynamics of the exocytotic event with the diffusion in the cell-chip junction. transistor recording of exocytosis does not depend on the electrochemistry of transmitters. as many kinds of exocytotic vesicles contain a large amount of buffered protons it can be applied to numerous kinds of exocytotic events, independent on the nature of the transmitter. we tried various solvents for the solubility of the uorescent product, and found that the product was insoluble in water and most organic solvents. a quite bright uorescence emitted by the particles was observed by uorescent microscope when emitted by uv nm. sem indicated that the size of the particles was µm µm, depending on the reaction time and phospholipid concentration in hexane solution. endothelial cells from human vein grew better on the surface prepared from the particles than the culture plate, implies a possible application as a new type of biomaterial as a coating material for medical devices, and as a uorescent tracer for human bodies. confocal microscopy of the phototactic ciliate f. salina. fabrea salina is a marine heterotrich ciliate, which dwells in salt ponds. in previous works we have described the phototaxis and the uorescence properties of a hypericin like endogenous pigment in an albino strain. we have recently obtained a heavily pigmented strain from the saline of torre colimena (taranto, italy). we have used confocal microscopy to characterize the uorescent properties of this strain and to compare them with those of the albino strain. the results obtained by one and two photon confocal microscopy show that, as in the albino strain, the uorescence intensity of the pigment is higher in dead cells than in the living cells. the excitation and emission spectra are quite similar in the two strains and this is also true for the uorescence lifetime, which is about ns. all together, these measurements indicate that the pigment of the new strain belongs to the family of hypericin-like chromophores. the analysis of different confocal planes shows that the pigment is localized not only in granules under the somatic membrane in the cellular body, as currently thought, but also in the cilia. some experiments of fotobleaching "in situ" con rm this result, that might have important implications in the understanding of the mechanisms of the photomotile responses of f. salina and probably of other heterotrich ciliates. there is no doubt that modern physician should have the knowledge of basic sciences as physics, chemistry and biology. furthermore, the biophysics is incorporated into the curriculum of most european medical schools. at medical school in zagreb, the course of physics and biophysics is positioned in rst and forth year of study. the students learn basic physics phenomena of structure of matter, mechanics, thermodynamics, electromagnetism, optics and acoustics applied on the human body. additionally, the interactions of the body with the surrounding are thoroughly discussed as the basis for different diagnostic methods. the arguing at our school is still going on where to include the content of this course. should it be the autonomous course or the part of physiology and radiology courses in the problem based learning approach?! so far at zagreb medical school the biophysical courses are autonomous structured according to the biophysics programs at other european universities. the highlighting is on seminar work and lab, encouraging the students for individual learning. the seminars are made more vivid and instructive for students by inclusion of different model devices constructed in our department learning on science is also learning how scienti c knowledge is produced. in this sense, issues related to the dynamics of science should be brought into focus in science education. the idea has support in the "declaration on science and the use of scienti c knowledge", particularly in the statement that "science curricula should include science ethics, as well as training in the history and philosophy of science and its cultural impact". thinking on rst year undergraduate students, we are interested in an integrated approach of that kind of themes. this presentation describes a practical teaching module included in the basic biophysics course for biochemistry majors. organized in case studies, it deals with stories of biophysics (and biochemistry), and addresses the role of the biophysical approach in the progress of the life sciences. the module follows the whole course, and consists in small exercises on the way a given understanding has been constructed explored within the practice trend of contemporary science studies. examples of the chosen stories -anchored in the subjects covered in the lectures of the course -relate to the search for the mechanism of energy production through atp-synthase, the development of radioactive labelling techniques, and the discovery of protein water channels. beyond their value as cultural legacy and as motivating tools, the insight they might provide is vast. axiomatic theory of biophysics q. zhao china rehabilitation research centre, beijing, china. until now, all approaches to interpret biology by applying principles of physics have been announced failure. the achievement of biophysics is limited in area to provide essential tools for biological research and biophysics is far from to be the basic theory of biology. to resolve it requires the fundamental research about the logic features of biophysical processes of biology. we promoted system logic and then protein thermodynamics structure theory. based upon it, the axiomatic theory of biophysics and biology could be developed. our result shows that the real understanding of biology or biophysics must be constructed based upon new thinking methods. a successful ligand-receptor docking methodology depends strongly in the ef ciency of the global optimization algorithm used to explore the ligand conformational space. in this work we have implemented and analyzed the performance of a new exible ligand-receptor docking methodology. this methodology uses as optimization method a multisolution version of the generalized simulated annealing algorithm adapted to problems with box constraints. a grid-based methodology, considering the receptor rigid, and the gromos classical force eld are used to evaluate the ligand-receptor scoring function. the methodology was tested in redocking (ligand within it own protein conformation) and cross-docking (ligand within another protein conformation) experiments for ve hiv protease-ligand complexes with known threedimensional structures. all ligands tested are highly exible, having to conformational degrees of freedom. the implemented docking methodology was able to redock successfully all exible ligands with a success ratio % and a mean rmsd lower than . Å with respect to the corresponding experimental structures. in the cross-docking experiments we observed a strong dependence of the mean success ratio with respect to the protein structure used as reference. in situations we observed a mean success ratio % and % in cases among the possible ones. s. aida-hyugaji , h. nakagawa , j. nomura , m. sakurai , d. tokushima , t. takada , u. nagashima , t. ishikawa tokai university, japan, tokyo institute of technology, japan, nec corporation, japan, national institute of advanced industrial science and technology, japan irinotecan is a widely-used antitumor drug that inhibits mammalian dna topoisomerase i. however, overexpression of abcg can confer cancer cells resistance to sn- , that is, the active form of cpt- . in the present study to develop a platform for the molecular modeling to circumvent drug resistance associated with abcg , we have characterized a total of fourteen new sn- analogues by some typical properties, which were evaluated by molecular orbital (mo) calculations and neural network (nn) analysis. the nn was rst applied to estimate hydrophobic properties (logp) of the analogues. thereafter, the electrostatic potential (esp) and the solvation free energy ( g) were evaluated by mo calculation. these indexes were found to be well correlated with the drug resistance ratio experimentally observed in abcg -overexpressing cells. it is suggested that hydrophilic analogues carrying oh-or nh -groups are good substrates for abcg and therefore exported from cancer cells. in contrast, sn- analogues with cl or br atom at those positions have similar logp values and high af nities toward the putative active site of abcg , however they were not substrates of abcg . from these results, it is strongly suggested that hydrogen bond formation with oh-or nh -groups are critically involved in the transport mechanism of abcg . a. agopian , j. depollier , e. gros , g. aldrian-herrada , p. clayette , n. bosquet , g. divita crbm-cnrs, route de mende, montpellier, france., spi-bio-cea fontenais aux roses, france reverse transcriptase (rt) plays an essential role in the replication of hiv and constitutes the main target for the development of aids therapies. the biologically active form of hiv rt is a heterodimer of two subunits, p and p , each consisting of distinct subdomains: the ngers, the palm, the thumb, the connection and the rnase h subdomain, the latter only present in p . we have demonstrated that formation of fully active rt is a two-step process involving rapid association of the two subunits (dimerization) followed by a conformational change (maturation). thanks to the crystal structure of rt we have identi ed a new class of inhibitors based on short peptide motifs derived from the dimer interface. we rst identi ed a short mer peptide (pep- ) derived from the tryptophanrich motif of the connection subdomain that blocks dimerization of rt and ef ciently abolishes hiv- replication. pep- interacts preferentially in a pocket involving residues trp and phe on p . we then designed mer peptides derived from the thumb domain which inhibit rt maturation as well as viral replication when delivered into cells. taking into account these results we propose that dimerization of rt constitutes a potential target for the design of more speci c new antiviral drugs. . the success of gene therapy largely relies on the availability of vectors that would deliver the genetic material ef ciently to the target cells with a minimal toxicity. in this context, our purpose was to evaluate as possible vectors a series of newly synthesized low molecular weight ( kda) chitosan derivatives grafted with dodecenoyl (ddc) groups at different percentages ( , , and %). in the absence of dna, the critical micellar concentration (cmc) of these derivatives in mm mes buffer ph . was found to be strongly dependent on the percentage of ddc but not on ph or salt concentrations. this indicates that the ddc groups confer to the chitosan derivatives the potency to self-assemble probably in micellar structures: a property that may dictate the formation and the structure of their complexes with dna. next, we investigated by quasielastic light scattering the size and the surface charge of complexes of plasmid dna with these derivatives at different ph, salt concentrations and n/p ratios (expressed in charged units of chitosan amines to dna phosphates). we found the smallest and more positively charged complexes were obtained at ph . and n/p= in the absence of salt: a condition where the chitosan derivatives were fully protonated and in excess over the dna phosphate groups. biophysical and biological examination of dna/lipids complexes particles of virus-like structure designed for in vivo gene transfer d. durand , m. schmutz , b. lebleu , a. r. thierry lure, centre universitaire paris sud, orsay, france, institut henri sadron, strasbourg, france, laboratoire des défenses antivirales et antitumorale, umr , montpellier, france the structure of complexes made from dna and suitable lipids (lipoplexes lx) was examined by cryo electron microscopy. we observed a distinct concentric ring-like pattern with striated shells when using plasmid dna. these spherical multilamellar particles have a mean diameter of nm with repetitive spacing of . nm with striation of . nm width. small angle x-ray scattering (saxs) con rmed cryoem data and revealed repetitive ordering of . nm, suggesting a lamellar structure containing at least a dozen layers. this concentric and lamellar structure with different packing regimes was also observed by cryoem with linear dsdna, ssdna and oligodeoxynucleotides. for the rst time, dna chains could be visualized in dna/lipid complexes. such speci c supramolecular organization is the result of thermodynamic forces, which cause compaction to occur through concentric winding of dna in a liquid crystalline phase. cryoem of t phage dna packed either in t capsides or in lipidic particles showed similar patterns. saxs suggested an hexagonal phase in lx-t dna. thus, both lamellar and hexagonal phases may coexist in the same lx preparation or particle and transition between both phases may depend upon equilibrium in uenced by type and length of the dna used. organization of such nucleotidic supramolecular assemblies is relevant for prebiotic chemistry. engineering self-assembly peptides for targeted delivery of therapeutics and imaging agents s. s. dhadwar, m. sung, k. kawamura, j. gariépy department of medical biophysics, university of toronto, canada peptide-mediated delivery systems have recently emerged as a means to substitute or augment conventional drug and gene delivery technologies. these approaches are versatile and easily designed to incorporate a number of speci c attributes required for ef cient delivery of therapeutic and imaging agents. in particular, self-associating peptide domains can be utilized to construct stable and structurally well-de ned protein-like assemblies displaying a series of cell-routing functions. more speci cally, a peptide-based self-assembling intercellular delivery vehicle was designed by incorporating the -residue long tetramerization domain of the human tumor suppressor protein p (hp tet). the resulting peptide tetramer displays termini within its structure that allows for the simultaneous presentation of distinct cell targeting signal or functional domains. the fusion of polycationic sequences to the hp tet domain promotes the cellular import of the resulting constructs into eukaryotic cells. this internalization event was dramatically enhanced for such multivalent peptides in relation to their monomeric counterparts. peptides containing a nuclear localization sequence along with a polycationic sequence were found to shuttle reporter plasmids ef ciently to the nucleus of cells. these results have important implications in the design and construction of novel targeted delivery vehicles. mechanisms of non-covalent peptide mediated cellular delivery of therapeutics: a biophysical study s. deshayes , m. c. morris , a. heitz , p. charnet , g. divita , f. heitz crbm -cnrs fre montpellier france, cbs -cnrs umr -inserm u montpellier france two different cell-penetrating peptides mpg and pep- were shown to promote non-endosomal intracellular delivery of non-covalent bound cargos, namely nucleic acids and proteins; respectively. in order to identify the peptide mediated internalization pathway, we undertook conformational investigations of both peptides with and without associated cargos and checked the conformational consequences of the presence of phospholipids. from the conformational point of view, pep- behaves differently from mpg. cd analysis revealed a transition from a non-structured to a helical conformation upon increase of the concentration while mpg remained nonstructured. determination of the structure by nmr showed that in water, it's a-helical domain extends from residue to . cd and ftir indicated that pep- adopts a helical conformation in the presence of phospholipids while mpg is in a -sheet form. adsorption measurements performed at the air-water interface were consistent with the helical form. pep- did not undergo conformational changes upon formation of a particle with a cargo peptide. in contrast, we observed a partial conformational transition when the complex encountered phospholipids. for mpg, interactions with nucleic acids generated a partial folding into -sheet which was more pronounced in the presence of lipids. electrophysiological measurements showed that both peptides, whether associated or not with their cargo, can induce transmembrane pore-like structures. self-assembly of hydrolysed alpha-lactalbumin into nanotubes j. f. graveland-bikker , k. g. de kruif nizo food reseach, ede, netherlands, van´t hoff laboratory, netherlands nanotubes are formed by self-assembly of partially hydrolysedlactalbumin, a kda milk protein. there are several promising applications of these -lactalbumin tubes, in food, pharmacy and nanotechnology. we studied the mechanism of self-assembly, the structure and the properties of the nanotubes. limited proteolysis of the -lactalbumin (by a serine protease) makes the molecule prone to self-assembly. in the presence of ca tubular structures are formed. other divalent ions like mn and zn can also induce tubular self-assembly, while mg leads to random aggregation. light scattering showed that the self-assembly is reversible, which is of relevance for controlled release applications. on the other hand, we could also make stable tubes by cross linking, which would be a requisite for several other applications. from afm and saxs measurements, we obtained values for the outer diameter: nm; and the inner diameter: nm. afm and cryo-em revealed the helical structure of the tube wall; it is a right-handed helix. by performing nano indentations with afm we determined mechanical properties of the tubes. the tubes were shown to be relatively resilient upon small deformations; the elastic modulus is of the order of . gpa. targeted delivery of photosensitizers into the cancer cell nuclei enhances their cytotoxic ef cacy the search for new pharmaceuticals has raised interest in locallyacting drugs which act over short distances within the cell, and for which different cell compartments have different sensitivities, e.g. photosensitizers used in anticancer therapy should be transported to the most sensitive subcellular compartments where their action is most pronounced. earlier we have produced a number of modular recombinant transporters for locally-acting drugs comprising several functional modules for cell-speci c targeting, internalization, escape from intracellular acidic vesicle, and targeting to the nuclei of melanoma cells overexpressing melanocortin receptors. here we describe new transporters on the basis of epidermal growth factor which are speci c for a wide variety of cancers. these transporters possess all necessary functional activities and deliver photosensitizers into the nuclei of human carcinoma cells to result in photocytotoxic effects almost orders of magnitude greater than those of nonmodi ed photosensitizers. characterization of mixtures of dna and nonionic polymeric agents for gene delivery in muscle j. m. gau , j. lal , l. auvray laboratoire mpi -lrp, umr cnrs , université d´Évry val d´essonne, Évry cedex, france, argonne national laboratory, ipns, south cass avenue, illinois , usa a strategy to cure muscle disease is to introduce genes (dna) into the muscle cell to correct or to add genes. nonionic polymeric agents have emerged as an ef cient vector to deliver dna in the muscle. these polymers protect dna from extracellular nuclease degradation by allowing the dna diffusion throughout the muscle tissue. there is at present no understanding about how nonionic polymers enhance transfection in the muscle. the kind of interactions between these nonionic agents and dna, dna-nonionic polymeric agent mixtures and cell membrane are currently unknown. also the structure of dna-nonionic polymeric agent mixtures is not yet well de ned. more information is needed to improve this delivery system. neutron scattering (contrast variation) and light scattering were used to investigate the interaction between: dna and nonionic polymers (pvp, di-and triblock copolymers). furthermore, electrical measurements with the same polymer complexes and black lipid membrane were also performed. depending on the polymer type there is either direct interaction with dna or in other cases polymers exhibit strong interaction with the lipid membrane. an explanation for transfection ef ciency of these nonionic agents in gene delivery to muscle will be given. high throughput in-silico screening against exible protein receptors b virtual screening of chemical databases to targets of known threedimensional structure is developing into an increasingly reliable method for nding new lead candidates in drug development. based on the stochastic tunneling method (stun) we have developed flexscreen, a novel strategy for high-throughput in-silico screening of large ligand databases. each ligand of the database is docked against the receptor using an all-atom representation of both ligand and receptor. in the docking process both ligand and receptor can change their conformation. the ligands with the best evaluated af nity are selected as lead candidates for drug development. using the thymidine kinase inhibitors as a prototypical example we documented the shortcomings of rigid receptor screens in a realistic system. we demonstrate a gain in both overall binding energy and overall rank of the known substrates when two screens with a rigid and exible (up to sidechain dihedral angles) receptor are compared. we note that the stun suffers only a comparatively small loss of ef ciency when an increasing number of receptor degrees of freedom is considered. flexscreen thus offers a viable compromise between docking exibility and computational ef ciency to perform fully automated database screens on hundreds of thousands of ligands. maturation and inhibitor design of sars-cov cl protease based on a product-bound crystal structure severe acute respiratory syndrome (sars) is an emerging infectious disease caused by a novel human coronavirus. here we report that the cl pro containing n-and/or c-terminal additional in-frame sequences underwent autoactivation to cleave the tags and yielded the mature protease in vitro. the -d structure of the c a mutant protease shows that the active site of one protomer of the dimeric protease is bound with the c-terminal six amino acids of the protomer in another asymmetric unit, suggesting a possible mechanism for maturation. the crystal structure of this product-bound form shows that the active site has a p pocket that binds the gln side chain speci cally. in addition, the p and p sites are clustered together to accommodate large hydrophobic side chains. the tagged c a mutant protein served as a substrate for the wildtype protease and the n-terminus was rst digested ( -fold faster) followed by the c-terminal cleavage as shown by the sds-page analysis. the analysis of t analytical ultracentrifuge experiments reveals the remarkably tighter dimer formation for the mature enzyme (k d = . nm) than for the mutant (c a) containing the n-terminal (k d = . nm) or the c-terminal extra amino acids (k d = . nm). taken together, the study here provides insights to the design of our new structure-based inhibitors. nevertheless, a signi cant proportion of patients do not respond to this therapy, and adverse effects are common. here we report the delivery and expression of recombinant mycobacterial dna vaccines in vivo and demonstrate the ability of multicomponent dna vaccines to enhance th -polarized immune responses. splenocytes from immunized groups of mice were re-stimulated in vitro and examined for cytotoxicity against bladder tumour cells. we used four combined recombinant bcg dna vaccines (multi-rbcg) for electroporative immunotherapy in vivo, and found that tumour growth was signi cantly inhibited and mouse survival was prolonged. increased immune cell in ltration and induction of apoptosis were noted after treatment with multi-rbcg alone, with the interleukin- (il- ) vaccine alone, and-most signi cantly-with their combinations. thus, electroporation immunogene therapy using multi-rbcg plus il- may be an attractive regimen for the treatment of bladder cancer. this approach presents new possibilities for the treatment of bladder cancer using recombinant bcg dna vaccines and il- dna vaccine. the cell-penetrating peptide (cpp) pep- is capable of introducing large proteins into different cell lines, maintaining their biological activity. two mechanisms have been proposed to explain the entrance of other cpps in cells, endosomal-dependent and independent. we evaluated the molecular mechanisms of pep- mediated cellular uptake of -galactosidase ( -gal) from e. coli, in large unilamellar vesicles (luv) and hela cells. fluorescence spectroscopy and immuno uorescence microscopy were used to study the translocation. internalization of -gal into luv and protein functionality in hela cells were detected by enzymatic activity. -gal translocated into luv in a transmembrane potentialdependent manner. likewise, -gal incorporation was extensively decreased in depolarized cells. furthermore, -gal uptake efciency and kinetics were temperature-independent and -gal did not co-localize with endosomes, lysosomes or caveosomes. therefore, -gal translocation was not associated with the endosomal pathway moreover transmembrane pores were not detected. these results indicated that the protein uptake in vitro and in vivo was mainly, if not solely, dependent on a physical mechanism governed by electrostatic interactions between pep- (positively-charged) and membranes (negatively-charged). peptide the dramatic acceleration in the identi cation of new nucleic acidbased therapeutic molecules has provided new perspectives in pharmaceutical research. however, the development of nucleic acidand peptide-based therapeutics is limited by their poor cellular uptake and traf cking. with the aim of addressing these issues, we have designed a family of short amphipatic peptides for delivery of nucleic acids (mpg) and of peptide/pna (pep). these carriers consist of a hydrophobic moiety and a nls-derived hydrophilic domain. they form stable non-covalent complexes with peptides, proteins, sirna or pna without any requirement for prior covalent cross-linking. both mpg and pep carriers enter cells rapidly, in a process involving membrane disorganization, independently of the endosomal pathway. mpg ef ciently delivers short odns and sirna into a wide variety of mammalian cell lines, without interfering with their biological function. pep signi cantly improves delivery of pna and peptides. both carriers were used for the delivery of sirna or antisense pna targeting the cell cycle regulatory protein cyclin b in an animal model and were found to block tumor growth upon intravenous injection. we believe that mpg and pepbased technologies will contribute signi cantly to the development of basic and therapeutic applications. probing the bound conformation of acetylcholinesterase (ache) inhibitor at the binding site c. g. kim, x. zhao, s. goodall, a. watts department of biochemistry, university of oxford, south parks road, oxford, ox qu, uk acetylcholinesterases are the enzymes which preferentially hydrolyze acetyl esters (such as ach or acetyl-â-methylcholine), containing amino acid residues in the eeache form and arranged as a -stranded â-sheet surrounded by á-helices. the protein is ellipsoidal in shape, with approximate dimensions of Å by Å by Å. inhibitors of acetylcholinesterase are of commercial and medical interest as pesticides and as therapeutics in the treatment of alzheimer's disease. an understanding of the conformation of inhibitors in the binding site enables the rational design of novel inhibitors with increased potency and speci city. interaction between the ligand, amino- -methyl- -( tri uoroacetylbenzyl-oxymethyl)quinoline (r ), and ache inhibitor has been studied by advanced solid-state nmr through double-quantum chemical shift and distance measurements. combining solid-state nmr data and docking simulations, conformation of the ache inhibitor at the active site has been predicted. in vivo, heat shock proteins (hsps) being stress-inducible chaperones can attenuate detrimental consequences of ischemic insults, inammation, neurodegenerative diseases, etc. also, intracellular accumulation and chaperone activities of some hsps may contribute to improved cell survival following uv or ionizing radiation. in models of pathological states and their treatment, we used special virusbased vectors for overexpression of hsp or hsp in cell cultures to confer cytoprotection under simulated ischemia/reperfusion. in parallel, similar cytoprotection was achieved after pretreatments of the cells with a pharmacological hsp inducer, geranylgeranylacetone. the cytoprotective effects were manifested in the lesser extent of oxidative modi cation and aggregation of cellular proteins, better preservation of the cytoskeleton, faster restoration of energy metabolism and the improved post-stress cell survival. in the other model, we treated normal and tumor cells with an inhibitor of the chaperone activity of hsp , geldanamycin. only the drug-treated tumor cells became more sensitive to gamma-irradiation; such results characterize this drug as a potentially selective radiosensitizer of tumors. taken together our data demonstrate promising approaches to clinically bene cial manipulating the levels of expression and/or chaperone activity of hsp(s) by means of gene therapy or pharmacotherapy. characterizaton of proteins from human pleural uid r. jain, s. kumar, n. singh, s. sharma, t. p. singh all india institute of medical sciences, new delhi ,india the samples of human pleural uid were obtained from both healthy subjects and patients infected by tuberculosis. after the preliminary processing these samples were run in independent lanes of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (sds-page). the two lanes indicated variations in the intensities of a few bands and some new bands were also observed in the infected samples. these were characterized by determining their nterminal sequences. the new bands which had low density were carefully identi ed and cloned. some of the common bands that showed intensity variations were characterized. these were matrix metalloproteins, secretory phospholipasea , transferrin and ceruloplasmin. they were also studied with maldi-tof and their molecular weights have been determined. some of these proteins have been crystallized and their detailed crystal structure determinations are in progress. biophysical study of non-lethal stress response of cultured dc f cells stress factors may induce two kinds of responses in living cells: either cell death or adapting mechanisms. our aim was to search for non-lethal effects of various stress conditions on cultured hamster lung broblasts (dc f cells) as well as to assess the recovery time after stress removal. dc f cells were cultured in standard conditions and were submitted to stress, either by incubation with chemical substances (sodium arsenate, sodium nitroprusiate) and drugs (bleomicine and statins) either by irradiation (uv, he-ne laser). the doses and exposure times were chosen as to avoid cell death. after stress removal, cells were allowed to recover and the recovery time period was measured. structural and functional parameters were evaluated before and after stress, as well as during recovery. by now, experimental models for the in vitro study of non-lethal stress inducing factors have been set up. severe acute respiratory syndrome (sars) is an emerging infectious disease caused by a novel human coronavirus. the viral maturation requires a main protease ( cl pro ) to cleave the virus-encoded polyproteins. we report here that the cl pro containing n-and/or c-terminal additional in-frame sequences underwent autoactivation to cleave the tags and yielded the mature protease in vitro. the -d structure of the c a mutant protease shows that the active site of one protomer of the dimeric protease is bound with the cterminal six amino acids of the protomer in another asymmetric unit, suggesting a possible mechanism for maturation. the tagged c a mutant protein served as a substrate for the wild-type protease and the n-terminus was rst digested ( -fold faster) followed by the c-terminal cleavage as shown by the sds-page analysis. the analysis of the quaternary structures for the tagged and mature proteases by analytical ultracentrifuge experiments reveals the remarkably tighter dimer formation for the mature enzyme than for the mutant (c a) containing the n-terminal or the c-terminal extra amino acids. taken together, the study here provides insights to the design of our new structure-based inhibitors. characterisation of macromolecular transport in physiologically relevant mixed ecm based gels s. lelu, a. pluen school of pharmacy and pharmaceutical sciences, university of manchester (uk) the extracellular matrix (ecm) a complex gel made of hyaluronic acid, collagen and proteoglycans (pg) impedes the penetration of macromolecules especially in tumours, and may compromise the success of novel therapies. though recent in vivo investigations pointed out that, not only ha but brillar collagen its content and organisation and its interactions with pg were involved in macromolecular transport hindrance, transport mechanisms relating the macromolecular drug and these ecm components are unknown. in this study we seek to evaluate the determinants of passive transport mechanisms of biomacromolecules in complex gels made of ha, collagen using uorescence techniques (frap and confocal re ection microscopy (crm)) and rheology. focus was on conditions relevant to tumours and, initially, on low collagen and relatively high ha content. rheology experiments showed that mixed systems containing less than mg/ml of ha present higher elastic modulus ge than pure ha network or pure collagen gels. interestingly crm and frap studies revealed similarities for collagen and mixed gels: the organisation and spacing of the collagen bres did not change and the ratio of the diffusivities (d/d ) of dextran m and igg were not different but higher than those in ha networks. systems with higher collagen content are under investigation to complete the characterisation of transport. encapsulation of clone vector dna by cationic diblock copolymer vesicles for gene delivery a. v. korobko , j. r. van der maarel leiden university, the netherlands, national university of singapore, singapore we will discuss the design, control, and structural characterization of cationic copolymer vesicles loaded with dna. these vesicles serve as a model system for diverse applications such as gene delivery, micro-arraying techniques and packaging of dna in congested states. encapsulation of dna was achieved with a single emulsion technique. for this purpose, an aqueous puc or pegfp-n plasmid solution is emulsi ed in an organic solvent and stabilized by an amphiphilic diblock copolymer. the neutral block of the copolymer forms an interfacial brush, whereas the cationic block complexes with dna. a subsequent change of the quality of the organic solvent results in a collapse of the brush and the formation of a capsule. the capsules are subsequently dispersed in aqueous medium to form vesicles and stabilized with an osmotic agent in the external phase. inside the vesicles, the dna is compacted in a liquid-crystalline fashion as shown by the appearance of birefringent textures under crossed polarisers and the increase in uorescence of labeled dna. the compaction ef ciency and the size distribution of the vesicles were determined by light and electron microscopy, respectively, and the integrity of the dna after encapsulation and subsequent release was con rmed by gel electrophoresis. we demonstrate the gene transfer ability of this new carrier system by the transfection of encapsulated pegfp plasmid into hela cancer cells. cellular transduction of nucleotide kinases to improve the activation of nucleoside analog prodrugs m. konrad , c. monnerjahn , s. ort , a. lavie max-planck-institute for biophysical chemistry, goettingen, germany, university of illinois at chicago, chicago, usa the objective of our study is to improve therapeutic enzymeprodrug systems by generating catalytically superior nucleoside and nucleotide kinases that are essential for activation of nucleoside analogs. compounds, such as azt for the treatment of hiv infections, acv and gcv used against herpes virus, or the anticancer compounds arac and gemcitabine, can enter cells only in the unphosphorylated state (prodrug) and need to be transformed by different kinases to their pharmacologically active triphosphate state that interferes with dna replication. we have rst designed mutants of the human tmp kinase (htmpk) that phosphorylate aztmp up to -fold faster than wildtype. expression of this enzyme in human cells leads to -fold higher intracellular concentrations of azttp and to enhanced hiv inhibition. second, the prodrugs acv and gcv are not phosphorylated by human kinases, but are converted to their monophosphate forms by hsv -tk which is used in enzyme/prodrug-dependent cancer suicide gene therapy. we generated enzyme variants which show selective and ef cient phosphorylation of gcv. third, an engineered human dck variant catalyzes more ef ciently the activation of the prodrugs arac and gemcitabine. thus, the concept of a gene (or enzyme) therapeutic treatment involving expression (or direct intracellular transduction) of a catalytically improved human enzyme may pave the way to the development of novel strategies in nucleoside prodrug-dependent cancer chemotherapy. docking-molecular dynamics studies on the peroxidase site of prostaglandin endoperoxide h synthase prostaglandin endoperoxide h synthases- and (pghs- and ) catalyze the rst step in the biosynthesis of prostaglandins, prostacyclins and thromboxanes. arachidonic acid is transformed into prostaglandin g (pgg ) at the cyclooxygenase site of the enzyme and the -hydroperoxide oxygen-oxygen bond of pgg is subsequently cleaved by reaction with haem at the distinct peroxidase site (pox) to produce prostaglandin h (pgh ). herein we present a plausible productive conformation obtained by docking calculations for the binding of pgg to the pox site of pghs- . the enzyme-substrate complex stability was veri ed by a -ps molecular dynamics simulation. structural analysis unveils the requirements for enzyme-substrate recognition and binding: the pgg -hydroperoxide group is in the proximity of the haem iron and participates in a hydrogen bond network with the invariant his and gln and a water molecule, whereas the carboxylate group establishes salt bridges with the remote lysines and . the interaction of the peptide lah with anionic lipids during dna/rna delivery to eukaryotic cells a. j. mason , a. martinez , c. leborgne , a. kichler , b. bechinger faculté de chimie, université louis pasteur, strasbourg, france, généthon, evry, france the histidine rich amphipathic peptide lah has antibiotic and dna delivery capabilities. the peptide has a strong af nity for anionic lipids found in the outer membrane of bacterial membranes and has shown evidence of higher transfection activity against transformed over healthy tissue in culture. it has been proposed that anionic lipids can ip-op to reach the cytoplasmic monolayer. here they neutralise the cationic transfection complexes thereby causing release of oligonucleotides into the cytoplasm. we were, therefore, particularly interested to test for the role of the acidic lipid phosphatidylserine (ps) in mediating lah -mediated delivery of dna ef ciency. to understand the potential peptide-lipid interactions in more detail, solid-state nmr experiments on model membranes have been performed. p mas nmr on mixed phosphatidylcholine (pc)/ps and pc/phosphatidylglycerol (pg) membranes has been used to investigate speci c lah interactions with anionic lipids. by using deuterated lipids and wide-line h nmr when probing lipid chain order, it is demonstrated that lah preferentially interacts with ps over pc. lah thereby effectively disorders the anionic ps lipid fatty acyl chains. the lipid chain destabilising effect of lah and also lah analogues can then be compared with their transfection ef ciency for dna or sirna in cell culture to aid in rational peptide vector design. virtual screening is now widely accepted as a basis for drug discovery thanks to signi cant improvement and good hit rates [ ]. however, it is still highly cpu-consuming. at the same time, the number of protein-ligand complexes described at the atomic level is rising and the sequence similarity is used for structure and function predictions. new approaches are being developed to take advantage of the available structural data and the huge number of protein sequences in order to allow better tuned virtual screening. new web servers are being built to ease and to speed up the whole process (http://abcis.cbs.cnrs.fr/kindock/). integrating these servers into a pipeline dedicated to molecular modelling (http://abcis.cbs.cnrs.fr/atome/) shall allow both the re ned validation of modelled active sites as well as the oriented screening for the primary caracterization of potential ligands. an ideal drug delivery system should own the following characteristics, the rst is the targeting of therapeutic agent to the speci c site of their action. the second is the controlled delivery of a therapeutic molecule or protein in a pulsatile or staggered fashion. the third is the achieving sustained zero-order release of a therapeutic agent over a prolonged period of time. in this study, a new drug delivery system combined these characteristics was provided, which contains azobenzene derivatives (ab lipid) as an on-off switch incorporated into liposomes. the drastic release of calcein was observed on the rst uv irradiation of ab lipid to the cis isomer, while a suppressed release was observed when irradiated with the rst visible light. after that, the slope of release pro le became coincident. furthermore, calcein release was greatly increased after uv irradiation of ab lipid to the cis isomer and the drug release was greatly suppressed after vis irradiation of ab lipid to the trans isomer. we can control the release rate of calcein from ab lipid/egg pc mixed liposomes by uv or vis light irradiation. tryptophanase (trpase), a bacterial enzyme with no counterpart in eukaryotic cells, produces l-trp pyruvate ammonia and indole. it was suggested that indole is essential for bacteria multiplication and bio lm formation. bio lms destroy equipment and food and cause many illnesses. most synthesized quasi-substrates inhibit trpase at mm range. an optimal and speci c inhibitor of trpase may eliminate indole production and prevent bio lm formation. x-ray crystallography of the holo-wt e. coli trpase soaked with l-trp and the known mechanism of trpase activity should provide the information for the design and synthesis of active-sitespeci c quasi-analogs. utilizing the chromogenic substrate s-(onitrophenyl)-l-cysteine, the following michaelis-menten kinetics analyses determined the mode of trpase inhibition by trp and quinone based quasi-analogues and the corresponding ki values (in µm): dl- -alanyl- , anthraquinone, noncompetitively, ; trypthophan ethylester, competitively, ; acetyltryptophan, uncompetitively, . ; s-phenylbenzoquinone-l-tryptophan, uncompetitively, . plp-l-trp, inhibited irreversibly only the apo form of trpase and may serve for structure-determination purposes. further attempts are being made to synthesize improved trpase inhibitors, i.e., in the nm range. polyelectrolyte multilayer lms (pem) adsorbed on biomaterial surfaces are a new way to create a controlled release system. using biodegradable polymers, the lms can be degraded in vivo and release active molecules. in this work, we demonstrate the possibility of tuning the degradability of polysaccharide pem in vitro and in vivo. chitosan and hyaluronan pem (chi/ha) were either native or cross-linked (cl) using a water soluble carbodiimide (edc) at various concentrations in combination with nhydroxysulfosuccinimide. the in vitro degradation of the lms in contact with enzymes was followed by quartz crystal microbalance measurements and confocal laser scanning microscopy after lm labeling with chi fitc . whereas the native lms were subjected to degradation, the cl lms were more resistant to enzymatic degradation. films made of chitosan of medium molecular weight were indeed more resistant than lms made of chitosan-oligosaccharides. in addition, macrophages could degrade all types of lms and internalize the chitosan in vitro. the native lms implanted in vivo in mouse peritoneal cavity for a week showed an almost complete degradation whereas the cl lms were only partially degraded. these results suggests that the polysaccharides pem are of potential interest for in vivo applications as biodegradable coatings and that degradation can be tuned by controlling lm cross-linking. membrane electroporation -tool for therapeutic electrotransfer of drugs and gene dna e. neumann, s. kakorin physical and biophysical chemistry, faculty of chemistry, university of bielefeld, germany membrane electroporation (mep) is a new electrical high voltage scalpel, transiently opening the cell membranes of tissue for the penetration of foreign substances. due to the enormous complexity of cellular membranes, many fundamental problems of mep have to be studied at rst on model systems, such as the curved bilayer membranes of unilamellar lipid vesicles. electrooptical and conductometrical data of unilamellar liposomes indicate that electric eld pulses cause not only the formation of membrane electropores but also shape deformation of the liposomes, both processes mutually affecting each other. the primary eld effects of mep and cell deformation can trigger a cascade of numerous secondary phenomena, such as pore percolation and transport of small and large molecules across the electroporated membrane. the chemical mep theory represents a molecular physico-chemical approach to electrochemomechanical pore formation, yielding transport parameters, such as permeation coef cients, pore fractions and pore sizes. the pore concept is successfully applied to rationalize optimization strategies for biotechnological and medical applications of mep. in silico elucidation of xenobiotic processing loops k. nakata , y. tanaka , t. nakano , t. ishikawa , h. tanaka , t. kaminuma national institute of health sciences, tokyo, japan, tokyo medical and dental university, tokyo, japan, tokyo institute of technology, yokohama, japan, hiroshima university, hiroshima, japan one of the important challenges for drug designers is to predict and analyze how drugs are absorbed, distributed, metabolized and excreted (adme) in the body. these processes highly correlated with toxicity of drugs and are actively studied in pharmacology. two classes of proteins, the drug metabolizing enzymes such as cytochrome p s (cyps) and transporters, are the target of such adme/tox research. it was relatively recent that these two classes of proteins are synthesized by the genes that are the target genes of the nuclear receptors. nuclear receptors are ligand-activated transcription factors that form a superfamily. in case of humans there are nuclear receptors almost half of whose ligands are identied, leaving some as true orphans. thus it was now recognized that these nuclear receptors play the role of sensors of drugs and other xenobiotic substances including environmental chemical pollutants and nutritional ingredients, while the drug metabolizing enzymes and the transporters are the processors which carry the actual cleaning jobs. we have started to elucidate the feedback loops that are formed by the xenobiotic ligands, nuclear receptors, their target genes, their product proteins, and their feedback actions on the ligands. the work is being carried out on our background database on the ligands and their receptors called kibank, and search programs for target genes of nuclear receptors algorithmically. the most recent results will be presented at the presentation. the major route for drug entry into cells is permeation across lipid bilayers. due to methodological limitations there are only few studies on permeation of drug-like molecules across lipid bilayers. an assay developed in our lab allows the direct measurement of lipid bilayer permeation of aromatic carboxylic acids (acas). tb , which forms a uorescent complex with acas, is entrapped in liposomes and aca entry is determined from luminescence increase. lipid bilayer permeation was ph-dependent, following a henderson-hasselbalch function with a plateau for the neutral and the anionic species, respectively. in contrast to the expectations of the ph-partition hypothesis, permeation of the anionic species was only to magnitudes lower than that of the neutral species, leading to anion-controlled permeation at ph . , independently of bilayer state and lipid composition. permeation across bilayers with a biologically relevant lipid composition was signi cantly slower than across egg-phosphatidylcholine membranes. the in uence of single lipids, such as cholesterol, was dependent on the structure and ionization state of the permeant. permeation coef cients of the neutral species correlated better with the polar surface area (psa) than logp oct , therefore psa is a better predictor for bilayer permeation of the neutral species of small acas than logp oct . interplay between polymerized liposomes physicochemical properties and composition and citotoxicity this study was aimed at investigating whether there is an interplay between diacetylenic polymerized liposomes physicochemical properties and lipid composition affecting citotoxicity in vitro. unsaturated , -bis( , -tricosadiynoyl)-sn-glycero- -phosphocholine with saturated , -dimiristoyl-sn-glycero- phosphocholine in molar ratio : , were combined to give a chemically modi ed membrane by uv-polymerization. biophysical characterization was carried out determining the hydrophobic factor and hydrodynamic radius. citotoxicity was evaluated through haemolytic capacity on bovine red blood cells and indirectly by capacity of induction of lipid peroxidation on microsomes or mitochondrial membranes. the haemolysis percentage in presence of dc , pc/dmpc is less than that induce by polymers used in dentistry. the data obtain suggests that the polymerized lipids can not induce lipid peroxidation on natural membranes. the polymerized diacetylenic liposomes showed less interaction with serum proteins than non polymerized and lower citotoxicity as compared with natural lipids. also cell viability was determined in cell line nih t after exposure to lipids systems under study. the hydrophobic factor showed further augmentation for polymerized liposomes and is discussed in relation to in vitro stability. the above results suggest that polymerized and non-polymerized liposomes would serve as an effective delivery vehicle. s. sonar, s. d´souza, k. p. mishra radiation biology and health sciences division,bhabha atomic research centre,mumbai- , india liposomes offer new approaches for drug delivery through their encapsulation to alter pharmacodynamic properties of loaded drug leading to reduction in toxicities and/ or improved ef cacy. for prolonged systemic circulation, the liposomes size has been shown to be limited to nm or less. the ethanol injection method is an excellent technique for the formation of liposomes of < nm without the need of sonication or extrusion. the present study was aimed to produce liposomes encapsulating doxorubicin in minimum procedural steps. liposomes were prepared using distearoyl phosphatidylcholine and cholesterol, distearoyl phosphatidylcholine, cholesterol and oleic acid. the effects of different operational conditions for vesicle production and drug encapsulation were evaluated, with a view to achieve process cost to a minimum, suitable size and high encapsulation ef ciency. although high ef ciency of doxorubicin encapsulation was obtained by 'active' or 'remote' loading process in dspc/chol system, it was poor in one-step injection method. oleic acid was included to cut down the active loading by ph-gradient. dspc/chol/oa systems spontaneously loaded doxorubicin with encapsulation ef ciency of % and nal drug to lipid molar ratio upto . . the mean diameter of the vesicles was + nm. the method offers liposomes of small size with high loading. design of peptides with consecutive dehydro phenylalanine residues r in order to develop general rules for the design of peptide conformations with consecutive alpha, beta-dehydro phenylalanine-residues, peptides were synthesized, crystallized and crystal structures and molecular conformations were determined. following conclusions were drawn based on the structural data: -peptide unit sequences with two consecutive dehydro-phe residues at (i+ ) and (i+ ) positions adopt an unfolded s -shaped structure with dihedral angles phi-psi centred at , . -the peptides containing two consecutive dehydro-phe residues at (i+ ) and (i+ ) positions • form two overlapping type iii beta -turns (incipient -helix). • with branched beta -carbon residue only at (i+ ) position adopt a conformation with two overlapping types ii and iii beta -turns. • with branched beta-carbon residues such as val and ile at both (i+ ) and (i+ ) positions form two overlapping types ii and i beta -turns. the consistency in the formations of these conformations makes the design of peptides with alpha,beta ?-dehydro -residues a useful and highly predictable method for developing speci c ligands for various biological applications including drug design. binding of cationic porphyrin to isolated double-stranded dna and nucleoprotein complex k. zupán , l. herényi , k. tóth , z. majer , g. csík institute of biophysics and radiation biology, semmelweis univ., budapest, hungary" biophysics of macromolecules, german cancer research center, heidelberg, germany, department of organic chemistry, eötvös loránd univ., budapest hungary the complexation of tetrakis( -n-methylpyridyl)porphyrin (tmpyp) with free and encapsidated dna of t bacteriophage was investigated. to identify binding modes and relative concentrations of bound tmpyp forms, the porphyrin absorption spectra at various base pair/porphyrin ratios were analyzed. spectral decomposition, uorescent lifetime, and circular dichroism measurements proved the presence of two main binding types of tmpyp, e. g., external binding and intercalation both in free and in encapsidated dna. tmpyp binding does not in uence the protein structure and/or the protein -dna interaction. concentrations of tmpyp species were determined by comprehensive spectroscopic methods. our results facilitate a qualitative analysis of tmpyp binding process at various experimental conditions. we analyzed the effect of base pair composition of dna, the presence of protein capsid and the composition of buffer solution on the binding process. protein crystallization and structural study of upa protease domain with active site serine mutation the urokinase (upa) system is composed of upa, its receptor (upar), and inhibitor (pai). it plays important role in various physiologic processes, including brinolysis, cell adhesion, and signal transduction, and has been recognized as a target for intervention in tumor growth and tumor metastasis. we constructed an active site mutant of upa protease domain ( - ) with three mutations (c a, n q, and s a) and expressed it as secreted protein in pichia pastoria with ppicza vector. the secreted mutant was captured from culture medium by a cation exchange column and then further puri ed on a gel ltration column. the puri ed mutant was then crystallized by sitting drop vapor diffusion method with several precipitant conditions: ( ) . - . m ammonium sulphate, - % peg , mm sodium citrate ph . or mm sodium phosphate ph . , . % sodium azide. ( ) . m ammonium sulphate, . m lithium sulphate, mm sodium acetate at ph . , ( ) . - . m sodium formate, mm sodium acetate at ph . . ( ) . - . m sodium chloride, mm sodium acetate at ph . . the crystals were of varying quality but generally diffracted from . Å- . Å with inhouse x-ray source. the structure of this upa mutant and its complex with various inhibitors will provide a platform for rational upa inhibitor design. abstract in this work, the linear interaction energy (lie) method was used to calculate the binding free energies of hiv- integrase (in) and a series of dicaffeoyl -or digalloyl pyrroliding and furan derivatives inhibitors. the model of binding free energy prediction for homogeneous inhibitors of hiv- in has been obtained with a root-mean-square deviation (rmsd) of . kj/mol and estimated to be a precise model with good prediction capability. in addition, the probable binding mode of this series of inhibitors with hiv- in was proposed by using molecular docking and molecular dynamics (md) simulation methods. our results indicate that caffeoyl -or galloyl group of inhibitors have close interaction with a hiv- in conservative dde motif. a speci c non-competitive inhibitor of a small g protein/gef complex on the protective role of selenium and catechin in cadmium toxicity s. Özdemir , s. dursun , s. toplan , n. dariyerli , m. c. akyolcu istanbul university, cerrahpasa medical faculty, department of biophysics, turkey, istanbul university cerrahpasa medical faculty, department of physiology, turkey cadmium as heavy metal is toxic and carcinogenic for organisms. cadmium perform their effects on living organisms by accumulation in blood and various tissues. due to their accumulation in various tissues and in blood, tissue antioxidant enzyme systems are affected. the present study was planned to determine the possible protective roles of selenium and catechin against the toxic effects of considered heavy metals. the study has been performed in wistar albino type rats which divided into four groups as control and cadmium, cadmium+selenium, cadmium+catechin received groups. besides cadmium as heavy metal, selenium concentration determinations were performed in blood, liver and kidney tissues of each group of rats. in the same tissue samples besides lipid peroxidation measurements, glutathione, glutathione peroxidase and superokside dismutase enzyme activity determinations were also performed. the accumulation of heavy metals was determined in blood, liver and kidneys after cadmium administration during experimental period. in the tissue of experimental group animals there was an increased lipid peroxidation but decreased antioxidant enzyme activities were observed. while effects of selenium in decreased toxicity of cadmium have been detected, there was no statistically signicant effect of catechin observed. proposed that motors could dynamically cluster at the tip of tubes when they are individually attached to the membrane. we demonstrate, in a recently designed experimental system, the existence of an accumulation of motors allowing tube extraction. we determine the motor density along a tube by using uorescence intensity measurements. we also perform a theoretical analysis describing the dynamics of motors and tube growth. the only adjustable parameter is the motor binding rate onto microtubules, which we measure to be . +/- . s . in addition, we quantitatively determine, for a given membrane tension, the existence of a threshold in motor density on the vesicle above which nanotubes can be formed. we nd that the number of motors pulling a tube can range from four at threshold to a few tens away from it. the threshold in motor density (or in membrane tension at constant motor density) could be important for the understanding of membrane traf c regulation in cells. kinesin and dynein move a peroxisome in vivo: a tug-ofwar or coordinated movement? c. kural, p. r. selvin, k. hwajin, g. goshima, v. i. gelfand university of illinois at urbana-champaign, usa we have used fluorescence imaging with one nanometer accuracy (fiona) for analysis of organelle movement by conventional kinesin and cytoplasmic dynein in a cell. we can locate a green uorescence protein (gfp)-tagged peroxisome in cultured drosophila s cells to within . nanometer in . milliseconds, a -fold improvement in temporal resolution, suf cient to determine the average step size to be nanometers for both dynein and kinesin. furthermore, we nd that dynein and kinesin do not work against each other in vivo during peroxisome transport. rather, we nd that multiple kinesins or multiple dyneins work together, producing up to times the in vitro speed. engineering a bio-molecular walker h. jankevics, j. e. molloy division of physical biochemistry, mrc national institute of medical research, the ridgeway, mill hill nw aa, london, uk in this work we describe the design and development of a biomolecular walker based on the motile system found in certain ciliated protists. the motile system is driven by the binding of ca ions and in contrast to other commonly studied motor proteins is independent of atpase (amos et al., ) . the motor protein is a kda ca-binding protein called spasmin (maciejewski et al., ; itabashi et al., ) which belongs to the ef-hand family of calcium binding proteins called calmodulins. upon calcium binding, spasmin is thought to undergo a large conformational change as it binds its own target peptide and we wish to exploit this in order to create our own novel molecular walker.we have created a recombinant spasmin with sequence tags to enable speci c immobilization and various conjugate chemistries. cystein mutants have been introduced at speci c points in the protein to enable attachment of other small molecules, for example uorophores. we are now optimising protein expression and puri cation to maximise the yield of active protein on which we can perform the conjugate chemistries. we will characterize the structural changes using biophysical methods such as circular dichroism, analytical ultracentrifugation, electron microscopy, afm and total internal re ection uorescence spectroscopy on single molecules. the force generation in muscle arises from direct interaction of the two main protein components of the muscle, myosin and actin. the process is driven by the energy liberated from the hydrolysis of atp by myosin. the interaction is performed by cyclic interaction of myosin with atp and actin, and at least six intermediates are proposed for actomyosin atpase in solution. the powerful dsc technique allows the derivation of heat capacity of proteins as a function of temperature. from the deconvolution of the thermal unfolding patterns it is possible to characterize the structural domains of the motor protein. in this work we tried to approach the temperature-induced unfolding processes in different intermediate state of atp hydrolysis in striated muscle bres. we have extended the experiments to study the ber system prepared from psoas muscle of rabbit in rigor, strongly binding and weakly binding states of myosin to actin where the inorganic phosphate (p i ) was substituted by the phosphate analogue orthovanadate. the dsc transitions were analyzed in different buffer solutions (tris and mops) to get information about the temperature dependence of ph on the conformational changes. single kinesin motor proteins walking through the searchlight s. verbrugge, l. c. kapitein, e. j. peterman vrije universiteit, de boelelaan , hv, amsterdam, the netherlands the dimeric motor protein kinesin steps by a hand-over-hand mechanism. this means that the centre of mass moves with nm steps, while the two motor domains, the one after the other, move nm to the next binding site on the microtubule. the molecular details of what happens during a step are not fully understood, partly because of lack of time resolution in wide-eld, single-molecule uorescence experiments. we set out to develop an approach to study the motility of kinesin with a time resolution below a millisecond (a single step takes on the order of milliseconds). this approach allows us to look into the mechanochemistry and coupling of the two kinesin motor domains while they are stepping. our method is based on confocal microscopy and we study the uorescent properties of single labeled motors while they walk through the confocal laser spot. we present the experimental details of our approach and show our results on human kinesin constructs that are speci cally labeled in the tail. we show that our approach enables us to study the mechanism of kinesin with a much higher time resolution than what was achieved before with single-molecule uorescence experiments. movement of coupled single-headed kinesins analysed by a brownian-ratchet model . the analysis of this system is expected to provide insights into the mechanism underlying the motility of conventional double-headed kinesin, espetially the roles played by individual heads. we would like to clarify whether the experimentally observed behaviors that are supposed to be caused by a pair of single-headed kinesins can be explained by a simple brownianratchet model, which is successful in describing the motion of an unconventional single-headed kinesin kif a. our model consists of two brownian motors (ratchets) separated by a xed distance r. the velocity and other quantities of the coupled motors are calculated by solving the fokker-planck equation with various choices of r and other parameters. then, assuming a certain probability distribution of r associated with random attachment of kinesin heads on a bead in the experiment, the statistical properties of the motion of the coupled brownian motors are analysed. the force-velocity relation observed experimentally is found to be consistent with the present model with appropriate choices of the model parameters. the adequacy of the parameter choice needs to be con rmed by other experiments. optical trap with fast programmable feedback loop to study rotary molecular motors t. pilizota, f. bai, r. m. berry clarendon laboratory, univeristy of oxford an optical trap with back-focal plane detection and fast programmable feedback has been developed for the study of rotary molecular motors. a helium-neon laser ( nm) is used for position detection and a solid state bre laser ( nm, w cw) forms the trap. acousto-optic de ectors (aods) controlled by a digital signalling processing board are used to achive programmable feedback loops with exible control options and speeds up to khz. several modes of feedback are demonstrated, controlling both bead position (x,y) and angle (r, ). polystyrene beads or bead pairs can be held at set (x,y) or , and the set-point can be changed while the program is running. for example, feedback can be used to move a bead or a bead pair in a circle. results of using the system to study the bacterial agellar motor are presented. a dimeric -d lattice gas as model for molecular motors collective dynamics p. pierobon , t. franosch , e. frey ludwing-maximilian universitaet, münchen, germany, hahn meitner institut, berlin, germany, arnold sommerfeld center for theoretical physics, münchen, germany the transport of molecular motors along microtubules closely resemble the dynamics of a driven lattice gas of dimers without conservation of particles. the unidirectionality, asymmetry and stochasticity of the motion are encoded in the well studied totally asymmetric simple exclusion process (tasep). we extend the model to a more realistic one, including attachment and detachment kinetics and extended (dimeric) particles. we study the stationary phase diagram by means of monte carlo simulations combined with a continuum description (based on an extended mean eld theory). we also evaluate the domain wall theory nding out the effective potential con ning the phase interface into the bulk. a. e. wallin , j. lisal , r. tuma department of physical sciences, pobox , fin- , university of helsinki, finland, institute of biotechnology, university of helsinki, finland molecular motors often consist of two or more subunits that cooperate to convert chemical energy into mechanical motion. hexameric helicases and viral packaging atpases constitute a special class of molecular motors that translocate along nucleic acids. recent structural and spectroscopic characterization of these motors revealed that their enzymatic cooperativity does not result from cooperative binding [ - ]. in order to understand this new type of cooperativity we simulated the kinetics of a single hexameric motor by multiple coupled stochastic reactions using the gillespie algorithm [ ] . in contrast to analytical methods, our direct simulation allowed us to investigate the kinetics with an arbitrary model for cooperativity between the subunits. simulations on the kinetics of the hexameric rna packaging motor p from dsrna bacteriophage [ ] with different cooperativity mechanisms provided insight into the rnamediated cooperativity and yielded a sound theoretical basis for the interpretation of experimental results [ ]. the viral infectivity factor (vif) encoded by hiv- is a small basic protein that strongly modulates the viral replication and is required for pathogenicity. vif is packaged into hiv- particles through a strong interaction with genomic rna and is associated with viral nucleoprotein complexes. moreover vif acts during the early stages of the viral infection (capsid disassembly, reverse transcription) as well as during the late stages of virus replication (virus assembly and maturation of the virion). however the effect on early stages is probably a consequence of a defective assembly and / or virion maturation. understanding the rna-binding properties of vif would contribute to elucidate the role played by vif in the regulation of the genomic rna traf cking in the cytoplasm to unable ef cient packaging, and the prevention of cellular inhibitors from altering hiv- rna. in this context, we have characterised the interactions of recombinant vif with hiv- genomic rna by uorescence spectroscopy, and determined the af nity of the protein for synthetic rnas corresponding to various regions of hiv- genome. taken together our results demonstrate cooperative and speci c binding. in particular, we showed that vif has a high af nity for the 'untranslated region of hiv- genomic rna. s. bernacchi , e. ennifar , k. toth , p. walter , j. langowski , p. dumas cnrs upr -strasbourg (france), dkfz -heidelberg (germany) we have used the dimerization initiation site (dis) of hiv- genomic rna as a model to investigate hairpin-duplex interconversion by using a combination of uorescence, uv-melting, gel electrophoresis and x-ray crystallographic techniques. fluorescence studies with molecular beacons and crystallization experiments with -nucleotide dis fragments showed that the ratio of hairpin to duplex formed after annealing in water essentially depends on rna concentration, and not on cooling kinetics. with natural sequences able to form a loop-loop complex (or 'kissing complex'), concentrations as low as µm in strands are necessary to obtain a majority of the hairpin form. on the contrary, when kissing-complex formation was made impossible by mutation in the loop, a majority of hairpins was obtained even at µm in strands. this mutated sequence also showed that kissing-complex formation is not a prerequisite for an ef cient conversion to duplex in presence of salts. we proved that this happens through hairpins engaged in a cruciform intermediate, but not from free strands after hairpin melting. supporting this view, the very rst step of formation of such a cruciform intermediate could be trapped in a crystal structure. such a mechanism might be biologically signi cant beyond the strict eld of hiv- rna dynamics. generation of rna dimeric form of the human immunode ciency virus type (hiv- ) genome is important for the viral replication. the dimerization initiation site (dis) has been identi ed as a short sequence that can form a stem-loop structure with a selfcomplementary sequence in the loop and a bulge in the stem. a mer dis rna fragment, dis , spontaneously formed "loosedimer" and was converted into "tight-dimer" by supplement of nucleocapsid protein ncp . nmr chemical shift analysis for dis in the kissing-loop and extended-duplex dimers revealed that three dimensional structures of the stem-bulge-stem region were similar between the two types of dimers. therefore, we determined the solution structures of two shorter rna molecules corresponding to the loop-stem region and the stem-bulge-stem region of dis , and the solution structures of dis in the kissing-loop and extendedduplex dimers were determined by combining the parts of structures. the mechanism of conformational conversion will be discussed based on the solution structures and the molecular dynamics analysis. nmr and molecular modelling studies of an rna hairpin containing a g-rich hexaloop the mrna of the pgy /mdr gene encoding the transmembrane p-glycoprotein (p-gp) contains a hairpin that is the target of antisense oligonucleotides, suppressing the p-gp function of multidrug resistance. the solution conformation of this hairpin constituted by the '(gggaug) ' loop closed by a g-u mismatch containing stem is studied by nmr and molecular dynamics in explicit solvent. special attention is given on the sugar and the backbone conformations and the hexaloop intrinsic properties of these two components are carefully investigated. the stem structures obtained by molecular dynamics with and without nmr constraints converge to the same a-type double helix. the wobble g-u mismatch moderately perturbs the overall conformation, despite of c '-endo sugars and unusual backbone conformations located between the mismatch and the loop. in the hexaloop part, the sugar puckers are in majority in c '-endo conformations, probably to extend the strand with the help of unusual backbone angles conformations. the loop appears stabilized by one hydrogen bond and stacking interactions. thus, from the ' to the '-ends, the four purine bases ggga are stacked together, then a u-turn like is observed, and nally, u stacks on the last g that remains rather far from the stem. nmr and molecular modelling studies of an rna hairpin containing a g-rich hexaloop the mrna of the pgy /mdr gene encoding the transmembrane p-glycoprotein (p-gp) contains a hairpin that is the target of antisense oligonucleotides, suppressing the p-gp function of multidrug resistance. the solution conformation of this hairpin constituted by the '(gggaug) ' loop closed by a g-u mismatch containing stem is studied by nmr and molecular dynamics in explicit solvent. special attention is given on the sugar and the backbone conformations and the hexaloop intrinsic properties of these two components are carefully investigated. the stem structures obtained by molecular dynamics with and without nmr constraints converge to the same a-type double helix. the wobble g-u mismatch moderately perturbs the overall conformation, despite of c '-endo sugars and unusual backbone conformations located between the mismatch and the loop. in the hexaloop part, the sugar puckers are in majority in c '-endo conformations, probably to extend the strand with the help of unusual backbone angles conformations. the loop appears stabilized by one hydrogen bond and stacking interactions. thus, from the ' to the '-ends, the four purine bases ggga are stacked together, then a u-turn like is observed, and nally, u stacks on the last g that remains rather far from the stem. aminoglycoside binding to hiv- dis kissing-loop complex: from crystals to cells e. ennifar , j.-c. paillart , a. bodlenner , p. pale , r. marquet , p. dumas cnrs upr , strasbourg -france, cnrs/université louis pasteur strasbourg umr , strasbourg -france all retroviral genomes consist in two homologous single stranded rnas. dimerization is an essential step for viral replication. hiv- dimerization initiation site (dis) is a strongly conserved stem-loop in the ' leader region of the genomic rna. it was shown in vivo that alteration of the dis dramatically reduces viral infectivity. we have previously solved crystal structures of the dis kissing-loop complex. analysis of these structures revealed an unexpected resemblance between the dis kissing-loop and the s ribosomal aminoacyl-trna site (a-site), which is the target of aminoglycoside antibiotics. we have shown that some aminoglycosides specifically bind to the dis kissing-loop complex with an af nity and geometry similar to that observed in the a-site. in agreement with these previous results, we have now solved highresolution crystal structures of the dis kissing-loop complex bound to four aminoglycosides. these structures show that, as expected, two aminoglycosides are bound per kissing-loop complex. importantly, the binding is observed not only in vitro on large hiv- genomic rna fragments, but also on infected cells. moreover, we showed that some of these aminoglycosides stabilize the kissingloop rna dimer, which is consistent with the observation in crystal structures of numerous direct and water-mediated drug-rna contacts. these structures are currently used as starting points for designing potential new drugs targeted against the viral rna. modeling the long range entropy of rna: w. k. dawson , k. fujiwara , k. yamamoto , g. kawai chiba institute of technology, - - tsudanuma, narashino, chiba, japan, international medical center of japan, - - toyama, shinjuku-ku, tokyo, japan non-coding rna appears to make up a large part of the human genome. a reliable rna structure prediction program is needed to understand the structure of this non-coding rna. we recently developed a new way to model the long range entropy in rna and applied it to rna secondary structure prediction. in some of instances, the new approach is able to achieve far better predictions than the state of the art secondary structure programs even given exactly the same parameters. predictions using this method tend to show distributions that are funnel shaped. a new and important parameter in these calculations is the persistence length (a measure of the correlation and exibility of the rna). (url: http://www.rna.it-chiba.ac.jp/ vsfold/vsfold /) this new approach has now been extended to prediction of pseudoknots. the method is a heuristic wherein the hierarchical folding hypothesis is used to nd the pseudoknots as the rna secondary structure is folding, and corrections to that secondary structure are made to accommodate the pseudoknot. it is able to do these searches in roughly n^ time. the model is consistent with the hierarchical hypothesis and it is possible to estimate rna folding times that are of the correct order of magnitude using this model. with further adaptations to account for the size, shape and variablity of amino acid residues (hydrophobicity etc.), the model also appears to be transferable to protein folding problems. a. v. melkikh ural state technical university, ekaterinburg, russia a model of the genome as a gene network capable of receiving information about the environment and performing some operations on genes has been considered. the evolution rate of replicators for the mechanism of random mutations has been estimated. it was shown that the evolution rate under random actions is negligibly small for real dimensions of genomes of replicators [ ]. it was inferred that only a deterministic mechanism of the evolution can explain the known evolution rate of replicators. a deterministic model of the evolution has been proposed. the basic principles of this model include: ) information about the replicators evolution is encoded in the conformational states of proteins; ) the conformational language of proteins is translated into the language of nucleotide sequences during the evolution; ) the structure of genes is controlled such that the transition to a nearest free ecological niche takes a minimum time (at a preset restriction on the control). transfer rnas are synthesized as part of longer primary transcripts that require processing of both their ' and ' extremities in every living organism known. the ' side is matured by the quasiuniversally conserved endonucleolytic ribozyme, rnase p, while removal of the ' tails can be either exonucleolytic or endonucleolytic. the endonucleolytic pathway is catalysed by an enzyme known as rnase z. rnase z cleaves precursor trnas immediately after the discriminator base in most cases, yielding a trna primed for addition of the cca motif by nucleotidyl transferase. rnase z is found in the vast majority eukaryotes and archaea and in about half of the sequenced bacteria. it is often essential for growth and mutations in one of the two genes encoding rnase z (elac ) have been linked with prostate cancer in man. in this poster we present the crystal structure of bacillus subtilis rnase z at . Å resolution ( ) resolved by mad method and propose a model for trna recognition and cleavage. the structure explains the allosteric properties of the enzyme and also sheds light on the mechanisms of inhibition.it also highlights the extraordinary adaptability of the metallohydrolase domain of the b-lactamase family for the hydrolysis of covalent bonds. ( )i. most rnas undergo several steps of post-transcriptional modi cation before carrying out their assigned functions. one of the major modi cations is the splicing process, by which non-coding introns are removed from the coding exons. splicing can be performed by autocatalytic, self-splicing introns (e.g. group ii introns), i.e. catalytic rna or ribozymes. group ii introns, which occur in bacterial genomes and in organellar genes of plants, funghi and lower eukaryotes, consist of a conserved set of six domains. domain recognizes the '-exon through a - base pairing interaction formed by two regions within the intron (exon binding sites, ebs and ebs ) and the last - nucleotides of the '-exon (intron binding sites, ibs and ibs ). as the correct recognition of ibs by ebs is crucial for a successful splicing event we are investigating the structural and metal ion requirements of this part by various spectroscopic techniques, e.g. nmr. our data shows that the hairpin including ebs consists of a helical region followed by an unstructured single stranded part, which is ready for splice site recognition. the results of the structure analysis will be presented. financial support by boehringer ingelheim fonds (fellowship to d. k.) and the swiss national science foundation (snf-förderungsprofessur to r. k. o. s.) is gratefully acknowledged. structural basis for the antigene and antisense properties of modi ed dna:dna and rna:dna duplexes e. c. m. juan , t. kurihara , j. kondo , t. ito , y. ueno , a. matsuda , a. takenaka graduate school of bioscience and biotechnology, tokyo institute of technology, graduate school of pharmaceutical sciences, hokkaido university, faculty of engineering, gifu university oligonucleotides containing polyamines are currently being evaluated as potential antigene and antisense compounds. those with -(n-aminohexyl)carbamoyl- '-deoxyuridine ( n u) and its '-omethyl derivative ( n u m ) exhibit improved nuclease resistance and form stable duplexes with their dna and rna targets. x-ray structures of these duplexes have shown good correlation between the conformational changes and the observed chemotherapeutic properties. the amide groups of the modi ed uracil bases form six-membered rings through the intra-molecular nh-o hydrogen bonds, so that the aminohexyl chains protrude into the major grooves. some of the terminal ammonium groups are involved in intra-duplex interactions with phosphate oxygen anions, whereas the others interact with those of the adjacent duplex. such interactions contribute to the stability of duplex formation. the '-o-methyl modi cation in n u m shifts the ribose ring toward the c '-endo conformation and in uences duplex stability. observed changes in the dimensions of the minor grooves and in the hydration structures are also well correlated to nuclease resistance and duplex stability. group ii intron ribozymes catalyze selfsplicing in bacterial genomes as well as in organellar genes of lower eucaryotes. for correct structure and function these ribozymes need speci c concentrations of monovalent and divalent cations such as k and mg . most of these ions are used for charge screening, but some are also bound to distinct sites ful lling various speci c tasks. the conserved secondary structure of group ii intron ribozymes consists of six domains grouped around a central wheel. here the focus is set on domain (d ) of the yeast mitochondrial intron ai , a hairpin of nucleotides, which is crucial for catalytic activity. the -nucleotide bulge in d is known to be exible and acts as a metal ion binding platform. we have investigated the binding of different metal ions (mg , ca , mn , cd ) to this platform by uorescence spectroscopy. for this the bulge site adenosine in d was replaced by the uorescent nucleotide base analogue aminopurine ( ap). the binding data ts to an equation describing a binding to a single class of sites. the titration experiments not only reveal different dissociation constants for the tested metal ions but also indicate different effects on the bulge structure. financial support by the swiss national science foundation (snf-förderungsprofessur to r.k.o.s) is gratefully acknowledged. modi ed nucleosides and across the anticodon loop interactions in trna u. b. sonavane, k. d. sonawane, r. p. tiwari national chemical laboratory, pune , india in several interesting trna molecules, the ( th ) as well as the ( th ) nucleoside are hyper modi ed. as an example, unique hypermodi ed nucleosides mcm s u and ms t a are crucial in human trna lys , which acts as a primer in hiv replication. modi ed nucleosides may facilitate or hinder across the loop interactions. large substituents in th and th modi ed nucleosides if oriented suitably may also interact with each other. across the loop interactions may lead to unconventional anticodon loop structures also affecting exibility of the anticodon loop. this may restrict or enlarge synonymous codon choice and decoding during protein biosynthesis. except for trna asn (with interacting q and t a ), our studies show conventional 'open' loop structure -free of across the loop interactions, for a number of interesting trna anticodon loops with diverse hyper modi ed nucleosides at both of these locations. molecular dynamics simulations of hydrated anticodon arm of trna asn show persisting interaction involving the diol group of q and carbonyl group of ureido linkage in t a . additionally, the hoogsteen edge of th adenine base participates in hydrogen bonding with watson -crick edge of rd base and thus contributes to unique loop structure of trna asn . resulting suboptimal q:c base pairing leads to unbiased reading of u or c as the third codon letter. absence of queuosine modi cation, q happens to be also associated with uncontrolled rapid proliferation of cells and malignant growth. structural properties of ctg/cag repeats, and preliminary x-ray analysis of cug repeats y. sato, k. kimura, a. takénaka graduate school of bioscience and biotechnology, tokyo institute of technology, yokohama, japan. the human genome contains so many different types of repetitive sequences. some of them are tandem repeats of trinucleotides. their unusual expansions cause genetic diseases such as type myotonic dystrophy (dm ) and huntington's disease (hd), the unit sequences being ctg and cag, respectively. the numbers of repeats of the two complementary sequences change independently during dna replication or repair. the direct origin of dm is, however, the transcribed rna fragments with cug repeats, which forms a speci c structure and inhibits other protein syntheses. in the present study, structural versatilities of such dna and rna fragments has been examined. native pages of (cug) n show that the hairpin structure with even number is more stable than that with odd number. this difference might be ascribed to the structural difference at the hairpin head. the pages also show that duplex formation is dependent on coexisting cationic species and their concentration. crystal data of (cug) (a=b= . , c= . Å, and the space group r ) suggest that the asymmetric unit contains the rna fragment. an approximate crystal structure solved by molecular replacement techniques at . Å resolution shows that the rna fragments form a duplex similar to an a-form rna. context-dependent selection of promoter in a natural selection-type evolution reactor h. nagayasu, y. ageno, x. t. ma, y. husimi saitama university, saitama, japan using an isothermal ampli cation of hairpin dna/rna (developed by g.joyce), we drove a natural selection-type evolution reactor taking the speci c growth rate as the tness. we used hiv- rt and thermot rnap at c. starting from the random promoter pool, we selected the strongest promoter at c, which was separated by hamming distance from the strongest promoter at c. the latter was found to be identical to the natural t promoter. when we used a simple random pool, the selection process showed one-step convergence. when we used a random pool with a speci c short sequence at the upstream anking region of the random region, we observed an evolution process as convergencedivergence-convergence of the promoter sequence, driven by deletion of the speci c short sequence. this context-dependent selection was found to come through a neutral path, judged from the tness measurement. intronic sirna and mirna, and dna methylation gif sur yvette, france. abnormal activation of small g proteins is involved in several human diseases. small g proteins are activated by gdp/gtp exchange, which is stimulated by their guanine nucleotide exchange factors (gefs). thus, small g protein/gef complexes appear as emerging targets for interrupting signalling networks regulated by small g proteins in pathological contexts. the activation of small g proteins of the arf family is initiated by exchange factors which carry a sec catalytic domain stimulating the dissociation of the bound gdp nucleotide. the structure of the arf -gdp-arno reaction intermediate, trapped by a mutation of the catalytic glutamate, was recently solved by x-ray crystallography (pdb code: r s) acknowledgements: the work was supported by contract k- j. q. yin, f. chen, y. tan, t. gu institute of biophysics, chinese academic sciences, beijing, china sirna/mirna can ef ciently induce mrna cleavage or translational repression at the posttranscriptional level in a sequencespeci c manner. recently, it has been shown that these small rnas guide genome modi cation in mammalian cells. however, their ability to direct cognate dna methylation has been con rmed so far only in plants, and their biogenesis, functions, and modes of silencing genes are yet elusive. here, we report that small rnas derived from intron regions of some genes can target homologous dna sequence in promoter, 'utr or 'utr regions of genes in different human tumor cells. surprisingly, we also discovered that endogenous sirnas from introns of genes possessed a large number of target mrnas by using bioinformatics, and con rmed their existence in human cells with northern blot analysis. intronic small rnas generated by sliceosomes can form mature mirnas or sirnas through the processing of drosha and /or dicer. rt-pcr analysis indicated that vector-based small rna repressed expression of homologous genes at the transcriptional and/or translational levels. western blotting demonstrated that the expression of some proteins was greatly reduced or completely inhibited owing to promoter methylation. these ndings reveal that the expression of some genes can incredibly control cell activities at both protein and rna levels. our results also suggest that these small rnas may regulate gene expression in different modes of action. role of stacking in speci c recognition of capped rna by the cbc protein the stacking interaction involving -methylguanine moiety of mrna ' terminal cap (m g) and aromatic amino acid side chains is a common feature of all known cap-binding proteins. the crystal structures of the human cap binding complex (cbc) showed its induced folding upon m gpppg cap analogue binding. stabilization of the cbc-m gpppg complex by sandwich stacking of m g in between y and y is additionally enhanced by stacking of the second base of capped mrna with y . gibbs free energy of the association of various cbc mutants with the synthetic cap analogues, m gpppg, m gpppu, and m gtp, has been determined by uorimetric titration. preference of the wild type (wt) cbc for the dinucleotide analogues is also observed for the y a mutant, with the energy loss of . - . kcal/mol. however, all proteins with the mutated second stacking partner y prefer to bind m gtp compared with m gpppg. the binding of m gtp to the y a mutant is only . kcal/mol less favourable compared with the wt cbc. these divergences may be ascribed to smaller entropic costs of conformational rearrangement of cbc in the case of a smaller ligand, which can nd more favourable contacts when its second part is not ef ciently 'constrained' by stacking with y . supported by kbn p a annealing of the tar dna hairpin to a complementary tar rna hairpin, resulting in the formation of an extended duplex, is an essential step in the minus-strand transfer process of hiv- reverse transcription. in this work, we use gel-mobility-shift analysis to follow the kinetics of this reaction in the absence or presence of hiv- nc prepared by solid-phase peptide synthesis. to elucidate the reaction pathway, we use either the complete -nt tar hairpins or truncated -to -nt minihelices (mini-tar) derived from the top part (i.e. hairpin loop) of tar. assays were also carried out with mutant tar constructs. the annealing kinetics were studied systematically as a function of dna concentration and temperature. we show that the annealing initiates through a weak loop-loop kissing interaction, followed by a much slower conversion step, which results in formation of the extended duplex. nc facilitates both reaction steps, resulting in the overall -fold and -fold rate enhancement for mini-tar and tar annealing, respectively. we show that the kissing step is facilitated by the nc-induced nucleic acid aggregation, which is more pronounced for the longer tar hairpins. at the same time, the conversion steps in tar and mini-tar appear to be very similar and are similarly facilitated by nc - -fold. the later effect relays on the ability of nc to destabilize nucleic acid duplexes, and is equivalent to destabilization of a few base pairs required for the conversion initiation. linking of the n-terminus of a peptide to its encoding mrna s. ueno, h. arai, y. husimi department of functional materials science, saitama university, saitama, japanin evolutionary protein engineering, in vitro selection using a cellfree translation system has advantages of large library size and also of applicability to cytotoxic protein. although many in vitro protein selection techniques such as in vitro virus, ribosome display are developed, most of these are the techniques that link the genotype molecule to the peptide at its cterminus. we developed a method to link the genotype molecules to the nterminus of its encoding peptide. the mrna has dna-linker hybridize region, translation enhancer, initiation codon, single codon, amber stop codon, n-terminus sequence in gfp gene, his-tag and ochre stop codon. the mrna is also linked at '-terminus to sup trna via spacer and the speci c amino acid. the mrna is translated in the cell-free translation system. thus, c-terminus of the protein becomes free. moreover, in this system, the free protein of the full length is never generated. for this reason, the problem in the conventional technique, that is, the competition between the protein displayed on the genotype molecule and the free protein is eliminated. we succeeded to turn one round of the " life cycle " of the in vitro virus, judged by his-tag selection. key: cord- - mvzkff authors: moore, john p.; robling, kristyn; romero, cristian; kiper, keturah; dachavaram, soma shekar; crooks, peter a.; hestekin, jamie a. title: oxone(®)-mediated tempo-oxidized cellulose nanomaterial ultrafiltration and dialysis mixed-matrix hollow fiber membranes date: - - journal: polymers (basel) doi: . /polym sha: doc_id: cord_uid: mvzkff recent exploration of cellulose nanomaterials has resulted in the creation of oxone(®)-mediated tempo-oxidized cellulose nanomaterials (oto-cnms). these materials, when incorporated into a polymer matrix, have properties showing increased flux, decreased membrane resistance, and improved clearance, making them an ideal material for dialysis. this study is the first to focus on the implementation of oto-cnms into hollow fiber membranes and a comparison of these membranes for ultrafiltration and dialysis. ultrafiltration and dialysis were performed using bovine serum albumin (bsa), lysozyme, and urea to analyze various properties of each hollow fiber membrane type. the results presented in this study provide the first quantitative evaluation of the clearance and sieving characteristics of oxone(®)-mediated tempo-oxidized cellulose-nanomaterial-doped cellulose triacetate mixed-matrix hemodialyzers. while the cellulose nanomaterials increased flux ( – %) in ultrafiltration mode, this was offset by increased removal of albumin. however, in dialysis mode, these materials drastically increased the mass transfer of components ( – %), which could lead to significantly lower dialysis times for patients. this change in the performance between the two different modes is most likely due to the increased porosity of the cellulose nanomaterials. according to the centers for disease control and prevention (cdc), chronic kidney disease is the th leading cause of death in the united states [ ] . there are an estimated eight million american adults with reported diagnoses of kidney disease [ ] . the cdc found that, in , over , americans died from kidney disease [ , ] . recent research has shown renal replacement therapy is essential in treating covid- [ ] . currently, the most common treatment technique is to filter the blood through hemodialysis or peritoneal dialysis. dialysis does not cure the disease, like kidney transplantation. still, it can remove contaminants from the blood, like the kidneys, to maintain the health of the individual for a significant period. the majority of patients will receive treatment three times per week, for to h. each treatment is based on how much blood can be filtered out of the body at a specific time, as well as the performance of the membranes. hemodialysis machines operate on the principle of diffusion [ ] . blood is pumped out of the body and passed through hollow fiber modules. the membranes are made of a semi-permeable material, usually cellulose or polysulfone, that has a pore size that is too small to allow blood cells and intermediary for nanocellulose production yields two forms of oxone ® -mediated tempo-oxidized cellulose nanomaterials (oto-cnms), form i and form ii [ ] . form i is partially oxidized, and form ii is fully oxidized. they were both found to have a novel crystalline structure [ ] . neither form i or form ii has been tested within polymer-based filtration studies. figure shows the general chemical structure of form i compared to form ii. the production of form i and form ii was also found to be an efficient method of crystalline nanocellulose production. utilizing the procedure from moore et al., oto-cnms form i and form ii were synthesized and used in conjunction with cellulose triacetate (cta) from acros organics (fair lawn, nj, usa), n-methyl pyrrolidone (nmp) from vwr (radnor, pa, usa), and deionized water to create novel membranes for filtration [ ] . the proteins used for ultrafiltration and dialysis characterization were bovine serum albumin (bsa), lysozyme, and urea supplied from vwr. these are common proteins found in the blood. bsa was chosen to represent large proteins in the blood; lysozyme was selected to represent middle molecular weight proteins in the blood, and urea was chosen because it is the primary waste product that is excreted from the kidneys. all membranes were housed in polyvinyl chloride pipes (pvc) supplied from lowe's (fayetteville, ar, usa) using underwater epoxy resin. ethanol for cleaning was also provided through vwr. four different solutions were created for membrane casting. the first, the control, was created using only cta and nmp. the casting solution was made using wt % cta and wt % nmp. these were mixed in a bottle and placed on a bottle roller for - days to allow for the complete incorporation of the cta. the second solution, form i, was created using cta, nmp, and oto-cnm form i. the casting solution was made using wt % cta, wt % nmp, and wt % form i. to incorporate the form i nanocrystals, nmp and form i were blended in a -ml beaker at rpms for min in a water bath. then, the solution was placed in a qsonica (newtown, ct, usa) sonicator at watts at khz for min with -s intervals of sonication followed by s of no sonication. after sonication, the oto-cnms were fully dispersed in solution. the solution was then run through a coarse filter using vacuum filtration to remove any large chunks. after filtration, it was added to a roller bottle with the cta and placed on a bottle roller for - days. this process was repeated with the other two types of solutions but with adjusted concentrations of oto-cnm form i and form ii. the third solution, / , was prepared using cta, nmp, and form i and form ii. the solution was made using wt % cta, wt % nmp, . wt % form i, and . wt % form ii. the same mixing procedure was followed that was used for form i. the fourth solution, form ii, was prepared using cta, nmp, and form ii. the solution was made with wt % cta, wt % nmp, and wt % form ii. this solution was mixed with the same procedure as form i and / . all compositions of oto-cnm/cta membranes based on wt % for form i to form ii can be found illustrated in supplemental table s . membranes were created with non-solvent-phase-induced separation hollow fiber casting. a water bath at • c was filled so the fibers would be fully immersed as they traveled the length of the bath. a bore solution containing wt % nmp and wt % deionized water was utilized. a spinneret was used to extrude the hollow fibers. it was set to obtain a -µm thickness of the membranes. the polymer solutions were passed through the outer layer of the spinneret at a pressure of psi, and the bore was passed through the spindle at a pressure of - psi to create a hollow tube. the spinneret was set at a -cm height above the water level to obtain a consistent time spent passing into the bath. as the solutions passed through the water bath, phase inversion occurred. the fibers were collected in a roll at a rate of . m per minute until casting was complete. immediately after completion, the fibers were placed in a hot water bath at - • c for min. this heat treatment sets the membranes to strengthen them and ensure high quality. after heat treatment, they were placed in a container filled with room temperature deionized water for storage until further use. no membrane was stored more than one month before use. for each dialysis run, a new module had to be created. for each module, pvc pipes were assembled in the fashion shown in figure to create complete hemodialysis modules for testing. each module contained fibers. the fibers were inserted into the pvc apparatus and glued into place with underwater epoxy resin. this glue was used to seal the ends of the device, so no leaking would occur during testing. about ml of glue was used for each end. after the glue fully solidified, the ends were cut to create an even surface, and threaded caps were added to the module to create the completed module. these were stored in a room temperature deionized water bath until ready for testing (up to a month). polymers , , x for peer review of was used to extrude the hollow fibers. it was set to obtain a -µ m thickness of the membranes. the polymer solutions were passed through the outer layer of the spinneret at a pressure of psi, and the bore was passed through the spindle at a pressure of - psi to create a hollow tube. the spinneret was set at a -cm height above the water level to obtain a consistent time spent passing into the bath. as the solutions passed through the water bath, phase inversion occurred. the fibers were collected in a roll at a rate of . m per minute until casting was complete. immediately after completion, the fibers were placed in a hot water bath at - °c for min. this heat treatment sets the membranes to strengthen them and ensure high quality. after heat treatment, they were placed in a container filled with room temperature deionized water for storage until further use. no membrane was stored more than one month before use. for each dialysis run, a new module had to be created. for each module, pvc pipes were assembled in the fashion shown in figure to create complete hemodialysis modules for testing. each module contained fibers. the fibers were inserted into the pvc apparatus and glued into place with underwater epoxy resin. this glue was used to seal the ends of the device, so no leaking would occur during testing. about ml of glue was used for each end. after the glue fully solidified, the ends were cut to create an even surface, and threaded caps were added to the module to create the completed module. these were stored in a room temperature deionized water bath until ready for testing (up to a month). dialysis and ultrafiltration module testing began with apparatus assembly. the filtration apparatus allowed solutions to pass through the membrane during ultrafiltration and two fluids to pass concurrently during dialysis. the transmembrane pressures were kept consistent with the use of pressure dampers. once the module was connected, water was passed through the dialysate side (outside) and the feed side (inside) of the membranes. the system was run for one hour prior to testing at - psi to stabilize the membrane. the dialysate side was run at a flow rate of ml/min, and the feed side was run at a flow rate of ml/min. the transmembrane pressure was kept consistent at psi. during the hour of stabilization, the feed solutions were prepared. for bsa and lysozyme experiments, the concentration was mg/ml; specifically, mg of protein was dissolved into ml of water. for the urea experiments, the concentration was mg/ml; specifically, mg of urea was dissolved into ml of water. once the pure water stabilization was finished, the setup was switched over to ultrafiltration, which involved turning off the dialysate. the feed was maintained at a flow rate of ml/min. three water samples were collected from the permeate (dialysate side) at psi transmembrane pressure. each sample was collected for s and then weighed for flux calculation. after three samples were collected at the , , and min marks, the dialysis and ultrafiltration module testing began with apparatus assembly. the filtration apparatus allowed solutions to pass through the membrane during ultrafiltration and two fluids to pass concurrently during dialysis. the transmembrane pressures were kept consistent with the use of pressure dampers. once the module was connected, water was passed through the dialysate side (outside) and the feed side (inside) of the membranes. the system was run for one hour prior to testing at - psi to stabilize the membrane. the dialysate side was run at a flow rate of ml/min, and the feed side was run at a flow rate of ml/min. the transmembrane pressure was kept consistent at psi. during the hour of stabilization, the feed solutions were prepared. for bsa and lysozyme experiments, the concentration was mg/ml; specifically, mg of protein was dissolved into ml of water. for the urea experiments, the concentration was mg/ml; specifically, mg of urea was dissolved into ml of water. once the pure water stabilization was finished, the setup was switched polymers , , of over to ultrafiltration, which involved turning off the dialysate. the feed was maintained at a flow rate of ml/min. three water samples were collected from the permeate (dialysate side) at psi transmembrane pressure. each sample was collected for s and then weighed for flux calculation. after three samples were collected at the , , and min marks, the feed solution was switched to the desired experimental solution to obtain ultrafiltration data. three more samples were collected and weighed at the , , and min marks. after ultrafiltration, the dialysate was turned back on to run dialysis with the desired component. three flow rates were used to model different rates of dialysis. the first flow rate was ml/min for the feed and ml/min for the dialysate. feed-in, feed-out, and dialysate-out samples were collected to measure mass transfer. the second flow rate was ml/min for the feed and ml/min for the dialysate. samples were gathered in the same manner as for the first flow rate. the third flow rate was ml/min for the feed and ml/min for the dialysate. samples were again collected in the same manner. after samples were collected, the machine was turned off, and the module was disconnected. a % ethanol/water mixture was run through the apparatus for min at ml/min to clean and sterilize the system after each use. the protein samples collected during dialysis and ultrafiltration were analyzed for their concentration using a biotek epoch multi-volume spectrophotometer (winooski, vt, usa). a uv-transparent -well plate was utilized. samples were pipetted in triplicate in -µl increments into the well plate. the absorbance was run at nm for the bsa and lysozyme samples. the urea samples were prepared with a quantichrom urea assay kit provided from vwr and run at nm in a standard polystyrene -well plate, both supplied through vwr. scanning electron microscope (sem) images were taken using a philips xl environmental scanning electron microscope machine (north billerica, ma, usa) to characterize the membrane morphology. they were taken at × and × magnification at kv. cross-sections of the fibers were prepared using a freeze-cracking method. a dialysis membrane must be able to operate in ultrafiltration mode (water removal) as well as dialysis mode. this first section will define some of the terms commonly used in dialysis as well as analyzing ultrafiltration performance. one of the first terms used, k uf, is the pure water flux of the membrane at a constant pressure, where q uf is the permeate flow rate, a is the membrane area, and p is the pressure on the inlet or outlet of the donating stream [ ] . the ultrafiltration mode sieving coefficient (s o ) of each membrane was determined using equation ( ), where c f is the concentration of the filtrate and c p is the concentration of the permeate [ ] . it must be noted that two types of sieving coefficients will be used in this paper. the sieving coefficient, as described in equation ( ), is the ultrafiltration sieving coefficient and refers to how much of a component passes through the membrane under a constant pressure, in this case, psi. an element with % rejection has a sieving coefficient of , and a component with a % rejection has a sieving coefficient of . three components of interest-bsa, lysozyme, and urea-were run in ultrafiltration mode to find their sieving coefficients ( figure ). supplementary table s shows the sieving coefficients in table form with standard deviation. polymers , , x for peer review of sieving coefficient data shows many things about the characteristics of these membranes. first, as would be desired in a dialysis membrane, the control and all modified membranes have urea sieving coefficients near or at unity, ensuring smooth passage of components like urea and salts. second, the sieving coefficients for the middle molecules range from . to . . as lysozyme is in the molecular weight range of uremic toxins, it shows that molecules within this size range can pass through the membrane. finally, the sieving coefficient for bsa under these conditions ranged from . to . , with the / membrane having a sieving coefficient significantly higher than any of the other materials. excessive loss of bsa may make the / membrane unsuitable for dialysis. to better understand how the additives were affecting the active membrane area, equations were used to estimate their pore sizes. a general set of equations used for calculated pore sizes are given below in equations ( ) and ( ) equation ( ) is a hydrodynamic model that uses the values calculated from the sieving coefficient, where λ is the ratio of the solute radius (rs) to the pore radius (rp) [ ] . the pore radius was determined by the minimization of the sum of the squared residuals between the model (equation ( )) and the data for bsa. the values obtained for pure water flux (kuf pure water), as well as pore size (rp) along with the area (a) in meters squared, hollow fiber radius (r) in microns, and hollow fiber thickness (t) in microns of the test membranes as measured through sem are displayed in table . when bsa was used to calculate pore radius, the data suggests that each of the modified membranes had a bigger pore radius than the control with / showing the most significant increase. the bsa numbers reported were used to estimate pore radius, because bsa is assumed to be spherical while lysozyme is not [ ] [ ] [ ] . the flux data of these membranes show that the control and form ii are about the same, with about a % increase for / and a % increase for form i. after analyzing all of this data, we conclude that, for pure components in water, it appears from sieving coefficient data shows many things about the characteristics of these membranes. first, as would be desired in a dialysis membrane, the control and all modified membranes have urea sieving coefficients near or at unity, ensuring smooth passage of components like urea and salts. second, the sieving coefficients for the middle molecules range from . to . . as lysozyme is in the molecular weight range of uremic toxins, it shows that molecules within this size range can pass through the membrane. finally, the sieving coefficient for bsa under these conditions ranged from . to . , with the / membrane having a sieving coefficient significantly higher than any of the other materials. excessive loss of bsa may make the / membrane unsuitable for dialysis. to better understand how the additives were affecting the active membrane area, equations were used to estimate their pore sizes. a general set of equations used for calculated pore sizes are given below in equations ( ) and ( ) equation ( ) is a hydrodynamic model that uses the values calculated from the sieving coefficient, where λ is the ratio of the solute radius (r s ) to the pore radius (r p ) [ ] . the pore radius was determined by the minimization of the sum of the squared residuals between the model (equation ( )) and the data for bsa. the values obtained for pure water flux (k uf pure water ), as well as pore size (r p ) along with the area (a) in meters squared, hollow fiber radius (r) in microns, and hollow fiber thickness (t) in microns of the test membranes as measured through sem are displayed in table . when bsa was used to calculate pore radius, the data suggests that each of the modified membranes had a bigger pore radius than the control with / showing the most significant increase. the bsa numbers reported were used to estimate pore radius, because bsa is assumed to be spherical while lysozyme is not [ ] [ ] [ ] . the flux data of these membranes show that the control and form ii are about the same, with about a % increase for / and a % increase for form i. after analyzing all of this data, we conclude that, for pure components in water, it appears from ultrafiltration data that form i modified is the best candidate, due to its high flux and relatively high rejections of bsa. cellulose triacetate membranes are used in dialysis to remove urea, and the rate at which urea is removed is still a major limiting factor in dialysis treatment time. while urea is not considered the most significant component to remove for a patient's health, it is a good indicator of dialysis membrane performance. (k oa ) is the mass transfer area coefficient, otherwise known as the theoretical clearance at infinite blood and dialysate flow rates. it can be used as a representative term to describe treatment capabilities, as well as to determine theoretical treatment times for patients [ ] . (k oa ) is defined using fick's law, equation ( ) [ , ] . (∆c m ) represents the change in concentration across the membrane, and (n) represents the solute transport rate in mg/min. the solute transport rate (n) was calculated using equation ( ), where (q f ) represents the flow rate, subscript (f ) represents feed, subscript (d) represents dialysate, subscript (o) represents out, and subscript (i) represents in. (a) represents the area, and (∆c m ) represents the log mean concentration difference, which was calculated using equation ( ) . subsequently, the clearance (k) can be determined from equation ( ). a series of triplicate dialysis experiments were performed with bsa, lysozyme, and urea, as shown in table . as can be seen by looking at urea, the clearance (k experimental ) is over % higher with form i as compared to the control. higher clearance values are also seen for form ii and the / mix. an increase in clearance was also seen with lysozyme (desirable) and bsa (undesirable) in the modified materials compared to the control. for bsa, the diffusion was calculated based on the sieving coefficient (sc in table ), and it ranged from × − to × − . the value × − is comparable with the membrane performance of commercial membranes, while the other sieving coefficient values fall slightly outside [ ] . however, another study suggests that, for high cut-off membranes, it may fall inside the range, which allows a sieving coefficient to go as high as . with a protein loss of fewer than grams per session [ ] . the process of membrane fabrication depends on the polymeric materials used and their specific interactions with the solvents upon phase separation [ , ] . this is never more evident than when observing the membranes through a scanning electron microscope (sem). a cross-sectional sem of each of the four types of membranes is shown in figure . while key parameters of membrane casting, such as spinneret size, bath temperature, uptake speed, drop height, etc., were kept consistent in this experiment, closer inspection with sem revealed a dramatic change in the internal pore morphologies. compared to the control (figure a) , each of the modified material membranes (figure b-d) showed alterations to the support material that increased porosity caused by the cellulose nanoparticle additive. a similar effect was demonstrated by bai et al. in the production of pvdf composite membranes using unmodified cellulose nanomaterials, leading to larger finger-like pores and more pores in the bottom surface. as a result, the cellulose nanomaterial composite membranes were improved, i.e., in pure water flux, mechanical properties, and maintained solute rejection [ ] . it has been shown that the support structures of membranes can lead to dramatically different separation performances under different operating conditions [ , ] . more importantly, the improvement in the dialysis performance of these membranes can be explained by looking at the backing, i.e., the macroporous support structure. manickam and mccutcheon derive a theory for transport with pressure-driven versus diffusion-based processes [ ] . their approach shows that backing is a significant source of resistance in a diffusion-based process, while it is not in a pressure-driven process. thus, more porous, spongy backings would be predicted to lead to a better dialysis transport rate while not necessarily affecting the ultrafiltration rate. the membrane performance of commercial membranes, while the other sieving coefficient values fall slightly outside [ ] . however, another study suggests that, for high cut-off membranes, it may fall inside the range, which allows a sieving coefficient to go as high as . with a protein loss of fewer than grams per session [ ] . the process of membrane fabrication depends on the polymeric materials used and their specific interactions with the solvents upon phase separation [ , ] . this is never more evident than when observing the membranes through a scanning electron microscope (sem). a cross-sectional sem of each of the four types of membranes is shown in figure . while key parameters of membrane casting, such as spinneret size, bath temperature, uptake speed, drop height, etc., were kept consistent in this experiment, closer inspection with sem revealed a dramatic change in the internal pore morphologies. compared to the control (figure a) while the experiments were conducted with a total volume similar to a dialysis system, the membrane area was approximately an order of magnitude smaller. therefore, calculations were utilized to determine how these membranes would act if a typical dialysis membrane area were used [ ] . the theoretical urea clearance was calculated by simultaneously solving fick's law, the log mean concentration difference, and a mass balance to express the ratio k/q f as a function of two dimensionless parameters, neither of which involves solute concentration. this is shown in equation ( ) , where z is the ratio of the feed flow rate to the dialysate flow rate and r = k oa a/q f [ , ] . the clearance values (k theoretical ) determined from the above dimensionless expression based on the experimental data can be found in table . from the theoretical clearance values, predicted treatment time was calculated following the us national kidney foundation (kt/v) target. in this equation, a -kg person, at % water weight, must reach a kt/v of [ ] . governmental standards are set on this premise to ensure patients are successfully treated during their time at the hospital [ ] . it was found that form i had the most drastically decreased treatment time, at a % reduction. form i showed a decrease in treatment time, while the control showed the typical treatment time (approximately four hours on average). furthermore, the membrane can also be observed as a function of size, as shown in figure , where the modified membranes show the ability to maintain appropriate treatment times at smaller sizes without a significant loss in performance. in other words, in the control, we see a % loss in performance in the control when moving from a -m membrane to one half that size, and, in the modified sample, oto-cnm form i mixed-matrix membrane, only an % loss in performance. this effect is exacerbated at smaller membrane sizes. some significant conclusions can be drawn from this data. first, the theoretical treatment time for the -m membrane area is in the range of what has been previously reported [ ] . this suggests that these membranes could make effective dialysis membranes. second, the differences in clearance from the control versus the modified membranes suggest a smaller membrane can be used to achieve the same results seen in the unmodified membranes. it is important to note that results may seem more significant in the smaller test modules than when compared at the one-meter square membrane area. this is because both sets of solutions are using the same volumetric flow rates. since dialysis is an unsteady process, larger membrane areas have the capacity for high urea removal rates in the beginning, returning the blood at almost urea. this makes the flow rate a major limiting step in the removal of urea. however, when using a smaller module, the membrane can be more fully utilized, and the gradients affecting transfer can remain larger. to fully realize the potential of these new membranes, less membrane area would need to be used in the same operating conditions as traditional hemofiltration membranes. smaller modules in the same operating conditions allow more effective and efficient treatment with less blood out of the patient. furthermore, it has been shown that less blood being removed from the body increases the health of the dialysis patient [ , ] . from the theoretical clearance values, predicted treatment time was calculated following the us national kidney foundation (kt/v) target. in this equation, a -kg person, at % water weight, must reach a kt/v of [ ] . governmental standards are set on this premise to ensure patients are successfully treated during their time at the hospital [ ] . it was found that form i had the most figure . dimensionless analysis of the time of clinical operation completion (kt/v= . ) as observed against membrane surface area, a (m ), for the control, form i, / , and form ii samples show membrane performance at different active membrane areas under the same conditions, i.e., a ml/min feed flow rate and ml/min dialysate flow rate using the same assumptions utilized in table . ) as observed against membrane surface area, a (m ), for the control, form i, / , and form ii samples show membrane performance at different active membrane areas under the same conditions, i.e., a ml/min feed flow rate and ml/min dialysate flow rate using the same assumptions utilized in table . in this study on the implementation of two derivatives of cellulose nanomaterials into hollow fiber membranes for ultrafiltration and dialysis, oto-cnm form i and form ii mixed-matrix membranes have been quantitatively evaluated for the first time by determining model molecule selectivity (bsa, urea, and lysozyme), as well as transport rates (flux and diffusion rates). each of the three membranes made from cellulose nanomaterials showed improvements when compared to the control (cellulose triacetate). all showed an increase in flux, mass transfer area coefficient, and urea clearance. the differences were more significant when in dialysis mode, which suggests that the porous, spongy backing layer reduced the resistance in the diffusion-based process. these membranes have characteristics that fall within the range of commercial dialysis membranes, indicating that they could be an attractive candidate for future hemofiltration membrane development. further analysis of dialysis membranes like these will/could show the possibility of the use of less membrane area and shorter treatment times, leading to better patient health. table s : the compositions of oto-cnm/cta membranes based on wt % for form i to form ii; supplemental table s : membrane sieving coefficient information. cardiovascular disease in patients with chronic kidney disease chronic kidney disease and the risks of death, cardiovascular events, and hospitalization behavioral counseling to promote a healthful diet and physical activity for cardiovascular disease prevention in adults without cardiovascular risk factors: us preventive services task force recommendation statement severe covid- : a review of recent progress with a look toward the future. front. public health hemodialysis: prescription and assessment of adequacy. renal urology news membranes for hemodialysis the future of hemodialysis membranes transport characteristics of asymmetric cellulose triacetate hemodialysis membranes preparation and preliminary dialysis performance research of polyvinylidene fluoride hollow fiber membranes requirements and pitfalls of dialyzer sieving coefficients comparisons membrane innovation: closer to native kidneys second generation nanoporous silicon nitride membranes for high toxin clearance and small format hemodialysis clinical implications of hemodialysis membrane biocompatibility importance of development of dialysis membranes for purification of small, middle, high molecular weight and protein-bound uremic toxins: the asymmetric cellulose triacetate (solacea™-h) nanomaterials for membrane fouling control: accomplishments and challenges tempo-oxidized cellulose nanofibrils dispersed in organic solvents thermal stabilization of tempo-oxidized cellulose mediated micron cellulose oxidation procedure: preparation of two nano tempo-cellulose forms oxone®-mediated tempo-oxidized cellulose nanomaterials form i and form ii a critical review of recent advances in hemodialysis membranes hemocompatibility and guidelines for future development simulating nephron ion transport function using activated wafer electrodeionization high-flux and low-flux membranes: efficacy in hemodialysis effect of dialysis dose and membrane flux in maintenance hemodialysis application note: viscosity measurement of a model protein solution of bsa light scattering of bovine serum albumin solutions: extension of the hard particle model to allow for electrostatic repulsion charge interaction and temperature effects on the solution structure of lysozyme as revealed by small-angle x-ray scattering cellulose triacetate: another membrane for continuous renal replacement therapy membrane filtration: a problem solving approach with matlab linear solutions of fick's law influence of non-solvent chemistry on polybenzimidazole hollow fiber membrane preparation cellulose nanocrystal-blended polyethersulfone membranes for enhanced removal of natural organic matter and alleviation of membrane fouling preparation and characterization of poly (vinylidene fluoride) composite membranes blended with nano-crystalline cellulose novel dual-layer hollow fiber membranes applied for forward osmosis process enhanced antifouling performance of pvdf ultrafiltration membrane by blending zinc oxide with support of graphene oxide nanoparticle understanding mass transfer through asymmetric membranes during forward osmosis: a historical perspective and critical review on measuring structural parameter with semi-empirical models and characterization approaches a non-dimensional analysis of hemodialysis mathematical modeling of membrane filtration porosity and pore size determination in polysulfone hollow fibers impact of blood and dialysate flow and surface on performance of new polysulfone hemodialysis dialyzers handbook of biomedical instrumentation hemodialysis system. in modelling and control of dialysis systems this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license characterization facility for the image support, as well as emily e. thompson for final grammatical corrections. it should be noted that four authors-john p. moore ii, soma shekar dachavaram, peter a. crooks, and jamie a. hestekin-have started an llc looking to market and develop cellulose materials such as the ones presented in this text. (a) membrane area, m (c f ) the concentration of the filtrate mg/ml (c p ) the concentration of the permeate mg/ml (k) clearance, ml/min (k experimental ) experimental clearance, ml/min (k oa ) is the mass transfer area coefficient ml/(m *min) (k theoretical ) determined clearance values, ml/min (k uf ) pure water flux of the membrane at constant pressure (l/hr/m /psi) (m w ) molecular weight, daltons (n) represents the solute transport rate mg/min (p) pressure, psi (q) represents the flow rate, ml/min (q uf ) permeate flow rate, ml/min (r) hollow fiber radius, µm (r p ) pore radius, µm (r s ) theoretical solute radius, µm r = k oa a /q f , dimensionless (s o ) sieving coefficient, dimensionless subscript (f ) represents the feed subscript (d) represents dialysate subscript (o) represents out subscript (i) represents in (t) hollow fiber thickness, µm (λ) the ratio of the solute radius to the pore radius, dimensionless (z) ratio of the blood flow rate, dimensionless key: cord- - zrbo w authors: wardhan, rashmi; mudgal, padmshree title: membrane transport date: - - journal: textbook of membrane biology doi: . / - - - - _ sha: doc_id: cord_uid: zrbo w every living cell has to exchange molecules across the membrane for cellular functions. the hydrophobic or lipophilic molecules do not require energy for crossing the membrane. they can diffuse freely from higher to lower concentration till equilibrium is established. this process is called passive transport or diffusion. every living cell has to exchange molecules across the membrane for cellular functions. the hydrophobic or lipophilic molecules do not require energy for crossing the membrane. they can diffuse freely from higher to lower concentration till equilibrium is established. this process is called passive transport or diffusion. this is not true for polar or lipophobic molecules because of the nonpolar nature of membrane lipid bilayer. for polar molecules, the cell recruits mediator or transporter proteins in the membrane to facilitate their movement. if the direction of transport is from higher to lower concentration region, the transporter proteins do not use external energy and the process is called facilitated transport, because the energy needed for transport is dispensed by molecule's own concentration gradient (unequal distribution across the membrane) in the favorable direction. the free energy is minimal in unequal distribution of polar molecules. if polar molecule is transported from lower to higher concentration region, against the concentration gradient, the energy has to be provided by external source to mediator protein to make the process spontaneous in the cell, and this process is called active transport. in the cell, the energy source is mostly adenosine triphosphate (atp). the facilitated transport unlike diffusion has shown speed and specificity, saturation kinetics, susceptibility to competitive inhibition, and susceptibility to chemical inactivation. the active transport of polar molecules against concentration gradient requires energy for transport. a large group of transporters that hydrolyze atp to transport molecules across the membrane are known as atpases. these carrier atpases are classified into five subclasses according to their structure and physiological functions as p-type atpases, v-type atpases, f-type atpases, ecto-type atpases, and a-type atpases. p-type atpase transporters are multidomain integral membrane proteins involved in the transport of cation and lipids. phylogenetically according to their function, these p-type atpases are divided into five major types and various subclasses. the vacuolar atpases are atp-dependent oligomeric protein proton pump, which regulate acidic ph in organelle compartment like phagosome and endosome for the separation of ligand from their receptors transport proton (h + ) across the plasma membrane. the secondary transport, also known as ion-coupled transport involves the use of electrochemical potential generated by an ion transport for the co-transport of another ion against the gradient across the membrane. atp-binding cassette (abc) transporters, importers, and exporters, the newly identified integral proteins, comprise a large superfamily of integral membrane proteins with diverse functions. there are three major transporters which are known as flippases, floppases, and scramblases. the flippases are also known as p -atpases, which transport phospholipids to inner leaflet of membrane and have substrate specificity to phosphatidyl serine and phosphatidyl choline. aquaporins (aqps), the inte-gral proteins of membrane, are small, hydrophobic, and homotetramer proteins, which are involved in bidirectional transport. the bacteria take nutrients from their surroundings by various modes of transport but the transport of sugars and its derivatives like sugar alcohols, amino sugars, glucuronic acids, and disaccharides is transported by group translocation with histidine heat resistance protein hpr as high-energy phosphate donor protein. the transporting sugar molecule is also phosphorylated during transport, which makes sugar negatively charged. the various organisms have specialized proteins of rhodopsin family to seize light energy for various physiological functions. the rhodopsin protein part, opsin, is covalently linked to retinal chromophore, which may be in trans-or cis-configuration. the animal rhodopsin (also known as type ii) are cell g-coupled receptors, while microbial rhodopsin (type i) may act as ion pump, ion channels, sensors, photosensory receptors and regulator for gene expression and kinases. pore-forming toxins (pfts), the bacterial virulence factor, single major family of proteins, are secreted by gram-positive and gram-negative bacteria. pfts may be present either in cytoplasm or in the membrane. the conformational change in cytoplasmic pfts may translocate them to membrane in need of hour. ionophores are natural and synthetic organic molecules of diverse type, which can transport cations by forming lipid ion-soluble complex. each ionophore possesses a unique property of changing transmembrane ion gradient and electrical potential, synthesized by bacteria, and acts as protector against competing microbes. the bacterial and eukaryotic membranes are found to have special proteins in their outer membrane for transport, which are known as porins and form channels for various small size solutes, ions, and other nutrients. the protein ion channels are passive transporters, are made up of transmembrane, are selective for their substrate ions, and have much faster rate of transport than pumps. these channel proteins do not interact with transporting ions and have two conformations, closed and open, to regulate flow of ions. the trimeric atp-activated channels are permeable to cations like sodium, potassium, and calcium. there are seven subclasses of the group, which are involved in various physiological functions like muscle contraction, neurotransmitter release and immune responses regulation. the gaba and glycine receptors are anion channels but inhibitory in nature. gaba receptors have two subdivisions as gaba receptors family of ligand-gated channel (ionotropic) gaba a and g receptor protein family (gaba b ). the n-methyl-d-aspartate (nmda) receptors are known as glutamate-gated cation channel for calcium permeation. the tetrameric ionotropic receptors include nmda, a-amino- -hydroxy- -methyl- -isoxazolepropionic acid (ampa), and kainate receptor as per their synthetic ligand binding. the voltage-gated ion channels open or close in response to change in voltage and the unit of membrane potential. in excitable cells like nerve and muscle, the chemical or electrical signaling is initiated through voltage-dependent sodium or calcium influx. the k p channels or voltage insensitive channels formerly referred to as leaky or resting channels, are ubiquitously found in all organisms. the functionally active k p channel has two subunits, and each subunit is made up of two pore regions (p and p ), four transmembrane domains (m -m ), and two characteristic extracellular helices cap c and c . this chapter explains all the mechanisms of membrane transport. while osmosis is the movement of solution from high concentration to low concentration without transporters across the lipid bilayer, the diffusion is a type of passive transport where small lipophilic or hydrophobic solute moves from higher to lower concentration. the membrane is made up of lipid bilayer to have hydrophobic interior in membrane, which is selectively permeable to small hydrophobic molecules but not to large polar ones. the passive diffusion of charged species across the membrane depends upon the concentration and also on charge of particle z and the electrical potential difference across the membrane dw. the diffusion of particle across the membrane is determined by fick's law of diffusion and affected by magnitude of concentration gradient, molecular weight of solute, distance, permeability, and surface area of membrane. the diffusion coefficient of any solute molecule of fick's law is proportionality factor, which can be defined as mass of solute diffusing through a unit area in a unit time (unit is square meter per second). the diffusion coefficient depends on the size, temperature, charge on the molecule because of difference between proton and electron and polarity (uneven distribution of charge in the molecule) ( fig. . ). according to fick's law, for membrane area a, thickness x, and concentration gradient c (higher) and c (lower) across the membrane, the diffusion rate will be proportional to probability coefficient and can be written as follows: the diffusion rate dx dt ¼ pa c Àc ð Þ ð : Þ the rate of diffusion is also proportional to partition coefficient k. so, where d is diffusion coefficient. from eqs. ( . ) and ( . ), equation ( . ) explains that the rate of diffusion is directly proportional to both partition coefficient and diffusion coefficient and inversely proportional to thickness of the membrane x; the thickness is constant factor for membrane; that's why diffusion largely depends on partition coefficient. the more hydrophobic molecule will move much faster ( fig. . ). as depicted in fig. . , the hydrophobic nonpolar molecule diffuses much faster than polar molecule. among the polar molecules, anionic molecules have higher permeability than cationic molecules. the gases like o and co are nonpolar and hydrophobic, so they easily diffuse. glycerol also diffuses across the membrane, but if this is phosphorylated, then this becomes polar. the urea (nh conh ) is less hydrophobic than diethyl urea (c h nhconhc h ), so urea diffuses slower than diethyl urea. the diffusion of molecules and gases from mother to fetus has been observed, and another molecule sodium thiopental is observed to diffuse through blood-brain barrier. facilitated transport is mediated by transporter protein, which is found in the membrane without using external source of energy. the transport occurs from higher concentration to lower concentration. in mammal's erythrocytes, the . passive diffusion glucose is transported by facilitated diffusion by glucose transporter protein. the facilitated transport unlike diffusion has shown speed and specificity, saturation kinetics, susceptibility to competitive inhibition, and susceptibility to chemical inactivation ( fig. . ). the permeability coefficient of the facilitated transport of glucose is much higher than permeability coefficient ( .  − cm/s) of diffusion. the glucose transporter proteins are divided into two major families like na + /glucose cotransporter (sglts) and facilitative glucose transporter (gluts). the sglt is found in specialized epithelial cells of intestine, proximal tubule of kidney, trachea, heart, brain, testis, and prostate. the na + /glucose cotransporters (sglts) are involved in secondary transport of glucose in intestine and nephrons. the members of sglt class are identified; out of them sglt , sglt , sglt , and sglt function as sugar transporter and sglt acts as glucose sensor. sglt is present in the kidney, brain, liver, thyroid, muscle, and heart. the facilitative glucose transporter (gluts) has fig. . diffusion of polar and nonpolar molecules in the artificial phospholipid bilayer. the small nonpolar molecules move much faster than polar and large molecules fig. . cellular uptake of glucose mediated by glut protein exhibits simple enzyme kinetics and greatly exceeds the calculated rate of glucose entry by simple diffusion members, and out of transport sugars. glut is present in erythrocytes, fetus placenta, brain, and kidney but low in liver and muscles. glut expressed in liver maintains glucose homeostasis. glut is neuron-specific glucose transporter with km of around mm for glucose. glut , a high-affinity, insulinresponsive transporter with approximately mm k m , is expressed in adipose tissue and muscle tissue. in the process of insulinstimulated glut translocation, the various proteins of signal pathway also participate. the high-affinity enzyme glut has km for glucose around - mm lower than blood glucose level ( - mm), hence transports glucose at significant level even in hypoglycemic conditions. the human erythrocyte glucose transporter (glut ) is a type iii transporter, having -residue glycoprotein with transmembrane (m -m ) spanning a helices (mr- , ). the n-terminal and c-terminal are located in cytoplasm. amino acids in the extra cellular loop between m and m , have n-linked glycosylation sites. the large hydrophobic amino acids are present between m and m . the , , , , and amphipathic helices are involved to form hydrophilic channel for glucose transport ( fig. . ) . the glucose transporter accounts for % of erythrocyte membrane proteins and runs as band . in sds-page gels of erythrocyte membranes. it is generally not visible on the gel because the heterogeneity of its oligosaccharides makes the protein band to diffuse (fig. . ). the glucose concentration is maintained at mm for all metabolic purposes inside the cell. the process of glucose transport can be compared to an enzymatic reaction, where the glucose concentration outside the cell is the substrate (s out ), glucose concentration inside the cell is the product (s in ), transporter t is the enzyme, initial velocity is (v ) and (kt) is the transport constant like michaelis constant. the graph between velocity of transport and extracellular glucose concentration [s] out shows saturation kinetics. v max and kt can be calculated from this double reciprocal plot between /v and /s out when [s] = kt, and the rate of uptake is / of v max . the rate equations for this process can be derived like enzyme-catalyzed reactions from michaelis-menten equation: the glucose molecule does not undergo any chemical change during transport across the fig. . structure of glut with transmembranes, and the alpha-helices , , , , and are involved to form hydrophilic channel for glucose . facilitated transport of glucose membrane making this a reversible process and helps to achieve equilibrium much faster in the presence of transporter glut transporter. the transporter protein undergoes a change in conformation on binding to glucose. it has a kt of . mm for d-glucose ( fig. . ). the carbon dioxide formed by cellular respiration of tissues is transported to lung as bicarbonate through erythrocytes and plasma. the hydration of co by carbonic anhydrase in erythrocytes forms bicarbonate, which is exchanged with chloride. the anion transporter involved in facilitated transport across the erythrocyte membrane is high-capacity transporter as the diffusion rate of chloride/bicarbonate is never rate limiting for co transport to lungs. this anion transporter constitutes % of erythrocyte membrane. the two identified families of anion exchanger proteins are known as slc/ a and slc a. the chloride-bicarbonate anion transporter belongs to slc a family. slc/ a has na + /bicarbonate cotransporter. the anion exchanger can transport bicarbonate in both directions. these transporters regulate ph of the cell, volume, and acid-base secretion. the carbonic anhydrase enzyme (pka of . , close to cellular ph) along with co /hco equilibrium acts as a buffer and maintains ph in the cell (fig. . ) . the human chloride-bicarbonate anion exchanger protein has residues with n-terminal domain, transmembrane, and c-terminal domain. the n-terminal and c-terminal are cytosolic. this comprises % of rbc protein. there are - transmembrane domains involved in transport. the protein is glycosylated at asparagine residue of extracellular domain four. the c-terminal domains of amino acids interact with carbonic anhydrase enzyme (transport metabolon). in erythrocyte, carbonic anhydrase reaction forms bicarbonate. during mitochondrial respiration, co is diffused in erythrocytes via capillaries, where it is converted into bicarbonate reaction as shown here. the bicarbonate formed in tissue erythrocytes is released in plasma for lungs. the process is reversed in lungs; the bicarbonate is transported back to lung erythrocytes, which is converted there into co by carbonic anhydrase ii isoform of lung and co is exhaled from lung by diffusion. in the exchange of one bicarbonate ion, chloride ion also moves in opposite direction; that's why this anion transporter is called electroneutral transport ( fig. . ). this anion transporter increases blood exchange capacity for co and provides flexibility of erythrocytes by interacting with rbc cytoskeleton. this transporter may be dimer or tetramer in erythrocytes. the n-terminal interacts with ankyrin, b-spectrin, . and . band proteins of rbc. the mutation in rbc anion exchanger results in defective interaction with cytoskeletal proteins to make rbc osmotic fragile and causes disease such as hereditary spherocytosis. in kidney, the anion exchanger protein controls acid-base homeostasis, electrolyte, and fluid concentration. the carrier protein as discussed earlier can be involved in active or passive transport. the active transport of polar molecules against concentration gradient requires energy for transport. a large group of transporters that hydrolyze atp to transport molecules across the membrane are known as atpases. these carrier atpases are classified into five subclasses according to their structure and physiological functions as p-type atpases, v-type atpases, f-type atpases, ecto-type atpaeses, and a-type atpases. . p-type atpases (e -e atpases): these atpases transport various cations and lipids across the cellular and organelle membrane to regulate cell functions. the various cations may be h + , na + , k + , ca + , cu + , cd + , hg + , zn + , ni, and pb + . the phylogenetic study has divided this group into five major types and more than subtypes. . e-type atpases: these are cell surface transporters involved in extracellular transport, which can use nucleoside diphosphates and nucleosides triphosphates including extracellular atp. these transporters depend upon ca + and mg + and are insensitive to specific inhibitor of p-type. examples are ecto-atpase and animal ecto-apyrase. the e-type atpases participate in termination of purinergic receptor-mediated responses. they are also involved in cell adhesion processes and are important for the maintenance of hemostasis in the cardio vasculature. . f-type atpases (f -f atpases): these atpases are involved in atp synthesis and localized on bacterial membrane, thylakoid membranes of chloroplasts, and inner membranes of mitochondria. the detail of these atpases is discussed in chap. . . v-type atpases (v -v atpases): these atpases act as proton pump and are found on various organelle membranes to regulate acidic ph in organelles. . a-type atpases (a -a atpases): these atpases are present in archaeal bacteria and in some other prokaryotes. they functionally resemble to f-type atpases and structurally to v-type atpases. . p-type (e -e ) atpases p-type atpase transporters are multidomain integral membrane proteins involved in transport of cation and lipids. phylogenetically according to their function, these p-type atpases are divided into five major types and various subclasses ( fig. . ). the p-type atpase or pump is a - kd protein having approximately around amino acids. p-type atpases may have maximum hydrophobic transmembranes from m -m with their n-terminal and c-terminal of proteins in cytoplasm. the cytoplasmic domain is highly conserved and inserted between m and m and m -m . these proteins have characteristic conserved amino acids dktgtlt, where aspartic acid (d) is reversibly phosphorylated during catalytic cycle. the five types of p-type atpases are divided as type i, type ii, type iii, type iv, and type v. type i p-type atpases are bacterial ion pump like escherichia coli-kdp atpases, which are made up of four different subunits with six transmembrane domains namely, kdp f, a, b, and c. the kdp b is the catalytic and ion-transporting subunit of the pump. type ib atpases of single peptide with eight transmembranes like znta and copa physiologically detoxify bacterial cell by throwing out toxic metals. type ii p-type atpases are majorly involved in membrane potential regulation in the cell. they are present ubiquitously in all the cells like na + /k + atpases and h + /k + atpases. type iia is most studied and diversified class that includes sarcoplasmic reticulum ca + atpase pump (serca). this calcium atpase transports two calcium ions from muscle to lumen of endoplasmic reticulum at the expanse of one atp and in exchange of two or three hydrogen ions. the muscle contraction is controlled by ca + atpases because the sudden calcium increase in muscle cell results in muscle contraction. the type ii a calcium atpases is inhibited by phospholamban, which is active in dephosphorylated form and blocks calcium entry by interfering in m transmembrane movement. the calcium atpases are inhibited by thapsigargin. type iib includes plasma membrane calcium atpases or pump (pmca). the type iib fig. . phylogenetic data of the p-type atpase. subfamily division according to their function and ion binding specificity type ia, bacterial kdp-like k + -atpases; type ib, soft-transition-metal translocating atpases; type iia, sarcoplasmic reticulum (sr) ca + -atpases; type iib, plasma membrane ca + -atpases; type iic, na + /k + -atpases and h + /k + -atpases; type iid, eukaryotic na + -atpases; type iiia, h + -atpases; type iiib, bacterial mg + -atpases; type iv, "lipid flippases"; and type v, eukaryotic p-type atpases of unknown substrate specificity. colour coded by species: green, genes from arabidopsis thaliana; orange, c. elegans; grey, e. coli; dark blue, homo sapiens; light blue, methanobacterium thermoautotrophicum; yellow, methanococcus jannaschii; purple, synechocystis pcc ; and red, saccharomyces cerevisiae atpases are regulated by their c-terminal or n-terminal calmodulin-binding domains. the type iic atpases are heteroligomers with aand b-subunits. the sodium-potassium atpases (na + /k + atpase) belong to this class. the a-subunit with maximum ten transmembranes (m -m ) is catalytic unit with autophosphorylation site and ion binding site, while b-subunit, glycosylated single transmembrane of kd, is required for insertion and assembly. the renal na + /k + atpase has additional c-subunit with fxyd motif (fig. . ) . the fxyd proteins act as regulator and stabilizer. the fxyd motifs provide stability to pump. the sodium-potassium pump is inhibited by digitalis glycosides and ouabain, and h + /k + atpases are inhibited by omeprazole, an antiulcer drug, so these pumps are the site of drug action also. the na + /k + atpases transport three na + ions out of the cell and two k + ions inside the cell per catalytic cycle. the type iid atpases are eukaryotic na + /k + atpases. the type iii atpases are prominently found in fungi and plants, where these pumps maintain intracellular ph of . against extracellular ph of . and maintain membrane potential in the similar way of animal na + /k + pump. the h + atpases are regulated by c-terminal autoinhibitory amino acids. fusicoccin is a fungal toxin, which activates h + atpases irreversibly. type iv and type v atpases' role is not very clear but structurally they resemble to type i and are found in eukaryotes. they are also known as flippases, which transport phospholipids from outer leaflet to inner leaflet and maintain lipid asymmetry of the membrane. the erythrocyte membrane has mg + -dependent flippases. these ion pumps may work in correlation with other lipid transport proteins to maintain asymmetry of membrane. . . structure of p-type atpases x-ray crystallography, nmr spectroscopy and electron cryo-microscopy (em) studies on various types of p-type atpases especially, calcium atpases and sodium-potassium atpases revealed that p-type atpases have four domains with conserved sequences, namely, actuator a, membrane m, nucleotide-binding domain nbd, and phosphorylation p-domain. the variation of amino acids has been observed only in extracytoplasmic loop. the most conserved phosphorylation domain is the catalytic domain with dktgtlt signature motif, where aspartic acid (d) is phosphorylated and dephosphorylated during catalytic cycle. it has rossmann fold (characteristics of many nucleotides proteins) made up of central seven-stranded b-sheet flanked by a-helices, including the cytoplasmic end of m . the nucleotide-binding domain inserts into p-domain through conserved hinge region of two antiparallel peptides. seven strands of b-sheet in central have lysine for nucleotide binding. in na + /k + atpases-n-domain phenylalanine clustering is observed whereas acidic residues are rich in calcium atpases. this might be site for regulator interaction, as observed with em. in these atpases are composed of two subunits (a and b). the a-subunit ( kd) binds atp and ions (na + and k + ) and contains an autophosphorylation site (p) but some isoforms like renal sodium-potassium have extra c subunit with fxyd the nucleotide binding site, adenosine base and phenylalanine interaction forms hydrophobic stacking and three phosphate group bulge out in solution so that c-phosphate could reach to p-domain aspartic acid for phosphorylation. the n-terminal actuator domain is highly conserved. similarly like p-domain, a-domain has two unequal divisions by m and m helices hairpin structure. the larger part has b-sheet secondary structure with conserved tge sequence. the tge sequence interacts with phosphorylation site during transport cycle. some of atpases like fungal h + -atpases have extra-long amino-terminal amino acids fused with actuator domain, which might play role in substrate sensing or in regulation. the membrane domain is the largest domain of approximately amino acids with ten transmembrane segments to enclose ion binding site. this domain is less conserved and is directly connected to catalytic core through m -m and m -m polar ionic side chains. as observed in calcium atpases, the m aspartic acid and m glutamate acidic side coordinate ca + ; the other amino acids such as valine , alanine , and isoleucine of m also participate in ion binding. m is the flexible region, which is arranged and rearranged during transport cycle for accommodating different sizes and charge ions and for catalysis ( fig. . ). the catalytic cycle of atp binding, phosphorylation, changes in conformation by arrangements of m-domain helices, hydrolysis of atp for energy are same in all p-type transporters. that's why p-, n-, and a-domains have conserved residues but m-domain with variable residues regions with ion binding site may change to accommodate various ions. the cycle starts with loading of ion (x + ) to high-affinity binding site and releasing of extracellular ion (y + ) in cytoplasm through cytoplasmic access channel. the interaction of ion (x + ) with polar group of amino acids near binding site at p-domain twists m , m , and m helices to move in e state. mg + /atp binding to nbd domain brings this close to p-domain through slight movement of b-strands. these movements result in correct positioning of c phosphate group of atp to p site, and e state of atpase is changed to e *p state. in e *p state, ion (x + ) is trapped in protein, which can be visible from any side of membrane. when ion (x + ) binding is complete, the phosphorylation takes place on aspartic acid residue of p-domain because the energy released by ion binding to e atpase favors the inclination of p-domain to bring more closure c phosphate group to aspartic acid for reaction ( fig. . ). the reaction may be explained as nucleophilic attack on aspartic acid in p-domain by c phosphate group. this may be the reason that sodium atpase needs three na + ions in comparison with two ca + ions in ca + atpases for p-domain movement. a-domain rotates parallel to membrane, and tge loop becomes closure to phosphorylation site. the e *p state changes to e *p state. simultaneously, m , m , and m helices of a-domain because of their rearrangement induced by phosphorylation may close polar ion access channel from cytoplasmic site. this might be understood that the inclination of p-domain induces the movement of helices in such a way that e *p state has low affinity to ion (x + ) and leaves this to extracellular site or into lumen of er. the e *p state has high affinity for ion (y + ), and binding to this ion may induce the hydrolysis of phosphorylated aspartic acid. the tge loop interaction with mg + ion will leave phosphate group unprotected, and this may result in hydrolysis by water. after this hydrolysis, e transforms into e state with open binding site in cytoplasm for next cycle of transport. the vacuolar atpases are atp-dependent oligomeric protein proton pump, which regulate acidic ph in organelle compartment like phagosome and endosome for the separation of ligand from their receptors and transport proton (h + ) across the plasma membrane. the v type atpases activity can be regulated by reversible disulphide bond formation between conserved cysteine residues at catalytic site. glutamate uptake in presynaptic vesicle is found to be dependent on v-type atpases. the v-type atpases regulate plasma ph by exporting proton through renal intercalated cells of kidney. the mutation in v-type atpases may cause renal tubular acidosis. the vacuolar atpases also regulates acidic ph in osteoclasts for bone degradation, and the genetic defect in v-type atpases may cause osteoporosis. v-type atpases are present at the surface of neutrophil and macrophage for maintaining ph. the sperm maturation and storage also need v-type atpases in epididymis of testis. the cancer cells are also found to manipulate v-type atpases. the v-type atpases are potential target for drug in various diseases. the prerenin receptors' study has indicated the role of renin/angiotensin signaling in v-type atpases' regulation. the v-type atpases are oligomeric protein complex with two different domains, which are known as peripheral (v ) of eight subunits and integral (v ) of six subunits. while peripheral proteins (v ) carry out atp hydrolysis, the v -domain of integral proteins transports proton ions. the peripheral proteins v are designated in bold alphabet like v a, b, c, d, e, f, g, and h, and integral membrane proteins are known as a, b, c, c", d, and v e. the various isoforms of these subunits are found in various tissues and organs. the peripheral proteins are present in stoichiometry concentration of a b c d e f g h - . the catalytic domain of vacuolar atpases contains heterohexamer of alternate a-and b-subunits with the atp binding site at the interface of a-and b-subunits. three subunits of a contribute majorly to three catalytic sites, and b-subunits are regulatory in functions. the remaining peripheral proteins form central and peripheral stalk, which is linked to v -and v domains. c-, e-, g-, and h-subunits form the peripheral stalk. the v -subunits are present in the ratio of a d e n c - c and c (n is variable number of e). the subunits c, c , and c have fig. . transport cycle of p-type atpases. in the e conformation, ion (x + ) binds to its high-affinity site in the membrane (m)-domain through cytoplasmic side. the binding of x + ion initiate phosphorylation of aspartic residue in e state (p-domain by atp). phosphorylated e p state transporter still is in same state. during transition of e to e change in conformation (regulated step), the p-domain rearranged itself for bringing tge loop near to phosphorylation site, which stabilizes phosphoryl group binding and adp dissociates. the a-domain m and m segments movement closes access to cytoplasmic ion access channel (light gray). the p-domain rotation disturbs the high-affinity x + binding site through its attachment to helices m -m (dark gray). x + is delivered to the outside (extracellular/lumenal side) through an exit channel. the ion binding site of e p now has a high affinity for second ion (y + ), which may bind from the outside. the aspartic acid is dephosphorylated leaving enzyme into e conformation. mg + and inorganic phosphate (pi) are dissociated, and the enzyme reverts back to the e state, in which y + is released into the cell. the atpases are ready for new cycle hydrophobic residues in high concentration and have one buried carboxyl group in one of the transmembranes as tm (for c and c ) and tm (for c ) for proton translocation. these buried carboxyl groups may be reversibly protonated during proton translocation. these hydrophobic subunits with single copy of c and c and four copies of c form proteolipid ring. the hydrophilic subunit d at the cytoplasmic side interacts with v subunits (d and f) of the central stalk complex, and this whole complex is referred as rotatory complex (fig. . ). the subunit a of v with eight transmembrane domains has buried arginine amino acid in seventh transmembrane domain, which participates in proton translocation by forming two hemichannels (one toward cytoplasmic side and other in opposite side). the cytoplasmic proton enters through cytoplasmic hemichannel of subunit a, which may protonate buried carboxyl group of v -subunit. the atp hydrolysis by a-subunit induces conformational change in heterohexamer of ab resulting in rotation of central motor with d-subunit as axle of the motor. f is regulatory unit. v d-subunit couples this rotation to ion-transporting proteolipid ring of band csubunits. the b-, c-, and a-subunits form channel for proton transport. the proton moves with rotation of b-and c-subunits. the v-type atpases function through rotatory mechanism, where v -induced atp hydrolysis results in rotation of central rotatory domain with ring of hydrophobic subunits (c, c , and c ) in relation to other subunits, which causes ion transport. this proton remains to interact with carboxyl group because of only polar group in the hydrophobic surroundings of v -subunits. when atp hydrolysis induces conformation change by rotation, the protonated carboxyl group is exposed to luminal hemichannel. these carboxyl groups interact with buried arginine residue of a-subunit and stabilize carboxyl group as carbonate ion, so proton gets disassociated and released into the luminal hemichannel. the continued movement of charged carboxyl group because of changes in conformation by rotation of subunit a places it once again in contact with the cytoplasmic hemichannel, where it is again available for protonation. regulation of v-type atpases can be observed through the following different ways either induced or natural. the v-type atpases may be regulated by ( ) reversible dissociation of v and v -subunit, ( ) by changing or uncoupling of proton transport and atp hydrolysis, ( ) by reversible disulfide bond formation between cysteine and cysteine of catalytic subunit a, ( ) by modulation proton pump density as in apical membrane of renal intercalated cell of kidney proton pump activity is regulated by reversible fusion of the pump with apical cells induced by bicarbonate-sensitive adenylate cyclase. the net ph of a compartment is the result of active proton transport and passive proton leakage. if passive proton ion diffusion is controlled, proton pump automatically will be regulated ( fig. . ). f-type atpases are discussed in detail in chap. . the secondary transport is also known as ion-coupled transport because the electrochemical potential generated free energy of an ion transport which is used by transporters for other substrate transports across the membrane. these transporters ubiquitously found in all cells constitute a major facilitator superfamily (mfs) or solute carrier group of transporters (slc). more than of these transporters are involved in lipid, amino acids, sugar transport, and antibiotics. the important examples may include na + /glucose (sglt ) transporter present in kidney proximal tubule and epithelial cells of intestine. oligonucleotide/proton transporter (facilitated proton transport and uphill peptide transport), proton/neurotransmitter transports in synaptic vesicle membrane of neuron (neurotransmitter transport against concentration gradient by using electrochemical energy generated by proton transfer), lactose permease lacy in bacteria, sodium taurocholate cotransporting peptide (oatp), apical sodium-dependent bile acids (apsbt). the major facilitator superfamily transporters have - transmembrane segments and fig. . v-type atpases may be regulated by a reversible disulfide bond formation between conserved cysteine residues of catalytic a-subunit, b controlling proton ion pump density through fusion with membrane, c regulation of other transporters for cl − or h + , d reversible dissociation of v and v domains and e uncoupling atp hydrolysis and transporting pump subunits. source cipriano et al. ( ) transport substrate by alternating conformations. the bacterial lactose secondary transporters (lacy) and sodium glucose transporters (sglt ) in human intestines are two well-explored transporters to understand secondary transport mechanism, which is discussed here. lactose permease (lacy) transporter of e. coli bacteria has selective affinity for disaccharides containing a d-galactopyranosyl ring, as well as d-galactose and symport d-galactopyranoside and hydrogen ion together in cell (symport). lacy protein is an integral protein ( amino acids) of transmembrane helices with a connecting loop between two pseudo-symmetrical six a-helice ( - and - amino acids) bundles. these a-helices form a central hydrophomation. the other conformation with open cavity toward periplasmic side is called outward-facing conformation. this alternate conformation of transporter (involved helices ii, iv, v in n-terminal and vii, x, xi in c-terminal) exposes bound sugar and proton to cytosol and periplasmic side alternatively; the basic principle of this type secondary transport is the conformation change in transporter by binding with substrate. the sugar-interacting residues are present in n-terminal region and the proton-interacting amino acids are present in c-terminal side ( fig. . ). the mutagenesis study has identified the role e of helix iv, r and w of helix v in substrate recognition and binding and r of helix ix, e and h of helix x in proton translocation. e is required for sugar identification and proton translocation. lacy has shown affinity toward numbers of dgalactopyranosyl disaccharide and with d-galactose also but not to d-glucose. the -hydroxyl group is found to have role in discrimination. the x-ray crystallography of lacy transporter has explained the mechanism. the transport cycle starts with inward open conformation. in the absence of sugar, lactose permease transporter is closed from periplasmic side. the binding of d-galactosyl pyranoside to lacy transporter induces changes in protein to open periplasmic side by closing cytosolic inward open cavity. the deprotonation of e causes closing of inward cavity and opening of periplasmic cavity. the membrane potential has no effect on substrate binding. the five residues r , w , e , n , and h bind the substrate binding and dissociation from transporter promote transition between inwardand outward-facing conformations. the lactose/proton symport has same mechanism in active or facilitative transport for galactose and proton but with the difference in rate limiting steps. the deprotonation step is rate limiting in favorable concentration gradient downhill transports, but in uphill transport, conformation changed induced protonation is rate limiting. the lactose permease (lacy) transfer potential energy, preserved in proton gradient facilitates galactose transport down the concentration gradient. hence, the transport is typically a thermodynamically driven and kinetically regulated secondary transport. there are various ways of glucose transporters as discussed earlier. the sodium/glucose (sglt ) transporter is present in epithelial cells of human intestine and also found on incretin (intestinal hormone, which enhances insulin secretion)-secreting vesicles and also in s and s segments of in kidney proximal renal tubule. sglt has transmembranes ( fig. . ). sglt has specificity for glucose and galactose. in intestine, the sodium/glucose transporters translocate one molecule of glucose per two sodium ions across the epithelial membrane of intestine by using electrochemical gradient of another ion (sodium/potassium). the electrochemical potential is generated by another sodium/potassium pump located in basolateral membrane of these polarized cells of intestine. the another sodium/potassium atpase pump is present in basolateral membrane to generate electrochemical gradient along with an independent glucose transporter, which also participates in translocation of glucose molecule from intestine to blood vessels. the detail mechanism of secondary transport is discussed above. the sodium-potassium atpase pump at basolateral membrane transports two potassiums inside the cell and three sodium ions out in blood in atp-dependent manner. the electrochemical gradient energy generated by this transport is used by sglt transporter in epithelial cell of intestine to import glucose against concentration gradient. when glucose concentration is accumulated in cell, the other facilitator transporter of summary of glucose transport in human intestine and then to blood vessels by secondary transporter sodium/glucose (sglt ). the mechanism is discussed in text glucose transports accumulated glucose from cell to blood (fig. . ). atp-binding cassette (abc) transporters, importers, and exporters, the newly identified integral proteins, comprise a large superfamily of integral membrane proteins with diverse functions. a total of transporters are identified in human. in bacteria % and in archaeabacteria % of the genome codes for abc transporters. they might transport various substrates like amino acids and peptides, monosaccharide, polysaccharides, nucleosides, oligonucleotides, vitamins, coenzymes, and different types of lipid. the abc transporter proteins are classified on the basis of highly conserved approximately amino acid residues with three motifs like walker a/p-loop ( amino acids), a signature motif/c-loop (lsggq), and the walker b motif (five amino acids), which participate in atp hydrolysis. abc exporters are physiologically involved in multidrug resistance and detoxification, while importers play role in nutrient uptake and also are used by some prokaryotic pathogens to show pathogenicity and virulence in plants, prokaryotes, and archaea. the abc exporters are also known as multidrug resistance transporters because of their role in detoxification. the other distinct classes of energy-coupling factor (ecf) transporters are also as class iii abc importers. the abc exporters and importers are further classified into subclasses as type i, ii, and iii. functionally, these all transporters may be divided into two major domains known as nucleotide-binding domain (nbd) and transmembrane domain (tmd). the nbd and tmd can be further divided into nbd and nbd and tmd into tmd and tmd . these domains may be separate, dimer, or fused peptides. in eukaryotes, the abc transporters are mostly multifunctional with single peptide with four domains but the assemble to form dimers or heteromers. in bacterial abc transporters nbd and tmd may be separate, dimer or fused. in some bacteria an extra subunit of - kd is found for binding to substrate and delivery to tmd. the bacterial abc transporters may be c the "half-transporter" with six transmembrane domains and one atp-binding region. the atp-binding camay be on the carboxy-terminal (c) or n-terminal localized in periplasm like gram-negative bacteria or may be membrane bound like gram-positive bacteria ( fig. . ). as mentioned above, the characteristic abc transporters conserved signature motif like walker a (gkt/s for phosphate binding domain), lsggq, and walker b motif in nbd are involved in nucleotide-induced conformation change in nbd region. the highly conserved aspartate/glutamate of the walker b motif forms hydrogen bonds with the threonine/serine of the gkt/s box and with a bound water molecule and facilitates nucleotide hydrolysis (fig. . ). abc transporters transport substrates against a chemical gradient, a process that requires atp hydrolysis as a driving force. under physiological conditions, abc transporters operate in a single direction (either import or export) except drug efflux pump lmra having reversible transport under acidic conditions. the transmembrane domain may face outside or inside by changing conformation. the energy for the conformational change in tmd in inward and out ward face is provided by the binding of substrate and mg + atp, followed by atp hydrolysis and substrate release. abc transporters have similarity to secondary transport in mechanism ( fig. . ) . the abc importers bind to substrate through mediator protein, while exporters directly interact with substrate through their tmd. the nbd forms catalytic domain with walker a or p-loop, walker b, q-loop and h motif (switch region) and conserved sequence lsggq in alpha-helical region. the relative orientation of catalytic sequence to alpha-helical region is decided by atp binding. usually, in active abc transporters, its subunits are arranged in head to tail in fig. . a abc transporters' various domain assemblies. the red half-circles and dashed lines indicate nbd interface, p-loops and conserved signature motifs. in all abc transporters, coupling helices transmit conformational changes between the nbds and the tmds. b in single nbd, various domains like p-loop, a-loop, catalytic glutamate, lsggq signature sequence is shown. c linear subunit presentation with conserved sequence opposite direction to each other. tmd and tmd heterogeneous helices are further divided into three separate fold as type abc transporter, type ii abc transporter, and abc exporters' folds. in outward positions, the tmd helices are in extended wing because of tm and tm from one subunit and tm and tm from other subunit. tmd primarily interacts with variable q-loop with conserved glutamine in a-helical domain. any change in q-loop is directly coupled to atp hydrolysis and conformation change of tmd. walker b-loop participates in binding. magnesium interaction with atp requires d of walker b. abc transporters utilize two atps per one molecule of substrate transport. in addition to substrate binding, two sets of amino acids of d-, q-, and h-loops are required for catalysis. one set acts as general base to activate water molecule to attack on c-phosphate group of atp, and other set of amino acids stabilizes phosphate oxygen. the amino acid e near walker b, q of q-loop and h in h motif essentially contributes to catalysis. atp molecule is bound at interface between tmd and nbd, and terminal c-phosphate is positioned between p-loop and lsggq signature sequence. the most important factor in catalysis is the relative position between nbd and tmd domain because of atp, adp, and substrate binding. the transport cycles move in order of substrate binding, hydrolysis, substrate release, which induces tmd conformation change from close form with bound atp to open form without atp. the change in conformation stimulates substrate translocation also. abc transporters are ubiquitously found in lung, brain, muscle, spleen, endosome, and rod photoreceptors in mammals. these transporters are classified into various classes as abca, b, c, d, e, f. the class abc a transporters of members are basically involved in transport of phospholipids and cholesterol to hdl. in lung, they protect cell surface (table . ). these are multidrug transporter and efflux n retinyl-diester-phosphatidyl ethanol amine. the abc b class of members are found in kidney, brain, er, liver, and mitochondria. these are involved in multidrug resistance. they are involved in peptide transport to er. the c class of members, present in all tissues, is involved in drug resistance, organic anion, nucleoside, chloride ion transport. abc d of four members are identified in peroxisomes. abc e class has only one member. the abc f is also present in all tissues. the abc g of eight members regulates transport of cholesterol and other sterols. the plasma membrane has to maintain lipid asymmetry for biological functions and requires constant and regulated transport of membrane components for the purpose. the nonpolar molecules can translocate easily from inner to outer in other direction but lipids with polar group are transported by transporters in atp-dependent manner. there are three major transporters which are known as flippases, floppases, and scramblases. the flippases are also known as p atpases, which transport phospholipids to inner leaflet of membrane and have substrate specificity to phosphatidyl serine and phosphatidyl choline. the flippases' transporters may cause change in shape of membrane like bending of the membrane for vesicle formation or vesicle budding. in erythrocyte, amino phospholipid flippases are identified for ps and pc transportation to inner membrane. atp b flippase is a phosphatidylserine translocase in human. the other enzyme, floppases, belongs to abc transporters' large family, which transport polar lipids to outer membrane in energy-dependent mode for transport. p-glycoprotein floppase is a multidrug resistance protein in human. the scramblases can transfer lipid in both the directions without requiring energy. these are activated by calcium during membrane injury, apoptosis, or coagulation and transport ps and pe to outer leaflet for destroying lipid symmetry. phospholipid scramblases (plscrs) are palmitoylated proteins of lipid raft, which is under regulation by phosphorylation and water is the main component of life, and its exchange is characteristic of any living being. first water channel proteins, known as aquaporins were first isolated from rbc and later from renal proximal tubule membrane peter agre group in . aquaporins (aqps), the integral proteins of membrane, are small, hydrophobic, and homotetramer proteins, which are involved in bidirectional transport. these proteins are present in all living beings. the aquaporins may facilitate the transport of urea, nitrate, no, hydrogen peroxide, carbon dioxide (co ), and ammonia (nh ) along with water. numerous identified isoforms are differentially expressed and modified by posttranslational processes for tissue-specific osmoregulation in various organisms. in mammal, aquaporins are involved in multiple physiological processes including urine concentration by kidney tubules, nerve transmission, metabolism of lipids, fluid secretion, tissue swelling, cell migration, and salivary gland secretion along with skin hydration. the genetic defects in aquaporins may lead to disorders like kidney dysfunction, loss of vision, and brain edema. according to their substrate specificity and localization, the aquaporins are divided into two subgroups: . classical aquaporins are exclusively water channels. . aquaglyceroporins are the proteins with broader specificity for nonpolar molecules like polyols, glycerol, ammonia, co , no, urea along with water and may participate in nutrient uptake and osmoregulation but bacterial glycerol facilitator glpf only allows the movement of polyols. the unusual aquaporin acts as anion channel. the aquaporins are mostly found in plasma membrane but may be localized inside the cell organelle membrane. the movement of water and other solutes through aquaglyceroporins (aqps) supports bidirectional downhill movement through pore or channel formed by aquaporins. the number of genes identified for coding aquaporins in various organisms is variable with one or two genes in bacteria and yeasts, around in mammals ( are for renal tubules to concentrate urine) and more than genes in plants. aquaporins are highly selective and efficient for the transport of water and glycerol but do not allow hydroxide, hydronium ion, and proton movement. this selective transport protects the membrane electrochemical gradient (figs. . and . ). the active form of aqps are homo-tetramers. each monomer of - kd contains amino acids with six transmembrane helices (tm - ) and five connecting loops (a-e). the primary structure of aquaporin oligomer is made of symmetrical two halves, hemipore- (transmembrane - ) and hemipore- (transmembrane - ), as shown in fig. . . the transmembranes are arranged into two halves of , , and , , , connected by loops to form channel. the four monomers together form a central pore or channel for ions and gases to move but water is not allowed through this central channel. the water diffuses through individual monomers of aquaporins. the three connecting loops a, c, e are externally located, while b and d face cytosolic side. the two loops, one from each side b and e, have signature motif of aquaporin family (major intrinsic family) asparagine, proline, and alanine (npa). there are two conserved sequences of npa in aquaporin channel with two asparagine side chains toward pore at the end of hemipore. all transmembranes have their n-terminal and c-terminal in cytoplasm. the b-loop entering from extracellular side and e from other cytosolic side create seventh alpha-helical domain to form a part of surface for water channel. if water is moving from outside to inside, it has to interact with side chain carboxylic group of amino acid at the surface of aquaporin. the presence of arginine and aromatic amino acids at the narrowest part of the pore is the final selection for any molecule to enter. the overlapping of asparagine, proline, and alanine (npa) in the center of pore reduces the size of pore for movement, which is important for water molecule interaction for selectivity. the water-specific aquaporin is observed with pore size of . Å, so bigger molecule or proton will not pass. after interaction with surface protein side carboxylic group, the water molecule will be selected by polar or nonpolar amino acid residues of pore wall proteins, which will provide channel specificity of molecule and will determine the rate of transport. figure . shows the various important amino acids found at pore site. the b and e short helices from opposite side of the pore with positive-charged amino acids with their side group projecting toward center play an important at the constricted site of npa with conserved aromatic and arginine amino acids, water ionic bonds become weak. the aromatic amino acids may vary in various aquaporins. in bovine aquaporin (aqp ), phenylalanine and arginine form constriction site. histidine at surface of pore provides ionic interaction to water molecule, while phenylalanine repels water. after clearing aro/r constriction site, the water molecule interacts with two conserved npa sequences and its neighboring amino acids, where protruding valine pulls water molecule to interact with asparagine and asparagine in npa segment. in the pore, the electrostatic interaction of the npa repeats and the aromatic/arginine (ar/r) constriction results in the exclusion of protons. these amino acids' segment of npa is strong lipid anchor for changing water orientation, which is also supported by strong dipole of b and e helices to change water dipole in opposite side to previous one. the npa amino acids are preceeded and followed by hydrophobic residues (isoleucine and phenyl alanine before and glycine , bovine aquaporin (aqp ) detail structure of pore region with conserved npa provides interaction mechanism of channel with water. half helices dipoles and the hydrophilic and hydrophobic residues lining the pore are shown in red and yellow, respectively; the aromatic/r and npa selective filters are shown as well alanine and valine after npa sequence). these hydrophobic residues stop water interaction with other amino acids of wall proteins for faster movement. tyrosine of d-loop is found responsible for closing of channel because of its insertion into npa site. the phosphorylation at serine of aquaporin induces change in conformation, which may open aquaporin channels, because serine is present in b-loop, which is directly connected to tyrosine . these channels are very fast to have permeability of water  mol/s. the aquaporin channels may be regulated by concentration gradient of water, phosphorylation and dephosphorylation, temperature, and ph. this has been observed in plants that decrease in ph may close the channel because of histidine protonation in d-loop. the mercury chloride binding to cysteine at npa site also closes aquaporins channel. vasopressin in kidney may activate channels through camp-induced phosphorylation. the genetic defect in human aquaporins aqp leads to diabetes insipidus. ca + is found to regulate translocation of aqp to the plasma membrane. aqp overexpression because of heart failure leads to water retention. aqps role in homeostasis of the cerebrospinal fluid (csf) and neuron excitability is well-established ( localized specifically to organelle membrane of endosome phosphorylated during transport, which makes sugar negatively charged. the phosphorylation of sugar does not change intracellular concentration gradient and also check sugar leakage from cell. the seven families of phosphotransferases have been identified in various groups of bacteria with mostly conserved sequences and dissimilarity in substrate specificity. during this transport, the sugar is chemically modified across the membrane and that's why this transport is also called as group translocation transport. the phosphortransferases complex has cytoplasmic, peripheral, and integral proteins. the cytoplasmic proteins are constitutive and nonspecific, which are involved in group translocation of various other proteins also. the cytoplasmic proteins comprise of heat-resistant histidine protein hpr and enzyme e . e protein has histidine at n-terminal, which participates in reversible phosphorylation of hpr, and its c-terminal domain is required for protein autophosphorylation by pep. hpr is small monomeric protein, which is phosphorylated at n-terminal histidine by e*p. the cytoplasmic pts proteins or membrane-associated hydrophilic pts domains undergo transient phosphorylation during transport of sugars. the integral membrane proteins are known as enzyme ii (eii), which participate in substrate translocation and its phosphorylation. the eii enzymes are inducible and substrate-specific proteins, which include eiia, cytoplasmic protein, a peripheral protein associated with inner membrane eiib, and carrier integral protein eiic. eiia and eiib are sugar-specific proteins. eiia for glucose is phosphorylated at histidine . the eii component may be separate or fused in different organisms. eiia is conserved; eiib is hydrophobic which is phosphorylated at conserved cysteine residue by eiia. eiic has - transmembrane regions with one conserved gxxe motif in hydrophobic loop. protein eiic has three periplasmic ( , , loops) and two cytoplasmic ( and % loops). in bacillus subtilis, oligo-b-glucoside-specific pts has three separate enzymes, while in the same organism, mannitol pts has eiib fused with eiic. the reaction cycle starts with transfer of phosphate group from pep to a histidine residue on enzyme i. the enzyme ei transfers this phosphate to hpr protein on histidine . in the next step, this phosphate group is transferred from hpr to eiia protein again on histidine residue. the specific enzyme eiia for sugar transfers phosphate to the cysteine residue of enzyme iib. finally, enzyme eiib transfers phosphate group to eiic bound sugar, which is phosphorylated and translocated to cytoplasm (fig. . ) . the majority of sugars are transported in phosphorylated form but fucosyl-a- , -n-acetylglucosamine in lactobacillus casei is translocated in unphosphorylated form exceptionally. some sugars are immediately dephosphorylated after translocation in cytoplasm like mannose in enterobacter faecalis. the glucose uptake is enhanced by the availability of nitrogen in bacteria through these phosphotransferases. the various organisms have specialized proteins of rhodopsin family to seize light energy for various physiological functions. the rhodopsin protein part, opsin, is covalently linked to retinal chromophore, which may be in trans-or cisconfiguration. the animal rhodopsin (also known as type ii) are cell g-coupled receptors, while microbial rhodopsin (type i) may act as ion pump, ion channel, sensor, photosensory receptor, regulator for gene expression or a kinase. halobacterium salinarum has normal respiration in the presence of o and high nutrients, but in the absence of nutrients, it survives by using light energy, which is identified with purple patch on the bacterial membrane because of retinal base. the three microbial pumps are well-characterized in their structure and function known as the bacterio rhodopsin h + proton pump, channel rhodopsin for chloride ion. the halobacterium salinarum has sensory rhodopsin i and sensory rhodopsin ii, which activate transducers for senses ( fig. . ) . the animal and microbial rhodopsins are transmembrane of a-helices, covalently linked to retinal through lysine residue and form protonated schiff base with it. the retinal chromophore may be in cis-or transconfiguration. the ground state of microbial and animal rhodopsins has all trans-and -cis-retinal configuration ( fig. . ) . thermodynamically, retinal is preferred in all transconfigurations in nonpolar environment with absorption maxima at nm wavelength but the protein interaction with retinal shifts this absorption maxima to broader range in visible light range from to nm. the light absorption by retinal stimulates isomerization from trans-cis to cis-trans in less than a fraction of second. the energy released by this transition isomerization induces change in opsin protein conformation for various activities. the sequences of animal and microbial proteins are quite dissimilar in spite of both having seven salinarum contains four rhodopsins, bacteriorhodopsin (br), halorhodopsin (hr), sensory rhodopsin i (sri), and sensory rhodopsin ii (srii, also called phoborhodopsin, pr). br and hr work as a light-driven transmembranes. bacteriorhodopsin translocates one proton inside to outside in cyclic reaction and generates membrane potential across the membrane for atp synthesis. the channel rhodopsin of h. salirium transports chloride ion from outside to inside of cell for atp synthesis and for osmotic balance. the sensory rhodopsin i (sri) acts as stimulator of positive phototaxis, while srii works oppositely. the bacterial movement toward particular wavelength light is positive phototaxis and moving away is negative. sri ( nm k max ) and srii ( k max ) perform through their transducers and cotransducers. the cotransducers are named as htri for sri and htrii for (srii). sr proteins with their cotransducers are localized in cell membrane in : ratio to form tetramer. the signal induced by retinal trans-cis isomerization by light is propagated to cytoplasmic transducers chea and chey resulting in phosphorylation and dephosphorylation for flagellar movement in desired direction ( fig. . ) . the crystallographic studies and electron density of catalytic side have explained the photocycle of ion transport. the seven transmembranes of opsin protein may be designated as a b c d e f g, which are arranged to cover centrally linked protonated retinal schiff base to g-helix by lysine . basically, the light absorption by retinal redistributes its own electrons in protonated base resulting in isomerization, proton transfer between various amino acids at catalytic side, water relocalization, and finally change in protein conformation for physiological function. the water molecule at position in catalytic site accepts proton from retinal and fig. . synthesis of retinal chromophore in microorganism and animal from b carotene. the ground state of microbial and animal rhodopsins possesses all-trans-and -cis-retinal as its chromophore, respectively, bound to a lys residue via a schiff base, which is normally protonated and exists in the -anti configuration. it should be noted that microbial rhodopsins depend exclusively on all trans-retinal, while some animal rhodopsins possess vitamin a (c =c double bond for fish visual pigments) and hydroxyl (c -oh for insect visual pigments) forms of -cis-retinal. usually, photoactivation isomerizes microbial rhodopsin selectively at the c =c double bond and animal rhodopsin at the c =c double bond. post-content transports it to aspartic acid and asp . the protonation of asp and retinal deprotonation induce proton movement to outside of membrane, and proton is released. cytoplasmic proton migration protonates retinal schiff base to its original position to complete light-induced cycle. the conserved residues involved in proton transfer are shown in figs. . and . . at the ground state of stable active site, the positively charged retinal schiff base interaction with three water molecules , , and aspartic acid (d) , d stabilize separate noninduced light changes fat active site. the h-bond interaction of aspartic acid d with threonine t asp d , tyrosine y , y , and water molecule keeps asp d in anionic form. the aspartic acid d and water also interact with positively charged arginine r . the transition of retinal base from trans to cis by light will dramatically destabilize this fig. . bacteriorhodopsin molecule is purple and is most efficient at absorbing green light (wavelength - nm, with the absorption maximum at nm). bacteriorhodopsin has a broad excitation spectrum fig. . a structure of bacteriorhodopsin (br), with conserved aromatic residues highlighted (pdb id: qm ). tyr , trp , and trp are strongly conserved among microbial rhodopsins (orange). aromatic amino acids are strongly conserved at the position of tyr , trp , and phe (yellow). in br, trp , trp , tyr , and trp constitute the chromophore binding pocket for all transretinal configuration (gray). b crystallographically observed internal water molecules of br (shown as green spheres) ground state. the hydrogen bond between these amino acids and water will be disturbed and rearranged, which will cause to disassociation of proton from schiff base. the asp d is first proton acceptor which is centrally located. water actively participate in hydrogen bond rearrangement because of its position its h + and oh − easily may protonate and deprotonate neighboring residues. the extracellular side of retinal linked to arginine r interacts with three-dimensional hydrogen bond mesh contributed by water molecule , , , and and amino acids. the retinal molecule is linearly connected to arginine through h bonds starting from water -d -water, -arg r , and other water -d -w -water -arg r nh /nh . the whole mesh is interconnected. the proton release complex with water , , , glu e , glu e is separated by isoleucine i and leul from hydrophilic environment. at the c-terminal of d helices transmembrane, a unpaired buried arginine r interacts with salt bridge to glutamate e and interacts with carboxylic group of amino acids at , , and positions. the transmembrane g at cytoplasmic side gets kink because of p bulging at alanine a , which results in peptide bending between alanine a and lysine k . this bending stimulates local rearrangement of h bonding between water and amino acids ( fig. . ) . the new hydrogen bonding between molecules and new local arrangements around g transmembrane helices slightly tilt this from canter, resulting in c-terminal displacement to outer side, and induce change in conformation. the water molecule at stabilizes this change through interactions with amino acids and carboxylic group of retinal schiff base. the rearrangement of g helices and water molecule amino acids interactions decrease pka of aspartic acid d , which donates proton in photocycle and control reprotonation of aspartic by isomerization of retinal schiff base from cis to all trans ( fig. . ). pore-forming toxins (pfts), the bacterial virulence factor, single major family of proteins, are secreted by gram-positive and gram-negative bacteria. pfts may be present either in in bacteriorhodopsin helices g at cytoplasmic side, a kink between alanine a and lysk is produced because of alanine p-bulging. the local hydrogen bonds between water and amino acids are rearranged to release proton cytoplasm or in the membrane. the conformational change in cytoplasmic pfts may translocate them to membrane in need of hour. the numbers of bacteria like staphylococcus aureus, e. faecalis, e. coli, and vibrio cholera had shown the presence of these proteins during pathogenesis. the size of pore may be small ( . - nm) or large (more than nm). mostly, pfts are water-soluble. binding of pore-forming toxin to host cell receptor through sugar, lipids, or protein leads to change in conformation for pore formation. the strength of pore formation is directly proportional to their virulence power. as per transmembrane structure of pfts in host cell membrane, the pfts are divided into two groups as a-pfts because of a-helical pore and b-pfts because of b-barrel pore (fig. . ) . the various molecules in the host membrane may act as receptor molecules like glycosyl phosphatidyl inositol (gpi), chemokine receptor (ccr or cd ) of white blood cells, disintegrin and metallo-protease (adam)-type proteins, cholesterol, and other lipids. pore-forming toxins may change their conformation to adjust in host environment. the pore formed by pfts may be continuous barrel layer of proteins or may be in toroidal form because of lipid protein interaction. the major pathogens producing different pfts proteins are shown in table . . the psts act first by binding to receptors, which are followed by oligomerization, change in conformation, and insertion into host membrane to form pore. till date, six families of pore-forming toxins, including three a-pfts and three b-pfts, are known. a-pore-forming toxins are heterogeneous group with various pfts like cytolysin of e. coli, exotoxin of pseudomonas aeruginosa, and diphtheria toxin from corynebacterium diphtheria. the various pfts may form various types of pores with different characteristics. the e. coli produced a-pfts colicins family produces pore in other bacterial inner membranes. the isolated colicin a and e pft, according to their function, may be divided into three domains for receptor binding, translocation, and pore formation. the pore-forming domain of colicin has ten alpha-helices. two of these hydrophobic helices are surrounded by eight amphipathic alpha-helices. the hydrophobic a-helices are inserted into inner membrane to make nonspecific voltage-gated ion channel. this pore destabilizes membrane potential of host cell. the other apft such as insecticidal cry toxin from bacillus thuringiensis, diphtheria toxin translocation domain, and eukaryotic protein bak, has shown similar structure like colicin for pore formation. the colicin pft of this group forms the pore by binding to receptor, translocates to periplasm, and causes cell death by forming pore in inner membrane. bcl- completes pore formation in mitochondria by releasing cytochrome c to activate caspases for apoptosis. a member of apft cytolysin family, cytolysin a(cly), hemolysin e(nhe) enterotoxin of salmonella enterica and shigella flexneri and e. coli forms pore of - nm diameter for cation in macrophage leading to apoptosis. cytolysin a is found to be a-helical protein in elongated shape with short hydrophobic b-strands in tongue shape (btongue). during formation after assembly, the b-strands become disassociated from the protein for insertion into lipid bilayer; this event causes the rearrangement of n-terminal region of amphipathic helices at the pore site. the n-terminal domain arrangement may vary in length but this variation cause is not clear till now. the conformational change by the n-terminal amphipathic alpha-helices catalyzes a very organized step-by-step oligomerization of monomers. there are various ways of protein-protein or lipid-protein interaction for pore formation as shown in fig. . . when toxin protein interacts with host lipid for pore formation, lipid may provide a curved nonbilayer shape at pore periphery. in this pore, the protein regulates stress caused by distortion of membrane. as seen in eukaryotic b-barrel actinoporin pft from sea anemones, in which cation selective pore is formed by interaction with sphingomyelin of host membrane. usually, the toxins recognize and interact with specific lipid domain of sphingomyelin clustering or cholesterol-sphingomyelin complexes, which support the accumulation of pfts locally to ease their function of pore formation. this may result in lamellar structure destabilization by lipid transmovement across the membrane (fig. . ) . the b-pore-forming toxins include aerolysin and cholesterol-dependent cytolysin family (cdc), and these are secreted majorly by gram-positive bacteria and minor by gram-negative bacteria. gram-negative aeromonas saps produces aerolysin as protoxin. the c-terminal cleavage converts protoxin into toxin protein, which is elongated multidomain protein. the aerolysin protein may be divided into four domains; the domains , , and are together, and domain is separated. the domain three has multiple short a helices with b-sheet. the domain four ( ) interacts with cholesterol for binding by inserting several loops. the domain four ( ) has conserved domain of tryptophan residues with three short hydrophobic regions known as l , l , and l . the domain has two transmembrane a-helices in soluble form, which are converted to b-hairpin transmembrane during pore formation. while a-pfts start pore formation by the insertion of hydrophobic helices, the b-barrel formation occurs in concerted mode (fig. . ) . . actinoporin and colicin family pfts pore formation by monomer alpha-helical hydrophobic segment extraction and insertion into host membrane, umbrella formation, oligomerisation either into two or more peptides as mentioned above, the other major family of b-pore-forming toxins is cholesterol-dependent cytolysins (cdc), which include perfringolysin o (pfo) of c. perfringens, streptolysin o (slo) of streptococcus pyogenes, pneumolysin of s. pneumoniae, and listeriolysin o (llo) of listeria monocytogenes belong to cholesteroldependent cytolysin family. these toxins are produced mostly by gram-positive bacteria and by few gram-negative bacteria. the pore formation is dependent on cholesterol for pore formation. in cholesterol-dependent cytolysin toxin, pore may contain from to proteins subunits, and each subunit donates b-hairpin for pore unlike other b-pfts. cytolysins are produced as monomer or dimer and have some similar features like eukaryotic membrane attack complex (mac). cytolysin also has four domains like aerolysin in elongated structure and makes well-defined prepore in the membrane. the cdc, unlike some other toxins, which rearranges their structure for pore formation, goes under transition from alpha-helices to their b hair pin secondary structure at membrane surface (prepore). the transmembrane interacts with lipid cholesterol and forms arc-like structure to create gap (semi pore) in the membrane. at this stage, oligomerization cytolysin monomers occur. the oligomerization transforms the surrounding arrangements of molecules to form functional pore. the conserved tryptophan in toxin and cholesterol play an important role in pore formation (fig. . ) . a-hemolysin (hla) of cholesterol-dependent cytolysin (cdc) family anchors through n-linked glycan proteins of hosts. aerolysin binds through glycosylphosphatidylinositol (gpi) anchors. colicins f a-pft family interact with lipopolysaccharides (lps) in the outer fig. . depiction of pore formation pathway of pore-forming toxins (pfts). soluble pfts are recruited to the host membrane by protein receptors and/or specific interactions with lipids (e.g., sphingomyelin for actinoporins or sterols for cholesterol-dependent cytolysins (cdcs)). upon membrane binding, the toxins concentrate and start the oligomerization process, which usually follows one of two pathways. in the pathway followed by most b-pfts, oligomerization occurs at the membrane surface, producing an intermediate structure known as a prepore (mechanism ), which eventually undergoes conformational rearrangements that lead to concerted membrane insertion. in the pathway followed by most a-pfts, pft insertion into the membrane occurs concomitantly with a sequential oligomerization mechanism, which can lead to the formation of either a partially formed, but active, pore (mechanism ) bacterial membrane. a-pft fragaceatoxin c (frac) binds to sphingomyelin clustering. the eukaryotic cell also has pore-forming toxins as the mechanism of their defense, which is known as membrane attack complex (mac) or performin toxins. the t cell secretes them to destroy pathogen. these toxins like cdc have two transmembrane domains. the hydrolysin from cnidaria (phylum of aquatic invertebrate animals), enterolobin toxin from enterolobium contortisiliquum, and human c are the examples of eukaryotic toxins. the various defense mechanisms against these virulent pore-forming toxins exit in host like human. the binding of toxin to cell membrane may also activate p mitogen-activated phosphokinase (mapk), c-jun n-terminal kinase like mapk-kgb pathway. the detail of receptor signaling is discussed in chap. . pfts pore formation may result sometimes in increase in extracellular potassium as a defense mechanism. the potassium efflux activates nucleotide-binding oligomerization domain (nod)-like receptors (nlrs), which have variable n-terminal domains such as caspase recruitment domain (card) and pyrin domain (pyd), which activate apoptosis and inflammation pathways. these receptors are found on immune cells like lymphocytes, macrophages, dendritic cells, and also in nonimmune cells and play a role in innate immunity, phagocytosis, and killing of pathogens. the caspase induces sterol regulatory element binding protein (srebps), the regulator of membrane lipid synthesis. the extracellular potassium increases in response to toxin interaction and also activates caspase for inducing apoptosis. the activation of p and extracellular response-activated kinase (erk) brings back potassium level to normal. a-defensin proteins are also detected in host to act against pfts (fig. . ) . calcium efflux in response to pfts activates the release of lysosomal enzymes such as sphingomyelinase and hydrolase to degrade pathogenic molecules endocytosis is another defense used by host cell. pfts also interact with cellular signaling, and molecules such as arthritis were caused by c-hemolysin in human. the hemolysin induced the expression of interleukin in osteoclasts. the downregulation of interleukin and nitric oxide synthetase is observed in macrophage by cytolysin toxin. ionophores are natural and synthetic organic molecules of diverse type, which can transport cations by forming lipid ion-soluble complex. each ionophore possesses a unique property of changing transmembrane ion gradient and electrical potential, synthesized by bacteria, and acts as protector against competing microbes. they disrupt membrane's ionic gradient required for proper transport; therefore, they have got antibiotic properties. ionophores increase the permeability of biological membranes to certain ions and antibiotics; that's why they are used as growth enhancers as additives in certain animal feed. the various ionophore antibiotics are macrolides. the two classes of ionophores are identified as carriers and channels. the carriers bind to specific ion, shield it from hydrophobic environment, leave the ion on other side of membrane by diffusing across the membrane, and come back without ion to original place. the carrier polyether ionophores include approximately more than types having antibiotic properties against bacteria, fungi, and virus. some of them are found to have antitumor activity also. the antibiotic property of these polyether ionophores structure specifies their cation metal binding and is directly correlated to their antibiotic property. the carrier ionophores are not found to be effective in gram-negative bacteria because of their cell wall but the opposite is true for gram-positive bacteria. the gram-positive bacterial membrane is permeable for these small molecules. because of this reason, e. coli is found to be important for food and human health. the carrier ionophore examples include valinomycin, calcimycin, salinomycin, monensin. valinomycin is produced by various strains of streptomyces species as s. tsusimaensis and s. fulvissimus. the valinomycin is highly selective for potassium and rubidium (rb + ) ion and does not allow sodium or lithium transport ( fig. . ) . the binding and affinity constant for the potassium-valinomycin complex are ten times higher than sodium. valinomycin was observed to downregulate the lymphocytes multiplication induced by phytohemagglutinins in human. valinomycin-metal ion interaction essentially requires dehydrating form of ions, and complex is formed in energy-dependent process. the amount of energy required is the deciding factor for forming valinomycin-potassium and rubidium complex but not with sodium (table . ). as shown in table . , because of unequal distribution of ions across the membrane, the electrical potential across the transmembrane is generated, which initiates binding of metal ion to ionophore for diffusion. the movement of ions may decrease or balance electrical potential of membrane. the ionophores may reduce membrane electrical potential through ions movement, which is the basis of their antibiotic property. these metal ion ionophores act as uncouplers like the cell's own uncouplers in mitochondrial respiration. the synthesis of atp becomes disassociated from electron transport chain. valinomycin acting as antibiotic may kill the cell by disturbing electrical gradient in transport and energy-generating process. in s. aureus, listeria innocua, l. monocytogenes, b. subtilis, and bacillus cereus, gram-positive bacteria the valinomycin activity is ph dependent. recently, the valinomycin is reported to inhibit severe acute respiratory-syndrome corona virus survival at . - lm (table . ). the structural study of carrier ionophore by x-ray has revealed the binding site of ions calcium-magnesium, calcium- hydrogen hydrogen > cesium (cs + ) % rubidium (rb) > potassium (k + ) > sodium (na + ) > lithium (li + ) nystatin monovalent cation and anion through oxygen atoms in the center of ionophore. ionophores are neutral complexes with monovalent cations (monensin, salinomycin) and with divalent metal cations (lasalocid, calcimycin). the carboxylic group is deprotonated at physiological ph, so more of neutral complexes are found in the cell. the pseudocyclic structure formed by cation may be stabilized by intramolecular hydrogen bonds of carboxylic group and hydroxyl groups. the hydrophobic residues of ionophore encircle the ionic interactions for the protection. the polyether ionophore anion (i-coo − ) binds the metal (m + ) cation or proton (h + ) and forms either (i-coo − m + ) or (i-cooh) depending on ph. this interaction changes na + /k + atpases activity to change in gradient of the cell. resultantly, this may cause the death of the bacteria cell because of increase in osmatic pressure, swelling and bursting of cell by disturbed atpases. the coordinated geometry between ionophores and ions is important for its function. the other examples of carrier ionophore, isolated from streptomyces chartreuses, are calcimycin (a ) and ionomycin calcium ionophore. ionomycin hydrolyzes phosphatidyl inositides in the t-cell membrane to activate phosphokinase c (pkc) through autophosphorylation by phosphorylating t helper cell (cd ) and t suppressor (cd ) cells. the hemisodium, synthetic ionophore, transports sodium ions. the calcium ionophore calcimycin, oxidative phosphorylation, and electron transport chain uncoupler are often used for enhancing thyroid and insulin secretion. calcimycin, because of fluorescent property, may be used as biomarker in membranes for transport study (fig. . ) . calcium ionophores promote three types of signal in calcium-resistant cell for cytosolic calcium increase: ( ) activate receptor-mediated calcium transport into cytosol by native ca + channels, ( ) phospholipase c-dependent calcium mobilization from intracellular stores like er, ( ) at low concentration of ca + , ionomycin in endothelial cell, like histamine, increases cytosolic calcium, while in fibroblast, only intracellular calcium from organelle store releases out. beauvericin ionophore has both antibiotic and insecticidal property. this has phenylalanine and hydroxyl-iso-valine amino acids alternatively to form cyclic hexodepsipeptide. it transports alkaline and alkali metals across the cell membrane. beauvericin, found in beauveria bassiana, wheat, barley, and corn, causes apoptosis in mammals additionally. the other class of enniatins ionophores are found in fusarium fungi. usually, proteins have the characteristic of forming channels in the membrane for transport of various molecules, but some of the ionophore also transport by forming a channel in the membrane. these ionophores have a few unusual amino acids normally not found in proteins. gramicidin, isolated from bacillus brevis, has amino acids, linearly arranged in d and l alternate fashion with amino-terminal formaldehyde and carboxyl terminal ethanol amine. the hydrophobic amino acids in gramicidin support its function in channel formation. these channels have outer lining of hydrophobic amino acids to interact with lipid bilayer of membrane with hydrophilic interior for ion interaction. these channels allow very fast diffusion of ions through them in very short period unlike carrier ions. their size is larger than fig. . crystal structure of : complex of calcimycin with the magnesium cation. https://www.hindawi.com/ journals/bmri/ / /fig / protein channel. gramicidin channels allow protons and other cation movements but not of divalent cations like ca , which block them. gramicidin has antibiotic property against gram-negative bacteria, gram-positive bacteria, and pathogenic fungi but sometimes its hemolytic activity may limit its use. in various solvents, gramicidin has stable amphiphilic antiparallel b-sheet structure with a polar and a nonpolar surface as a-helix is not possible because of alternate d-and l-amino acid residues. gramicidin has . residues per turn in b-sheet structure with channel diameter approximately . nm. the ethanol residue is projected on the surface and formyl group inserted in membrane. the nonpolar amino acids play a role in microbial specificity, and disrupted beta-sheet structure adds to its antimicrobial activity ( fig. . ) . the natural mixture of gramicidin, known as gramicidin a, is observed fluorescent because of tryptophan amino acid. its antimicrobial activity may be through gene regulation as e. coli rna polymerase was found to be inhibited by gramicidin ( fig. . ) . gramicidin is not synthesized by ribosomes but by cellular enzymes, and at position, it has tryptophan which is replaced by phenylalanine in gramicidin b and tyrosine in gramicidin c, respectively. the interior of the channel is formed by the polar amino acids as discussed above. gramicidin has affinity for ions in the following order as maximum for cesium and minimum for lithium cs + > rb + > k + > na + > li + . gramicidin is also used to stimulate the production of cytokines interferon. the property of ionophore to induce apoptosis in cancer cells is being used for drug targeting especially sodium and calcium, because ionophore has capacity to disrupt lysosomal function and can inhibit proteasome function for apoptosis ( fig. . ). the bacterial and eukaryotic membranes are found to have special proteins in their outer membrane for transport, known as porins which form channels for various small size solutes, ions, and other nutrients. the porins found in mitochondria are also known as voltage-dependent anion channel (vdac). the mitochondrial conserved porin proteins are tissue specific, but in bacteria, these porins constitute a diverse group because of bacterial variable environment. the porins are made of , , , or b-barrel strands in slightly tilted form. the porins may be monomer but mostly are trimers. based on their structure and function, porins are broadly classified as -stranded nonspecific b-barrel such as omp , oprp, and phoe, -stranded specific b-barrel (lamb from e. coli), b-strands and monomeric such as ompg and multidomain like cyma from bacteria. t. thermophilus which is p porin protein with n-terminal peptidoglycan binding domain and a coiled coil domain with large pore size. nmr and crystallographic studies have revealed mitochondrial vdac pore size of . nm height and nm diameter. the vdac has b-strands with n-terminal helix buried in pore. the sugar-specific porins (fig. . ) may allow solute up to kd. the bacterial porins are oval in shape with pore size - . and nm height. the porin first and last b-strands are joined in antiparallel mode to form complete pore. these b-strands are connected to each other from cytoplasmic side by - long loops and - small turns from periplasmic side. the loop from the cytosolic side in all porins is folded in side in barrel as shown in fig. . and regulates pore size. usually, loop is found between and color -blue (n terminus) , red (c terminus) with approximate molecular dimensions. the first and last b-strands (b and b ) are paired in a parallel way (yellow arrows). the upper view highlights n-terminal a-helix localized inside the barrel strands of barrel. this has mostly polar amino acids aspartic acid and glutamic acids. the l , l , and l interact with each other monomer to form trimer. l , l , and l are present on the surface of porin. the loop l being folded into interior contributes in channel opening toward extracellular side (fig. . ) . the aromatic amino acids like tyrosine and phenylalanine are predominantly present in inner side and outer side of pore. the tyrosine is more toward outer side and phenylalanine to cytosolic side. all porins are found to have phenylalanine at their c-terminal which is required for proper folding and import. the alge porin from p. aeruginosa ( b-strands) has positive amino acids like arginine inside the pore to translocate negatively charged alginate polymer. the mitochondrial porins have three isoforms and are of round shape. the first and last b-strands of strands pair parallel teach other unlike bacterial porins. all strands are connected through short loops, little elongated to cytoplasmic side ( - number) (fig. . ) . the mitochondrial porins are imported by tom protein and inserted by sorting and assembly machinery (sam complex) bacterial porins are inserted by b-barrel assembly machinery (bam) complex. in -stranded b-barrel porins of nonspecific class, the transport depends on molecular mass and rate of transport is directly proportional to concentration gradient. the specific b-barrel porins like lamb maltoporin of e. coli have specific binding site for substrate inside channel and shows michaelis-menten kinetics of transport. many prokaryotic porins are closed at high voltage of - mv. vdac also has voltage dependence and can be easily gated by change in ph, and their permeability is controlled by membrane potential. they are involved in transport of atp and adp; that's why a large number of positively charged amino acids are present in interior wall of pore. the protein ion channels are passive transporters, are made up of transmembrane, are selective for their substrate ions, and have much faster rate of transport than pumps. these channel proteins do not interact with transporting ions and have two conformations closed and open, to regulate flow of ions. the channels may be classified into ligand-gated channel and voltage-gated channel. ligand-gated channel opening and closing depends on the interaction with positive or negative ligand. the binding of ligand induces conformational change in protein for opening or closing. on structural basis, ligand channel (lgic) is classified into three classes. (nicotinic ach) superfamily, which has pentamer cysteine loop such as nicotine acetyl receptor hydroxyl tryptamine, zn-activated channel cation transport, gama-aminobutyrate (gaba a ), strychnine-sensitive glycine receptors for anions, glutamate-gated chloride channel of c. elegans. which has four subunits divided into three subclasses like n-methyl d-aspartate (ndma) receptor, a-amino- -hydroxy- -methyl- isoxazolepropionic acid (ampa) and kainate receptors. . p x receptor family, which has three subunits of cysteine loop. these are cation permeable-gated channels and opened or closed by atp interaction (fig. . ). the trimeric atp-activated channels are permeable to cations like sodium, potassium, and calcium. there are seven subclasses of the group, which are involved in various physiological functions like muscle contraction, neurotransmitter release, immune responses' regulation. p x receptors are present only in eukaryotes and ubiquitously expressed. p x are trimers of any three from seven variable subunits (p x - ) and exist as homomer or heteromer. each of the subunit may have two transmembranes. the detail is discussed in chap. . in nerve transmission, the neurotransmitter is released in synaptic cleft by presynaptic vesicles. these neurotransmitters have to be internalized by postsynaptic membrane for the propagation of impulses. for internalization, these neurotransmitters bind to receptors on postsynaptic membrane and induce conformational change in receptor to open channel for translocation. the gaba and glycine receptors are anion channels but inhibitory in nature. gaba receptors have two subdivisions as gaba receptors family of ligand-gated channel (ionotropic) gaba a and g receptor protein family (gaba b ). the g receptors are discussed in chaps. and . the ligand for gaba a receptor is c-aminobutyrate. the cysteine loop family in eukaryotes has conserved motif of two cysteine separated by other amino acids. the gaba a receptors are multisubunits with major isoform of gaba a. receptors are a b and c . the various isoforms of these proteins are found in human as asubunits with six isoforms, b-subunits three, c-subunits three, q-subunits three, and -, d-, h-, and p-subunits one copy. the single subunit of gaba a receptor has around amino acids with both c-terminal and n-terminal that are extracellular. its half hydrophilic n-terminal has signature motif of cysteine, and other c-terminal has four transmembrane helices from m to m . the m transmembrane forms lining of channel. the large intracellular loop between m and m constitutes modulation site for phosphorylation. the m and m domains of receptor may interact with other regulatory proteins. the functional receptors are made up of five subunits as homopentamer or heteropentamer. the -and d-subunits enhance polymeric association of monomer ( fig. . ). ( ) the pentameric cysteine loop superfamily receptor superfamily. ( ) the tetrameric ionotropic glutamate receptors are subdivided into n-methyl-d-aspartate (nmda), a-amino- -hydroxy- -methyl- -isoxazolepropionic acid (ampa), and kainate receptor subfamilies. the highly schematic topography of each receptor category indicates the locations of the extracellular and intracellular termini, the number of transmembrane spans (large colored cylinders), and cysteine residues participating in disulfide bond formation (yellow circles). red cylinders indicate a-helical regions participating in ion conduction/selectivity the density of these receptors varies from cell to cell. the gabaa are found in nerve cells, muscle, and liver and on immune cells. the gaba receptors initially were thought to be only on postsynaptic membrane for opening of gaba chloride channel for hyperpolarization of a depolarized membrane but recent studies have confirmed their presence on dendrites, and other tissues also for the regulation of synapses. the gaba a receptors are gated chloride anion channels and occasionally may translocate bicarbonate anions. gaba binds to two binding sites at the interface of a-/b-subunit of receptor. the important region for ion translocation is a-subunit segment (f loop), which bulges out from ligand binding site toward the ion channel, and in c-subunit, this segment modulates channel activity. the second segment, a lysine residue of the short loop between m and m , is important for the formation of a salt bridge during the process of gating. the third segment resides within the b -b linker of the b-subunit. gaba binding to binding sites at the interface of a-/ b-subunit stimulates conformational change around binding site which passes through open and many closed states (flipped states) for final gate opening. glutamate receptor channels n-methyl-d-aspartate (nmda) the n-methyl-d-aspartate (nmda) receptors are known as glutamate-gated cation channel for calcium permeation. the tetrameric ionotropic receptors include nmda, a-amino- -hydroxy- methyl- -isoxazolepropionic acid (ampa), and kainate receptor as per their synthetic ligand binding. these are excitatory neurotransmitters. in excitatory transmission, glutamate released from presynaptic membrane binds to ndma or ampa receptor of postsynaptic membrane. the ndma receptor family have three different subunits known as glu n - . the crystallographic and functional studies have revealed detail structure and mechanism of channel opening. active nmda receptors are tetramer with two glun and two glun . the monomer subunit may be divided into four distinct domains as extracellular n-terminal domain, ligand binding domain, pore-forming membrane domain, and c-terminal intracellular. the extracellular n-terminal domain is linked to ligand binding domain through small linker, which is also attached to pore-forming transmembrane ( fig. . ). the transmembrane is liked to c-terminal domain. the channel opening starts in sequential order of ligand binding ! change in conformation ! channel opening. two separate ligands are required for nmda receptors activation. the ligand binding domain of subunit has two subdomains s and s . the s domain of two neighboring subunits, at their interface, creates ligand binding site and is rigid in structure but lower one s domain is mobile without any interaction. in typical ndma receptor, glun and glun will interact with two molecules of glutamate and two molecules of glycine. the receptor with glu n and glu n will require only glycine for the activation. the nmda receptors have high affinity to glutamate, high calcium (ca + ) permeability, which may be blocked by magnesium ion in voltage-dependent manner. the nmda differs from ampa receptor of the same family in its function. the n-methyl-d-aspartate (nmda) receptors unlike a-amino- -hydroxy- -methyl- -isoxazolepropionic acid (ampa, activation time ms) are slow in activation (app. - ms) and deactivation (app. - ms). ampa receptor initiates rapid polarization in response to neurotransmitter release during synaptic transmission but nmda receptors only care for time span of synaptic currents. the glycine is coagonist for nmda receptor. two molecules of glycine and two molecules of glutamate bind on separate binding site in receptor for the activation and opening of channel, which results in influx of calcium and sodium with efflux of potassium. the electrical synapses are converted into chemical synapses through nmda receptor because influx of a the arrangement of nmdar subunits with four hydrophobic domains (m through m ), two ligand binding domains (s and s ), and amino and carboxyl terminal domains (ntd, ctd). b monomer subunit with n-terminal signal peptide on extracellular side, transmembrane helices m , m , and m , m , a cytoplasmic reentrant hairpin loop a link between m and m and ligand binding domains s and s , c twodimensional membrane folding model of glycine binding nr and glutamate binding nr subunits . tetrameric ionotropic glutamate receptor channels … calcium in postsynaptic membrane activates calcium-dependent signaling pathway for the modulation of synaptic structure, plasticity, and connectivity. the signaling is discussed in chap. . the independent binding of two ligands, glycine and glutamate, collectively induces change in conformation to open channel. the closing of channel may result in glutamic acid release and desensitization of receptors. it has been observed that the nmda receptors may be in desensitized form with bound glutamate for long period. the other function of nmda receptor in learning and memory can be inhibited by magnesium ion in voltage-dependent mode. in close state of receptor, the magnesium is bound to receptor in inhibition mode, which can be released only by sufficient depolarization of the membrane. this kind of interaction empowers nmda receptor to act as coincidence detector for pre-and postsynaptic changes. the slow kinetics of nmdar-mediated excitatory postsynaptic current (epsc) supports temporal summation and decreases dendritic filtering of synaptic input. the receptors provide strength to synapses through long-term potentiation (ltp) and weakness by long-term depression (ltd) for learning and memory. through calcium influx, the overstimulation of nmda receptors may cause alzheimer, parkinson, huntington, and amyotrophic lateral sclerosis (als). physiologically, nmda receptors may be desensitized by unfavorable interaction between glycine and glutamate to cause close conformation of receptor or calcium influx activate calmodulin to remove actin from receptor to keep receptor in desensitized mode. the receptors activity may be decreased by zinc, polyamines, and proton. the phosphorylation of receptor protein enhances open form of channel by removing magnesium and inducing favorable conformation. the voltage-gated ion channels open or close in response to change in voltage and the unit of membrane potential. in excitable cells like nerve and muscle, the chemical or electrical signaling is initiated through voltage-dependent sodium or calcium influx. the sodium channel protein in human is composed of large a-subunits ( kd with amino acids) and small b-subunits ( - kd); the small b-subunit has two isoforms b- and b- . the sodium channel b-subunits have n-terminal extracellular domain into immunoglobulin-like fold, a single transmembrane segment, and a short intracellular segment. these units may form heterodimer or heterotrimer. the a-subunits are linked noncovalently to b- and through disulfide linkage to b- . the voltage-gated sodium ion channels are also known as nav channels. in human, a-subunit has various isoforms like nav . , nav . , nav . , nav . , nav . , nav . , nav . , and nav . . the nav . , nav . , and nav . are found in peripheral nervous system and nav . , nav . , nav . , and nav . in central nervous system, and the nav . resides in heart muscle and nav . in skeletal muscle. the a-subunit is coded by ten different genes in human but one of these codes for nonchannel a-protein is known as sensing helix. b-subunits have four isoforms as b- , b- , b- , and b- . the b- and b- associate with a-subunit noncovalently and b- and b- through disulfide linkage. navb-subunits resemble cell adhesion molecules in structure and may have role in localization. the three-dimensional structure of voltage-gated sodium channel has revealed structural detail, mechanism and catalytic amino acids involved, drug target site, and regulatory site. the bacterial sodium channel (navab) is seen to have central pore surrounded by four pore-forming helix segments s and s segments and the intervening pore loop. the four voltage-sensing elements made up of s -s segments are symmetrically associated with the peripheral pore elements. the structural analysis of bacterial sodium channel (navab) has shown the swapping of functional domain of one subunit to other like all individual voltage-sensing elements are found in close associated with the pore-forming element to neighbor as shown in fig. . in dark colors. this type of arrangement and association is also observed in potassium channel (fig. . ). the navab sodium channel are found to have large outer entrance with narrow selectivity filter, a large central cavity filled with water and layered by the s segments and an intracellular activation gate at the crossing of the s segments and intracellular surface of the membrane. sodium and potassium channel has similar type of structure but differs significantly in permeation mechanism. the potassium ion channels discriminate k + by direct interaction with a series of four ion coordination sites created by the carboxyl group of ion selectivity filter amino acid residues in the zone. water is excluded and does not interact with selection filter, while the navab ion selectivity filter has a high-field-strength site for ions at its extracellular end because of the side chains participation of four highly conserved and key determinants glutamate residues. the sodium ion with two planar water molecules for hydration could fit in this high-field-strength site. on outer side of the field, two carboxylic groups of peptide create coordination site for sodium binding with four planar waters of hydration. these negatively charged group interaction with sodium removes water of hydration partially for permeating sodium as a hydrated ion, which interact on the way with the pore residues through its inner shell of bound waters. the local anesthetics, antiarrhythmic and antiepileptic drugs may inhibit this channel by binding to inner side of segment (drug receptor site) but binding to this site navab channels is solely controlled by the side chain of phe , single amino acid residue. the sodium channels open in response to depolarization and within fraction of second are closed also for repetitive firing of action potential in synapse. the short intracellular loop connecting a-subunit iii and iv domain acts as inactivation gate in channel inactivation. this acts as an intracellular blocking particle by folding into the channel structure and blocks the pore during inactivation. the nmr study of channel inactivation gate has shown the presence of a rigid a-helix followed by two loops of protein with ifm motif and a neighboring threonine residue on their surface for interaction participate in closing. the inactivation gate bends at conserved pair of glycine residues to fold into the intracellular mouth of the pore. the fast inactivation gate is not found in homotetrameric bacterial sodium channels. the voltage sensing is executed by the s transmembrane segments of sodium channels, which has four to eight repeated motifs of a positively charged amino acid residue (usually arginine), followed by two hydrophobic residues, and shifts the gating charges of sodium channels in the sliding helix in voltage sensing. the gating charges are stabilized in their transmembrane position by ionic interacting pairing with neighboring negatively charged residues, which can exchange their ion partners during outward movement. fig. . subunit primary structures of the voltage-gated sodium channels y, sites of probable n-linked glycosylation; p in red circles, sites of demonstrated protein phosphorylation by pka (circles) and pkc (diamonds); green, pore-lining segments; white circles, the outer (eeee) and inner (deka) rings of amino residues that form the ion selectivity filter and the drug binding site; yellow, s voltage sensors; h in blue circle, inactivation particle in the inactivation gate loop; blue circles, sites implicated in forming the inactivation gate receptor the formerly referred leaky channels, resting, back ground, or constitutive channels are now referred as k p or voltage insensitive channels. the k p channels are ubiquitously found in all organisms. the functionally active k p channel has two subunits, and each subunit is made up of two pore regions (p and p ), four transmembrane domains (m -m ), and two characteristic extracellular helices cap c and c . the yeast tok , kp channel with eight transmembrane helices (m -m ). the tok ( p/ tm) is exceptionally outward rectifier. these channels are highly regulated and functionally important in nervous system, muscles, blood vessels, endocrine system, and kidney. according to sequence homology, eukaryotic k p channels are classified into six groups; ( ) the twik clade is weak inward rectifying channels, ( ) trek clade is mechanosensitive and also regulated by lipids, ( ) task clade channels are regulated by acid ph, ( ) talk clade is alkaline ph-sensitive, ( ) thik clade is inhibited by halothane, and ( ) tresk clade is present in spinal cord. unlike voltage-dependent channels (nav, kv, cav), these k p channel do not have voltage-sensing domain and have two pores. the k p are not inactivated during depolarization except twik- . the voltage-dependent channels have one pore made by four transmembranes (fig. . ) . k p-channels in the cell membrane can be regulated at the transcriptional level. the presence of cap helices c and c is unique to these channels, which vertically extends * Å on the extracellular side of membrane to cover entrance with two lateral portals for k + ions. the pore-lining helices, m and m , traverse obliquely through the membrane and m and m , and the outer helices are more vertically oriented. the transmembrane segments of the channel have lateral openings between m and m facing toward lipid environment. the selectivity filter and the pore helices have fourfold symmetry with inner wide open cavity. the extracellular cap, the central cavity, and the inner opening of channel are found in twofold symmetry unlike other potassium channels. the two subunits were found to be assembled in a simple antiparallel manner. the c -c linkers are linked via a disulfide bridge. the two opposite pore helices and the corresponding pore loops contribute to forming the selectivity filter. the m -helix of one subunit is adjacent to the end to m -helix of the other subunit. the c-helix is amphipathic with charged residues facing the cytoplasmic side. the human k p channel cytosolic end of m has two to four charged residues and forms an amphipathic helix. the cytosolic part of the pore participates in gating and permeation. the crystallized k p-channel has been found to have lateral opening at the interface between the m -helix of one subunit and the m -helix of the other subunit to connect the inner cavity with the hydrophobic core of the cell membrane. the lateral cavity, occupied with the hydrophobic tails of membrane phospholipids, may be important in channel gating because of lipid interaction with m and m domains and alkyl chains of membrane lipids into the cavity through the lateral opening influences the conductance and the pharmacological properties. like all voltage-dependent channels, the glycine residues are conserved m and the m helices of all k p-channels for creating kink in m and the m domain around the hinge of g for moving m -helix from a "down" to an "up" configuration for transport, where lateral fenestration is closed and ions are passed through the selectivity filter. in down conformation, the central cavity m -helix crosses the membrane in such a way that lipids of the central cavity blocks the permeation. k p-channels are lined up with hydrophobic residues in layer. the opening of lower activation gate promotes the opening of upper inactivation gate, and c-type inactivation is promoted unlike voltage-dependent gate. during depolarization, all k p-channels have shown immediate current component and following time-dependent component of outward current. this instantaneous component supports all k p channels to have open probability at negative potentials, and the time-dependent current changes support the regulation of k p-channels by voltage changes. the steady-state current-voltage relation under physiological conditions (with - mm k + extracellular concentration) of k p channel, studied at depolarized potentials, is much larger than the current flowing through k p channel at resting membrane potential to support role of k p channel in action potential configuration. the anesthetics enhance the leakage of ions in neural tissue and decrease excitability. in various tissues, k p channels are present to perform various functions and may also play role in apoptosis subjected to further analysis (figs. . and . ). structure and regulation of the vacuolar atpases assembling the puzzle: oligomerization of alpha pore forming protens in membrane structure of a bacterial multidrug abc transporter the bacterial phosphoenolpyruvate: carbohydrate phosphotransferase system: regulation by protein phosphorylation and phosphorylation-dependent -protein interactions aquaporins are multifunctional water and solute transporters highly divergent in living organisms understanding poly specificity of multidrug abc transporters: closing in on the gaps in abcb structure of an abc transporter in complex with its binding protein structural mechanism for light-driven transport by a new type of chloride ion pump, non labens marinus rhodopsin- the gramicidin ion channel: a model membrane protein gating of two pore domain potassium channels a structural overview of the plasma membrane na + , k + -atpase and h + -atpase ion pumps lactose permease h -lactose symporter: mechanical switch or brownian ratchet? p-type atpases phosphoenolpyruvate: carbohydrate phosphotransferase systems of bacteria abc transporters: the power to change much more than a leak: structure and function of k p-channels the mechanism of pore formation by bacterial toxins aquaporins: important but elusive drug targets structure, function, and pharmacology of nmda receptor channels biology of human sodium glucose transporters the role of mitochondrial porins and the permeability transition pore in learning and synaptic plasticity structure and mechanism of abc transporters structure and evolution of mitochondrial outer membrane proteins of b-barrel topology key: cord- -mmkrwj t authors: snijder, eric j.; limpens, ronald w.a.l.; de wilde, adriaan h.; de jong, anja w. m.; zevenhoven-dobbe, jessika c.; maier, helena j.; faas, frank f.g.a.; koster, abraham j.; bárcena, montserrat title: a unifying structural and functional model of the coronavirus replication organelle: tracking down rna synthesis date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: mmkrwj t zoonotic coronavirus (cov) infections, like those responsible for the current sars-cov- epidemic, cause grave international public health concern. in infected cells, the cov rna-synthesizing machinery associates with modified endoplasmic reticulum membranes that are transformed into the viral replication organelle (ro). while double-membrane vesicles (dmvs) appear to be a pan-coronavirus ro element, studies to date describe an assortment of additional coronavirus-induced membrane structures. despite much speculation, it remains unclear which ro element(s) accommodate viral rna synthesis. here we provide detailed d and d analyses of cov ros and show that diverse covs essentially induce the same membrane modifications, including the small open double-membrane spherules (dmss) previously thought to be restricted to gamma- and delta-cov infections and proposed as sites of replication. metabolic labelling of newly-synthesized viral rna followed by quantitative em autoradiography revealed abundant viral rna synthesis associated with dmvs in cells infected with the beta-covs mers-cov and sars-cov, and the gamma-cov infectious bronchitis virus. rna synthesis could not be linked to dmss or any other cellular or virus-induced structure. our results provide a unifying model of the cov ro and clearly establish dmvs as the central hub for viral rna synthesis and a potential drug target in coronavirus infection. epidemic, cause grave international public health concern. in infected cells, the cov rna- synthesizing machinery associates with modified endoplasmic reticulum membranes that are transformed into the viral replication organelle (ro). while double-membrane vesicles (dmvs) appear to be a pan-coronavirus ro element, studies to date describe an assortment ro [ , , ] , was entirely possible and started to attract attention. notably, dmvs can be also formed in the absence of vrna synthesis by expression of key transmembrane nsps [ ] [ ] [ ] [ ] [ ] . moreover, several studies suggested a lack of direct correlation between the number of dmvs and the level of cov replication in the infected cell [ , ] . the interpretation of the cov ro structure and function was further compounded by the discovery of different ro double-membrane spherules (dmss) we first set out to analyse the ultrastructure of mers-cov-infected huh cells under sample preparation conditions favourable for autoradiography (see materials and methods) (fig , s video). strikingly, in addition to the dmvs and cm that are well established hallmarks of beta-cov infections, the presence of small spherules, occasionally in large numbers, was readily apparent (fig a and b) . these spherules were notably similar to the dmss previously described for the gamma-cov ibv [ ] . their remarkably regular size of ~ nm (average diameter . ± . nm, n= ), a delimiting double membrane and their electron- dense content, made these spherules clearly distinct from other structures, including progeny virions, which had comparable diameter (fig c and d ). the double-membrane spherules (dmss) generated during ibv infection were previously described as invaginations of the zippered er that remain open to the cytosol [ ] . in mers-cov-infected cells, the dmss were connected to the cm from which they seemed to derive ( fig e) . clear openings to the cytosol could not be detected for the large majority (~ %, n= ) of the fully reconstructed dmss, which suggests that the original invagination may eventually transform into a sealed compartment. this type of apparently closed dmss were also present, though in a lower proportion (~ %, n= ), in ibv-infected cell samples processed in an identical manner (s the d architecture of mers-cov-induced ro aligned with previous observations for other cov [ , ] . no clear openings connecting the interior of the dmvs and the cytosol could be detected. all three types of mers-cov-induced membrane modifications appeared to be interconnected, either directly or indirectly through the er. while dmss were connected to cm, and cm to er, er membranes were often continuous with dmvs (fig f, arrowheads) . therefore, like other covs, mers-cov infection appears to induce a network of largely interconnected modified er membranes that, as a whole, can be considered the cov ro. quantification of the autoradiography signal per subcellular structure (see also s table) . labelling densities and relative labelling indexes (rli) in different subcellular regions of (c) vero e cells infected with sars-cov (moi ) or (d) huh cells infected with mers-cov (moi ). radioactively-labelled uridine was provided for the indicated periods of time immediately before fixation at hpi and hpi, respectively. control mock-infected cells are excluded from the rli plots, as rli comparisons between conditions require the same number of classes (subcellular regions) and these cells lack ros and virions. budding vesicles (fig a-c) .the m and s proteins also localized to the golgi complex, aligning with previous observations for other covs [ , , , ] . the mers-cov n protein was found in regions with cm and dmss, though the distribution of signal was homogenous and dmss were not particularly densely labelled (fig d) . the presence of the assembly, or the s protein (fig e and f ). previously, the cm induced by sars-cov and mhv were shown by iem to accumulate viral nsps, while dsrna signal was primarily found inside the dmvs [ , ] . similarly, nsp mapped to the cm induced in mers-cov infection, but also to the dmss to a comparable extent (fig g) . our attempts to combine dsrna antibody labelling with thawed cryo-sections were unsuccessful, which made us resort to hpf-fs samples. in these, however, while dmvs were easily detected, the morphology of cm and dmss was less clearly defined. nevertheless, dsrna signal was clearly associated with dmvs, while the dark membranous regions between dmvs that we interpreted as cm and dmss clusters appeared devoid of signal (fig h and i ). in summary, for the antibodies tested (recognizing n, m, s, nsp , and dsrna), the labelling pattern in mers-cov-induced dmss closely resembled that of the cm, from which they seem to derive. the absence of labelling for key proteins in virus assembly, like the m and s proteins, strongly suggest that dmss do not represent (spurious) virus assembly events. the comprehensive analysis presented here demonstrates that viruses across different cov genera induce essentially the same type of membrane structures. after somewhat disparate observations [ , , , , , ] , the unifying model that emerges from our study is that our results add to studies that, in the last years and after much speculation, have started to provide experimental evidence that the dmvs induced by +rna viruses are active sites of vrna synthesis [ , [ ] [ ] [ ] . however, it is not clear that dmvs always play the primary role in virus replication that we demonstrate here for cov. for picornaviruses, for example, virus- were freeze-substituted in a leica afs system with . % (wt/vol) uranyl acetate as previously described [ ] , with the only modification that acetone was replaced by ethanol from the last washing step before lowycril infiltration onwards. cell sections ( nm thick) were incubated with the primary mouse antibody, then with a bridging rabbit anti-mouse-igg antibody (dako cytomation), and finally with protein a coupled to -nm gold particles. after immunolabelling, samples were additionally stained with % uranyl acetate and reynold's lead citrate. large mosaic em maps containing dozens of cell profiles were used for the quantitative analysis of the newly-synthesized rna autoradiography signal (see s table) . for each cov, different conditions (infected and mock-infected cells, plus different labelling times) were compared using only samples developed after the same period of time. the analysis of the signal in different subcellular regions was carried out using home-built software. areas of µm were randomly selected from the mosaic em maps and the autoradiography grains present in those areas were manually assigned to the underlying cellular structures. the abundance of the different types of subcellular structures was estimated through virtual points in a × lattice superimposed to each selected area, which were also assigned to the different subcellular classes. regularly along the process, the annotated data per condition was split into two random groups and the kendall and spearman coefficients, which measure the concordance between two data sets [ ], were calculated. new random regions were added until the average kendall and spearman coefficients resulting from random splits were higher than . and . , respectively (maximum value, ). labelling densities and relative labelling indexes (rli) were then calculated from the annotated points [ ] . for the analysis of the association of vrna synthesis with each of the different ros motifs, the specific dmvs, dmss and cm included in the analysis were carefully selected. only individual dmvs that were at least one micron away from any other virus-induced membrane modification were included in the analysis. for every grain present in an area of nm radius around each dmv, the distance to the dmv centre was measured. in the case of dmss, which were always part of clusters of virus-induced membrane structures, only dmss in the periphery of these clusters were selected. the quantified signal was limited to sub- areas devoid of other ro motifs, which were defined by circular arcs (typically o to o , radius nm) opposite to the ro clusters. cms are irregular structures that appear partially or totally surrounded by dmvs. only large cm (> . µm across) were selected in order to make more apparent (if present) any decay of the autoradiography signal as the distance to the surrounding dmvs increased. for each autoradiography grain, both the distance to the closest cm boundary (d ) and the distance to the opposite cm edge (d ) were measured. the relative distance to the cm edge was then calculated as d /(d +d ) and expressed in percentages. all the measurements in different dmvs, dmss and cm were made using aperio imagescope software (leica) and pooled together into three single data sets. autoradiography is a classic technique that allows the em visualization of a radioactive marker, usually targeting a certain process, and thus reveals the subcellular localization of that process [ , ] . tritiated uridine, for example, can be used to locate active rna synthesis [ ] [ ] [ ] , as shown also in this study. a clear advantage over the use of alternatives for metabolic labelling of newly-synthesized rna (e.g. br-uridine, br-utp, -ethynil uridine) is that the radioactive precursor is chemically identical to the natural substrate. after labelling, the samples are immediately fixed and processed for em. the location of the radioactive marker can then be made apparent by applying a highly-sensitive photographic emulsion (a nuclear emulsion) on top of the cell sections and exposing it for several weeks to months. the beta particles that are emitted as a result of tritium disintegrations generate electrons that get trapped in the silver halide emulsion and create a "latent image". when the emulsion is developed, these negative charges promote the reduction to metallic silver, generating electron-dense grains that are visible by em. in principle, given enough time to accumulate enough radioactive disintegrations, even low levels of the radioactive marker could be detected. in practice, other factors (e.g. background radiation, emulsion aging) set some limits to autoradiography, which is nonetheless a very sensitive technique. the resolution of em autoradiography is limited by the fact that radioactive disintegrations generate beta particles that are emitted in random directions. importantly, the probability of giving rise to signal degreases with the distance from the radioactive source; however, some beta particles may travel up to a few hundred nanometers before striking the photographic emulsion [ ] . therefore, it is important to keep in mind that the silver grains may not directly overlay the structure containing the radioactive source. quantitative analyses of the signal that take this factor into account, like those presented in this study, become indispensable to maximize the information 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and pi p-independent formation of encephalomyocarditis virus replication organelles. mbio sars-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro early endonuclease-mediated evasion of rna sensing ensures efficient coronavirus replication mechanisms and enzymes involved in sars coronavirus genome expression a new virus isolated from the human respiratory tract reverse genetics system for the avian coronavirus infectious bronchitis virus the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex monoclonal antibodies to double-stranded rna as probes of rna structure in crude nucleic acid extracts towards a solution to mers: protective human monoclonal antibodies targeting different domains and functions of the mers-coronavirus spike glycoprotein resolution of a gold latensification-elon ascorbic acid developer for ilford l emulsion correlated fluorescence and d electron microscopy with high sensitivity and spatial precision a rapid method for assessing the distribution of gold labeling on thin sections dmss (white arrowheads) and zippered er (white arrows). most zippered er consists of long stretches of though branching zippered er, closer to the cm described for beta-cov, was also present (b) virus particles (black arrowheads) budding into the er membranes were often observed. scale bars analysis of previously described samples of cov-infected cells, prepared for em either by hpf(a) or cryo-plunging (b). a targeted search revealed the presence of dmss (white arrowheads) in close association with cm. in comparison with the chemically fixed samples used in this study, the superior ultrastructural preservation of cryo-fixation results in less distorted membranes, but also in a denser cytoplasm and darker cm that makes dms less apparent. (a) example from a mers-cov-infected huh cell sars-cov-infected cell ( hpi) metabolic labelling of newly-synthesized vrna in ibv-infected cells and analysis of the autoradiography signal. vero cells infected with ibv were pre-treated with actinomycin d for hour, then labelled for or min with tritiated uridine a) overview of an ibv-infected vero cell ( min labelling). the areas containing dmvs and zippered er are outlined in yellow and blue, respectively, and other subcellular structures annotated (n, nucleus; m, mitochondria; au, autophagosome; vcr, virion-containing regions). the autoradiography signal accumulates in areas of virus-induced membrane modifications that often only contain dmvs close-up of the area boxed in black in (a), which contains dmvs the contrast between the densely labelled dmvs and the zippered er and dmss largely lacking signal is apparent and suggests that the autoradiography grains sometimes present on the latter structures arose from radioactive disintegrations in the surrounding active dmvs. (c) in agreement with this possibility, most of the dmss ( %) were devoid of signal, and most of those that contained label where close to an active dmv (n dms = ). (d) furthermore, the distribution of autoradiography grains around dmss resembled that of a random distribution dmvs proved that these structures are associated with vrna synthesis, as the signal reaches maximum values in the proximity of the dmvs (n dmvs = ). ((c, d) see materials and methods for the selection criteria and details) electron tomography of the membrane structures induced in mers-cov infection animation illustrating the tomography reconstruction and model presented in fig b. the video first shows the tomographic slices ( . nm thick) through the reconstructed volume, and then surface-rendered models of the different structures segmented from the tomogram cm (blue) and dmvs (yellow and lilac, outer and inner membranes), er (green), and a vesicle (silver) containing virions (pink). the movie highlights the dms association with cm techniques and applications of autoradiography in the light and electron microscope autoradiography & radioautography. electron microscopy: principles and techniques for biologists association of polioviral proteins of the p genomic region with the viral replication complex and virus-induced membrane synthesis as visualized by electron microscopic immunocytochemistry and autoradiography escaping host factor pi kb inhibition: enterovirus genomic rna replication in the absence of replication organelles the origin, dynamic morphology, and pi p-independent formation of encephalomyocarditis virus replication organelles. mbio mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-alpha treatment sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum table data sets and sampling for the quantifications of the autoradiography signal presented in fig c and key: cord- -pk snuy authors: suh, hwal (matthew) title: collagen fabrication for the cell-based implants in regenerative medicine date: journal: bioengineering in cell and tissue research doi: . / - - - - _ sha: doc_id: cord_uid: pk snuy though transplantation of cells, tissue or organ has been regarded as an ideal approach, scarcity of donor is a practical barrier in clinics. current progresses in cell engineering has opened a new era, providing tools for host-regeneration by implanting manipulated cells in forms of cell therapy, which includes delivery of single cells or multicellular structural support of hybridized cells, as a representative individualized treatment method. this chapter mainly concerns on the cellbased implant made of cells and collagen, the main structural protein in extracellular matrix in mammalian tissue, as it has been regarded as a promising method for manufacturing a biologically mimicked artificial tissues. regenerative medicine is to repair, replace and/or modify the disordered or damaged human body, either from a disease and/or an injury, via functional regeneration of the host cells, tissues and organs by placing appropriate cells of optimal quantity into the damaged body and maintaining the cellular functions that provide expected efficacy. outcome of the regenerative medical treatment completely depends on the viability of delivered cells. embedding and planting, referred to as implantation, of devices made of biocompatible materials has been widely applied. metals, ceramics, synthetic and natural polymers are the fundamental biomaterials that are convenient for processing and fabricating into various forms of tissue supporting structures. however, lack of their biological function is too far from the ideal goal for treatment. from this perspective, transplantation which involves transferring procured cells, tissue or organ from a donor and planting into a recipient to replace the damaged lesion has been regarded as an ideal approach that provides biological restoration with recovery of physiological functions. nevertheless, the opportunity for selection of the completely perfect donor for a patient is critically limited in practice. the gene-homogeneity between the donor and recipient, mainly in human leukocyte antigen (hla) types, is the first factor to avoid any post-operative complication oriented from the immune responses. although autologous tissue is the most appropriate if damage or defect is not related to immune disease, applicable size and volume of donor site are extremely limited. allogeneic tissue has been the second choice for transplantation since several immune-suppressive agents have been introduced, but these agents also lead to other immune-depressed diseases by breaking the natural immune homeostasis. xenogeneic tissue and organ transplantation have been suggested by a number of researchers but longevity difference between human and donor animal is a great barrier that cannot be overcome at present. an approach to treat gene-defect oriented congenital disease by delivering correct genes directly to a patient, known as gene therapy, has been introduced as an individualized treatment tool. selected genes hybridized with a carrier vector are injected into a patient, and the vector may infect patient's cells and leave the selected genes within patient's cells. then, the delivered genes may express its genetic characteristics necessary to cure the disease. however, the gene therapy bears several problems: ( ) there is no perfect method to deliver genes into appropriate cells, ( ) efficiency of gene expression varies, and ( ) the gene delivering viral vector may induce unpredictable and unknown complications in body. another individualize treatment tool called cell therapy is planting therapeutic cells. instead of transplanting tissue or organ, therapeutic cells are delivered in forms of either transplantation of natural cells or implantation of artificially manipulated cells. in addition, resource of applicable tissues from which therapeutic cells could be prepared is expanded. conventional transplantation mainly consists of matured tissue or organ, but it is now possible to obtain therapeutic cells from any tissue at any growth stage, including embryonic blastocyst. through in vitro gene modification of cells, it is possible to select only the cells which possess appropriate genes for treatment purposes from outside of the human body. theoretically, in case of autologous cell therapy, patient's cells are harvested through autopsy or procurement, and the treatment gene for correction is transfected into the cells. after culture, only the completely gene-transfected, healthy cells are collected, and the selected cells are further cultured to obtain abundant number of cells to deliver in forms of cell suspension for injection. as the whole procedure is performed in vitro environment, only perfectly modified therapeutic cells are selected. in case of allogeneic cell therapy, donor's hla could be also replaced by the recipient's to escape from the post-operative immune rejection. furthermore, risk of inducing uncontrollable oncogenesis oriented from the mutated cells is avoidable during the cell selecting procedure in vitro [ ] . to deliver the selected cells into the selected site in recipient's body, it is necessary to fabricate cell delivery vehicle with biomaterials that supply agents to maintain cell-viability and act as probes for piloting cells toward appropriate site. the cell-based implant is defined as any implant in combination of cells and biomaterials which are intended to repair, modify and/or regenerate human body, through expected regulation and control of cellular functions and/or behaviors after implantation. the cell-based implant consists of artificially manipulated therapeutic cells hybridized with biomaterials in vitro. for cell therapy, each cell is individually hybridized with biomaterials, but in forms of tissue engineered implants, cells are hybridized with scaffold-biomaterials that act as structural support in the same way as the extracellular matrix in tissue does [ ] (fig. . ). substances applied to control biological events of cells may have biological risks. in manipulating cells, various kinds of substances functioning chemically at molecular level are used in cell-modifying procedures. biological agents, such as enzymes in the nucleic acid recombination procedure, cytokines in cell differentiation and proliferation control, and peptides in cell culture media, are regarded as biologic substances, and they must not produce any biological hazard during the manipulation procedures. in gene modification process, to avoid adverse reactions oriented from viral vectors, such as retrovirus, adenovirus and adeno-associate virus, non-viral vectors are designed to avoid biological risks and to manipulate easily. capability of non-viral vectors is mainly dependent on endocytosis mechanism, but the extent of development in an artificial vector that provides efficient endosomal escape to deliver the gene finally into nucleus is still questionable at present. biocompatibility is the primary requirement for any material used in manufacturing implant. in the cell-based implants, the material hybridized with cells shall either biologically or mechanically be compatible with the neighboring cells at the delivered site. for cell therapy, each cell would be hybridized with nano-sized particles that have specific affinity to the treatment target site and self-driving ability. particles shall be dissociated from the delivered cell after landing at the site and be completely excreted without accumulation in the recipient's body and any side effect. size of a tissue cell is about × − m (= μm), a virus is × − m (= nm), diameter of a dna is × − m (= nm), diameter of a hydrogen atom is × − m (= nm), and hybridizing substances are smaller than a μm. however, there is no accurate tool to detect these extremely small, nano-scale particles that exist in living body for safety evaluation of a substance at present. the cell-fabricating operator might be exposed to the substances during the whole manipulating procedure, and further investigation is necessary to avoid hazards. otherwise, currently introduced biomaterials for cell encapsulation are designed to protect therapeutic cells from immune rejection while providing the excretion of biologics from the cell, and mostly to manufacture macromolecules with less difficulty to characterize [ ] . in producing multicellular structural cell-based implants, so called tissue engineered implants, various biomaterials of either synthetic or natural substance are applied. basically, conventionally available biomaterials can also be adopted as long as the permanent biocompatibility is approved. synthetic polymeric biomaterials are the representatives, and can be non-biodegradable or biodegradable after implantation. non-biodegradable materials are usually intended for use in cell therapy where cell-encapsulation is required to provide and maintain optimal cellular function (e. g., alginate, liposome, etc.) by protecting the cells from host immune reaction, and/or for a tissue engineered implant, which requires the physiological load-bearing compliance (e. g., polyurethane scaffolds for blood vessels, tendons, ligaments, etc.) after implantation. they permanently remain at the planted place in recipient's body. meanwhile, biodegradable materials are usually intended for use in implants which restore the histological structure and replace the cellular function of recipients. they are gradually degraded in recipient's body through hydrolysis or enzymatic function after implantation (e. g., poly l-lactic acid, poly glycolic acid, etc.). in cases of using synthetic polymeric biomaterials, behavior of hybrid cells is mainly dependent on surface characteristics of material. especially, the cellular events such as adhesion, differentiation, proliferation and migration on the non-biological synthetic biomaterial are important for maintaining viability of cells implanted in bio-inert materials. furthermore, an increased regional acidity induced by dissolved acidic component through hydrolysis from the biodegradable materials demonstrates the limitation in mimicking natural biological environment with synthetic biomaterials. natural biomaterials are mainly composed of extracellular matrix (ecm) components (including structural components and biomolecules) originated from autologous, allogeneic and xenogeneic tissues of mammalians. collagen, elastin, chondroitin- -sulphate and hyaluronic acid are structural components. biomolecules, such as peptides, fibronectin, laminin, vitronectin and fibrin, and cytokines as growth factors and apoptosis signal promoters, are biologically active substances produced by nature. non-mammalian substances such as silk fibroin, crab chitin and chitosan and agar are also included in this criteria. at this scope, it can be recognized that, although any xenogeneic cells are still not permitted, xenogeneic ecm components are permitted for the "trans-" or "im-"plantable biomaterials. for cell-based implants that utilize xenogeneic tissues or their derivatives as biomaterials, secure risk controls shall be applied on sourcing, collecting and handling xenogeneic ecm, on the validation of elimination and/or inactivation of adventitious agents, such as transmissible spongiform encephalopathy (tse) agents in case of using bovine tissue, in products. non-comparable biological superiority of natural ecm components are applied for improving biocompatibility of synthetic polymeric biomaterials as grafting materials onto the surface of material. also, bioactive agents (including biologics, antibiotics, and antimicrobials) and/or synthetic drugs can be medicinal components in biomaterials, and they shall be assessed, in the context of their integration with the cell-based implant, according to pharmaceutical principles. this assessment shall consider the effects of medicinal components on product and vice versa. furthermore, the medicinal components could be an additive to treat the recipient's disease [ ] . in mammalians, collagen comprises about % of total proteins and exists as a main structural component in ecm and supports anatomical morphology of every tissue and organ. although more than types of them are informed, type i collagen, which has a specific molecule of the super-coiled triple helical peptide chains, is the most abundant in body. in brief, each peptide chain is a specific left-handed helix of , molecular weight and consists of repeating "-glycine-x-y-" amino acid sequence. as the arginine-glycine-aspartic acid (rgd) sequence is a typical cell adhesive ligand, collagen demonstrates strong cell adhesion property. left-handed helical peptide chains are integrated by intramolecular bindings of hydrogen bonds at glycines to glycines and hydroxyl bonds at hydroxyprolines to hydroxyprolines in each chain to form a right-handed triple helical collagen molecule. in a chain, amino acids are involved in the formation of a molecule, and each free-end of a chain that is not integrated with the others, is made of - amino acids and named as telopeptides. each of these at the c-and n-terminals of molecule binds to another and forms a long linear chain. collagen is easily denatured at temperature over • c through disintegration of the intermolecular bonds, and becomes gelatinized to form a randomly coiled chain with less viscosity than that of collagen. once denatured, gelatin is amorphous and does recover the intermolecular bonds in nature and is more easily digested by metaloprotease than collagen. a type i collagen fibril consists of collagen linear chains with strong intermolecular hydrogen bonds at lysine to lysine in each molecule. in nature, ε-nh of lysines and hydroxylysines at the both extremities of telopeptide is converted to aldehydes by lysineoxidase, and the schiff base formation by binding aldehyde with residual ε-nh or by aldol condensation between aldehyde to aldehyde occur. this strong molecular binding is called as the crosslinking and introduces strong mechanical characteristics to collagen to act as the structural matrix in tissue and provide organ morphology. as hydrogen concentration of body fluid is ph . , collagen is regarded as a weak base substance, and is generally extracted from mammalian tissue. collagen is the hydrophobic protein and is generally insoluble in a neutral solution, but non-crosslinked collagen is soluble in the neutral base solution such as nacl or na hpo . on the other hand, weak acid such as hcl, citric acid and acetate breaks the intermolecular schiff base bonds and the acid soluble collagen molecules are extracted. after extracting acid soluble collagen, non-acid soluble remnants remain, and these are strongly crosslinked collagen fibrils. when additional process using pepsin that digests the linear intermolecular bonds at each telopeptide is performed, telopeptide-free collagen molecules, known as the atelocollagen, can be obtained from remaining collagen fibrils. because telopeptide demonstrates individual gene-characteristics, these atelocollagen molecules are recognized as immune-free substance and are applicable to medical and pharmaceutical purposes. high concentration of bases such as nacl, na so , na hpo aggregate the acid-soluble collagen molecules in solution, and this phenomenon is applied to produce collagen membranes, threads and hollow fibers. however, randomly reconstructed intramolecular and/or intermolecular bonds do not provide sufficient mechanical properties compared to natural collagen in tissue. hence, in order to fabricate collagen with higher mechanical strength, re-crosslinking methods are introduced [ ] (fig. . ). collagen has been regarded as the first candidate for hybridization with cells as it exists in every part of body as the main extracellular matrix component. although implantation of xenogeneic cells is not practically permitted, immunefree atelocollagen is generally extracted from mammalians for medical use. bovine is the most popular resource provided if cows are grown at the officially recognized region that is free from tse. as atelocollagen has no telopeptides that integrate with other atelocollagen molecules, basic technology to produce intramolecular and intermolecular bonds for construction of the extracellular matrix with adequate mechanical properties required by tissue where the artificial cell-based implant is delivered is the recrosslinking method using chemical reagents or physical dynamics. most common chemical reagent adopted for this process is glutaraldehyde that introduces stable covalent nh to nh bindings between the molecules. as amine to amine (nh-nh) bonding exists in every amino acid in molecules, the chemically crosslinked collagen demonstrates high strength, but irregular patterns of the crosslinked fibers and protracted resorption are general disadvantages. furthermore, in case of using glutaraldehyde, complications due to residual aldehydes often conduct calcification that directly leads to the loss of mechanical strength. to overcome these disadvantages, -ethyl- ( -dimethylaminopropyl) carbodiimide (edc), which introduces nh to cooh covalent bonding and has no toxicity, has been applied to the procedure. to avoid any disadvantage arising from the use chemical reagents, cross-linking by physical method is employed. irradiating with gamma or ultraviolet rays produces radicals in the form of unpaired electrons in the nuclei of aromatic residues such as those in tyrosine and phenylalanine, and binding of these radicals results in the collagen cross-linking through random nh to cooh bonding. even though the physical method is safe as it does not involve the use of chemicals, stability of the crosslink is less than that of chemically induced crosslink. collagen can be fabricated into various forms of gel, fiber, membrane with or without pores, and it can even be grafted onto the non-viable metal, ceramic and synthetic biomaterials to introduce biological layer on surface. atelocollagen gel is easily prepared by dispersing in weak acidic solutions. in general, collagen is dispersed in a weak acid solution of ph - . and finally adjusted to required ph by adding base for medical purpose. additive substance to ultimate tensile stress of dense membrane in relation to uv irradiation time. each value represents the mean ± sd in five samples, * and †: significantly different compared to the control non-treated group for each type of membrane, * : p < . and †: p < . . control : nontreated group, uv / : uv-irradiated group for mins, uv : uv-irradiated group for hours, uv : uv-irradiated group for hours, uv : uv-irradiated group for hours, ga: . % glutaraldehyde pretreated group for hours (y -axis: maximum tensile stress (kpa)) provide and/or control the biological function of the collagen can be introduced at this stage utilizing the high viscosity property. biochemical property of collagen molecule can be modified by varying the surface electric charge by altering the molecular side chain which leads to the adjustment of hydrophilicity-hydrophobicity balance. succinylization of amines (nh-co(ch ) -cooh) by using anhydrate succinyl acid provides abundant (−) charges, and the succinylated collagen becomes soluble in neutral solution, which finally results in translucent viscous collagen gel with hydrophilicity. meanwhile esterization (-cooch ) of carboxyl group by methanol produces abundant (+) charges on the collagen and provides a favorable hydrophobic niche for protein adhesion. atelocollagen gel is fabricated into fiber form by using an electro spinning. for example, a collagen solution dissolved in , , , , , -hexafluoro- -propanol (hfp) having concentration over % is useful to produce a diameter controlled nanofiber by passing it through a diameter adjustable nozzle under the condition of a high voltage at kv, flow velocity at . m/h, metal collector of cm width with rotating speed at rpm, and distance between the metal collector and spinner of cm, and finally removing organic solute for hours by drying in a vacuum chamber. the produced nanofiber is crosslinked by either edc or uv irradiation later in order to reinforce the fiber strength, and demonstrates the typical triple helical structure of collagen molecule. freeze-drying the gel in vacuum condition is a simple procedure for fabrication of a porous membrane, as lower concentration and higher freezing temperature leads to smaller and bigger pores [ ] [ ] [ ] [ ] [ ] , (figs. . - . ). collagen contains much of the cell adhesive rgd sequences in molecule, thus has cell conductive characteristics and is applied either in the independent form, in form of hybridization with biofunctional agents and cells, or, occasionally, in form of copolymer by introduction of synthetic polymers which is mainly aimed to enforce mechanical property. collagen gel is usually applied to cell culture. a cell culture plate whose bottom surface is coated by collagen gel and dried makes it possible for suspended cells to produce a monolayered cell cluster by attaching them to the coated collagen layer. this technique is directly applied to produce skin wound dressing membranes. exposure of dermis to the open air, the grade skin defect, in which the basal epithelial layer that prevents direct contact of inner body to the outer bodily environment and supports epithelium as the bed for keratinocytes is destroyed from burn, and sore spots induced by diabetes mellitus or accident are critical emergency in clinics. they evolve the direct contamination of dermis which usually progresses toward fatal septicemia or skin necrosis. the conventional treatment procedure mainly consists of complete irrigation and debridement of the wound, topical administration of broad spectrum antibacterial and antifungal agents, covering the wound by oily ointment to protect it from air contact, painful daily dressing change, and observation of the auto-regeneration of wound by proliferation of both dermal fibroblasts and epithelial keratinocytes and basement membrane reconstruction. promoting proliferation of these skin cells with protection from air contact until complete healing is the key technology, and collagen membrane has been applied for this. for example, freeze dried atelocollagen in vacuum forms a membrane with random pores, and the pore size into which the cells may proliferate is controlled by the gel concentration and temperature at which it is frozen. freeze dried collagen gel of , , and % in concentration at − , − , and − • c for / , , , and hours demonstrated that higher concentration produced less and smaller sized pores. also, the lower temperature and prolonged freezing time decreased viscosity. to produce a collagen membrane with optimal pore size of over μm which permits penetration and proliferation of dermal fibroblast proliferation, the % collagen gel freeze dried at − • c for hours and crosslinked by edc or uv irradiation for hours was found to be recommendable to use as a dermal cell conductive membrane that provides the appropriate viscosity for initial anchorage, and any inflammatory exudate escapes through pores. a collagen bi-layered membrane is applied to skin in forms of either wound dressing or extracellular structural supporting matrix for artificial skin. laminin, which is a cell adhesive protein and a main component of the basement membrane between dermis and epithelium, is mixed with % collagen gel. a porous collagenlaminin membrane is fabricated and freeze-dried as previously described and additional % collagen gel is coated on the porous membrane and crosslinking is promoted by edc treatment or uv irradiation. through this procedure, a bi-layered collagen matrix that consists of dense collagen layer overlaid on the prefabricated porous collagen-laminin membrane was produced. in use as a wound dressing, the upper dense layer prohibits direct wound contact with the open air and plays a role as a bed for keratinocyte proliferation, while the porous layer conducts dermal fibroblast proliferation. thus, the painful daily dressing change becomes unnecessary. antibacterial agents encapsulated by hyaluronic acid can be incorporated into the collagen gel and the drug may be released from the collagen membrane to avoid periodic topical drug application. fabrication of the bi-layered collagen membrane is the basic technology applicable to manufacture of artificial skin. in the s, bell introduced a method in fig. . collagen-laminin hybridized porous membrane for dermis which dermal fibroblasts and collagen gel mixture were cultured at • c, and when the cells were confluent in the gradually shrinked gel, the collagen-fibroblast composite is produced. keratinocytes were overlaid on the composite to mimic anatomy of the dermal cellular structure, but it required about days for the manufacture to be completed and it was therefore not applicable to patients with grade skin wound. on the other hand, green introduced an advanced method in which dermal fibroblasts were cultured to form monolayer on a collagen coated culture plate and then keratinocyte was directly co-cultured on the fibroblast monolayer in a media containing epithelial growth factor. nonetheless, this method still required at least a week for the complete fabrication, and employment of growth factors holds a risk of oncogenesis induction. to produce an artificial skin, autologous dermal fibroblasts of rat were seeded into the edc crosslinked porous collagen-laminin membrane and cultured in minimum essential medium (mem) for days to provide the cell-niche adaptation period. -day culture is not enough for complete proliferation of cells into pores, but cells were firmly attached and anchored onto the superficial layer of the porous membrane due to specific cell adhesive characteristics of -rgd-sequences in col- lagen and laminin. after weeks of coverage onto a grade skin wound, dermis was completely replaced, and basement membrane lined beneath the dense layer as the seeded dermal fibroblasts were cultured in situ. epithelial regeneration upon the dense layer surface was poor and partially occupied by the keratinocytes, but full coverage by epithelium was observed after weeks. this phenomenon can be understood as the porous collagen based membrane can play as a vehicle for dermal autologous cell delivery, and the reconstructed basement membrane, which is a keratinocyte supplying bed in nature, has driven epithelial conduction. later, a collagen-elastin nanofiber was fabricated as previously described, and the extruded fibers were brought onto collagen gel and crosslinked by edc. using nanofiber made it possible to produce a lattice controlled matrix, and uniformly aligned lattices conducted cells to proliferate into the designed lattice pattern [ ] [ ] [ ] [ ] [ ] [ ] [ ] (figs. . - . ). collagen gel hybridized with calcium phosphate is applicable as a bone conductive substitute. apatite ( ca (po ) ++ ), a typical inorganic component of skeleton, was synthesized and heated at • c after which apatite crystals were not sintered but degradable by hydrolysis. % collagen gel was mixed with the apatite in : w/v, and the mixture was crosslinked. then the produced apatite-collagen pellet was implanted into rabbit's resected tibiae. weeks after implantation, the resected defect was completely regenerated by host bone with cortical bone continuity and cancellous bone stroma, and after that, typical bone remodeling process followed to produce natural bone. however, mechanical strength of the regenerated area was less than general bone. although collagen based gel is favorable to fabricate cell conductive substitute, weak mechanical property is a barrier for application in the physiological stress bearing tissues, and, to resolve this problem, hybridization of collagen with polymeric biomaterials has been suggested. introducing functional groups on esters, such as polyurethane (pu) and poly lactic-glycolic acid (pl-ga), through treatment with ozone induces surface oxidization that produces carboxyl groups on the surface which react with amines in collagen. as the apatite-collagen provided insufficient strength despite promotion of a favorable osteogenesis, collagen was grafted onto the biodegradable pla membrane to reinforce the strength. pla of m.w. , was resolved by % chloroform solution to produce % (w/v) pla solution, and nacl crystals ( - μm) were mixed to the pla solution with adjusting nacl:pla ratio to : (w/w). the prepared solution was cast into an appropriate shape, and the solvent was allowed to evaporate over hrs to produce a porous membrane after leaching out the nacl particles with distilled water. to induce molecular bindings between the collagen within apatite-collagen composite and the pla membrane surface, pla membrane was oxidized by ozone to produce reactive carboxyl and hydroperoxide groups on the pla surface. after the ozone treatment at v for min, apatite-collagen was delivered onto the ozone treated pla membrane to produce an apatite-collagen grafted pla membrane. the grafted membrane was pressed ( bar at • c) from vertical direction, and then edc collagen cross-linking was performed to integrate the mixed atelocollagen fibers in the grafted material. the final product was im- collagen grafted synthetic polymer is applicable to soft tissue replacement as well. except the air passages such as trachea and bronchus which require almost no repeating dilatation-shrinkage action of compliance, most of the tubular-shaped tissue requires various level of compliance against the mass passing through lumen, as arteries to pulsating blood flow and esophagus to swallowing dietary mass. in general, tubular walls of those tissues consist of multiple-layered smooth muscle cells to resist against pressure at luminal wall exerted by transporting mass, and appropriate flexibility is required. to replace esophagus, polyurethane, a bioinert biomaterial with mechanical advantage of high durability against continuous bending stresses, was employed. an approach is presented for graft copolymerization of type i atelocollagen onto surface of polyurethane (pu) treated with ozone. through surface oxidization by ozone to modify the pu surface, peroxide groups are easily generated. those peroxides are broken down by redox-polymerization and provide active species which initiate graft polymerization by reacting with amines in the collagen molecules. ozone oxidation time and voltage could readily control the amount of peroxide production. maximum concentration of peroxide was about . × − mol/cm when ozone oxidation was performed at v for min. after the reaction of pu by ozone oxidation, type i atelocollagen gel was graft copolymerized onto the pu. all the physical measurements on the collagen grafted surface indicated that the pu surface was effectively covered with type i atelocollagen. interaction of the collagen grafted pu surface with fibroblasts could be greatly enhanced by the surface graft polymerization with type i atelocollagen. attachment and proliferation of fibroblasts on the grafted type i atelocollagen were significantly enhanced, and it is assumed that the atelocollagen matrix supported the initial attachment and growth of cells. in the early stage of proliferation, collagen synthesis in fibroblasts was not activated and remained at a relatively low level due to the grafted type i atelocollagen, increasing only fibroblast differentiation. the mechanical property of tubular tissue was oriented from the alignment of cells that consists of repeated overlayers of longitudinal and circumferential layers and forms wall thickness. regarding this result, a double layered pu tubular scaffold grafted with collagen was fabricated to produce an artificial esophagus. pu purification was performed by diluting pu particles in dmac(dimethyl-acrylamide) solution then precipitating in non-solvent methanol, and drying the precipitate under vacuum for days to remove residual methanol. after purification, pu was dissolved in dmac solution at the final concentration of %w/w. glass rod, with diameter of mm and length of cm, was worn with % pu solution and then air-dried for hours. this process was repeated twice. after wearing, an end part of the glass rod was cut, and then the rod was worn again in reverse direction by the same process to be uniformly worn. it was dried under vacuum for hours afterward. completely dried sam- ple was swelled in benzene solution for hour and washed with distilled water for hours and ethyl alcohol for hour. after swelling and wearing process, a porous pu tube was separated from glass rod and dried under vacuum. to graft collagen, ozone treatment was performed as described above. the tube was immediately immersed in % type i atelocollagen gel, and mohr's salt [feso (nh ) so h o] was added to the gel to decompose peroxides. then polymerization was allowed to proceed at • c and ph . . type i atelocollagen grafted porous pu tube was dried in vacuum at • c. the tube was dipped in a % collagen gel and crosslinked by edc later. by this procedure, a porous collagen-grafted tube with inner diameter of mm, thickness of . mm covered by dense collagen layer was produced. rabbit's esophageal smooth muscle cells were seeded and cultured in a mechanically stressful environment of % strain magnitude and hz frequency. culturing for hours of mechanically stretching condition and hours of stationary condition on every -hour period, the cultured cells aligned in the perpendicular direction to the strain direction after days. the collagen grafted pu tube with cell alignment control was subcutaneously implanted in nude mice for weeks, and a histological finding demonstrated that a well-aligned smooth muscle cells reconstructed the tube which is applicable for replacement of esophagus [ ] [ ] [ ] [ ] [ ] the idea of collagen grafting was also applied to produce a healing-promoting antiadhesive membrane that is particularly necessary in peritoneal surgery to prevent postoperative adhesion. at first, glycolide and d,l-lactide were recrystallized from ethyl acetate and dried under vacuum before use. lactide and glycolide, at a molar ratio of : , were put into a glass ampoule, and mpeg was added for preparing the mpeg-plga block copolymer through ring-opening polymerization. . %(w/w) of stannous octoate added to the solution as a catalyst. the ampoule was evacuated by a vacuum pump, sealed with a torch, and was heated in an oil bath at • c for hrs. after the reaction was complete, resulting polymers were purified by dissolving in methylene chloride and then precipitated in excess methanol. obtained polymers were dried in vacuum. for the preparation of polymer film, % solution of mpeg-plga in chloroform was cast on a glass plate, and the solvent was evaporated in a vacuum oven for days. on the other side, a porous collagen-hyaluronic acid (ha-col) membrane was fabricated and crosslinked. % of hyaluronic acid (ha) (sodium salt, mw = , − , ) as an aqueous so- to prepare ha-col and mpeg-plga bi-layered composite membrane, chloroform was sprayed on the surface of mpeg-plga film, and then cross-linked ha-fn-col membrane was loaded on the slightly dissolved surface of mpeg-plga film. in vitro adhesion test revealed that fibroblasts attached better on ha-col membrane compared to those on mpeg-plga film, plga film or oxidized cellulose film. mpeg-plga film had the lowest cell adhesive property. in confocal microscopic observation, the actin filaments were significantly more polymerized when or μg/cm fibronectin was incorporated on the ha-col membranes. after -day culture, fibroblasts penetrated throughout the hafn-col network and the cell density increased whereas very few cells were found attached on surface of the mpeg-plga film. in vivo evaluation by implantation test for days in rabbit's peritoneal wound showed that the composite membrane could protect tissue collagen based biomaterial was also applied as a mesenchymal stem cell (msc) delivery vehicle. liver hepatocyte is a representative stable cell that has tremendous proliferative capacity and ability to differentiate and proliferate when injury or damage occurs in liver. in case of no-proliferation of hepatocytes, oval cells are stimulated to divide and eventually differentiate into mature hepatocytes; therefore, an oval cell is regarded as a compensatory cell in liver injury, and has been concerned to be equivalent to liver stem/progenitor cells. oval cell is oriented from bone marrow mesenchymal cell, and is a facultative bipotential precursor cell that differentiates into hepatocytes or bile duct cells. human mscs harvested from tibial bone marrow and co-cultured with hepatocytes for days in medium with additive hepatocyte growth factor (hgf) produced a confluent mixture of undifferentiated mscs and oval cells. to purify the oval cells from nonparenchymal cells, cell cloning method was applied. characterization of oval cells was performed by a double immuno- observed fiblasts on the peg-plga surface were significantly fewer than on the plga membrane surface. collagen-ha membrane demonstrated high affinity to cell attachment fluorescence method using alpha-fetoprotein (afp) and cytokeratin- monoclonal antibody expecting co-expression. isolated human mscs oriented oval cells were seeded on an edc crosslinked porous type i atelocollagen matrix, and cultured in medium containing insulin, dexamethasone, and hydrocortisone as the hormone stimulators and additive cytokines of hgf and epidermal growth factor (egf). for co.) is a commercially available anti-adhesive membrane made by oxidized cellulose weeks, albumin secretion and urea detoxification rate continuously increased either in the hormone additive or the cytokine additive groups, and this was directive knowledge that avoidance of using growth factors in committing msc-oriented oval cell toward hepatocyte can escape from risky induction to liver cancer. the collagen scaffold was able to foster long-term viability and protection of the cells, and this -dimensional culture of oval cells is considerable for designing a cell-delivering tool for hepatic disease [ ] (figs. . - . ). asymmetric collagen-fibronectin grafted peg-plga copolymer membrane was appeared to successfully reduce the incidence of postoperative adhesion formation although collagen is a favorable biomaterial to be employed in a wide range of fabrication procedures with cells, there is no established tool to isolate and produce absolute atelocollagen, the immune-free substitute, at present. even a single remaining collagen dimer or trimer may cause immune reactions in future. the resource of collagen, especially for clinical applications is also important, because unknown viruses that produce future unpredictable diseases from xenogeneic extracellular matrix may exist. if single-cell encapsulation by collagen containing signal transduction agents and ligands attracting specific cell adhesive receptors is systemized, commitment of stem cell differentiation and proliferation toward the designated target tissue will be achieved, and this may contribute to the future progress of stem cell-based implant. tissue restoration fundamental concepts for tissue engineering biocompatibility assessment of medical devices and materials extracellular matrix components anti-infection treatment of a transcutaneous device by a collagen-rifampicine composite treatment of collagen for tissue regenerative scaffold reaction monitoring of succinylation of collagen in matrix-assisted desorption/ionization mass spectrometry characterization of uvirradiated dense/porous collagen membranes: morphology, degradation, and mechanical properties characterization of porous collagen/hyaluronic acid caffold modified by -ethyl- ( -dimethylaminopropyl)carbodiimide cross-linking preparation of collagen modified hyaluronan microparticles as antibiotics carrier behavior of fibroblasts on a porous hyaluronic acid incorporated collagen matrix laminin modified infection-preventing collagen membrane containing silver sulfadiazine-hyaluronan microparticles an infection-preventing bilayered collagen membrane containing antibiotic-loaded hyaluronan microparticles: physical and biological properties biological characterization of edc-crosslinked collagen-hyaluronic acid matrix in dermal tissue restoration evaluation of antibiotic-loaded collagen-hyaluronic acid matrix as a skin substitute electrospun nanofibrous membrane for the engineering of cultured skin substitutes biodegradable ceramic-collagen composite implanted in rabbit tibiae behaviors of osteoblasts-like cell (mc t -e ) on collagen grafted poly l-lactic acid (plla) membranes with various pore sizes a bone replaceable artificial bone substitute: morphological and physicochemical characterization behavior of osteoblasts on type i atelocollagen grafted ozone oxidized poly l-lactic acid membrane a bone replacable artificial bone substittue: cytotoxicity, cell adhesion, proliferation, and alkaline phosphatase activity a bone replaceable artificial bone substitute: osteoinduction by combining with bone inducing agent interaction of mesenchymal stem cells and osteoblasts for in vitro osteogenesis ex vivo mechanical evaluation of carbonate apatite-collagen grafted porous plla membrane in rabbit calvarial bone type i atelocollagen grafting on polyurethane tube and its mechanical property compliance of surface modified polyurethane tubular scaffold for artificial esophagus type i atelocollagen grafting onto ozone treated polyurethane films: cell attachment, proliferation, and collagen synthesis time-dependent modulation of alignment and differentiation of smooth muscle cells seeded on a porous substrate undergoing cyclic mechanical strain construction of functional soft tissues from premodulated smooth muscle cells using a bioreactor system evaluation of tissue adhesion preventive surface modified natural and synthetic polymeric materials preparation and characterization of biodegradable anti-adhesive membrane for peritoneal wound healing behaviors of isolated rat oval cells in porous collagen scaffold key: cord- -p oae e authors: o'callaghan, barbara; synguelakis, monique; le gal la salle, gildas; morel, nicolas title: characterization of aminopeptidase n from torpedo marmorata kidney date: - - journal: biol cell doi: . /s - ( ) - sha: doc_id: cord_uid: p oae e a major antigen of the brush border membrane of torpedo marmorata kidney was identified and purified by immunoprecipitation. the sequence of its n terminal amino acids was determined and found to be very similar to that of mammalian aminopeptidase n (ec . . . ). indeed aminopeptidase n activity was efficiently immunoprecipitated by monoclonal antibody k . the purified antigen gives a broad band at kda after sds-gel electrophoresis, which, after treatment by endoglycosidase f, is converted to a thinner band at kda. this antigen is therefore heavily glycosylated. depending on solubilization conditions, both the antigen and peptidase activity were recovered either as a broad peak with a sedimentation coefficient of s ( % chaps) or as a single peak of . s ( % chaps plus . % c( )e( )), showing that torpedo aminopeptidase n behaves as an oligomer stabilized by hydrophobic interactions, easily converted into a kda monomer. the antigen is highly concentrated in the apical membrane of proximal tubule epithelial cells ( gold particles/μm( ) of brush border membrane) whereas no labeling could be detected in other cell types or in other membranes of the same cells (basolatéral membranes, vacuoles or vesicles). monoclonal antibodies prepared here will be useful tools for further functional and structural studies of torpedo kidney aminopeptidase n. aminopeptidase n (ec . . . ) is an abundant membrane bound peptidase of kidney and intestinal microvilli (for reviews, see [ , ] ). it hydrolyses the n-terminal amino acid of short peptides, with preference for amino acids with uncharged side chains [ ] . while it completes digestion of oligopeptides in the intestine [ ] , its function in kidney brush border membrane remains unclear. this enzyme has also been reported in the plasma membrane of various cell types like hepatocytes [ , ] or myeloid cells [ ] . the complete amino acid sequences of human intestinal [ ] and rat kidney [ ] aminopeptidases n have been determined. northern blot analysis revealed the existence of two human rna transcripts, both encoding for the same protein but under the control of different promoters. however, intestinal and kidney epithelial cells express the same mrnas, which are shorter than the transcripts of myeloid or fibroblastic cells [ ] . aminopeptidase n is composed of a single type of subunit, a -kda glycosylated polypeptide (for reviews, see [ , ] ). this subunit possesses a single transmembrane domain near its n terminus while the major part of the enzyme is ectocellular, including the catalytic sites [ , , ] . in the present work, we have characterized the aminopeptidase n from torpedo marmorata kidney and found it to be very similar to the mammalian enzyme. torpedo marmorata were provided by the marine station of areachon (france). crude fractions of kidney membranes were prepared from pooled whole kidneys which had been stored at - °c for seve .r'al days. the tissue was homogenized ( % w/v) in buffer a ( mm naci, mm na phosphate buffer, mm edta, . mm dithiothreitol, % glycerol, final ph . ) to which . mm pmsf was added. the homogenate was centrifuged at gm~ for min. membranes were pelleted ( gm~x for rain) from the low speed supematant. balb/c mice were immunized by three intraperitoneal injections (at -day intervals) of kidney membrane proteins (about /.tg emulsified proteins in freund's adjuvant). three days after a final booster intraperitoneal injection (without adjuvant), immunized spleen cells were fused with the non-secreting myeloma clone p x -ag . cells using polyethylene glycol as the fusing agent [ , ] . hybrids were selected in hypoxanthine, aminopterin and thymidine medium and superuatants from the culture wefts containing hybrid cells were tested for the presence of antibodies binding to the apical membrane of tubular epithelial cells on torpedo kidney frozen sections. clones were selected, some of which were cloned by limiting dilution. crude kidney membranes ( mg proteinhrd) were solubilized in % chaps and . % ci e at °c for min and non-solubilized material was pelleted ( gmx for rain). for screening experiments, proteins (about mg/ml) were biotinylated using immunopure nhs-lc-biotin (pierce, /zg/mg protein) for h, at °c. the reaction was stopped by addition of glycine ( mm final concentration). then, immunoprecipitation was performed in one step: . ml of solubilized proteins ( mg protein/ml), ml of culture supernatant containing monoclonal antibodies, . ml of a suspension of protein a sepharose- b (pharmacia, mg dry powder/ml of mm naci, mm tris buffer ph . ) and /.tl of antimouse igg antibodies raised in rabbit ( mg ig/ml, biosys)were mixed and incubated overnight at °c. beads were recovered by low speed centrifugation ( g for rain) and washed extensively. bound proteins were eluted by resuspension of the beads in p.i sample buffer ( % sds, % fl-mercaptoethanol, m sucrose, mm tris buffer (ph . ), mm bromophenol blue), and boiled for rain. proteins were immunoadsorbed as above and eluted from the protein a beads in i /al sds sample buffer. they were then diluted in volumes of mm na acetate buffer, mm edta, mm fl-mercaptoethanol, mg/ml chaps, final ph . and incubated at °c for h with or without . u endoglycosidase f/n-glycosidase f (boehringer). after concentration under vacuum, samples were resuspended in sds sample buffer and submitted to gel electrophoresis. electrophoresis was performed in - % linear acrylamide gradient gels according to [ ] . proteins in acrylamide gels were either stained with coomassie blue, or electrotransferred onto nitrocellulose as described by [ ] . biotinylated proteins on the blots were indirectly visualized using streptavidin-biotinylated peroxidase complex (amersham). the peroxidase substrates were h and diaminobenzidine. immunoprecipitation was performed as above but starting from mg solubilized kidney membrane proteins, and using ml of culture supernatant (mab k ), / of rabbit antibody to mouse ig ( mg ig/ml, biosys), and ml of a suspension of protein a sepharose- b beads ( mg dry powder/ml). eluted proteins were dialysed against . % sds (overnight) and " . % sds for h (spectrapor ), and concentrated to / under vacuum. electrophoresis was performed as above except that . mm thioglycolate was added to the migration buffer and that a prerun ( min at ma) was performed [ ] . proteins bands were transferred onto 'problott' membranes (applied biosystems) overnight at v in transfer buffer ( mm boric acid, mm tris-base). the kda band was stained by amidoschwartz and cut out. electroblotted proteins were sequenced by dr jp le caer (institut alfred fessard, cnrs, gif-sur-yvette) in a a gaz-liquid protein sequencer (abi) using the problott cart.ridge. phenylthiohydantoin amino acids were analysed on a a pth analyser (abi, foster city, ca). kidney membrane proteins ( mg protein/ml) were solubilized for h at c in one of the following detergent-containing buffers: ) mm naci, mm phosphate buffer, . mm mgci , % glycerol, % chaps; ph . ; ) mm naci, mm phosphate buffer, mm edta, % glycerol, % chaps; ph . ; ) mm naci, mm phosphate buffer, . mm mgci , % glycerol, % chaps, . % c e ; ph . . after eentrifugation ( gmax for h), an aliquot ( or /tl supernatan of solubilized proteins was layered on top of ml sucrose gradient. we used - % linear sucrose gradients in the solubilization buffer but with a -fold lower detergent concentration. proteins markers: alcohol deshydrogenase ( . s, boehringer), catalase ( . s, boehringer), and fl-galactosidase ( s, sigma) were solubilized and submitted to centrifugation in parallel linear sucrose gradients. the kda antigen content in the fractions was estimated after / to / dilution in /.d transfer buffer ( mm tris, . % triton x- , % methanol; ph . ) and blotting onto nitrocellulose, using a dot-blot apparatus. dots were probed with mab ki and antimouse ig antibodies conjugated to peroxidase (institut pasteur productions). intensity of the dot staining was quantified using a scanning densitometer (hoeffer scientific instruments) and compared to that of known amounts of solubilized kidney membrane proteins. enzymatic marker activity was measured as previously described [ , ] . aminopeptidase n activity was followed at nm using l-alanine p-nitroanilide (sigma) as the substrate in a mm tris phosphate buffer (ph . ) [ ] . protein content was determined by the method of lowry [ ] . kidney pieces were fixed immediately after dissection in % paraformaldehyde in mm naci, i mm na phosphate buffer (ph . ) for several hours. they were then incubated in the saline containing increasing sucrose concentrations (up to %) and frozen in isopentane cooled on dry ice. sections ( /.tm thick) were mounted on gelatin coated microscope slides and stored at - °c. primary antibodies (or culture supernatants) were diluted in mm naci, mm tris buffer (ph . ), . % tween and % bovine serum albumin (bsa) and allowed to bind overnight. bound antibodies were visualized using fluorescein conjugated antimouse ig antibodies (institut pasteur productions). kidney pieces were immersed in . % glutaraldehyde in mm naci, mm na phosphate buffer (ph . ), for h at room temperature. tissue pieces were permeabilized by . % saponin in the same buffered saline for rain and preincubated in . by indirect immunofluorescence on frozen kidney sections, clones were selected which produced antibodies that bound to the apical membrane of proximal tubule epithelial cells. each of these culture supernatants was used to immunoprecipitate kidney membrane proteins that had been in some experiments, when kidney membranes were pre-pared in the absence of the protease inhibitor pmsf, an additional kda band was immunoprecipitated by each of the anti- kda antibodies (not shown). this kda can be very easily removed from membranes by a single washing step. most probably, it is a proteolytic fragment of the kda antigen bearing the immunogenic regions but lacking the membrane domains of the protein. the kda antigen was immunoprecipitated by monoclonal antibody k , submitted to gel electrophoresis and electroblotted (see materials and methods). the band at kda was cut out and the n-terminal amino acid sequence was determined using automated edman degradation. starting from about pmol of the kda antigen, pmol pth-aminoacids were obtained in each of the successive cycles. the n-terminal amino acid sequence of the kda antigen (table i) turned out to be very similar to the corresponding sequence of aminopeptidase n (ec . . . ) which has been determined in man [ ] , rat [ ] , rabbit [ ] and pig [ ] . this sequence begins with eight hydrophilic table i . n-terminal amino acid sequence of the kda antigen. " for comparison, the n-terminal amino acid sequences of aminopeptidase n from rat kidney [ ] and from intestines of man [ ] , rabbit [ ] and pig [ ] are shown. ratkidney akg f y i s k s lg i lg illg human intestine ak g f y i s k s lg i lg illg rabbit intestine akg f y i s ka lg i lg fxlg porcintestine akg f y i s l a lg i agxlxv ) . immunopreeipitated proteins were directly submitted to sds gel electrophoresis (-) or submitted to endoglycosidase f/n-glycosidase f action ( . u at °c for h) before electrophoresis. gels were stained with coomassie blue. to check whether the kda antigen was indeed aminopeptidase n, we measured aminopeptidase n activity using alanine p-nitroanilide as the substrate [ ] . ~ln the course of immunoprecipitation experiments, enzyme activity was determined both in non-retained sampies (incubation mixture after removal of immunoadsorbed proteins by low speed centrifugation of protein a beads) and in immunoadsorbed protein samples (washed protein a bead suspensions just before sds elution). a significant proportion ( %) of peptidase activity was immunoprecipitated in a single step by monoclonal antibody k (table ii) . an unrelated monoclonal antibody k [ ] was unable to precipitate any detectable enzyme activity. in the same experiment, the antigen precipitated by antibody k was visualized as a kda band after sds-gel electrophoresis, which was absent from samples incubated with the control antibody k (fig ) . proteins immunoabsorbed by antibodies k or k , and eluted in sds, were treated by endoglycosidase f (see materials and methods). the kda band was converted to a lower molecular mass polypeptide, at kda (fig ) . this demonstrates that torpedo zminopeptidase is heavily glycosylated, deglycosylation reducing its apparent molecular mass by about %. note, for compar-ison, a small effect of endoglycosidase treatment on the heavy immunoglobulin chain migration (at about kda) in both k and k samples. bands at kda most probably correspond to endoglycosidase f ( kda molecular mass). to investigate whether the torpedo kidney aminopeptidase n was a monomer or an oligomer, we studied the velocity sedimentation in sucrose gradients of both the kda antigen and the aminopeptidase n activity, solubilized in nondenaturing conditions. kidhey membranes were treated by % chaps + . % c e . solubilized proteins were sedimented in - % linear sucrose gradients at rpm for h. fractions were collected from the bottom of the gradients and their kda antigen content and peptidase activity were estimated as described in materials and methods. both antigen and activity were recovered as a single peak, with a sedimentation coefficient of . s (fig ) . considering that aminopeptidase n is a hydrophilic membrane protein with a single transmembrane domain [ , ] , we can assume that it binds only a small amount of detergent and behaves as a globular protein in sucrose gra- dients. taking the approximation of martin and ames [ ] , this would give an approximate molecular mass of kda for the kda antigen, which therefore behaves, in these solubilization conditions, as a monomeric protein. then kidney membranes were solubilized by % chaps alone, in the presence of . mm mgc (fig a) or of mm edta ( fig b) . after velocity sedimentation in sucrose gradients containing either mgc or edta, a broad peak of antigen was found, with a sedimentation coefficient around s. in the presence of mgci , the peak of activity was also broad and it did not coincide with that of the antigen ( fig a) : it was shifted to lighter fractions. in addition, a second smaller peak of activity was recovered around . s. in the edta containing gradients, the total peptidase activity was much lower than in the mgc conditions. it corresponds to edta insensitive peptidase activity. its distribution ( fig b) revealed a peak at . s, very similar in amplitude and position to the . s noticed in mgci . some activity was also detected in denser fractions. by difference, it was possible to estimate the distribution of edta sensitive peptidase activity, which is similar to that of the antigen (fig c) . aminopeptidase n has been shown to be a zinc metalloprotein [ , ] and inhibition of its activity by the divalent cations chelator edta was not surprising. when solubilized by chaps alone, torpedo aminopeptidase behaves as a large size oligomer. this oligomer is dissociated in the presence of . % ci eg, and appears therefore to be stabilized by hydrophobic interactions. the presence of dithiothreitol during solubilisation and centrifugation does not modify the sedimentation profile of torpedo aminopeptidase (data not shown), showing that disulfide bonds are not necessary for its oligomerization, as reported for mammalian aminopeptidase [ ] . torpedo kidney organization has been schematically described by gfrard [ ] . a thin tubular segment, with a cili- ated epithelium, connects the glomerule to the proximal convoluted tubule, characterized by its large epithelial cells with their typical brush border apical membrane. the distal tubule follows, with a flat epithelium and epithelial cells devoid of flagella or microvilli. in the large field view presented in figure (upper panel), proximal tubule sections are concentrated on the left whereas distal tubule sections are mainly located on the right. anti- kda antibodies labeled very intensely the apical membrane of proximal tubule epithelial cells. no staining of the basolateral membranes was observed, even at higher magnification (lower panel). distal tubules and glomerules were not labeled. in sections of boundary segments of the proximal tubule, the staining was restricted to the apex of the larger epithelial cells. the distribution of the kda antigen was studied at the subeellular level using monoclonal antibodies k and k (figs and ) . these antibodies were chosen because their binding to kidney membranes adsorbed onto nitrocellulose was not affected by incubation of the blots in % glutaraldehyde (dot blot experiment not shown). binding of the mabs k or k was indirectly visualized using antimouse igg antibodies conjugated to nm gold particles. a general view of the apical portion of a proximal tubule epithelial cell is presented in figure . the apical membrane covers numerous microvilli, about . #m large and several microns long. this membrane is homogeneously decorated with numerous gold particles. in contrast, no labelling was associated to the basolateral membrane, or to the numerous vesicular or vacuolar membranes located under the brush border. in higher magnification views ( fig ) the abundance and selectivity of the kda antigen distribution are demonstrated. the density of gold particles associated to the apical membrane (about particles/btm ) was estimated on membrane profiles perpendicular to the planes of the section. gold particles are located extracellularly, at some distance (about nm) from the membrane, except of course on tangential sections (fig , top panel) . in some sections of proximal tubules, ciliated cells were found intercalated between epithelial cells (fig , lower panel) . no gold particles were associated to their apical membrane, nor to basolateral membranes, in contrast to the microvilli plasma membrane of the adjacent epithelial cells. working on torpedo kidney membranes, we have characterized a major immunogen of the brush border membrane of proximal tubules. in a fusion experiment, we selected hybridoma clones secreting antibodies which bound to these membranes. % of these clones produced antibodies which precipitated the same kda antigen, a protein which was only a minor component of kidney membranes. this antigen was highly purified in a single immunoprecipitation step, followed by gel electrophoresis. this purification allowed us to determine the n-terminal amino acid sequence of the antigen, which turned out to be very similar to that of aminopeptidase n from various mammals including man. the first eight amino acids, which probably correspond to the cytoplasmic domain, are identical in all sequences determined; the following amino acids are hydrophobic and some c o n s e r v a t i v e changes are observed. in addition to the kda antigen, monoclonal antibody k immunoprecipitated efficiently aminopeptidase n activity, showing that the kda antigen carries the activity. mammalian forms of aminopeptidase n are heavily glycosylated membrane proteins composed of a single type of subunit (m r of kda, detergent form [ ] ) which possess a single transmembrane domain near its n terminus (see [ , ] for reviews; [ ] ). the torpedo protein was not extracted from membranes at high ionic strength or by alkaline (ph ) treatment, demonstrating that it is an intrinsic membrane protein. it gave a broad band after sds gel electrophoresis and migrated in these conditions with an apparent molecular mass of kda. when deglycosylated by endoglycosidase f/n-glycosidase f, the antigen gave a thinner band at kda. thus, the torpedo enzyme is also a heavily glycosylated protein; this would explain its high immunogenicity. when solubilized in % chaps alone, torpedo aminopeptidase n was recovered in large protein complexes with a sedimentation coefficient around s. dithiothreitol was unable to dissociate these complexes (not shown), demonstrating that disulfide bonds were not necessary for the oligomerization of the enzyme, as previously reported [ ] . in contrast, low concentrations of ci e were sufficient to dissociate the oligomers showing that they were stabilized by hydrophobie interactions. in some experiments (not shown), when proteases were not inhibited by pmsf, a kda polypeptide was immunoprecipitated in addition to the kda antigen. this polypeptide, which most probably is a proteolytie fragment of aminopeptidase n, could be eliminated by a simple washing of the crude membranes. this shows that it has lost the transmembrane anchoring domain of the molecule and that it does not remain membrane associated through an interaction with an intact aminopeptidase n monomer, as has been reported for proteolytic digests of porcine [ , ] and bovine [ ] aminopeptidase n. in t o r p e d o kidney, a high density of the antigen (about gold particles/pan ) was detected in the brush border membrane in proximal tubule epithelia. no gold particles were associated with the apical membrane of other cells, either ciliated cells in proximal tubules or epithelial cells in distal tubules (not shown). no antigen was detected in other membranes of proximal tubule epithelial cells, neither basolateral membranes nor intracellular membranes such as vacuoles or vesicles membranes were labeled. mammalian peptidase n is concentrated in microvillar apical membranes of both kidney proximal tubules and intestinal epithelia. but in enterocytes, enzyme was found associated to purified basolateral membrane fractions, where its specific activity is times lower than that of the brush border membrane fractions [ ] . similarly, in madin-darby kidney cells transfected with human aminopeptidase n edna, this enzyme was predominantly recovered in the apical membrane but some of it (about %) was associated to basolateral membranes [ ] . it was therefore surprising that we could not detect any enzyme in basolateral membranes of torpedo kidney, especially considering the high density of gold particles associated with the apical membrane and the absence of background staining. sorting of aminopeptidase n has been extensively studied in various cell types: hepatocytes [ ], enterocytes [ , ] and kidney epithelial cells [ ] . in madin-darby canine kidney cells, aminopeptidase n is directly sorted to the apical plasma membrane [ ] . no label was detected in intracellular vesicles in our experiments, suggesting a reduced accessibility of antibodies to these structures. this could also reflect a low turnover of the antigen or possibly that antibodies bind only to mature forms of tl~ enzyme and not to its precursor forms, present in the endoplasmic reticulum and the golgi apparatus [ , ] . the major part of mammalian aminopeptidase n, including the catalytic domain, is located on the extracellular surface of the epithelium [ , ] . this is also the case for the torpedo kidney enzyme. indeed, epitopes labelled by antibodies k and k are exposed in the extracellular medium since gold particles were on the external side of microvilli membranes, located at some distance (about nm) from the membrane. considering the size of gold particles ( rim) and that of immunoglobulins g ( nm), we can estimate that the epitope is about nm outside the plane of the membrane and therefore that a large part of the antigen is localized in the tubular lumen. in conclusion, we have identified the kidney aminopeptidase n from torpedo marmorata. to our knowledge, it is the first characterization of this enzyme from torpedo and from fish in general. monoclonal antibodies prepared here will be useful tools for further functional studies since they bind to the solubilized protein without loss of its enzymatic activity. fine ultrastructural studies will also be possible since these antibodies are able to probe aminopeptidase n in its membrane environment, even after glutaraldehyde fixation of tissues. by its subcellular distribution, its biochemical properties and its n-terminal amino acid sequence, this enzyme closely resembles the mammalian enzymes. biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation protein-electroblotting and microsequencing strategies in generating protein data bases from two-dimensional gels rabbit intestinal aminopeptidase n. purification and molecular properties a simple method for polyethylene glycol-promoted hybridization of mouse myeloma cells isolation and characterization of membrane bound arylamidases from human placenta and kidney topology of microvillar membrane hydrolases of kidney and intestine rat intestinal brush border membrane peptidases. i. solubilization, purification and physicochemical properties of five different forms of the enzyme continuous culture of fused cells secreting antibodies of predefined specificity cleavage of structural protein during the assembly of the head of bacteriophage t human myeloid plasma membrane glyeoprotein cd (gpl ) is identical to aminopeptidase n protein measurement with folin phenol reagent the aminopeptidase from hog intestinal brush border a method for determining the sedimentation behavior of enzymes: application to protein mixtures evidence for the transit of aminopeptidase n through the basolateral membrane before it reaches the brush border of enterocytes l'acetylcholinesttrase des organes ectriques des poissons complexes membranalres sorting of endogenous plasma membrane proteins occurs from two sites in cultured human intestinal epithelial cells (caco- ) subcellular fractionation and subeellular localization of aminopeptidase n in rabbit enterocytes immunological identification of a new x m r membranebound protein in torpedo electric organ molecular and cellular basis of digestion complete amino acid sequence of human aminopeptidase n deduced from cloned edna the oligomerie structure of the renal aminopeptidase n from bovine brush border membrane vesicles anchoring and biosynthesis of stalked bursh border membrane proteins: glycosydases and peptidases of erythrocytes and renal tubuli look at ( ) separate promoters control transcription of human aminopeptidase n gene in myeloid and intestinal epithelial cells a human liver aminopeptidase n. the amino acid and carbohydrate contents, and some physical properties of sialie acid containing glycoprotein electrophoretie transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some application on the subunit structure of particulate aminopeptidase from pig kidney amino acid sequence deduced from a edna suggests it encodes the zn-peptidase aminopeptidase n aminopeptidase n is directly sorted to the apical domain in mdck cells human aminopeptidase n is a receptor for human coronavirus e the amino acid sequence of the hydrophobic anchor of rabbit intestinal brush border aminopeptidase n we thank drs jp le caer and j rossier (institut alfred fessard, cnrs, gif-sur-yvette) for amino acid sequencing and dr s o'regan for improving our manuscript. this work was supported by a grant from dret (n ° / ). ° key: cord- -ogu gmcb authors: da cunha, nicolau b.; cobacho, nicole b.; viana, juliane f.c.; lima, loiane a.; sampaio, kamila b.o.; dohms, stephan s.m.; ferreira, arthur c.r.; de la fuente-núñez, césar; costa, fabrício f.; franco, octávio l.; dias, simoni c. title: the next generation of antimicrobial peptides (amps) as molecular therapeutic tools for the treatment of diseases with social and economic impacts date: - - journal: drug discov today doi: . /j.drudis. . . sha: doc_id: cord_uid: ogu gmcb anti-infective drugs have had a key role in the contemporary world, contributing to dramatically decrease mortality rates caused by infectious diseases worldwide. antimicrobial peptides (amps) are multifunctional effectors of the innate immune system of mucosal surfaces and present antimicrobial activity against a range of pathogenic viruses, bacteria, and fungi. however, the discovery and development of new antibacterial drugs is a crucial step to overcome the great challenge posed by the emergence of antibiotic resistance. in this review, we outline recent advances in the development of novel amps with improved antimicrobial activities that were achieved through characteristic structural design. in addition, we describe recent progress made to overcome some of the major limitations that have hindered peptide biosynthesis. most drugs currently used therapeutically were obtained as naturally occurring molecules purified from microorganisms, plants, and animals [ ] . it is estimated that, from to , anti-infective agents based on natural product scaffolds accounted for up to % of all approved antibacterial new chemical entities (nces), thus attesting for the importance of natural products as the main source of therapeutics against pathogenic bacteria [ ] . in this context, microorganisms, notably members of the gram-positive phylum actinomycetes, have been the workhouse source of clinically approved antibacterial agents. the high diversity of candidate molecules extracted from microorganisms frequently leads to the discovery of new compounds with distinct mechanisms of action compared with drugs currently used for clinical purposes. however, in recent years, the traditional screening of drug candidates belonging to completely new antibacterial classes from microbial sources has suffered a considerable decline, mostly because of the presence of numerous, already well-characterized, molecules [ ] . this scenario is particularly worrisome because of the increasing emergence of bacterial resistance to antibiotics, notably in developing countries, where there is a widespread and indiscriminate use of antibiotics for clinical and veterinary purposes. the search for new therapeutic molecules for commercial applications is an ongoing in the pharmaceutical industry, which now is focusing on not only drug prospection, but also the modification of existing antibiotics in a timely fashion that meets the customer's needs [ ] . among the diverse naturally occurring anti-infective agents that have been discovered to date, amps are particularly important [ ] . these molecules are versatile, highly specific antimicrobial compounds that constitute promising candidates for commercial and clinical uses. amps are essential components of the innate immune system of vertebrates and the nonspecific host defense system of plants, fungi, and invertebrates that evolved over . billion years ago as potent anti-infective agents against different viruses, bacteria, fungi, and parasites. these small peptides usually comprise - amino acid residues and are - kda in size [ ] . commonly, they show a cationic structure rich in positively charged arginine and lysine residues, which favors the interaction between these peptides and microbial cytoplasmic membranes. however, although less common, there are also anionic amps, notably in plants. amps usually kill pathogens by interacting with membrane phospholipids, resulting in membrane permeabilization and subsequent disruption. these peptides also show different secondary structures, such as b-sheets stabilized by two or three disulfide bridges and often display a helical amphipathic structure [ ] [ ] [ ] . amps can target a variety of essential metabolic processes in the plasma membrane or at extra-and intracellular sites, and frequently exhibit immunomodulatory properties that stimulate cytokine production while repressing inflammation, can kill cancer cells, and promote wound healing [ ] . in eukaryotes, most natural amps are encoded by specific genes, which are constitutively expressed at basal levels and rapidly transcribed after induction by contact or exposition to invading pathogens. thereby, a variety of different amps can be found simultaneously in organisms, such as plants, in response to pathogen stimuli at different organs, such as roots, seeds, flowers, stems, and leaves [ ] . recent advances in in silico drug design and high-throughput screening of compound libraries based on protein-protein interactions have triggered a growing interest in the discovery of new antibacterial drugs from microbial, plant, and animal origins. such efforts have contributed to uncover a variety of molecules with novel structures and improvement of the currently available classes of antibiotic, such as b-lactams, ketolides, macrolides, glycopeptides, aminoglycosides, oxazolidinones, and other antiinfective agents [ ] . the use of viral vectors based on the tobacco mosaic virus (tmv) and potato virus x (pvx) for cloning and massively expressing amp genes in nicotiana benthamiana appears to be an interesting new approach to considerably enhance recombinant peptide production before structural and functional characterization. the rapid, high-throughput staggered biosynthesis of peptides using agroinfiltrated leaves of n. benthamiana constitutes the most promising strategy for delivering new antigenic peptides and vaccine candidates, addressing the interest of many private and state health institutes [ ] . finally, gene editing using the clustered regularly interspaced short palindromic repeats (crispr)-crispr-associated protein (cas) system is another molecular tool that could revolutionize the recombinant biosynthesis of amps [ ] . this approach specifically targets dna sequences by adding, removing, or replacing dna segments mediated by cellular dna repair mechanisms of site-specific double-stranded breaks. it allows the edition of specific genes within the host genome to integrate the fragments containing the coding sequence of a particular amp in a hotspot site to enable increased amp biosynthesis. working on a case-tocase scenario, this expression strategy could overcome major drawbacks in the production of amps, notably the low biosynthesis levels presented by plant and animal amps [ ] . the antimicrobial activity of a given amp is specifically related to its amino acid composition and physical chemical properties, such as positive net charge, flexibility, size, hydrophobicity, and amphipathicity [ , ] . marginal changes in peptide residue sequence are normally followed by major changes in antimicrobial activity [ ] . amps present different mechanisms of action, many of them well described in the literature. the molecular interactions of some amps and microorganisms rely on the variety of targets presented by microorganisms, particularly gram-positive and gram-negative bacteria. [ ] . generally, the mechanisms of action of amps can be classified into two basic groups: (i) the disruptive mechanisms, which are associated with membrane lysis; and (ii) the membrane undisruptive mechanisms, which focus on neutralizing intracellular targets [ ] . independently of the proposed group, the first step of any mechanism is the molecular interaction between the amp and the cytoplasmic membrane. the driving force of such interaction is the electrostatic force presented by the amp, which is normally cationic, and the polyanionic surface of bacteria [ , ] . differences in the cell wall composition among bacterial groups affect directly the mode of action of amps. gram-negative bacteria present three major layers in the cell envelope: (i) the outer membrane (om), comprising a lipid bilayer, mostly of lipopolysaccharide (lps) interlaced by teichoic acid, which has a major role in protection against the environment; (ii) the peptidoglycan cell wall, comprising repeated units of a disaccharide (n-acetyl glucosamine-nacetylmuramic acid) linked by pentapeptide side chains; and (iii) the cytoplasmic membrane-phospholipid bilayer. by contrast, in gram-positive bacteria, there is no om, but the peptidoglycan layer is thicker than in gram-negative bacteria [ ] . the disruptive mechanisms are presented in four classical models: (i) the toroidal model; (ii) the carpet model; (iii) the aggregate model; and (iv) the barrel model. recently, new disruptive models or models indirectly associated with membrane disruption were described: (i) the disordered toroidal model; (ii) the membrane thinning/thickening model; (iii) the charged lipid clustering model; (iv) the non-bilayer intermediate model; (v) the oxidized lipid targeting model; (vi) the anion carrier model; (vii) the nonlytic membrane depolarization mode; and (viii) the electroporation model. all the models are non-mutually exclusive, allowing that a single amp might present a multiple-hit strategy based on two or more simultaneous mechanisms. in the toroidal model, the amp binds the membrane and forms a 'flip-flop' translocation channel that opens the membrane vertically, with the amp remaining closely associated to the lipid head-groups throughout the process [ , ] . the carpet model provides the disruption of the membrane without the internalization of the amp. by contrast, the amp remains associated with the membrane until a crucial concentration of the peptide contributes to weaken the hydrophobic interactions of structural phospholipids and amps form a carpet structure that increase membrane disruption [ ] . in the aggregate model, the amps have a detergent-like role by interweaving the phospholipids and disaggregating them, similar to a true detergent. depending on the amp and the membrane composition, this mode of action does not provide the membrane rupture. instead, it just forms a thin channel for the crossing of amp and other molecules into the cell [ ] . the barrel model provides the formation of a regularly organized aggregate of amps that interacts and associate with the membrane. the amp oligomer leads to the formation of pores in the lipid bilayer by intimately interacting its hydrophobic side chains with hydrophobic parts of the membrane. the transmembrane pores allow the internalization of the hydrophilic part of the amp that faces the internal region of the membrane [ ] . lastly, there is the model that shares the most similarities with the four classic models: the disordered toroidal model. this mode of action provides a stochastic pore formation after inward lipid distortion that allows the aggregation of a maximum of two peptides in the center of the pore. however, in the external peripheral region of the pore, many peptides are set up before translocation [ , ] . the undisruptive mechanisms are based on the amp crossing the membrane because of the combined features of amp sequence and membrane composition, and the inhibition of some reactions of cellular metabolism, causing cell death. there are two different ways that an amp enters the cell. the first is an obscure spontaneous translocation across the membrane; the other is mainly the result of the presence of a secondary structure in the amp that causes membrane permeabilization. in this method, named the shai-matsuzaki-huang method, a a-helical amp binds parallel to the membrane. hydrophobic residues facing the membrane permit the internalization of part of the amps and the change of their organization to a transversal mode by forming toroidal pores [ , , ] . the other model suggests that the b-sheet amps are organized into a flat-aggregate form that allows the insertion of some aromatic residues in the membrane and the opening of thin translocational spaces in which the amps can cross the membrane [ ] . once across the membrane, the amps can target a variety of intracellular sites, such as gene promoters and coding sequences, mrna-binding sites, enzyme regulatory sites, and protein prefolding sites. such inhibitory interactions involve blocking both dna transcription and/or rna translation, or incorrect protein folding, triggering the failure of metabolic pathways and cell death [ ] . recently, a new undisruptive mechanism was discovered in amp-sensitive bacteria. species such as escherichia coli and salmonella spp. have evolved the ability to detect and prevent amp antibacterial activities by triggering pathways involved in sensing and amplifying resistance to cationic amps. resistance is provided by the phoq/phop system of e. coli, which comprises the activation of the amp detector kinase phoq by mg + and ca + , which phosphorylates and activates the transcription factor phop [ ] . the active phop activates pagp, which encodes a resistance enzyme against amps; mgta, encoding a transporter of mg + ; and hdea, encoding a chaperone responsive to acidic conditions. phosphorylated phop activates the transcription of the quee gene, encoding an enzyme that controls the biosynthesis of hypermodified guanosine found in rare trnas and, once overexpressed, inhibits cell division by downregulating the bacterial divisome [ , ] . sublethal concentrations of the c g amp, a highly cationic, amphiphilic peptide derived from the c-terminal sequence of human protein platelet factor , can trigger the phoq-dependent filamentation of wildtype e. coli by the overexpression of quee. the bacterial filamentation (tens to hundreds of microns in length) prevented fully bacterial growth and was detectable in genetically engineered strains with a phop-regulated promoter driving the transcription of a yellow fluorescent protein [ ] . another good example is buforin ii, a potent amp from chinese toad bufo gargarizans, with antimicrobial activity against a range of microorganisms. once inside the cell, buforin ii inhibits gene expression by associating with dna and mrna during transcription and translation. the same occurs with melittin, a bee venom peptide active against both gram-positive and gram-negative bacteria, as well as some pathogenic fungi, which is a typical example of nondisruptive amp, discovered during the late s, that supported the carpet model of microorganism inhibition detailed in [ , , ] . the development of antibiotics has had a major impact on modern medicine. however, the increasing emergence of antibiotic resistance and the limited development of novel classes of antibiotic over the past four decades has led to a scenario in which some infections are no longer treatable with available antibiotics [ ] . antimicrobial resistance (amr) is defined as the resistance of microorganisms to an antimicrobial against which they were once sensitive [ , ] . amr is an inevitable evolutionary outcome once all organisms develop genetic mutations that can improve fitness and lead to selection as a response to the selective pressure of the environment. in fact, more than % of pathogenic bacteria are resistant to at least one type of antibiotic [ ] . undesired selection of microbial cells with resistance-conferring mutations or other resistant elements represents the main drawback in long-term treatment efficiency of patients, leading to intensive medical research to discover new targets to overcome multidrug resistance (mdr) [ , , ] . the current classes of antibiotic face a constant threat represented by the diverse bacterial resistance mechanisms. the eskape pathogens (enterococcus faecium, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa, and enterobacter species) represent the most dangerous pathogens to immunocompromised patients because they are commonly isolated as drug-resistant (dr) or mdr microorganisms [ ] . there are two major ways in which common bacteria evolve to become resistant: by intrinsic or acquired resistance against amps. intrinsic resistance occurs as a natural consequence of the presence of amps in the natural environment of bacteria, which develop mechanisms to resist antibiotic action [ ] . this can happen via passive or inducible mechanisms. passive resistance is always associated with less tight interactions between bacteria and amps because of the inherent accumulation of more positive charges in lipid a, and is more common in genera such as proteus, providencia, burkholderia, morganella, and serratia [ ] . inducible resistance is a consequence of perennial, reversible modifications at the molecular level in both gram-positive and gram-negative bacteria [ ] . acquired resistance is a product of high-fitness mutants that often contain more than one mutation that is unrelated to the amp selection. bacterial mutated genes provide altered genetic systems that allow bacterial growth in the presence of amps and can be identified by experimental procedures with and without amp in the culture media [ ] . the bacterial om and inner membrane architectures can be altered and sites for the ligation of amps can be protected in response to reduced levels of ca + , mg + , and specific proteins, and changes in lipid composition. this considerably reduces membrane fluidity and permeability to polymyxins, defensins, and cathelicidins [ , ] . the bacterial membrane phospholipid content, such as phosphatidylethanolamine (pe), phosphatidylglycerol (pg), and cardiolipin (cl), reflects a global state of protection against pore formation [ ] . the biosynthesis, turnover, and translocation of phospholipids to target sites in the membrane can be modulated by the expression of proteins closely related to resistance against amps [ , ] . profiles of cationic amp resistance can be shown by the % increase in the total content of cl in liposome model membranes [ ] . strains of s. aureus resistant to methicillin, for example, can increase lipid biosynthesis and translocation to the membrane by simply activating genes that encode multiple peptide resistance factor (mprf), cardiolipin synthase (cls), and phosphatidylglycerol synthase (pgsa) [ , ] . the affinity between the cell membrane and amps can be considerably reduced by minimizing the negative charge of the phospholipid bilayer (i.e. the lipid composition). in addition, twocomponent signal regulatory systems (tcs), such as the phoq/ phop system in p. aeruginosa and the apsr/apss in staphylococcus epidermidis, can act together with lipid modifications to enhance resistance against cationic amps [ ] . the cell wall is the outer barrier that acts as a secondary physical protection structure against pore formation in bacteria. it gives strength to the bacterial cell and influences the final cell format. amps often establish ionic and/or hydrophobic interactions with the cell wall. modifications of the polysaccharide bilayer of peptidoglycan and teichoic acids in the cell wall are particularly interesting in gram-positive bacteria. the amp-cell wall interactions can be avoided in s. aureus and staphylococcus xylosus, which present multiple copies of the dlt operon, a regulatory sequence that, when active, promotes d-alanylation of teichoic acids in the cell wall, reducing their anionic charges [ ] . in some gram-negative bacteria, the affinity of lipopolysaccharides (lps) in the om by cationic amps can be reduced by the increase of positively charged lipid a with substituents such as palmitate, phosphoethanolamine, and -amino- -deoxy-l-arabinose at -or -phosphate groups [ ] . these modifications in lipid a are also induced by the phoq/phop system and appear to be closely related to cationic amp resistance, mainly against polymyxin b, in gram-negative bacteria. glycosylation of lipid a was recently reported as another lipid modification that results in polymyxin resistance in ei tor vibrio cholerae [ ] . the addition of glycine to lipid a is regulated by the almefg operon, which controls the expression of several proteins (aime, almf, and almg) related to glycine activation and transfer to lipid a. glycine is activated by adenylation by alme, which transfers active glycine to the -phosphopantetheine group of almf. once active, almf donates the glycine to the hydroxylauryl chain of lipid a, a transfer reaction performed by almg [ ] . in bacteria, is commonly stated that the thicker the cell wall, the less efficient an antibiotic is, and, by extension, an amp will be. improving the cell wall thickness is a strategy adopted by s. aureus against erythromycin, vancomycin, acriflavine, and many amps. the cell wall peptidoglycan layer can increase in thickness by the upregulation of glutamine synthase in e. coli strains that are resistant to magainin ii, a characteristic absent in susceptible strains [ ] . some bacteria have an arsenal of molecules that prevent cellular metabolism from suffering stress caused by amps. several pathways can be up-or downregulated to increase the biosynthesis of proteases, modification of membrane sites recognizable by amps, overproduction of biofilms, and suppression of superficial elements related to pore formation [ ] . an inherent mechanism of amp resistance by bacteria is the partial or total proteolytic cleavage of amps. strains of s. aureus overproducing the metalloprotease aureolysin are resistant to cathelicidin. proteus mirabilis producing high amounts of the metalloproteases zapa and ll- can avoid the antimicrobial activity of b-defensin (hbd ) simply by breaking down the protein into six or nine innocuous peptides [ ] . other systems for the upregulation of the biosynthesis of bacteria proteases have been discussed elsewhere. the nisin resistance gene (nrs) of resistant strains of lactococcus lactis is a central element of nisin-controlled gene expression systems (nice). a protease encoded by the nrs gene cleaves the c terminus of nisin, conferring in vitro resistance for non-nisin-producing l. lactis [ ] . by analogy, the speb cysteine protease of streptococcus pyogenes catalyzes the proteolysis of ll- in vitro and in patients infected with resistant bacteria. under stress caused by amps, s. pyogenes synthesizes a g-related a -macroglobulin-binding (grab) protein that acts as a potent inhibitor of the protease inhibitor a -macroglobulin, forming a cluster named the 'grab-a -macroglobulin complex' that retains an active speb on the bacterial surface for posterior ll- cleavage, causing bacterial resistance [ ] . molecular traps that capture amps and biofilm production are also important resistance mechanisms evolved by different species of bacteria. for example, l. lactis expressing pilb, a pilus backbone protein in gram-positive bacteria, can trap cathelicidin along with the cell wall, avoiding contact with the cytoplasmic membrane. biofilm-producing bacteria can resist many different antibiotics. biofilm-mediated resistance results mainly from extracellular polymeric substance (eps), a liquid comprising mainly amyloid and adhesive fimbriae, extracellular dna, and exopolysaccharides that embeds multiple cells throughout the biofilm matrix [ ] . the eps extracellular dna of p. aeruginosa can induce resistance against polymyxin b and colistin in response to the chelating of environmental cations. the decrease in the concentration of biofilm cations modulates the upregulation of lps modification genes associated with resistance, a typical mechanism in resistant strains of p. aeruginosa [ ] . in addition to their natural antimicrobial activities, amps also have other potential applications in the therapy and treatment of disease. for example, some amps have antitumoral and immunomodulatory activities and these peptides are important drug candidates known as anticancer peptides (acp) and host defense peptides (hdp), respectively. several acps show improved absorption and higher specific cytotoxicity to tumor cells and fewer adverse effects compared with chemical agents. the high number of interactions between acps and tumor receptors could result from the presence of abundant anionic sites dispersed on the tumoral cell, resulting in rapid and selective binding and cell death [ ] . by contrast, hdps frequently show weak antimicrobial activity in mammals, but are potent triggers of the immune response through a variety of mechanisms that affect the innate immunity of hosts. these peptides have diverse structures and sequences because of constant interactions with different microbial cells presenting multiple infective strategies [ , ] . the innate immune systems of mammals show characteristics of nonspecific, quick antimicrobial therapies mediated by the activation of elements of resistance against pathogens [ ] . hdps act as triggers of immune responsive elements by several mechanisms, such as the upregulation of the expression of hundreds of genes in monocytes and epithelial cells, the induction of differentiation responses and chemokine synthesis, and promotion of angiogenesis, and inflammatory and wound-healing responses [ ] . some therapeutic peptides have already reached market status, with more than peptides currently available on the market in the usa. it is estimated that, in , approximately therapeutic peptides reached preclinical trials and were included clinical trials in the usa [ ] . despite the development of many potential pharmaceutical peptides, the road to market is restrictive because drug candidates must meet several requirements, such as similar or higher efficacy and tolerability compared with already existing analogous drugs, improved pharmacodynamics and pharmacokinetics, low toxicity, and safe use [ , ] . economic issues must also be satisfied, mainly relating to market competition, scalable production, and intellectual property. for these reasons, more than % of novel therapeutic candidates fail to achieve marketable status [ ] . regulations surrounding the use of such molecules are also determined based on the physicochemical properties and manufacturing of each peptide. the us food and drug administration (fda) usually ranks peptides as conventional drugs mostly because their chemical structures exceed residues, although exceptions are made mostly in the case of vaccines, which are ranked as biological products [ ] . in europe, the european medicines agency (ema) evaluates the source of the peptide [ ] . if the molecule was screened from a natural biological source, it is treated as a biological entity. by contrast, chemical entities are those that were chemically synthesized in vitro. in addition, manufacturing must guarantee the identity, purity, potency, and individual lot consistency [ , ] . examples of drug candidate failure are perhaps more common than might expected. the most famous case was the peptide magainin (pexiganan), a potent amp isolated from the african clawed frog xenopus laevis. after completion of phase clinical trials in , the fda did not approve its commercialization because the drug was not proved to be more effective compared with the antibiotics utilized during the trials [ ] . notable expansion in the peptide-based drug discovery field over the past years, encouraged the screening and testing of new drug candidates. the approval rate since for peptides is around %. this reflects an increasing number of peptides entering annual clinical trials, one in compared with in . most candidates that enter phase clinical trials are painkillers (> %), or anticancer and anticardiovascular disease agents. acps dominate phase ( %) and ( %) clinical trials, followed by painkillers, anti-infectious disease and antiallergen agents [ ] . historically, the search for new efficient amps was based on the high-throughput screening (hts) of biologically active molecules. this concept relies on the discovery of naturally occurring peptides using classic purification and in vitro and in vivo techniques for checking antimicrobial activity. many amps have been identified and tested against clinical and natural strains of pathogens using this approach [ ] . bioactive peptides obtained from natural sources have been under evolutionary pressure for millennia, and consistently show high stability and target affinity and/or specificity. however, naturally occurring amps are normally synthesized at low rates by their biological sources, many are susceptible to protease degradation, and have low bioavailability (i.e. the presence of bioactive molecules at usual low levels). despite the recent advances in hts techniques, this approach is laborious and it is difficult to produce high yields of peptides in a scalable fashion [ , ] . new amps with potent antimicrobial activities and lower propensity to select for drug resistance have been intensely investigated. in silico methodologies for the rational design of peptides aim to improve the biological activities and increase production efficiency, speeding up biosynthesis and decreasing production costs. these rational tailored peptides represent a new generation of designer drugs to simultaneously overcome pathogen resistance and enhance microbial killing [ ] [ ] [ ] . amp design is based primarily on structure-function relations of host-derived synthetic amps and computational analysis of surface interactions between the peptide and pathogen structures. there are three main ways to enhance peptide activities through computational design: (i) epitope and net charge engineering by de novo sequence optimization of amp motifs; (ii) changes in posttranslational patterns of glycosylated peptides by amino acid substitution in glycosylation sites; and (iii) engineering different peptides as chimeric molecules and/or biomaterial surfaces with amp properties [ , , ] . sequence optimization of motifs is mainly applied for the design of cationic a-helical amps with improved and specific antimicrobial activities and low toxicity to mammalian cells. diverse engineered cationic antimicrobial peptides (ecaps) have been synthetically produced in laboratories worldwide, and show a range of in vitro and in vivo antimicrobial activities. examination of structure-function relations is primarily performed for the prediction of protein interfaces to infer protein-protein and protein-lipid interaction networks [ , ] . interface prediction is based on the physicochemical properties of residues in interfaces of protein complexes and by overlapping interface and non-interface segments of protein motifs. the most prominent characteristics of amps for peptide design are the sequence conservation of interface residues, proportion of the types of amino acid residue, relative presence of secondary structures, solvent accessibility, and sidechain conformational entropy [ ] . some amps are synthesized as glycoconjugates, peptides carrying a variety of n-or o-linked glycans that are crucial for peptide recognition, binding, and protein-protein interactions. most surface glycans are post-translationally added in the golgi after the peptide has passed through the secretory pathway [ ] . plantderived amps are the main primary targets for glycosylation engineering that aims to improve biological activity against phytopathogens and to humanize glycosylation for clinical use in humans. plant n-glycans differ considerably from those in mammals. typically, mammalian a , fucose (n-acetylglucosamine of the core), b , n-acetylglucosamine (b-mannose of the core), and b , galactose combined with sialic acid and linked to the terminal n-acetylglucosamine are substituted in plants, by an a , fucose, a bisecting b , xylose, and a b , galactose and fucose a , -linked to the terminal n-acetylglucosamine, respectively [ , ] . another important issue concerning the humanization of amp glycosylation is avoiding the addition of allergenic glycoepitopes to the peptide surface, because humans are frequently allergic to plant a , fucose and b xylose. currently, the main efforts to minimize undesirable plant glycosylation of amps rely on avoiding the complete transit throughout the secretory pathway by confining peptides inside the endoplasmic reticulum, using n-or c-terminal retention signals, such as kdel. advances in the humanization of the glycosylation of proteins (i.e. the full substitution of non-mammalian host n-linked glycans for typical human glycans by knocking down plant xylosyl and fucosyltransferases and yeast mannosidases and expression of human glycosidases) is already a reality in the production of plant and yeast antibodies and interferons, but has not yet been fully applied to amps [ , ] . chimeric ecaps are another major group of engineered amps. they are synthesized chemically or recombinantly as fusion peptides or as antimicrobial surface-coating agents, based on the potential synergistic effect of multiple active epitopes that considerably enhances their antimicrobial activities [ ] . these features reveal an interesting aspect of chimeric ecaps, namely the potential to prevent bacterial colonization and biofilm formation, a promising approach to eliminate implant infections. after synthesis, the individual domains of chimeric ecaps must present solidbinding kinetics to nanoformulated surfaces or substrates without the loss of antimicrobial properties. these peptides remain one of the most promising engineered anti-infective agents to be popularized against opportunistic pathogens in postoperative care settings [ ] . naturally occurring amps are typically subject to proteolysis, because they comprise l-amino acids recognizable by proteases. to minimize peptide degradation, the rational design of sequences comprising analogous d-amino acids substituted for l-amino acids can consistently increase the peptide post-translational stability without altering biological function. given that the interactions between amps and the bacterial membrane are not strictly dependent on interactions mediated by specific receptors, the d-enantiomers of a peptide often retain the antimicrobial activity [ ] . another interesting modification of ecaps is the addition of polyalanine tails in the n or c terminus of the peptide. the amino acid alanine is moderately hydrophobic and, when polymerized as a repetitive peptide tail, can exceed the hydrophobic limit for insertion mechanisms in the cytoplasmic membrane. polyalanine peptides can not only be inserted into the membrane of microbes without causing significant phospholipid displacement, maintaining the bilayer integrity, but also be internalized within the cell for further modifications in metabolic pathways. therefore, polyalanine tails can induce undesirable peptide configurations that lead to the formation of peptide clusters that are unable to anchor lipid bilayers [ , ] . one of the most promising modifications of the ecap structure is peglyation: that is, the covalent addition of polyethylene glycol (peg) chains to peptides. peglyation provides improved structural stability and higher bioavailability of modified peptides and proteins. pegylated synthetic ecaps can retain their antimicrobial activity with higher target specificity, although superfluous covalently attached peg moieties can reduce the interactions between ecap and the target sites of the cytoplasmic membrane of bacteria [ , ] . when the peptide shows therapeutic functions, it must be injected into the bloodstream either in its pure form or nanoencapsulated. in such cases, the stability of the peptide must be preserved to maintain a high level of bioactivity. peptide cyclization is a major strategy to improve the serum stability of synthetic peptides. the joining of the n and c terminus backbone or the formation of internal cross disulfide bridges cyclizes the peptide, hiding proteolytic cleavage sites from specific cellular aminopeptidases [ , ] . the amidated c terminus of peptides appears to generally improve antimicrobial activity and stability. in general, amidated peptides exhibit higher antimicrobial activity. amidation of the c terminus affects the hydrophobic moment of synthetic peptides, corroborating to enhance interactions with the membrane. although this has been an important strategy to improve microbial death, it also appears to improve hemolytic activity, requiring case-by-case studies to evaluate the pros and cons of such modifications [ , ] . following the accurate prediction of structural changes, the engineered peptides can be chemically synthesized or routinely produced by many genetic engineering strategies (fig. ) . in this context, in silico interaction databases are the most valuable tools to predict the sites where peptides physically interact with the microbial cell and the optimization of motif architecture and net charge by amino acid replacements. many examples of rationally designed amps have already been described in the literature, most of which show true potential for future use in clinical settings. often, these ecaps present not only enhanced biological activities against mdr microorganisms, but also lower propensity to select for resistant bacteria in vitro compared with native analogs. table lists promising ecaps for therapeutic use against mdr microbes [ , ] . after prospection or improvements in silico, a selected amp must be produced on a large scale at a consistently high quality and under good manufacturing practice rules (gmp). the size and chemical properties of a given peptide will directly influence the choice of production strategy [ ] . there are at least four major strategies to achieve satisfactory yields of high-quality products: (i) solid-state chemical synthesis; (ii) recombinant microbe platforms; (iii) transgenic plants and animals; and (iv) cellfree expression systems. regardless of the method used, downstream processing is the crucial step before the commercialization of any peptide. this is the most expensive, time-consuming and testing phase of the production pipeline [ , ] . specially designed strains of bacteria or fungal accumulating induced mutations are typically utilized as reactors of naturally occurring amps. these strains can enhance protein synthesis and secretions, as well as upgrade peptide folding. under these circumstances, peptide yields can increase by three orders of magnitude, but in many cases, the production levels will still be below the standard production levels required [ ] . as an alternative to natural sources, the production technology can result in the chemical synthesis of partial or full peptide chains. there are three types of chemical synthesis: (i) the solution phase; (ii) the solid phase; and (iii) hybrid approaches [ ] . most of the commercially approved peptides, which are frequently small to medium in size, are synthesized by solution phase approaches [ , ] . this methodology provides standard protocols for the isolation, characterization, and purification of peptides. solidphase synthesis provides platforms for the production of large and structurally complex peptides on a large scale. the hybrid method combines characteristics of the two previous methodologies. although efficient for the production of active peptides, the three approaches are expensive because they enable prolonged to maximize peptide biosynthesis, genetically engineered bacteria and yeast cells are frequently explored as vehicles for the recombinant production of bioactive amps [ ] . many different amps have been synthesized in e. coli and pichia pastoris [ ] . despite the high therapeutic potential of recombinant amps, limited investment of companies and drawbacks in terms of poor yield, low quality, and unsatisfactory in vivo activity have restricted commercial development to only a few promising amps. regardless of these production limitations, some of these therapeutic peptides have reached advanced clinical trials before commercialization, as detailed in table . among the most important factors that limit the recombinant production of amps in microbial systems is the inner toxicity of the peptide toward host cells; however, this is not typically a limitation because many amps kill bacteria at very low, nontoxic concentrations. another concern is the low quality of the peptide product following post-translational modifications. under such circumstances, plants appear to be an interesting and promising alternative host system for the production of recombinant amps [ ] [ ] [ ] . although plants perform a range of post-translational modifications, low levels of recombinant biosynthesis of peptides are common, resulting in low quantities of purified products. however, a new transformative technology, called magnifection, has emerged as a platform for the fast production of large numbers of plant-derived recombinant proteins and peptides [ , , ] . developed by gleba and collaborators at the german biotech company icon genetics, magnifection is a transient expression platform that utilizes nicotinia tabacum or nicotinia benthamiana plants as efficient reactors for the production of massive yields of recombinant proteins, in a rapid, scalable fashion [ , ] . the process is based on the infiltration of whole plants with a suspension of transgenic agrobacterium tumefaciens cells carrying plasmids that encode viral rnas replicons (fig. ) . these gram-negative soil bacteria have key roles in infection and movement throughout plant tissues because they systemically spread through the plant to eventually reach most leaves and stems [ , ] . infiltrated plants contain viral vectors based on tobacco mosaic virus (tmv) or potato virus x (pvx) carrying amp-coding sequences, delivered by bacteria to be transiently expressed and amplified [ ] . these potent machines of transcript production use the viral machinery to enhance the production of viral proteins along with the selected amp, without stable transgene integration, resulting in massive yields of amps [ ] . speed is one of the main advantages of magnifection, because it provides expression kinetics that frequently reach the peak of peptide production - days after infiltration. such conditions allow the scale-up of plant infiltration by vacuum and peptide biosynthesis, combining elements of three biological systems (i.e. viral potent transcription, bacterial systemic spread, and plant accurate post-translational modifications) in a single expression strategy [ , ] . therefore, magnifection significantly reduces amp production costs because of a rapid and straightforward approach that results in the production of the first milligrams of amps in just weeks and up to kg in year, using a much-diluted a. tumefaciens suspension to infiltrate completely an entire green house. the high biomass of tobacco (> . kg ha À ) also contributes to the scaling up of peptide production, while reducing overall costs [ , ] . using magnifection, protein and peptide amounts frequently are - -fold higher than those observed for stable genetically transformed plants, yielding up to % of the total soluble protein (tsp). using this technology, amounts of up to g of peptides per kilogram of fresh agroinfiltrated leaves have been obtained routinely [ , , ] . the magnifection platform has experienced considerable success in the production of a variety of proteins and peptides, notably vaccines [ ] . the small size of antigen peptides and relatively simple chemical structures of some amps appear to fulfill the requirements of the platform. since , the magnifection system has been explored by the canadian biotech company medicago (http://www.medicago.com/) for the industrial production of a vaccine against h n flu in the usa. a us$ million budget financial deal was signed between medicago and the us department of defense agency defense advanced research projects agency (darpa) to develop million doses per month to avoid the risk of epidemic flu outbreaks [ , [ ] [ ] [ ] . the vaccine is currently in phase clinical trials, as is another vaccine against the variant virus h n , synthesized using the same platform [ ] . table details other vaccines already synthesized in tobacco using the magnifection system. although efficient for the transient biosynthesis of peptides, the addition of plant-specific post-translational modifications, notably the addition of n-glycans to peptides, is a potential limitation of the system and could lead to nonfunctional products or highly immunogenic vaccines. the threshold of economically accepted expression levels using magnifection is a restrictive factor similar to other previously explored recombinant platforms. although a good producer of full antibodies, the size of multimeric proteins constitutes another important challenge for the magnifection system, requiring the manipulation of two viral vectors to express two or more assembled polypeptides [ , ] . genome editing by crisprs and amp biosynthesis: advances, implications, and challenges screening and assembly is technically challenging. typically binds to short - -bp sequences. replacement of fragments longer than kb is difficult. off-target effects. target events in animals require screening. larger than zfns (coded usually by -kb dna). evidence that larger talens can lead to less specificity. off-target effects. crisprs are recently discovered bacterial adaptive immune systems that use a combination of short rnas and associated proteins to target specific sequences of dna for the generation of dsbs. crisprs revolutionized genome editing standards by presenting several technical advantages compared with previous systems, although they still have some disadvantages. easily adapted system to target and modify any genomic sequence. the cas protein, the main protein for dna recognition and cleavage, remains unchanged for any crisprs. easy to use to target numerous sites or across genomic libraries. use of multiplex-based guide rnas to simultaneously edit multiple sites. size of cas (cdna approximately . kb). expression levels and improve product quality. however, frequent gene silencing at the transcriptional level and instability of genes cloned in vectors for stable or transient expression remain major challenges limiting the efficient production of amps. another persistent issue is the poor quality of the peptides endogenously synthesized in bacteria, yeast, and plants, particularly resulting from undesirable post-translational modifications [ , ] . improved targeted genome engineering represents a sophisticated approach that could help minimize such limitations. over the past decade, alternative genome-editing tools, such as artificial engineered enzymes, zinc-finger nucleases (zfns), and transcription-activator-like effector nucleases (talens), have been successfully utilized to modify the genome of microbes, plants, and animals by adding, removing, or replacing segments of dna [ ] . a more efficient and less time-consuming technology for genome engineering was developed more recently, and appears to considerably expand the possible modifications of target sites in almost any sequenced genome: the clustered regularly interspaced short palindromic repeats system (crisprs) [ ] (box ). conventional genome-editing systems use synthetic nucleases to induce genomic double-stranded breaks (dsbs) at target sites. dsbs are targets of the imprecise cellular repair machineries either mediated by non-homologous end-joining (nhej) or homology directed repair (hdr), which require a donor dna template [ ] . crisprs constitute an incredibly versatile genome-editing platform derived from the s. pyogenes crispr-associated protein (cas ) [ , ] . there are three types of crisprs. type ii is the simplest, presented only by bacteria and comprises four proteins (i.e. cas , cas , cas , and cas ); it is the most widely used system for gene engineering. the most popular crispr-cas system is constituted by cas endonuclease proteins and crispr rnas [ ] . the nuclease cas can catalyze the precise cleavage of target sites assisted by two short helper rnas: crna and tracrna. the fusion of both rnas forms a hybrid single guide rna (sgrna) that binds to cas to form a supramolecular complex named rna-guided endonuclease, a sequence-specific recognizer complex that cleaves at specific sites in the genome, resulting in dsbs that are then repaired preferentially by hdr (in the case of bacterial genomes), thus altering the genome in a precise manner by site-specific modifications [ ] . the crispr-cas system has been used to introduce point mutations, modify gene function, generate gene knockouts, integrate foreign genes, repress and/or activate specific genes, deliver epigenetic modifications, and aid genomic loci accession by proteins. although limited studies report the utilization of the type ii crisprs-cas system to edit plant genomes, it has been exploited for editing other organisms [ ] [ ] [ ] . crispr/cas system ii technology description for amp biosynthesis mediated by genome edition. spacer acquisition: (a) formation of crisprs array by recognition and integration of foreign dna as spacer within the crispr locus, or fully synthesized by genetic engineering. the protospacers are non-coding region inserted in the bacterial dna with - bp. adjacent to each protospacer are found - bp short dna sequences termed protospacer adjacent motifs (pam) crrna processing: (b) the crispr array is transcribed as a long rna (pre-crrna) that is cleaved into crrnas with the help of cas proteins. an extra small rna (tracrrna) complementary to the repeat sequence is also synthesized. (c) the tracrrna pairs with the repeated region of crrna and helps in the processing of pre-crrna into crrna with the help of rnase iii for cleavage. interference stage: crrnas binds to ca proteins (d) to form a complex that recognizes foreign dna (e). a single multifunctional protein, cas , recruits crrna and tracrrna to cleave the recognized foreign dna using internal endonuclease domains (f). all the process is based in the recognition and pairing of the pam and the foreign dna. double strand breaks (dsbs) are generated (g). a donor dna containing the coding sequence of an amp of interest is integrated in the site of the dsbs by homologous recombination (i), and after gene expression, the amp is extracted and purified (h). in addition genome editing, the crispr-cas system can be utilized for other purposes within the cell. for instance, gene silencing by crispr interference (crispri) has proved to be a powerful tool to complement (and, in many cases, improve) previously described rnai, by the double formation of cas bound to sgrna and attached to the nontemplate strand of dna, blocking transcription [ ] . this approach can also be utilized to simultaneously silence multiple target genes (a process called multiplexing), thus overcoming one of the limitations of rnai in terms of its gene silencing potential. another interesting application of crispr-cas is to block transcription initiation in a specific and reversible manner [ ] . there is immense potential for the utilization of crisprs to improve the recombinant biosynthesis of amps in almost any organism. tailored genomes modified by crisprs can harbor amp coding sequences within the donor dna fragment inserted in a genomic expression hot spot, potentially boosting heterologous production of the peptide to unprecedented levels [ , ] (fig. ) . to reduce undesirable post-translational modifications of amps, it might be possible to knockout host glycosylase genes or substitute them with coding sequences of human glycosylases to humanize glycopeptides. this could contribute to enhance antimicrobial activity and therapeutic peptide quality (purity, lack of undesirable post-translational modifications, high levels, among others). in addition glycosylation, other post-translational modifications and peptide-processing pathways could be manipulated with crispr-cas. crispr-cas represents a promising technology to improve peptide engineering and biosynthesis, pushing the boundaries of the development of clinical drugs to new levels of sophistication [ , , ] . the major concern for the utilization of crisprs as a genomeediting tool is its potential secondary mutations or off-targets effects. this is a common scenario in human edited cells (however, on-target efficiency has been recently improved), but still rare in plants. to avoid off-target effects, it is imperative to properly design sgrnas that direct cas to an exact target in the genome, and efforts along these lines have been published recently. in addition, high levels of the nuclease cas relative to sgrna helps reduce the incidence of off-target effects [ ] . the cas nuclease can be further engineered into a nickase, which only cleaves a single strand of dna and the same strategy on the other strand enhances the specificity of site recognition and consistently minimizes off-target effects [ , [ ] [ ] [ ] [ ] . the prospect of novel, efficient amps is a crucial starting point to combat antibiotic-resistant microbial pathogens. the emergence of mdr bacteria is a tremendous global health problem that has been predicted to lead to the death of million humans per year by [ ] . microbes, plants, and animals are natural sources of therapeutic drugs and could also be a source of novel, biologically inspired, previously unknown antibiotics for human applications. in addition to their obvious importance, novel amps with high anti-infective properties are difficult to prospect and are naturally synthesized only at low levels in their respective host organisms. therefore, there is an urgent need to increase the efficiency and final yield of peptides during their production to allow their economic exploitation. new insights into the rational design of peptides are required to maximize amp variability and increase specificity and antimicrobial activity. currently, examples of synthetic and recombinant amps with tailored domains are now facing advanced clinical trials, with promising results. these rationally designed molecules present modified epitopes with improved net charge and enhanced antimicrobial activity, and represent a new generation of anti-infective agents with the potential to overcome mdr. the transient expression of amp genes in tobacco is a new technology with immense potential to considerably boost the heterologous production of new antibiotics and vaccines, in a fast, cheap, and efficient way. the use of potent viral vectors associated with the bacterial delivery of transcriptional units provides a scalable platform for the massive production of diversified amps, with potential desirable improvements in terms of processing and production costs. some drawbacks presented by plant expression systems are solved by the crispr system, a revolutionary genome-editing technology that presents myriad possibilities for genetic manipulation at the genomic level and provides unprecedented tools (crispri and crispra) to precisely control gene expression and the structural modification of amps. despite its current minor limitations (e.g. off-target effects), crispr could have a key role in the future development of clinical drugs as biotechnological antiinfective agents, including the rational biosynthesis of next-generation antimicrobials. overview: global and local impact of antibiotic resistance the emergence of peptides in the pharmaceutical business: from exploration to exploitation mechanisms and consequences of bacterial resistance to antimicrobial peptides antibiotic strategies in the 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specificity screening and paired nickases for cooperative genome engineering antimicrobial resistance: tackling a crisis for the health and wealth of nations. hm government and the welcome trust the developing world urgently needs phages to combat pathogenic bacteria isolation and characterization of recombinant antibody fragments against cdc a from arabidopsis thaliana development of a recombinant vaccine against japanese encephalitis production of a modified peptide clavanin in pichia pastoris: cloning, expression, purification and in vitro activities cm-p : an antifungal hydrophilic peptide derived from the coastal mollusk cenchritis muricatus (gastropoda: littorinidae) design of short membrane selective antimicrobial peptides containing tryptophan and arginine residues for improved activity, salt-resistance, and biocompatibility corn as a production system for human and animal vaccines a plant signal peptide-hepatitis b surface antigen fusion protein with enhanced stability and 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spectrum of antimicrobial activity and measurements of bactericidal activity histatins: antimicrobial peptides with therapeutic potential the big and small of drug discovery can innate immunity be enhanced to treat microbial infections? suspension-cultured by- tobacco cells produce and mature immunologically active house dust mite allergens plant-derived recombinant f , v, and f -v fusion antigens of yersinia pestis activate human cells of the innate and adaptive immune system immunogenicity of transgenic plant-derived hepatitis b surface antigen production of hiv- p protein in transgenic tobacco plants cloning of helicobacter pylori urease subunit b gene and its expression in tobacco (nicotiana tabacum l.) translational fusion of chloroplast-expressed human papillomavirus type l capsid protein enhances antigen accumulation in transplastomic tobacco plant-based vaccine: mice immunized with chloroplastderived anthrax protective antigen survive anthrax lethal toxin challenge severe acute respiratory syndrome (sars) s protein production in plants: development of recombinant vaccine protection against tetanus toxin using a plant-based vaccine induction of oral tolerance to prevent diabetes with transgenic plants requires glutamic acid decarboxylase (gad) and il- expression of viral capsid protein antigen against epstein-barr virus in plastids of nicotiana tabacum cv. sr expression of foot-and-mouth disease virus epitopes in tobacco by a tobacco mosaic virus-based vector key: cord- -gf asni authors: galdiero, stefania; falanga, annarita; morelli, giancarlo; galdiero, massimiliano title: gh : a milestone in understanding the many roles of membranotropic peptides date: - - journal: biochim biophys acta biomembr doi: . /j.bbamem. . . sha: doc_id: cord_uid: gf asni here, we review the current knowledge about viral derived membranotropic peptides, and we discuss how they may be used for many therapeutic applications. while they have been initially discovered in viral fusion proteins and have been involved in the mechanism of viral entry, it is now clear that their features and their mode of interaction with membrane bilayers can be exploited to design viral inhibitors as well as to favor delivery of cargos across the cell membrane and across the blood–brain barrier. the peptide gh has been extensively used for all these purposes and provides a significant contribution to the field. we describe the roles of this sequence in order to close the gap between the many functions that are now emerging for membranotropic peptides. over the past few decades peptides have progressively achieved increased value in drug design and pharmaceutical delivery. moreover, great interest has been dedicated to the identification of peptides as drug candidates. the number of peptides in the pharmaceutical industry is continuously growing and about % of the entire drug market is represented by peptide based drugs [ , ] . bioactive peptides can be derived from natural sources or can be discovered through rational engineering, high-throughput screening, or structure-based design starting from defined protein regions [ ] . among the many peptides playing a relevant role in biology, some show a high propensity for binding to lipid membranes due to their simultaneous hydrophobic and amphipathic nature. this class of hydrophobic peptides is characterized by the presence of unusual conspicuous amounts of alanine and glycine residues and sometimes also prolines. such a degree of ala/gly content is uncommon for hydrophobic domains such as signal sequences and transmembrane anchors; in fact, their presence may account for the intrinsic conformational flexibility which is a typical feature of membrane interacting peptides. also aromatic residues are generally present and dominate the interactions that take place at this unique physical-chemical environment of the water-membrane interface [ ] . the favorable interactions of aromatic side chains with phospholipid moieties located at the membrane interface contribute to the insertion of the peptide into the bilayer. amphipathicity is a key feature of these peptides. the term amphipathicity generally refers to molecules with both hydrophilic and hydrophobic faces [ ] . peptides can be amphipathic in their primary structure or secondary structure. primary amphipathic peptides correspond to the sequential assembly of a domain of hydrophobic residues with a domain of hydrophilic residues divided by a spacer domain; while secondary amphipathic peptides are generated by the conformational state which allows positioning of hydrophobic and hydrophilic residues on opposite sides of the same molecule. in particular, amphipathic, hydrophobic peptides present one face with large and aromatic residues and the other with small residues such as ala/gly. this distribution of amino acid residues facilitates the membrane interaction and peptide insertion into the bilayer [ ] . conformational polymorphism plays a key role; in fact, the ability to shift from random to α/β conformations as a consequence of membrane composition and peptide concentration has emerged as a common structural pattern for this class of peptides [ ] . there are several types of membrane active peptides which can be roughly divided in antimicrobial peptides [ ] , viral peptides [ ] and cell penetrating peptides [ ] . although very different in primary sequence one from the other, it may be hypothesized that their common physical features could result in a shared mechanism of action and essentially determines the many roles that they can play in nature. among the hydrophobic peptides with a propensity for membrane binding, characterized by a high interfacial hydrophobicity or amphipathicity, the ones derived from enveloped virus glycoproteins are attracting considerable attention. these peptides can interfere with enveloped virus entry by direct physical interaction with the hydrophobic surfaces present on membranes and/or fusion proteins and are, thus, critical for both fusion and entry. viral glycoproteins undergo conformational changes as a consequence of either low endosomal ph or receptor binding which leads to the exposure of hydrophobic peptides, loops or patches, which then interact with and destabilize one or both the opposing membranes. crystallographic data on the post-fusion structures of viral fusion proteins have allowed the identification and characterization of three different classes [ , ] . class i fusion proteins are characterized by trimers of hairpins with a central α-helical coiled-coil structure and have been identified in orthomyxoviruses, paramyxoviruses, retroviruses, filoviruses and coronaviruses [ ] [ ] [ ] [ ] [ ] . class ii fusion proteins are present on viral envelopes as pre-fusion dimers which convert into post-fusion trimers of hairpins composed of β structures and have main representatives in the flaviviridae and togaviridae families [ , ] . class iii fusion proteins are characterized by a central α-helical trimeric core similar to class i and two fusion loops located at the tip of an elongated β-sheet similar to class ii fusion proteins and members are present in herpesviridae and rhabdoviridae families [ ] . despite several differences in the mechanism of entry elicited by the three classes of fusion glycoproteins, they all induce membrane fusion in a similar manner through the formation of an analogous hairpin structure which allows fusion peptides to insert into cell membranes and to drive membrane destabilization. thus, during the viral entry process, the hydrophobic surfaces that become exposed are characterized by somewhat variable but at the same time detailed physical characteristics which include the size, shape and secondary structure of exposed hydrophobic patches, as well as the nature of the neighboring polar or charged residues. the wimley-white interfacial hydrophobicity scale (wwihs) is an experimentally-determined free energy scale that calculates the propensity of individual amino acids in peptide sequences to partition from water into a phosphatidylcholine interface [ , ] and has been effectively used to identify fusion peptides in viral glycoproteins. the hydropathy analysis allows calculating a hydrophobicity score along the sequence of a protein, identifying segments with a propensity to interact with membrane interfaces. wwihs values are calculated assuming random coil peptides partitioned into the bilayer interface; the values are minimum possible values and the Δg may get more favorable if peptide binding also promotes an increase in secondary structure [ , ] . the interfacial helical hydrophobic moment (ihhm) is a further physico-chemical factor that is important for membrane interactions and secondary structure formation of peptides bound to membrane interfaces. the ihhm describes the degree to which a peptide sequence would have segregated hydrophobic and hydrophilic faces if it folded into an α-helix [ ] . a peptide with a large ihhm can interact strongly with membranes as a helix due to partitioning-folding coupling [ , ] even with a wwihs score that is not positive overall. the presence of the fusion peptide within the ectodomain exposed to the aqueous phase is a feature shared by all viral fusion proteins and constitutes an absolute requirement for their fusogenic activity. fusion peptides are typically - residues long and potentially fold into amphipathic helices and are rich in glycines and alanines, providing them a high degree of conformational flexibility. thus, their structure is polymorphic and strongly dependent on the environment. the fusion peptide of influenza virus, for example, has been observed in random coil, α-helical and β-sheet conformations in different environments [ ] (fig. ) . it has been proposed that all three forms have some physiological relevance; the peptide may be unstructured in solution on the way to the target membrane; it may be helical at low concentrations but may self-associate in β-sheets at higher concentrations in the membrane interface. the fusion peptide of hiv also undergoes conformational transitions; it adopts α helical or β sheet structures depending on concentration, lipids and ionic conditions [ ] . the helical form of the influenza fusion peptide is probably a key determinant to promote fusion. the structure of the peptide in membrane shows a kink which separates the n terminal and the short c terminal helices which together form a boomerang shaped structure [ ] . both helical arms are amphipathic with bulky hydrophobic residues facing the membrane interior. the conserved n-terminal glycine residue is critical for fusion and for the correct structure of the peptide inside the membrane; in fact, when it is mutated to a valine the n-terminal helix is partially unwound and the fusion peptide is inactive [ ] . actually, there are numerous studies demonstrating that a delicate balance between α and β structures, is essential for membrane fusion and is influenced by environmental conditions such as ph, ionic strength, peptide sequence, presence or absence of divalent cations, cholesterol content and also by the lipid/peptide ratio. for instance, studies performed on the ebola fusion peptide, show that the conformational transition from an α-helix to a β-sheet is induced by a change in the peptide to lipid ratio in the membrane [ ] . at low peptide concentration in lipids, it is essentially an α-helix; while as the local peptide concentration increases in the membrane, the proportion of α-helix drops off in favor of a mainly antiparallel β-sheet structure. this concentration dependent effect on peptide conformation might be of biological relevance [ ] . the functional meaning of the conformational polymorphism is unclear, although it is believed to be fundamental to enable backbone reorientation of the fusion protein; therefore, the ability to insert at various levels might be required for the evolving of final stages of the fusion cascade [ ] . independently from the principal conformational organization, the degree of insertion plays a key role for inducing membrane fusion. it has also been hypothesized that [ ] fusion domains first assemble as β-sheets on the surface of the membrane and later convert into α-helices to complete fusion. aromatic residues are generally present in fusion peptides and may help in overcoming the energy cost of peptide bond partitioning into membranes. the interactions with phospholipid moieties located at the membrane interfaces may also help in stabilizing the insertion into just one leaflet of the bilayer. the initial interaction with the external leaflet is thought to generate elastic stresses which drive to bilayer fusion, helping to overcome the hydration repulsion forces between approaching bilayers by orienting the poorly solvated face toward the external medium [ ] . the asymmetric insertion into one membrane monolayer may promote expansion of the polar head region and determine a curvature stress onto the overall lipid bilayer; the created bulges that protrude from the membrane can facilitate the formation of lipid contacts between fusing bilayers [ ] . particular attention has also been devoted to the effect of additional membranotropic sequences on the overall fusogenicity. the presence of additional fusogenic sequences was evidenced in sendai f [ ] , measles f [ ] , sars-cov s [ ] , hepatitic c virus e and e [ ] , dengue e [ , ] and herpes virus gb and gh [ ] [ ] [ ] . the idea that a single fusion peptide is the solely responsible for the complete membrane fusion event has been substituted by the assumption that a concerted action of different membranotropic regions is necessary for membrane interacting/perturbing activity. as a matter of fact, also membrane proximal regions (pre-tm) play a key role in fusion [ , [ ] [ ] [ ] . the pre-tm domains are particularly rich in aromatic residues which enable them to insert into the membrane interface. effective therapeutics against enveloped viruses are still scarcely represented. a few drugs have been developed against hiv, influenza virus, hepatitis virus and a few other viruses but they are still not ideal and in some cases have proved to induce resistance [ ] . as a consequence, for most enveloped viruses, there are no effective therapies and entry inhibitors represent an interesting and underutilized target. peptides with a propensity for membrane binding can also interfere with enveloped virus entry by direct physical interaction with the hydrophobic surfaces present on cell membranes and/or fusion proteins. as recently reviewed by badani et al. [ ] , there are many peptide inhibitors that are somewhat hydrophobic and/or amphipathic with a propensity to bind to bilayer membrane interfaces and other hydrophobic surfaces. it is not known whether membrane binding directly affects viral fusion, or whether interaction with the fusion protein itself is an absolute requirement for entry inhibition. it is widely accepted that membrane binding of an inhibitory peptide will greatly increase the effective concentration of the peptide close to the fusion protein, indicating that the interaction with membrane and the interaction with the fusion protein may be effectively coupled [ ] . as a matter of fact, the potential of numerous fusion peptides and/or membranotropic peptides derived from proteins of enveloped viruses as entry inhibitors has been widely described in literature [ ] [ ] [ ] [ ] . the accepted view is that the inhibition of infectivity may be due to the formation of inactive aggregates between the fusogenic stretches present in both the viral protein and the synthetic peptides. these aggregates are formed as a consequence of their ability to oligomerize or to mimic the modes of binding of their original domains in their partner protein. it has been hypothesized that they stabilize a pre-fusion intermediate and prevent merging of the bilayers. it is now evident that several domains are essential for membrane fusion and thus peptides involved in the fusion mechanism may all interfere with the intramolecular interactions between the several domains and may represent interesting targets for the design of entry inhibitors. thus, membrane physical properties could be critically important to the events that drive viral entry and it is conceivable that peptides interfere with the function of viral fusion proteins by changing the physical chemistry of the membrane itself by direct interaction. many laboratories are working to unravel the mechanism of action of viral membranotropic peptides and several hypotheses have been proposed. many studies suggest that multiple mechanisms may take place simultaneously. indirect ways in which interfacial binding peptides can affect viral entry have also been hypothesized. selfoligomerization of membrane embedded fusion peptides has been proposed to be responsible of inhibition [ , ] . the most studied case is that of hiv, where the inhibition has been attributed to the formation of structurally defined oligomeric complexes [ , ] ; while mutants with a lower helical content and tendency to self-associate into β-sheets [ ] are able to inhibit membrane fusion at various stages. it is interesting to note that there are clinical studies on a peptide called virip, which is designed as an inhibitor of the hiv fusion peptide [ , ] . this sequence was able to block hiv- infection by targeting gp fusion peptide [ ] and optimized versions of this sequence proved to be as potent as inhibitors targeting the coiled coil sequences and moreover were devoided of cellular toxicity. a -day monotherapy clinical trial enrolling hiv- infected patients [ ] showed that the drug can be well tolerated by patients and reduces their plasma viral load. its identification and clinical evaluation represent the first proof of concept that membranotropic sequences could suppress viral replication in infected individuals and have potential clinical effectiveness. the hypothesis that peptide entry inhibitors act by a physicalchemical interaction with hydrophobic surfaces exposed during the fusion process suggests that this novel approach may be a general rule; moreover, instead of focussing on the structure-based design, it would be possible to design novel hydrophobic/amphipathic inhibitors which could be easily made protease resistant by the introduction of nonnatural or d-amino acids. the membrane bilayer represents a semi-permeable barrier, defining the interior of an individual cell; its existence confers cells their potential to survive and function properly. nevertheless, crossing of the cellular membranes remains one of the major obstacles for the proper delivery of therapeutics [ , ] . the lipophilic nature of biological membranes restricts the direct intracellular delivery of most compounds; whereas small molecules and ions can diffuse across the bilayer, larger molecules are generally excluded from simple diffusion into the cell. the differing hydrophobicity/hydrophilicity of the lipid membrane renders the transfer across this barrier extremely difficult due to differences in solubility. notwithstanding the therapeutic potential of a number of novel molecules, their pharmaco-distribution properties hamper the possibility to reach the stage of pharmaceutical preparations and stimulate industrial interest; in fact, these molecules need to be delivered intracellularly to exert their therapeutic action inside the cytoplasm or onto individual organelles. it is, thus, evident that the therapeutic potential of a drug is largely dependent on the development of delivery tools able to selectively and efficiently carry it to target cells with minimal toxicity. the translocation across the membrane is by far less well understood than the binding step. there is a significant similarity in the physico-chemical parameters between membrane partitioning peptides and membrane translocating peptides [ ] . a novel intriguing hypothesis is that hydrophobic peptides that partition into membranes may also be able to cross cell membranes and enter cells. therefore, these peptides may also cross endothelial layers in vivo, including the blood-brain barrier [ , ] . delivery across cellular membranes involves several membrane reorganization processes such as transient permeabilization of the cell membrane, which are similar to the ones involved in the entry of viruses. membrane fusion and its disruption are related processes, although leakage and fusion capacities of peptides do not always correlate and the features/activities of membranotropic peptides may depend on particular environmental and temporal conditions. since not all membranotropic peptides are able to cross the membrane bilayer, it is essential to identify structural characteristics of hydrophobic peptides know to enter the cell membrane to highlight any feature that is involved in the penetration which may help in the design of novel delivery tools. thus, an important feature to consider is the structural requirements for cellular uptake and the ability of membranotropic peptides to interact with the cell surface and lipid moieties of the cell membrane. a very complete review describing the binding and translocation of membrane active peptides has been recently published [ ] which highlights the fact that peptide translocation is not coupled with dye flux. graded dye flux would occur concomitant with peptide translocation, which would explain incomplete dye release; whereas all-or-none flux would occur with peptides unable to translocate, and therefore these peptides accumulate on the membrane until a rupture point is reached, resulting in complete dye release [ ] . cell-penetrating peptides (cpps) have been widely used due to their capability to transport several kinds of macromolecules across the membrane bilayer in vitro and in vivo [ ] [ ] [ ] . cpps are short and usually basic amino acid rich peptides originating from proteins that are able to cross biological barriers, such as the viral tat protein. although the uptake mechanism of cpps is still debated, it seems to involve mainly the endocytic pathway, trapping the conjugated cargo in endosomes eventually ending in lysosomes where common enzymatic degradation mechanisms take place, therefore leading to a limited delivery of therapeutic agents to the intracellular target. hydrophobic peptides that efficiently traverse biological membranes, promoting lipid-membrane reorganizing processes, such as fusion or pore formation and involving temporary membrane destabilization and subsequent reorganization [ , ] , may be able to circumvent the endosomal entrapment either favoring the escape from the endosome or by translocating a cargo through the plasma membrane directly into the cytosol. this idea has been exploited to design the drug delivery tool called mpg. mpg is an amphipathic peptide whose primary sequence is composed of the hydrophobic amino acids of the hiv- fusion peptide (galflgflgaagstmga) associated to a hydrophilic domain derived from the nuclear localization sequence (nls) of simian virus (sv ) large t antigen (pkkkrkv). these hydrophilic and hydrophobic segments are separated by a three amino-acid spacer (wsq) [ , ] . this peptide exploits the known properties of the glycine-rich hiv fusion peptide essential for membrane fusion activity and the nls of the sv large t antigen to improve the nuclear addressing of the peptide [ , ] . at the moment this is the only viral fusion peptide that has been widely exploited for applications in drug delivery. a milestone in understanding the role of hydrophobic viral peptides is represented by the sequence "gh " derived from glycoprotein h of herpes simplex virus type i. herpes simplex virus (hsv) is an important human pathogen, responsible for significant morbidity and mortality worldwide and is characterized by a complex multi-component entry machinery. hsv enters host cells by fusion of the viral envelope with either the plasma membrane or an endosomal membrane, and the entry pathway is likely determined by both virus and host cell factors and involves multiple viral glycoproteins and cellular receptors in a cascade of molecular interactions [ ] [ ] [ ] [ ] . the envelope glycoproteins gh/gl, gb and gd are all essential for the entry process and their expression is able to induce the fusion of cellular membranes in a virus-free system [ , ] . both gh/gl and gb constitute the core fusion machinery and cooperate to induce the initial lipid destabilization that ends in fusion [ ] and both gb and gh contain several membranotropic sequences [ ] [ ] [ ] , , [ ] [ ] [ ] . although it has recently become available the crystal structure of the gh-gl complex [ ] , it is still debated whether gh is merely a fusion regulator or it plays a more direct role in the fusion process and many studies suggest that the gh-gl complex may undergo dynamic rearrangements [ , ] . in particular, some peptides derived from the gh ectodomain block virus entry, while others have the ability to bind and disrupt model membranes. gb is considered a canonical class iii fusion protein and has been demonstrated to be involved in virus attachment, penetration and cell-to-cell spread. the crystal structure of gb is a trimer in which multiple contacts between protomers throughout the molecule contribute to its stability [ ] . it has been hypothesized [ ] that gb refolds similarly to class i fusion proteins and that the packing of the c-terminal arm against the coiled-coil provides the driving force for gb refolding from the prefusion to the post-fusion conformation. the gb structure corresponds to a post fusion conformation and it is now widely accepted that gb undergoes conformational changes upon variations of ph in order to bring about fusion [ ] [ ] [ ] [ ] . several synthetic gb peptides induced the fusion of large unilamellar vesicles and inhibited herpes virus infection [ , ] . when the crystal structures of gb and of gh/gl were not yet available, we reported the identification of several sequences in gh and gb with the ability to interact with the membrane and among these sequences there was also the canonical fusion peptide of gb [ , , , ] . although it is not yet well understood the role played by the other membranotropic sequences and in particular by the glycoprotein gh in the whole fusion process, it is now widely accepted that several regions in the fusion glycoproteins are involved in the local destabilization of the membrane bilayer which ends in the fusion of the viral envelope with the host cell membrane. gh was first selected and characterized by our group in [ ] and still is the hsv- peptide with the highest fusion capability and the most widely studied. it was initially identified using the wwih scale and subsequent works allowed determining the many applications of this sequence from membrane fusion, to viral inhibition and drug delivery [ , , ] . the twenty residue peptide gh (from aa to aa ) is a membrane-perturbing domain, (fig. ) which interacts with biological membranes and is implicated in the merging of the viral envelope and the cellular membrane [ , ] . the peptide contains residues crucial for its capacity to interact and destabilize target lipid membranes. it is rich in hydrophobic residues including glycines, leucines, alanines, and aromatic residues such as tryptophan and tyrosines, which are known to be located preferentially at the membrane interface. the peptidelipid interactions are initiated by the arginine residue located at the c-terminus; in fact, when the arginine is mutated, the fusogenic activity of the peptide is strongly impaired. the hydrophobic domain is also crucial for its insertion into the membrane and further supports the view that hydrophobic interactions between fusion proteins and cellmembrane phospholipids initiate membrane perturbation in the early stages of viral fusion. the many biophysical experiments performed on gh have shown that the peptide interacts with model membranes, penetrates the bilayer from its n-terminal side, has a tryptophan residue buried inside the bilayer, and adopts a helical conformation with its hydrophobic residues on one face of the helix and polar or charged residues on the opposite face [ , , ] . the analysis of peptides with longer and shorter sequences derived from this region and of their interactions with membranes clearly demonstrated that the activity of this region depends on the amino acid sequence and on its length. the presence of a histidine residue at the n-terminus of the native sequence strongly increases the fusion activity [ ] . the importance of a single histidine residue as a switch for triggering viral fusion was also reported for other viruses [ ] such as paramyxoviruses, therefore supporting the importance and specificity of the histidine moiety in activating fusion. furthermore, a conserved histidine in one of the fusion loops of semliki forest virus e protein was found to be fundamental [ ] . the histidine in gh both helps the initial interactions with the membrane and the oligomerization process [ ] . this hypothesis is further supported by the fact that the histidine is located at the n-terminus and correct configuration of the n-terminal fusion peptide appears to be crucial for the fusogenic function of several fusion proteins as well as its location in the membrane core after peptide-bilayer interaction. the addition of one histidine at the n-terminus of gh is sufficient to make the peptide approximatively -fold more active. in particular, the addition of any other residue at the n-terminus impaired the fusion ability of the sequence (data not published). gh strongly interacts and spontaneously penetrates the lipid-phase and inserts into membranes with a α-helical structure [ , , ] . both the tryptophan and tyrosine are on the same side of the helix in the three-dimensional structure, forming an amphiphilic helix in which one side is constituted by aromatic and hydrophobic residues, whereas the other side is formed by hydrophilic or small residues. the interaction between the aromatic ring of tryptophan and the side chain of tyrosine is important for maintenance of structural stability during the interaction with the membrane. an amphipathic α-helix is believed to be an important feature of membranotropic peptides playing a crucial role for mediating lipid-protein interactions during the binding of proteins to membranes and once bound, the hydrophobic face of the amphipathic peptide would allow the peptide to enter the membrane interior, thereby triggering local fusion of the gh has the ability to penetrate deep into the bilayer as a helix without causing significant bilayer perturbations which may help explaining its ability to perform several different roles. gh showed a significant inhibitory effect and this effect appears conditioned by its ability to partition into membranes and aggregate within them. since the peptide self-associates in aqueous and lipid solutions, it is possible that it binds to its counterpart in the gh protein. gh may also interact with the host cell membrane, therefore its ability to moderately inhibit viral entry when cells are treated first, is dependent on the possibility that the virus will find a modified cell membrane still exhibiting on its surface the peptide [ ] . moreover, gh does not have any activity in virus preincubation experiments, indicating that an eventual binding partner site on the pre-fusion gh protein is probably hidden and not available to interactions with free peptides, as also demonstrated by the analysis of the gh crystallographic structure. while the n-terminal histidine residue was proven to be fundamental for the interaction with the membrane bilayer and for translocation across the membrane, the absence of this residue induced similar levels of viral inhibition when compared with the full length peptide. the substitution of leu with a valine residue does not alter the hydrophobicity of the peptide, and does not influence its infectivity inhibition properties while its substitution with a polar residue (serine) substantially reduces its inhibitory activity [ ] . recently, poly(amide)-based dendrimers functionalized at their termini with gh were shown to inhibit both hsv- and hsv- at a very early stage of the entry process, most likely through an interaction with the viral envelope glycoproteins; thus, preventing the virus from coming into close contact with cellular membranes, a prerequisite for viral internalization [ ] . the % inhibitory concentration was and nm against hsv- and hsv- respectively, with no evidence of cell toxicity at these concentrations, indicating that the functionalization of a dendrimer with a membranotropic peptide represents a promising strategy for inhibition of viruses of the herpesviridae family. the multivalent display of gh on the dendrimer scaffold results in an almost six fold increase of antiviral activity for hsv- and two fold for hsv- in comparison to the activity of the dendrimer itself, and more than -fold increase in the activity of the unsupported peptide. the cytotoxicity profile measured by the mtt assay showed that the peptidodendrimer is not toxic to vero cells up to the highest concentration investigated in antiviral testing, while some toxicity was observed for the unfunctionalized dendrimer, especially at higher concentrations, demonstrating another advantage of the peptide functionalization [ ] . any inhibitory activity was excluded when the compounds were added at a post-entry step and also when cells were pre-treated with the dendrimer derivatives, indicating that both the peptidodendrimer and the dendrimer are not able to interfere with viral replication once the virus has gained access to the cellular milieu. the peptidodendrimer might sterically hinder the gh relative domain, either in a pre-fusogenic or in an intermediate conformation, preventing a complete and functional interaction between gh and the membrane to fuse. the mechanism of inhibition may involve binding to gh itself through oligomerization of the gh domain present on the glycoprotein or interaction with other glycoproteins present on the virion envelope, such as gb or gd. the modification of a dendrimer scaffold with membranotropic peptides represents an attractive strategy for the design of a new class of antiviral drugs that exert their effect, coupling the intrinsic anti-viral properties of the dendrimer with the activity of membranotropic peptides and have the potential of being developed as multifunctionalized scaffolds to provide a therapeutic molecule to directly deliver to its target [ ] . the inhibition of membrane fusion represents an attractive target for drug design and although further studies are needed to better define the exact mechanism of inhibition by hydrophobic peptides and the specific nature or location of their interactions with viral targets, the data obtained for gh suggest that hydrophobic domains play a significant role in membrane fusion and provide an alternative approach to the development of viral peptide inhibitors outside of the classical inhibitory heptad repeat regions. gh cellular uptake is associated with its hydrophobic and amphipathic characters which provide the necessary ability to interact with membrane lipids and to form a transient helical structure that temporarily affects membrane organization, thereby facilitating insertion into the membrane and translocation [ ] . compared to tat peptide (a positively charged cpp) which mainly exploits the endocytic pathway, gh crosses membrane bilayers mainly through a translocation mechanism. a one amino acid shorter version of this fusogenic peptide was also found to improve the endosomal release of dna/lipofectamine lipoplexes and transgene expression up to -fold in human cell lines [ ] . it has been recently demonstrated that gh is able to traverse the membrane bilayer and to transport into the cytosol several compounds, such as qds [ ] , liposomes [ ] , nps [ ] , dendrimers [ ] , and proteins [ ] . examples of using gh as an intracellular delivery enhancer are provided in the remaining part of the paragraph (fig. ) . qds are fluorescent probes under intense research and development for broad applications in molecular, cellular and in vivo imaging [ ] . although considerable success has been achieved in using qds for labeling fixed cells and for imaging cell membrane proteins, only limited progress has been made for molecular imaging inside living cells because of their insufficient ability to traverse cell membranes. several authors have recently reported on the functionalization of qds with positively charged cpps and established that the main route of entrance is via endosomal uptake, therefore, escape from the endosomal system is of paramount importance [ ] [ ] [ ] . gh -qd internalization was demonstrated to be highly successful and to involve the endocytic pathway only to a minor extent [ ] . liposomal aggregates have also attracted great attention due to their success as in vivo carriers of drugs [ ] . to enhance the antitumor efficacy of liposomal drugs, the efforts of many research groups are directed toward the improvement of cellular internalization of liposomes through the addition of surface ligands and cell penetrating peptides. liposomes decorated with gh and loaded with doxorubicin (dox) [ ] , were able to penetrate inside living hela cells. the results obtained suggest that the functionalization of liposomes with gh could affect the uptake mechanism of liposomes and their intracellular distribution and dox release. this evidence could be useful in the design of carriers for a controlled delivery and release of dox in order to avoid side effects associated to dox itself. dendrimers [ , ] also represent a very promising tool for drug delivery, combining the advantageous features of nanoparticles (ideal size as in vivo carriers, multivalency), of polymeric materials (low cost, tunable properties, biocompatibility) and of small molecules (monodispersity and detailed control of their properties) [ , ] . their surface modification by means of conjugation or adsorption of a biospecific ligand, may allow their delivery to specific sites and modulation of drug release minimizing toxic effects and increasing intracellular bioavailability [ ] . thus, the dendrimeric scaffolds may be a promising tool for an efficient drug delivery engine. little information is available on the mechanism of dendrimer uptake and intracellular trafficking [ ] . studies performed on pamam dendrimers [ ] and pamam dendrimers functionalized with the tat [ ] indicate that endocytosis mechanisms contribute to the internalization and intracellular trafficking and that adding the tat failed to enhance delivery efficiency. the attachment of gh to the termini of a poly(amide)-based dendrimer allows the conjugate to penetrate into the cellular matrix, whereas the unfunctionalized dendrimer is excluded from translocation. the peptide-functionalized dendrimer is rapidly taken into the cells mainly through a non-active translocation mechanism [ ] . the combination of the benefits of dendrimers and peptides chemistry could be useful for the development of a selective carrier which could cross the membrane and be efficiently internalized into the cellular targets. many therapeutic drugs are excluded from entering the brain, due to their lack of transport through the blood-brain-barrier (bbb) [ ] . the development of new strategies for enhancing drug delivery to the brain is fundamental in diagnostics and therapeutics of central nervous diseases (cns). most strategies to transport drugs inside the cns cause disruption of the anatomical texture of the bbb, therefore impairing its natural function; as a consequence, effective delivery approaches should be cautiously assessed considering their impact on the overall protective function of the bbb [ ] . targeted delivery of a therapeutic cargo to the intended site of action in the brain appears to be one of the most promising non-invasive approach to overcome the bbb, combining the advantages of brain targeting, high incorporation capacity, reduction of side effects and circumvention of the multidrug efflux system [ ] [ ] [ ] [ ] . polystyrene nanoparticles (nps) decorated on their surface by gh showed that the uptake of nps with gh by brain endothelial cells was greater than that of the nps without the peptide and functionalized nps were free to move intracellularly [ ] . most importantly, gh decreased np intracellular accumulation as large aggregates and enhanced the np bbb crossing. the surface functionalization with gh may change nps fate and provides a good strategy for the design of promising carriers to deliver drugs across the bbb for the treatment of brain diseases. whether multifunctional nanosystems, designed and tested in vitro, are able to properly work in vivo into mammalian hosts, is not fully granted. to address this issue, in vivo studies are necessary, thus validating design strategies and facilitating optimization and further functionalization. although numerous studies showed that gh is an efficient carrier for bioactive cargoes in vitro [ ] , these results did not guarantee that it can be developed into a useful pharmaceutical delivery platform. the ability of gh to cross the bbb in vivo was also recently evaluated [ ] . gh was administered in vivo to rats and its presence in the liver and in the brain was detected. within . h from its i.v. administration, gh can be found beyond the bbb in proximity fig. . the many applications of gh to drug delivery. confocal microscopy images showing the internalization of gh functionalized: a) proteins [ ] ; b) liposomes [ ] ; c) qdots [ ] ; d) dendrimers [ ] . e) scanning electron microscopy images of functionalized polystyrene nanoparticles [ ] . of cell neurites. gh has no toxic effect in vivo, since it does not affect brain maximal oxidative capacity and mitochondrial respiration rate. the data suggest that gh , for its ability to cross the bbb, represents a novel nano-carrier system for drug delivery to the central nervous system. these results open new possibilities for direct delivery of drugs into patients in the context of theranostics and might address the treatment of several human diseases. other peptides have been proposed as a drug delivery system; it was demonstrated that tat was able to enter tissues in vivo in mice [ ] ; antp was able to activate endogenous t cells in mice [ ] . these peptides are highly positively charged, and absorptive-mediated transcytosis has been proposed for their transport across the bbb. a bradykinin analogue has also been reported to increase the penetration of small molecules by transitory opening of the bbb [ ] . gh is the first viral membranotropic peptide which was shown to be a potential delivery system for macromolecules in vivo; these results coupled with previous in vitro data support the view that gh enters the bbb without involving endocytic processes. hence, the eventual cargo may be immediately and completely available [ ] . the presence of multiple metabolic barriers may restrict the application of such peptide-based ligand for targeted drug delivery in vivo. peptides alone or conjugated on the surface of nanocarriers are subject to proteolysis in the blood after systemic administration. in addition, the bbb is also a metabolic barrier due to the presence of various enzymes in brain capillary endothelial cells. gh starts to be degraded after h of incubation but the intact peptide is still present after . h of incubation and thus holds the potential for extending brain targeting efficiency due to its resistance to proteolysis for . h [ ] . it is still under-recognized that some amino acid sequences in virtue of their specific features can play many different roles in nature. membranotropic viral peptides derived from fusion glycoproteins are widely studied especially for their ability to fuse membranes but there are many literature data also describing other roles besides membrane fusion. gh is an example of how these sequences can be employed for completely different purposes: fusion of membranes, viral inhibition and drug delivery. till now, gh is the only membranotropic peptide that has been extensively used for many applications and among them as a drug delivery system for the brain. in the development of new therapies to treat brain pathologies, the bbb represents a major obstacle against the use of potential drugs for treating disorders of the cns due to the impermeable nature of the cell membranes of this compartment to several molecules [ , ] . the data reported on the in vivo application of gh for brain delivery, support the novel view that synthetic peptides derived from viral membranotropic sequences can be used successfully to deliver biologically active substances inside the bbb. the exact molecular mechanism of gh entry remains to be established but it appears to be a general feature of membranotropic peptides, which may be used for mediating delivery to virtually any tissue and in particular across the bbb, conveying a wide variety of cargoes with intact bioactivity into virtually any tissue or organ. other sequences have been found to be useful for drug delivery which happens to have the same features of the viral fusion peptide, indicating that it may be possible to design novel sequences with pre-determined characteristics which can be useful to treat many diseases. however, still much work has to be done on this type of peptides; we should not forget that their mechanism of perturbation of membrane bilayers may also allow the design of new membranotropic peptides with the ability to denature the membrane bilayer of bacteria and thus we may add to their many roles also the antibacterial activity which may represent an alternative to classical antibiotics in order to combat the antibiotic resistance problem. much of the vast literature on membranotropic peptides is devoted to single activities of these sequences; yet compelling structure-function relationship studies bridging the gap among all these activities are necessary. the future of peptide-based drugs synthetic therapeutic peptides: science and market therapeutic peptides: technological advances driving peptides into development the preference of tryptophan for membrane interfaces use of hydrophobic moment plot methodology to aid the identification of oblique orientated α-helices antimicrobial peptides and viral fusion peptides: how different they are? antimicrobial peptides: promising compounds against pathogenic microorganisms membrane fusion and fission: enveloped viruses recent progress of cellpenetrating peptides as new carriers for intracellular cargo delivery mechanism of membrane fusion by viral envelope proteins viral membrane fusion structure of the haemagglutinin membrane glycoprotein of influenza virus at a resolution retrovirus envelope domain at . angstrom resolution crystal structure of the ebola virus membrane fusion subunit, gp , from the envelope glycoprotein ectodomain structure of the parainfluenza virus f protein in its metastable, prefusion conformation structural basis for coronavirus-mediated membrane fusion. crystal structure of mouse hepatitis virus spike protein fusion core the envelope glycoprotein from tick-borne encephalitis virus at Å resolution the fusion glycoprotein shell of semliki forest virus: an icosahedral assembly primed for fusogenic activation at endosomal ph class iii viral membrane fusion proteins hydrophobic interactions of peptides with membrane interfaces experimentally determined hydrophobicity scale for proteins at membrane interfaces folding of β-sheet membrane proteins: a hydrophobic hexapeptide model folding of amphipathic α-helices on membranes: energetics of helix formation by melittin amphipathic helix motif: classes and properties ph-dependent self-association of influenza hemagglutinin fusion peptides in lipid bilayers conformational transitions of membrane-bound hiv- fusion peptide membrane structure and fusiontriggering conformational change of the fusion domain from influenza hemagglutinin structure and function of membrane fusion peptides structure and orientation study of ebola fusion peptide inserted in lipid membrane models the three lives of viral fusion peptides fusion activity of hiv gp fusion domain is related to its secondary structure and depth of membrane insertion in a cholesterol-dependent fashion lipid tail protrusion in simulations predicts fusogenic activity of influenza fusion peptide mutants and conformational models protein-driven membrane stresses in fusion and fission paramyxovirus f protein has two fusion peptides: implications for the mechanism of membrane fusion participation of two fusion peptides in measles virus-induced membrane fusion: emerging similarity with other paramyxoviruses identification of the membrane-active regions of hepatitis c virus p protein: biophysical characterization of the loop region the membraneactive regions of the hepatitis c virus e and e envelope glycoproteins the membrane-active regions of the dengue virus proteins c and e hydrophobic segment of dengue virus c protein. interaction with model membranes fusogenic domains in herpes simplex virus type glycoprotein h evidence for a role of the membrane-proximal region of herpes simplex virus type glycoprotein h in membrane fusion and virus inhibition the identification and characterization of fusogenic domains in herpes virus glycoprotein b molecules interfacial pre-transmembrane domains in viral proteins promoting membrane fusion and fission viral fusion proteins: multiple regions contribute to membrane fusion interaction of a peptide derived from glycoprotein gp of feline immunodeficiency virus and its lipoylated analogue with phospholipid membranes peptide entry inhibitors of enveloped viruses: the importance of interfacial hydrophobicity a synthetic peptide corresponding to a conserved heptad repeat domain is a potent inhibitor of sendai virus-cell fusion: an emerging similarity with functional domains of other viruses peptides containing membrane-interacting motifs inhibit herpes simplex virus type infectivity analysis of a membrane interacting region of herpes simplex virus type glycoprotein h the presence of a single n-terminal histidine residue enhances the fusogenic properties of a membranotropic peptide derived from herpes simplex virus type glycoprotein h computational identification of self-inhibitory peptides from envelope proteins self-association of glutamic acid-rich fusion peptide analogs of influenza hemagglutinin in the membrane-mimic environments: effects of positional difference of glutamic acids on side chain ionization constant and intra-and inter-peptide interactions deduced from nmr and gel electrophoresis measurements oligomerization of fusogenic peptides promotes membrane fusion by enhancing membrane destabilization fusion peptides derived from the hiv type glycoprotein associate within phospholipid membranes and inhibit cell-cell fusion. structure-function study a synthetic all d-amino acid peptide corresponding to the n-terminal sequence of hiv- gp recognizes the wildtype fusion peptide in the membrane and inhibits hiv- envelope glycoproteinmediated cell fusion thermodynamics of fusion peptide-membrane interactions short-term monotherapy in hiv-infected patients with a virus entry inhibitor against the gp fusion peptide discovery and optimization of a natural hiv- entry inhibitor targeting the gp fusion peptide multifunctional nanocarriers endocytic mechanisms for targeted drug delivery direct cytosolic delivery of polar cargo to cells by spontaneous membranetranslocating peptides shuttle-mediated nanoparticle delivery to the blood-brain barrier exploitation of viral properties for intracellular delivery membrane-active peptides: binding, translocation, and flux in lipid vesicles twenty years of cell-penetrating peptides: from molecular mechanisms to therapeutics truncated hiv- tat protein basic domain rapidly translocates through the plasma membrane generation of endosomolytic reagents by branching of cell-penetrating peptides intracellular delivery: exploiting viral membranotropic peptides a novel potent strategy for gene delivery using a single peptide vector as a carrier a new peptide vector for efficient delivery of oligonucleotides into mammalian cells detection of a fusion peptide sequence in the transmembrane protein of human immunodeficiency virus in vitro mutagenesis of a putative dna binding domain of sv large-t nectin- -mediated entry of a syncytial strain of herpes simplex virus via ph-independent fusion with the plasma membrane of chinese hamster ovary cells structure-function analysis of herpes simplex virus glycoprotein b with fusion-from-without activity entry of herpes simplex virus and other alphaherpesviruses via the paired immunoglobulin-like type receptor alpha glycoprotein d receptor-dependent, low-ph-independent endocytic entry of herpes simplex virus type fusing structure and function: a structural view of the herpesvirus entry machinery glycoproteins gb, gd, and ghgl of herpes simplex virus type are necessary and sufficient to mediate membrane fusion in a cos cell transfection system herpes simplex virus glycoproteins gb and gh function in fusion between the virion envelope and the outer nuclear membrane biophysical characterization and membrane interaction of the two fusion loops of glycoprotein b from herpes simplex type i virus role of membranotropic sequences from herpes simplex virus type i glycoproteins b and h in the fusion process structure and orientation of the gh - membrane interacting region of herpes simplex virus type in a membrane mimetic system crystal structure of the conserved herpesvirus fusion regulator complex gh-gl crystal structure of the epstein-barr virus (ebv) glycoprotein h/glycoprotein l (gh/gl) complex crystal structure of glycoprotein b from herpes simplex virus residues within the c-terminal arm of the herpes simplex virus glycoprotein b ectodomain contribute to its refolding during the fusion step of virus entry conformational modifications of gb from herpes simplex virus type analyzed by synthetic peptides structure of a trimeric variant of the epstein-barr virus glycoprotein b crystal structure of the low-ph form of the vesicular stomatitis virus glycoprotein g the postfusion structure of baculovirus gp supports a unified view of viral fusion machines analysis of synthetic peptides from heptad-repeat domains of herpes simplex virus type glycoproteins h and b multiple peptides homologous to herpes simplex virus type glycoprotein b inhibit viral infection peptide inhibitors against herpes simplex virus infections lipid composition modulates the interaction of peptides deriving from herpes simplex virus type i glycoproteins b and h with biomembranes a histidine switch in hemagglutinin-neuraminidase triggers paramyxovirus-cell membrane fusion a conserved histidine in the ij loop of the semliki forest virus e protein plays an important role in membrane fusion dendrimers functionalized with membrane-interacting peptides for viral inhibition a peptide derived from herpes simplex virus type glycoprotein h: membrane translocation and applications to the delivery of quantum dots a fusogenic segment of glycoprotein h from herpes simplex virus enhances transfection efficiency of cationic liposomes clickable functionalization of liposomes with the gh peptide from herpes simplex virus type i for intracellular drug delivery dendrimer functionalization with a membrane-interacting domain of herpes simplex virus type : towards intracellular delivery pedone, gh is a viral derived peptide for effective delivery of intrinsically disordered proteins probing cellular events, one quantum dot at a time intracellular delivery of quantum dot-protein cargos mediated by cell penetrating peptides intracellular trafficking and unpacking of sirna/quantum dot-pei complexes modified with and without cell penetrating peptide: confocal and flow cytometric fret analysis spatiotemporal multicolor labeling of individual cells using peptide-functionalized quantum dots and mixed delivery techniques the benefits and challenges associated with the use of drug delivery systems in cancer therapy dendrimers and dendrons: concepts, synthesis, applications designing dendrimers for biological applications dendrimers and dendritic polymers in drug delivery surface modifications of nanocarriers for effective intracellular delivery of anti-hiv drugs crossing cellular barriers using dendrimer nanotechnologies comparison of the endocytic properties of linear and branched peis, and cationic pamam dendrimers in b f melanoma cells tat-conjugated pamam dendrimers as delivery agents for antisense and sirna oligonucleotides current approaches to enhance cns delivery of drugs across the brain barriers astrocyte-endothelial interactions at the blood-brain barrier nanoparticle-mediated targeted delivery of antiretrovirals to the brain nanobiotechnology-based strategies for crossing the blood-brain barrier biomaterial-based technologies for brain anti-cancer therapeutics and imaging receptor-mediated delivery of magnetic nanoparticles across the blood-brain barrier review of a viral peptide nanosystem for intracellular delivery the peptide gh enters into neuron and astrocyte cell lines and crosses the blood brain barrier in rats tat-mediated delivery of heterologous proteins into cells introduction of exogenous antigens into the mhc class i processing and presentation pathway by drosophila antennapedia homeodomain primes cytotoxic t cells in vivo facilitation of drug entry into the cns via transient permeation of blood brain barrier: laboratory and preliminary clinical evidence from bradykinin receptor agonist, cereport getting into the brain: approaches to enhance brain drug delivery the authors thank luca de luca for excellent technical assistance. key: cord- -l cn n authors: ohnishi, shun-ichi title: chapter fusion of viral envelopes with cellular membranes date: - - journal: curr top membr transp doi: . /s - ( ) - sha: doc_id: cord_uid: l cn n this chapter reviews some characteristic features of membrane fusion activity for each virus and discusses the mechanisms of membrane fusion, especially low ph-induced membrane fusion. it concentrates on the interaction of the hydrophobic segment with the target cell membrane lipid bilayer and suggests the entrance of the segment into the lipid bilayer hydrophobic core as a key step in fusion. the envelope is a lipid bilayer membrane with the virus specific glycoproteins spanning it. the bilayer originates from the host cell membrane and has a lipid composition and transbilayer distribution quite similar to the host's. the viral glycoproteins have the functions of binding to the target cell surface and fusion with the cell membranes. the two functions are carried by a single glycoprotein in influenza virus (ha), vesicular stomatitis virus (vsv) g glycoprotein, and semliki forest virus sfv e glycoprotein. in sendai virus (hvj), the functions are carried by separate glycoproteins, hemagglutinin-neuraminidase (hn) for binding and fusion glycoprotein (f) for fusion. when viruses encounter target cells, they first bind to the cell surface through an interaction of the viral glycoprotein with receptors. many families of viruses have an envelope wrapping their genome, rna or dna. the envelope is a lipid bilayer membrane with the virusspecific glycoproteins spanning it. the bilayer originates from the host cell membrane and has a lipid composition and transbilayer distribution quite similar to the host's. the viral glycoproteins have the functions of binding to the target cell surface and fusion with the cell membranes (see table i ). the two functions are carried by a single glycoprotein in influenza virus (ha), vsv (g), and sfv (e).' in hvj, the functions are carried by separate glycoproteins, hn for binding and f for fusion. when viruses encounter target cells, they first bind to the cell surface through interaction of the viral glycoprotein with receptors ( fig. la) . sialoglycoproteins andlor sialoglycolipids are known to be the receptors for hn in hvj and ha in influenza virus. fusion of the virus envelope with target cell membranes is an essential initial step in infection since the virus can transfer its genome into the cytoplasm by this event (fig. lc) . envelope fusion is induced by the action of the virus glycoprotein on target membranes. some fusion proteins are initially produced in precursor forms and then cleaved posttranslationally by proteolytic enzymes. f in hvj and ha in influenza virus are such examples. they are produced as precursor forms fo and ha and then cleaved into f and f (homma and ohuchi, ; scheid and choppin, ) and ha and ha (klenk et al., ; lazaro-witz and choppin, , linked by disulfide bonds between the subunits, respectively. the precursors are inactive, but the cleaved forms are active in both fusion and infectivity, strongly suggesting a causal relationship between them. other examples are gpr in mmtv and e in mouse hepatitis virus which are cleaved into gps and gp (redmond and dickson, ) and a and b (sturman et al., , respectively, to become active. e in sfv is cleaved into e , e , and el (garoff et d., ) . on the other hand, g in vsv is not subject to cleavage. the amino-terminal segment of ha , f , and gp consists of or more hydrophobic amino acid residues ( table ) . that of ha also contains two or three acidic residues. these segments are produced on posttranslational proteolytic activation. they are assumed to be responsible for fusion activity because of the hydrophobicity and also because of the conservation of sequence among various strains of hvj and influenza virus (gething el a!., ) . however, the sequence homology is not always observed; for example, influenza c virus and pneumovirus have sequences different from other viruses except for the hydrophobicity. el in sfv and g in vsv do not contain such amino-terminal hydrophobic segments but have internal hydrophobic stretches. residues - in el and residues - in g, which have sequence homology among the strains, may be such stretches though not strongly hydrophobic (table shun-ichi ohnlshl and the putative fusogenic segment should be able to interact with the target membrane, inducing some disturbance eventually leading to fusion (fig. ib) . generally, proteins may have similar hydrophobic segments in them. even acqueous enzymes have such segments: two in porcine trypsin and one in hepatic alcohol dehydrogenase (asano and asano, ) . virus fusion proteins can also have more than one hydrophobic segment. for example, residues - in g is another hydrophobic sequence (table ). these hydrophobic segments can be fusogenic only when they approach and interact with target membranes. envelope fusion was first shown to occur for hvj by electron microscopy (howe and morgan, ) . such fusion was not clearly demonstrated for other viruses, however, and, instead, uptake of viruses into intracellular coated vesicles and smooth vesicles was observed. this raised a question of the mechanism of virus genome transfer into the target cell cytoplasm for these viruses. ten years later, the induction of envelope fusion activity in mildly acidic media was discovered for sfv (vmnanen and kaariainen, , ) , influenza virus (maeda and ohnishi, ; huang ef al., ; lennard and miller, , and later vsv (white et al., ; mifune et al., ; matlin et al., ) . the ph dependence of fusion was markedly different; while hvj can fuse at neutral as well as acidic ph values, sfv, influenza virus, and vsv can fuse only at acidic ph (fig. ) . a new virus entry mechanism was proposed for these viruses on the basis of these findings, sfv by helenius et ul. ( ) , influenza virus by maeda and ohnishi ( ) , matlin et al. ( , and yoshimura et al. ( ) , and vsv by matlin et al. ( ) . after uptake into intracellular vesicles, these viruses fuse with vesicle membranes when the intravesicular lumen becomes acidic and release their genome into the cytosol. at first, lysosomes were proposed as the acidic compartment, since they are well known to have a ph of . (ohkuma and poole, ) . after the proposal, however, a rapid acidification of prelysosomal endocytic vesicles (endosomes) was discovered in by tycko and maxfield ( ) and van renswoude et al. ( ) . genome transfer by fusion with endosomes was shown shortly afterward by marsh et al. ( b) for sfv and yoshimura and ohnishi ( ) for influenza virus. in this chapter, i first review some characteristic features of membrane fusion activity for each virus and then discuss the mechanisms of membrane fusion, especially low ph-induced membrane fusion. i concentrate on the interaction of the hydrophobic segment with the target cell membrane lipid bilayer and suggest the entrance of the segment into the lipid bilayer hydrophobic core as a key step in fusion. for the hydrophobic segments containing a few acidic residues, i emphasize protonation of those residues as a requirement for the entrance. finally, i briefly review (yamada and ohnishi. ) and fusion with mdck cells (white et ( i / . , ) . (modified from ohnishi and yoshimura. x .) the entry pathway of virus into cells leading to infection. several review articles have been published on virus membrane fusion activity (white r t a / . , ; ohnishi and yoshimura, ; asano and asano, ; ohnishi, a) and virus entry mechanisms (helenius t r f i l . , : dimmock, marsh, ) . fusion of hvj with erythrocyte membranes was first observed by electron microscopy as a dispersal of viral antigen into the cell membrane (howe and morgan, ) . virus-induced hemolysis has been used as a shun-chi ohnlshl rapid, sensitive, and convenient assay for envelope fusion. however, this assay is indirect since it measures a result of envelope fusion. a different approach based on intermixing of viral lipids with target cell membrane lipids was developed (maeda et al., ; ohnishi, a) . virus is incubated with spin-labeled phospholipids to incorporate them into its envelope. the spin-labeled virus is then incubated with target cell membranes. the esr peak height increases on fusion due to dilution of spin-labeled phospholipids with cell membrane lipids. assays of fusion using fluorescent probes based on the same principle were also developed (wyke at al., ; struck et al., ) . a recent example is to use octadecyl rhodamine b chloride loaded into virus envelopes and to measure the relief of fluorescence quenching on fusion (hoekstra at al., ) . these spectroscopic methods provide rapid, continuous, and quantitative information on fusion. a drawback is the difficulty in discriminating fusion from exchange of lipids between two membranes without fusion, if it occurred. envelope fusion is a rapid reaction reaching a saturation level within - min at °c. it does not require ca + or mg +. it has a characteristic temperature dependence; practically no fusion below °c and progressively faster fusion at higher temperatures. the temperature dependence was correlated with the onset of the segmental and rotational motion of the viral glycoproteins in the envelope as measured by the decay of transient dichroism of eosine triplet probes (lee et al., ) . a discontinuity in the fluidity of target erythrocyte membranes was also observed near °c using spin-labeled pc (tanaka and ohnishi, ) . the fusion reaction follows first-order kinetics. the rate constant of fusion with erythrocyte membranes was obtained as . min-' at °c by the spin-label assay (kuroda et al., ) . kinetic analysis suggested that not all virus particles bound on cells fuse at this rate constant but only about - particles per cell do. kinetic analysis of the fluorescence dequenching data based on the mass action law gave . - . min-' for the rate constant (nir et al., b) , which is around - times greater than that obtained by the spin-label method. the ph dependence of fusion is rather broad in the range from ph to (fig. a) . the fluorescence assay also showed a broad ph dependence, although the rate constant as well as the efficiency of fusion are considerably low on either sides of neutral ph . hvj can fuse with liposomes containing as well as not containing receptors (glycophorin or gangliosides). fusion with liposomes containing anionic phospholipids is more efficient. the effect of cholesterol on fusion is controversial. while haywood and boyer ( ) observed no effect of cholesterol, hsu et al. ( ) showed a requirement in fusion and kundrot (duzgiines, ) . liposomes reconstituted with the viral glycoproteins hn and f have hemolytic and fusogenic activities (hosaka and shimizu, ; volsky and loyter, a) . ozawa and asano ( ) showed that cholesterol was required for the functional reconstitution. hvj causes hemolysis. the virus appears to have an inherent defect in the envelope membrane which creates pores in the cell membrane after fusion. accelerated water inflow causes cell swelling and lysis after a certain threshold. pore formation is observed as the permeability to low molecular weight (< , ) compounds increases (pasternak, ) and the membrane potential or resistance decreases (okada et al., ) . membrane damage in cultured cells is repaired on further incubation at °c (okada et a/., ) . ca + inhibits pore formation, thus inhibiting virus-induced hemolysis (pasternak, ) . virus harvested i day after infection of embryonated eggs (one cycle of reproduction) is nonhemolytic, in marked contrast to hemolytic viruses obtained after days (homma ei ul., ) . in electron micrographs, uranyl acetate stain does not penetrate the early harvested viral envelope, whereas it penetrates the late harvested viruses (shimizu et ul., ) . the early harvests have fusion activity as well as infectivity, in spite of the lack of hemolytic activity. the virus therefore appears to have less damage in the envelope and cannot produce pores in the cell membranes. interestingly, early harvests become hemolytic on freeze-thawing, incubation at "c, or sonication . a morphological study was carried out to obtain the structural basis for the difference (kim et al., ) . both early and late harvests have closely packed spikes on the envelope consisting of hn and f. however, there is a marked difference between t em in freeze-fracture electron micrographs. while late harvests have large imps with diameters of nm on the e-face, early harvested viruses have no visible particles on either fracture face. large imps appeared on the e-face on incubation at °c. nucleocapsid strands are regularly folded under the envelope in early harvests but become irregular and detached from the envelope on incubation. the invisible to visible transformation of imps could be due to aggregation of viral glycoproteins in the envelope. the peripheral matrix (m) proteins may also be involved in the transformation in this case. p shun-ichi ohnlshl . cell fusion cell fusion induced by hvj was discovered by okada ( ) (see okada, chapter , this volume) . cell fusion is initiated by viral envelope fusion with the cell membrane, but requires factors, such as atp, in addition to the latter process. it is inhibited by cytochalasins and high concentrations of mono-and disaccharides (maeda et a / . , ) , whereas envelope fusion is not affected by these agents. envelope fusion produces intercellular cytoplasmic bridges, and the ensuing osmotic swelling of cells causes expansion of the locally fused sites to form spherical polykaryons (knutton, ) . this sequence was clearly shown using nonhemolytic viruses. the fusion reaction stopped at the initial stage with these viruses because of the lack of cell swelling. when the cells were subsequently swollen by lowering the osmolarity of the medium, they formed spherical polyerythrocytes (knutton, ; knutton and bachi, ) . polyerythrocyte formation is very efficient with erythrocytes but extremely inefficient with erythrocyte ghosts because of the lack of osmotic swelling in the latter. sekiguchi and asano ( ) have succeeded in attaining a large enhancement of polyerythrocyte formation by preloading bovine serum albumin ( %) into ghosts. hvj causes clustering of imps with the accompanying formation of a naked lipid bilayer area in erythrocyte (bachi et al., ) as well as in cultured cell membranes (kim and okada, ) . the clustering in cultured cell membranes disappears on continued incubation at °c but persists in erythrocyte membranes. the clustering is dependent on the temperature at which the membrane specimen was frozen. it is observable when quenched from lower temperatures ( - °c) but not on quenching from higher temperatures ( - °c) (volsky and loyter, b; kim and okada, i) . this is essentially a reversible thermotropic phenomenon triggered by mobilization of the membrane proteins by the action of the virus (see section ii,b, ). the clustering of imps is well correlated with cell fusion, and it is not observed under conditions where the fusion of cultured cells is inhibited (kim and okada, ) . for erythrocytes, this correlation is further confirmed by an experiment using ghosts loaded with bovine serum albumin. hvj caused efficient fusion of these ghosts, but, when antispectrin antibody was additionally loaded in ghosts, both cell fusion and imp clustering were inhibited (sekiguchi and asano, ) . this result also indicates a correlation between imp clustering and the cytoskeletal spectrin meshwork as well. the amino-terminal segment of fl consists of about hydrophobic amino acids, hydrophobic residues with an anionic asp at position and a positive arg at in hvj, and hydrophobic residues with a positive lys at in respiratory syncytial virus (a pneumovirus) (see table ). the sequence is well conserved among paramyxoviruses, but the homology with pneumovirus is poor. these hydrophobic segments should be able to approach target membranes in the fusion reaction. partial exposure of this segment to external media was shown by using various proteolytic enzymes (asano er al., ) . treatment of hvj with aminopeptidase m resulted in a change of the f amino terminus from phe to ala, yet in no apparent change in the mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page), suggesting that (or ) amino-terminal residues were cleaved off. since the treated virus retained infectivity, these terminal amino acids may not be essential for fusion activity. presence of receptors for the amino-terminal segments in target membranes has been suggested from studies on inhibition of virus replication by small peptides with amino acid sequences similar to that of the viral amino terminus (richardson et al., ; richardson and choppin, ) . carbobenzoxy (z)-d-phe-l-phe-gly-d-ala-d-val-d-ile-g y and z-d-phe-l-phe-gly are the most potent inhibitors of measles virus replication. other peptides with - amino acids are also inhibitory, though weaker. these peptides bind to and express their inhibitory activity on cells but not on virus. however, the recognition is limited to only - residues. even z-d-phe and z- .-phe had inhibitory activity. peptides with natural amino termini have much weaker activity, / to / , of that of peptides with the artificial carbobenzoxy group. d-amino acids are not contained in the viral peptides. the results therefore do not appear to prove the presence of a specific receptor. moreover, the results are not compatible with the observation that the cleaving off of afew residues from the amino terminus does not affect infectivity (asano et al., ) . asano and asano ( ) proposed a specific binding of the amino-terminal segment of f to cholesterol in target membranes. a. virus. low ph-induced envelope fusion, hemolysis, and cell fusion were observed by ohnishi ( ), huang et al. ( , lennard and miller ( ) , white et al. ( ) , and yoshimura et ul. ( ) . fusion is activated at ph values lower than and becomes optimum at ph s (fig. b ). an electron micrograph showing fusion with erythrocytes at acidic ph is given in fig. b . proteolytic cleavage of ha into ha and ha is required for fusion activity (huang et a/., ; maeda et al., , indicating an essential role for the low ph fusion activity in infection. envelope fusion is a rapid reaction, reaching a saturation level of - % fusion in - min at °c at ph . . temperature dependence of the hemolytic activity correlated well with that of the rotational mobility of ha glycoproteins. the mobility thus appears to be required for the molecular rearrangements necessary for fusion activity (junanker and cherry. ). not only various strains of type a and b influenza viruses (shibata et d., ) but also type c viruses (ohuchi r' t a/., ; kitamae ef a / . , ) have low ph-induced fusion activity. the ph dependence is a little different among them. as a conventional measure to show the difference in the ph dependence, the ph at half-maximal fusion, ph,,?. is taken (table ). it ranges from . to . for most strains. wild-type viruses may contain variants with different ph characteristics. such variants were isolated from the x- i strain. variants with a common substitution at position in hai, arg for his, showed a large shift (+ . unit) in the phli value (rott et uf., ) (see table ). another variant showed a smaller shift ( + . unit). a substitution at position in ha , asn for asp, is responsible for this shift (doms et d . , ) . daniels rt al. ( ) selected mutant viruses with altering ph dependence by growing virus in cells treated with amantadine chloride, which raises the endosomal ph. thirty-eight mutants had phli values from . to a maximum of . unit higher than the parent viruses, x- and somes, but their genome has already been released into the cytoplasm to initiate reproduction of viral materials. bar, . p m . (photographs a-f are rrom yoshimura c i ol., ( ) yoshimtiria c'/ rrl. ( x ) . ( ) kitdniilc c/ d. ( x ) . chicken/germany/ strains. mutants with the highest phliz value ( . and . ) are listed in table . destabilization of the location of the ha amino-terminal segment and alteration of the intersubunit contacts are related to the altered ph dependence. gething et al. ( ) constructed three mutants by site-specific mutagenesis that introduced amino acid changes in the ha amino-terminal segment (see table ). a mutant substituting glu for gly at position i i had the same fusion activity as the parent virus, japan/ / . substitution of gly for glu at position raised the threshold ph by . unit and decreased fusion efficiency. substitution of gly for glu at position i destroyed fusion activity down to ph . . c. ha and bha. ha solubilized from the virus with detergent forms rosettelike micelles on removal of the detergent. ha rosettes bind to the cell surface receptor and induce hemolysis at acidic ph in a similar manner to the parent virus (sato et al., ) . they can also cause fusion of erythrocyte membranes at low ph, when the membranes are brought into close contact by poly(ethy ene glycol) ( %) (s. b. sato et al., unpublished) , as well as fusion of liposomes (wharton et al., ) . recently, it has been shown that a synthetic amino acid peptide with the same sequence as that of ha n-terminal segment induced fusion of egg pc vesicles at acidic ph (murata et al., b; see section iii,b, ,b) . ha is a member of a family of glycoproteins which contain a covalently linked fatty acid (schmidt, ) . a possible involvement of the fatty acid in the hemolytic or fusion activity was shown by schmidt and lambrecht ( ) . these authors observed loss of hemolytic activity of influenza virus and ha rosettes on removal of fatty acid with hydroxylamine. bromelain treatment of the virus releases the ectodomain of ha by cleaving the peptide bond at residue in ha . bha is soluble in water but aggregates to form micelles in acidic media. bha binds to the cell surface receptor but cannot induce hemolysis at acidic ph (sato et al., ) , d. interaction of ha with liposomes. interaction of ha with receptor (gang ioside)-containing liposomes was studied by measuring the fluorescence of trp residues (ohnishi, %; kobayashi et al., unpublished) . after allowing the binding of ha to liposomes and washing, the liposome suspension was acidified, neutralized, and treated with semialkali protease. ha was digested and ha remained bound. liposomes were isolated, subjected to further extensive proteolysis by bromelain or proteinase k, and centrifuged in a sucrose density gradient. isolated liposomes still contained a small peptide(s) which gave fluorescence due to the trp residue at nm. the fluorescence was not quenched by the aqueous quencher acrylamide, but was greatly quenched by spin-labeled stearates incorporated into liposome membranes. these results suggest that a peptide portion of ha containing a trp residue(s) has entered into the lipid bilayer at acidic ph. e . ha-reconstituted vesicles. ha reconstituted in pckholesterol ( : ) vesicles can have the same fusion activity as the parent virus (kawasaki et al., ) . fusion efficiency was dependent on the spike density in the reconstituted membranes. preparations at appropriate protein-lipid ratios produced vesicles with a high spike density that fused with erythrocyte membranes at ph . as rapidly and efficiently ( - %) as the parent virus. influenza virus can fuse with liposomes, either containing or not containing receptors, in the same way as with cell membranes. fusion does not require specific classes of phospholipids, although fusion is more efficient with ps-containing liposomes than with pc alone. cholesterol does not significantly affect fusion (maeda et ai., ; white et al., ) . fusion with cardiolipin liposomes was more efficient than that with ps (stegmann et al., ) . the fusion rate constant was obtained as sec-' at °c at ph . from the analysis of fluorescence dequenching data (nir et al., a) . liposomes made only of cardiolipin could be considered to be artificial and to have different characteristics than natural membranes as target membranes for fusion (stegmann et al., ) . the three-dimensional structure of bha at neutral ph was determined to a resolution of a (wilson et ul., ) (see fig. ). ha molecules form trimers and extend . nm from the envelope surface. the receptor binding site is located in ha near the upper surface of the molecule. the amino-terminal hydrophobic segment in ha is located in a different domain near the envelope surface and hidden inside the trimer. when the virus binds to the cell surface receptor, the two membranes are still far apart. a large conformational change is therefore required at acidic ph to bring the two membranes into close contact and to make the hydrophobic segment exposed and approach the target membrane. such a structural change has been observed by various methods. bha and ha are resistant to trypsin at neutral ph but become susceptible after low ph treatment. the ph dependence of the susceptibility agrees well with that of envelope fusion (skehel et al., ; sat et al., ) . the cleaved sites were determined to be at lys and lys in the ha component (skehel et al., ) . the conformational change appears to expose the ha amino-terminal hydrophobic segment. aggregation of bha molecules to form micelles at acidic ph is likely to take place via hydrophobic association, placing the amino-terminal segments near the center. treatment with thermolysin removed the amino-terminal residues and caused resolubilization (daniels et al., ) . bha binds to phospholipids or detergents at acidic ph when these amphiphilic molecules coexist, probably by association with the exposed hydrophobic segment. the ph dependence of the binding is the same as that of envelope fusion. electron microscopic observation of the isolated virus and of ha showed a thinning and marked elongation of ha in acidic media. trypsin treatment reversed the elongation, resulting in a shortening of ha (ruigrok et al., ) . low ph treatment of bha did not signficantly affect the cd spectrum in the far uv region, indicating a very small, if any, effect on the secondary structure. therefore, the structural change may not involve a gross denaturation but a relative movement of domains which maintain their individual structures (skehel et al., ) . the low phinduced phenomena are apparently irreversible on restoring the ph to neutral. ha molecules form micelles at neutral ph. this association is probably due to hydrophobic clustering of the membrane-spanning hydrophobic segments near the ha carboxy terminus. low ph causes aggregation of these micelles, probably via hydrophobic association of the ha aminoterminal segments. ha molecules interact strongly with target membranes probably via the exposed hydrophobic segment, and cause lysis and fusion. entrance of a trp-containing peptide segment in ha into the lipid bilayer at acidic ph has been suggested (section ii,b, i ,d). influenza virus causes imp clustering in erythrocyte membranes when incubated at ph . but not at ph . (see figs. c-e) (yoshimura et al., ) . clustering was dependent on the temperature of quenching, as in the case of hvj (section ii,a, ) . at "c, protein distribution was more random. the clustering is a reversible thermotropic aggregation of membrane proteins, and the effect is similar to that of hvj, but much more extensive (see, for example, fig. e) . the difference is not due to the incubation of erythrocytes at acidic ph, since the clustering induced by hvj at ph . was similar to that at ph . . ha rosettes also cause imp clustering in erythrocyte membranes at ph . , although the degree of clustering is not so large (fig. f) , similar to that caused by the virus after incubation for a short period, e.g., i min (fig. d) . imp clustering is probably a result of virus-induced mobilization of target membrane proteins as observed by fluorescence photobleaching recovery measurements (yoshimura et ul., ) . lateral mobility of band proteins in erythrocyte membranes is largely restricted under physiological conditions; the diffusion constant is of the order of cm? sec-i. incubation of erythrocytes with influenza virus or ha rosettes at ph . caused a large mobilization of band proteins, the diffusion constant increasing or -fold, respectively. no such mobilization was observed when the cells were incubated at ph . or when ghosts were incubated at ph . without virus. the mobilization may be related to modification of the cytoskeletal meshwork underneath the membrane, consisting of spectrin-actin-band . peripheral proteins. a diffuse fibril structure was converted into aggregated dense spots, and membrane regions lacking the meshwork were produced after incubation with virus at ph . . a model for the restriction by the cytoskeletal network has recently been presented (tsuji and ohnishi, ) . there are two populations of band proteins. about lo-i % of them, calculated as band monomers, are anchored to the cytoskeletal network via ankyrin and are immobile. ankyrin-free band proteins are mobile, but the mobility depends on the spectrin associated state (tetramer or dimer). band cannot move when its cytoplasmic domain is surrounded by the tetrameric spectrin network, but it can cross the network when spectrins are in dissociated dimers. the virus-induced mobilization could therefore arise from modification of the spectrin association state by direct or indirect action. low ph-induced hemolytic activity of sfv was first observed by vaananen and kaariainen ( ; ) . envelope fusion with various cultured cells was very rapid (within sec) and efficient ( %) at ph . at °c (white ef d., ) . virus-induced cell fusions were also observed (white et ul., ) . the ph dependence of fusion activity is shifted a little higher than that of influenza virus (fig. c) . the phi, value ranges from . to . (table ). a fusion mutant had a lower value of . . sindbis, another alphavirus, had a phi of . . low ph-induced fusion activity was also observed for yellow fever virus (cammack and gould, ) and west nile virus (gollins and porterfield, ) , both belonging to a different genus, flauiuirus (table ) . sfv can fuse with simple liposomes in a way similar to fusion with cell membranes. in these fusions, cholesterol in the target membranes is required, a characteristic difference from many other viruses . maximum fusion was observed with more than mol % cholesterol. the phospholipid requirement was not strong, although fusion with pc was less efficient compared with other classes of phospholipids such as pe and ps. ca ' or mg + was not required for fusion . isolated e glycoproteins form octameric micelles which, however, do not have hemolytic activity (vaananen and kaariainen, ) . the glycoproteins reconstituted into liposomes showed low ph-induced hemolytic and fusion activities (marsh et al., a) . the fusion efficiency was lower ( %) than that of the parent virus, possibly due to lower spike density on the reconstituted membranes. cholesterol was not required for reconstitution. of the three components of e, el was shown to be active for hemolysis and hemagglutination. yamamoto et al. ( ) solubilized e glycoproteins from western equine encephalitis virus, an alphavirus, and isolated the components. reconsituted vesicles containing e i alone but not those containing e alone showed fusion activity. it is suggested that e may cooperate with el in the hemagglutination activity. both el and e span the viral envelope membrane, with the hydrophobic segment near their carboxy termini. another hydrophobic segment near but not at the amino terminus of e l , residues - or - , may be fusogenic (table ). there are a few acidic residues in the segment. sindbis el has a quite similar sequence at the same site. a low phinduced conformational change in sindbis el was detected by the change in susceptibility to tryptic cleavage (edwards et al., ) . low ph-induced hemolysis and cell fusion were observed in vsv by white et al. ( ) . the ph range for fusion activity is similar to sfv (fig. d) , the phli value ranging from . to . (table ) . a characteristic of vsv different from other viruses is that protease pretreatment of target cells does not destroy but rather enhances virus binding and fusion. the human erythrocyte is an extreme example: the virus cannot agglutinate nor fuse with intact cells but shows enhanced hemagglutination and fusion activities to trypsin-treated cells (mifune el al.. ) . fusion with trypsinized erythrocytes is very rapid (within - min) and efficient (- % fusion) at ph . at °c (yamada and ohnishi, ) . pretreatment of l cells with trypsin or neuraminidase also results in increased binding and plaque formation (schloemer and wagner, ) . these results strongly suggest that sialoglycoproteins and sialoglycolipids as well as proteins are not receptors. instead, lipids may be the direct target site. inhibition of virus binding and infection by ps liposomes supports this idea (schlegel et d., ) . vsv can bind and fuse with simple liposomes (yamada and ohnishi, ) . fusion with liposomes is quite similar to that with trypsinized erythrocyte membranes, in the rate, efficiency, and ph dependence. binding to liposomes is also dependent on ph; the threshold ph ( . ) is about i unit higher than that for fusion. binding and fusion with liposomes did not decrease at lower ph values, while those with cells decreased. binding to liposomes was dependent on the head groups of the phospholipids, being stronger to phosphoserine than to phosphocholine. on the other hand, the head group requirement was not strong for fusion, but the presence of cis-unsaturated fatty chains in phospholipids was required. for example, efficiency of fusion with cis-unsaturated dioleoyl pc was , but that with saturated dimyristoyl pc was negligibly small, %. cholesterol enhanced fusion further but was not an absolute requirement. efficient fusion with various natural phospholipids and lipids is probably due to the presence of cis-unsaturated fatty acids. lsolated g glycoproteins do not have hemolytic activity by themselves; however, g glycoproteins reconstituted into vesicles have fusion activity (eidelman et a/. , ) . reconsituted vesicles prepared at high protein/ lipid ratios (- . mol protein) and by slow removal of detergent had high fusion activity. fusion with liposomes containing ps or pe are much faster than that with pc alone. inclusion of cholesterol did not affect the fusion. however, it is noteworthy that the ph range for fusion shifted largely to lower values, with a threshold at ph . . a n alternative reconstitution procedure has been used recently to produce reconstituted vesicles whose fusion activity has a ph dependence similar to that of the intact virus (metsikko et al., ) . the amino-terminal segment of g protein is not hydrophobic, containing basic lys and his residues. residues - have sequence homology between lndiana and new jersey serotypes and appear more hydrophobic (table ) . however, a thermodynamic estimate shows that they are not hydrophobic as a whole (section iil,b, ). residues - can be hydrophobic at acidic ph (see table iv ). peptides of amino acid residues corresponding to the amino-terminal segment of g can cause hemolysis at low ph, with a ph dependence similar to that for the virus (schlegel and wade, , ) . even much smaller peptides ( amino acid residues) caused more efficient hemolysis. however, the activity was independent of ph. therefore, the activity may not reflect that of the parent virus. low ph-induced fusion activity has also been observed for other enveloped viruses, a retrovirus (mmtv) and a bunyavirus (la crosse) ( table ) . a low ph-induced conformational change in la crosse virus gi glycoprotein has been reported (gonzales-scarano, ) . in the fusion reaction, virus first binds to a target cell membrane through interaction of the viral glycoprotein with the receptor (fig. la) . the virus and cell membranes would still be far apart since the viral glycoproteins usually extend externally some nm or more (fig. ) . closer apposition of the two membranes is required for further interactions. in influenza and other viruses, the close apposition may be achieved by a conformational change of the viral glycoprotein induced at acidic ph. the conformational change would cause mobilization and rearrangement of target cell membrane proteins to produce naked lipid bilayer domains assisting the close apposition. the conformation change would also cause exposure of the hydrophobic segment in the glycoprotein to make it approach and interact with the lipid bilayer in target membranes. (fig. ib) . morphological observation of isolated influenza virus and ha showed a thinning and marked elongation of ha at low ph (ruigrok et al., ) . however, this is the change induced between ha spikes in the absence of binding to target membranes, and change in ha when bound to target membranes may be different. the conformational change may be triggered by protonation of some acidic amino acids. the conversion of charged residues into protonated neutral ones would greatly alter interresidue interactions. the pk, value of mildly acidic residues in proteins lies in a range . - . for his, . - . for asp (p-carboxyl), - . for glu (y-carboxyl), and . - . for the carboxy-terminal a-carboxyl group (dawes, ) . the pk, value may deviate for some specific residues. for example, in lysozyme, glu has a much higher value of - . and asp a much lower value, . - , while the other asp residues and have ordinary values, - . and . - . . respectively (imoto ef ul., ) . since the conformational change of viral glycoproteins occurs at ph values lower and higher than . , asp, glu, and his residues are possible sites for the protonation. viruses can also bind and fuse with simple liposomes lacking receptors. the mode of binding should be different from that of binding to the receptor-containing membranes. for example, the site in the viral glycoprotein used for the binding should be different. the ph dependence may therefore be different between these two types of binding. such differences have been demonstrated: while binding of influenza virus to cell membranes occurs in a wide range of ph values from to ( m a t h et al., : yoshimura et al., ) , binding to liposomes occurs only below ph . , in parallel to the fusion activity (doms et al., ) . the binding to liposomes may involve interaction of the ha amino-terminal hydrophobic segment, exposed at the low ph, with the lipid bilayer. both events are thus effected by the same cause. on the other hand, binding to receptors on target membranes occurs at neutral ph and also at acidic ph so long as the conformational change in ha does not destroy the binding site in ha . no gross denaturation of ha at low ph was indicated by cd measurements (skehel et a / . , ) . binding of hvj to liposomes may be ascribed to the partially accessible hydrophobic segment in f . the hydrophobic segment in virus fusion proteins probably attacks the lipid bilayer domain in the target cell membrane. such domains may be formed as a result of virus-induced mobilization of target membrane proteins (yoshimura et d., ) . formation of lipid domains has been detected by freeze-fracture electron microscopy. ability of viruses to fuse with liposomes either containing or not containing receptors supports the idea, provided that such fusions mimic those with cell membranes. the presence of a specific receptor to the amino-terminal segment has been suggested but not fully proved (section i ,a, ). the hydrophobic segment may enter the target lipid bilayer hydrophobic core. the free energy for transfer of the segment from aqueous to lipid bilayer phases will be negative. an estimate for the free energy value was made as shown in table iv . this analysis gave large negative values for many viral hydrophobic segments. a segment containing charged residues is not favorable for transfer because of the work necessary to bring a charge into hydrocarbon media (parsegian, ) . when these charged residues are neutralized by protonation or deprotonation, transfer would be much easier. it has been shown that a single substitution of a hydrophobic amino acid residue (e.g.,ala) for a charged residue (e.g., glu) in the signal peptide of secretory proteins blocks transfer of the proteins across the inner membrane of mutant e. coli (bedouelle et al., ; emr er al., ) . " free energies were calculated for transfer of a-helical segments, using parameter values assigned to each amino acid residue by von heijne f ). the values for transfer at neutral ph include the work to protonate acidic residues at neutral ph, while the work is omitted from the values at acidic ph since protonation occurs spontaneously. for the amino-terminal hydrophobic segments, free energy values are given for transfer of the internal segment (e.g.. - ) and the whole segment (e.g., - ), corresponding to models a and b in fig. . respectively. in the latter, a contribution from the work to deprotonate the aminoterminal charge at the respective ph values is included. the hydrophobic segment in viral glycoproteins which have fusion activity only at acidic ph often contains a few acidic residues. we have proposed that the low ph is required for protonation of the acidic residues, as well as for the conformational change, to neutralize the charge so that the hydrophobic segment may interact more easily with the target lipid bilayer (maeda and ohnishi, ; ohnishi, sa) . the protonation occurs spontaneously at acidic ph, but requires work at neutral ph since the latter is higher than the pk, value of the acidic residues. this extra work, - kj/mol per acidic residue, is included in the free energy for transfer at neutral ph but omitted at acidic ph. the free energy values at acidic ph are therefore much lower ( - kj/mol) than those at neutral ph (table iv) , although the latter values are also negative in most cases. the hydrophobic segment in gp of mmtv, another low ph-active virus, does not contain acidic residues, however. in this case, the low ph may be required only for the conformational change to expose the hydrophobic segment which is ready for interaction without protonation. the free energy value for transfer is largely negative, independent of ph (table iv). our point is that when the segment has acidic residues, they should be neutralized for the interaction. this leads to a hypothesis that the hydrophobic segment in viruses with fusion activity at neutral ph should not contain acidic residues, which so far holds. there are two possiblities for the entrance of the neutralized hydrophobic segment into the lipid bilayer (fig. ) : entrance of the internal hydrophobic segment between two charged residues (a) or entrance of the whole hydrophobic segment (b). extra work is required to neutralize the aminoterminal charge in b, when it has a charge, and the free energy value for transfer is correspondingly high ( table iv) . the internal segments in sfv el and vsv g proteins listed in table i are not so hydrophobic ( table a t the segment - in el between two positively charged residues has a positive free energy value at neutral and acidic ph. a shorter segment ( - ) can have a negative value at acidic ph, though small. the well-conserved sequence - in vsv g protein is not hydrophobic. another segment can have a negative free energy value at acidic ph. whether these segments are actually involved in fusion of these viruses requires investigation. b. ph-dependent fusion activity of a peptide with the same sequence as that of the ha amino-terminal segment. in order to investigate that the putative fusogenic segment actually has membrane fusion activity and also that protonation of the acidic residues is required for activity, we synthesized a -amino-acid peptide with the same sequence as that of the ha amino-terminal segment and studied its fusion activity (murata et al., b) . the synthetic peptide caused a rapid and efficient fusion of egg pc vesicles at acidic ph, but not at neutral ph, in a manner quite similar to that of the parent virus (fig. a) . the threshold ph was around . , and the phllz value was . . fusion efficiency was dependent on the peptide to phospholipid ratio in the mixture, increasing with increases in the ratio. the low ph-induced fusion can be stopped immediately by shifting the ph to neutral and started again by readjusting to acid. the peptides with acetylated or succinylated amino termini also had similar fusion activity. the ph range was, however, shifted about ph unit to the acidic side. these results clearly demonstrate a direct involvement of the fusogenic segment in fusion and strongly suggest the requirement of protonation for activity since there are no other acid-sensitive groups in this simplified fusion system. c. ph-dependent fusion activity of succinylated melittin. hydrophobic peptides can have membrane fusion activity. the natural hydrophobic peptide bee venom melittin is one such example. this peptide actually caused fusion of egg pc vesicles at neutral as well as acidic ph (fig. b ) (murata et al., a) . a succinylated derivative of this peptide was synthesized in which all four amino groups, one a-amino group and three eamino groups at lysines, were converted to carboxyl groups. this derivative caused fusion of pc vesicles only at acidic ph, in marked contrast to the parent peptide (fig. b) . the threshold ph for fusion was . and the phllz . . the ph characteristics of fusion can thus be modified to acid sensitive by introducing acidic groups. the low ph-induced fusion by succinylated melittin can be stopped immediately by shifting the ph to neutral and started again by readjusting to acid. protonation of the introduced carboxyl groups was studied by c-nmr spectroscopy using melittin succinylated with [ i , -i c] succinic anhydride. protonation in the presence of pc vesicles occurred in the same ph range as that for fusion. the pk, value for the four carboxyl groups was obtained as . , in close agreement with the phliz value. the agreement strongly indicates that fusion activity is induced by protonation of acidic residues. the pk, value was higher than that in the absence of vesicles, . . the upward shift will occur when the protonated form is stabilized in the presence of vesicles. a probable model is entrance of the protonated hydrophobic segment into the lipid bilayer as b in fig. , since it has no charge at the amino terminus when protonated. such stabilization will cause a shift of the acid-base equilibrium in favor of the protonated form. in the i c-nmr spectrum of succinylated melittin both in the presence and absence of vesicles, only a single peak was observed for each carboxyl group during the titration. this indicates fast exchange between the protonated and deprotonated forms, faster than msec. if the pro-tonated segment entered into the lipid bilayer, then the fast exchange would include the entrance into and returning back to the surface of bilayer membranes. such a dynamic nature of interaction can well explain the rapid on/off switching of fusion on shifting ph to neutral and acid. d . primury sequence versus hydrophobicity. the above discussions are solely thermodynamic, considering only the hydrophobicity of the segment which does not require any specific primary sequence. is the primary sequence also essential for the fusion function? needless to say, the primary sequence determines the three-dimensional structure and the conformational change of ha at low ph. the ha amino-terminal segment is also involved in secondary structure formation; the terminal nitrogen and the amide nitrogen of residues , , and form hydrogen bonds to oxygen atoms of residue (asp) in the long helix of ha (wilson et al., ) . mutations at these sites should alter interresidue interactions to affect stability of the structure and ph for the conformational transition (daniels et al., ) . however, the primary sequence may not be important for interaction with the lipid bilayer. for example, influenza c virus has an amino-terminal sequence with very little homology with a and b viruses, but has a common feature of hydrophobicity. a . membrane perturbation. studies on the fusion of phospholipid vesicles by hydrophobic peptides containing acidic residues indicated that fusion activity was induced by protonation of the acidic residues (section iii.b, ). at neutral ph. these peptides bind to the vesicle surface but do not cause aggregation of vesicles. at acidic ph, when charges in the hydrophobic segment are neutralized by protonation, vesicles may be brought into close contact owing to the increased surface hydrophobicity, which could overcome the surface dehydration energy (ohki, ) . vesicles aggregate under such conditions. moreover, the neutralized hydrophobic segment may enter into the lipid bilayer. the segment would then compress the lipid molecules surrounding it. since the interaction can be dynamic, the rapid entrance into and returning back to the surface would cause density fluctuations in the membrane. such destabilization would trigger fusion of two apposed bilayer membranes. the hydrophobic segment may produce some perturbed structures in the target lipid bilayer membrane by binding to lipid molecules. we have recently attempted to detect the possible intermediate structure in liposomes incubated with influenza virus at acidic ph using the quick-frozen replica technique. on replicas made sec after acidification at °c of receptor (g ycophorin)-containing egg pc liposomes bound with influenza virus, we observed very close apposition of the virus particle and liposomes and also small - protrusions with a diameter of about nm on the convex fracture face, with a complementary pit on the opposite concave face, of liposomes. no close apposition nor perturbed structures were observed on replicas obtained after incubation at neutral ph. the protrusiordpit structure may be the fusion point because of disturbance due to the high curvature (kawasaki et a / . , ) . cullis and hope ( ) have proposed inverted micelles and other nonbilayer structures as the intermediate in membrane fusions. phospholipids with larger tail to head volume ratios such as pe or cardiolipin spontaneously form nonbilayer structures under appropriate conditions (cullis and de kruijff, ) . even pc can have a nonbilayer structure under specific conditions (gruner et ul., ) . it is interesting to note that, in fusion of vsv with liposomes, the presence of cis-unsaturated fatty acyl chains in target phospholipids was required (yamada and ohnishi, ) . also, phospholipids used in fusion studies with various enveloped viruses have been those from natural sources which contain large fractions of cisunsaturated fatty acyl chains. cis-unsaturated phospholipids have larger tail to head volume ratios. h. p h dependence of virus memhriinr fusion. the ph characteristics of virus membrane fusion activity will be determined by the ph dependencies of the conformational change of the fusion protein and the neutralization of the fusogenic segment. the two processes may occur in the same or different ph ranges. if they are different, the process occurring at lower ph would determine the overall ph profile of fusion. for example, the phliz for mmtv may represent that of the conformational change of gp + gp . its fusogenic segment does not contain acidic residues and can interact with target membranes at neutral as well as acidic ph. influenza virus variants and mutants have been isolated and studied (section ii,b,i ,a). in these mutants, modification of fusion activity can be caused by changes in the ph ranges both for the conformational change and also for neutralization of the fusogenic segment. for example, a mutant with gly at positon i for glu in the ha amino-terminal segment showed the same fusion activity as the wild type (gething et al., ) . in this case, the fusogenic segment can interact with the target lipid bilayer at the higher ph because of the lack of one acidic residue in the arm, but the conformational change of the mutant ha was shown to occur in the same ph range as the wild type. substitution of gly for glu at position in the arm destroyed fusion activity down to ph . (gething et a / . , ) . however, the ph range for fusion activity of the mutant segment may have shifted lower than . . the ha amino-terminal peptide with a chemically modified amino terminus had such a shifted fusion activity (section iii,b, ,b). the response of cells to viruses is quite similar to that in the receptormediated endocytosis of physiological ligands such as low density lipoprotein, asialoglycoprotein, and epidermal growth factor (for review, see goldstein et al., ) . a characteristic difference is that the viruses fuse with cell membranes to release their genome into the cytoplasm during endocytic processing. the fusion site depends on the ph characteristics of the virus membrane fusion activity. paramyxovirus can fuse with the cell surface plasma membrane because of its ability to undergo fusion at neutral ph. on the other hand, most other viruses fuse with the membrane of acidic vesicles after entering the cell because of their restriction to fusion at acidic ph. various types of viruses bind to the target cell surface and coated pits, often near the foot of microvilli, and are taken up into coated and smooth vesicles after brief warming (see figs. g, h for influenza virus) (for reviews, see marsh, ; dimmock, ) . uptake of hvj by endocytosis was also observed, but the number of virus particles in intracellular vesicles was smaller, less than half as compared with a fusion-inactive cell-growth hvj (yasuda et a[., ) . on further incubation at "c, the endocytosed viruses are transported into secondary lysosomes and subject to degradation by the action of various lysosomal hydrolytic enzymes. excretion of the degraded materials into the external medium is observed after - min. the degradation is inhibited in the presence of lysosomotropic reagents for sfv; matlin et al., , and yoshimura et al., , for influenza virus; matlin et al., , for vsv). the intracellular fusion site for these viruses was initially assigned to lysosomes because of the well-known acidity of the lysosome lumen with a ph value of . (ohkuma and poole, ) , which induces envelope fusion (sfv, helenius et al., ; influenza virus, matlin et al., , and yoshimura et al., ; vsv, matlin et al., ) . inhibition of virus replication by "lysosomotropic" weak bases, which raise the lysosomal ph quickly, supports the idea. these reagents do not affect the binding and uptake of viruses. they inhibit virus replication when added in the early phases of infection, up to min after infection, but do not inhibit when added after min. the relationship between the lysosomal ph and virus replication has been studied. the intralysosomal ph can be adjusted were measured using dextran tagged with fluorescein isothiocyanate according to ohkurna and poole ). (from ohnishi and yoshimura, .) by changing either the concentration of the reagents or the ph of the extracellular medium, as shown in fig. . influenza virus replication was assayed under various lysosomal ph conditions. inhibition of the replication was observed when the lysosomal ph was greater than . but not at ph values below . (fig. ) (yoshimura r t al., ; ohnishi and yoshimura, ) . the ph dependence agrees well with that of envelope fusion. just after the publication of these studies, a rapid acidification of prelysosomal endocytic vesicles was discovered by tycko and maxfield ( ) and van renswoude et al. ( ) . the acidification is caused by a proton pump on the vesicle membrane, and the ph value can be as low as . . envelope fusion is thus possible when the viruses arrive in endosomes before reaching the secondary lysosomes. the release of the virus genome into the cytoplasm by fusion in endosomes has been shown for sfv by marsh et al., ( b) and for influenza virus by yoshimura and ohnishi ( ) . these authors showed that the virus replication had already started after - min at "c, or after hr at t , under which conditions the viruses are still in endosomes and not yet transported into secondary lysosomes, as confirmed by biochemical assays. yoshimura and ohnishi ( ) measured the ph of the virus environment using ha tagged with fluorescein isothiocyanate. the ph was lowered to . - . after min at oc, or after i hr at "c, while the viruses were in endosomes. the ph was raised to . by addition of a "lysosomotropic" reagent, nh,ci ( mm), in the medium. virus replication was inhibited in the presence of the reagent. previous data on the inhibition by "lysosomotropic" reagents are not inconsistent with genome release from endosomes, since the reagents rapidly penetrate and raise the endosomal ph as well as the lysosomal ph as shown above. the ph value in the ordinate in fig. may well represent the endosomal ph. the lysosomotropic reagents are also endosomotropic, and thus are more generally termed acidotropic reagents (de duve et d., ) . some populations of viruses are transported to secondary lysosomes where they can also fuse to release the viral genome, in competition against degradation. however, fusion in endosomes would contribute more to infection simply because it occurs earlier. hvj fuses with the plasma membranes, but it can also fuse with endosomes after endocytosis since it can fuse at acidic ph as well. other viruses may also take the intracellular uncoating route. for mmtv, la crosse virus, the west nile virus, the low ph-induced fusion activity and the inhibition of virus replication by acidotropic reagents (andersen and nexo, , for murine type c retrovirus; gollins and porterfield, , for wnv) have been observed, supporting the intracel-mar route. enveloped viruses thus utilize the host cell machinery, developed for the uptake and processing of biological materials, for their entry. the viral genome enters the cytoplasm from the acidic endosomes where the cells separate ligands from receptors and send these materials to their respective destinations. not only enveloped viruses but also naked viruses may enter cells from acidic endosomes, as suggested for adenovirus (fitzgerald e t a / . , ; yoshimura, ) and poliovirus (madshus et al., ) . a polypeptide toxin, diphtheria toxin, also enters the cytoplasm through acidic endosomes (sandvig and olsnes, ) . membrane fusions and splittings occur frequently in endocytic processing and in the transport of newly synthesized proteins, lipids, and other materials to organelles, the plasma membrane, and the external media. these membrane fusions may be mediated by some specific proteins. it would not be unreasonable to imagine that the virus fusion proteins originate from such cellular fusion proteins. entry of murine retrovirus into mouse fibroblasts molecular mechanisms of virus entry to target cells why is a specific amino acid sequence of f glycoprotein required for the membrane fusion reaction between envelope of hvj (sendai virus) and target cell membranes? structural requirements for hemolytic activity of f-glycoprotein of hvj (sendai virus) studied by proteolytic dissection fusion of erythrocytes by sendai virus studied by immuno-freeze-etching mutations which alter the function of the signal sequence of the maltose binding protein of esc/ieric,hia w l i . ntr/rrrc sequence determination of the sendai virus fusion protein the biologically active proteins of influenza virus: neuraminidase conditions for haemolysis by flaviviruses and characterization of the haemolyh nucleotide sequence of the gene encoding the fusion (f) glycoprotein of human respiratory syncytial virus lipid polymorphism and the functional roles of lipids in biological membranes studies of the influenza virus haemagglutinin in the ph s conformation. i n "the origin of pandemic influenza viruses fusion mutants of the influenza virus hemagglutinin glycoprotein quantitative problems in biochemistry lysosomotropic agents initial stages in infection with animal viruses variant influenza virus hemmagglutinin that induces fusion at elevated ph cholesterol and membrane fusion. i n "cholesterol in biological systems sindbis virus-mediated cell fusion from without is a two step event conformational changes in sindbis virus envelope protein accompanying exposure to low ph ph-dependent fusion induced by vesicular stomatitis virus glycoprotein reconstituted into phospholipid vesicles sequence analysis of mutations that prevent export of a receptor, an escherichiu coli outer membrane protein adenovirus- shun-ichi ohnlshl induced release of epidermal growth factor and pseudomonos toxin into the cytosol of kb cells during receptor-mediated endocytosis nucleotide sequence of a cdna clone encoding the entire glycoprotein from the new jersey serotype of vesicular stomatitis virus nucleotide sequence of cdna coding for semliki forest virus membrane glycoprotein purification of the fusion protein of sendai virus: analysis of nh -terminal sequence generated during precursor activation cloning and dna sequence of double-stranded copies of haemagglutinin genes from h and h stains elucidates antigenic shift and drift in human influenza virus studies on the mechanism of membrane fusion: site-specific mutagenesis of the hearngglutinin of influenza virus receptor-mediated endocytosis: concepts emerging from the ldl receptor system flavivirus infection enhancement in macrophages: radioactive and biological studies on the effect of antibody on viral fate flavivirus infection enhancement in macrophages: an electron microscope study of viral cellular entry ph-dependent fusion between the flavivirus west nile and liposomal model membranes la crosse virus g glycoprotein undergoes a conformational change at the ph of fusion la crosse bunyavirus can mediate ph-dependent fusion from without lipid polymorphism: the molecular basis of nonbilayer phases effect of lipid composition upon fusion of liposomes with sendai virus membranes the entry of viruses into animal cells on the entry of semliki forest virus into bhk- cells fluorescence method for measuring the kinetics of fusion between biological membranes characterization of the fusogenic properties of sendai virus: kinetics of fusion with erythrocyte membranes trypsin action on the growth of sendai virus in tissue culture cells on the study of sendai virus hemolysis i. complete sendai virus lacking in hemolytic activity artificial assembly of envelope particles of hvj t sendai virus) . assambly of hemolytic and fusion factor\ from envelopes solubilized by nonidet p . l'irolog? interaction hetween sendai virw and human erythrocytes analysis of sendai virus mrna with cdna clones of viral genes and sequence of biologiciilly imporlanl regions of the fusion protein fusion of sendai virus with lipowmes: dependence on the viral fusion protein tf) and the lipid composition of liposomes x i ) . lnfleunza viruses cause hemoly\is and fusion of cells phillips. . c., and rupley vertebrate lysozymes temperature and ph dependence of the haemolytic activity of influenza virus and of the rotational mobility of the spike glycoprotein membrane fusion activity of reconbtiluted vesicles of influenza virus hemagglutinin glycoprotein membrane fusion of influenza virus and liposomes studied by quick-frozen replica techniques membrane fusion mutants of semliki forest virus morphological chmges in ehrlich ascites tumor cells during the cell fusion reaction with hvj (sendai virus) . cluster formation of iniramembrane particles in the early stage of cell fusion. exp. c d l hrs transformation of intramembrane particles of hvj (sendai virus) envelopes from an invisible to visible form on aging of virions proteolytic activation of hemolysis and fusion by influenza c virus activation ofinfluenza a viruses by trypsin treatment studies of membrane fusion. v. fusion oferythrocytes with non-haemolytic sendai virus. j . c d l s c i the role of cell swelling and haemolysis in sendai virusinduced cell fusion and in the diffusion of incorporated viral antigens enhancement of the infectivity of influenza a and b viruses by proteolytic cleavage of the hemagglutinin polypeptide correlation of rotational mobility and flexibility of sendai virus spike glycoproteins with fusion activity ph-dependent hemolysis by influenza. semliki forest virus, and sendai virus nucleotide sequence of the akv enu gene mechanism of entry into the cytosol of poliovirus type : requirement of low ph activation of influenza virus by acidic media causes hemolysis and fusion of erythrocytes a spin-label study on fusion of red blood cells induced by hemagglutinating virus of japan modification of cell membrane with viral envelopes during fusion of cells with hvj (sendai virus). ill. effects of mono-and disaccharides on cell fusion and membrane movement of fused cells interaction of influenza virus hemagglutinin with target membrane lipids is a key step in virus-induced hemolysis and fusion at ph . the entry of enveloped viruses into cells by endocytosis interactions of semliki forest virus spike glycoprotein rosettes and vesicles with cultured cells penetration of semliki forest virus from acidic prelysosomal vacuoles infectious entry pathway of influenza virus in a canine kidney cell line pathway of vesicular stomatitis virus entry leading to infection reconstitution of the fusogenic activity of vesicular stomatitis virus hemolysis and cell fusion by rhabdovirus membrane fusion activity of succinylated melittin is triggered by protonation of its carboxyl groups ph-dependent membrane fusion activity of a synthetic twenty amino acid peptide with the same sequence as that of the hydrophobic segment in influenza virus hemagglutinin influenza c virus hemagglutinin: comparison with influenza a and b virus hemagglutinins fusion of influenza virus with cardiolipin - fusion of viral envelopes with cellular membranes liposomes at low ph: mass action analysi\ of kinetics and extent kinetics and extent offusion between sendai virus and erythrocyte ghosts: application of ti mas\ action kinetic model surface tension. hydration energy and membrane fusion press. plenum fluorescence probe measurement of the intl-alysosomal ph in living cells and the perturbaof ph by various agents membrane fwion: mechanism and role in cellular response to enveloped virus interaction of hemagglutinin with target membranes infectious cell entry mechanism of enveloped vi demonstration of hemolytic and fusion the fusion of ehrlich's ascites tumor cells caused by hvj virus in u i~r o modification of cell membranes with viral envelopes during fusion of cells with hvj (sendai virus) i. interaction between cell membranes and virus in the early stage i). the preparation of cell fusion-inducing proteoliposomes from purified glycoproteins of hvj (sendai virus) and chemically defined lipids energy of an ion cros\ing b low dielectric membrane: solutions to four relevant electrostatic problems how viruses damage cells: alterations in plasma membrane function complete nucleotide sequence of an influenza virus haemagglutinin gene from cloned dna sequence and expression of the mouse mammary tumor virw enu gene mouse mammary tumor virus can mediate cell fusion at reduced ph nucleotide sequence of the s mrna of sindbis virus and deduced sequence of the encoded virus structural proteins oligopeptides that specifically inhibit membrane fusion by paramyxoviruses: studies on the site of action specific inhibition of paramyxovirus and myxovirus replication by oligopeptides with amino acid sequences similar to those at the n-termini of the f, or h a viral polypeptides nucleotide sequences of the mrna's encoding the vesicular stomatitis virus g and m proteins determined from cdna clones containing the complete coding regions studies on the adaptation of influenza viruses to mdck cells electron microscopy of low ph structure of influenza virus huemagglutinin diphtheria toxin entry into cells is facilitated by low ph hemolytic activity of influenza virus hemagglutinin glycoproteins activated in mildly acidic environments identification of biological activities of paramyxovirus glycoproteins. activation of cell fusion, hemolysis and infectivity by proteolytic cleavage of an inactive precursor protein of sendai virus a synthetic peptide corresponding to the nh terminus of vesicular stomatitis virus glycoprotein is a ph-dependent hemolysin biologically active peptides of the vesicular stomatitis virus glycoprotein inhibition of vsv binding and infectivity by phosphatidylserine: is phosphatidylserine a vsv binding site? cellular adsorption function of the skdloglycoprotein of vesicular stomatitis virus and its neuraminic acid fatty acid binding: a new kind of posttranslational modification of membrane protein on the structure of the acyl linkage and the function of fatty acyl chains in the influenza virus haemagglutinin and the glycoproteins of semliki forest virus human adult t-cell leukemia virus: complete mucleotide sequence of the provirus genome integrated in leukemia cell dna participation of spectrin in sendai virus-induced fusion of human erythrocyte ghosts role of viral glycoproteins in hemolysis by influenza b viruses on the study of sendai virus hemolysis . morphological study of envelope fusion and hemolysis nucleotide sequence of moloney murine leukemia virus changes in the conformation of influenza virus haernagglutinin at the ph optimum of virus-mediated membrane fusion kinetics of ph-depen-, dent fusion between influenza virus and liposomes fusion activity of influenza virus. a comparison between biological and artificial target membrane vesicles use of resonance energy tr;insfer t o monitor membrane fusion proteolytic cleavage of the e? glycoprotein of murine coronavirus: activation of cell-fusing activity of virions by trypsin and separation of two different yok cleavage fragments heterogeneity in the tiuidity of intact erythrocyte membrane and its homogenization upon hemolysih rewiction ofthe liitcriil motion of hand in the erythrocyte membrane by the cytoskcletiil network: dependence on speclrin association state rapid iiciditication of endocytic vesicles containing a~-inacroglobulin haemolysis hy two iilphaviruses: semliki forest and sindhis virus fuhion hind haeniolysih of erythrocytes caused by three togaviruses: semliki forest sindbih, ;ind rubella receptoi--mediated endocytosis o f transferrin and the uptake of fe in ks cell\: identification of a nonlysosomal acidic compartment. pro(.. nor antigenic drift between the heemagglutinin of the hong kong influenza striiins aiaichii i and aivictoriai i , nirrrrrc an cflicient method for r e a w m h l y of fusogenic sendni virus envelopes after solubili/ation of intact virions with triton x- role ofc;i'* in virus-induced membrane fu\ion. ca" accumulation and ultrastructural changes induced by sendai viru\ in chicken erythro ). c. ( iyx ). studies ofinlluenza haemagglutininmediated membrane fusion ph-dependent fusion between the semliki forest virus membrane and liposomes. /'roc fusion olsemliki forest virus with the plasmit membrane can be induced by low ph. . coil e i~l cell fuhion by semliki forest. influenzit kind vesicular stomatitis viruh membrane fusion activity of influenza virus membrane fusion protein of enveloped animal viruses structure of the haemagglutinin memhrane glycoprotein of influenza virus at a resolution. nurrtrr~ ( f m i c l m i ) h nucleotide sequence o f the haemagglutinin gene of a human influenza virus h i subtype components bicjf'/icr!li.y/r\ vesicular stomatitis virus binds and fuses with phospholipid domain in target cell membranes hemolytic activity of the envelope glycoproteins of western equine encephalitis virus in reconstitution experiments immunoelectron microscopic study on interactions of noninfectious sendai virus and murine cells adenovirus-induced leakage of co-endocytosed macromolecules into the cytosol uncoating of influenza virus in endosomes. j . v i r d infectious cell entry mechanism of influenza virus mobilization and aggregation of integral membrane proteins in erythrocytes induced by interaction with influenza virus at acidic ph key: cord- -aqtceqqo authors: hunter, eric title: membrane insertion and transport of viral glycoproteins: a mutational analysis date: - - journal: protein transfer and organelle biogenesis doi: . /b - - - - . -x sha: doc_id: cord_uid: aqtceqqo nan the eukaryotic cell faces a continual problem of partitioning a variety of enzyme activities into different subcellular organelles where specific macromolecular reactions can occur. this subcellular organization requires specific intracellular targeting of macromolecules through the cytoplasm, by as yet ill-defined mechanisms, and through the secretory pathway via a more clearly defined vesicular transport mechanism (sabatini et al. y ) . as obligate intracellular parasites with only limited genetic complexity, viruses must utilize the existing cellular transport mechanisms to colocalize their virion components in a part of the cell where assembly can take place. this problem of intracellular targeting is compounded for the enveloped viruses because they must transport capsid polypeptides through the cytoplasm and envelope components through the secretory pathway to a common point of assembly. since these viruses depend on the preexisting host cell processes and possess lipid envelopes that are biochemically similar to cellular membranes, they can provide ideal systems for probing the cellular mechanisms involved in glycoprotein biosynthesis and transport. the relatively simple structure of virions, the high level of expression of viral genes, the ease of molecularly cloning and manipulating those genes, together with the availability of conditional lethal mutants with defects in viral protein transport, confer several additional advantages for such studies. we have chosen an enveloped, rna-containing virus, rous sarcoma virus (rsv), for an analysis of viral glycoprotein biosynthesis and transport, because in addition to the aspects delineated above, the availability of molecularly cloned, infectious dna copies of the genome of this retrovirus has also allowed us to pose questions about the role of the glycoproteins in virus assembly and virus infectivity. like most simple enveloped viruses, the retroviruses consist of a host membrane-derived, lipid bilayer that surrounds (envelopes) a protein capsid structure (fig. ) . the icosahedral capsid of rsv assembles as the virus particle buds from the plasma membrane of the cell, and so these two events are linked both temporally and spacially. glycoprotein knobbed spikes extend from the surface of the virion, and it is generally thought that during virus assembly a specific interaction between the glycoproteins and capsid (and/or "matrix" protein) is required, since cellderived polypeptides are for the most part excluded from the budding structure. the envelope glycoproteins span the lipid bilayer and are thereby divided into three distinct domains: an external, hydrophilic receptor-bind- ing domain that functions in virus-cell attachment; a hydrophobic membrane-spanning domain; and a hydrophilic cytoplasmic domain. in rsv two polypeptides, gp and gp , make up this structure (fig. ) ; the external domain is primarily made up of the amino acid long gp which contains regions that define the host-range and neutralization properties of the virus. the amino acid long gp polypeptide, on the other hand, is a bitopic protein that anchors the envelope glycoprotein complex into the virion via disulfide linkages to gp . it is less heavily glycosylated than gp (having only versus potential glycosylation sites) and contains two apolar regions in addition to the hydrophilic cytoplasmic domain. one apolar region is located near the amino terminus of gp and may be analogous to the fusion peptide of the hemagglutinin ha polypeptide of influenza virus that mediates viral entry into the cell. the hatched regions represent the highly hydrophobic signal and anchor sequences found at the n and c terminus, respectively. the location of a nonpolar region that may be analogous to the fusion peptide of ortho-and paramyxoviruses is shown by the stippled box. the aug at the start of the env orf was used to initiate translation of transcripts expressed in an sv vector (wills et ai, ) , but during a virus infection the genomic length transcript is spliced such that the aug and first codons from gag (black box) are spliced into the env orf . in both cases the signal peptide is removed during translation, and cleavage of the polyprotein precursor to gp and gp occurs in the golgi at the basic tetrapeptide, -arg-arg-lys-arg-. branched structures denote potential n-linked oligosaccharide addition sites (asn-x-ser or asn-x-thr). (b) orientations of gp and pg in the viral membrane are depicted schematically. in the electron microscope this structure is seen as a spiked knob, where gp is the spike and gp is the knob. second apolar region in gp consists of a amino acid long stretch of hydrophobic residues near the carboxy terminus that functions during translation to stop the movement of the protein into the lumen of the rough endoplasmic reticulum (rer) and to anchor the complex in the membrane. the general orientation and structure of the rsv glycoprotein is thus similar to that of the influenza virus hemagglutinin (ha) (porter et al, ; gething et al, ) , the vesicular stomatitis virus (vsv) g protein (gallione and rose, ) , and several membrane-spanning cellencoded glycoproteins. the two viral glycoproteins of rsv are encoded by a single viral gene, env, and are translated in the form of a heavily glycosylated precursor polypeptide, pr e/ii; . since several processing and maturation events occur during the transport of the env gene products to the plasma membrane, they provide excellent markers for the subcellular compartments of the cell. a long ( amino acid) amino-terminal signal peptide, which mediates translocation of the env gene product across the rough endoplasmic reticulum (rer), is removed cotranslationally from the precursor protein, and in the lumen of the rer - high-mannose core glycosylation units are added to the nascent pr . the marked increase in molecular weight (mw) (approximately k) that results from the addition of this endo-ß-tv-acetylglucosaminidase h (endo h) sensitive carbohydrate provides a clear indicator for the translocation event. removal of glucose residues and some mannose moities appears to occur prior to transport of the protein to the golgi, where further trimming of the mannose residues and addition of glucosamine, galactose, and fucose are observed. cleavage of pr to gp and gp takes place after galactose and prior to fucose addition; it thus provides an excellent marker for trans-golgi locations. while the transit time of pr from the rer to golgi is quite long (t = min) compared to other viral glycoproteins, movement through the golgi appears rapid and precludes any dissection of individual compartments within this organelle. these major biochemical modifications to the env glycoprotein coupled with sensitive immunological probes allow a fairly accurate mapping of the cell's secretory pathway. several viruses assemble at points within the secretory pathway, and since in many cases the location of the viral glycoproteins defines the virus maturation point (roth et al., b; jones et al., ; gottlieb et al., ; gahmberg, ; kabcenell and atkinson, ) these systems are proving useful in investigating the signals that target proteins to specific subcellular locations. figure is a schematic summary of the assembly points for the major groups of enveloped viruses. herpes simplex virus (hsv), a complex enveloped dna virus that encodes several glycoproteins (gb, gc, gd, ge) , buds into the nuclear envelope (spear, ) . virion glycoproteins synthesized on the rer appear to be transported to the nuclear membrane in an endo h-sensitive form where they are incorporated into nascent virions (compton and courtney, ) . it has been postulated that intact hsv virions traverse the secretory pathway thereby exposing the glycoproteins to the entire array of carbohydrate-modifying enzymes such that the mature virion contains glycoproteins with complex carbohydrate side chains (compton and courtney, ; spear, ) . a majority of the hsv glycoproteins can be found on the surface of infected cells, suggesting that targeting to the nuclear membrane is not absolute. expression of the cloned hsv gd gene in the absence of other viral components resulted in more rapid alphaviruses ortho-, paramyxoviruses transport to the plasma membrane and reduced accumulation on the nuclear membrane, indicating that interactions with other viral-encoded proteins may be required for normal nuclear membrane localization (johnson and smiley, ) . the corona-, flavi-, and rotaviruses have been reported to undergo assembly at the rer (dubois-dalcq et al, ) , but the rotaviruses appear to target this organelle most specifically. while the mature rotavirus is not enveloped, it contains a glycosylated capsid protein in its outer shell that is derived from a transient membrane during the assembly process. the inner and outer protein shells of these viruses form sequentially and by very different mechanisms. the inner capsids, containing the genomic rna segments, are the equivalent of nucleocapsids of other viruses and assemble in the cytoplasm at the edge of electron-dense inclusions called "viroplasm" (petrie et al, ) . they acquire a transient envelope, or pseudoenvelope, by budding at rer membranes adjacent to the viroplasm. further maturation of rotaviruses occurs within the cisternae of the rer where enveloped particles are converted to mature double-shelled virions and the lipid bilayer is removed by a process that remains to be elucidated (dubois-dalcq et al, ) . vp , the glycosylated protein found in the outer capsid, has been shown to have carbohydrate structures consistent with its rer location and to target specifically to this organelle when expressed in the absence of other viral proteins from a recombinant expression vector (kabcenell and atkinson, ; poruchynsky et al, ) . the golgi body is the site of assembly for several viruses. coronaviruses, for example, mature by budding into the lumina of rer or golgi cisternae; virions form as the intracytoplasmic, helical neucleocapsids align under regions of intracellular membranes containing viral proteins. two glycoproteins, el and e , comprise these membrane-associated polypeptides. e forms the large peplomers or spikes characteristic of coronaviruses and is a multifunctional molecule, being responsible for virus-induced cell fusion, binding of the virion to receptors on the plasma membrane of susceptible cells, and for inducing neutralizing antibody (dubois-dalcq et al, ) . el, in contrast, is an unusual polypeptide; it has only a short amino-terminal domain, which contains the glycosylation sites of the protein, and two long stretches of hydrophobic amino acids, suggesting that it may traverse the membrane more than once (dubois-dalcq et al, ; boursnell et al, ) . during infection, e can be transported through the secretory pathway to the plasma membrane, whereas el is transported only as far as the golgi apparatus, where it accumulates during the infection cycle (sturman and holmes, ) ; it has been suggested that this restricted intracellular movement of el ac-counts for the intracellular budding site of coronaviruses (sturman and holmes, ) . members of the bunyavirus family also mature intracellularly, by budding at the golgi complex (bishop and shope, ; dubois-dalcq et al., ) . by immunofluorescent microscopy, kuismanen and colleagues showed that in cells infected with uukuniemi virus the golgi region underwent an expansion and became vacuolized . both glycoproteins, gl and g , accumulated in the golgi region during virus infection; neither polypeptide could be chased out of the golgi even after a -hr treatment with cycloheximide (gahmberg et al., ) , conditions that would allow complete transport of the semliki forest virus membrane proteins from the golgi (green et al., a) . furthermore, the glycoproteins of a temperature-sensitive strain of uukuniemi virus were retained in the golgi even under conditions where no virus maturation took place and no nucleocapsids accumulated in the golgi region (gahmberg et al., ) . thus intracellular targeting of these viral components appears to be independent of other viral components and of the assembly process itself. moreover, it supports the concept that it is the glycoproteins themselves that dictate the cellular site of virus maturation. for several virus groups, virion assembly does not occur until the envelope components have traversed the entire secretory pathway. thus the ortho-and paramyxoviruses, rhabdoviruses, alphaviruses, and retroviruses mature at the plasma membrane. even these proteins, however, possess additional membrane-targeting information such that in polarized epithelial cells, where different cell proteins are inserted in the apical and basolateral membranes, the different viruses assemble from distinct membranes; for example, the ortho-and paramyxoviruses bud from apical membranes, rhabdo-and retroviruses from basolateral membranes (rodriguez-boulan and sabatini, ; herrler et al., ; roth et al., a; rindler et al., ) . as with those viruses that mature at points within the secretory pathway, it is the glycoproteins themselves that appear to specify the specific plasma membrane location for virus assembly, since glycoproteins expressed from recombinant expression vectors are transported in a polarized fashion (roth et al., b; jones et al., ; gottlieb et al., ; . the problem of polarized expression will be dealt with in more detail later in this chapter (see section ii, b, ) . from the brief outline presented above it is clear that viral envelope components provide a plethora of systems for studying intracellular protein targeting. recombinant dna approaches, described below, are already providing information on the role of the different glycoprotein domains in this important aspect of viral and cell biology. in addition to utilizing the secretory pathway of vertebrate cells for transporting viral components to the point of virus maturation, several enveloped viruses take advantage of a second vesicle-mediated transport system, the endocytic pathway, to gain entry into susceptible cells. these viruses, such as orthomyxoviruses, rhabdoviruses, and togaviruses, in contrast to paramyxoviruses, such as sendai virus, which bind and fuse with the plasma membrane of the host cell, bind to the host cell surface and are subsequently internalized by endocytosis. this latter process serves an important role in the normal uptake of nutrients and in the internalization of receptor-bound ligands such as hormones, growth factors, lipoproteins, and antibodies (mellman et al., ; hopkins, ) . bound virions are carried into clathrin-coated pits, which form continually on the surface of the cell, and which fold inward and pinch off into the cytoplasm to form "coated vesicles." as the coated vesicle moves into the cytoplasm, it loses its clathrin and fuses with an endosome, a large acidic vacuole with a smooth outer surface. for viruses entering by this pathway, membrane fusion occurs in the endosomal compartment (marsh, ; yoshimura and ohnishi, ) . fusion is triggered by the mildly acidic endosomal ph and is catalyzed by the virally encoded glycoproteins which undergo a low ph-dependent configurational change (skehel et al., ; kielian and helenius, ) . the ph dependence of fusion varies among virus types, with the optimal ph for fusion generally falling within the range of ph . - . for endocytosed viruses (white et al., ) . in order to obtain an understanding of the molecular mechanisms involved in these low ph-induced fusion reactions, virus mutants have been isolated which fuse with ph optima different from those of their respective parents. through the use of an elegant selection scheme, in which mutagenized virus was allowed to fuse with nuclease-filled liposomes at a ph below . , kielian et al. ( ) isolated the first such fusion mutant of semliki forest virus. this virus, fus- , fused at a ph optimum . ph units lower than that of the wild type (ph . versus . ). the mutant was, nevertheless, fully capable of infecting cells under standard infection conditions and even under conditions that prevent fusion of endosomes with lysosomes. on the other hand, the fus- mutant showed increased sensitivity to lysosomatropic agents that increase the ph in acidic vacuoles of the endocytic pathway. in addition to proving that alterations within viral structural components can significantly affect the ph at which virus-induced fusion can occur, these results showed that a ph below . exists within the endosomal compartment and thereby demonstrated the usefulness of mutant viruses as biological ph probes of this pathway. in parallel studies, rott and co-workers ( ) have shown that variants of the x strain of influenza virus, selected for their ability to undergo activation cleavage and growth in madin-darby canine kidney (mdck) cells, have an elevated fusion ph threshold (approximately . ph units higher than the wild type). similar virus variants have been selected by growth of influenza virus in the presence of amantadine, a compound that raises endosomal ph (daniels et al., ) . in this latter study, viruses were obtained that fused at ph values . - . units higher than the parental strain. analogous mutants have been reported to occur naturally within stocks of the x strain of influenza virus (doms et al., ) . such mutants should provide useful probes for elucidating the endocytic pathway. the mechanisms by which cells send membrane-bound and secreted proteins to their proper subcellular locations remain a central problem in cell biology. it has been postulated to involve the specific interaction of "sorting signals," located within the structure of the newly synthesized proteins, with membrane-bound receptors in the rer and golgi apparatus of the cell (for review, see sabatini et al., ; silhavy et al., ) . this concept is supported by the facts that cell, as well as viral, glycoproteins can be retained at or targeted to specific points in the secretory pathway and that cells can transport and secrete a variety of glycosylated and nonglycosylated proteins at distinctly different rates (strous and lodish, ; fitting and kabat, ; gumbiner and kelly, ; ledford and davis, ; , kelly, . very little is known about the composition(s) or indeed the exact role of sorting signals, but it is generally thought that they are composed of protein. clearly, the initial step that introduces polypeptides into the secretory pathway is mediated by the interaction of a sequence of amino acids (the signal sequence) within the polypeptide and the signal recognition particle (srp)/docking protein (dp) complex (blobel and dobberstein, ; blobel, , a,b; walter et al., ; meyer and dobberstein, ; meyer et al., ; gilmore et al., a,b; . mutants of secreted proteins that are defective in later stages of transport have been identified that differ from the wild-type forms by one (mosmann and williamson, ; wu et al., ; shida and matsumoto, ) or two (yoshida et al., ; hercz et al., ) amino acid substitutions, supporting the concept that sorting signals are composed of protein. also, several conditional transport-defective mutants of membrane-bound viral glycoproteins have been identified (for example, knipe et al, b; zilberstein et al, ; pesonen et al, ) . studies using tunicamycin, an inhibitor of glycosylation, suggest that carbohydrate moieties are not recognized directly by the sorting machinery but may be important for maintaining the proper secondary or tertiary structures of (protein-composed) sorting signals (struck et al., ; gibson et al, gibson et al, , leavitt et al, ; hickman et al, ; roth et al, ; strous et al, ; green etal, b) . the lack of a direct role for carbohydrate moieties in the sorting process is perhaps to be expected in view of the fact that many secreted proteins are not glycosylated at all (for example, strous and lodish, ; underdown et al, ) . nevertheless, addition of carbohydrate to molecules that are unable to be transported through the secretory pathway can release the block to their transport (guan et al, ; machamer et al, ) . in addition, the transport of certain hydrolases to the lysosome (and away from the secretory pathway) does appear to require the addition of a carbohydrate moiety (mannose -phosphate) (hasilik and neufeld, ; sly and fisher, ; creek and sly, ) , but these additions in turn must require the recognition of signals within the polypeptide chains. even less is known about the intramolecular location(s) of sorting signals. in the case of the membrane-spanning glycoproteins, three protein domains exist which together or separately may harbor sorting signals: ( ) the internal or cytoplasmic domain, ( ) the hydrophobic or transmembrane domain, and ( ) the extracytoplasmic or external domain. since most secreted proteins (which may be cotransported with membranebound glycoproteins, strous et al, ) possess only external domains, it might be reasonable to expect the transmembrane and cytoplasmic domains to be unimportant to the sorting process. we have tested this hypothesis by introducing genetic lesions into the gene encoding the envelope glycoproteins of rsv (wills et al, (wills et al, , hardwick et al, ; , as have others for the vsv g protein bergmann, , ; rose et al, ; adams and rose, a,b) , the influenza virus hemagglutinin protein (sveda et al, gething and sambrook, ; doyle et al, doyle et al, , gething et al, ) , and the glycoproteins of semliki forest virus (garoff et al, ; garoff, ; . genetic analyses of protein transport in prokaryotic systems have provided both support for the role of the signal peptide in protein translocation and valuable insights into the polypeptide interactions that are required for the intracellular targeting of bacterial secreted and membrane proteins (reviewed by michaelis and beckwith, ; silhavy et al, ; benson et al, ; oliver, ) . while similar experiments are more difficult to perform in eukaryotic cells with the enveloped virus systems described here, both the classic and molecular genetic approaches outlined below are providing information on the role of different protein domains in the transport process. during the genetic analysis of enveloped virus replication through the isolation and biochemical characterization of spontaneous and mutageninduced variants, complementation groups were established for several viruses that contained mutants defective in normal transport of the viral glycoproteins (knipe et al., a,b; zilberstein et al., ; pesonen et al, ; gahmberg, ; ueda and kilbourne, ) . the existence of conditional lethal mutants that were blocked at different stages of virus glycoprotein maturation suggested that the viral polypeptides themselves might contain the signals necessary for normal sorting by the cells' transport machinery and raised the possibility that such mutants could be used tq dissect the maturation pathway of a glycoprotein. since it is impossible in this chapter to provide a detailed review of the characterization of mutants in each of these systems, and since the transport of the influenza virus glycoproteins has recently been discussed in detail by , this section will concentrate primarily on mutants of the vsv g protein gene as an example of these approaches. temperature-sensitive (ts) mutants in complementation group v of this vsv have defects in the structural gene for the viral glycoprotein, g, and cells infected at the nonpermissive temperature with such mutants produce markedly reduced yields of virus like particles which are noninfectious and specifically deficient in g protein. at the nonpermissive temperature the mutant g polypeptide is synthesized normally; however, it does not accumulate on the cell surface, nor is it incorporated into virions (knipe et al., b; zilberstein et al., ) . the ts(v) mutants can be subdivided into two subclasses with respect to the stage of posttranslational processing at which the block occurs (zilberstein et al., ) . three mutants, tsl \ , tsm l, and ts , encode g proteins that at the nonpermissive temperature are blocked at an early, pre-golgi step of the secretory pathway. while insertion into the er membrane, removal of the amino-terminal hydrophobic signal sequence, and addition of the two n-linked high-mannose core oligosaccharides occur in a way that is indistinguishable from the wild type, all subsequent golgi-mediated carbohydrate processing reactions are blocked (zilberstein et al., ) . these results are consistent with subcellular fractionation and immunoelectron microscopy studies which indicated that the g protein in tam or ta -infected cells was arrested in its transport from the rer to the golgi complex at the nonpermissive temperature (zilberstein et al., ; bergmann et al., ; bergmann and singer, ) . the defect in transport in these mutants is a reversible phenomenon, thereby excluding irreversible denaturation as the basis for lack of movement; proteins synthesized at the nonpermissive temperature rapidly move by stages to the plasma membrane upon shift to the permissive temperature bergmann and singer, ) . within min after shift to °c, g protein of tsoa could be seen by immunoelectron microscopy at high density in saccules at one face of the golgi complex and by min later was uniformly distributed through the complex (bergmann and singer, ) . movement of the mutant proteins to the cell surface occurred rapidly and was accompanied by incorporation into virions . the second class of ts mutants of vsv is represented by tal . g protein encoded by this mutant is transported normally through most of the golgi-mediated functions involved in the processing of carbohydrate side chains, including addition of the terminal sialic acid residues. however, this molecule does not undergo two posttranslation modification reactions that take place with the wild-type g protein. the first is the addition of fucose to the tel oligosaccharide chains, which is reduced at both permissive and nonpermissive temperatures (zilberstein et al., ) . in the second, a molecule of palmitic acid (a -carbon fatty acid) is covalently attached to the wild-type g polypeptide near the membranespanning region rose et al., ) . attachment occurs at a late stage of maturation, just before oligosaccharide processing is completed (schmidt and schlesinger, ) , probably in the eis compartment of the golgi complex (dunphy et al., ) . this modification of the g protein does not occur at the nonpermissive temperature in cells infected with the tsl ll mutant. taken together with the almost complete processing of the mutant's oligosaccharide side chains, this suggests that at the nonpermissive temperature the asl glycoprotein accumulates at a specific region within the golgi complex. the mutants of vsv, together with equivalent mutants in other viral systems that were blocked at different stages of the secretory pathway (pesonen et al., ; gahmberg, ; kuismanen et al., ) , raised the possibility of identifying and understanding the nature of sorting signals in secreted polypeptides. with the advent of recombinant dna and rapid nucleotide sequence techniques, it has been possible to determine at the amino acid level the basis for these defects (gallione and rose, ; arias et al., ) , but the interpretation of this information with regard to protein transport has been less than straightforward. gallione and rose ( ) determined the nucleotide sequence of the ts mutant of vsv and compared it to that of the parent and a wildtype revertant. the mutant and revertant differed in three amino acid residues, and through the construction and expression of hybrid genes it was possible for these investigators to demonstrate that the basis of the temperature-sensitive phenotype was a single amino acid change of phenylalanine to serine. since this polar substitution occurred within a very hydrophobic region of the g protein, it was suggested that it might significantly affect protein folding in this region such that reversible denaturation of the protein might occur at the nonpermissive temperature. this denaturation could prevent further transport to the golgi apparatus and the cell surface. alternatively, gallione and rose ( ) pointed out that the conformational change at the nonpermissive temperature might be more subtle, perhaps preventing recognition by a component of the protein transport machinery. however, since hydrophobic residues are generally buried within a protein (kyte and doolittle, ) , it is unlikely that the mutated sequence itself would play a direct role in such an interaction. nothing is known about the -dimensional structure of the g protein or whether accessory proteins are involved at this stage of protein transport, thus both suggestions remains viable possibilities. a similar analysis has been carried out by arias et al. ( ) , who sequenced the genes encoding the viral glycoproteins of tslo and ts , mutants of sindbis virus defective in the intracellular transport of their glycoproteins, and of revertants of these mutants. these investigators found ts to have a double mutation in glycoprotein el, while ts was a single mutant in the same glycoprotein. in each case reversion to temperature insensitivity occurred by changes at the same site as the mutation, in two cases restoring the original amino acid and in the third case substituting a homologous amino acid (arginine in place of lysine). since the three mutations were far apart from each other in the protein, these authors concluded that the -dimensional conformation of el was very important for the correct migration of the glycoproteins from the er to the plasma membrane. similarly, two ts mutants in the ha gene of influenza virus that result in ha protein transport being arrested in the rer are also caused by single point mutations that probably disrupt the tertiary structure of the molecule (nakajima et al., ) . in summary, several conditional mutants have been isolated by classic genetic approaches that at the nonpermissive temperature disrupt the normal transport of viral glycoproteins through the secretory pathway. while these mutants carried the promise of defining specific protein domains that might interact with components of the transport machinery, the evidence from the nucleotide sequencing experiments outlined above suggests that many or all of the mutations may exert their phenotype through distortion of the -dimensional shape of the molecule. while this mechanism does not preclude a role for specific protein-protein interactions in the secretory pathway, it provides no direct evidence for it at this time. the recent development of cdna cloning, gene sequence manipulation, and gene expression technologies has opened up new approaches for localizing and characterizing those structural features of a protein that act as sorting signals. these new methodologies have allowed investigators to delete or modify potentially important structural regions of a protein at the nucleotide level and then to determine the effect of such changes by expressing modified genes in suitable eukaryotic expression vectors. furthermore, in some instances it has been possible to test directly the functional role of a particular peptide region by fusing it to another protein and analyzing the behavior of the chimera. since these general approaches to protein sorting have been reviewed recently (garoff, ; gething, ) , this chapter will describe our recent recombinant dna analyses of the biosynthesis and transport of the rsv envelope glycoproteins, within the context of similar analyses in other viral systems. as we have discussed earlier, the rsv env gene encodes two viral glycoproteins, gp and gp , that mediate recognition of, attachment to, and penetration of the susceptible target cell. these proteins are synthesized as a glycosylated precursor protein, pr , that is proteolytically cleaved in the golgi complex. the coding sequences for gp and gp have been placed in an open reading frame that extends from nucleotide to nucleotide , and predict sizes of amino acids ( , mw) for gp and amino acids ( , mw) for gp ( fig. ) . carbohydrate makes up a significant contribution to the observed molecular weights of these polypeptides-the predicted amino acid sequence contains potential glycosylation sites (asn-x-ser/thr) in gp and in gp . experiments aimed at determining the number of carbohydrate side chains yielded results consistent with most or all of the sites being occupied . although an initiation codon is located early (codon ) in the open reading frame, during a viral infection splicing yields an mrna on which translation initiates at the same aug as the gag gene to produce a nascent polypeptide in which gp is preceded by a amino acid long leader (signal) peptide (fig. ) . this peptide contains a hydrophobic sequence that we have shown (see below) is necessary for translocation across the rer and is completely removed from the env gene product during translation . it represented one of the longest signal peptides described to date, and we were therefore interested in determining the signal peptide requirements for normal biosynthesis of gp and gp . for these studies the env open reading frame was excised from the rsv genome and inserted into an sv expression vector under the control of the late-region promoter (wills et al., ; . in this construction translation is initiated at the aug present at the start of the open reading frame (at nucleotide of the rsv genome) and results in the synthesis of an even longer ( amino acid) signal peptide; nevertheless, biosynthesis of pr and signal peptide cleavage occur normally. furthermore, expression of the rsv env gene in african green monkey (cv- ) cells parallels that seen in a normal virus infection in avian cells (wills et al., ; hardwick et al., ) , making it an excellent system for the analysis of mutant env genes. a. deletion/substitution of the signal peptide. in order to examine the role of the signal peptide in rsv glycoprotein biosynthesis we constructed a series of deletion mutations within the ' coding region of the env gene using the double-stranded exonuclease babl. oligonucleotide linkers of the sequence catcgatg were ligated to the ends of the truncated molecules to introduce a unique restriction endonuclease cleavage site and to replace the deleted in-frame aug. the mutants were then sized and their nucleotide sequence determined to find those with a suitable deletion and an in-frame aug. one such mutant, al, contained a deletion of nucleotides within the env coding sequences and encoded an env product that completely lacked an aminoterminal hydrophobic sequence (fig. ) . expression of this gene from the sv vector resulted in the synthesis of a nonglycosylated, kda cytoplasmic protein that was similar in size to the nonglycosylated wild-type env gene product produced in the presence of the glycosylation inhibitor, tunicamycin. in contrast to the tunicamycin product, however, the al protein was not associated with membrane vesicles and was rapidly degraded (half-life < min; e. hunter, k. shaw, and j. wills, unpublished) . thus the signals for initiating translocation of the rsv env gene product must reside within the cotranslationally removed amino-terminal sequence, and in their absence the molecule is synthesized as an unstable cytoplasmic protein. this result is similar to those obtained by gething and sambrook ( ) and by sekikawa and lai ( ) with the influenza virus ha gene product. influenza hemagglutinin sequences are depicted by italicized text. underlined text denotes amino acid residues encoded by oligonucleotide linkers. arrows depict the signal peptidase cleavage site at which the signal is removed cotranslationally from each of the constructs. plus symbols indicate that translocation, glycosylation, or transport to a plasma membrane location is observed; minus symbols mean that the properties above are not observed. since the signal peptide of the rsv env gene product is exceptionally long, it was of interest to determine whether another signal peptide could substitute for it. for these experiments we have utilized the signal sequences of the influenza virus ha gene (a/jap/ / ; gething et al., ) . two constructions were made: in the first of these the al deletion mutant coding sequence was fused in-frame to the ha signal coding sequence at the signal peptidase cleavage site of the latter (fig. ) and in the second, we made use of a sail restriction enzyme site in the ha gene and an xhol site in the rsv env gene, so that the signal sequence and amino acids of ha were fused with env amino acids into gp (fig. ) . expression of these hybrid genes in cv- cells resulted in the biosynthesis of a glycosylated pr protein that was transported to the golgi complex, cleaved to gp and gp , and displayed on the cell surface (e. hunter, k. shaw, and j. wills, unpublished) . to demonstrate that the pr molecules expressed from the ha-a fusion gene had undergone signal peptide cleavage, pr was immunoprecipitated from [ h]leucine, pulse-labeled cells and analyzed by sequential edman degradation, in order to determine the amino-terminal sequence. to our surprise the signal peptidase had cleaved at the ha cleavage site, despite the fact that according to the analyses of von heijne ( ) only the rsv cleavage sequence should have been recognized. thus the h a -al fusion protein contains two potential signal peptidase cleavage sites (that from the ha and that remaining in the env sequences), but only the first of these is utilized. both gene fusions, therefore, result in the synthesis of aberrant gp proteins-that from the ha-a fusion having an amino acid amino-terminal extension, and that from the sallxho fusion having lost amino-terminal amino acids and gained from ha which nevertheless can be transported to the plasma membrane (wills et al., unpublished data) . reciprocal gene fusions, in which the env gene signal peptide was fused to the structural sequences of ha (fig. ) , also resulted in translocation of the ha molecule across the rer membrane, supporting the concept that this transient sorting sequence is not polypeptide specific. however, only in the construction where the signal sequence of env was precisely fused to the amino terminus of ha was transport beyond the rer observed (wills et al., unpublished data) . in constructions where the amino-terminal sequence of ha was perturbed, the recombinant protein was apparently prevented from assembling into trimers and its transport was blocked in the rer . thus, while signal peptides may be capable of mediating the translocation of foreign polypeptides across the rer, other sorting "signals" must be active for transport of the molecule to continue. site. the experiments described above have been extended to determine the following: (a) whether the hydrophobic region of the signal peptide carries all the information required for transfer of the env gene product into the rer; (b) what the structural specificities of the signal peptide are; and (c) where the specificity for signal peptidase cleavage is located. more than prokaryotic and eukaryotic signal peptides have been sequenced (watson, ) . comparison shows that most extensions comprise - amino acid residues; one of the longest being that of the rsv envelope glycoprotein. there is no homology between sequences, but a characteristic distribution of amino acid chains is observed. three structurally distinct regions have been observed so far: a positively charged aminoterminal region, a central region of or more hydrophobic residues, and a more polar carboxy-terminal region that appears to define the cleavage site (von heijne, (von heijne, , (von heijne, , perlman and halvorson, ) . the importance of these general features has been supported by the genetic studies in prokaryotic systems (reviewed by silhavy et al., ; benson et al, ) . to investigate these questions we initially constructed a series of internal deletion mutants that initiated within the amino-terminus of gp and extended into the signal peptide. the deletion mutations were introduced into the coding region for the envelope glycoprotein by digestion of a plasmid containing the env gene at a unique xhol site located base pairs (bp) from the ' end of the coding sequence for gp , followed by digestion with the double-stranded exonuclease bal l. potential mutants were identified by restriction enzyme analysis and dna sequencing, and those of interest were engineered into the sv expression vector. these are depicted in figs. and . mutants x -a -b, and -c were derived from a single out-of-frame parent, and they represent a nested set of mutants in which the hydrophobic sequence varies from the wild-type length of to only amino acids. expression of these mutant genes in cv- cells gave the results summarized in fig. . mutant polypeptides with the shortest hydrophobic domain (x -c) resembled the al mutant polypeptides in that they had a cytoplasmic location, were nonglycosylated, and were rapidly degraded. they differed from the al mutant in length ( versus kda) confirming that the mutated signal was not removed. mutant x -a polypeptides, on the other hand, were translocated and glycosylated with an efficiency equivalent to wild type, despite the substitution of serine and isoleucine for leucine and cysteine residues within the hydrophobic domain. mutant x -b expressed a phenotype intermediate between that of x -a and x -c, approximately % of the polypeptides being translocated and glycosylated. none of the mutants contain the sequences that specify the signal peptidase cleavage site, and molecules of x -a/x -b that were translocated across the rer retained an uncleaved signal peptide. the data from these mutations suggest the following: ( ) that the length, rather than the amino acid composition, of the hydrophobic domain of the env signal peptide is critical for translocation across the rer and ( ) that signal peptide cleavage is not a requirement for translocation. the first of these conclusions is supported by genetic experiments in prokaryotic systems, where a requirement for secondary structure in the signal peptide was suggested bankaitis et al., ) . the second is consistent with the presence of permanent insertion sequences in secreted and membrane-spanning proteins that are translocated across the rer membrane without removal of an amino-terminal signal peptide (palmiter et al., ; bos et al., ; markoff et al., ; zerial et al., ; spiess and lodish, ) . several membrane-spanning proteins are anchored in the membrane by an amino-terminal anchor/signal domain and display what is termed group ii protein topology (garoff, ; wickner and lodish, ) , where the amino terminus of the protein is cytoplasmic and the carboxy terminus is luminal. after translocation of a nascent chain across the endoplasmic reticulum has been initiated, the signal peptide is removed. this cleavage is carried out by signal peptidase, a cellular gene product. two classes of signal peptidases have been described. a signal peptidase of escherichia coli (spase i) has been cloned into pbr (date and wickner, ) , and has been shown to accurately cleave eukaryotic precursor proteins as well as bacterial protein precursors (talmadge et al., ) . conversely, the eukaryotic signal peptidase will accurately cleave prokaryotic proteins (watts et al., ) . the latter enzyme has been studied, using detergentsolubilized dog pancreas signal peptidase (jackson and white, ) and hen oviduct signal peptidase (lively and walsh, ) , and been demonstrated to be an integral membrane protein that can be solubilized only when the lipid bilayer is dissolved. a second prokaryotic signal peptidase (e. coli spase ii) has been described that is specific for prolipoproteins (hussain et al., ; tokunaga et al., ) and membrane-bound penicillinases (nielsen and lampen, ) . this enzyme maps to a different locus on the e. coli genome and requires a glyceride-modified cysteine for cleavage. perlman and halvorson ( ) and von heijne ( ) have examined sequences of a number of membrane proteins and have described amino acid sequence patterns that allow prediction of signal peptidase cleavage sites with greater than % accuracy. the most striking feature of signal peptidase cleavage sites is the presence of an amino acid with a small, uncharged side chain at the carboxy terminus of the signal peptide. the most common amino acids found at this position are alanine and glycine. from statistical analyses, the peptidase cleavage site appears to be determined by sequences within the signal peptide and not by sequences beyond the cleavage site. this is in contrast to the observations that mutations within the structural protein itself prevent signal peptidase cleavage of the lamb gene product and the m coat protein (emr and bassford, ; benson and silhavy, ; rüssel and model, ) . to investigate this question with regard to the rsv env gene product, the deletion mutants shown in fig. were constructed as described above. mutant xi has a amino acid deletion encompassing residues - of gp , which results in the loss of one potential glycosylation site. this deletion resulted in the synthesis of a slightly smaller precursor polypeptide that lacked one carbohydrate side chain but was otherwise glycosylated normally. based on size estimations in pulse-labeling experiments, in the presence and absence of tunicamycin, the precursor polypeptide lacked the long, signal-containing leader peptide. thus, although the mutation in xi significantly alters the sequences near the signal peptidase site, the signal peptidase still recognized and removed the signal peptide. in mutants x and x the amino-terminal nine and six amino acids, respectively, of gp are deleted. they therefore encode gp poly- xhol site in dna r\- | °| asp val his leu leu glu gin pro gly asn leu trp ile thr trp ala asn arg . fig. . amino acid sequence deduced from dna sequence of mutants xi, x , and x . the rsv glycoprotein is schematically represented: the location of the hydrophobic signal sequence within the long ( amino acid) leader peptide is denoted by a stippled bar, and the mature gp glycoprotein by a hatched bar. the signal peptidase cleavage site in both the cartoon and the numbered amino acid sequence is denoted by a long arrow. the potential glycosylation site in the amino terminus of gp is shown as a cho. the amino acid sequence of the last amino acids of the signal peptide and the amino-terminal amino acids of gp are shown for the wild-type gene product. the solid black bars show the lengths and positions of the deletions in mutants xi, x , and x . peptides with novel amino termini that alter the signal peptidase cleavage site from ala/asp-val-his to ala/asn-leu-trp and ala/gln-pro-gly, respectively. thus both the charge and secondary structure of the cleavage site would be predicted to be altered by the loss of asp and his (x ) and by the relocation of proline near the cutting site (x ). nevertheless, the signal peptidase efficiently cleaved the leader peptide from the nascent polypeptide, and its specificity of cleavage was unaffected by these alterations (hardwick et al, ) . these experiments then support the generalized conclusion from statistical analysis, that the sequence to the right of the signal peptidase site is not critical for signal peptidase cleavage. none of the deletions generated in the rsv glycoprotein gene resulted in a loss of recognition and cleavage by the signal peptidase. this result contrasts with previously described prokaryotic mutants. a amino acid deletion starting at the fifth residue beyond the signal peptidase site of the lamb gene product blocked cleavage of the signal (emr and bassford, ; emr et al, ) , and a deletion of amino acids beginning amino acids downstream from the signal abolished signal cleavage although the shortened protein was localized correctly . in addition, the substitution of a leucine, in place of the glutamic acid, at residue of the mature m coat protein also inhibited signal peptidase cleavage; however, in this latter instance the procoat protein was transported inefficiently across the inner membrane (boeke et al., ; rüssel and model, ) . although more mutants will be required to properly define these systems, the prokaryotic cleavage site appears to be more sensitive to manipulation than that of eukaryotes. there is accumulating evidence that transported prokaryotic proteins, unlike those of eukaryotes, may not be transferred across membranes in a strictly cotranslational manner (randall and hardy, ) . thus, altered regions within the structural protein portion of a molecule would have the opportunity to interact and interfere with signal peptidase cleavage; such as interaction would not be possible in the cotranslational system described for eukaryotes. although the mutant xi polypeptides were translocated across the rer membrane in a normal fashion, immunofluorescence experiments and posttranslational modification probes indicated that the transport and maturation of the xi glycoprotein was halted shortly after exiting the rer, perhaps within pre-or cis-golgi vesicles. cells synthesizing the mutant protein showed no surface immunofluorescence, no cleavage of the pr to gp /gp , and no terminal sugar additions (hardwich et al., ) . the basis for this block appears to be the altered amino acid sequence rather than the loss of the carbohydrate side chain since by using a mutagenic oligonucleotide we have modified the amino terminus of the xi gp from asp-val-his-arg-thr-to asp-val-asn-arg-thr-, thereby reinserting the glycosylation site missing from this mutant. the derivative mutant, x a, is glycosylated at this site but remains blocked at the same intracellular point in the secretory pathway (k. shaw, k. kervin, and e. hunter, unpublished) . a second deletion mutant of the rsv env gene is also blocked in intracellular transport. this mutant, c , has an engineered deletion at the carboxy terminus of gp that removed the cytoplasmic tail and transmembrane region (see below and wills et al., ) . its transport is clearly blocked at an earlier stage than that of the xi mutant since it was localized to the er and never reached the golgi apparatus, whereas by immunofluorescent staining of fixed cells the xi protein appeared to colocalize with the golgi complex (see fig. ; hardwick et al., ) . although the xi and c mutants contain alterations at opposite ends of the env gene product, they both appear to lack an element that normally signals their transport to and beyond the cis-golgi. while there may be a specific amino acid sequence (analogous to the amino-terminal signal sequence) that is required for these transport steps, it is as likely that a correctly aligned tertiary structure is the critical factor. just as small changes as the amino terminus of ha can disrupt assembly and transport un rhodamine fig. . intracellular immunofluorescence of cells expressing wild-type and mutant polypeptides. fixed infected cv- cells were stained to detect the intracellular localization of wild-type and mutant glycoproteins. rabbit anti-glycoprotein antibodies were tagged with fluorescein-conjugated goat anti-rabbit antibodies which in wild-type and xi-infected cells could be localized on the nuclear membrane (nm), endoplasmic reticulum (er), and golgi apparatus (g). the golgi was localized by staining the same cells with rhodamine-conjugated wheat germ agglutinin. in mutant c -infected cells neither the nuclear membrane nor the golgi apparatus stained with the antiglycoprotein antiserum. magnification is . of the ha trimer, the deleted amino acids unique to the xi mutation may similarly play a critical role in the tertiary structure of the env complex, such that sorting signals required for transport through the golgi complex are lost. mutants x and x have deletions that begin at the amino terminus of gp and extend nine and six amino acids into this structural protein; thus these mutations overlap with the deletion in xi. nevertheless, both mutant proteins were transported to the cell surface and were indistinguishable from the wild type. mutants x and x thus indicate that the terminal nine amino acids of pr are not required for normal intracellular transport and define the critical region in gp as the seven amino acids that are uniquely deleted in the xi mutant. a. truncation of the cytoplasmic sequences. dna and protein sequence studies demonstrated the presence of a amino acid long hydrophobic (and presumably membrane-spanning) domain and a amino acid long cytoplasmic domain at the carboxy terminus of gp ; see fig. ). comparison of these domains with those of other exogenous and endogenous strains of rsv has revealed that the sequence within the hydrophobic domain is highly conserved and that within the cytoplasmic domain the sequence of the first amino acids (adjacent to the hydrophobic domain) is also highly conserved while those at the carboxy termini diverge greatly (hughes, ; hunter et al., ) . these results raised the possibility that the conserved region of the cytoplasmic domain might play a functional role in either transport of the env gene product through the secretory pathway or in virus assembly. to investigate this question we initially altered the cytoplasmic domain by introducing deletion mutations into the molecularly cloned sequences of the proviral env gene and examined the effects of the mutations on transport and subcellular localization in cv- cells. we found that replacement of the nonconserved region of the cytoplasmic domain with a longer unrelated sequence of amino acids from sv vector sequences (mutant cl) did not alter the rate of transport to the golgi apparatus nor the appearance of the glycoprotein on the cell surface. larger deletions, extending into the conserved region of the cytoplasmic domain (mutant c ), however, resulted in a -fold slower rate of transport to the golgi complex, but did not prevent transport to the cell surface (wills et al., (wills et al., , . these results were thus consistent with the cytoplasmic domain of the rsv env gene product playing some role in transport to the golgi complex. similar results were obtained by rose and bergmann ( ) who introduced into the cdna clone encoding the vsv g protein a series of deletions that affected the cytoplasmic domain. these mutants fell into two classes; the first was completely arrested in their transport at a stage prior to the addition of complex oligosaccharides (presumably the rer) and the second showed severely reduced rates of transport to the golgi complex although the proteins were ultimately transported to and expressed on the cell surface. the method by which these mutants were constructed (as with the rsv env mutants) meant that the truncated g proteins terminated in sv sequences, and in at least one case the block to transport could be alleviated by substitution of a termination codon for these "poison" sequences. even in these constructions, however, three foreign amino acids were translated prior to termination (rose and bergmann, ) . the concept that the cytoplasmic domain might influence or govern the rate at which membrane-spanning proteins were transported to the golgi complex was supported by similar studies of doyle et al. ( ) on the ha polypeptide. while the ha cytoplasmic domain could be replaced by the equivalent region from the rsv env gene product without affecting the rate of transport of the hybrid ha from the rer to the golgi complex, truncation of the ha cytoplasmic domain or addition of the amino acid long cytoplasmic domain from gp slowed transport significantly, and addition of amino acids encoded by pbr sequences blocked transport of the ha from the rer. on the other hand, studies on the class i histocompatibility antigens (zuniga et al., ; murre et al., ) , the p of semliki forest virus (garoff et al., ) , and additional studies on the ha of influenza indicated that the cytoplasmic domains of these proteins could be truncated without affecting transport to the cell surface, although the kinetics of transport were not determined in every case. b. substitution mutations. it should be noted that in most of the mutant constructions described above, the carboxy-terminal region contained one or several aberrant amino acids as a result of the recombinant dna approach. thus in order to determine more directly the role of the cytoplasmic domain of gp , we have used oligonucleotide-directed mutagenesis to introduce an early termination codon in the coding sequences of gp such that the arginine residue that represents the first amino acid of the cytoplasmic domain is changed to an opal terminator. this mutation creates a truncated viral glycoprotein lacking specifically the cytoplasmic domain of gp . the biosynthesis and transport of the products of this mutant viral glycoprotein gene were analyzed by expression from an sv late-region replacement vector, and its ability to be active in viral assembly was investigated by substitution of the mutated gene for the wild-type gene in an infectious avian retrovirus vector. in contrast to our previous results, deletion of the entire cytoplasmic domain alone had no effect on the biosynthesis or rate of intracellular transport of the env glycoprotein. thus it seems unlikely that the conserved amino acids present in this region play a role in intracellular transport. although the cytoplasmic domain contains several charged, hydrophilic residues, it does not appear, by itself, to be required for anchoring the complex in the membrane, since molecules lacking the cytoplasmic domain were expressed stably on the plasma membrane and were not shed into the cell culture medium . a recent study by gething et al. ( ) has demonstrated that mutations within the cytoplasmic domain of the influenza virus ha can affect the conformation of the extracellular domain by preventing assembly and trimerization of the ha molecule, thereby resulting in a failure of those mutants to be efficiently transported. a similar requirement for the assembly of oligomeric forms of the vs v g protein prior to its transport to the golgi complex has also been reported (kreis and lodish, ) . the inconsistency of our previous results with the ones obtained with the opal mutant could be explained in a similar way. we cannot rule out the possibility that in our earlier experiments the extra amino acids, added as a consequence of the loss of the env termination codon, created a conformational change in the extracellular domain of pr and slowed its transport from the rer. our present results indicate that the cytoplasmic domain of gp is neither a recognition signal for transport to the plasma membrane nor a requirement for anchoring the molecule to it. these findings also support the idea that the charged amino acids present in most of the cytoplasmic domains of many transmembrane proteins (garoff et al., ; sabatini et al., ) are dispensable for anchor function (davis etal., ) . this latter question has also been addressed by cutler and co-workers , who mutated the cytoplasmic domain of the p polypeptide of semliki forest virus. this region, which normally contains a charge cluster (arg-ser-lys) flanking the hydrophobic domain, was changed to a neutral (met-ser-gly) or an acidic (met-ser-glu) one using oligonucleotide mutagenesis. expression analyses of these mutant proteins confirmed that the basic amino acids were not required for cell surface transport since they reached the surface in a biologically active form. nevertheless, both mutant polypeptides showed reduced stability when membranes containing them were extracted with high-ph buffer . charged residues within the cytoplasmic domain may thus provide an additional measure of stability to the membrane-bound complex. since the conserved residues in the cytoplasmic domain of gp were not required for protein transport it seemed possible that this region might play a role in the process of infectious virus assembly. the fact that the mutant protein was efficiently transported to the cell surface allowed us to analyze this potential role for the cytoplasmic domain in the process of virus budding. chemical cross-linking experiments have demonstrated an interaction between gp and pl , one of the gag gene products that structure the viral core of rsv (gebhardt et al, ) . while it is clear that virus assembly can occur in the absence of glycoproteins, it was suggested that the pl /gp interaction may be part of the driving force for the process of viral assembly and budding. furthermore, since host membrane glycoproteins are excluded from the viral membrane there must be some positive signal for inclusion of the viral env gene products in the budding virion. to determine whether the cytoplasmic domain is involved in this interaction and required for infectious virus assembly, we reconstructed a retrovirus genome carrying the "tail(-)" env gene mutation. surprisingly, such mutant viruses were infectious on avian cells and spread through the culture with similar efficiency to those containing a native env glycoprotein complex. furthermore, this truncated env gene complex was incorporated as efficiently into virus particles as the wildtype complex . this fact suggests that if an interaction between gp and pl is required to mediate the incorporation of the glycoproteins into the envelope of the budding viral particle, it must occur within the lipid bilayer, presumably with the hydrophobic anchor domain. it is thus unlikely that interactions between viral capsid proteins and the cytoplasmic domain of the env complex constitute a driving force for preferential incorporation of the viral glycoproteins in the avian retroviral envelope. what then is the function of the cytoplasmic domain of the env glycoprotein? since this segment of the viral polypeptide does show a region of conserved sequence, it is possible that it has evolved to facilitate transport to the plasma membrane without being a requirement for it; clearly, randomly inserted alterations within this domain can exert a negative effect on the transport process. while we have observed normal assembly and infection by virus encoding a "tail(-)" env product, it will be of interest to determine whether continued growth of the virus results in the dominant appearance of revertants that encode a functional cytoplasmic domain. many cell surface and membrane proteins of animal viruses are bound to the lipid bilayer by a membrane-spanning hydrophobic peptide close to the carboxy terminus of the polypeptide (reviewed by warren, ; armstrong et al, ) . experimental evidence for this first came from deletion mutants of the influenza virus ha (gething and sambrook, ; sveda et al, ) , the vsv g protein (rose and bergmann, ) , and the minor coat protein of phage fl (boeke and model, ) in which removal of sequences that encoded the cytoplasmic and membrane-spanning domains resulted in secretion of the protein. the hydrophobic membrane-spanning peptide of these polypeptides is thought to be an essential component of the cotranslational signal that results in the arrest of chain transfer across the rer membrane during synthesis. these stop-translocation sequences have been proposed to be a region of the nascent protein molecule which halts insertion through the membrane by disassembling the translocation apparatus and thereby creates proteins with three topological domains (blobel, ) . they appear to be inseparable from the anchor sequences (yost et al, ; rettenmier et al., ) since the transfer of intact transmembrane domains to normally secreted proteins has caused translocation of the constructed hybrid molecules to stop at the added sequences (yost et al., ; guan and rose, ) . however, the precise structural and physical properties of the stop-translocation sequences have not been defined. wold et al. ( ) have suggested that the cytoplasmic domain of membrane-spanning proteins might act to interrupt translocation; however, this seems unlikely since deletion mutants lacking this domain are found to be associated with the membrane in a normal manner (see above; garoff et al., ; zuniga et al, ; murre et al, ; doyle et al, ; . while length and sequence vary widely among regions described as transmembrane anchors, they do have characteristics in common. most often, they are long stretches ( - residues) of predominantly nonpolar and hydrophobic amino acids, bounded by charged residues, at the carboxy terminus of membrane proteins. membrane-spanning sequences have also been described, however, at the amino terminus of some viral proteins (blok etal.,a s ; palmiter et al, ; bos et al, ; markoff et al, ; zerial et al., ; spiess and lodish, ) and in the middle of other proteins (rettenmier et al, ; kopito and lodish, ; finer-moore and stroud, ) . we have investigated the structural requirements for a functional anchor/stop-translocation sequence in the rsv env system by constructing both deletion and point mutations in this region. a. deletion of the anchor domain. during our studies on the role of the cytoplasmic domain in env product transport, we characterized a mutant (c ) in which the entire cytoplasmic and transmembrane domains were deleted. this mutant, in contrast to those described for the influenza virus ha and the vsv g protein, was arrested in its transport at the rer and thus was not secreted from the cell (wills et al., ) . pulse-chase experiments coupled with oligosaccharide precursor labeling experiments showed that the c polypeptide was not transported to the golgi complex, even though it accumulated in a soluble, nonanchored form in the lumen of the rer; the mutant thus appeared to lack a functional sorting signal. surprisingly, immunofluorescent labeling studies showed that the c protein (unlike the wild type) did not accumulate on the nuclear membrane but rather in vesicles distributed throughout the cytoplasm (fig. ) , suggesting that movement to the nuclear membrane, blocked in c , may require a specific transport event, even though the rer and nuclear membranes appear to be continuous. this hypothesis is supported by studies on the vsv g protein that indicate that transitional vesicles (for the transport of glycoproteins to the golgi apparatus) may be derived from "blebs" in the nuclear membrane (bergmann and singer, ) . although these studies raised the possibility that sorting signals might exist within the deleted region of c , the amino acid deletion in this mutant, which extends into the external domain of gp , would be expected to prevent normal folding of the glycoprotein. to determine whether the transmembrane domain was required for intracellular transport, we have modified the env gene by oligonucleotide-directed mutagenesis, changing the lysine (aaa) codon, which precedes the hydrophobic domain of gp , to an ochre nonsense codon (taa). this modified gene thus encodes a protein consisting of the entire external domain of pr and lacking precisely the hydrophobic membrane-spanning and hydrophilic cytoplasmic domain. the biosynthesis and intracellular transport of the truncated protein in cv- cells was not significantly different from that of the wild-type glycoprotein, suggesting that any protein signals for biosynthesis and intracellular transport of this viral glycoprotein complex must reside in its extracellular domain. in contrast to the case of the c mutant, this complex lacking just the transmembrane and cytoplasmic domains is secreted as a soluble molecule into the culture medium . since the glycoprotein complex lacking only the cytoplasmic domain of gp is stably expressed on the cell surface, in a manner similar to the wild-type complex, it can be concluded that the transmembrane domain alone is required for anchoring the rsv env complex in the cell membrane. b. requirements for a functional stop-translocation/anchor sequence. we have approached the question of the compositional requirements for membrane anchoring and orientation (stop-translocation) of a membrane-spanning protein by substituting an arginine for a centrally positioned leucine in the hydrophobic anchor region of the rsv env gene product (fig. ) . the arginine substitution is one of the most drastic ^ membrane-spanning mutant « domain * wt his leu leu lysjgly leu leu leu gly leu val val ile leu leu leu leu val cys leu pro cys leu leu gin phe val ser ser ser ile|arg lys met μΆ^ his leu leu lys|gly leu leu leu gly leu val val ile leu leu leu leu val cys|arg|pro cys leu leu gin phe val ser ser ser ile) arg lys met t his leu leu lys|gly leu leu leu gly leu val val ile leu leu leu leu val cysj ;;;-::;;;: : :;|i|leu leu gin phe val ser ser ser ile|arg lys met t his leu leu lys|gly leu leu leu gly leu val val ile leu leu leu leu val| |ser ser se Üe|arg lys met t his leu leu lys|gly leu leu leu gly leu val val ile leu leu[ jval ser ser ser ile| arg lys met t his leu leu lys|gly leu leu leu gly leu val| |ser ser ser ile|arg lys met t his leu leu lys|gj leu||| |ser ser ile|arg lys met compositional point mutations that could be made since it is only rarely found buried in hydrophobic environments (kyte and doolittle, ) and has a high predicted potential for terminating membrane-buried helices (rao and argos, ) . the substitutions we have made fall within the conserved leucine-rich " i c " region proposed by patarca and haseltine ( ) and near the two cysteine residues where palmitate may be covalently added (gebhardt et al, ; kaufman et al., ) . by changing the anchor's hydrophobic integrity through the insertion of point mutations we hoped to define better what constituted a functional anchor sequence. placing a highly charged basic side chain into the hydrophobic core of the membrane might be expected to either ( ) terminate the membrane-spanning helix, thereby partitioning the charged residue to one side or the other of the membrane, or ( ) destroy the stop-translocation signal, causing the protein to be secreted. the results of these experiments showed that a single amino acid substitution in the transmembrane anchor did not affect membrane association or its orientation in the membrane; unexpectedly, however, it affected targeting of the protein at a stage late in the transport pathway, such that the mutant protein was rapidly degraded in lysosmes . the early translation products of both the arginine-mutant and wild-type genes behaved normally: they were synthesized with equal efficiency, had normal bitopic symmetry, and were glycosylated. the kinetics for the turnover of these precursors were nearly identical to those previously reported in infected chicken embryo fibroblasts (bosch and schwarz, ) and in sv expression vectors (wills et al., ) . furthermore, in the golgi, palmitate was added to the precursors, they were cleaved to gp -gp , and they received terminal sugars. only after this last stage did the presence of the charged side chain of the substituted arginine alter expression. at the level of the trans golgi, a post-golgi compartment (saraste and kuismanen, ) , or at the cell surface, the gp -gp complex was rapidly shunted to lysosomes and degraded-as shown by the protection afforded the terminally glycosylated env proteins by the lysosomatropic agent, chloroquine . the exact pathway that the molecules take to the lysosome is not known. they may be transported directly from the trans golgi or first to the surface where they are rapidly endocytosed. discriminating between the alternate pathways has not been possible from current data. why the insertion of an arginine into the anchor should result in targeting to lysosomes is not obvious. a possible explanation is that we have introduced a specific sorting signal into the molecule; however, this is unlikely since other polypeptides with charged residues in the membranespanning domain are not so targeted (kabcenell and atkinson, ; saito et al., ; hayday et al., ) . the arginine's charge is incompatible with the hydrophobic environment of the lipid bilayer; to achieve stability, the charged guanidinium group needs to be neutralized, and how this is done inside the bilayer is not clear. parsegian ( ) has postulated that a lone charge sequestered in a membrane must form a pore or tunnel along with localized membrane thinning to acheive the lowest energy state. if the charged residue in the gp anchor causes the mutant molecules to aggregrate and form channels in the membrane in an analogous manner, it would likely kill the cell unless there was a mechanism to remove it rapidly. alternatively, since the env protein is not an isolated entity in the membrane, it is conceivable that it aggregates with other components of the membrane to reduce net charge cooperatively, and thereby triggers the endocytotic machinery (mellman and plutner, ) . charged residues are found in several proposed membrane-spanning helices (kabcenell and atkinson, ; saito et al., ; hayday et al., ; reviewed by rao and argos, ) . the charged residues in bacteriorhodopsin membrane-spanning a helices have been suggested to be neutralized by forming ion pairs (engelman et al., ) . this is likely a special case, however, since the energy required to bury an ion pair in the membrane is not much different from that required to bury the free charged group itself (parsegian, ) . neutralization of strong charges, particularly of lysine and arginine, may occur through the formation of strong hydrogen bonds with tyrosine (kyte and doolittle, ) ; however, no tyrosine residues are present in the anchor domain of the env gene product which could participate with the arginine. the t cell a, ß, and y gene products and the rotavirus vp protein have a putative structure similar to that of the arginine mutant, with a lysine centered within the transmembrane anchor; however, unlike the molecule we created, they invariably have tyrosine residues adjacent to the lysine (saito et al., ; kabcenell and atkinson, ; hayday et al., ) which could stabilize the charge through hydrogen bonds (kyte and doolittle, ) . adams and rose ( a) have described the similar insertion of an arginine (and glutamine) residue at the center of the transmembrane domain of the vsv g protein. their mutant protein, like the env mutant we have characterized, was bitopic, could be seen localizing in the golgi, and did not accumulate on the cell surface. since these investigators observed a "lower level of protein expression" with their mutant g protein, it is possible that it also was rapidly degraded in lysosomes following terminal glycosylation. in contrast, observed no alteration in the biosynthesis and transport of a mutant p polypeptide of semliki forest virus in which the hydrophobic domain was interrupted by insertion of a glutamic acid residue. in this protein, however, the outer boundary of the hydrophobic domain is not delineated by a charged residue and so it is possible that additional uncharged residues from the external domain were pulled into the membrane. since the insertion of a charged polar residue into the transmembrane region of the rsv env gene product did not interfere with its anchor/stoptranslocation function, we have investigated the requirement for the long ( amino acid) hydrophobic domain in arresting translocation and anchoring the env complex. a series of deletion mutations was generated by progressively removing base pairs to either side of a unique sph\ restriction site that had been previously engineered into the center of the anchor coding region. this produced env proteins with truncated transmembrane anchors that ranged in length from (t ) to a single apolar amino acid (tl) summarized in fig. ; . while the effects of the deletions on the transport and subcellular localization of the env gene product appeared to be a complex function of the length and composition of the remaining anchor, the mutants appeared to fall into three broad phenotypic classes (summarized in table i ). even the smallest deletion (t ), which removed only three amino acids, greatly reduced the surface expression of the mature env proteins. t and mutants t , t , and t had a normal bitopic orientation in the membrane but appeared to be cleared from the cell surface and degraded in lysosomes, since they accumulated only in the presence of choroquine, an inhibitor of lysosomal degradation. the reduced surface expression of the as determined by the distance between charged residues. b hydrophobicity score as determined using the hydropathicity values of kyte and doolittle ( ) . mean hydrophobicity is hydrophobicity score divided by the apparent anchor length. c symbols: + denotes either a wild-type or positive response; ± denotes an intermediate response; -denotes a negative response. d sec, secreted. e nd/na, not determined/not applicable. f with chloroquine treatment, pr appears in the medium with gp and gp . « anchorless and tailess deletion mutant . largest deletion mutant (t ) was surprising since the effective hydrophobic peptide remaining in this construct was as long or longer than functional anchors reportedly present in other integral membrane proteins [e.g., amino acids: m protein of influenza (lamb, ) ; amino acids: vsv g protein (rose et al., ) and adenovirus e protein (wold et al., ) ]. deletions which reduced the size of the transmembrane anchor to seven amino acids (t ) or less resulted in the secretion of mature glycoproteins into the medium. a third class of mutants with hydrophobic regions of (t ) and (til) amino acids, respectively, while remaining membrane associated, no longer appeared to span the rer as bitopic proteins. neither mutant could be found at the surface of cells, nor could their degradation be arrested by chloroquine treatment. from the sum of the data obtained with these mutants, it would appear that bitopic insertion of the env gene product is possible with effective anchor domains of at least amino acids; if additional amino acids are removed from the domain, the protein can no longer exist bitopically, and it either partitions monotopically to the luminal side of the rer membrane or withdraws amino acids from the cytoplasmic side of the membrane into the bilayer. nevertheless, such sequences from the cytoplasmic domain are not able to stabilize the shortest anchors (tl, t , and t ) since these are secreted from the cell. davis and model ( ) have investigated the requirements of a functional anchor domain by inserting artificial hydrophobic peptides of varying length into the membrane-associated pill protein of the bacteriophage fl. their results show that hydrophobic amino acids are sufficient to maintain the protein in a bitopic configuration; however, the amino acid anchor was "deleterious to the cell" presumably because it was too short to assume a stable conformation compatible with existence in the bilayer and thereby destabilized the membrane. a construct with an anchor of hydrophobic amino acids was membrane associated but showed an intermediate phenotype in its sensitivity to solubilization by alkali. in contrast, a construct with only hydrophobic amino acids-also membrane associated-was completely released into the supernatant at high ph. adams and rose ( b) reduced the anchor domain of vsv g by precisely deleting amino acids from within the hydrophobic core. when the length of the anchor was reduced from amino acids to as few as , the protein was normally membrane associated and expressed in a bitopic fashion on the cell surface. on the other hand, proteins with an anchor domain of or amino acids, while spanning the membrane, appeared to be transported only as far as the golgi where they accumulated; the surface expression of these proteins was greatly reduced ( amino acid anchor) or undetectable ( amino acids). doyle et al. ( ) have characterized a series of carboxy-terminal deletion mutants of the ha polypeptide in which the amino acid anchor domain was truncated to , , and amino acids, respectively. in this case, molecules with a amino acid long transmembrane domain were stably anchored but were transported less efficiently to the plasma membrane. truncation of the hydrophobic anchor to or residues resulted in ha proteins that were unstable and whose transport appeared to be blocked in the rer or in a pre-golgi compartment-resembling the til mutant of the rsv env gene described above. from the results of these different systems and approaches it would seem that in strictly physical terms, anchors may be significantly reduced in their length without any consequence to the membrane association. the limits for this length ap-pear to be about - amino acids, but it must be appreciated that the mere presence of a stretch of hydrophobic amino acids within a protein does not serve to constitute an anchor. several mammalian virus envelope proteins contain in their external domain long hydrophobic amino acid regions that are equivalent to the truncated bitopic anchors have described here (gething et al, ; white et al, ) . indeed, gp contains a strongly hydrophobic amino acid region that clearly does not act as a stop-translocation sequence or play a role in membrane association . the work of davis and model ( ) , on the other hand, implies that the length of a hydrophobic region is the major determinant as to whether or not it will confer membrane association properties to a protein, although they point out that the position of such sequences within the molecule may play a role. most eukaryotic membrane-spanning polypeptides have a complex tertiary structure that is stabilized by multiple disulfide linkages, and it is possible that the entropy of a correctly folded molecule is sufficient to pull potential stop-transfer regions through the membrane. a corollary of this hypothesis, therefore, is that, once folding is complete, short hydrophobic regions that can potentially span the membrane as an a helix would stop translocation. the length requirements for such a region could be shorter than the residues predicted from a-helix dimensions if the region were flanked by arginines or lysines. since the latter have long side chains, equivalent in length to a single turn of an a helix, a stretch of hydrophobic amino acids - amino acids long might be sufficient. such a prediction fits well with the data we have obtained and with those of adams and rose ( b) and davis and model ( ) . it will be of interest to determine what effect inserting the truncated anchors of mutants t and t into the middle of the env precursor has on translocation; if the above speculations are correct they should be extruded into the external domain. finally, it should be reemphasized that merely providing a bitopic membrane anchor/stop-translocation is not sufficient to confer wild-type biological activity on a polypeptide. mutant t of the rsv env gene has a hydrophobic domain which might be expected to be sufficiently long and hydrophobic in character to span the membrane stably; it is modified normally by palmitic acid and can clearly be transported to its targeted cellular location. nevertheless, it is degraded rapidly by the cell. these results imply that hydrophobic transmembrane domains contain additional (and perhaps subtle) signals that remain to be deciphered, a conclusion that is supported by the finding that deletion of the anchor domain of the rotavirus vp protein abolishes its specific targeting and retention in the rer (poruchynsky et al, ) . polarized epithelial cells exhibit apical and basolateral membrane domains that are separated by well-defined tight junctions. each membrane domain has a unique protein composition (louvard, ; reggio et al., ) , indicating that mechanisms must exist to specifically target membrane proteins to different surfaces. the sorting process occurs during or shortly after passage of the glycoproteins through the golgi complex pfeiffer et al., ; rindler et al., rindler et al., , . however, the mechanisms determining this directed transport to either the apical or basolateral membranes are not understood. their study has been facilitated by the use of cultured epithelial cell lines, such as the mdck cell line, and by the observation that certain rna viruses bud exclusively from apical or basolateral domains of these polarized cells in culture (rodriguez-boulan and sabatini, ; herrler et al., ; roth et al., a; rindler et al., ) . avian and mammalian retroviruses together with rhabdoviruses such as vsv mature from the basolateral surface, while ortho-and paramyxoviruses bud from the apical surface. the carbohydrate residues present on the different proteins do not appear to play a role in this sorting process, since tunicamycin does not interfere with the polarized release of the viruses (roth et al., ; green et al., b) . as with the maturation of viruses that assemble at intracellular locations within the secretory pathway, polarized budding of enveloped viruses is dependent on the site to which viral glycoproteins are transported. expression of cloned viral glycoprotein genes from both sv -based and vaccinia expression vectors in polarized cells has demonstrated that the ha and neuraminidase polypeptides of influenza virus are targeted to the apical surface (roth et al., b; jones et al., ; gottlieb et al., ) while the g protein of vsv and the gp /pl e complex of murine leukemia virus (mulv) are transported exclusively to the basolateral membranes . in an attempt to locate the signals which direct these glycoproteins to the apical or basolateral domains, recombinant dna techniques have been employed to construct chimeric proteins and express these in polarized cells in culture. in experiments where sequences encoding the external domain of ha were fused to those encoding the transmembrane and cytoplasmic domains of the vsv g protein, the hybrid glycoprotein behaved in the same manner as wild-type ha and was transported to the apical domain of polarized cells (mcqueen et al., ; roth et al., ) . conversely, fusing the external domain of g protein to the anchor/cytoplasmic domain of ha results in basolateral transport (mcqueen et al., ) . these experiments thus suggest that the ectodomains of ha and g protein contain signals for apical and basolateral transport, respectively. while expression of a secreted form of the ha glycoprotein in an apical polarized manner supports this conclusion (roth et al. y ) , the unanchored ectodomain of the mulv gp /pl e complex, which is normally targeted to the basolateral membrane, is secreted in a nonpolarized fashion . it is possible that this soluble protein is improperly folded and thus is unable to interact with the sorting machinery, alternatively it also raises the possibility that targeting signals may be located in more than one domain of these molecules. further analyses should shed light on this problem. the studies described in this chapter demonstrate the breadth of information that has been and can be obtained from studies on enveloped virus glycoprotein biosynthesis. many of the studies were performed at a time when the cloned genes and molecular probes for cellular glycoproteins were unavailable and thus provided valuable insights into the manner in which cells compartmentalized and transported membrane proteins. the exciting possibility of utilizing viral glycoprotein genes for genetic analyses of the transport pathway, in a way analogous to that pursued in prokaryotic systems (michaelis and beckwith, ; silhavy et al. y ; oliver, ) , has led to a plethora of studies that utilized both classic and recombinant dna genetic approaches. these investigations have resulted in great progress in our understanding of the general processes involved in intracellular transport of proteins through the secretory pathway but at the same time have raised difficult questions about the molecular interactions required for protein sorting. the initial observation that proteins destined for secretion contain an amino-terminal sorting sequence provided a precedent on which to build models for protein targeting based on topogenic sequences (blobel, ) . to a large extent the identification of signal sequences was facilitated by their transient nature, not by a conserved primary sequence. indeed, while some common characteristics of signal peptides can be recognized (von heijne, ) , the identification of signal peptides in proteins where they are not removed has proved difficult and has required the use of sophisticated recombinant dna technology [e.g., ovalbumin (tabe et ah, ) ]. the requirement for a signal sequence to initiate translocation across the er membrane has been clearly confirmed through the isolation and construction of mutants which lack this functional region (as discussed above) and by fusion of this sequence to proteins that are not normally translocated (lingappa et al., ) . these approaches have also been facilitated by the transient, nonstructural nature of many aminoterminal, translocation signals. recent experiments by friedlander and blobel ( ) and kaiser et al. ( ) , however, raise questions about the informational content of signal sequences. in particular kaiser and colleagues showed that several random amino acid sequences derived from human genomic dna fragments could act as signal sequences for translocation of the yeast invertase enzyme. thus even in this well-defined situation, where an amino acid sequence is known to play a functional role in the sorting process, it can be impossible to predict with confidence its location in the protein; how then might we expect to identify additional sorting sequences that may or may not exist within the structural domain of a transported protein? the possibility that additional sorting sequences might be involved in steering the transport of a membrane-spanning or secreted protein through the vesicular maze of the secretory pathway remains open. at the present time the necessity for a native tertiary structure cannot be separated from the possibility of such additional sorting sequences. it is clear that disruption of a poly peptide's normal folding can completely prevent its transport from the er kreis and lodish, ) , and the simplest explanation for the phenotypes of a variety of conditional and nonconditional transport mutants would be that they alter the tertiary structure of the mature protein (see section ii, a, above). a -dimensional structure has been determined for only a few molecules that traverse the secretory pathway, and even with these proteins the current, predictive algorithms are insufficiently accurate to model potential changes in molecular shape in response to mutations. the question of a direct role for tertiary structure in protein transport thus represents a major challenge to molecular biologists. furthermore, one might argue that a change in protein shape could also mask or distort a necessary (peptide) sorting sequence. this possibility is supported by the observation that for a majority of eukaryotic proteins the amino-terminal signal peptide is unable to initiate translocation across the er if translation is allowed to proceed to completion, presumably because the tertiary structure of the nascent polypeptide precludes the interaction of the signal peptide with the translocation machinery. it is quite feasible that sorting signals and targeting signals could be represented by different entities within a single polypeptide, particularly if the latter were required to fix the intracellular location of a protein. for example, the rotavirus vp polypeptide accumulates within the er un-less its amino-terminal hydrophobic anchor region is deleted, whereupon it is transported to the cell surface and secreted (poruchynsky et al., ) ; in this instance the deleted region presumably contains a sequence that can fix the intracellular location of the protein despite the fact that the molecule has the potential to be exported from the cell. since most translocated polypeptides appear to follow a common pathway to a late compartment of the golgi (kelly, ) , it might be argued that a native conformation is the sole requirement for transport to this organelle and that the observed differences in the transport rates of proteins to the golgi merely reflect the time necessary for completion of the folding process. nevertheless, proteins leaving the golgi appear to be sorted into specific pathways; for example, in secretory cells proteins may follow either the constitutive pathway or be sequestered in secretory granules (moore and kelly, ; reviewed by kelly, ) , and in epithelial cells specific proteins appear to be transported directly to either the apical or basolateral membranes (see above). thus, it would seem likely that some form of sorting signal must be present in the polypeptide at this point in the secretory pathway in order to correctly direct its transport; initial results from viral glycoprotein expression studies indicate that at least in polarized cells the ectodomain of the sorted protein plays a dominant role (mcqueen et al., ; . additional studies should provide a clearer picture of this complex process. in summary, studies on the biosynthesis and transport of enveloped virus glycoproteins have provided important insights into the general processes involved in the intracellular movement of these membrane-associated molecules. the specific questions that remain to be answered are many and difficult, but it is likely that these viral systems will continue to play a vital role by providing clues and direction in this important area of cell biology. incorporation of a charged amino acid into the membrane spanning domain blocks cell surface transport but not membrane anchoring of a viral protein structural requirements of a membrane-spanning domain for protein anchoring and cell surface transport sequence analysis of two mutants of sindbis virus defective in the intracellular transport of their glycoproteins domain structure of bacteriophage fed adsorption protein intragenic suppressor mutations that restore export of maltose binding protein with a truncated signal peptide information within the mature lamb protein necessary for localization to the outer membrane of eseherichia coli k- genetic analysis of protein export in escherichia coli k- immunoelectron microscopic studies of the intracellular transport of the membrane glycoprotein (g) of vesicular stomatitis virus in infected chinese hamster ovary cells passage of an integral membrane protein, the vesicular stomatitis virus glycoprotein, through the golgi apparatus en route to the plasma membrane bunyaviridae. in "comprehensive virology intracellular protein topogenesis transfer of proteins across membranes. i. presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma studies on the size, chemical composition and partial sequence of the neuraminidase (na) from type a influenza viruses show the nterminal region of na is not processed and serves to anchor na in the viral membrane a prokaryotic membrane anchor sequence: carboxyl terminus of bacteriophage fl gene iii protein retains it in the membrane processing of filamentous phage precoat protein: effect of sequence variations near the signal peptidase cleavage site nh -terminal hydrophobic region of influenza virus neuraminidase provides the signal function in translocation processing of gpr env, the precursor to the glycoproteins of rous sarcoma virus: use of inhibitors of oligosaccharide trimming and glycoprotein transport sequence of the membrane protein gene from avian coronavirus ibv virus-specific glycoproteins associated with the nuclear fraction of herpes simplex virus type -infected cells mutants of the membrane-binding region of semliki forest virus e protein. i. cell surface transport and fusgenic activity mutants of the membrane-binding region of semliki forest virus e protein. ii. topology and membrane binding fusion mutants of the influenza virus hemagglutinin glycoprotein isolation of the escherichia coli leader peptidase gene and effects of leader peptidase overproduction in vivo a charged amino acid substitution within the transmembrane anchor of the rous sarcoma virus envelope glycoprotein affects surface expression but not intracellular transport altered surface expression, membrane association and intracellular transport result from deletions within the transmembrane anchor of the rous sarcoma virus envelope glycoprotein an artificial anchor domain: hydrophobicity suffices to stop transfer fine structure of a membrane anchor domain variant influenza virus hemagglutinin that induces fusion at elevated ph mutations in the cytoplasmic domain of influenza virus hemagglutinin affect different stages of intracellular transport analysis of progressive deletions of the transmembrane and cytoplasmic domains of influenza hemagglutinin assembly of enveloped rna viruses early and late functions associated with the golgi apparatus reside in distinct compartments localization and processing of outer membrane and periplasmic proteins in escherichia coli strains harboring export-specific suppressor mutations suppressor mutations that restore export of a protein with a defective signal sequence importance of secondary structure in the signal sequence for protein secretion path of the polypeptide in bacteriohodopsin amphipathic analysis and possible formation of the ion channel in an acetylcholine receptor evidence for a glycoprotein "signal" involved in transport between subcellular organelles bovine opsin has more than one signal sequence characterization of two recombinant-complementation groups of uukuniemi virus temperature-sensitive mutants uukuniemi virus glycoproteins accumulate in and cause morphological changes of the golgi complex in the absence of virus maturation nucleotide sequence of a cdna clone encoding the entire glycoprotein from the new jersey serotype of vesicular stomatitis virus a single amino acid substitution in a hydrophobic domain causes temperature-sensitive cell-surface transport of a mutant viral glycoprotein using recombinant dna techniques to study protein targeting in the eucaryotic cell expression of semliki forest virus proteins from cloned complementary dna. ii. the membrane-spanning glycoprotein e is transported to the cell surface without its normal cytoplasmic domain rous sarcoma virus pl and gp can be chemically crosslinked to high molecular weight complexes. an insight into viral association cold spring harbor laboratory, cold spring habor construction of influenza haemagglutinin genes that code for intracellular and secreted forms of the protein purification of the fusion protein of sendai virus: analysis of the nh -terminal sequence generated during precursor activation cloning and dna sequence of double-stranded copies of haemagglutinin genes from h and h strains elucidates antigenic shift and drift in human influenza virus expression of wild-type and mutant forms of influenza hamagglutinin: the role of folding in intracellular transport synthesis and infectivity of vesicular stomatitis viruses containing nonglycosylated g protein the nonglycosylated glycoprotein of vesicular stomatitis virus is temperature-sensitive and undergoes intracellular aggregation at elevated temperatures protein translocation across the endoplasmic reticulum. i. detection in the microsomal membrane of a receptor for the signal recognition particle protein translocation across the endoplasmic reticulum. ii. isolation and characterization of the signal recognition particle receptor the mechanism of protein translocation across the endoplasmic reticulum membrane sorting and endocytosis of viral glycoproteins in transfected polarized epithelial cells passage of viral membrane proteins through the golgi complex glycosylation does not determine segregation of viral envelope proteins in the plasma membrane of epithelial cells conversion of a secretory protein into a transmembrane protein results in its transport to the golgi complex but not to the cell surface glycosylation allows cell-surface transport of an anchored secretory protein two distinct intracellular pathways transport secretory and membrane glycoproteins to the surface of pituitary tumor cells amino-terminal deletion mutants of the rous sarcoma virus glycoprotein do not block signal peptide cleavage but block intracellular transport biosynthesis of lysosomal enzymes in fibroblasts: phosphorylation of mannose residues structure, organization, and somatic rearrangement of t cell a genes antitrypsin: the presence of excess mannose in the z variant isolated from liver isolation and structural analysis of influenza virus c virion glycoproteins studies on the mechanisms of tunicamycin inhibition of iga and ige secretion by plasma cells the importance of the endosome in intracellular traffic sequence of the long terminal repeat and adjacent segments of the endogenous avian virus rous-associated virus complete sequence of the rous sarcoma virus env gene: identification of structural and functional regions of its product mechanism of signal peptide cleavage in the biosynthesis of the major lipoprotein of the escherichia coli outer membrane phospholipid is required for the processing of presecretory proteins by detergent-solubilized canine pancreatic signal peptidase intracellular transport of herpes simplex virus gd occurs more rapidly in uninfected cells than in infected cells surface expression of influenza virus neuraminidase an amino-terminally anchored viral membrane glycoprotein, in polarized epithelial cells processing of the rough endoplasmic reticulum membrane glycoproteins of rotavirus sah many random sequences functionally replace the secretion signal sequence of yeast invertase cysteines in the transmembrane region of major histocompatability complex antigens are fatty acylated via thioester bonds pathway of protein secretion in eukaryotes ph-induced alterations in the fusogenic spike protein of semlilci forest virus membrane fusion mutants of semliki forest virus separate pathways of maturation of the major structural proteins of vesicular stomatitis virus maturation of viral proteins in cells infected with temperature-sensitive mutants of vesicular stomatitis virus primary structure and transmembrane orientation of the murine anion exchange protein oligomerization is essential for transport of the vesicular stomatitis virus glycoprotein to the cell surface uukuniemi virus maturation: an immune fluorescence microscopy study using monoclonal glycoprotein-specific antibodies a simple method for displaying the hydropathic character of a protein influenza m protein is an integral membrane protein expressed on the infected-cell surface impaired intracelluiar migration and altered solubility of nonglycosylated glycoproteins of vesicular stomatitis virus and sindbis virus kinetics of serum protein secretion by cultured hepatoma cells: evidence for multiple secretory pathways determinants for protein localization: / -lactamase signal sequence directs globin across microsomal membranes hen oviduct signal peptidase is an integral membrane protein reversible block in intracellular transport and budding of mutant vesicular stomatitis virus glycoprotein hepatoma secretory proteins migrate from rough endoplasmic reticulum to golgi at characteristic rates apical membrane aminopeptidase appears at site of cell-cell contact in cultured kidney epithelial cells l ). a single n-linked oligosaccharide at either of the two normal sites is sufficient for transport of vesicular stomatitis virus g protein to the cell surface polarized expression of a chimeric protein in which the transmembrane and cytoplasmic domains of the influenza virus hemagglutinin have been replaced by those of the vesicular stomatitis virus g protein basolateral expression of a chimeric protein in which the transmembrane and cytoplasmic domains of vesicular stomatitis virus g protein have been replaced by those of the influenza virus hemagglutinin glycosylation and surface expression of the influenza virus neuraminidase requires the n-terminal hydrophobic region the entry of enveloped viruses into cells by endocytosis internalization and degradation of macrophage fc receptors bound to polyvalent immune complexes acidification of the endocytic and exocytic pathways identification and characterization of a membrane component essential for the translocation of nascent proteins across the membrane of the endoplasmic reticulum secretory protein translocation across membranes-the role of the "docking protein mechanism of incorporation of cell envelope proteins in escherichia coli secretory protein targeting in a pituitary cell line: differential transport of foreign secretory proteins to distinct secretory pathways structural mutations in a mouse immunoglobulin light chain resulting in failure to be secreted construction, expression and recognition of an h- molecule lacking its carboxyl terminus identification of the defects in the hemagglutinin gene of two temperature-sensitive mutants of a/wsn/ influenza virus membrane-bound penicillinases in grampositive bacteria carbohydrate moieties of glycoproteins, a reevaluation of their function protein secretion in escherichia coli ovalbumin: a secreted protein without a transient hydrophobic leader sequence energy of an ion crossing a low dielectric membrane: solutions to four relevant problems similarities among retro virus proteins mutations within the proteolytic cleavage site of the rous sarcoma virus glycoprotein precursor block processing to gp and gp mutants of the rous sarcoma virus envelope glycoprotein that lack the transmembrane anchor and/or cytoplasmic domains: analysis of intracellular transport and assembly into virions a putative signal peptidase recognition site and sequence in eucaryotic and procaryotic signal peptides reversible defect in the glycosylation of the membrane proteins of semliki forest virus tsl mutant localization of rotavirus antigens in infected cells by ultrastructural immunocytochemistry intracellular sorting and basolateral appearance of the g protein of vesicular stomatitis virus in mdck cells complete nucleotide sequence of an influenza virus haemagglutinin gene from cloned dna deletions into an nh -terminal hydrophobic domain result in secretion of rotavirus vp , a resident endoplasmic reticulum glycoprotein export of protein in bacteria a conformation preference parameter to predict helices in integral membrane proteins surface and cytoplasmic domains in polarized epithelial cells transmembrane orientation of glycoproteins encoded by the w-fms oncogene viral glycoproteins destined for apical or basolateral plasma membrane domains traverse the same golgi apparatus during their intracellular transport in doubly infected madin-darby canine kidney cells polarized delivery of viral glycoproteins to the apical and basolateral plasma membranes of madin-darby canine kidney cells infected with temperature-sensitive viruses asymmetric budding of viruses in epithelial monolayers: a model system for study of epithelial polarity intracellular transport of influenza virus hemagglutinin to the apical surface of madin-darby canine kidney cells expression from cloned cdna of cell-surface secreted forms of the glycoprotein of vesicular stomatitis virus in eukaryotic cells altered cytoplasmic domains affect intracellular transport of the vesicular stomatitis virus glycoprotein vesicular stomatitis virus is anchored in the viral membrane by a hydrophobic domain near the cooh terminus the presence of cysteine in the cytoplasmic domain of the vesicular stomatitis virus glycoprotein is required for palmitate addition polarity of influenza and vesicular stomatitis virus in mdck cells; lack of a requirement for glycosylation of viral glycoproteins influenza virus hemagglutinin expression is polarized in cells infected with recombinant sv viruses carrying cloned hemagglutinin dna basolateral maturation of retroviruses in polarized epithelial cells heterologous transmembrane and cytoplasmic domains direct functional chimeric influenza virus hemagglutinins into the endocytic pathway membrane insertion and intracellular transport of influenza virus glycoproteins the large external domain is sufficient for the correct sorting of secreted or chimeric influenza virus hemagglutinins in polarized monkey kidney cells studies on the adaption of influenza viruses to mdck cells a mutation downstream from the signal peptidase cleavage site affects cleavage but not membrane insertion of phage coat protein mechanisms for the incorporation of proteins in membranes and organelles complete primary structure of a heterodimeric t-cell receptor deduced from cdna sequences pre-and post-golgi vacuoles operate in the transport of semliki forest virsus membrane glycoproteins to the cell surface fatty acid binding to vesicular stomatitis virus glycoprotein: a new type of posttranslational modification of the viral glycoprotein relation of fatty acid attachment to the translation and maturation of vesicular stomatitis and sindbis virus membrane glycoproteins evidence for covalent attachment of fatty acids to sindbis virus glycoproteins defects in functional expression of an influenza virus hemagglutinin lacking the signal peptide sequences analysis of the hemagglutinin glycoprotein from mutants of vaccinia virus that accumulates on the nuclear envelope mechanisms of protein localization changes in the conformation of influenza virus hemagglutinin at the ph optimum of virus-mediated membrane fusion the phosphomannosyl recognition system for intracellular and intercellular transport of lysosomal enzymes glycoproteins specified by herpes simplex viruses an internal signal sequence: the asialoglycoprotein receptor membrane anchor nonpolarized expression of a secreted murine leukemia virus glycoprotein in polarized epithelial cells polarized transport of the vsv g surface expression of viral glycoproteins is polarized in epithelial cells infected with recombinant vaccinia viral vectors intracellular transport of secretory and membrane proteins in hepatoma cells infected by vesicular stomatitis virus vesicular stomatis virus glycoprotein, albumin, and transferrin are transported to the cell surface via the same golgi vesicles effect of tunicamycin on the secretion of serum proteins by primary cultures of rat and chicken hepatocytes the molecular biology of coronaviruses cell surface expression of the influenza virus hemagglutinin requires the hydrophobic carboxy-terminal sequences influenza virus hemagglutinin containing an altered hydrophobic carboxy terminus accumulates intracellularly segregation of mutant ovalbumins and ovalbumin-globin fusion proteins in xenopus oocytes: identification of an ovalbumin signal sequence eukaryotic signal sequence transports insulin antigen in escherichia coli prolipoprotein signal peptidase in escherichia coli is distinct from the m procoat protein signal peptidase temperature-sensitive mutants of influenza virus: a mutation in the hemagglutinin gene structural studies of iga myeloma proteins having anti-dnp antibody activity patterns of amino acids near signal-sequence cleavage sites how signal sequences maintain cleavage specificity signal sequences, the limits of variation purification of a membrane-associated protein complex required for protein translocation across the endoplasmic reticulum signal recognition protein (srp) mediates the selective binding to microsomal membranes of m-i>//ro-assembled poly somes synthesizing secretory protein translocation of proteins across the endoplasmic reticulum iii. signal recognition protein (srp) causes signal sequence-dependent and sitespecific arrest of chain elongation that is released by microsomal membranes translocation of proteins across the endoplasmic reticulum i. signal recognition protein (srp) binds to m-wvro-assembled polysomes synthesizing secretory protein membrane proteins: structure and assembly compilation of published signal sequences m procoat and a preimmunoglobulin share processing specificity but use different membrane receptor mechanisms cell fusion by semliki forest, influenza, and vesicular stomatitis viruses membrane fusion proteins of enveloped animal viruses multiple mechanisms of protein insertion into and across membranes alterations in the transport and processing of rous sarcoma virus envelope glycoproteins mutuated in the signal and anchor regions mutations of the rous sarcoma virus env gene that affect the transport and subcellular location of the glycoprotein products the -kda glycoprotein precursor coded by region e of adenovirus secretion of a x immunoglobulin chain is prevented by a single amino acid substitution in its variable region molecular abnormality of human a r antitrypsin variant (pi-zz) associated with plasma activity deficiency uncoating of influenza virus in endosomes a stop transfer sequence confers predictable transmembrane orientation to a previously secreted protein in cell-free systems the transmembrane segment of the human transferrin receptor functions as a signal peptide mutants of vesicular stomatitis virus blocked at different stages in maturation of the viral glycoprotein expression and function of transplantation antigens with altered or deleted cytoplasmic domains key: cord- - cg z authors: chakraborty, hirak; bhattacharjya, surajit title: mechanistic insights of host cell fusion of sars-cov- and sars-cov- from atomic resolution structure and membrane dynamics date: - - journal: biophys chem doi: . /j.bpc. . sha: doc_id: cord_uid: cg z the emerging and re-emerging viral diseases are continuous threats to the wellbeing of human life. previous outbreaks of severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers had evidenced potential threats of coronaviruses in human health. the recent pandemic due to sars-cov- is overwhelming and has been going beyond control. vaccines and antiviral drugs are ungently required to mitigate the pandemic. therefore, it is important to comprehend the mechanistic details of viral infection process. the fusion between host cell and virus being the first step of infection, understanding the fusion mechanism could provide crucial information to intervene the infection process. interestingly, all enveloped viruses contain fusion protein on their envelope that acts as fusion machine. for coronaviruses, the spike or s glycoprotein mediates successful infection through receptor binding and cell fusion. the cell fusion process requires merging of virus and host cell membranes, and that is essentially performed by the s domain of the s glycoprotein. in this review, we have discussed cell fusion mechanism of sars-cov- from available atomic resolution structures and membrane binding of fusion peptides. we have further discussed about the cell fusion of sars-cov- in the context of present pandemic situation. membrane fusion, one of the most fundamental processes for the survival of eukaryotes, occurs when two closely apposed lipid bilayers merge into a continuous single bilayer and the inner contents are mixed with each other. several cellular events such as endocytosis, exocytosis, cellular trafficking, compartmentalization, import of nutrients and export of waste, vesiculation, inter cellular communication, fertilization and many others involve membrane fusion, though they vary vastly in space and time. the fusion of synaptic vesicles is almost , -fold faster than the fusion of vacuoles. despite many diversities all fusion processes follow a common route that includes membrane contact (docking), membrane merger (stalk and transmembrane contact formation), and opening of pore to transfer the intracellular material (pore formation). there are several models that describe the mechanism of membrane fusion, out of which the stalk model is the most accepted one [ ] . the continuum model of membrane fusion or stalk model proposes that the fusion starts from a point-like membrane protrusion that transform into an hourglass like connection between two apposed monolayers [ ] . this early hemifusion connection is called as the fusion stalk, which further expanded to form the hemifusion diaphragm [ ] . the further expansion of the hemifusion diaphragm opens the pore either on the diaphragm or its perimeter [ , ] . fusion pore can also be formed directly from stalk by avoiding the hemifusion state [ , ] . though membrane fusion is an integral event for the survival of eukaryotes, enveloped viruses utilize this process to enter the host cell [ , ] . the emerging and reemerging viral pandemics prompted us to understand the mechanism of fusion for better intervention against viral diseases. the recent worldwide outburst of coronavirus- (cov- ) causing severe acute respiratory syndrome (sars), covid- , has created havoc in terms of morbidity and mortality. middle east severe acute respiratory syndrome (mers) coronavirus and sars coronavirus- (sars-cov- ), which belong to the same family, coronaviradae, were confined in a certain part of the world [ ] . sars-cov- has shown much higher infectivity, and has been spread all over the world. there is no vaccine for mers and sars, and now the whole world is eagerly waiting for covid- vaccine. the fusion between the host cell and virus being the first step of infection process, it is important to understand the fusion mechanism. this review discusses the mechanistic insights of the fusion of sars-cov- and sars-cov- from their atomistic structure, and membrane interaction. enveloped viruses exploit membrane fusion for their entry to the host cell. the enveloped viruses cover their genetic material with the host cell derived lipid bilayer, which houses the fusion protein that is instrumental in the fusion between virus and host cell [ ] . the receptor binding domain of the fusion protein finds its interaction partner on the cell surface and the interaction between these two allows the virus to dock on the host cell surface. several viruses such as human immunodeficiency virus and coronavirus fuse at the cell surface to transfer the genetic material in the host cell, whereas viruses like influenza enters the host cell through endocytosis and then fuses with the endosome upon ph trigger [ ] . therefore, the viral fusion with the host cell is considered as the first step of viral infection. the fusion protein presents on the viral envelope is called fusion machine as it orchestrates the fusion process between the virus and host cell. interestingly, there is no sequence homology among the fusion proteins of different viruses however, all the fusion proteins share several common features [ ] . now we have a decent understanding on the fusion mechanisms aided by three dimensional structures of fusion proteins and peptides of several viruses with the help of nmr spectroscopy, cryo-electron microscopy and x-ray crystallography. in recent years, the fusion proteins are considered as the target for antiviral intervention due to our growing understanding on the proteins and protein complexes that mediate fusion between virus and host cell. the amino acid sequences of fusion proteins are remarkably different among the viruses, but all initiates membrane fusion through trimer formation, and a common pathway of membrane dynamics [ ] . the trimeric glycoproteins of class i virus generally contain a signal peptide, a receptor binding domain, a fusion domain, and a cytoplasmic tail (figure ) . the signal peptide is being cleaved by the signal peptidase but remains in contact with the lipid and subunit of peptidase complex before it is released in the cytosol. the signal peptide of hiv fusion protein, gp is known to interact with a calcium binding cytosolic protein, calmodulin [ ] . however, the direct role of signal peptide in membrane fusion is not yet clear. the receptor binding domain (rbd) is extremely critical as it provides host tropism and zoonotic transmission of the virus [ ] . there are specific receptors on the host cell surfaces, which are being recognized by the receptor binding proteins to dock on the host cell surface. the cd receptor binds to the rbd of hiv (gp ) [ ] , whereas the rbd of influenza hemagglutinin (ha ) binds to the cell surface sialic acid to anchor on the host cell [ ] . the spike protein (s ) of coronaviruses utilizes host angiotensin converting enzyme (ace) to find the host cell for infection [ ] . any small alteration in the rbd leads to inefficient binding resulting in reduced infectivity of the virus [ ] . interestingly, binding of virus glycoprotein with cell surface proteins other than the specific receptor does not promote entry of the virus to the cell [ ] . the interaction between rbd and the cell surface receptor induces dramatic conformational changes in the fusion protein leading to exposure of the fusion domain [ ] . [ ] . mutations in this stretch of amino acids have been shown to block fusion mediated viral infection for many viruses [ ] [ ] [ ] . interestingly, fusion peptide itself is capable to induce fusion between lipid vesicles, and several putative mechanisms have been proposed on how fusion peptide promotes membrane fusion [ , [ ] [ ] [ ] [ ] . the α-helical trimeric heptad region plays a crucial role in inducing fusion by the formation of six helix bundle. the six helix bundle formation, a trademark of class i viral fusion protein, brings two apposing membranes close to each other [ ] . the heptad region is further divided into two regions, portions close to the n-terminal (or near the fusion peptide) and the c-terminal (or near transmembrane domain) are termed as fusion peptide proximal region (fppr) and membrane-proximal external region (mper), respectively [ ] . the interaction between fppr and mper promotes the formation of six helix bundle. the transmembrane domain is a stretch of - hydrophobic amino acids that remains anchored to the viral envelope. it is hypothesized that the fusion peptide (partitioned in the host membrane) interacts with the transmembrane domain (anchored in the viral envelope) to facilitate pore formation [ ] . residue influenza virus ha peptide into suv at ph . [ ] . in addition, it had been proposed that binding of - fps of ha would yield required free energy change, - kcal/mol, for the stabilization of curved membrane lipid membrane which is initially essential for fusion process [ ] . insertion of fps into lipid bilayer is known to support stable structures with propensity for stable oligomerizations [ ] . the fps of influenza virus ha peptide, hiv- gp belonging to the type i fusion protein have been serving as an archetypal example for mechanistic studies with model membranes. both ha and hiv- gp fps assume monomeric -helical structure, determined by solution nmr methods, in detergent micelle (figure ) [ , , ] . a large part (residues ile -ala ) of helical structure of the fp of hiv- gp in sds micelle was deduced to be deeply inserted into the micelle core akin to the orientation of transmembrane helices [ ] . initial solution nmr and epr studies determined an inverted "v" shaped or boomerang like helical structure of the -residue long ha fp in dpc micelle [ ] . the non-polar face of the amphipathic helical structure probably be embedded into the monolayer of the lipid bilayer. interestingly, a tightly packed helical hairpin structure of ha fp was determined in dpc micelle by solution nmr. this -residue construct of ha fp contains additional three conserved residues trp -tyr -gly at the c-terminus [ ] . the helical hairpin structure of ha fp might undergo a structural change to a continuous long helix during fusion pore formation. it is conceivable that virus fusion would require higher order oligomerization or association of the fusion proteins. these oligomeric states are likely to confine fusion- solid state nmr methods showed transition of -helical to -sheet conformations of fps of hiv- gp and ha in membrane bilayers [ , ] . structures of fps could significantly be influenced depending on membrane mimetic environments. as stated above, the fp of hiv- gp assumed helical structure whereas aggregated -sheet conformations were deduced in cholesterol containing lipid bilayers. these observations appear to indicate that multiple conformations of fps may be playing important roles in membrane fusion. although, determination of atomic resolution structures is challenging in lipid bilayers, therefore, structural works utilizing systems of lipid nanodisks or bicelles could be employed for better understating on the effect of membranes [ ] [ ] [ ] [ ] . it is worthy to mentioned that host protein is known to be important to aid in membrane coronaviruses (covs) are zoonotic pathogens subdivided, based on genomes and serology, into four different categories alpha, beta, gamma and delta [ , ] . covs are enveloped viruses with a positive sense single stranded rna as genetic element [ ] . covs the trimeric s or spike glycoprotein at the envelop of the covs appears to be determining in host specificity [ , ] . the s protein of covs including sars-cov- and mers helps the virus to bind to the receptor of the host cells and host-virus fusion. the s protein can be divided into two domains s and s which can be proteolytically cleaved in some covs [ , ] . the s domain of s is largely involve in receptor binding whereas the s domain is responsible for host-virus membrane fusion [ ] [ ] [ ] [ ] [ ] . the full-length s of sars-cov- is amino acid long with several functional domains and potential multiple proteolytic cleavage sites at the boundary of s and s , and also s ' site (figure ) . cryo-em structures of s proteins from mouse coronavirus (mhv), human coronavirus khu , sars-cov- and mers-cov are reported in the prefusion state [ ] [ ] [ ] . these structures revealed trimeric architecture of the s protein with extensive packing j o u r n a l p r e -p r o o f between s and s domains. further, the fusogenic components of the s domain including hr , fusion peptides are found to be buried within the core of the trimeric structure, whereas, the receptor binding domain of s can be amenable for interactions with the host cell. the s protein is expected to undergo a large conformational change from the latent prefusion state to fusion active state upon receptor binding and priming by host proteases. the x-ray structures of the s domain of s protein largely on hr , hr and hr /hr complex revealed canonical trimeric coiled-coil helical structures akin to type i fusion system [ , ] . the hr /hr complex forms a bundle of trimeric six helix whereby three short helices of hr can be found to be tightly packed along the long three helices of hr . the quaternary association of hr and hr would bring the fusion peptides and viral tm helix at proximity, presumably facilitate membrane fusion process (figure ). although membrane fusion of sars-cov- belongs to the type i fusion system, the large s protein of sars-cov- encodes number of regions in the s domain with membrane binding and/or membrane fusion activity [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the existence of multiple potential fps in the s domain of s protein in sars-cov- is unique in comparison to type i fusion proteins of hiv and influenza viruses. notably, the single fp, located at the n-terminus, has been known to be responsible for membrane fusion of hiv- gp and ha influenza viruses [ ] [ ] [ ] . intuitively, membrane fusion mechanism of sras-cov- and other covs may be complex involving several distinct stages. the upstream part of the hr (residues - ) of s domain of s protein of sars-cov- appears to be involved in membrane fusion (figure ) . several research groups have identified membrane binding and fusogenic peptides or potential fps from the upstream region (residues - ) of hr ( table ) . (table ) , also termed as internal fusion peptide or ifp , demonstrating high binding affinity and leakage from model membranes of various compositions [ ] . the ifp appeared to be binding with negatively charged phospholipids with higher affinity compared to zwitterionic lipids [ ] . it may be noteworthy that the ifp is located at the c-terminus of a proteolytic cleavage at residues / at s ' site. this would suggest that after proteolytic priming of s protein at s /s and s ' sites, the ifp would remain a part of the fusion protein whereas fp, residues - , will not be covalently bonded to the rest of the s domain (figure ) . in a later study, whittaker and co-workers has defined a yet another segment of residues - in proximity to the s 'cleavage site as a fusion peptide or ifp [ ] . mutagenesis studies on the full-length s protein identified residues l , l and f that are critically involved in fusion. biochemical analyses with synthetic peptide of ifp demonstrated ability of this peptide in membrane fusion. in addition to these fusogenic peptides, a c-terminal segment adjacent to tm or ptm (residues - ) is known to confer strong membrane partitioning (figure ) . further, ~ amino acids long polypeptide between hr and hr also appeared to be containing peptide motifs with membrane interacting properties and virus-cell fusion inhibiting activity [ ] . gaalqipfamqmayrf (ifp ) alisgtatagwtfgagaalqipfamqmayr j o u r n a l p r e -p r o o f we have determined -d structures of fps and ptm of sars-cov- in solution of dpc detergent micelle by nmr methods [ , ] . the atomic resolution structure of the fp ( myktptlkyfggfnfsqil ) revealed a bend helical structure presumably resulting from two gly residues g and g at the center of the sequence (figure ) . lgkyeqyikwpwyvwlgf ) of s protein has also been determined in dpc micelle. the ptm is a conserved sequence among sars-covs, and also observed in other viruses like hiv- and eob. it has been postulated that the ptm segment could be involved in viral membrane fusion process [ ] . micelle-bound ptm assumes a fold of helix (residues k -y )-loop (i -k )-helix (w -f ) structure whereby the two helices were found to be independently oriented (figure ) . observations of multiple adjacent fusogenic peptides in the s domain prompted us to examine structure and membrane localization of a -residue long, residues r -e , or lfp (long fusion peptide) in detergent micelle solution [ ] . the primary structure of lfp contains fp (residues - ) and ifp (residues - ) with additional residues at the n and c-termini. lfp was over-expressed in e. coli as a fusion protein containing an -resiude long prodomain of human furin followed by an asp-pro sequence for formic acid digestion [ , ] . the construct also contains an additional six residue his-tag at the n-terminus for affinity purification resulting a fusion protein his -prodomain-d-p-lfp. atomic resolution structure, n relaxation and micelle localization were investigated by heteronuclear nmr methods in dpc micelle solution (figures , ) . -d structure of lfp demonstrated existence of discretely folded helices connected by several loops (figure a ). the cterminal region of lpf defines a long -helix including residues t -y with a kink at residue d . it may be noted that ifp , residues - , is included within the c-terminal j o u r n a l p r e -p r o o f helix. the fp segment (residues - ) in lfp assumed a helix-loop-helix structure, although, the isolated fp delineated a bend helical structure (figure ) . interestingly, the nterminal residues r -q of lfp assumed an amphipathic helical conformation ( figure a ). the helical structures of lpf were found to be motionally rigid experiencing fast motion in ns-ps time scale. the fusogenic property of the membrane generally depends on the its composition and curvature [ ] . protein undergoes oligomerization and may be important for infection process [ , ] . pandemic due to coronavirus was predicted after sars and mers outbreaks. however, no vaccines or effective drugs were developed for the mitigation of the threats. a new strain of coronavirus called sars-cov- or covid- is demonstrating a rapid spread j o u r n a l p r e -p r o o f all over the world which was initially found in wuhan, china. sars-cov- pandemic, so far, has caused nearly , deaths globally with an infection of over million people. the high level of infectivity of sars-cov- compared to sars-cov- could be related to an efficient cell entry of the virus. although, the molecular mechanism of the cell entry process remains unclear, binding of the virus to the host cell receptor is an important step in successful infection. studies have shown that the isolated rbd of sars-cov- binds to ace with tighter affinity compared to sars-cov- , indicating potential for higher infectivity [ , [ ] [ ] [ ] [ ] . however, paradoxically, experiments with the full-length s protein have evidenced either similar binding affinity or even lower affinity of rbd to human ace in comparison to sars-cov- [ ] [ ] [ ] . the cryo-em structure of sars-cov- showed that the rbd domain could be hidden within the s protein structure for immune evasion [ ] . therefore, other factors might be responsible for high infectivity of sars-cov- . protease cleavage of s protein has been postulated to be one of the factors responsible for high infectivity of sars-cov- [ ] [ ] [ ] . an efficient membrane fusion mechanism between sars-cov- and host cell could also be responsible for the high level of infection. sequence comparison of s proteins domain between sars-cov- and sars-cov- indicated high level of conservation both for the s and s domains [ ] . nevertheless, variations can be observed for the fusogenic regions of the s domain between two viruses. although, effect of these variations for an efficient membrane fusion remains to be examined. emerging and re-emerging viral infections are continuous threats to human kind. mechanics of membrane fusion point-like protrusion as a prestalk intermediate in membrane fusion pathway on the theory of membrane fusion. the stalk mechanism stalk mechanism of vesicle fusion. intermixing of aqueous contents lipid intermediates in membrane fusion: formation, structure, and decay of hemifusion diaphragm a quantitative model for membrane fusion based on low-energy intermediates energetics of intermediates in membrane fusion: comparison of stalk and inverted micellar intermediate mechanisms the many mechanisms of viral membrane fusion proteins receptor binding and membrane fusion in virus entry: the influenza hemagglutinin epidemiology, genetic recombination, and pathogenesis of coronaviruses role of lipids in virus replication mechanisms of coronavirus cell entry mediated by the viral spike protein structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme signal peptide fragments of preprolactin and hiv- p-gp interact with calmodulin recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission atomic structure of the ectodomain from hiv- gp structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid genetic analysis of the sars-coronavirus spike glycoprotein functional domains involved in cell-surface expression and cell-to-cell fusion the sweet spot: defining virus-sialic acid interactions cellular entry of the sars coronavirus receptor-induced conformational changes in the su subunit of the avian sarcoma/leukosis virus a envelope protein: implications for fusion activation studies on the mechanism of membrane fusion: site-specific mutagenesis of the hemagglutinin of influenza virus wild-type and mutant hemagglutinin fusion peptides alter bilayer structure as well as kinetics and activation thermodynamics of stalk and pore formation differently: mechanistic implications a specific point mutant at position of the influenza hemagglutinin fusion peptide displays a hemifusion phenotype hiv- fusion peptide decreases bending energy and promotes curved fusion intermediates fusion peptide from influenza hemagglutinin increases membrane surface order: an electron-spin resonance study the gaussian curvature elastic energy of intermediates in membrane fusion energetics of vesicle fusion intermediates: comparison of calculations with observed effects of osmotic and curvature stresses crystal structure of hiv- gp including both fusion peptide and membrane proximal external regions gp transmembrane domain interacts with the fusion peptide: implication in lipid mixing and inhibition of virus-cell fusion fusion peptides and the mechanism of viral fusion viral fusion proteins: multiple regions contribute to membrane fusion physiological and molecular triggers for sars-cov membrane fusion and entry into host cells hypothesis: spring-loaded boomerang mechanism of influenza hemagglutinin-mediated membrane fusion the three lives of viral fusion peptides hiv entry and envelope glycoprotein-mediated fusion the polar region consecutive to the hiv fusion peptide participates in membrane fusion a host-guest system to study structure-function relationships of membrane fusion peptides early and late hiv- membrane fusion events are impaired by sphinganinelipidated peptides that target the fusion site sendai virus internal fusion peptide: structural and functional characterization and a plausible mode of viral entry inhibition are fusion pepties good model to study viral cell fusion lipids in biological membrane fusion relationship between the infectivity of influenza virus and the ability of its fusion peptide to perturb bilayers membrane structure and fusiontriggering conformational change of the fusion domain from influenza hemagglutinin the complete influenza hemagglutinin fusion domain adopts a tight helical hairpin arrangement at the lipid:water interface structure and function of the complete internal fusion loop from ebolavirus glycoprotein the amino-terminal region of the fusion peptide of influenza virus hemagglutinin ha inserts into sodium dodecyl sulfate micelle with residues - at the aqueous boundary at acidic ph. oligomerization and the conformational flexibility nmr structure, localization, and vesicle fusion of chikungunya virus fusion peptide interaction of the dengue virus fusion peptide with membranes assessed by nmr: the essential role of the envelope protein trp for membrane fusion solid-state nmr spectroscopy of the hiv gp membrane fusion protein supports intermolecular antiparallel beta sheet fusion peptide structure in the final six-helix bundle state fully hydrophobic hiv gp adopts a hemifusion-like conformation in phospholipid bilayers structurerelated roles for the conservation of the hiv- fusion peptide sequence revealed by nuclear magnetic resonance conformation and lipid interaction of the fusion peptide of the paramyxovirus piv in anionic and negative-curvature membranes from solid-state nmr a strong correlation between fusogenicity and membrane insertion depth of the hiv fusion peptide structure and dynamics of micelle-associated human immunodeficiency virus gp fusion domain ph-dependent self-association of influenza hemagglutinin fusion peptides in lipid bilayers permeabilization and fusion of uncharged lipid vesicles induced by the hiv- fusion peptide adopting an extended conformation: dose and sequence effects solid-state nmr spectroscopy of human immunodeficiency virus fusion peptides associated with host-cell-like membranes: d correlation spectra and distance measurements support a fully extended conformation and models for specific antiparallel strand registries nanodiscs in membrane biochemistry and biophysics stabilization and structural analysis of a membrane-associated hiapp aggregation intermediate a. ramamoorthy, ph resistant monodispersed polymer-lipid nanodiscs spontaneous lipid nanodisc formation by amphiphilic polymethacrylate copolymers a seminal finding for understanding hiv transmission human prostatic acid phosphatase: structure, function and regulation bacterial curli protein promotes the conversion of pap - into the amyloid sevi: cross-seeding of dissimilar amyloid sequences semen-derived amyloidogenic peptides-key players of hiv infection helical conformation of the sevi precursor peptide pap - , a dramatic enhancer of hiv infectivity, promotes lipid aggregation and fusion coronavirus diversity, phylogeny and interspecies jumping epidemiology and clinical presentations of the four human coronaviruses e, hku , nl , and oc detected over years using a novel multiplex real-time pcr method planning for epidemics -the lessons of sars isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus: transmission and phylogenetic evolution broad reception for coronavirus sars-cov and emergent coronaviruses: viral determinants of interspecies transmission coronavirus spike proteins in viral entry and pathogenesis different host cell proteases activate the sars-coronavirus spike-protein for cell-cell and virus-cell fusion evidence that tmprss activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response structure of sars coronavirus spike receptorbinding domain complexed with receptor severe acute respiratory syndrome coronavirus (sars-cov) infection inhibition using spike protein heptad repeat-derived peptides crystal structure of severe acute respiratory syndrome coronavirus spike protein fusion core structure of a proteolytically resistant core from the severe acute respiratory syndrome coronavirus s fusion protein bat to human: spike features determining host jump of coronaviruses sars-cov, mers-cov and beynod pre-fusion structure of a human coronavirus spike protein cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains identification and characterization of the putative fusion peptide of the severe acute respiratory syndromeassociated coronavirus spike protein identification of the membraneactive regions of the severe acute respiratory syndrome coronavirus spike membrane glycoprotein using a / -mer peptide scan: implications for the viral fusion mechanism glycoprotein internal membrane-active peptide. biophysical characterization and membrane interaction the aromatic domain of the coronavirus class i viral fusion protein induces membrane permeabilization: putative role during viral entry interaction of a peptide from the pre-transmembrane domain of the severe acute respiratory syndrome coronavirus spike protein with phospholipid membranes structural and dynamic characterization of the interaction of the putative fusion peptide of the s sars-cov virus protein with lipid membranes membrane insertion of the three main membranotropic sequences from sars-cov s glycoprotein characterization of a highly conserved domain within the severe acute respiratory syndrome coronavirus spike protein s domain with characteristics of a viral fusion peptide inhibition of severe acute respiratory syndrome-associated coronavirus infectivity by peptides analogous to the viral spike protein identification of the fusion peptide-containing region in betacoronavirus spike glycoproteins nmr structures and localization of the potential fusion peptides and the pre-transmembrane region of sars-cov: implications in membrane fusion nmr structure and localization of a large fragment of the sars-cov fusion protein: implications in viral cell fusion ph-induced conformational transitions of a molten-globule-like state of the inhibitory prodomain of furin: implications for zymogen activation designed di-heme binding helical transmembrane protein chembiochem membrane composition modulates fusion by altering membrane properties and fusion peptide structure structural and dynamic characterization of the interaction of the putative fusion peptide of the s sars-cov virus protein with lipid membranes sars-cov fusion peptides induce membrane surface ordering and curvature membrane cholesterol modulates oligomeric status and peptide-membrane interaction of severe acute respiratory syndrome coronavirus fusion peptide organization of higher-order oligomers of the serotonin( )(a) receptor explored utilizing homo-fret in live cells excitements and challenges in gpcr oligomerization: molecular insight from fret cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding structural basis of receptor recognition by sars-cov- structural insights of a self-assembling -residue peptide from the c-terminal tail of the sars corona virus e-protein in dpc and sds micelles: a combined high and low resolution spectroscopic study self-assembly of a nine-residue amyloid-forming peptide fragment of sars corona virus e-protein: mechanism of self aggregation and amyloid-inhibition of hiapp structure, function, and antigenicity of the sars-cov- spike glycoprotein cryo-em structure of the -ncov spike in the prefusion conformation sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor characterization of spike glycoprotein of sars-cov- on virus entry and its immune cross-reactivity with sars-cov cell entry mechanisms of sars-cov- structural, glycosylation and antigenic variation between novel coronavirus ( -ncov) and sars coronavirus (sars-cov) mechanistic insights of host cell fusion of sars-cov- and sars-cov- from atomic resolution structure and membrane dynamics hirak chakraborty , and surajit bhattacharjya odisha , india centre of excellence in natural products and therapeutics key: cord- -h fevqcw authors: compans, richard w.; kemp, maurice c. title: membrane glycoproteins of enveloped viruses date: - - journal: curr top membr transp doi: . /s - ( ) - sha: doc_id: cord_uid: h fevqcw this chapter focuses on the recent information of the glycoprotein components of enveloped viruses and points out specific findings on viral envelopes. although enveloped viruses of different major groups vary in size and shape, as well as in the molecular weight of their structural polypeptides, there are general similarities in the types of polypeptide components present in virions. the types of structural components found in viral membranes are summarized briefly in the chapter. all the enveloped viruses studied to date possess one or more glycoprotein species and lipid as a major structural component. the presence of carbohydrate covalently linked to proteins is demonstrated by the incorporation of a radioactive precursor, such as glucosamine or fucose, into viral polypeptides, which is resolved by sodium dodecyl sulfate (sds) polyacrylamide gel electrophoresis. enveloped viruses share many common features in the organization of their structural components, as indicated by several approaches, including electron microscopy, surface-labeling, and proteolytic digestion experiments, and the isolation of subviral components. the chapter summarizes the detailed structure of the glycoproteins of four virus groups: ( ) influenza virus glycoproteins, ( ) rhabdovirus g protein, ( ) togavirus glycoprotein, and ( ) paramyxovirus glycoproteins the information obtained includes the size and shape of viral glycoproteins, the number of polypeptide chains in the complete glycoprotein structure, and compositional data on the polypeptide and oligosaccharide portions of the molecules. search. the most important advantage is structural simplicity, in that lipid-containing viruses possess a small number of virus-coded polypeptides and glycoproteins as membrane components. sufficient quantities of many enveloped viruses can be purified for detailed biochemical analysis, including primary structure studies of viral membrane proteins like those now underway. purification procedures are rapid and simple, and a fairly homogeneous population of particles can be obtained free of contaminating cellular membranes. for the simpler lipid-containing viruses, the possibility therefore exists for complete structural determination of a biological membrane, and such information should contribute greatly to an understanding of the molecular details of membrane structure and interactions among membrane components in general. other advantages of enveloped viruses in studies of membrane structure and biogenesis include the ease of biosynthetic labeling of viruses grown in cell culture with specific radioactive precursors and the availability of mutants in defined gene products, some of which are proving to be useful in the analysis of viral membrane assembly. since envelope components are integral parts of cellular membranes during the assembly of virus particles (compans et al., ; compans and choppin, ) , studies of the synthesis and mechanism of insertion of these components into membranes should provide useful information pertaining to the biogenesis of cellular membranes. many viral systems offer the additional advantage that host cell biosynthesis is inhibited during infection, so that it is possible to analyze the synthesis of membrane components in a cell in which only a small number of virus-coded membrane components are being produced and to follow the intracellular migration of specific membrane glycoproteins by radiolabeling procedures. finally, the ability to prepare viruses with specific modifications in either lipid or protein composition arises from the fact that viral envelope lipids are derived from the plasma membrane of the host cell, whereas viral proteins are entirely virus-coded. thus by growing different virus types in the same cell it is possible to prepare a membrane in which the protein composition can be varied while the lipid composition remains constant. similarly, by growing the same virus in a series of different cells, viral membranes are obtained which possess the same set of proteins but vary in lipid composition. these approaches have been useful in studies of lipid-protein interactions in viral membranes (landsberger et al., ) . several recent reviews are available concerning both the experimental and theoretical aspects of viral envelope structure and assem-bly (lenard and compans, ; blough and tiffany, ; wagner, ; choppin and compans, ; compans and choppin, ; klenk, ) . in this chapter, we emphasize recent information on the glycoprotein components of enveloped viruses and endeavor to point out specific findings on viral envelopes which we believe to be of broad significance. in table i are listed the established major groups, or families, of lipid-containing viruses of vertebrates, and examples of the best studied members of certain groups. for several families, only limited information is available on viral envelope structure, whereas other groups have been the object of intensive study. we concentrate our discussion on the glycoproteins of the most intensively studied virus groups, which are the first four groups of rna viruses listed in table i : myxoviruses, paramyxoviruses, rhabdoviruses, and togaviruses. we also discuss selected findings for other virus groups where these have revealed unusual or important aspects of viral membrane structure or assembly. although enveloped viruses of different major groups vary in size and shape, as well as in the mws of their structural polypeptides, there are general similarities in the types of polypeptide components present in virions. the types of structural components found in viral membranes are summarized briefly in the following discussion, and further detailed information is available in the recent reviews cited above. all the enveloped viruses studied to date possess one or more glycoprotein species. the presence of carbohydrate covalently linked to proteins has usually been demonstrated by the incorporation of a radioactive precursor such as glucosamine or fucose into viral polypeptides, which is resolved by sodium dodecyl sulfate (sds) polyacrylamide gel electrophoresis. the specificity of labeling by these precursors is shown by the lack of their incorporation into carbohydrate-free polypeptides of the virion (straws et al., ) . the number of distinct glycoprotein species present in the virion varies for various virus groups. as shown in table , there is also marked variation in the glycoprotein content of virions when expressed as a percent of (gard et al., ) , whereas the data given are for pichinde virions which contain two glycoproteins. the values represent our calculations from the autoradiograph scan data presented in the reference. the values given represent our calculations from the data presented in the reference. total viral protein. as discussed in section v, these data suggest that glycoproteins of the various virus groups may play a greater or lesser role in assembly and maintenance of the viral envelope structure. however, glycoproteins are essential components for viral infectivity even where they constitute only a minor fraction of the mass of the virion, since they are necessary for the initial events in the viral replication cycle. the fine structure, biological functions, and orgapization of glycoproteins in the viral envelope are discussed in detail in section iv. the carbohydrates of viral glycoproteins are specified in large part by host cell transferases, whereas the amino acid sequences are coded b y the viral genome. glycoproteins of the same virus may exhibit host cell-dependent differences in electrophoretic mobility, which are due to differences in the carbohydrate components (haslam et al., ; compans et al., ; schulze, ) . thus viral glycoproteins may be useful probes to detect differences in glycosylation among various cell types. an important exception to the general finding of host cell-specified carbohydrates is found in myxoviruses and paramyxoviruses. glycoproteins of these viruses lack sialic acid (klenk et al., a,b) , which is believed to be a result of the virus-specified neuraminidase incorporated as a structural component of these virions. all enveloped viruses contain lipid as a major structural component, and available evidence indicates that all the lipid is contained in a single bilayer structure which forms the matrix of the limiting membrane of the virion. investigations using biophysical methods including xray diffraction (harrison et al., ) and electron spin resonance (landsberger et al., (landsberger et al., , landsberger and compans, ) indicate that viral lipids are arranged in a bilayer. with electron microscopy, a well-defined unit membrane is observed at the location of the bilayer. the virus derives its lipids from the host cell membrane where virus maturation occurs, which is the plasma membrane in most instances. therefore a given virus can exhibit marked variation in lipid composition when grown in different cell types, which reflects the composition of the host cell plasma membrane in each instance (klenk and choppin, , ; quigley et al., ) . apart from minor differences in carbohydrates of glycoproteins, virion proteins are indistinguishable when the virus is propagated in a variety of cells; therefore there appears to be little or no determining influence of viral proteins on the composition of the lipid bilayer. since the viral envelope is continuous with the host cell membrane during morphogenesis, and membrane lipids are characterized by a high rate of lateral diffusion in the plane of the membrane, the similarity in lipid composition between viral envelopes and plasma membranes is not surprising. the distribution of various lipid classes on the internal and external sides of the bilayer has recently been investigated in influenza virions using phospholipase digestion and phospholipid exchange proteins (tsai and lenard, ; rothman et al., ) . the results indicate that lipids are distributed asymmetrically, with the majority of the phosphatidylinositol and about half of the phosphatidylcholine in the external half of the bilayer and most of the phosphatidylethanolamine and phosphatidylserine in the inner half. these distributions are thought to reflect a similar distribution in the host cell plasma membrane. since less than half of the total phospholipids were accessible b y either approach, it was suggested that glycolipids constitute a significant part of the outer monolayer of the viral membrane. the presence of glycolipids in various enveloped viruses has been demonstrated by chemical analyses , and the types of glycolipids present also reflect those of the host cell. myxoand paramyxoviruses lack neuraminic acid residues in their glycolipids, which again is a likely result of their neuraminidase activity klenk et al., b) . the reactivity of viral glycolipids toward specific lectins demonstrates that their carbohydrates are exposed on the external surface of the bilayer (klenk et al., a) . all polypeptides located internal to the viral lipid bilayer are devoid of carbohydrate, and they may be subdivided into several types according to their structural location and functions. all enveloped viruses possess at least one polypeptide species closely associated with the nucleic acid to form a nucleocapsid or core structure, which is termed the nucleocapsid protein. in addition, viruses with helical nucleocapsids possess another major internal protein, termed the membrane protein, that appears to be associated with the internal surface of the lipid bilayer. most nucleocapsid proteins are not directly involved in interaction with the lipid bilayer, although the nucleocapsids of togaviruses are roughly spherical structures that appear to be closely apposed to the inner surface of the bilayer. the internal membrane (m) proteins of myxo-, paramyxo-, and rhabdoviruses occupy a similar location in the viral envelope. it appears that these proteins are primarily responsible for conferring considerable rigidity on the viral envelope, as compared to that of host cell membranes of similar lipid composition (stoeffel and bister, ; landsberger and compans, ; lenard et al., ) . more complex viruses possess additional internal polypeptides as major components, but their precise locations in the virion have not been established. in addition, minor internal protein components are frequently observed in virions which possess enzymic functions, such as transcriptase activity. such minor polypeptides are not thought to play a direct role in the structure or assembly of the viral envelope. it is well established that carbohydrate chains are covalently linked to glycoproteins and glycolipids of the viral envelope, and both of these components were described above. however, it is uncertain whether or not other complex carbohydrates serve a function in viral envelope structure. the presence of sulfated mucopolysaccharides in highly purified virus preparations of several major groups was recently observed by radiolabeling virions with sulfate pinter and compans, ) . these sulfated components are derived from the host cell, since they can be labeled selectively by the growth of cells in s ,-containing medium prior to virus infection. in the case of influenza virus, it is possible that these components represent a host cell antigen described to be associated with purified virus particles (knight, ) . labeled sulfated mucopolysaccharides can be removed from virus preparations by digestion with hyaluronidase or trypsin without any effect on viral infectivity, suggesting that these components are not essential . it is likely that they associate with the external surface of the virion during the process of maturation. enveloped viruses share many common features in the organization of their structural components, as indicated by several approaches which include electron microscopy, surface-labeling and proteolytic digestion experiments, and isolation of subviral components. much of the evidence pertaining to the arrangement of membrane components has been discussed in other reviews cited above and is only briefly summarized in this section. the organization of the viral membrane corresponds in many respects to the fluid mosaic membrane model . viral glycoproteins appear to be integral membrane proteins which are exposed on the external surface of a lipid bilayer. the external location of the glycoproteins is indicated by their sensitivity to proteolytic digestion and reactivity toward surface-labeling reagents (see review in lenard and compans, ) . partial penetration of the glycoproteins into the bilayer is suggested by the fact that segments of the glycoproteins of several virus groups are found to be resistant to protease (gahmberg et al., ; mudd, , schloemer and wagner, b; lenard et al., ) . in contrast to the glycoproteins, the carbohydrate-free polypeptides of enveloped viruses are located internally to the lipid bilayer, as indicated by their resistance to protease treatment and lack of reactivity toward surface-labeling reagents. these internal proteins are also not reactive toward specific antibodies without disruption of the viral envelope. the best examples of nonglycosylated proteins which appear to be intimately associated with the internal surface of the envelope are the m proteins of myxo-, paramyxo-, and rhabdoviruses. however, whether these polypeptides associate with the bilayer as integral membrane proteins, or as peripheral membrane proteins such as spectrin in erythrocyte membranes, has not been clearly established. in fig. the schematic cross section of an influenza virion shows the general arrangement of its structural components. the extent of penetration of the glycoproteins into the bilayer is not indicated but, fig. . schematic cross section of an influenza virion, depicting the arrangement of the major structural polypeptides. the rodlike ha spikes and the more complex na spikes are composed of ha and na polypeptides, respectively. these glycoproteins are exposed at the external surface of the lipid bilayer, and segments may penetrate the bilayer (not shown). the internal membrane-associated m protein appears to form a closely packed layer beneath the bilayer. the helical nucleocapsids are found as multiple discrete segments, each containing an rna molecule coated with a major nucleocapsid protein, (np). the nucleocapsids probably interact directly with the m protein during virion assembly. (from compans and choppin, .) as discussed in section iv, it is likely that a hydrophobic segment penetrates into the bilayer and may even traverse it. a closely packed layer of m protein is depicted on the internal surface of the bilayer in accordance with available information, and the helical nucleocapsids are depicted as twisted hairpinlike structures . it is likely that the nucleocapsids interact with the m proteins during assembly and in so doing participate in determining virion size and shape. little is known about these interactions, and they are not indicated in fig. . in this section we summarize the available information on the detailed structure of the glycoproteins of four virus groups. the infonnation obtained includes the size and shape of viral glycoproteins, the number of polypeptide chains in the complete glycoprotein structure, and compositional data on the polypeptide and oligosaccharide portions of the molecules. detailed structural information on glycoproteins has been obtained for only a few virus types and is obviously incomplete even for the best studied viral systems. influenza viruses possess two glycoproteins with distinct biochemical and morphological properties: hemaggluti-nin (ha) and neuraminidase (na). the ha of influenza virus iii one of the best studied viral glycoproteins. electron microscope studies of the isolated spike structure have shown that it is a rod-shaped triangular prism approximately nm long and nm wide (laver and valentine, ) . the ha spike projects radially from the virion envelope, and it is probably a trimer having a mw of about , daltons (schulze, ; wiley et al., ) . ha glycoproteins can exist in one of two alternative forms: a single polypeptide with a mw of , daltons designated ha, or two polypeptides with mws of approximately , and , daltons designated ha and ha , respectively. ha, and ha, are proteolytic cleavage products of ha, which remain cross-linked by disulfide bonds (lazarowitz et al., ; laver, ) . the extent of cleavage of ha to ha, and ha may vary from none to complete cleavage of all ha polypeptides (lazarowitz et al., ) . the extent of cleavage de-pends upon several factors including the host cell, the virus strain, and the presence or absence of proteases in the culture medium. the cleavage of ha may not be a requirement for infectivity of all virus strains, and spike morphology is not altered detectably by cleavage of ha to ha, and ha,, but recent studies have shown that cleavage enhances the infectivity of some influenza virus strains (klenk et al., ; lazarowitz and choppin, ) . the tryptic peptide maps of ha, and ha, are distinct, and the only notable difference in the amino acid composition of the two polypeptides is the proline content (laver, ) . the proline content of ha, is at least six times higher than that of ha,. ha, is tightly folded on itself, probably reflecting the high proline content. a portion of the ha spike is released by protease treatment as a soluble protein which can be crystallized (brand and skehel, ) . functional ha spikes have been isolated from several influenza strains by treating virions with ionic or nonionic detergents. after removal of the detergent, the spikes aggregate, forming rosettelike structures, suggesting that the base of the ha spike is hydrophobic in nature (laver and valentine, ) . available evidence indicates that the ha spike is amphipathic and that a portion of it is buried within the lipid bilayer of the viral envelope. this is indicated b y the finding that protease-treated virions retained ha, or portions thereof while having no identifiable spikes (compans et nl., ; lenard et al., ) . the ha, glycoprotein therefore contains the hydrophobic end of the spike. this glycoprotein aggregates even in the presence of guanidine hydrochloride (laver, ) . the composition of a -residue peptide associated with the lipid bilayer, which is removed from the carboxyl terminus of ha by bromelain, has been estimated, and of the amino acids are hydrophobic in nature, whereas may be charged (skehel and waterfield, ) . the carboxyl terminus of this peptide is thought to be buried within the lipid bilayer. the aminoterminal end of ha, seems to extend radially from the virion surface, and a sequence of residues from the amino-terminal end of ha, is highly conserved in both type-a and type-b influenza virions (skehel and waterfield, ) . the hydrophobicity of ha, suggests that it is similar to other membrane-associated glycoprotein segments (segrest et al., ; nakashima et al., ) , but freeze-fracture studies of the influenza virion indicate that extensive portions of ha and na do not penetrate through the lipid bilayer (bachi et al., ) . circular dichroism studies of ha spikes isolated intact and those cleaved from the virion surface may be useful in further defining the secondary structure of the hydrophobic regions. until recently, little information was available concerning the carbohydrate portion of the ha glycoprotein. about % of the carbohydrate attached to viral proteins was shown to be linked to ha; the gly-cosy moieties were shown to be comprised of glucosamine, mannose, galactose and fucose at a molar ratio of : : : , respectively, and the total carbohydrate of ha, determined by chemical means, was estimated to be approximately , daltons (laver, ) . it was also found that ha, of influenza a virions contains fucose, whereas ha, of influenza b (gl ) does not . recently the glycopeptides obtained by pronase digestion of influenza a viruses have been characterized (schwarz et al., ; nakamura and compans, b) . both groups showed that ha possesses type i (oligosaccharide side-chain comprised of glucosamine, mannose, galactose, fucose and sialic acid) and type i (oligosaccharides comprised of glucosamine and mannose) glycopeptides, but the distribution of each type on ha, and ha, was shown to depend upon the virus strain. schwarz et al. ( ) showed that ha, of two avian influenza viruses possessed only type i glycopeptides, whereas both type i and i glycopeptides were shown to be present on ha,. however, nakamura and compans ( ) using the wsn strain of influenza showed that ha, possessed both type i and i glycopeptides whereas ha, contained only type i glycopeptides. since it is likely that ha, occupies a similar structural location in wsn and avian hemagglutinin proteins, these results suggest that specific amino acid sequences determine whether a type i or type i oligosaccharide is added at a particular site on the glycoprotein. the host cell type may also determine the type of oligosaccharide chains added to a given viral glycoprotein. thus, although ha, of wsn strain influenza virions grown in mdbk cells contained only type i glycopeptides, both type i and i glycopeptides were found in ha, when grown in cef cells (nakamura and compans, ~) . schwarz et al. ( ) showed that the type i ha glycopeptides of fowl plague and n influenza strains grown in chicken embryo fibroblasts (cef) had a molecular weight of approximately , daltons, whereas type i glycopeptides from the same source has a molecular weight of approximately , daltons. the molecular weights of type i and i glycopeptides of the wsn strain of influenza virus were also found to have approximately this size (nakamura and compans, b) . ha glycopeptides of virus grown in mdbk cells were slightly larger in size than those of virus grown in cef cells. based upon the estimated carbohydrate content ( , daltons) of the ha glycoprotein obtained by laver ( ) and schwarz and klenk ( ) and the size estimates of the type i and i glycopeptides of influenza virus grown in mdbk cells, it was estimated that ha, contains a single type i glycopeptide whereas ha, possesses two type i and one or two type i oligosaccharide side-chains for the wsn strain (nakamura and com pans, b) . the na spike, as shown in fig. , also projects radially from the lipid bilayer of the influenza virion but is morphologically distinguishable from the ha spike (laver and valentine, ) . the na spike is elongated and has a square knoblike structure at one end. the oblong head is approximately x . nm, and it is attached to a fiber approximately nm long. the na spike has a mw of approximately , daltons and is comprised of four na polypeptides each having a mw of approximately , daltons; two na polypeptides appear to be linked by disulfide bonds to form dimers, which are thought in turn to aggregate by noncovalent bonds to form the tetrameric spike (bucher and kilbourne, ; lazdins et al., ) . the amino acid composition of the na has been determined, and it has a cysteine content significantly higher than that of the other viral polypeptides (laver and baker, ) . influenza na is in some ways morphologically similar to enzymes involved in sugar metabolism in the gut, as discussed by forstner and riordan in this volume. after trypsin treatment a tetramer of four coplaner subunits of x x nm was isolated (wrigley et al., ) . this structure appears to correspond to the knob, and it is enzymically active. the knob structure can no longer aggregate with itself or with ha molecules, indicating that the hydrophobic regions of the molecule have been removed by proteolysis. the mw of the portion of the na monomer which remains associated with the envelope after trypsin treatment was estimated to be daltons by lazdins et al. ( ) and , daltons by wrigley et al. ( ) . presumably, this portion functions to attach the na molecule to the virion and plays no role in the enzymic properties. furthermore, the portion of the spike which remains associated with the viral envelope is more highly glycosylated than the knob-shaped portion of the na spike with enzymic activity (lazdins et al., ) . na is present in influenza virions in smaller amounts than ha, in a ratio of about three to four ha polypeptides for each na polypeptide. little information is available on the carbohydrate components of the na. recently, both the ha and na of influenza virions were shown to be sulfated glycoproteins . the sulfate appears to be covalently linked to the oligosaccharide chains of viral gly-coproteins compans, , a) . glycoproteins of enveloped viruses of all the other major groups studied also are sulfated, whereas carbohydrate-free polypeptides are not kaplan and ben-porat, ). rhabdoviruses are covered with closely spaced glycoprotein spikes approximately nm in length as shown by electron microscope studies. rhabdoviruses possess only a single glycoprotein species termed the g protein, and for vesicular stomatitis virus (vsv) it has a mw of approximately , daltons (wagner, ) . carhvright et d . ( ) have postulated that only a single glycoprotein molecule constitutes the spike structure. the g proteins of rhabdoviruses are amphipathic, like the glycoproteins of influenza virions. after protease treatment of vsv a fragment of the g protein was demonstrated to be associated with the intact virion (mudd, ) . more recently, schloemer and wagner ( a) isolated a small nonglycosylated portion of the g protein from the envelope of protease-treated vsv virions. the fragment was found to have a mw of daltons, approximately equivalent to amino acids. this is similar to the estimated size of the portion of the ha protein of the influenza virion thought to be buried within the lipid bilayer. amino acid analysis of the g-protein fragment showed that it contained a preponderance of hydrophobic amino acids. as is the case for the membrane-associated portions of influenza ha, the hydrophobic fragment of the g protein is long enough to penedate the lipid bilayer. conclusive evidence for such penetration has not been obtained, but cross-linking experiments with glutaraldehyde suggest that interactions may occur between g proteins and internal m proteins (brown et d . , ) . the carbohydrates linked to the g protein of vsv have been studied in detail. the glycoprotein is - % carbohydrate by weight and contains mannose, galactose, n-acetylglucosamine, and neuraminic acid as the major sugar components, with lesser amounts of n-acetylgalactosamine and fucose (mcsharry and wagner, ; huang, . etchison and holland, a) . the size and composition of the carbohydrate moieties per vsv glycoprotein are variable, depending upon the cell type in which the virions are grown (burge and huang, ; etchison and holland, a) . likewise, the sequence of the carbohydrates within the glycosyl side-chains may exhibit cell depen-dence (moyer and summers, ) . the monosaccharide composition of the oligosaccharide side-chains is similar to that of influenza virus (etchison and holland, b) , with the exception that sialic acid is present on the termini. klenk et az. ( b) demonstrated the presence of sialic acid on the envelope of vs virions by treating the virus with colloidal iron hydroxide which stains sialic acid residues. the stain was shown to bind to the envelope of vsv, whereas it did not bind to influenza virions or paramyxoviruses as shown by electron microscopy. more recent studies have shown that the glycosyl moieties are not terminated by sialic acid when vsv is grown in mosquito cells, because these cells lack sialyl transferase (schloemer and wagner, b) . preliminary studies indicated that from - cyanogen bromide peptides of the vsv g-protein may be glycosylated (wagner, ) . however, etchison and holland ( a,b) have calculated that there are only glycopeptides of - daltons in each g protein molecule. more recent studies by etchison et al. ( ) have indicated that the vsv g-protein possesses two identical glycopeptides. the number average molecular weight of the glycopeptides was estimated to be by gel filtration analysis and based on composition of the amino acid and sugar residues. additional information concerning the structure of the glycopeptide was obtained by sequential chemical and enzymatic degradation. these results indicate that the glycopeptides are acidic type i glycopeptides with two or three mannose branches terminating in sialic acid. moyer and co-workers ( ) have obtained evidence that the glycosyl residues are covalently linked to an asparagine residue of the g protein, and the sequence of the monosaccharides that constitute the oligosaccharide side-chains has been determined (hunt and summers, b) . togavinises are small, spherical enveloped viruses - nm in diameter with a core structure that appears to be icosahedral. although there are many members of the togavirus group, the best studied are sindbis virus and semliki forest virus (sfv). sindbis virus possesses two glycoproteins designated el and e,; both have a mw of approximately , daltons, and they are not linked by disulfide bonds because they can be separated under nonreducing conditions and without alkylation (schlesingeret al., ) . sfv is similar to sindbis virus in that it also has glycoproteins of similar molecular weight designated el and e , but a third glycoprotein designated e, has also been detected. the molecular weights of el, e,, and e, were estimated to be , , , and , daltons, respectively (garoffet al., ) . e, and e, are synthesized as a common precursor protein (nvp ) that is cleaved to yield e, and e,. el, ep, and e, of sfv are present in equimolar ratios as are e, and e of sindbis virus. the arrangement and relationship of the glycoproteins within the spike structure of the sindbis and sfv virion are yet to be resolved. and garoff ( ) have used the cross-linking agent dimethyl suberimidate to study the interrelationships of the glycoproteins of sfv. el and e, were most readily cross-linked, but steric hindrance may have prevented the cross-linking of el, e , and e,. recent studies by jones et al. ( ) have suggested that el and e, of sindbis virus, like el, ez, and e, of sfv, probably constitute the glycoprotein spike, since the cleavage of pep, a precursor of e,, does not occur in temperature sensitive mutants of complementation groups including that thought to represent el. furthermore, cleavage is inhibited by antibodies directed against either el or e,. these data suggest that pe, and el may exist as a complex in the membrane of the infected cell, and presumably also that el and e, remain as a complex in the viral envelope. protease treatment of sfv cleaves the glycoproteins, el and e,, and residual segments can be isolated from the envelope of spikeless particles (utermann and simons, ) . the amino acid composition of each of these peptides demonstrates that they are enriched in hydrophobic amino acids. the residual peptides have a mw of approximately daltons; hence they are comprised of about amino acids. thus, like the glycoproteins of influenza virus and vsv, togavirus glycoproteins are amphipathic in nature and appear to possess a peptide of similar size embedded within the viral envelope. when sfv was treated with high concentrations of dimethyl suberimidate, both the tail fragments of el and ez were cross-linked with the nucleocapsid protein, which supports the conclusion that the hydrophobic segments, of el and e, may penetrate through the lipid bilayer and interact with the nucleocapsid . carbohydrate analysis of sfv showed that el contains about moles of monosaccharide, e about moles and e, about moles . e seemed to be particularly rich in mannose. it has recently been reported that the sfv glycoproteins el, e,, and e, are differentially glycosylated (mattila et al., ) . el and e, were shown to contain, on the average, one type a glycosyl side chain. e was shown to contain one type a glycosyl side chain and possibly one or two b-type glycosyl side chains. a-type oligosaccharides are complex structures containing fucose, galactose, mannose, and n-acetyl-glucosamine, whereas b-type oligosaccharides contain only mannose and n-acetylglucosamine (johnson and clamp, ( ) showed that one type-a and one type-b residue were attached to each of the two different glycoproteins. the extent of completion of the glycosyl side chains was shown to be dependent in part upon the cell in which the virus was grown (burge and huang, ; keegstra et ul., ) . bhk- cells were able to complete type-a side chains (i.e., to add galactose and terminal sialic acid), whereas chicken embryo fibroblasts were quite inefficient in adding sialic acid (keegstra et al., ) . both sfv and sindbis virus possess hemagglutinating activity. recent studies by dalrymple et al. ( ) localized this activity to the el glycoprotein of sindbis virus, whereas the e glycoprotein possesses the antigenic determinants which react with neutralizing antibody. the virions of paramyxoviruses are covered with glycoprotein surface projections approximately nm in length. two functionally distinct types of glycoproteins have been isolated from several members of the paramyxovirus group (scheid et al., ; scheid and choppin, ; . in contrast to the situation in influenza viruses, which possess hemagglutinating and neuraminidase activities on distinct glycoprotein molecules, both of these activities are associated with the larger of the two glycoprotein species in paramyxoviruses, which is designated the hn glycoprotein. the smaller glycoprotein component in paramyxoviruses is associated with cell fusion and hemolysis activities and is designated the f glycoprotein. purified glycoproteins of sv form rosettelike clusters in the absence of detergents, suggesting that the spikes have hydrophobic bases (scheid et al., ) . the aggregates formed are morphologically distinguishable, the hn protein forming berrylike aggregates while the f protein forms distinct rosettelike clusters consisting of radiating spikes - nm in length with distinct terminal knobs. in the three best studied paramyxoviruses, sv , newcastle disease virus (ndv), and sendai virus, the h n glycoprotein has a mw range of , - , daltons, and the hn spike solubilized by detergent treatment sediments at . s (scheid et al., ) . although the exact size and fine structure of the hn spike remain to be determined, it has been suggested that each morphological spike contains at least two glycoprotein monomers . in some strains of ndv, an , -dalton precursor of the hn glycoprotein designated hno is incorporated into virions . these particles have reduced hemagglutinating and neuraminidase activities which are activated upon proteolytic cleavage, which produces the , dalton hn molecule. in svs, ndv, and sendai virions, the f glycoprotein has a mw of - , daltons, and the f spike that is solubilized by treatment of sv virions with triton x- has a sedimentation coefficient of approximately . s (scheid et al., ) . in sendai virions (homma and ohuchi, ; scheid and choppin, ) and certain strains of ndv (nagai et al., ) grown in some cell types, the f glycoprotein is not found, and a larger precursor molecule designated fo is observed. recent studies have indicated that proteolytic cleavage of the fo glycoprotein yields two cleavage products, which have bekn designated f and f, (shimizu et al., ; nagai et al., ; scheid and choppin, ) . the f cleavage product is more highly glycosylated than f , and appears to be located on the distal end of the spike; it contains a blocked n-terminal as is also found in the uncleaved f, glycoprotein (scheid and choppin, ) . thus the cleavage of f, generates a free n-terminal on the f, segment of the glycoprotein, and this appears to expose a new hydrophobic region of the molecule which may be important for virus-induced cell fusion. the f, glycoprotein is inactive in cell fusion and hemolysis and can be converted into the active f glycoprotein by proteolytic cleavage, with concomitant activation of cell fusion and hemolysis. it is of particular interest that virions containing the fo precursor are not infective and gain infectivity upon such proteolytic cleavage. in the case of enveloped viruses, glycoproteins located on the surface of the virion are the components involved in adsorption to cellular receptors, which may or may not be host cell glycoproteins. hemagglutination by influenza virus has been studied as a model system for the adsorption of a virus to receptors, and considerable information has been obtained. adsorption to the erythrocyte occurs by the ha spike binding to sialic acid-containing components on the cell surface. the receptor molecule has been isolated from chick red blood cells, and it is a major glycoprotein containing m and n blood group anti-gens. a detailed summary of the properties of this receptor is given by schulze ( ) , as well as by tanner in this volume. removal of sialic acid from the receptor molecule by na prevents the agglutination of erythrocytes by influenza virus. hemagglutination can also be inhibited by pretreating the virus with specific antibodies. while the model for adsorption of influenza virus to erythrocytes seems straightforward, its applicability to the adsorption of viruses to other cell types is uncertain, and little information is available on the nature of receptors for viruses. liposomes containing gangliosides may be capable of acting as receptors for sendai virus, a paramyxovirus (haywood, (haywood, , . sialyoglycoproteins inserted in liposomes can also act as receptors, but the fact that gangliosides can act as receptors raises the possibility that glycolipids may be involved in viral attachment. further evidence that viral glycoproteins specify virus-host cell interactions has been obtained from the studies of bishop et al. ( ) . they produced spikeless particles of the indiana serotype of vsv b y treating the virus with bromelain or pronase. these particles were shown to be noninfectious but, when they were reconstituted with purified g protein isolated from the same strain or from the new jersey serotype, infectivity was restored. antibody directed against the homologous g protein used for reconstitution effectively neutralized the virus, but antibody directed against the serotype of the spikeless particle was ineffective in neutralization when glycoproteins from a different serotype were used for reconstitution. the mechanism of adsorption of other enveloped viruses has not been studied to the same degree as that of ortho-and paramyxoviruses. herpes viruses do not exhibit hemagglutinating activity, but they possess at least glycoproteins which are asymmetrically located on the external surface of the envelope (roizman and furlong, ; o'callaghan and randall, ) . removal of the envelope by nonionic detergents irreversibly alters the infectivity of these viruses (abodeely et al., ) . oncornavinis glycoproteins react specifically with host cell receptors and in the case of avian leukosis viruses they define the host range as well as the classification into subgroups based on interference and neutralization properties ishizaki and vogt, ; duff and vogt, ) . in addition, tozawa et al. ( ) showed that the viral glycoproteins absorbed homologous neutralizing antibody but not antisera prepared against heterologous virions. the purified glycoproteins were shown to interfere with the early steps in infection by the homologous virus. the determination of host range by the glycoproteins of avian leukosis viruses was further demonstrated by phenotypic mixing experiments. defective rous sarcoma virions that lack the envelope gene cannot infect cells. however, when grown in the presence of an avian leukosis virus, they acquire the glycoproteins of that virus, and the host range of the sarcoma virus reflects that of the leukosis virus (hanafusa, ; vogt, ; kawai and hanafusa, ) . the major glycoprotein of rauscher murine leukemia virus (gp ) binds specifically to receptor molecules found on murine cells but not on other mammalian cells (delarco and todaro, ). the phenomenon of cell fusion may be caused by members of several groups of enveloped viruses of which paramyxoviruses are the best studied. the properties of hemolysis and cell fusion are associated with the f glycoprotein as described above. virus-induced cell fusion may occur in the absence of virus replication, in the presence of high concentrations of virus particles. both infectious and noninfectious viruses are equally adept at causing such cell fusion which sometimes has been termed fusion from without (bratt and gallaher, ) . in contrast, fusion with low multiplicities of virus has been called fusion from within and is dependent upon replication of the virus. it is likely that the f glycoprotein is involved in both types of fusion phenomena. the process of fusion through the direct action of concentrated virus has been studied intensively since it was first observed by okada ( ) . sendai virus-induced fusion involves several separate identifiable steps, as indicated by maeda et al. ( ) : ( ) adsorption of the virus to the cell, which seems to occur at the tips of the spikes located on the virion surface; ( ) aggregation of cells; ( ) fusion of the viral envelope with the cell membrane and finally fusion of the cells. these studies suggest that the envelope bilayer of the virion and the membrane of the target cell are brought into contact by the action of the viral hn glycoprotein. the f protein then causes destabilization of the lipids, and an intermixing of lipids occurs. the mechanism of action of the f protein remains to be determined. a neuraminidase activity has been shown to be associated with orthomyxoviruses and paramyxoviruses. the functional role of this enzyme was uncertain for many years, and there was even doubt at some point that it was a viral gene product, since similar enzyme ac-tivity is present in normal cells (white, ); however, these doubts have been resolved by studies of the biochemical, genetic, and antigenic properties of viral neuraminidase (see review in bucher and palese, ) . several distinct functions have been proposed for this enzyme. it is postulated that neuraminidase-containing virus lodges in the upper respiratory tract and binds to mucin via ha. the glycosyl residues of muchin are terminated by sialic acid (n-acetylneuraminic acid) to which ha binds, and one role of the neuraminidase may be to cleave the sialoglycoprotein bond, freeing the bound virion. it is presumed that in this way the virus is released, and that underlying cell receptors are exposed to which ha can attach (davenport, ). it has also been postulated that neuraminidase is involved in an early event such as penetration, but this is unlikely because virions remain infectious after inhibition of neuraminidase activity by specific antibody (bucher and palese, ) . however, the possibility that neuraminidase participates in release of the budding virion from the infected cell surface has gained support from several types of experiments. by using influenza strains with different levels of enzyme activity, it was shown that virus strains having low activity were released from cells more slowly than strains with higher enzyme activity (palese and schulman, ) . further, in the presence of antibody to viral neuraminidase, which inhibited enzyme activity, virions were formed but release of virus into culture media was inhibited (seto and rott, ; compans et al., ; webster, more conclusive information on the function of the enzyme has been obtained with temperature-sensitive mutants of influenza virus which are defective in neuraminidase activity . at the nonpermissive temperature, no neuraminidase activity is detected; virus particles are produced by cells, despite the fact that infectivity titers are markedly reduced. however, the virus particles form large aggregates, and in contrast to wild-type virions these particles contain sialic acid as shown by colloidal iron hydroxide staining. since influenza virions bind to sialic acid residues, these results indicate that the mutant virus particles aggregate to each other, because sialic acid is added to viral carbohydrates, and that the essential function of viral neuraminidase is to remove or prevent the addition of such sialic acid. in support of this conclusion, the addition of bacterial neur-aminidase to cells infected with these mutants cause a marked enhancement of virus release. further evidence supporting this role for the neuraminidase was obtained with a neuraminidase inhibitor, -deoxy- , -dehydro-ntrifluoracetylneuraminic acid (fana). influenza virus grown in the presence of fana contains neuraminic acid on its envelope, and the particles undergo extensive aggregation (palese and compans, ) . a marked reduction in virus yield is observed because of this aggregation, and treatment with purified neuraminidase results in a marked enhancement in progeny virus yields, apparently through disaggregation of virus. thus viral neuraminidase is not required for assembly of progeny virions but appears to be essential for the removal of sialic acid from the surface of the virion itself. from the studies described above it is evident that the membranes of enveloped viruses are asymmetrically constructed, with the glycoproteins that comprise the spikes or surface projections physically located on the exterior of the viral envelope. thus the glycoproteins are exposed to the immune surveillance system of the host and, being good immunogens, may elicit humoral and cellular immune responses (evans, ) . the classification of virus isolates into specific strains depends largely upon serological procedures, and for enveloped viruses, surface glycoproteins are usually the relevant antigens in such tests. neutralizing antibodies are usually directed against the viral proteins involved in attachment to receptors, e.g., ha glycoprotein of influenza virus (webster and laver, ) , g protein of vsv (wagner, ) , gp / of murine leukemia viruses (fischinger et az., ; strand and august, ) , gp of avian leukosis viruses (bolognesi, ) , and e, of sindbis virus (dalrymple et d., ) . high concentrations of antibody can prevent attachment of the virus to receptors, but low concentrations can also neutralize infectivity by a mechanism that is not understood. the specific determinants of viral glycoproteins recognized by virus-neutralizing antibodies have not been chemically characterized. it has been postulated that antibody molecules may recognize only determinants on the tip of the ha spike of the influenza virion (white, ) . in reaching these conclusions, it has been assumed that the size of the antibody molecule precludes the possibility that it could make contact with any other part of the spike, since the spaces between the spikes are too small for the immu'noglobulin to interact with other regions. electron microscopic observations (lderty and oertilis, ) indicate that antibody molecules interact with the tips of surface spikes, and analysis of the tryptic peptides of ha molecules isolated from *closely related influenza strains has shown that they rarely differ b y more than one or two peptides webster and laver, ) , suggesting that the strain differences are indeed restricted to small regions or determinants of the ha spike. the antigenic character of many viral glycoproteins is stable, but the determinants exhibited by the glycoproteins of influenza virions are characteristically variable, as evidenced by the many different strains of type-a and -b influenza. the ha and na glycoproteins of influenza viruses are antigenically distinct, and they undergo antigenic changes independently of each other. the antigenic changes that occur may be gradual, in which case the different virus strains are clearly related to each other with respect to both surface antigens. antigenic changes of this nature are termed antigenic drift, and they result from the interplay of viral mutability and immunological selection (webster and laver, ) . the presence of antibody of low avidity may select for single-step mutants, which have an altered amino acid in the key area of the antigenic determinant, giving rise to a new viral strain. at intervals of - years sudden and complete changes in the determinants of type-a influenza glycoproteins occur; the changes are such that the viruses that arise possess glycoproteins that appear completely distinct on peptide mapping . dramatic changes in the antigenic determinants of viral glycoproteins are termed antigenic shifts, and it is these new viruses that cause worldwide influenza pandemics. it has been postulated that antigenic shift may occur because type-a strains of human origin may undergo recombination with type-a strains of avian and animal origin. the antigenic determinants of the new ha or na are sufficiently different that the human host does not possess immunity, and the virus gains a selective advantage. interestingly type-b influenza viruses do not undergo antigenic shift. the reason for this may lie in the fact that type-b influenza viruses have not been isolated from other animal species; thus it is probable that the type-b viruses can not undergo similar recombination events (webster and laver, ) . viral glycoproteins provide excellent systems for analysis of the function of carbohydrates in membrane glycoproteins. several approaches have been used to modify the carbohydrates, including treatment of virions with specific glycosidases, growth of virus in cells with specific sugar transferase defects, and treatment of virus-infected cells with inhibitors of glycosylation. removal of sialic acid from the g protein of vsv has been reported to reduce the infectivity of vsv virions (schloemer and wagner, ) , whereas the infectivity of sfv (kennedy, ) and friend leukemia virus (schafer et al., ) were reportedly unaltered by such treatment. moreover, enzymic addition of sialic acid to the glycoproteins of sindbis virus did not alter the infectivity of this virus (stollar et al., ) , while enhancing the infectivity of influenza virus (schulze, ) and restoring infectivity to neuraminidase-treated vsv (schloemer and wagner, ) . treatment of influenza virions with glycosidases alter hemagglutinating activity, but neuraminidase activity was unaffected; however, similar treatment of ndv, a paramyxovirus, resulted in no alteration in hemagglutinating or neuraminidase activities (bike and knight, ) . schiifer et al. ( ) showed that the indirect hemagglutinating activity of friend leukemia virus was inhibited by glycosidase treatment but that viral infectivity and determinants involved in viral interference and absorption of neutralizing antibody were unaltered. sindbis virus were grown in these cells, the apparent mws of their glycoproteins were lower. the infectivity of vsv and sindbis virus grown in these cells was not altered from that of fully glycosylated virions. however, glycosidase treatment of sfv was shown to decrease the infectivity of this virus (kennedy, ) . the apparent difference between these results remains to be resolved. it is possible that the core of the glycosyl side chain is added to the sindbis virus glycoproteins in the enzyme-deficient cells, whereas the glycosidase treatment used by kennedy may have removed more of the glycosyl moieties from the sfv glycoproteins. inhibitors of glycosylation have also been used to assess the role of glycosyl side chains of glycoproteins. primarily, three different inhibitors have been used for this purpose: -deoxy-~-glucose ( -dg), an analog of glucose which substitutes for mannose, preventing the further addition of monosaccharides to the glycosyl side chain; dglucosamine, which is thought to inhibit glycosylation at high concentrations by decreasing utp pools in the cell and subsequent activation of other sugars (scholtissek, ) ; and tunicamycin (tm), a glucosamine-containing antibiotic that inhibits the formation of n-acetyl-glucosamine-lipid intermediates which serve as donors for the synthesis of the oligosaccharide side chains of glycoproteins (tkacz and lampen, ) . kilbourne ( ) and kaluza et al. ( ) were among the first investigators to utilize inhibitors of glycosylation to study the role of carbohydrates in influenza virions. they showed that -dg and d-glucosamine inhibited the biosynthesis of active ha, na, and mature infectious influenza virions. subsequent biochemical studies revealed that high concentrations of -dg or dglucosamine prevented the synthesis of influenza virus glycoproteins (klenk et al., b nakamura and compans, a) . instead, an unglycosylated or incompletely glycosylated hemagglutinin precursor ha,, was detected. once synthesized, hao was found associated with cytoplasmic membranes, as is the case with the normal glycoprotein. hao glycoprotein in cells infected with the fowl plague strain (fpv) was cleaved by cellular proteases to yield a heterogeneous product . however, the comparable protein synthesized in cells infected with the wsn strain was processed and incorporated into virions (nakamura and compans, a) . because -dg and d-ghcosamine interfere with metabolic reactions other than the glycosylation of glycoproteins, they may cause side effects that can affect virus replication. therefore tm, a compound that seems to affect only the glycosylation of glycoproteins, has been employed to extend these studies. t m inhibited virion formation in fpvinfected cells and the unglycosylated glycoprotein ha,, appeared to be degraded by cellular proteases (r. t. ; ha,, of the wsn strain synthesized in the presence of tm also appeared to be completely unglycosylated, whereas even at high concentrations of -dg and dglucosamine some glycosylation occurred (nakamura and compans, a) . nonetheless, tm did not inhibit virion formation to the same extent as -dg (nakamura and compans, ) . the surface spike layer of wsn virions produced in the presence of -dg, d-glucosamine, and tm was altered morphologically, and the hemagglutinating activity of the virions was significantly reduced. these results suggest that glycosylation of virion glycoproteins is not required for influenza virion formation but is needed for biological activity of viral glycoproteins. these glycosylation inhibitors have also been used with several other enveloped viruses. when herpes viruses are grown in the presence of -dg, the infectious virus yield is decreased by greater than %, but the yield of viral particles is not reduced (courtney et al., ) . the reduction in infectivity was attributed to an inability of the virions to attach to the host cell and penetrate, implying that oligosac-charide residues of herpes virus glycoproteins play a role in the attachment and recognition of host cell receptors. the glycoproteins of vsv, sindbis virus, and sfv are not glycosylated in the presence of tm (r. t. leavittet al., ) . mature vsv and sindbis virions are not released from tmtreated cells, but nonglycosylated precursors are synthesized and seem to be stable within the cell (leavitt et al., ) . similarly, r. t. schwarz et al. ( ) showed that unglycosylated sfv glycoprotein precursors were synthesized in tm-treated cells. moreover, they were not degraded by host cell proteases and virion assembly was completely inhibited. it is interesting that, as noted above, ha, of fpv is degraded when it is grown in tm-treated chicken embryo fibroblasts, but the glycoprotein precursors of sfv synthesized in the same cells are stable. these results may be due to greater release of protease by fpv infection or, alternatively, the polypeptide backbone of sfv glycoproteins may be less susceptible to proteolytic degradation. high concentrations of glucosamine rapidly shut off the production of infectious avian sarcoma virus particles (hunter et al., ) . however, avian sarcoma virus particles were assembled and released at about % of the control level in tm-treated cells, and such particles appeared to lack glycoproteins (r. t. . the infectivity titer of the virus produced in tm-treated cells decreased by only %. these results indicate that the effects of glycosylation inhibitors may vary with the virus and host cell, as well as with the specific inhibitor used. side effects of some inhibitors may have marked effects on virus replication. however, the fact that in some systems virus particles are produced with unglycosylated or incompletely glycosylated glycoproteins clearly demonstrates that the complete glycosylation process is not essential for intracellular migration of glycoproteins or their incorporation into the plasma membrane and subsequently into virus particles. the biological activities of some glycoproteins such as those of influenza virus and sfv appear to require glycosyl moieties, whereas for other viruses, such as murine leukemia virus, this may not be the case. the membranes of enveloped viruses, particularly those of singlestranded rna viruses, are unique in their simplicity of construction and are useful systems for studying the interactions of specific proteins with the lipid bilayer. hence comparative studies of the effects of viral proteins of the lipid bilayer structure have been made using the techniques of electron spin resonance (esr), nuclear magnetic resonance (nmr), and fluorescence polarization (fp). esr studies have provided evidence that the lipids of influenza virus, sv , rauscher leukemia virus, vsv, and sfv (landsberger et uz., (landsberger et uz., , (landsberger et uz., , ; sefton and gaffney, ) are bilayer structures with fluid lipid phases similar to those observed for other biological membranes, but all viral membranes were shown to be substantially more rigid than the corresponding host cell plasma membrane. proteolytic removal of the glycoprotein spikes from the surface of sv and influenza virions did not appreciably alter the fluidity of the lipid bilayer of these viruses. lenard et al. ( ) have provided further evidence that the glycoproteins of the influenza virion contribute little to the rigidity of the lipid bilayer. the phospholipid composition of standard influenza particles, and "incomplete" virus produced upon serial undiluted passage, were compared and found to be indistinguishable, as were the esr spectra of the two types of particles. the incomplete particles were shown to contain approximately twice the amount of glycoproteins relative to the complete particles. thus it was concluded that the rigidity of viral membranes may be determined b y the m protein and not by the viral glycoproteins. this may not be entirely the case for vsv and sfv, since sefton and gaffney ( ) and landsberger and compans ( ) observed that, when the glycoproteins of these viruses were removed by proteases, the envelope became more fluid, indicating that the viral glycoproteins may contribute to the rigidity of the envelope. however, landsberger and compans ( ) postulated that the major effect on vsv bilayer fluidity was exerted by the m protein, since the fluidity of the lipid bilayer was altered only slightly when the g protein was removed by protease, whereas vesicles prepared from extracted viral lipids were much more fluid than lipids in virions. using the technique of fluorescence depolarization, moore et az. ( ) and barenholz et al. ( ) showed that the envelope of sfv, sindbis virus, and vsv has a higher microviscosity than that of the plasma membranes from which the virions budded. the increased microviscosity was attributed in part to insertion of the hydrophobic regions of the glycoproteins into the envelope bilayer. stoffel and bister ( ) , using nmr spectra of ' c-labeled lipids, also demonstrated that the envelope lipids of vsv are highly rigid, as a result of either lipid-lipid or lipid-protein interactions. in general it may be concluded therefore that the lipid bilayer of enveloped virions is more rigid than the host cell membrane from which the virion buds. while virion glycoproteins do interact with the lipid bilayer and may affect the rigidity of the membrane to some extent, the internal membrane protein(s) may also be of equal or greater importance in determining membrane rigidity. most enveloped viruses form by a process of budding at the plasma membrane, with little or no participation of other membrane structures in the final steps of maturation. however, there are important exceptions in the case of certain virus groups. the capsids of herpes viruses are assembled in the nucleoplasm and are observed to bud through the inner nuclear membrane, acquiring their envelopes in the process (roizman and furlong, ; o'callaghan and randall, ) . they appear to be transported as enveloped particles through cytoplasmic channels to the cell surface. bunyaviruses (murphy et az., ) and coronaviruses (oshiro, ) appear to form primarily by budding into cytoplasmic cisternae, and extracellular virions are observed associated with the plasma membrane. for the rhabdovirus group, several modes of maturation have been reported. vsv, the most widely studied member, usually forms by budding at the cell surface, but maturation at intracellular membranes has been observed. the new jersey strain of vsv was observed to bud primarily from plasma membranes of l or vero cells, and almost entirely at intracytoplasmic membranes of pig kidney cells (zee et al., ) . these reports indicate that the site of maturation of a specific virus type may vary depending on the host cell. rabies virus, which resembles vsv morphologically and biochemically, is exceptional in that assembly of the virion appears to occur through a process involving de n o w formation of membranes in the cytoplasmic matrix (hummeler et al., ) . de novo formation of membranes is the usual process of assembly for members of the pox virus group (dales and mosbach, ) . the appearance of viral proteins on the cell surface, as well as the cellular site of virus assembly, can be modified by external agents including antibodies and lectins, the phenomenon of antigenic modulation involves altering the expression of cell surface antigens by specific antibody, and such antigens may be of viral origin (lampert et d . , ) . this undoubtedly has an effect on viral maturation, although the precise effects have not been determined. exposure of influenza virus-infected cells to convanavalin a appears to alter the site of virus maturation (stitz et al., ) . in the presence of this lectin, normal maturation at the plasma membrane is not observed, but instead large numbers of virions are observed in intracellular vacuoles. it is evident therefore that the maturation site for enveloped viruses can vary with the virus type as well as the host cell, and can be altered in response to specific stimuli. the process is likely to be determined as a result of interactions between virus-specific proteins and host cell membranes. i t is remarkable that in most instances only a single type of cellular membrane is selected as the site of virus assembly in a particular virus-infected cell. an understanding of the mechanisms which govern the selection of the assembly site may provide new insights into the assembly of cellular membrane components and organelles, since similar interactions are likely to be involved in determining the location of subsets of cellular proteins in specific cellular organelles. the synthesis and assembly of viral membrane components have been analyzed in cells in which host cell synthesis is inhibited as a result of virus infection. similar conclusions have been made for several virus types. viral glycoproteins appear to be synthesized on membrane-bound polyribosomes and remain associated with various cellular membranes (spear and roizman, ; compans, a,b; stanley et al., ; klenket al., ; david, ; hay, ; atkinsonet al., ; nagai et al., ; knipe et al., ) . glycosylation occurs in association with cytoplasmic membranes. glycoproteins appear to migrate from rough endoplasmic reticulum to smooth or golgi complex membranes to the cell surface and are then incorporated into virions; at no time are they found as ''soluble'' cytoplasmic components. this general scheme for the synthesis and migration of proteins through intracellular membranes to the cell surface has been termed membrane flow. although the precise intracellular location of viral glycoproteins remains to be established, a scheme may be envisaged in which these components remain associated with the cisternal side of membranes of the endoplasmic reticulum and golgi complex after synthesis. incorporation into the plasma membrane by a process of vesicle fusion would then result in the correct orientation on the cell surface for viral as-sembly. a similar scheme has been suggested for the incorporation of glycoproteins into the plasma membrane, based on the distribution of oligosaccharides in different membrane fractions (hirano et al., ) . viral systems allowed investigators to follow the intracellular migration and glycosylation of specific glycoprotein species for the first time, whereas previous studies with cellular glycoproteins kad dealt with cell fractions essentially uncharacterized as to the specific proteins present. recently, katz et al. ( ) and rothman and lodish ( ) have described a new model system for studies of the incorporation of viral glycoproteins into membranes. katz et al. ( ) have shown that the mrna for vsv g-protein translated in vitro by wheat germ extracts in the presence of dog pancreas rough endoplasmic reticulum is associated with the membrane. furthermore, the newly synthesized g protein was shown to span the membrane with the amino-terminal asymmetrically located. in addition, the polypeptide portion which penetrated the membrane was shown to be glycosylated. glycosylation did not occur in the absence of membranes. these studies were extended by rothman and lodish ( ) , who showed that the insertion of g-protein into the membrane begins when or fewer amino acid residues are polymerized. evidence was also obtained that the nascent chain is glycosylated while still attached to the ribosomes on the cytoplasmic side of the endoplasmic reticulum vesicle. since the mechanism by which viral glycoproteins are inserted into membranes is in all probability similar to that of the insertion of host cell glycoproteins, further studies using viral systems may provide more insight into the mechanism of insertion of glycoproteins into membranes. the process of glycosylation does not appear to play an important role in determining the intracellular migration of glycoproteins. although this has been suggested as a possible function for carbohydrate components of glycoproteins, the available data using inhibitors of glycosylation suggest that it is possible to inhibit or extensively modify the gl ycosylation process without preventing the migration of viral glycoproteins to the cell surface (courtney et al., ; nakamura and compans, a) . the incorporation of carbohydrate-free m proteins into membranes appears to involve a distinct pathway in which cytoplasmic synthesis is followed by rapid association with the plasma membrane (lazarowitz et d., ; meier-ewert and compans, ; hay, ; nagai et al., ; knipe et al., ) . no evidence for migration through cytoplasmic membrane structures has been obtained' for these components, aad it has been suggested that they are inserted directly into membranes after synthesis. cytoplasmic synthesis followed by direct insertion into membranes has also been proposed for some classes of cellular membrane proteins (lodish and small, ) . although there are several unanswered questions concerning the precise steps in assembly even for the best studied enveloped viruses, the available information from electron microscope studies as well as the biochemical approaches described above suggest a scheme like that depicted in fig. for influenza virus. glycoproteins appear to be inserted into the plasma membrane as the first step in assembly, after migration through cytoplasmic membranes. after they are inserted into the plasma membrane, initial random distribution may occur in which the proteins are free to undergo lateral diffusion in the plane of the membrane. such random distribution is illustrated for the glycoproteins of parainfluenza virus in fig. , and similar observations have been reported for other viruses (birdwell and strauss, ; h. schwarz et al., ) . random distribution of viral antigens (fig. ) is observed only when ferritin-antibody conjugates are applied to cells after glutaraldehyde fixation. in previous studies of the distribution of antigen, in which unfixed cells were used ), antigens were observed in discrete patches. it is likely that under these conditions they undergo lateral redistribution and aggregation into patches as a result of bivalent antibody. schematic diagram of the assembly process of an influenza virion. clycoproteins are thought to be inserted into the plasma membrane by a process called memhrane flow and are initially found randomly dispersed in the membrane. following in--sertion of the m protein, glycoproteins are thought to accumulate in discrete regions from which host cell membrane proteins are excluded. the association of the ribonucleoprotein (rnp) with such regions of modified membrane is followed by budding and release of the completed virion. in the case of influenza virus, the m protein may form a domain on the internal surface of the plasma membrane, stabilized by proteinprotein interactions. specific recognition of the m protein by the glycoproteins could then produce an accumulation of glycoproteins in a circumscribed region of the cell surface. alternatively, it is possible that m protein monomers associate with glycoprotein monomers at the plasma membrane, and that these complexes undergo lateral diffusion and aggregation into domains. in either case the lack of host cell membrane proteins in the viral envelope indicates that cellular proteins are efficiently excluded from the region of the plasma membrane which becomes the viral envelope. association of the nucleocapsid with regions of the cell surface containing viral envelope protein may stimulate the process of budding. fig. . the presence of virus-specific glycoproteins on the external surface is indicated by tagging with ferritin-conjugated antibody. in fig. , the emerging virus particles tagged with ferritin-antibody are shown at higher magnifica- for viruses with icosahedral nucleocapsids and no m protein, a similar mechanism for virus assembly has been proposed in which the nucleocapsid itself binds to glycoproteincontaining membranes, with subsequent lateral diffusion and clustering of the glycoproteins in this region. although information is accumulating about the pathways by which viral membrane proteins are incorporated into plasma membranes, there is little direct evidence concerning the precise interactions which lead to the formation of domains on the cell surface which contain virus-specific proteins and lack host cell proteins. the lack of significant amounts of host cell protein in the virion indicates that such domains must he intermediates in assembly. further, the available data on viral protein composition (table ) and morphology suggest that assembly interactions may differ for various virus groups. in all cases it is likely that protein-protein interaction serves to create a patch of virus-specific proteins in a cellular membrane, but this interaction may occur on the external or internal surface of the lipid bilayer. as discussed above, for viruses that contain m proteins or welldefined icosahedral nucleocapsids, some evidence has been obtained for the initial random distribution of glycoproteins on the cell surface, which may be followed by lateral diffusion and accumulation of glycoproteins in juxtaposition to the m protein or icosahedral nucleocapsid, with transmembrane interactions between the external and internal proteins serving to anchor the viral glycoproteins in place. with other virus types that contain a large amount of glycoprotein and no obvious m protein or well-defined icosahedral capsid, it is possible that lateral interactions between the glycoproteins are important in assembly. the glycoproteins of togaviruses and bunyaviruses have been observed in a regular surface arrangement (von bonsdorff and harrison, ; von bonsdorff and pettersson, ) . the latter viruses lack an m protein or an icosahedral internal component, and it has been suggested that direct interactions between glycoproteins may be involved in assembly and maintenance of the viral structure (von bonsdorff and pettersson, ) . similar interactions may be important in other virus groups in which glycoproteins are major protein constituents of the virion and there is no known internal membrane protein, such as b-type oncomaviruses and coronaviruses. pox viruses are unique in that formation of their lipid-containing membrane occurs de novo in the cytoplasm, rather than on a preexisting membrane structure. these viruses thus provide an unusual system for investigation of the molecular interactions involved in the formation of a highly organized structure within the cytoplasmic matrix (dales and mosbach, ). however, these viruses are structurally very complex, and limited information has been obtained on their molecular organization. shape and size determination may also be controlled by viral proteins at various levels; nucleocapsids, m proteins, or glycoproteins could be the determining factor for different virus groups. the phenomenon of phenotypic mixing of envelope glycoproteins in cells doubly infected with vsv and the parainfluenza virus sv clearly demonstrates that the internal proteins and not the glycoproteins determine the particle size and shape of rhabdoviruses mcsharry et al., ). this conclusion is supported by the observation that particles with the internal proteins of vsv possess the characteristic bullet shape of vsv while containing mixtures of envelope glycoproteins derived from vsv and sv . a similar analysis of phenotypically mixed particles produced by dual infections with viruses of other major groups may provide further insights into the macromolecular interactions involved in virion assembly. the fixed shape and size of many 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(fana): mechanism of action isolation and characterization of influenza virus recombinants with high and low neuraminidase activity: use of -( '-methoxy-pheny )-n-acetylneuraminic acid to identify cloned populations characterization of temperature sensitive influenza virus mutants defective in neuraminidase sulfated glycoproteins and polysaccharides of enveloped viruses phospholipid composition of rous sarcoma virus, host cell membranes, and other enveloped viruses the replication of herpes viruses transbilayer phospholipid asymmetry and its maintenance in the membrane of influenza virus role of carbohydrate in biological functions of friend murine leukemia virus gp lj isolation and purification of the envelope proteins of newcastle disease virus identification of biological activity of paramyxovirus glycoproteins: activation of cell fusion, hemolysis and infectivity by proteolytic cleavage of an inactive precursor protein of sendai virus isolation of paramyxovirus glycoproteins: association of both hemagglutinating and neuraminidase activities with the larger sv glycoprotein identification of a second glycoprotein in sindbis virus grbwth of enveloped rna viruses in a line of chinese hamster ovary cells with deficientn-acetylglucosaminyl transferase activity sialoglycoprotein of vesicular stomatitis virus: role of the neuraminic acid in infecti n mosquito cells infected with vsv yield unsialylated virions of low infectivity association of vesicular stomatitis virus glycoproteins with virion membrane: characterization of the lipophilic tail fragment detection of an unstable rna in chick fibroblasts after reduction of the utp pool by glucosamine the struchire of influenza virus. i. the polypeptides of the virion the biologically active proteins of influenza virus: the hemagglutinin properties of mouse leukemia virus. xi. immunoelectron microscopic studies on viral antigens on the cell surface inhibition of glycosylation of influenza virus hemagg utinin carbohydrates of influenza virus i. glycopeptides derived from viral glycoproteins after labeling with radioactive sugars suppression of glycoprotein formation of semliki forest, influenza, and avian sarcoma virus by tunicamycin effect of the viral proteins on the fluidity of the membrane lipids in sindbis virus red cell membrane glycoprotein: amino acid sequence of an intramembranous region functional significance of sialidase during influenza virus multiplication isolation and characterization of two distinct types of hvj (sendai virus) spikes the fluid mosaic model of the structure of cell membranes studies on the primary structure of the influenza virus hemagglutinin. proc. n a t l the proteins specified by herpes simplex virus. iv. the site of glycosylation and accumulation of viral membrane proteins the polypeptides of influenza virus. vii. synthesis of the hemagglutinin studies on the inhibitory effect of lectins on myxovirus re ease sc nuclear magnetic resonance studies on the lipid organization of enveloped virions (vesicular stomatitis virus) sialic acid content of sindbis virus from vertebrate and mosquito cells structural proteins of ribonucleic acid tumor viruses carbohydrate content of membrane protein of sindbis virus tunicamycin inhibition of polyisoprenyl n-acetylglucosaminyl pyrophosphate formation in calf liver microsomes strain-specific antigen of the avian leukosis sarcoma virus group asymmetry of influenza virus membrane bilayer demonstrated with phospholipase c studies on the amphipathic nature of the membrane proteins in semliki forest virus structural components of the arenavirus pichinde a heterogeneity of rous sarcoma virus revealed by selectively resistant chick embryo cells patterns of viral interference in the avian leukosis and sarcoma complex sindbis virus glycoproteins form a regular surface lattice surface structure of uukuniemi virus reproduction of rhabdoviruses estimation of the molecular weights of the polypeptide chains from the isolated hemagglutinin and neuraminidase subunits of influenza viruses studies on the origin of pandemic influenza. i. antigenic analysis of a influenza viruses isolated before and after the appearance of hong kong influenza using antisera to the isolated hemagglutinin subunits antigenic variation of influenza viruses influenza viral proteins: identification and synthesis evidence from studies with a cross-linking reagent that the haemagglutinin of influenza virus is a trimer structural components of mouse mammary vesicular stomatitis virus maturation og!/ , - . shape of influenza virus neuraminidase research by the authors was supported by grants no. a and ca from the usphs, pcm - from the national science foundation, and vc b from the american cancer society. m.c.k. was supported b y a fellowship from the anna fuller fund. key: cord- -y lewk t authors: zhang, li-zhi title: fabrication of a lithium chloride solution based composite supported liquid membrane and its moisture permeation analysis date: - - journal: j memb sci doi: . /j.memsci. . . sha: doc_id: cord_uid: y lewk t a novel composite supported liquid membrane has been prepared for ventilation air moisture recovery. the membrane is composed of three layers: two hydrophobic protective layers and a sandwiched hydrophilic support layer in which licl solution is immobilized to facilitate water vapor transfer. a test is conducted to measure the moisture permeation rate through the composite membrane. various resistances in the cell and in the composite membrane are clarified. linear equilibrium relations between humidity, temperature, and licl concentration in the liquid solution layer are obtained to aid in the model set-up. it has been found that the mean moisture permeation rate through the composite membrane is around . × (− ) kg m(− ) s(− ), almost two times higher than that through a solid hydrophilic cellulose acetate membrane with comparative thickness. further, the supported liquid layer only accounts for % of the total moisture transfer resistance in the cell, indicating that there is much potential for further performance improvement. people's concern on indoor air quality has greatly deepened since the outbreak of the severe acute respiratory syndrome epidemic (sars) that devastated south china and some other parts of the world in the spring of . increased fresh air ventilation is the most efficient and necessary way to have a better indoor air quality, either in eliminating disgusting odors, diluting vocs, or decreasing the possibility of being infected by viruses. increased ventilation rates usually lead to high-energy consumption in air-conditioning. this is because the relative humidity in an occupied building must be controlled to within - % for health and comfort reasons. it has been proved that ventilation air constitutes about % of the total moisture load in most commercial buildings [ ] . as a consequence, treatment of the latent load from the ventilation air is a difficult and imminent task for hvac engineers, especially in hot and humid climates. * tel.: + ; fax: + . e-mail address: lzzhang@scut.edu.cn. to save energy in treating fresh air, various techniques for independent air dehumidification have been proposed [ , ] . among those drawn much attention is one that involves membrane technology-the so called membrane based total heat recovery (mthr) [ ] [ ] [ ] [ ] . mthr is an air-to-air heat exchanger where the two incoming streams (fresh air and exhaust air) exchanges heat and moisture simultaneously through membranes. the fresh air represents outdoor air (fresh air intake for a hvac system). the exhaust air represents stale room air that would normally be exhausted to the outside. the core material of an mthr ventilator are vapor-permeable membranes, therefore both heat and moisture are transferred between these two air streams when they flow through the unit. thus, heat and humidity would be recovered from the exhaust stream in winter and excess heat and moisture would be transferred to the exhaust in order to cool and dehumidify the incoming air in summer. in this way, large quantities of energy in hvac could be saved. hydrophilic polymer membranes that are permeable to vapor, but impermeable to air, have been considered for mthr ventilators. nafion [ , ] , regenerated cellulose [ ] , cellulose triacetate [ ] , sulfonated poly(phenylene oxide) [ ] , polyether-polyurethane [ ] , siloxane-amido copolymer [ ] , polystyrene-sulfonate [ ] , polyvinylidene fluoride and polyethersulfone [ ] , and cellophane [ ] are good choices since they have already been used in air dehumidification applications which have similar moisture transfer mechanisms to moisture recovery. however, moisture diffusion coefficients in such polymer membranes are usually very low, in the order of − to − m s − [ , ] , while mthr ventilators only have limited transmembrane vapor partial pressure difference, consequently performances are quite limited currently. in contrast to solid membranes, moisture diffusion in liquid membranes (∼ − m s − [ , ] , diffusion coefficients) is several orders higher than that in solid membranes. due to this reason and the inherent high selectivity, in recent years, there has been much effort in progressing the researches of supported liquid membranes (slm) in various fields: air dehumidification [ ] , so /co separation [ ] , h s/ch separation [ ] , wastewater treatment [ ] , metal ions concentration (uphill transport) [ ] , separation of isomeric amines between two organic phases [ ] , to name but a few. to improve the performances of mthr ventilators, in this study, a novel membrane, a composite slm, which employs licl liquid solution immobilized in a porous support membrane to facilitate the transport of moisture, is prepared. to protect the slm, two hydrophobic polyvinylidene fluoride (pvdf) layers are formed on both surfaces of the slm. the concept is shown in fig. . the sweep represents exhaust air. moisture permeation through this membrane is of great interest. moisture transfer characteristics will be the focus of this study. three types of commercial membrane were obtained from a supplier. very hydrophilic cellulose acetate (ca) membranes with nominal pore diameter . m a thickness - m are used as the support media to immobilize licl solution. two hydrophobic pvdf membranes (equal nominal pore diameter . m, thickness m) are used as the protective layer. crystals of licl·h o with laboratory class purity is used as the solute. before the preparation of composite membrane, each membrane is experimented and observed for their basic microstructures. figs. and show the scanning electron photomicrograph (sem) graphs of the ca membrane and pvdf membrane, respectively. under room temperature, well-stirred licl solution with % mass fraction is first prepared in a closed glass container. vacuum degassing is applied for h for the three membranes, after which, the ca membrane is dipped into the licl solution. after h, the ca membrane is moved from the solution and placed onto a clean glass plate which is cleaned by alcohol. surplus licl solution on surfaces of ca membrane is blotted off with paper tissue. to be sure that no ionic liquid is removed from the membrane pores, the cleaning procedure is very gentle. at this stage, pvc glue is brushed on one surface of the two pvdf membranes, and at the same time on both surfaces of the ca membrane. after a few seconds, the two pvdf membranes are glued to the ca membrane and are pressed together gently for a few seconds. the prepared composite membrane is placed in a constant-temperature-constant-humidity chamber for another h, before experiment is performed. for comparison, a composite membrane with no licl solution immobilized in the ca membrane is also made with the same procedure. the cross-section sem views of the two composite membranes are shown in figs. and , respectively. to prevent the microstructure being destroyed by knife crushing when preparing cross-section samples, the membranes are first frozen in liquid nitrogen before they are broken off to see the cross-sections. as seen from fig. , there are some gaps between different layers. some big cavities in the support layers are also observed, which are presumed to be imperfections in membrane fabrications. however, they have no adverse effects for this study because during operation, they will be filled with liquid solution. in the preparation process, some thickness of the ca membrane is dissolved by the glue, resulting in a lesser support layer thickness than raw material. fig. with licl solution shows that the ca layer and pvdf layer connect to each other very closely and have a dense and continuum interface. there are more big cavities in the support layer. the reason behind this may be that with licl solution soaked, the wetted molecular chains in ca membrane structure become more flexible and they will swell and expand to two sides. the boundaries between different layers are pressed together and linked to each other closely. the final ca layer thickness is m. the membrane module is a circular cell having an exchange area of . cm . it is composed of two parts: the lower chamber and the cap, as shown in fig. . when testing, the flat sheet membrane is placed on the lower chamber inside which saturated salt solution is contained. the cap is then covered on the membrane surface and forms a sandwiched structure. the membrane and the inner surface of the cap form a cone-shaped cavity. the air is supplied through the air slits in the cap. it is introduced through two diametrically positioned inlets (symmetrically placed) into a circular-shaped channel at the perimeter, from where the air is distributed over the membrane surface through the circular air slit. the air flows inward radially, until it exits the cap outlet in the center. the cap is designed that a constant axial air velocity is realized. when flowing across the membrane, the air stream exchanges moisture with the salt solution through the composite slm, and is humidified. this test uses nacl solution since it can ensure outlet humidity not saturated, for the protection of rh sensors. the whole experimental set-up is shown in fig. . the cell is supplied with clean and humidified air from an air supply unit. the supply air flows from a compressed air bottle and is divided into two streams. one of them is humidified through a bubbler immersed in a bottle of distilled water, and then re-mixed with the other dry air stream. the humidity of the mixed air stream is controlled by adjusting the proportions of air mixing. the air flow rates are controlled by two air pumps/controllers at the inlet and outlet of the cell. the humidities and temperatures to and from the cell are measured by the built-in rh and temperature sensors, which are installed in the pumps/controllers. a detailed description of the test procedure is given in [ ] . in the test, the vapor evaporation is slow, and the cell is well conductive. therefore, only moisture transfer is considered, by neglecting thermal influences. moisture transfer from the saturated nacl solution to air stream above membrane, is depicted in fig. . variations of air humidity along the transfer path are shown in fig. to simplify the mass transfer model. there are totally five resis- tances that can be clarified: resistance in the lower chamber air gap ( - ); resistance in the first protective layer l ( - ); resistance in the supported liquid membrane l ( ) ( ) ; resistance in the other protective hydrophobic layer l ( ) ( ) ; and resistance in the air stream h d ( ) ( ) , as demonstrated in fig. . the moisture permeation rate from the solution surface to the air stream can be summarized by where r tot is the total resistance from the solution surface to air stream and ω is the humidity difference between the solution in lower chamber and air stream above membrane. where subscripts l and d represent solution in lower chamber and air in air duct, respectively. the mean moisture permeation rate across the whole membrane surface in the cell is calculated by where ω lm is the logarithmic mean humidity difference between the solution surface and air stream, and it is calculated by where subscripts o and i represent outlet and inlet of air stream, respectively. the total resistance is comprised of five parts as where r l , r , r , r , r d are resistances in air gap, in the first protective layer, in the liquid membrane layer, in the second protective layer, and in air stream, respectively. moisture resistance below the membrane can be represented by the vapor diffusion distance from solution surface to the membrane lower surface. where l is the height of air gap (m), ρ a the dry air density (kg m − ), and d va is the vapor diffusivity in dry air (m s − ). moisture transfer resistance (m s kg − ) in the composite membrane comprises three layers: where where δ i is membrane thickness in ith layer (m) and d ei is the equivalent diffusivity of moisture in ith membrane, i = - . the two protective layers on both sides of the liquid membrane are highly hydrophobic. the established theory of gas diffusion in such membranes considers three mechanisms: poisseuille flow, ordinary molecular diffusion, and knudsen diffusion, or a combination of them. the governing quantity that provides a guideline in determining which mechanism is operative in a given pore under given operating conditions is the ratio of the pore size to the mean free path λ, which is calculated for a species i using the following expression [ ] : where σ i is the molecular collision diameter (m), . and . Å for water vapor and air, respectively [ ] , k b the boltzmann constant, . × − j k − , p m the mean total pressure within the membrane pores (pa), and t is the absolute temperature (k). for gaseous mixtures of two components, the mean free path and the collision diameters are different from the corresponding quantities for the pure component. the following relationship can be applied for vapor-air mixtures [ ] : under room temperature and atmospheric pressure, calculated λ for air is . m; while under vacuum conditions, mean free path for air may be several microns to several meters. knudsen number, where d p is mean pore diameter (m). when kn ≥ , the knudsen flow is dominant, the poisseuille mechanism may be neglected [ ] . actually, in most cases for hvac industry with microporous membranes, knudsen number is larger than , and poisseuille flow can be neglected, then the flow is considered to be combined knudsen and ordinary diffusion. ordinary diffusion coefficient of water vapor molecule in air is expressed by ref. [ ] d = c a t . where c a = . × − . the terms v v and v a are molecular diffusion volumes and are calculated by summing the atomic contributions: v a = . , and v v = . [ ] . m v and m a are molecular weights of vapor and air in kg mol − . m is . kg mol − for water vapor and . kg mol − for air, respectively. knudsen diffusion coefficient [ ] where r is gas constant, . j mol − k − . the effective diffusivity of combined knudsen and ordinary flow is [ ] moisture flux (kg m − s − ) is expressed by where ω is humidity difference between the two sides of l or l . consequently, water transfer in liquid membrane [ ] : where d wl is water diffusivity in liquid membrane (m s − ) and c w is the difference of water concentration in liquid membrane solution (kg m − ) between the two sides of liquid membrane. water vapor partial pressure, temperature, and licl solution concentration are governed by a thermodynamic equation [ ] log where in this equation p v , is in kpa, t in k, and m is molality of the electrolyte (mol licl/kg water). water concentration in solution is where ρ sol is solution density (kg m − ), and it is calculated by the following equation [ ] : where ρ w is pure water density at temperature t, and ρ i are given below [ ] : in moist air, water vapor partial pressure is calculated by ref. [ ] p v = ωp ω + . ( ) humidity ratio in ambient air is in the range of . - . kg/kg, therefore the above equation can be simplified to p v = . ωp ( ) as can be seen, the relations between the air humidity and water concentration in solution are rather complicated and need iterations to find solution. calculations of thermodynamic equilibrium chart of licl solution with eqs. ( )- ( ) found that under isothermal conditions, a linear equation similar to henry's sorption law can be used to express the water concentration in the solution as where k p is called the henry coefficient (kg m − pa − ) and c w is a constant (kg m − ). table lists the curve regressed values of k p and c w under different temperatures. this table gives the following correlations to estimate k v and c w from temperature: table values of k p and c w for licl solution the moisture diffusion resistance in the liquid membrane layer can be expressed by where the equivalent diffusion coefficient of vapor in liquid membrane is convective moisture resistance on air stream side is where k is convective mass transfer coefficient (m s − ). convective mass transport in the cell has been investigated by the author and co-workers previously [ ] and is expressed in terms of a correlation by where sh, re, and sc are sherwood number, reynolds number, and schmidt number, respectively. they are defined as where v is the kinematic viscosity of air (m s − ), r the radial coordinate (m), r the radius of the cell (m), and u a is the air velocity (m s − ) in radial direction. moisture conservation in air stream is represented by a onedimensional steady-state equation: boundary conditions: ref. [ ] . others are from manufacturer's specifications. mean moisture permeability across the whole membrane surface (kg m − s − )/(kg/kg), is calculated by where a c is the cross-section area of air duct (m ) and a t is the transfer area of membrane in the cell (m ). the permeability pe here represents moisture transfer rate (kg s − ) for unit area of membrane under unit transmembrane humidity difference (kg vapor/kg dry air). it reflects the performance of membrane. dimensionless radius table lists the values of operating conditions and system configurations. for each test, several minutes are needed for the system to become steady state. after outlet rh reaches steady state, water vapor permeability can be calculated with eq. ( ). this is the experimental data. the outlet rh can also be predicted with eq. ( ) . this is the model prediction. in calculations, the cell radius is divided into grids. fig. plots the relative humidity of outlet air under different air flow rates. the model predictions are also plotted in the figure. they are in agreement. maximum difference is %. fig. shows the distributions of equivalent air relative humidity on both surfaces of liquid membrane and in air stream, along cell radius. they have lower values at inlet and higher values at outlet, indicating moisture emits continuously from salt solution in lower chamber to air stream. fig. plots the distributions of equilibrium mass fraction of licl in liquid on both surfaces of liquid membrane, along cell radius. before the test, liquid membrane has a uniform mass fraction of %. during the operation, new equilibrium between liquid membrane and surrounding air relative humidity has been set-up. as a result, licl concentration in the liquid membrane re-distributed and forms a non-uniform mass fraction field. under the gradients of licl (or water) concentrations, moisture is transferred from the lower gap to the air stream. the liquid solution layer also acts as a barrier to air transfer since little air is dissolved in licl solution. fig. shows the local vapor emission rate along cell radius. as seen, the emission rate exhibits a non-uniform distribution on membrane surface. it decreases from × kg m − s − at air inlet to . × − kg m − s − at air outlet. the mean moisture emission rate is . × − kg m − s − , which is two times higher than the performance of ca membranes in our previous study with the same system ( . × − kg m − s − ) [ ] . the resulted mean permeability is . (kg m − s − )/(kg/kg). with models just proposed, various resistances in moisture transfer can be estimated. fig. shows the percentages of various resistances to total resistance, . m s kg − . as seen, the fig. . percentages of various resistances to total moisture transfer resistance, v = l min − . current cell fluid dynamics has a relatively larger convective moisture transfer resistance, accounting for % of the total resistance. the two protective layers account for % of the total resistance each. the air gap diffusion resistance amounts to less than % of the total resistance. the supported liquid layer, licl solution layer, only accounts for % of the total resistance. in real applications in mthr ventilators, only membrane resistance and convective resistance are considered. as a result, to further improve performances in future, the resistances in protective layers and flow channels should be lowered as a priority. one efficient measure may be to lower the thickness of protective layers-to a dozen micrometers, for instance. using turbulent flow arrangement such as cross-corrugated parallel plates may be the good way to lower resistance in flow channels. a composite supported liquid membrane for moisture recovery has been developed. the supported liquid layer and the two protective layers are in good contact, from sem observations. the microstructure and the performances are good, though there are some undesired cavities formed in the support layer. the measured water vapor permeation rates are two times higher than a hydrophilic solid membrane with comparative thickness. various resistances in the composite membrane and in the test cell have been clarified. it is found that the liquid layer only accounts for % of the total resistance, therefore there should be much potential for further performance improvement in future, by decreasing other resistances. that is the direction currently being pursued. dehumidification equipment advances energy wheel effectiveness. part i. development of dimensionless groups performance comparisons of desiccant wheels for air dehumidification and enthalpy recovery membrane modules for building ventilation heat and mass transfer in a membrane-based energy recovery ventilator effectiveness correlations for heat and moisture transfer processes in an enthalpy exchanger with membrane cores membrane-based enthalpy exchanger: material considerations and clarification of moisture resistance water transport in ionic polymers water transport properties of nafion membranes. part i. single-tube membrane module for air drying removal of water vapor and vocs from nitrogen in a hydrophilic hollow fiber gel membrane permeator permeation of water vapor through cellulose triacetate membranes in hollow fiber form studies on the sulfonation of poly(phenylene oxide) (ppo) and permeation behavior of gases and water vapor through sulfonated ppo membranes. ii. permeation behavior of gases and water vapor through sulfonated ppo membranes interaction of polyetherpolyurethane with water vapor and water-methane separation selectivity hollow fiber air drying water transport across polystyrenesulfonate/alumina composite membranes membrane porosity and hydrophilic membrane based dehumidification performance temperature influence on moisture transfer through synthetic films water vapor permeability and diffusivity through methylcellulose edible films diffusion-mass transfer in fluid systems on the application of a membrane air-liquid contactor for air dehumidification dehumidification of air by a hygroscopic liquid membrane supported on surface of a hydrophobic microporous membrane liquid membranes for flue gas desulfurization hydrogen sulfide separation from gas streams using salt hydrate chemical absorbents and immobilized liquid membranes equilibrium and mass transfer characteristics of -chlorophenol removal from aqueous solution by liquid membrane instability mechanisms of supported liquid membranes supported liquid membranes using ionic liquids: study of stability and transport mechanisms evaluation of moisture diffusivity in hydrophilic polymer membranes: a new approach pervaporation and vacuum membrane distillation processes: modelling and experiments mass transfer of hcl and h o across hydrophobic membrane during membrane distillation thermodynamic properties of aqueous electrolyte solutions. . vapor pressure of aqueous solutions of licl, libr, and lii properties of aqueous solutions of lithium and calcium chlorides: formulations for use in air conditioning equipment design laminar fluid flow and mass transfer in a standard field and laboratory emission cell (flec) this project is supported by national natural science foundation of china. the author also acknowledges help from graduate students-miss zhang mingrui and mr. xu xueli in doing experiments. knudsen number l height of air gap (m) m molality of electrolyte (mol licl/kg water) m molecule weight (kg mol − ) p partial pressure (pa) key: cord- -zjfhpum authors: patangi, sanjay orathi; shetty, riyan sukumar; shanmugasundaram, balasubramanian; kasturi, srikanth; raheja, shivangi title: veno-arterial extracorporeal membrane oxygenation: special reference for use in ‘post-cardiotomy cardiogenic shock’ — a review with an indian perspective date: - - journal: indian j thorac cardiovasc surg doi: . /s - - - sha: doc_id: cord_uid: zjfhpum the ultimate goals of cardiovascular physiology are to ensure adequate end-organ perfusion to satisfy the local metabolic demand, to maintain homeostasis and achieve ‘milieu intérieur’. cardiogenic shock is a state of pump failure which results in tissue hypoperfusion and its associated complications. there are a wide variety of causes which lead to this deranged physiology, and one such important and common scenario is the post-cardiotomy state which is encountered in cardiac surgical units. veno-arterial extracorporeal membrane oxygenation (va-ecmo) is an important modality of managing post-cardiotomy cardiogenic shock with variable outcomes which would otherwise be universally fatal. va-ecmo is considered as a double-edged sword with the advantages of luxurious perfusion while providing an avenue for the failing heart to recover, but with the problems of anticoagulation, inflammatory and adverse systemic effects. optimal outcomes after va-ecmo are heavily reliant on a multitude of factors and require a multi-disciplinary team to handle them. this article aims to provide an insight into the pathophysiology of va-ecmo, cannulation techniques, commonly encountered problems, monitoring, weaning strategies and ethical considerations along with a literature review of current evidence-based practices. inability to wean off cardio-pulmonary bypass (cpb) is a morbid condition associated with cardiogenic shock secondary to impaired myocardial contractility. once the vicious cycle sets in, vital organ perfusion is compromised, culminating in severe metabolic derangement. the incidence of refractory cardiogenic shock post cardiotomy ranges from . to % [ , ] with a mortality rate as high as % [ ] . veno-arterial extracorporeal membrane oxygenation (va-ecmo) has gained popularity over the years as a 'bailout' option after conventional circulatory support methods have proved refractory in the operating room (or)/intensive care unit (icu). va-ecmo facilitates luxurious end-organ perfusion and adequate gas exchange and supports organ functionality allowing time for recovery/ bridge to decision. however, its usage has not been directly linked with early positive outcomes with few articles reporting increased mortality [ ] [ ] [ ] . post-cardiotomy va-ecmo is used in both adult and paediatric populations [ ] . the worldwide incidence of instituting post-cardiotomy va-ecmo varies between . and . % [ ] . the extracorporeal life support organization (elso) database states a substantial increase in its use over the last decade. the patient population in which this therapy has been used for dealing with post-cardiotomy cardiogenic shock (pccs) included those with renal insufficiency, prior myocardial infarction, critical left main coronary artery disease, redo-surgery and severe left ventricular (lv) dysfunction [ ] . age is not a contraindication for this therapy. va-ecmo has been used to tide over patients with pccs covering the ambit of cardiac surgery and transplantation. cannulation is the first decisive step for a smooth ecmo run that can be either central (atria-aortic) or peripheral (femoral veinfemoral artery/axillary artery) (fig. ) . a meta-analysis favours the peripheral route due to lesser transfusions, bleeding/ tamponade events and lower mortality [ ] . however, these observational studies were based on a small sample size without accounting for confounding factors like the patient pre-initiation condition, effect of lv unloading and the timing of ecmo initiation. notably, the use of temporary mechanical support following pccs is associated with higher vascular complications when the duration exceeds days [ ] and incidence of amputation even with distal limb perfusion is . % [ ] . offloading the lv reduces the myocardial oxygen demand and allows for quicker myocardial recovery with higher survival rates. the decision on cannulation should be expeditious with minimal blood loss ensuring the myocardium is fully rested to hasten recovery and is fundamental in ensuring survival following pccs. techniques of venting include insertion of an intra-aortic balloon pump (iabp), atrial septostomy, percutaneous ventricular assist device (vad) or direct cannulation of the lv apex [ ] . monitoring and maintenance on va-ecmo standard monitoring includes mean arterial pressure (map), central filling pressures, temperature, pulse oximetry and urine output. cardiac output (co) measurement by thermodilution technique is overestimated whereas pulse contour analysis is unreliable due to non-pulsatility [ ] . abnormal rhythm leads to ineffective lv ejections resulting in lv distension and myocardial injury. presence of an lv vent prevents distension while reversal to sinus rhythm can be achieved by cardioversion, antiarrhythmics, pacing or ablation [ ] . lv pulsatility is assessed by arterial pressure waveform. map is maintained in a range of - mmhg to ensure vital organ perfusion [ ] . temperature monitoring is crucial as ecmo has cooling effects and temperature elevations beyond the 'defined temperature regulated range' signal infection. vascular access, urinary catheter, pneumonia and surgical wound sites are potential sources of infection [ ] . hypothermia worsens coagulopathy and platelet dysfunction [ ] . transcranial doppler, near-infrared spectroscopy (nirs) and cerebral oximetry monitoring can detect cerebral hypoperfusion and upper body hypoxaemia. nirs helps to identify lower-extremity ischaemia in peripheral arterial cannulation [ ] . in va-ecmo, total systemic flow equals the sum of pump flow plus native co. cannulae selected should provide - ml/kg/min of flow, and when flows are maintained at %, it avoids stasis in the pulmonary vasculature [ ] . sweep gas flow titration regulates carbon dioxide (co ) levels while oxygenation depends on the fraction of delivered oxygen (o ), oxygenator blood flow and exposed surface area [ ] . va-ecmo is not an indication for mechanical ventilation, and patients may be extubated on va-ecmo support [ ] . for patients who require mechanical ventilation, there is limited data on optimal ventilation strategies. extrapolating veno-venous ecmo (vv-ecmo) literature, lung protective ventilation is commonly recommended, with - ml/kg tidal volume, cm h o positive end expiratory pressure (peep) and plateau pressure < cmh o to maintain < cmh o driving pressure (inspiratory pleural pressure-peep) [ ] . unfractionated heparin (ufh) remains the mainstay of anticoagulation due to quick onset of action and rapid reversibility. heparin monitoring relies on activated partial thromboplastin time (aptt) and anti-xa activity while activated clotting time (act) is unreliable in low to moderate doses of heparin ( . to iu/kg/h) [ ] . echocardiography enables assessment of biventricular function, aortic valve opening, cannula position, pericardial and pleural effusions and ventricular thrombus formation [ ] . mixed venous saturations (scvo ) and lactate levels are monitored for adequacy of tissue perfusion and o delivery to end organs. elevated lactate levels reflect tissue hypoxia and are associated with mortality [ ] . plasma free haemoglobin (pfhb) monitoring indicates haemolysis occurring in the ecmo circuit. pfhb levels > mg/dl increase risk of thrombosis due to affinity of the von willebrand factor to platelet glycoprotein gpib [ ] . no guidelines exist to decide the optimal time of weaning. recovery of adequate cardiac and respiratory function is a pre-requisite. some authors suggest weaning as early as - h [ ] but rarely beyond days, except in cases of postheart transplantation for resolution of pulmonary hypertension [ ] . recovery of cardiac function in the post-cardiotomy setting is limited and rare beyond days [ , ] . a longer duration of support is associated with increased complications and mortality [ ] . weaning is by a multidisciplinary consensus. cardiac index, pulmonary capillary wedge pressure, central venous pressure, pulse pressure > mmhg and map > mmhg with minimal haemodynamic support are indicators of cardiac function recovery [ ] . resolution of pulmonary oedema, lung recruitment manoeuvres and clearing of airway secretions by bronchoscopy to ensure partial pressure of o (pao )/fraction of inspired o (fio ) ratio > , fio < % on the ventilator and fio < % on ecmo circuit are desirable [ , ] . although end-organ recovery to pre-ecmo levels is important [ ] , complete recovery from acute tubular necrosis can take weeks; hence, complete resolution of renal function is not mandatory before weaning. the patient can be supported with haemodiafiltration during this period [ ] . a weaning trial assesses suitability to separate from the ecmo machine. assessment of right ventricular (rv) function is crucial [ ] . ecmo flows are reduced gradually with inotropic and respiratory support whilst being monitored by clinical and echocardiographic variables. cavarocchi et al. [ ] described a -stage weaning trial using a miniaturized transoesophageal echocardiogram (tee) with % positive predictive value. lv ejection fraction (lvef) > - %, aortic velocity time integral (vti) > cm, mitral lateral annulus systolic velocity > cm/s, rv ejection fraction > . %, no lv or rv distension and ability to maintain map with minimal inotropic supports indicate the possibility of a successful wean. assessments of tricuspid regurgitation, tricuspid annular plane systolic excursion or lv filling parameters are not reliable [ ] . among clinical variables analysed, pulsatility and lactate clearance predicted a successful wean. however, no threshold was identified [ , ] . biomarkers such as nterminal fragment of the b-type natriuretic peptide (bnp), troponin i, the mid regional fragment of the proatrial natriuretic peptide, proadrenomedullin and copeptin have poor predictive ability in va-ecmo weaning [ ] . fast and slow weaning strategies have been proposed [ , ] . westrope et al. described a unique technique of pump-controlled retrograde trial off. here, the pump speed is gradually decreased to encourage reversal of flow into the ecmo circuit which creates a controlled arterio-venous (a-v) shunt without a steep drop in systemic vascular resistance [ ] . the pulmonary artery catheter is a useful monitoring tool in the weaning/post-weaning phase [ ] . levosimendan improves weaning success. it works via non-adrenergic pathways with active metabolites producing effects that last - days [ ] . inhaled nitric oxide improves rv function by reducing pulmonary vascular resistance [ ] . total vessel density and perfused vessel density monitoring of sublingual microcirculation have been found to have a good association with aortic vti, lvef parameters during ecmo wean [ ] . pccs is an uncommon occurrence with high morbidity and mortality in cardiac surgery. this low co state is refractory to high inotropic and iabp use. in this scenario, va-ecmo is a bridge to decision/recovery. va-ecmo drains blood from the venous system and inputs into the arterial system causing a reduction in rv and lv preload and improving endocardial blood flow by decreasing lv end diastolic pressure. lv afterload is increased in higher map states which distends the lv and leads to pulmonary congestion [ ] . the higher flows via ecmo improve macroand micro-circulation [ ] . non-pulsatile flow produces atrophic changes in the medial layer of the aorta and reduces vascular contractility [ ] . the ecmo oxygenator is made of microporous polypropylene membrane containing hollow fibre bundles. the input gas flows within these bundles while blood passes over it. gas exchange is by diffusion. the o uptake is determined by fio , gradient across membrane fibres, surface area of oxygenator and ecmo flow while sweep gas controls co elimination. as co transfer is six times faster compared to o , failure to clear co indicates oxygenator failure [ ] . ischaemic damage due to shunting of pulmonary blood flow, inflammatory activation, collapsed lungs, ischaemiareperfusion damage and passive congestion from lv distension contribute to ecmo-induced lung damage [ ] . extubation is recommended to reduce lung damage. however, patients have reduced alveolar ventilation secondary to co clearance by ecmo. to prevent post-extubation atelectasis, it is important to maintain co and ph levels by non-invasive ventilation along with sweep gas flow titration [ ] . although va-ecmo provides gas exchange in addition to circulatory support, native lung function is important in peripheral ecmo as myocardial and cerebral oxygen delivery is determined by o content of blood exiting the lv [ ] . studies comparing pulsatile with non-pulsatile flows have found maintained cerebral metabolism and autoregulation with both patterns provided map is > mmhg [ ] . varying levels of organ damage prior to ecmo improve after establishing adequate flows. effective venous drainage relieves congestion, further improving organ circulation. pulsatile flow enhances end organ recovery and splanchnic circulation compared to non-pulsatile flows although microcirculation is maintained in both patterns [ ] . rate of renal recovery is comparable in either flow patterns [ ] . there is no difference in clinical outcomes in either pattern provided adequate flows are maintained [ ] . lymph flow is dependent on muscle activity and pulsatility. arterial pulsation is a primary determinant of lymphatic drainage in supine patients. non-pulsatile flows lead to peripheral oedema and intestinal congestion [ ] . the blood-material interface activates coagulation, fibrinolytic and inflammatory systems releasing proinflammatory mediators that lead to endothelial injury and neutrophil activation affecting other organ systems. mast cell degranulation produces vasoplegia requiring vasoconstrictors to maintain map [ ] . major complications can be broadly divided into circuitrelated and patient-related factors ( table ). the commonest mechanical complication is thrombosis within the circuit [ ] . thrombosis starts in areas of low flow and increases turbulence. clots on the arterial side of the circuit have a risk of embolization into systemic circulation and should be immediately addressed. clots on the venous side can cause coating of oxygenator and its failure [ ] . even when visible clots are absent, microthrombi and fibrin deposits on the oxygenator reduce its efficiency over time due to suboptimal gas exchange. this is more common, but not limited to long ecmo runs. air can enter into the circuit via loosely attached connectors, inadvertently open access ports or tube defects. this can be catastrophic by bringing the pump to a halt. air on the arterial side can embolize as well. in view of the critical nature of the patient subset undergoing ecmo therapy, complications can have a significant impact on outcomes [ ] . disseminated intravascular coagulation and acquired von willebrand disease are seen in patients on ecmo due to activation of the coagulation cascade with resultant consumption coagulopathy with a - % incidence of bleeding [ ] . the commonest sources of bleed are surgical and cannulation sites. intra-thoracic, intra-abdominal and intra-cranial bleeds can also occur [ ] . incidence of reopening for tamponade or haemorrhagic complications can be as high as %, paving the way for low-dose heparin protocols during the maintenance phase of ecmo [ ] . lower antithrombin iii levels are associated with higher transfusion requirements and mortality rates [ ] . replacement of blood components is based on haematocrit (hct), act, prothrombin time (pt) and aptt ratios. point-of-care testing like thromboelastography (teg) and rotational thromboelastometry allows for quick intervention and specific corrections thereby reducing risk of volume overload and inflammatory/immunological activation. the recommended target act is - s which is reduced to - s in the event of bleeding. an international normalized ratio (inr) of > . warrants correction with fresh frozen plasma, and maintaining platelet count of > , /mm is recommended. activated factor vii (viia) is used as a last measure when other modalities have failed. extreme care is exercised when viia is used and a lower dose of - μg/kg is recommended as opposed to a conventional dose of - μg/kg to avoid an inadvertent pro-thrombotic state [ ] . ufh is the anticoagulant recommended for initiating and maintaining ecmo support as per elso guidelines [ ] . heparin-induced thrombocytopenia (hit) is associated with usage of ufh. hit is an immune-mediated pro-thrombotic condition characterized by antibodies to the heparin-pf complex on platelet surfaces which induces thrombosis. it has an incidence of . - % and mortality rate of % [ ] . bivalarudin, a direct thrombin inhibitor, is an alternative anticoagulant for hit-positive patients. teg and aptt ratio are used to monitor anticoagulation [ ] . the incidence of gastrointestinal (gi) bleeding is - . % secondary to reduced gut perfusion, decreased gastric ph leading to stress ulcers and a-v malformations in the small bowel as a result of non-pulsatile flows [ ] . other contributing factors for gi bleeding include anticoagulation, coagulopathy, thrombocytopenia, platelet dysfunction, acquired von willebrand syndrome and hyperfibrinolysis. gi bleed commonly occurs around the th day of ecmo [ ] . elderly patients and need of high-volume red blood cell transfusion were associated with higher mortality [ ] . elso registry data showed high mortality from gi bleed in contrast to recent studies [ , ] . no specific guidelines for prevention of gi bleeding are available. a meta-analysis in critically ill patients showed that proton pump inhibitor prophylaxis reduces incidence of gi bleed, albeit with higher risk of ventilatorassociated pneumonia (vap) [ ] . elso guidelines recommend correction of coagulation followed by endoscopy and endotherapy. endotherapy using haemospray, fibrin glue, cyanoacrylate, cautery and clips has been used to control gi bleeding successfully [ ] . hyperbilirubinaemia and elevated liver enzymes are commonly seen in patients on ecmo and are challenging to manage due to limited therapeutic options. pre-existing liver disease can manifest as acute liver dysfunction on ecmo. when associated with cardiorespiratory problems, passive hepatic congestion can lead to chronic changes and fibrosis [ ] . the current concept of 'two hit' ischaemic liver injury happens when a liver primed by such chronic congestion experiences acute hypoperfusion causing a rapid spike in aspartate transaminase (ast) and alanine transaminase (alt) levels followed by hyperbilirubinaemia [ ] . hyperbilirubinaemia, an independent predictor of poor outcomes, occurs due to a combination of extracorporeal haemolysis and liver dysfunction [ ] . elevated alkaline phosphatase, lactate and bnp are associated with poor outcomes [ ] , while elevated ast and alt levels do not seem to be predictive. [ ] . in cases where va-ecmo was used as a bridge to heart transplant, pre-existing liver dysfunction with total bilirubin ≥ μmol/l and inr ≥ . was a predictor of mortality [ ] . similarly, a meld unos score > has also been noted to be associated with high mortality [ ] . ischaemic hepatitis and liver congestion are self-limiting, and measures which improve cardiac function can restore liver perfusion and aid recovery [ ] . molecular adsorbent recirculation system therapy may be considered in the setting of alf as it has been noted to accelerate recovery of liver function and improve survival [ ] . acute kidney injury (aki) is seen in % of patients undergoing ecmo with complex multifactorial aetiology and pathophysiology with majority of them requiring renal replacement therapy (rrt) [ ] . the most common indication for initiating rrt is to achieve fluid balance in patients unresponsive to diuretic therapy. intermittent therapies of rrt are effective in haemodynamically stable patients; however, the most common modality used is continuous rrt (crrt). a large meta-analysis has shown higher mortality when rrt was used sparingly in patients on ecmo [ ] . rrt can be provided using either an 'integrated system' or a 'parallel system'. the integrated system could be an 'in-line' haemofilter or an rrt circuit incorporated into the ecmo circuit. however, when an integrated rrt circuit is used, there is a risk of micro-clot formation within the circuit clogging the oxygenator. hence, knowledge of intra-circuit pressures, appropriate modifications in connections of the rrt circuit and additional regional anticoagulation would be necessary. on the other hand, a 'parallel system' involves a separate indwelling vascular catheter to provide rrt, obliviating the need for additional anticoagulation as patients are already anticoagulated for ecmo. close review and readjustment of prescription is crucial as per changing needs of the patient. most studies have shown that use of crrt with ecmo is not associated with increased mortality, and in fact, when used, these subset of patients needed shorter duration of ecmo. data from large ecmo centres have shown that ecmo survivors who have received rrt have similar renal outcomes with no increase in incidence of endstage renal disease in comparison to patients who did not receive rrt [ ] . neurological complications in va-ecmo are associated with high mortality and morbidity, with high incidence ( . - %) across all age groups [ ] . this has been attributed to nonpulsatile flow, low arterial o saturation in the upper half of the body and entrainment of unfiltered thrombi into the systemic circulation [ ] . risk factors associated with neurological injury in neonates are low birth weight < kg, gestational age < weeks, pre-ecmo cardio-pulmonary resuscitation (cpr), metabolic acidosis, bicarbonate use and prior ecmo exposure [ ] . neonates are more susceptible to intracranial haemorrhage (ich) ( . %) as opposed to the paediatric group ( . %) and adults ( . %) [ ] . risk factors in adults include female gender, central cannulation during cardiac surgery, thrombocytopenia, serum creatinine > . mg/dl, hypercapnia while initiating ecmo, duration of ecmo and use of anticoagulants. incidence of acute ischaemic stroke is . - % across all age groups which is multifactorial in origin [ ] . thrombocytosis at ecmo initiation is a potentially modifiable factor [ ] . brain computed tomography scan (fig. ) is the recommended imaging modality when neurological deficits are identified since magnetic resonance imaging, despite being more sensitive, is contraindicated during ecmo [ ] . use of va-ecmo is associated with a higher incidence of electrographic seizures (fig. ) in neonates ( . %) and children ( . %) as compared to adults ( . %) [ ] . nosocomial infection is a major cause of morbidity and mortality on ecmo, with an incidence of - % [ ] [ ] [ ] . the fig. non-contrast computed tomography of the brain showing rightsided intra-parenchymal haemorrhage with peri-haemorrhagic cerebral oedema and midline shift on day of starting va ecmo rate of infection was highest in adults, followed by paediatric and neonatal age groups [ , ] . it is crucial to practice meticulous infection prevention measures. longer duration of ecmo support is an independent risk factor for infection [ ] . bizzarro et al. reported a prevalence of . % in patients on ecmo > days compared to . % in patients with < days support [ ] . bloodstream infections and vap were commonly encountered [ ] . the median time interval between initiation of ecmo and occurence of a bloodstream infection was - days [ ] . these infections were predominantly caused by gram-positive organisms (coagulase-negative stapylococcus, enterococcus and staphylococcus aureus), candida and pseudomonas species [ , ] . vascular complications stem from difficult cannulation, low flow states and high use of vasoconstrictors. percutaneous cannulation techniques in peripheral va-ecmo can be associated with posterior vessel wall perforation resulting in inadequate perfusion and subsequent development of a compartment syndrome/retro-peritoneal haematoma [ ] . larger-size cannulae (> f) usage, female gender and associated peripheral vascular disease are proven risk factors [ ] . insertion of a distal perfusion cannula should be considered to augment perfusion [ ] . differential hypoxia, north-south/harlequin syndrome, occurs in peripheral va-ecmo when the heart has recovered on the backdrop of a lung still lagging behind. the peripheral ecmo cannot compete with native co, which causes poorly oxygenated blood supply to the upper half of the body while the lower half of the body receives well-oxygenated blood from the circuit [ ] . monitoring the patient's arterial saturation in the right upper limb helps in diagnosis. remedial measures include advancement of inferior vena caval cannula and delivery of oxygenated blood into the right atrium by venoarterial-venous ecmo/ hybrid circuit [ ] . the incidence of pccs va-ecmo is . % in paediatric cardiac surgery [ ] . operative stress and residual lesions added on to a physiologically compromised heart in congenital heart disease can predispose to poor cardiac function post-operatively. there is no consensus on the timing to initiate ecmo in the paediatric population. the indications and contraindications of post-cardiotomy ecmo are listed in table . oxygen extraction ratio (o er) is the ratio between oxygen consumption (vo ) and o delivery (do ). normal o er is : or %, derived from scvo . do reduces in low perfusion states. up to a certain point, tissues maintain aerobic metabolism by extracting o from blood, increasing the central venous extraction and o er. at an o er of : , there is an imbalance between metabolic demand and aerobic metabolism thereby initiating anaerobic metabolism, producing lactate and metabolic acidosis. o consumption in infants and children ( - ml/kg/min) is more in comparison to that in fig. an excerpt of continuous eeg monitoring of the patient depicted in fig. showing right hemispheric epileptic activity adults ( - ml/kg/min); hence, anaerobic metabolism and organ damage occur earlier [ ] . ufh is the mainstay for anticoagulation in children on ecmo. as hit is rare in children [ ] , the need for alternative anticoagulants seldom arises. elso guidelines [ ] suggest a bolus dose of - units/kg of ufh before cannulation and an infusion of - units/kg/h for maintenance. besides act, aptt ratio, teg, antithrombin and activated factor xa levels are used for titration of anticoagulant. transthoracic echocardiography (tte), and catheterbased diagnostic studies aid in detection of postoperative residual lesions which, when addressed, help in weaning from ecmo [ ] . the decision to vent the heart is based on tte findings. tte assists in documenting serial ventricular function improvement, identifying pericardial/ pleural effusions, assessing pulmonary hypertension and shunting at atrial or ventricular levels [ ] . all inotropes are stopped when complete myocardial rest is indicated. map targets are achieved using vasoconstrictors and vasodilators. although there is limited experience, levosimendan ( . - . mcg/kg/min for h) can be used during ecls to aid weaning [ ] . map, capillary refill time, urine output, lactate levels and scvo trends are reliable monitoring tools. a hct of % is targeted if scvo > %. in situations of do /vo mismatch/ palliated single-ventricle patients, a higher hct (> %) is targetted. gentle ventilation to achieve - ml/kg tidal volume and a peep of up to mmhg is preferred to avoid barotrauma [ ] . ecmo circuitry triggers an inflammatory response and capillary leak, causing fluid to shift out of the intravascular compartment. in the backdrop of intravascular volume depletion secondary to bleeding, patients are predisposed to prerenal aki aggravating a pre-existing hypoxic kidney injury. once volume status is normalized within - h, the capillary leak subsides and diuresis improves [ ] . treatment options for inadequate diuresis are diuretics, peritoneal dialysis (pd) and modified ultrafiltration on the circuit. rrt has not shown to improve mortality in children on ecmo [ ] . bowel hypoperfusion secondary to low co, sedative and paralytic agent usage, vasoconstrictive drugs and gut inflammation can predispose to gastric dysmotility and feed intolerance in a child on ecmo [ ] . regional gi ischaemia causes hyperlactaemia. withholding enteral feeds translocates gut bacteria thereby increasing the risk of sepsis. starting of trophic feeds once lactate has normalized is a recommended strategy [ ] . parenteral nutrition is considered when there is persistent hyperlactataemia or feed intolerance taking on board the risk of infection [ ] . severe irreversible brain injury extremely low gestational and weight (< weeks gestation or < . kg) uncontrollable haemorrhage fig. a-v bridging on ecmo consists of a circuit that runs parallel to the patient with the cannulae in situ but clamped. heparin flush is constantly circulated through the cannulae during the clamped interval to prevent clotting. if there is haemodynamic or respiratory instability, va-ecmo is recommenced by just removing the clamps on the venous and arterial cannulae the timing and sequence of weaning off ecmo is not standardized. once the myocardium shows signs of recovery, organ functions have improved and residual lesions are ruled out or corrected, full ventilatory support is commenced, inotropes started to augment cardiac contractility and ecmo flows are gradually reduced. the left-sided vent, if present, is removed when sustained ejections are seen. on further weaning to minimal ecmo flows ( ml/min), tte is done. decision is then taken to decannulate the patient. in borderline cases, a-v bridging with a trial period off ecmo ( - h) is an option. this consists of a circuit that runs parallel to the patient with the cannulae in situ but clamped (fig. ) . during a-v bridging, if there is haemodynamic or respiratory instability, va-ecmo is recommenced. if the trial period off ecmo is uneventful, the patient is decannulated [ ] . tte plays a pivotal role during various stages of weaning and in the post-wean phase [ ] . palliative surgery for univentricular hearts poses special challenges on ecmo. in infants with single ventricular physiology and systemic to pulmonary shunts, the shunt has to be partially occluded to counter pulmonary steal. the alternative is to target higher flows [ ] . the success rate for ecls, post glenn and fontan procedures is lower. the reasons are multifactorial, including complex physiology and altered cardiac anatomy, the presence of atrioventricular valve regurgitation, previous surgeries, the need for multiple drainage cannulae and the inability to achieve full flows on ecmo [ ] . success after ecmo typically is defined as survival to hospital discharge after a successful wean [ ] . for pccs va-ecmo, the average survival rate across all age groups is . % [ ] . the indicators of poor outcome have been outlined as advanced age, redo-valve surgery and climbing lactate levels. pre-operative pulmonary hypertension had no bearing on mortality [ ] . the -year survival rate was % for patients discharged from the hospital while % for patients surviving at days [ ] . discharge from hospital is the most important predictor of successful outcome [ ] . lai et al. concluded that a small percentage of patients especially in a high-volume centre would benefit from additional ecmo therapy if their cardiopulmonary function declined. additionally, a higher incidence of infection and need for rrt was reported in this subset [ ] . literature per se is nebulous about the quality-of-life predictors after ecmo. studies have shown % mortality within months of hospital discharge and this increases to % over a -year period [ ] . on the contrary, studies have depicted . % survival at months without elaborating on the quality of life [ ] . the new york heart association (nyha) class is a good tool for ascertaining functional status in ecmo survivors [ ] . functionality status of survivors have been diverse, ranging from nyha class i-iv indicating survivors can achieve a reasonable quality of life. chen et al. have reported a higher readmission rate during the first year of follow-up resulting in higher medical expenditure. infections and cardiac events were stated as common causes of mortality/morbidity in the follow-up period [ ] . literature has extensively debated on the cost-effectiveness of ecmo. several analyses have deemed ecmo to be an expensive therapy amounting to an average of $ , per case in the usa and have recommended package payments to buoy individual institutes delivering this service [ ] . in the uk, mechanical support is funded only when it has been advocated as a bridge to transplantation [ ] . there has been no such cost analysis done in the indian subcontinent. the average cost of initiating ecmo in india is meagre compared to the west. however, additional costing gets added on to patient maintenance in the icu, which could include rrt, medication, imaging, laboratory and transfusion costs. by advocating a wholesome package module, the implications of cost can be negated with the outcome benefits. from an ethical viewpoint, three perspectives have to be considered: surgeons, the patient's family and financial implications. a surgeon would look forward to successful outcomes, but, when faced with a situation of inability to wean off cpb, it is normal to consider options for recovery even if chances of successful outcome is low. the patient's family should be briefed about the condition and given an opportunity to partake in the decision-making. financial implications of ecmo need be explained as healthcare in india is not funded. though the cost of initiation of ecmo is finite, duration and end-point are difficult to ascertain and costs are impractical to calculate. there are situations where hospitals or other payers like insurance will meet the additional expense, but that is the exception rather than the rule. these nuances should be discussed and documented to enable the family to make an informed decision. counselling and communication is key to supporting the family through this tumultuous phase. va-ecmo has made inroads into the management of pccs in india. bearing its cost implications, it has yet to make a significant impact in the management of these subset of patients. at narayana institute of cardiac sciences (nics), bangalore, india, we on an average perform about - ecmo runs a year, a majority of which are post-cardiotomy ecmo runs. considering the factors influencing cannulation, our institutional preference is the central route. if the patient presents with pccs, our cannulation sites are the right atrial appendage and the ascending aorta close to the sino-tubular junction. we have observed harlequin syndrome in those patients with high aortic cannulation, and these patients presented with persistent ventricular fibrillation and altered sensorium. this was due to deoxygenated blood from the lv selectively streaming into coronaries and the right innominate artery. this was promptly reversed by changing to a lower cannula position. cannulae are snugged using rubber spigots, following which they are tunnelled sub-xiphoid and chest closed with sternal wires. absolute haemostasis is imperative. a common bleeding point is the aortic cannulation site. for this, rows of purse strings are used that encompass the cannula twice causing a cuff of adventitia to evert around the cannula to stop further bleeding. for lv venting, our strategy is to cannulate the lv apex directly with a separate limb to the inflow circuit (fig. ) . we keep a dedicated flow sensor on this limb to detect low flow. an obvious disadvantage of this is formation of a lv clot around the cannula inflow. this is prevented by maintaining a higher aptt ratio and using a larger-sized cannula. the decision to vent the left heart is based upon the disappearance of ejections on the arterial trace, presence of spontaneous echo contrast on tee, inadequate flows with obvious signs of lv distention and to prevent harlequin syndrome in peripheral cannulation. we strongly believe in central cannulation for the following reasons: at our institute, the weaning process starts in the icu and the final stage happens in the or under tee guidance. the patient is primed with levosimendan overnight without a loading dose. the ventilator is adjusted for optimal gas exchange. haemodynamic variables are monitored using echocardiography and a pulmonary artery catheter. metabolic stability is monitored by trends in lactate level, base excess in arterial gases and scvo monitoring. anticoagulation is maintained with aptt - s or act - s. flows are reduced by . l/min every nd hourly till a flow of % is achieved. if a lv vent is in situ, a gated clamp regulates the lv vent flows. flows are reduced in aliquots of ml every second hour till a flow of ml is reached. the patient is idled at this flow till transfer to the or for final wean and decannulation. weaning is aborted at any stage if there is ventricular distension, increase in inotropic levels to maintain haemodynamics, worsening gas exchange or metabolic parameters. we performed a total of , cardiac surgeries in and . pccs va-ecmo was instituted in ( . %) of these patients. our results are broadly outlined in table . bleeding was a significant problem. major transfusions were needed in the initial -h period. the transfusion requirements were monitored with th hourly clotting screens and teg. the incidence of aki with rrt requirements were comparable across age groups which is similar to published data [ ] . our preference for instituting rrt is using a parallel circuit to prevent possible air embolism. rrt in the paediatric patients was provided by pd. a rising trend of total bilirubin was associated with failure to wean and mortality. overall mortality of ecmo patients with limb ischaemia was . %. literature has shown higher incidence of neurological [ ] [ ] [ ] in contrast to our data. the incidence of gi bleed in our cohort was . % which compares favourably with published data [ , ] . better weaning results were observed between and (table ) a review of the bloodstream infections in patients on pccs va-ecmo revealed a predominance in gramnegative bacilli (gnb) ( %), followed by candida species ( %) and gram-positive organisms ( %). klebsiella species constituted the majority of the gnb bacteraemias. carbapenem resistance was present in % of the gramnegative organisms isolated ( % carbapenem resistance in klebsiella species alone). most of the blood culture isolates grew within the first h of incubation. however, % of the organisms grew only after days of incubation, highlighting the 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complications during extracorporeal membrane oxygenation in children: a single-center experience in runs arterial complications in patients undergoing extracorporeal membrane oxygenation via femoral cannulation thromboembolic events in patients on extracorporeal membrane oxygenation without anticoagulation hypoxia and complications of oxygenation in extracorporeal membrane oxygenation perioperative mechanical circulatory support in children: an analysis of the society of thoracic surgeons congenital heart surgery database physiology of gas exchange during ecmo for respiratory failure heparin-induced thrombocytopenia in paediatrics: clinical characteristics, therapy and outcomes pediatric cardiac failure, extracorporeal life support organization outcomes of pediatric patients undergoing cardiac catheterization while on extracorporeal membrane oxygenation the role of echocardiography in neonates and pediatric patients on extracorporeal membrane oxygenation cardiopulmonary resuscitation in infants and children with cardiac disease: a scientific statement from the the complex relationship of extracorporeal membrane oxygenation and acute kidney injury: causation or association? outcome of pediatric patients treated with extracorporeal life support after cardiac surgery nutritional practices and adequacy in children supported on extracorporeal membrane oxygenation guidelines for the provision and assessment of nutrition support therapy in the pediatric critically ill patient: society of critical care medicine and american society for parenteral and enteral nutrition a comparison of pumpcontrolled retrograde trial off to arterio-venous bridging for weaning from venoarterial extracorporeal membrane oxygenation extracorporeal membrane oxygenation for infant postcardiotomy support: significance of shunt management fontan physiology revisited clinical outcomes in patients after extracorporeal membrane oxygenation support for postcardiotomy cardiogenic shock: a single-centre experience of cases outcomes of venoarterial extracorporeal membrane oxygenation patients requiring multiple episodes of support longterm survival in adults treated with extracorporeal membrane oxygenation for respiratory failure and sepsis long-term prognosis after extracorporeal life support in refractory cardiogenic shock -results from a real-world cohort patients' self-assessed functional status in heart failure by new york heart association class: a prognostic predictor of hospitalizations, quality of life and death long-term outcomes of extracorporeal membrane oxygenation support for postcardiotomy shock financial and clinical outcomes of extracorporeal mechanical support the comparative use of ventricular assist devices: differences between europe and the united states publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements we would like to acknowledge the help and insights provided by dr. julius punnen, dr. varun shetty, dr. rammohan sripad bhat, dr. vijay samuel richard, dr. rangarajan kasturi, dr. radhika manohar, dr. shrinivas hittalamani and dr. anup daniel varghese who are specialists in their fields in writing this document. we would also like to mention the assistance provided by mr. selvakumar, senior key: cord- -choywmwd authors: nan title: membrane protein sorting in the yeast secretory pathway: evidence that the vacuole may be the default compartment date: - - journal: j cell biol doi: nan sha: doc_id: cord_uid: choywmwd the targeting signals of two yeast integral membrane dipeptidyl aminopeptidases (dpaps), dpap b and dpap a, which reside in the vacuole and the golgi apparatus, respectively, were analyzed. no single domain of dpap b is required for delivery to the vacuolar membrane, because removal or replacement of either the cytoplasmic, transmembrane, or lumenal domain did not affect the protein's transport to the vacuole. dpap a was localized by indirect immunofluorescence to non-vacuolar, punctate structures characteristic of the yeast golgi apparatus. the -amino acid cytoplasmic domain of dpap a is sufficient for retention of the protein in these structures, since replacement of the cytoplasmic domain of dpap b with that of dpap a resulted in an immunolocalization pattern indistinguishable from that of wild type dpap a. overproduction of dpap a resulted in its mislocalization to the vacuole, because cells expressing high levels of dpap a exhibited vacuolar as well as golgi staining. deletion of residues of the dpap a cytoplasmic domain resulted in mislocalization of the mutant protein to the vacuole. thus, the cytoplasmic domain of dpap a is both necessary and sufficient for golgi retention, and removal of the retention signal, or saturation of the retention apparatus by overproducing dpap a, resulted in transport to the vacuole. like wild type dpap b, the delivery of mutant membrane proteins to the vacuole was unaffected in the secretory vesicle-blocked sec mutant; thus, transport to the vacuole was not via the plasma membrane followed by endocytosis. these data are consistent with a model in which membrane proteins are delivered to the vacuole along a default pathway. ar~y proteins that reside in the organelles of the secretory pathway of eukaryotic cells have targeting information that directs retention in or sorting to the appropriate compartment (pfeffer and rothman, ) . in the absence of retention or sorting signals, soluble proteins of the secretory pathway are secreted; thus, the default pathway for these proteins is secretion (burgess and kelly, ; pelham, ) . in saccharomyces cerevisiae, mutations in the targeting signal of the soluble vacuolar protein, carboxypeptidase y (vails et al., ) , or the retention signal of the soluble er protein, bip (hardwick et al., ) , result in the secretion of these proteins. likewise, the flow of membrane proteins to the cell surface of nonpolarized mammalian cells is apparently by default, because mutations that disrupt the retention of er or golgi-retained membrane proteins (machamer et al., ; machamer, ; jackson et al., ) or the sorting of a lysosomal membrane protein (williams and fukuda, ) result in localization to the plasma membrane. little is known regarding membrane protein sorting in s. cerevisiae, although a previous study suggested that the cell surface is the default compartment for membrane proteins (fuller et al., b) . in this paper, we characterize the targeting of two membrane proteins of the yeast secretory pathway, dap dipeptidyl aminopeptidases (dpaps) ~ dpap a and dpap b of the golgi apparatus and vacuole, respectively. the biogenesis of two membrane proteins of the yeast vacuole, dpap b (see fig. a), and alkaline phosphatase (alp), has been characterized (klionsky and emr, ; roberts et al., ) . both dpap b and alp are type ii membrane glycoproteins (nomenclature of singer, ) , consisting of nh -terminal cytoplasmic domains of approximately amino acids, single hydrophobic membrane anchors, and cooh-terminal lumenal catalytic domains. these proteins transit the early compartments of the secretory pathway (i.e., er and golgi), but not the later compartments (i.e., secretory vesicles), indicating that the proteins do not transiently reside at the plasma membrane before delivery to the vacuole. the localization signals of these two proteins have not been identified, although the lumenal domain of alp has been shown to be unnecessary for vacuolar targeting (klionsky and emr, ) . the biosynthesis of several membrane proteins that reside this paper is dedicated to the memory of david merrill stevens. in the yeast golgi apparatus has also been examined. in yeast, three membrane-bound proteases, kex p, kexlp, and dpap a (see fig. a) process the mating pheromone a-factor precursor polypeptide as it traverses the secretory pathway (bussey, ; fuller et al., ) . the biosynthetic pathways of kex p and kexlp have been characterized (fuller et al., a,b; cooper and bussey, ) , and kex p has been shown to function in a late golgi compartment (julius et al., ; graham and emr, ) . kex p has been localized by indirect immunofluorescence to three to six punctate structures per cell that exhibit a somewhat random distribution within the cytoplasmic compartment (franzusoff et al., ; redding et al., ) . thus, the golgi apparatus of yeast is not localized to a perinuclear location, as in mammalian cells, but rather is dispersed throughout the cell. both kexlp (a. cooper and h. bussey, manuscript submitted) and dpap a (see below) have been immunolocalized to punctate bodies that are similar to those containing kex p in size, abundance, and distribution. in this paper, a combined gene fusion and mutational analysis was used to show that no single domain of dpap b is required for vacuolar localization. furthermore, both overproduction of dpap a and a mutation in the cytoplasmic domain of dpap a resulted in mislocalization of this protein to the vacuole. finally, the fusion proteins analyzed in this work are shown to be transported directly to the vacuole from the golgi, and not to the plasma membrane. the bacterial strain mc (casadaban and cohen, ) was used for all subcloning steps. oligonucleotide-directed mutagenesis was carded out in strain cj (kunkel et al., ) . the yeast strains used were jhry - aaia (mata, dap a: : h s , stel a : :leu , ura - , leu - , eu , his -a , pep - ; roberts et al., ) , sey zs (match, dap :: leu , emr et al., ) , cjry - b (mata dap a : :leu mnn ura - leu - leu , and sey- ( mata, s e cl-l , dap : : le u , ura - , le u - , leu ) . yeast cultures were grown in yepd or minimal (sd) medium supplemented with the appropriate nutrients as previously described (sherman et al., ) . for the simultaneous induction of the gal/ promoter and the secl secretion defect, ceils were grown to log phase in minimal media plus raltinose and then harvested and resuspended in yep-raitinose. after a -h incubation at ~ galactose was added directly to the cultures and at the same time the cultures were shifted to ~ after h, the cells were fixed immediately and prepared for indirect immunofluorescence as described below. oligonucleotides for mutagenesis were prepared by the university of oregon biotechnology laboratory on an applied biosystems b dna synthesizer (foster city, ca) as described (ito et al., ) . tran s-label and zymolyase t were from icn biomedicals (irvine, ca), endo h was from boehringer marmheim (indianapolis, in), ultra-pure sds was from bdh biochemicals (san francisco, ca), glusulase was from dupont pharmaceuticals (wilmington, de), and all antibodies (except anti-dpap b, anti-dpap a, anti-alp, and anti-vat p antibodies) used for indirect immunofluorescence were from jackson immunoresearch (west grove, pa), cappel products (malvern, pa), or promega biotech (madison, wi). all other reagents were from sigma chemical co. (st. louis, mo). dpap and invertase assays were performed as previously described (gildstein and lampen, ; roberts et al., ) . restriction endonuclease digests and ligations were performed as recommended by the suppliers. plasmid purification, agarose gel electrophoresis, fill-in reactions of sticky-ended dna fragments using t dna polymerase, and dna-mediated transformation of escherichia coli were done according to standard procedures (maniatis et al., ) . lithium acetate transformations of yeast were performed as described (jto et al., ) . a disruption of the chromosomal ste/ locus was constructed by onestep gene disruption (l~thstein, ) , using the plasmid pslk (kindly provided by dr. george sprague). psl consists of pbr containing a . kbp bamhi ste/ fragment, from which a . -kbp bcli fragment within the coding region of the dpap a lumenal domain (c. a. flanagan, d. a. barnes, m. c. flessel, and j. thorner, manuscript submitted for publication) was replaced by the . -kbp bglii leu fragment. to create a strain lacking both dpap a and dpap b, a disruption of the chromosomal dap locus with the his gene was made using the plasmid pgp , which contains the . -kbp ecori-bamhi his fragment (sikorski and hieter, ) in place of the l -kbp bsteii-kpni portion of the coding region of dap (roberts et al., ) . the c~-factor signal sequence was fused to the lumenal domain of dpap b as follows: a sail linker was inserted at the hincll site of plasmid p , which contains a portion of the ' region of the mfcd gene (kurjan and herskowitz, ) , including bp of non-coding region (not including the uas) and bp of coding region, including the signal sequence, signal peptidase cleavage site (waters et al., ) , and nh -terminal residues of pro-a-factor, fused to the kre/gene (boone et ai., ) . the acci site at position + of the dap gene was changed to a sali site using oligonucleotide-directed mutagenesis (kunkel et al., ) . mutagenesis of dap was performed using the vector pcjr , which contains the . -kbp bamhi-hindiii da/' fragment in the plasmid ks + (stratagene, san diego, ca). the . -kbp sali-hindui fragment, encoding the lumenal domain of dpap b, was inserted into the sali-hindiii sites of p /sali, fusing the coding regions of mfal and dap in frame, c~fss-b was placed under the control of the ga/_j promoter (johnston and davis, ) by inserting the . -kbp bamhi-hindiii fragment from this plasmid into the bamhi-hindiii sites of pc jr , which contains the -bp ecori-bamhi ga/_,/ promoter fragment inserted into ecori-bamhi sites of the cen plasmid pseyc ca modified version of pseyc ; emr et al., ) . the resulting fusion protein consists of the nh -terminal residues of prepro-afactor and two residues arising from linker sequences fused to residue of the lumenal domain of dpap b (fig. l b ; amino acid sequence nh -mrfpsiftavlfaassala-apvgrphh . . . ). the bb-inv fusion protein expression vectors, pcjr and pc jr ( p and cen plasmids, respectively), were constructed by inserting the . -kbp bamhi-acci fragment (acci blunt ended) into the invertase fusion vectors psey (a derivative of the /z plasmid psey ; emr et al., ) and the cen plasmid pseyc (johnson et al., ) at the bamhi and hin-diii sites (hindlij blunt ended). the resulting fusion protein contains the nh -terminal residues of dpap b fused to residue three of cytoplasmic invertase ( fig. b) . bb-inv was placed under the control of the gal/promoter by cutting pcjr with ecori and hiediii and ligating in the -bp gal/fragment (johnston and davis, ) . this fused the ' end of the gal/fragment at nucleotide - of the dap sequence. vectors encoding a -bb and a -bb were constructed as follows. oligonucleotide-directed mutagenesis (kunkel et al., ) was used to create -and -bp deletions in dap (a -bb and a -bb, respectively) in pcjr . the ~ . kbp bamhi-hindiii mutant dap fragments were inserted into the bamhi-hindiii sites of the cen plasmid pseyc , creating pmnh ( -bb) and pc jr (a -bb). & -bb and a -bb were over-produced by replacing the dap promoter with the gal/promoter as follows. the hindiii site at - of dap was blunt ended and religated, creating an nhei site. the ~ -kbp nhei-hindiii fragments of pc jr and pc jr were inserted at the xbai-hindiii sites of pc jr , creating pc jr and pc jr , respectively. the a and a deletions removed residues - and - of dpap b, changing the nh -terr~nal acid sequence of dpap b from nh -meg-geeeveripdelfdtkkkhlldklirv to nh -mhlldklirv and nh -mirv, respectively ( fig. b) . the pt plasmid encoding dpap a, pcjr , was constructed as follows: the . -kbp xbai-bamhi fragment, containing the stf_j gene (c. a. flanagan, d. a. barnes, m. c. flessel, andj. thorner, manuscript submitted for publication) was isolated from the plasmid p - (julius et al., ) and inserted into puc . the . -kbp sali-bami-ii fragment from this vector was inserted into the xhoi-bglii sites of the # plasmid pckr (c. raymond and t. stevens, unpublished results) . the cen plasmid pcjr was made by inserting the . -kbp eagi-pvuii ste/ fragment into the eaglecorv sites of prs (sikorski and hieter, ) . the plasmid pc jr , encoding the fusion protein aa-b, was constructed by inserting the . -kbp bamhi-mlui (mlui blunt ended) fragment from p - , encoding the stf.j promoter and the cytoplasmic and trans- membrane domains of dpap a, and the . -kbp sall-hindlii fragment of dap (sail blunt ended), encoding the lumenal domain of dpap b, into the bamhi-hindlii sites of pseyc . the . -kbp bamhi-hindlii fragment of the resulting vector (pcjr ) was cloned into the bamhi-hindlii sites of the # plasmid, psey (emr et al., ) . the resulting plasmid encodes aa-b, which consists of the nh -terminal residues of dpap a to residue of dpap b (fig. c) . for the construction of the fusion protein a-bb, xbai sites were created in both the dap and ste/ genes just upstream of the coding regions of the transmembrane domains of dpap b and dpap a, respectively, using oligonucleotide-directed mutagenesis (kunkel et al., ) . nucleotides and of the dap gene were changed from gt to tc (roberts et al., ) , and nucleotides - of ste/ were changed from gcc to aga (with the a of the initiation codon as ) (c. a. flanagan, d. a. barnes, m. c. flessel, and j. thorner, manuscript submitted for publication). a . kbp saci-xbai fragment (encoding the ste promoter and the cytoplasmic domain of dpap a) and a . -kbp xbal-hindlii dap fragment (encoding the transmembrane and lumenal domains of dpap b) were ligated into the saci-hindlii sites of psey . the resulting plasmid (pcjr ) encodes a protein consisting of residues of the nh -terminal cytoplasmic domain of dpap a fused to amino acid of dpap b (fig. c) . the fusion protein b-a-b was constructed as follows: the . -kbp bamhi-hindui dap fragment, including the xbal site at + , was ligated into the bamhi-hindlii sites of pseyc . a . -kbp xbal-hindlii fragment of ste/ , encoding the transmembrane and lumenal domains of dpap a, was inserted into the xbai-hindlii sites, resulting in the plasmid pc jr . the . -kbp sali-hindiii dap fragment (sali blunt ended), encoding the dpap b lumenal domain, was inserted into the mlui-hindlii sites (mlui blunt ended) of pcjr , resulting in pcjr , a cen plasmid encoding b-a-b. the . -kbp bamhi-hindlii fragment from pcjr was inserted into the same sites of psey , resulting in pcjrt . b-a-b was placed under the control of the gal/promoter by cloning the nhei-hindlii b-a-b fragment into the xbai-hindlii sites of pcjr , creating pc jr . the b-a-b fusion protein consists of residues - of dpap a in place of residues - of dpap b ( fig. c) . oligonucleotide-directed mutagenesis of the portion of ste/ encoding the amino acid cytoplasmic domain of dpap a ( fig. a) was performed in pcjr , which consists of the . -kbp eagi-psti fragment of ste/ inserted into the eagi-psti sites of ks +. a -bp in-frame deletion, removing the amino acids - (z~ ), was created, and the sacl-mlui fragment from this plasmid was inserted into the saci-mlui sites of pcjr , creating the # plasmid psn , which encodes a -aab ( fig. c) . the same saci-mlui fragment was ligated with the -kbp mlui-hindlll (encoding the lumenal domain of dpap a) into the saci-hindiii sites of psey , creating psn , which encodes a -aaa ( fig. c) . a -aa-b was placed under the control of the gal/promoter by fusing a saci-eagi fragment from pcjr (contains the bp hindlii-ecorl gal/fragment in pgem- zf) to an eagl-hindlii fragment from psn , both of which were ligated into the saci-hindlii sites of psey , creating psn . the fusl-laczp expression plasmid pcjrll as follows: a -kb nhei-hindlli fragment, encoding the nh -terminal amino acids of the fus protein fused to ~-galactosidase, under the gall promoter (isolated from pcjrll , a derivative of psb (trueheart and fink, ) ) was cloned into the xbal-hindlll sites of pvt u, a # plasmid (vernet et al., ) , creating pcjr . for the production of dpap a antigen to be used to generate dpap a antiserum, the plasmid pcjr was constructed by inserting the . -kbp mlui-hpal (mlui blunt ended) ste/ fragment into the sinai site of pexp , an e. coli expression vector containing the tac promoter just upstream of the translational start codon of t lysozyme and a multiple cloning site (raymond et al., ) . a amino acid peptide, corresponding to the nh -terminal residues of dpap b followed by a cooh-terminal cysteine residue, was synthesized on an applied biosystems peptide synthesizer. the peptide was coupled to carboxymethylated bsa through the cooh-terminal cysteine with the hifunctional crosslinking agent, mbs, following the manufacturer's recommendations (pierce chemical co., rockford, il), and through lysine residues with gluteraldehyde as previously described (kagen and glick, ) . a : mixture of bsa-peptide conjugates prepared by the two methods was used to immunize rabbits as described previously (vaitukaitis, ) . for affinity purification of the antibody, the poptide was cross-linked to tresyl-activated sepharose b (pharmacia fine chemicals), and affinity purification was carded out as described (raymond et al., ) . dpap a antigen was produced in e. coli cells containing the plasmid pc jr . induction with iptg resulted in the production ofa -kd protein that corresponds to the nhe-terminal portion of the lumenal domain of dpap a (see fig. ) fused to seven residues of t lysosyme and seven residues encoded by the pexpi polylinker. antigen purification was performed as described previously (raymond et al., ) . immunoprecipitations were performed by growing cultures to log phase in supplemented minimal media lacking methionine and cysteine, then pulse labeling in the same media with tran ss-label and chasing by adding /~g/ml methionine and /~g/ml cysteine, followed by the addition of sodium azide to ram. the cells were converted to spheroplasts (stevens et al., ) which were lysed in % sds, m urea plus a protease inhibitor cocktail ( . mm pmsf, /~g/ml leupeptin, and /~g/ml pepstatin) for rain at ~ and adjusted to ml in ip buffer (pbs, . % sds, . % triton x- , mivl edta). for precipitating ~fss-b from the extracellular fractions, the medium was supplemented with m ,,/ml bsa and mm potassium phosphate, ph . . after spberoplasting, the periplasmic and media fractions were pooled and adjusted to ml in ip buffer plus protease inhibitors. after pre-adsorbtion to . % iggsorb, anti-dpap b coohterminal antibody (roberts et al., ) , or anti-dpap a antibody was added, and samples were incubated one hour on ice. iggsorb was added to . %, followed by h on ice, and the immune complexes were precipitated and washed twice with ip buffer. the immune complexes were solubilized, and half of the samples were treated with endoglycosidase h (endo h) overnight at ~ as described (orlean et al., ) . samples were analyzed by sds-page and fluorography as described previously (stevens et ai., ) . the secretion of t~fss-b was quantified using an ambis radioanalytic imaging system (ambis systems, inc., san diego, ca). the fractionation of membranes in the presence of high ph sodium carbonate was performed as described (roberts et al., ) . preparation of fixed, spheroplasted cells for indirect immunofluorescence was carried out essentially as previously described (roberts et al., ) , except that the fixed spheroplasts were treated with % sds for - minutes. antibody adsorption against fixed spheroplasts harboring null mutations in either dap or stej was performed as described elsewhere (raymond et al., ; roberts et ai., ) . the fixed spheroplasts were stained with a : dilution of adsorbed anti-dpap a or anti-dpap b affinity-purified antibody in pbs-bsa (roberts et al., ) . for co-localization with the -kd subunit of the vacuolar h+-atpase (vat p, the product of the vat gene; yamashiro et al., ) , the antibody solution also contained a : dilution of the mab, dll (kane et al., ) . the dpap b or dpap a staining pattern was amplified by subsequent incubations with goat antirabbit antibody conjugated to biotin, followed by a streptavidin-fitc conjugate. the last antibody incubation also contained goat anti-rabbit antibody conjugated to rhodamine. co-detection of alp and fusl-laczp was performed by staining cells with a : dilution of a rabbit polyclonal anti-alp antibody (raymond et al., ) and a : , dilution of a mouse monoclonai anti-/~-gaiactusidase antibody, followed by antibody amplification identical to that used for dpap a and dpap b. the cells were mounted in media containing dapi for staining nuclei, and photomicrographs were made as described previously (roberts et al., ) . the role of the lumenal domain in the sorting of dpap b to the vacuole was addressed in two ways. first, to determine if the lumenal domain was sorted to the vacuole when expressed in the secretory pathway as a soluble protein, a gene fusion was used to create the protein ctfss-b, consisting of the nh -terminal er-targeting signal sequence of prepro-t~ factor fused to the lumenal domain of dpap b at residue ( fig. b) . the signal sequence should direct the translocation of the protein into the er lumen and be cleaved, rendering the lumenal domain a soluble protein in the secretory figure . immunoprecipitations of c~fssb from mnn and mnn strains. jhry - a ( dap a mnn ) or cjry - b ( dap a mnn ) cells were labeled with tran ss-label for rain and chased for min in the presence of izg/ml methionine and /~g/ml cysteine. cultures were separated into two fractions, intracellular (i.e., spheroplasts) and extraceuular (i.e., periplasmic and media), and immunoprecipitated with affinity purified dpap b antibody. half of each immunoprecipitation sample was treated with endo h, and equal amounts were analyzed by sds-page and fluorography. positions of the molecular weight standards are indicated (in kd). pathway. the construct was analyzed in a strain which contains a null allele of dap , the structural gene for dpap b (suarez rendueles and wolf, ) . fig. shows the results of immunoprecipitating o~fss-b from intraceuular and ex-traceuular (i.e., combined periplasmic and medium) fractions of s-labeled cells using an antibody that recognizes the cooh-terminal half of dpap b (roberts et al., ) . analysis of the samples by sds-page and fluorography showed that % of txfss-b was secreted as a heterogeneous population of highly glycosylated species (fig. , lane ) , similar to the secreted protein invertase (esmon et al., ) , whereas the intracellular fraction contained a tightly migrating species (fig. , lane /). treatment of the immunoprecipitates with endo h to remove n-linked carbohydrate demonstrated that the difference in the apparent mobilities was due to glycosylation (fig. , lanes and ) . the glycosylation pattern of the secreted material differs from that of wild type dpap b, which receives only modest glycosyl modifications of the core oligosaccharides in the golgi apparatus (roberts et al., ) . to test whether the alteration in glycosylation caused the secretion of the lumenal domain, otfss-b was expressed in an mnn mutant, which is deficient in the addition of the extensive t~ , outer chain glycosyl groups (kukuruzinska et al., ) . fig. , lanes and , show that t~fss-b was secreted to the same extent ( %) from mnn cells, even though the protein was not aberrantly glycosylated. indirect immunofluorescence microscopy showed that the portion of the lumenal domain that remained intracellular was retained in the er, and no staining of the vacuole was observed (data not shown). unlike the secreted material, the er-retalned material was enzymati- (suc -a ) cells containing a plasmid encoding bb-inv (pcjr ) were labeled with tran s-label for rain and chased for rain in the presence of /~g/ml rrw, thionine and /~g/ml cysteine. the cells were converted to sphemplasts, and extracts immunoprecipitated with either affinity-purified dpap b antibody (lanes - ) or affinity-purified invertase antibody (lanes and ) . half of each immunoprecipitation sample was treated with endo h, and equal amounts were analyzed by sds-page and fluorography. positions of the molecular mass standards for lanes - are indicated (in kd) on the left, whereas standards for lanes and are indicated on the right. cally inactive (data not shown), and thus may be unfolded and incompetent to exit the er (rose et al., ; rothman, ) . the role of the lumenal domain of dpap b in vacuolar targeting was also tested by constructing the fusion protein bb-inv, in which the nh -terminal residues of dpap b were fused to the non-vacuolar protein, invertase (fig. b) . immunoprecipitations of bb-inv using anti-invertase antibody, followed by sds-page and fluorography, showed that the fusion protein was glycosylated, and treatment with endo h showed that the protein was of the expected size (fig. , lanes and ) . bb-inv fractionated with membranes under high ph carbonate conditions, consistent with bb-inv being an integral membrane protein (data not shown). invertase enzyme assays of either permeabilized or non-permeabilized whole cells deleted for the invertase structural gene and expressing bb-inv from a cen plasmid showed that < % of the total invertase activity was extracellular. the subcellular localization of bb-inv was determined by indirect immunofluorescence microscopy using an antibody that recognizes the cytoplasmic domain of dpap b (see materials and methods). fig. shows the staining pattern of wild type dpap b (fig. , a-c) and bb-inv (fig. , g-i) when expressed from the high copy number ( #) plasmids, pgp (roberts et al., ) and pcjr , respectively. for these and the other # plasmid constructs used in this study, the proteins were overproduced - -fold as determined by enzyme activity. both dpap b and bb-inv were localized to the vacuolar membrane as judged by differential interference contrast (nomarski) optics and co-localization with a marker for the vacuolar membrane, the -kd subunit of the vacuolar h+-atpase (vat p; yamashiro et al., ; table i ). dpap b and bb-inv were also localized to the vacuolar membrane when expressed from a single copy (cen) plasmid (data not shown). aside from the differences in signal intensity, no difference in subcellular localization was observed when bb-inv was expressed from cen or # plasmids. the results from the afss-b and bb-inv fusions indi-cate that, similar to alp (klionsky and emr, ) , the lumenal domain of dpap b is neither necessary nor sufficient for vacuolar targeting. the role of the transmembrane domain in the vacuolar targeting of dpap b was tested by constructing the fusion protein b-a-b (fig. b) , in which the membrane anchor of dpap b was replaced by that of the non-vacuolar membrane protein, dpap a. the role of the cytoplasmic domain of dpap b was tested by constructing in-frame deletions in this domain using oligonucleotide-directed mutagenesis of the dap gene. two deletion variants of dpap b were constructed, a -bb and a -bb, in which and amino acids, respectively, were removed from the residue cytoplasmic domain (fig. b; see materials and methods). immunoprecipitations of b-a-b, a -bb, and a -bb from s-labeled cells, followed by sds-page and fluorography, showed that the mutant proteins were glycosylated, and that the deglycosylated proteins were of the predicted size ( fig. , lanes - ) . as with bb-inv, these proteins behaved as integral membrane proteins in high ph carbonate fractionation experiments (data not shown). dpap activity assays of cells expressing b-a-b, a -bb, and a -bb showed that the proteins were fully enzymatically active, and that - % of the total activity was intracellular. indirect immunofluorescence microscopy of ceils expressing b-a-b, a -bb, and a -bb from either # plasmids (fig. , c-h) or cen plasmids, using an antibody that recognizes the cooh-terminal half of dpap b, showed that each of the mutant proteins was predominantly localized to the vacuolar membrane as determined by nomarski optics (fig. ) and co-localization with vat p (data not shown). no staining of the plasma membrane was observed for any of these proteins. thus, the cytoplasmic and transmembrane domains of dpap b are not necessary for targeting to the vacuolar membrane. a significant fraction of cells expressing a -bb and a -bb also showed some er localization, as judged by staining of the perinuclear space and long cisternal compartments (table i; rose et al., ) , whereas b-a-b showed only vacuolar labeling. the z~ -bb and ~ -bb cells in fig. show predominantly vacuolar staining. in many cases, the plane of focus had to be adjusted to observe staining of both vacuolar and er structures within a given cell. the increased residence time of a -bb and a -bb in the er was corroborated by immunoblotting analysis, which showed that a small amount of the er forms of a -bb and a -bb were seen in the steady state, along with the golgi-modified forms, whereas only the golgi-modified form was seen for wild type dpap b (data not shown). the increased er retention of a -bb and a -bb could be due to an impaired ability to achieve a native conformation competent for exiting the er, as has been observed with mutant membrane proteins in mammalian cells (gething et al., ; doms et al., ) . upon exiting the er, however, a -bb and a -bb were transported through the golgi complex to the vacuole, indicating that the cytoplasmic domain of dpap b is not necessary for vacuolar localization. these results, combined with those from the analysis of eefss-b, bb-inv, and b-a-b, demonstrate that, aside from a membrane anchor, no single domain of dpap b is required for transport to the vacuole. * for each construct, the percentages refer to the fraction of stained cells that exhibited staining of a particular organelle. depending on the protein being monitored, some cells showed staining of more than one class of organdie; thus, the percentages for a given protein may add up to more than . w vacuolar localization was determined both by coincidence of staining with the vacuole membrane as determined by nomarski optics, and by co-localization with vat p. i) golgi localization was defined as punctate, non-vacuolar, and non-er staining, characteristic of dpap a, kex p , and kexlp (cooper and bussey, manuscript submitted for publication). ** er localization was determined by staining of perinuclear (as determined by dapi staining of nuclei) and extended cisternal structures. several different models can explain the data presented above (see discussion), including a simple model in which the vacuolar membrane, not the plasma membrane, is the default compartment for membrane proteins of the yeast secretory pathway. to distinguish among these models, we analyzed the retention signal of the golgi membrane protein, dpap a. mutations in the retention signal of this protein should result in dpap a becoming localized to the default compartment for membrane proteins. dpap a is a protein that resides in the golgi apparatus, where it processes the c~-factor precursor polypeptide (julius et al., ) . the structural gene for dpap a (ste ) has been cloned and sequenced (julius et al., ; c. a. flanagun, d. a. barnes, m. c. flessel, and j. thorner, manuscript submitted for publication), and the predicted structure of dpap a is similar to that of dpap b in several regards. both are type ii integral membrane proteins, and both have lumenal enzymatic domains of ,x, residues that share a high degree of sequence identity, including % identical residues over the cooh-terminal residues (fig. a) ; however, there is no significant sequence similarity between the cytoplasmic and transmembrane domains of dpap a and dpap b. to assess the subcelhlar distribution of dpap a, an antibody specific for dpap a was generated (see materials and methods). the specificity of the antibody is demonst-rated in fig. . immunoprecipitation of dpap a was carried out with s-labeled cells that varied with regard to the dosage of the ste/ gene. sds-page and fluorography showed that the antibody immunoprecipitated a protein of kd from wild type cells but not from ste a ceils, and that this polypeptide was overproduced ~ -fold in a strain containing the ste/ gene on a t~ plasmid (fig. a) . a protein of * kd was also immunoprecipitated; however, the level of the -kd protein did not vary with the dosage of ste/ . the ste/ dna sequence predicts three possible sites for addition of n-linked carbohydrate (c. a. flanagan, d. a. barnes, m. c. flessel, and j. thorner, manuscript submitted for publication). treatment of dpap a with endo h resulted in an ~, kd decrease in apparent molecular weight (fig. b) , suggesting that at least two of the three asn-x-ser/thr sites of dpap a were modified, assuming that the core oligosaccharides added were of the typical structure (kukuruzinska et al., ) , and that the carbohydrate moieties were only slightly modified in the golgi apparatus, as is the case for the glycosyl groups of kex p (fuller et al., a,b) and kexlp (cooper and bussey, ) . the apparent molecular weight of deglycosylated dpap a, kd, is consistent with the molecular weight of dpap a predicted from the ste dna sequence, , d (c. a. flanagan, d. a. barnes, m. c. flessel, andj. thorner, manuscript submired for publication). indirect immunofluorescence microscopy of cells containing the $ f,/ gene on a # plasmid showed that dpap a was localized to several non-vacuolar puncture patches dispersed throughout the cell, as judged by nomarski optics and in double staining experiments with vat p antibody (fig. , d-u). this signal was absent in a stei a strain (fig. , a-c). this staining pattern is typical of the golgi apparatus, as determined by the localization of kex p and kexlp (a, cooper and h. bussey, manuscript submitted for publication). attempts to localize dpap a in cells containing only the chromosomal copy of ste/ were unsuccessful due to the low abundance of the protein. because the copy number of yeast /~ plasmids can vary from - copies/cell within a population (rose and broach, ) , the fluorescence signal corresponding to dpap a when expressed from a # plasmid also varied from cell to cell, ranging from a weak signal to a very strong signal, whereas the intensity of the vat p signal was consistent from cell to cell. all of the cells that showed a signal with the dpap a antibody displayed a golgi-staining pattern (fig. , d-u; table i ); however, cells exhibiting a very strong signal, presumably due to a high dosage of the ste/ gene, also showed vacuolar staining ( % of the cells ofapep - strain; table i ). an example of this is shown in fig. , (~ s, and u) . a single cell exhibiting a strong dpap a signal is shown, and the signal stains both vacuolar and punctate non-vacuolar structures, as judged by co-localization with vat p. the percentage of cells showing vacuolar localization of dpap a decreased significantly in an otherwise isogenic pep strain, which contains the full complement of vacuolar proteases (i %; table i) . thus, cells producing high levels of dpap a showed mislocalization of the protein to the vacuolar membrane, with the mislocalized protein degraded in a vacuolar proteasedependent fashion. dpap enzyme assays on permeabilized and non-permeabilized cells expressing dpap a from either cen or g plasmids showed that , % of the total activity was intracellular; thus, it is possible that a small percentage of dpap a is present at the cell surface, even though no plasma membrane staining was detected by indirect immunofluorescence, however, the apparent extraceuular dpap a enzyme activity could in part be due to a small degree of cell lysis during the assay. to determine whether a specific domain of dpap a con-the journal of cell biology, volume , tained the signal for golgi retention, the fusion proteins aa-b and a-bb were constructed ( fig. c) , in which the lumenal domain of dpap a was exchanged for that of dpap b (aa-b), and the cytoplasmic domain of dpap b was exchanged for that of dpap a (a-bb). immunoprecipitation of aa-b and a-bb from s-labeled cells, followed by sds-page and fluorography, showed that both fusion proteins were glycosylated and treatment of the immunoprecipitates with endo h showed that the proteins were of the expected size (fig. b) . the broadly migrating species observed for aa-b and a-bb indicate that the proteins are more extensively modified in the golgi apparatus than dpap a. dpap activity assays on permeabilized or non-permeabilized whole cells demonstrated that the aa-b and a-bb proteins were enzymatically active, and that ~ % of the activity was intracellular. a-bb fractionated with membranes in the presence of high ph sodium carbonate, and thus behaves biochemically as an integral membrane protein (data not shown). indirect immunofluorescence microscopy using anti-dpap b lumenal domain antibody showed that aa-b and a-bb exhibited non-vacuolar punctate staining patterns indistinguishable from wild type dpap a (fig. , a-f) . again, no plasma membrane staining was detected. as with dpap a, all cells that showed a signal displayed a punctate staining pattern, and a subset of those cells that showed a strong signal also exhibited vacuolar localization (table i ). an example is shown in fig. (a-c) for cells expressing aa-b. the cell in the bottom right corner shows a very intense fluorescein signal relative to the other cells in the panel, and clearly exhibits co-localization with vat p. thus, as was seen for wild type dpap a, overproduction of these proteins resulted in some mislocalization to the vacuole. these experiments demonstrate that the cytoplasmic domain of dpap a is sufficient for the retention of an otherwise vacuolar membrane protein in punctate structures typical of the golgi apparatus in s. cerevisiae. the cytoplasmic domain of dpap a also has been shown to be sufficient for golgi retention when fused to the transmembrane and lumenal domains of the vacuolar membrane protein alp (s. nothwehr and ". stevens, unpublished data). to determine if the cytoplasmic domain of dpap a was also necessary for the retention of dpap a in the golgi, a series of in-frame deletion mutations in the cytoplasmic domain were generated using oligonucleotide-directed mutagenesis (see materials and methods). deletion variants that lacked (fig. ) , although both proteins also showed a small amount of er staining (table i). immunoblotting analysis of crude extracts prepared from cells expressing a -aa-b showed a broadly migrating species on sds-page similar to aa-b and a-bb (data not shown; fig. b) , suggesting that the a -aa-b protein transits the golgi apparatus before its transport to the vacuole. similar to the deletions in the cytoplasmic domain of figure . immunolocalization of aa-b and a-bb. jhry' - a (ste a dap a) cells containing plasmids expressing either aa-b (pcjr , a-c) or a-bb (pcjr , d-f) were fixed, spheroplasted, and stained with dpap a antibody and vat p mab. the cells were viewed by nomarski optics (a and c) and by epifluorescence using filter sets specific for fluorescein (b and e) and rhodamine (c and f) fluorescence. dpap b, this deletion delays exit of the fusion proteins from the er; however, upon exit from the er, the enzymatically active a -aaa and a -aa-b fusion proteins were transported to the vacuole. thus, the cytoplasmic domain is both necessary and sufficient for the retention of dpap a in the golgi apparatus, and the signal for the retention of dpap a maps to a -amino acid segment within the ll -residue cytoplasmic domain. we have previously shown that the transport of dpap b to the vacuole does not involve delivery to the plasma membrane followed by endocytic targeting to the vacuole (roberrs et al., ) . this was demonstrated by expressing dpap b in a secl mutant, which at ~ is blocked at a late stage of the secretory pathway and accumulates secretory vesicles salminen et al., ) . to ad-dress the possibility that the mutant constructs analyzed in this study were mislocalized to the plasma membrane followed by rapid endocytic uptake to the vacuole, indirect immunofluorescence experiments were performed on bb-inv, b-a-b, a -bb, and/x -aa-b expressed in a secl strain at ~ as a positive control for accumulation in secretory vesicles, the localization of the fusion protein fusl-laczp was analyzed (trueheart et al., ) . the fus gene product is required for the breakdown of the cell walls after the fusion of a and ct cells during conjugation (mccaffrey et al., ; trueheart et al., ) , and has been shown by protease treatment of whole yeast cells to be a plasma membrane protein (trueheart and fink, ) . the fusion protein fusl-laczp, consisting of the nh -terminal amino acids of fuslp fused to the/acz gene product (/ -galactosidase), has been shown by immunofluorescence microscopy to be localized to the plasma membrane after induction during mating (trueheart et al., ) . for these experiments, the expression of these proteins was under the control of the inducible gal/promoter (see materials and methods; johnston and davis, ) . neither fusl-laczp, bb-inv, b-a-b, or a -bb showed any signal before addition of galactose (data not shown). fig. (a-f) shows that fusl-laczp was localized to the plasma membrane in a sec § strain after h of induction at ~ but accumulated intracellularly, presumably in secretory vesicles, in a secl strain under the same conditions. no colocalization with the vacuolar marker, alp, was detected. figure . immunolocalization in sec* and secl strains. jhry - a (sec*) and sey- (secl) cells containing plasmids encoding fusl-laczp (pcjrll ) or b-a-b (pcjr ) under the control of the gal/ promoter were grown to log phase in media containing raffinose, then shifted to ~ at the same time that galactose was added to the cultures. after h, the cells were fixed, spheroplasted, and prepared for immunofluorescence. cells expressing fusl-laczp were stained with anti-/~-galactosidase mab and anti-alp polyclonal antibody, and were viewed using filter sets specific for fluorescein and rhodamine fluorescence, respectively. cells expressing b-a-b were stained as in fig. . typically, fusl-laczp accumulated in a large patch in the bud and bud neck region of budded cells, and near the cell periphery of unbudded cells. bb-inv, b-a-b, and a -bb were each localized to the vacuole under these conditions (e.g., fig. , g-l; table ii) , as defined by co-localization with vat p. the intraceuular, non-vacuolar signal occasionally observed for b-a-b ( %; table ii ) was different from that of fusl-laczp, in that the signal looked reminiscent of the golgi signal observed for dpap a (data not shown). this was also observed for b-a-b in sec § cells at ~ suggesting that the kinetics of delivery of this protein is slowed at elevated temperatures. the analysis of a -aa-b in the secl strain was complicated by the fact that expression of the protein was leaky; ,~ % of the cells in the population showed a weak vacuolar staining pattern before galactose addition. however, addition of galactose to the culture after shifting the cells to ~ resulted in '~ % of the cells exhibiting an intense vacuolar signal (data not shown). these data show that the bb-inv, b-a-b, a -bb, and a -aa-b proteins are targeted directly from the golgi apparatus to the vacuole without transient appearance at the cell surface; thus, these proteins follow the same route to the vacuole as wild type vacuolar membrane proteins (roberts et al., ) . the search for the vacuolar localization determinant of dpap b led to the surprising conclusion that neither the cytoplasmic, transmembrane, nor lumenal domain of the protein was necessary for vacuolar delivery. analysis of the golgi retention signal of dpap a led to the equally surprising observations that both overproduction of the protein and mutations in the cytoplasmic domain resulted in mislocalization of dpap a to the vacuole, not the plasma membrane. these results were unexpected, given that the targeting of soluble vacuolar proteins in yeast and soluble and membrane proteins of the lysosomes of animal cells require targeting information to prevent delivery to the cell surface (vails et al., ; kornfeld and mellman, ; williams and fukuda, ; machamer, ) . the fusion proteins analyzed in this study are appropriate tools for these experiments for the following reasons. all of the proteins were stable, enzymatically active, membranebound, and glycosylated when expressed in yeast, indicating that they have the correct membrane topology. in addition, all of the fusion proteins containing the dpap b lumenal domain, i.e., a -bb, a -bb, b-a-b, and a -aa-b, were transported to the vacuole after receiving glycosyl modifications in the golgi apparatus. finally, each of these fusion proteins was transported to the vacuole directly from the golgi apparatus, without prior delivery to the plasma membrane followed by endocytic uptake to the vacuole. other golgi membrane proteins have also been shown to be mislocalized to the vacuole rather than the plasma membrane. both mutations in the cytoplasmic domain of kexlp as well as its overproduction result in mislocalization of the protein to the vacuolar membrane (a. cooper and h. bussey, manuscript submitted for publication). similarly, a single amino acid change in the cytoplasmic domain of kex p results in its transport to the vacuole (c. wilcox, k. redding, r. wright, and r. fuller, manuscript submitted for publication). whereas this result might appear to conflict with earlier published data on kex p, more recent analysis indicates that membrane-bound forms of kex p that fail to be retained in the golgi are found in the vacuole rather than the plasma membrane, when these proteins are expressed at wild type or modestly elevated levels (c. wilcox, k. redding, r. wright, and r. fuller, manuscript submitted for publication). given that several golgi membrane proteins all show missorting to the vacuole when either mutant or overproduced, the model we favor to explain these data is the vacuolar default model, which states that vacuolar membrane proteins do not require sorting information, because the default pathway for membrane proteins of the secretory pathway leads to the vacuole. alternatively, one could argue that, given the similarity between dpap b and dpap a, both proteins could contain vacuolar targeting information in their transmembrane domains, even though no significant similarity is apparent from the amino acid sequence data (c. a. flanagan, . a. barnes, m. c. flessel, and j. thorner, manuscript submitted for publication). mutations in the golgi retention signal of dpap a, or saturation of the retention apparatus, would then result in the delivery of dpap a to the vacuole. this model requires that both kex p and kexlp must also have cryptic vacuolar targeting signals. with regard to the cryptic vacuolar targeting signal model, we have found that replacement of the membrane spanning domains of dpap a and a -aaa with a residue hydrophobic sequence, l(lalv)~, creating the proteins axa and a -axa, results in the retention of axa in the golgi and the transport of a -axa to the vacuole (s. nothwehr and t. stevens, unpublished data). these data suggest that the transmembrane domain of dpap a does not contain vacuolar targeting information, and thus support the vacuolar default model. the vacuolar default model is directly testable by analyzing potential localization signals of yeast plasma membrane proteins. in apparent conflict with the vacuolar default model is the observation that the majority of kex p ( %) is missorted to the plasma membrane in cells deficient for clathrin heavy chain (payne and schekman, ) . however, dpap a is missorted to the cell surface to a lesser extent ( %; seeger and payne, a) . it is possible that the majority of dpap a and a significant percentage of kex p are missorted to the vacuole in cells lacking clathrin heavy chain. mutations in clathrin may affect both the retention of golgi membrane proteins and the functional integrity of the sorting pathway (seeger and payne, b) . this could obscure the default pathway for membrane proteins, resulting in the transport of golgi membrane proteins to the plasma membrane as well as the vacuole. an alternative explanation of the data is that one or more of the dpap a and dpap b fusion or mutant proteins analyzed in this study were in a partially unfolded state, and thus recognized as abnormal and transported directly from the er to the vacuole via a "garbage" pathway. there is no clear evidence for the existence of such a pathway in yeast or animal cells. however, several cases have been described in animal cells in which non-lysosomal membrane proteins were transported to lysosomes, either due to mutations (armstrong et al., ) , or in the case of the heptameric t-cell antigen receptor, complexes lacking the ~" subunit are degraded in lysosomes (minami et al., ) . in these cases, the proteins were transported through the golgi apparatus, and it is unclear whether the proteins were transported to lysosomes by a "garbage" pathway, or by the uncovering of a cryptic lysosomal targeting signal. in the great majority of cases, mislocalized membrane proteins of the secretory pathway in animal cells accumulate at the plasma membrane, which is presumed to be the default destination for these proteins (e.g., williams and fukuda, ) . it is well established that proteins that are slow to reach the folded state are retained in the er due to the action of proteins such as bip (pelham, ; rothman, ) , and proteins that are unable to fold or oligomerize properly are degraded in the er (klausner and sitia, ) . several of the proteins analyzed in this study (i.e., a -bb, a -bb, a -aaa, and a -aa-b) showed increased retention in the er. however, all of these proteins eventually exited the er, were enzymatically active, and were transported through the golgi complex to the vacuole. the -amino acid segment of the cytoplasmic domain of dpap a presumably contains the recognition domain for a "retention protein" of the golgi apparatus. this protein could be a permanent resident of the golgi apparatus; alternatively, the "retention protein" could reside in a post-golgi compartment and function as a salvage receptor, such as the proposed receptor for soluble er proteins (pelham, ) . it is interesting to note that the -amino acid stretch identified as the dpap a golgi retention signal contains five phenylalanine residues (fig. ) . in animal cells, one or more aromatic amino acids in the cytoplasmic domains of cell surface receptors have been shown to comprise part of the signal for the clustering into coated pit regions of the plasma membrane (chen et al., ; johnson et al., ; lobel et al., ; mcgraw et al., ) . that the phenylalanine residues in the a region may play a direct role in golgi retention is supported by the observation that mutations in just two of these residues result in a substantial level of missorting of dpap a to the vacuole (s. nothwehr and t. stevens, unpublished data) . the results of this study suggest that the default pathway for membrane proteins of the yeast secretory pathway may be different from that of certain mammalian cell lines that have been examined, where mutant forms of er, golgi, and lysosomal membrane proteins are mislocalized to the plasma membrane with the bulk flow of membrane (machamer and rose, ; jackson et al., ; williams and fukuda, ; wieland et al., ; karrenbauer et al., ; machamer, ) . however, in polarized epithelial ceils, it remains unclear which membrane serves as the default destination for membrane proteins (simons and wandinger-ness, ; mostov et al., ) . even if the default compartments for membrane proteins of the secretory pathways of yeast and certain animal cells are different, the same mechanistic considerations apply, that is, positive sorting information is required for proteins to avoid delivery to the default compartment. according to the vacuolar default model for yeast membrane protein sorting, not only must er and golgi membrane proteins have sorting signals specifying their retention in the appropriate compartment, but plasma membrane proteins are predicted to have sorting information that prevents their localization to the vacuole. we are currently investigating whether yeast plasma membrane proteins have targeting signals that prevent their transport to the vacuole. lysosomal sorting mutants of coronavirus e protein, a golgi membrane protein yeast kre genes provide evidence for a pathway of cell wall/~ glucan assembly constitutive and regulated secretion of proteins proteases and the processing of precursors to secreted proteins in yeast npxy, a sequence often found in cytoplasmic tails, is required for coated pit mediated internalization of the ldl receptor characterization of the yeast kex gene product" a carboxypeptidase involved in processing secreted precursor proteins differential effects of mutations in three domains on folding, quaternary structure, and intracellular transport of vesicular stomatitis virus g protein an mfod-suc (~-factor-invertase) gene fusion for study of protein localization and gene expression in yeast the amino terminus of the yeast ft-atpase ~-subunit precursor functions as a mitochondrial import signal compartmentalized assembly of oligosaccharides on exported glycoprotein in yeast localization of components involved in protein transport and processing through the yeast golgi apparatus enzymes required for yeast prohormone processing yeast prohormone processing enzyme (ke, x gene product) is a ca~+-dependent serine protease intracellular targeting and structural conservation of a prohormone-processing endoprotease expression of wild type and mutant forms of influenza hemagglutinin: the role of folding in intracellular transport / -d-fructofuranoside fructohydrolase from yeast compartmental organization of golgispecific protein modification and vacuolar protein sorting events defined in a yeast secl (nsf) mutant erdi, a yeast gene required for the retention of luminal endoplasmic reticulum proteins, affects glycoprotein processing in the golgi apparatus solid phase synthesis of polynucleotides vi. further studies on polystyrene copolymers for the solid support transformation of intact yeast ceils treated with alkali cations identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum cation-dependent mannose -pbosphate receptor contains two internalization signals in its cytoplasmic domain distinct sequence determinants direct intracellular sorting and modification of a yeast vacuolar proteinase sequences that regulate the divergent gal -galio promoter in saccharomyces cerevisiae yeast a factor is processed from a larger precursor polypeptide: the essential role of a membrane-bound dipeptidyl aminopeptidase glycosylation and processing of prepro-a-factor through the yeast secretory pathway assembly and targeting of peripheral and membrane subunits of the yeast vacuolar h+-atpase the rate of bulk flow from the golgi to the plasma membrane protein degradation in the endoplasmic reticulum membrane protein sorting: biosynthesis, transport, and processing of yeast vacuolar alkaline phosphatase a new class of lysosomal/vacuolar protein sorting signals protein glycosylation in yeast rapid and efficient sitespecific mutagenesis without phenotypic selection structure of a yeast pheromone gene (mfa): a putative a-factor precursor contains four tandem copies of mature c~-factor mutations in the cytoplasmic domain of the kd mannose -phosphate receptor differentially alter lysosomal enzyme sorting and endoeytosis golgi retention signals: do membranes hold the key? a specific transmembrane domain of a coronavirus el glycoprotein is required for its retention in the golgi region molecular cloning: a laboratory manual. cold spring harbor laboratory identification and regulation of a gene required for cell fusion during mating of the yeast saccharomyces cerevisiae mutaganesis of the human transferrin receptor: two cytoplasmic phenylalanines are required for efficient internalization and a second-site mutation is capable of reverting an internalization-defective phenotype building a multichain receptor: synthesis, degradation and assembly of the t-cell antigen receptor plasma membrane protein sorting in polarized epithelial cells order of events in the yeast secretory pathway analysis ofglycoproteins from saccharomyces cerevisiae clathrin: a role in the intracellular retention of a golgi membrane protein control of protein exit from the endoplasmic reticulum biosynthetic protein transport and sorting by the endoplasmic reticulum and golgi molecular analysis of the yeast vps gene and the role of its product in vacuolar protein sorting and vacuolar segregation during the cell cycle immunolocalization of kex protease identifies a putative late golgi compartment in the yeast saccharomyces cerevisiae structure, biosynthesis, and localization of dipeptidyl aminopeptidase b, an integral membrane glycoprotein of the yeast vacuole methods for studying the yeast vacuole cloning genes by complementation in yeast kar , a karyogamy gene, is the yeast homolog of the mammalian bip/grp gene polypeptide chain binding proteins: catalysts of protein folding and related processes in ceils one step gene disruption in yeast a ras-like protein is required for a post-golgi event in yeast secretion selective and immediate effects of clathrin heavy chain mutations on golgi membrane protein retention in saccharomyces cerevisiae a role for clathrin in the sorting of vacuolar proteins in the golgi complex of yeast methods in yeast genetics a system of shuttle vectors and yeast host strains designed for efficient manipulation of dna in saccharomyces cerevisiae polarized sorting in epithelia the structure and insertion of integral proteins in membranes gene dosage-dependent secretion of yeast vacuolar carboxypeptidase y identification of the structural gene for dipeptidyl aminopeptidase yscv (dap ) of saccharomyces cerevisiae the yeast cell fusion protein fusi is o-glycosylated and spans the plasma membrane two genes required for cell fusion in yeast: evidence of a pheromone-induced surface protein yeast carboxypeptidase y vacuolar targeting signal is defined by four propeptide amino acids a family of yeast expression vectors containing the phage fl intergenic region prepro-a-factor has a cleavable signal sequence the rate of bulk flow from the endoplasmic reticulum to the cell surface accumulation of membrane glycoproteins in lysosomes requires a tyrosine residue at a particular position in the cytoplasmic tail we acknowledge cathy flanagan, jeremy thorner, antony cooper, howard bussey, celeste wilcox, robert fuller, and greg payne for communication of results prior to publication; scott ernr, charlie boone, and george sprague for plasmids; margaret ho and joe horecka for help in the construction of the a -bb and fusl-laczp constructs, respectively; and jerry gleason and scan poston for the photographic work. we especially thank christopher raymond for numerous insightful contributions to this work, and nick davis, charlie boone, george sprague, antony cooper, carol vater, cynthia bauerle, and margaret ho for comments on the manuscript. received for publication february and in revised form june . key: cord- -m zgn authors: stokes, john w.; gannon, whitney d.; sherrill, wren h.; armistead, leslie b.; bacchetta, matthew; rice, todd w.; semler, matthew w.; casey, jonathan d. title: bleeding, thromboembolism, and clinical outcomes in venovenous extracorporeal membrane oxygenation date: - - journal: crit care explor doi: . /cce. sha: doc_id: cord_uid: m zgn objectives: bleeding and thromboembolism are common during venovenous extracorporeal membrane oxygenation. the relative frequency of these complications and their impact on clinical outcomes have not been described, and no randomized trials exist to guide anticoagulation strategies in extracorporeal membrane oxygenation. our objective was to examine the relative frequencies of bleeding and thromboembolic events and their associations with survival among a cohort of consecutive patients receiving venovenous extracorporeal membrane oxygenation. design: retrospective cohort study. setting: a single academic medical center. patients: adult patients receiving venovenous extracorporeal membrane oxygenation and anticoagulation. eligibility criteria for this analysis were selected to emulate the population that would be recruited for a randomized trial of anticoagulation strategies during venovenous extracorporeal membrane oxygenation. patients were excluded if they had active bleeding or thromboembolism prior to extracorporeal membrane oxygenation initiation, a history of trauma or surgery in the days prior to extracorporeal membrane oxygenation initiation, an arterial extracorporeal membrane oxygenation cannula, or if they received greater than hours of extracorporeal membrane oxygenation support at another institution interventions: none. measurements and main results: outcomes included bleeding and thromboembolic events, duration of extracorporeal membrane oxygenation support, hospital length of stay, and in-hospital survival among patients receiving venovenous extracorporeal membrane oxygenation. bleeding events occurred in patients ( . %), and thromboembolism occurred in eight patients ( . %). bleeding events were associated with longer duration of extracorporeal membrane oxygenation support (p = . ) and worse in-hospital survival (p = . ). thromboembolic events did not appear to be associated with clinical outcomes. conclusions: in this cohort of patients receiving venovenous extracorporeal membrane oxygenation and anticoagulation, bleeding occurred more frequently than thromboembolism and was associated with worse survival. these results highlight the need for randomized trials to evaluate the safety and efficacy of continuous iv anticoagulation among patients receiving venovenous extracorporeal membrane oxygenation. objectives: bleeding and thromboembolism are common during venovenous extracorporeal membrane oxygenation. the relative frequency of these complications and their impact on clinical outcomes have not been described, and no randomized trials exist to guide anticoagulation strategies in extracorporeal membrane oxygenation. our objective was to examine the relative frequencies of bleeding and thromboembolic events and their associations with survival among a cohort of consecutive patients receiving venovenous extracorporeal membrane oxygenation. design: retrospective cohort study. setting: a single academic medical center. patients: adult patients receiving venovenous extracorporeal membrane oxygenation and anticoagulation. eligibility criteria for this analysis were selected to emulate the population that would be recruited for a randomized trial of anticoagulation strategies during venovenous extracorporeal membrane oxygenation. patients were excluded if they had active bleeding or thromboembolism prior to extracorporeal membrane oxygenation initiation, a history of trauma or surgery in the days prior to extracorporeal membrane oxygenation initiation, an arterial extracorporeal membrane oxygenation cannula, or if they received greater than hours of extracorporeal membrane oxygenation support at another institution interventions: none. measurements and main results: outcomes included bleeding and thromboembolic events, duration of extracorporeal membrane oxygenation support, hospital length of stay, and in-hospital survival among patients receiving venovenous extracorporeal membrane oxygenation. bleeding events occurred in patients ( . %), and thromboembolism occurred in eight patients ( . %). bleeding events were associated with longer duration of extracorporeal membrane oxygenation support (p = . ) and worse in-hospital survival (p = . ). thromboembolic events did not appear to be associated with clinical outcomes. conclusions: in this cohort of patients receiving venovenous extracorporeal membrane oxygenation and anticoagulation, bleeding occurred more frequently than thromboembolism and was associated with worse survival. these results highlight the need for randomized trials to evaluate the safety and efficacy of continuous iv anticoagulation among patients receiving venovenous extracorporeal membrane oxygenation. key words: adult; critical care; extracorporeal membrane oxygenation; hemorrhage; respiratory distress syndrome; thromboembolism b leeding and thromboembolism are common during venovenous extracorporeal membrane oxygenation (ecmo) ( ). reported frequencies and associations with clinical outcomes vary and available data are limited by heterogenous study populations ( ) ( ) ( ) . multiple anticoagulation strategies have been proposed to balance the risks of bleeding and thromboembolism during venovenous ecmo ( , ) , but which strategy is most effective remains unknown. data on the relative frequencies of bleeding and thromboembolism during venovenous ecmo, and their respective associations with survival, are needed to provide preliminary data and inform equipoise for future randomized trials. our objective was to evaluate the frequency and clinical significance of bleeding and thromboembolic events during venovenous ecmo. we hypothesized that bleeding events, but not thromboembolic events, would be associated with worse in-hospital survival. we performed a retrospective cohort study examining data from all patients who received venovenous ecmo at the adult hospital at vanderbilt university medical center between january , , and may , . prespecified exclusion criteria were used with the goal of including a patient population similar to those who would be included in a randomized trial comparing anticoagulation strategies, a technique known as "target trial emulation" ( ) . we excluded patients who had active bleeding or thromboembolism prior to ecmo initiation, experienced trauma or surgery in the days prior to ecmo initiation, received greater than hours of ecmo support at another institution, or received arterial cannulation. the study was approved by the vanderbilt university medical center institutional review board (irb no ). we collected the following data from the electronic health record: patient characteristics in the hours prior to ecmo initiation; bleeding and thromboembolic events during venovenous ecmo as previously defined ( ); and clinical outcomes, including in-hospital survival, ecmo duration, and hospital length of stay. bleeding events were defined as overt bleeding associated with either a drop in hemoglobin concentration by g/dl or a transfusion of at least two units of packed rbcs in hours, bleeding at any critical site (e.g. intracranial bleeding), or bleeding requiring a procedural intervention ( ) . thromboembolic events were defined as cerebral stroke, intracardiac thrombus, acute pump head thrombosis, acute oxygenator failure, pulmonary emboli, or deep vein thrombosis (dvt) ( ) . cannula-associated dvts following decannulation did not meet the composite definition for thromboembolic event and were omitted from the survival analysis to limit immortal time bias. continuous variables are presented as median with interquartile range (iqr). categorical variables are summarized as frequencies and percentages. differences between groups were compared using a chi-square test, fisher exact test, or wilcoxon rank-sum test as appropriate. log-rank tests were used to compare time with hospital discharge between groups. all analyses were performed using stata . (statacorp, college station, tx), and a twosided p value of . was considered to be statistically significant. no adjustments were made for multiple testing. of the patients who received venovenous ecmo during the study period, met exclusion criteria. a total of patients were excluded for recent trauma or surgery, patients were excluded for active bleeding, patients were excluded for recent thromboembolism, five patients were excluded for receiving ecmo at an another institution for greater than hours, and three patients were excluded for arterial cannula placement while receiving venovenous ecmo. a total of patients were included in the analysis. the median age was years (iqr, - yr), and % were women. according to institutional protocols, all patients received a continuous infusion of unfractionated heparin following ecmo cannulation, titrated to either antifactor xa levels of . - . u/ml or a partial thromboplastin time of - seconds. a total of bleeding events occurred among patients ( . %), including eight gastrointestinal bleeds, seven intracranial hemorrhages, four cannula site bleeds, four episodes of hemoptysis, three tracheostomy bleeds, two hemothoraces, and two episodes of epistaxis. of these, six ( intracranial hemorrhages and gastrointestinal bleed) were considered the primary cause of death. the median time from ecmo cannulation to first bleeding event was days (iqr, - d). eight patients ( . %) experienced a thromboembolic event during ecmo, including five deep venous thromboses (dvt), two acute circuit thromboses requiring circuit exchange, and one brachial artery thrombosis. the median time from ecmo cannulation to first thromboembolic event was days (iqr, - d). no thromboembolic events were considered the primary cause of death. a total of additional cannula-associated dvts were identified on protocolized ultrasound screening following decannulation. baseline characteristics and serum markers of coagulation and thrombocytopenia were similar between groups ( table ) . anticoagulation monitoring did not vary between groups. rbc transfusion requirement was greater among patients with a bleeding event than patients without a bleeding event (p = . ). in univariate analysis, patients who experienced a bleeding event had a longer duration of ecmo support (p = . ) and worse inhospital survival compared with patients who did not experience a bleeding event (p = . ) ( table and fig. ) . thromboembolic events did not appear to be associated with any differences in duration of ecmo support, hospital length of stay, or in-hospital survival (table and fig. ). in this retrospective cohort study of patients receiving venovenous ecmo for respiratory failure, all of whom received continuous anticoagulation, nearly half of patients experienced a bleeding event. patients who experienced a bleeding event experienced worse survival than patients who did not experience a bleeding event. in contrast, thromboembolic events were less frequent and did not appear to affect survival. this is the first study to examine the relative impact of bleeding or thromboembolism during venovenous ecmo only. these results should prompt further research to evaluate the safety and efficacy of continuous iv anticoagulation in such patients. several factors may contribute to bleeding and thromboembolism in patients receiving ecmo. the interface of blood and nonbiologic circuit components causes activation of the coagulation system and the consumption and degradation of hemostatic factors ( , ) . underlying critical illness compounds the risks of bleeding and thromboembolism ( ) . continuous anticoagulation during ecmo may increase the risk of bleeding ( ), and prior retrospective data suggest a dose-response relationship between anticoagulation and bleeding events ( , ). conducting venovenous ecmo without continuous systemic anticoagulation has been proposed ( , ). although confounded by indication bias, recent observational studies suggest that strategies using only prophylactic doses of anticoagulation appear safe in venovenous ecmo ( , ) . further, a recent systematic review suggested that the rates of thromboembolism and circuit thrombosis among patients who did not receive systemic anticoagulation during venovenous ecmo were comparable with the rates reported among patients treated with systemic anticoagulation ( ) . it is possible that avoidance of systemic anticoagulation might improve outcomes for some patients receiving venovenous ecmo. our study has several strengths. by including only patients without a pre-existing indication or contraindication to anticoagulation, the population in our study emulates the population that would be recruited for a randomized trial of anticoagulation strategies during venovenous ecmo. further, we used previously published, objective criteria to define bleeding and thromboembolism. our study has several limitations. the study was conducted at a single center using a retrospective design. although we used structured and prespecified eligibility criteria, selection bias remains possible. the risks of bleeding and thromboembolism may be confounded by severity of illness and immortal time bias. finally, this study was largely conducted prior to the coronavirus disease (covid- ) pandemic. only one patient in the study cohort experienced respiratory failure as a consequence of covid- . emerging data describe both hypercoagulability ( ) and a higher risk of bleeding for patients receiving venovenous ecmo for covid- ( ) . it is unknown if the results of this analysis would be different if conducted entirely among a population of patients receiving venovenous ecmo for covid- . our data include only patients who received anticoagulation and do not inform the risks of thromboembolism among patients receiving venovenous ecmo without anticoagulation or with prophylactic-dose anticoagulation. this purely descriptive univariate analysis does not attempt to account for potential confounders and does not infer a causal relationship between bleeding or thromboembolism and survival. in this cohort of patients receiving venovenous ecmo and anticoagulation, bleeding occurred more frequently than thromboembolism and was associated with worse survival. these results provide preliminary data for a randomized trial examining the safety and efficacy of systemic anticoagulation in select patients receiving venovenous ecmo. drs. stokes and gannon contributed equally to this work. drs. stokes, gannon, sherrill, bacchetta, rice, semler, and casey contributed to conception and design of the study. drs. stokes, gannon, sherrill, and armistead contributed to data acquisition. drs. stokes, gannon, and sherrill contributed to analysis of the data. drs. stokes and gannon drafted the initial article. all authors contributed to the data interpretation and edited the article for important scientific content. all of the authors agree to be accountable for all aspects of the work in regards to accuracy and integrity. bacchetta was supported, in part, by the national institutes of health (nih) jr chair in surgery no . dr. rice was supported, in part, by the nih (ul rr ). dr. semler was supported, in part, by the national heart, lung, and blood institute (k hl ). dr. casey was supported predictive factors of bleeding events in adults undergoing extracorporeal membrane oxygenation severe respiratory failure, extracorporeal membrane oxygenation, and intracranial hemorrhage anticoagulation practices during venovenous extracorporeal membrane oxygenation for respiratory failure. a systematic review extracorporeal life support organization: elso anticoagulation guideline low-dose versus therapeutic anticoagulation in patients on extracorporeal membrane oxygenation: a pilot randomized trial using big data to emulate a target trial when a randomized trial is not available clinical controversies in anticoagulation monitoring and antithrombin supplementation for ecmo kaplan-meier in-hospital survival curves from time of venovenous extracorporeal membrane oxygenation for patients who did and did not experience a bleeding event and for patients who did and did not experience a thromboembolic event current understanding of how extracorporeal membrane oxygenators activate haemostasis and other blood components feasibility of veno-venous extracorporeal membrane oxygenation without systemic anticoagulation venovenous extracorporeal membrane oxygenation with prophylactic subcutaneous anticoagulation only: an observational study in more than patients thrombosis and bleeding in extracorporeal membrame oxygenation (ecmo) without anticoagulation: a systematic review covid- : the vasculature unleashed extracorporeal membrane oxygenation for severe acute respiratory distress syndrome associated with covid- : a retrospective cohort study key: cord- -bli hwda authors: lipp, joachim; dobberstein, bernhard title: the membrane-spanning segment of invariant chain (iγ) contains a potentially cleavable signal sequence date: - - journal: cell doi: . / - ( ) - sha: doc_id: cord_uid: bli hwda abstract the human invariant chain (iγ) of class ii histocompatibility antigens spans the membrane of the endoplasmic reticulum once. it exposes a small amino-terminal domain on the cytoplasmic side and a carboxyterminal, glycosylated domain on the exoplasmic side of the membrane. when the exoplasmic domain of iγ is replaced by the cytoplasmic protein chloramphenicol acetyltransferase (cat), cat becomes the exoplasmic, glycosylated domain of the resulting membrane protein iγcat∗. deletion of the hydrophilic cytoplasmic domain from iγcat gives rise to a secreted protein from which an amino-terminal segment is cleaved, most likely by signal peptidase. we conclude that the membrane-spanning region of iγ contains a signal sequence in its amino-terminal half and that hydrophilic residues at the amino-terminal end of a signal sequence can determine cleavage by signal peptidase. the human invariant chain (ly) of class ii histocompatibility antigens spans the membrane of the endoplasmic reticulum once. it exposes a small amino-terminal domain on the cytoplasmic side and a carboxyterminal, glycosylated domain on the exoplasmic side of the membrane. when the exoplasmic domain of ly is replaced by the cytoplasmic protein chloramphenicol acetyltransferase (cat), cat becomes the exoplasmic, glycosylated domain of the resulting membrane protein i$at*. deletion of the hydrophilic cytoplasmic domain from l$xt gives rise to a secreted protein from which an amino-terminal segment is cleaved, most likely by signal peptidase. we conclude that the membranespanning region of ly contains a signal sequence in its amino-terminal half and that hydrophilic residues at the amino-terminal end of a signal sequence can determine cleavage by signal peptldase. translocation of proteins across the membrane of the endoplasmic reticulum (er) requires signal sequences and specific receptors that recognize them (see recent reviews by hortsch and meyer, ; walter et al., ; rapoport and wiedmann, ; wickner and lodish, ) . signal sequences have been found at the amino-terminal end of precursors for secretory and transmembrane proteins. in many cases they are cleaved during their translocation across the membrane by a specific protease (signal peptidase). signal sequences are quite variable in length, ranging from to more than amino acid residues (von heijne, ) . they all have a central core of hydrophobic amino acid residues, and most of them have a positively charged amino-terminal segment (von heijne, ) . signal sequences on nascent polypeptides are recognized by the signal recognition particle (srp), a ribonucleoprotein complex that mediates the interaction with the membrane by the selective binding to docking protein (or srp receptor) (walter et al., b; meyer et al., ; gilmore et al., ) . membrane proteins are also inserted into the er membrane by an srp-mediated mechanism (anderson et al., ; rottier et al., ; spiess and lodish, ; lipp and dobberstein, ) . those spanning the membrane once have either the carboxyl terminus (type i membrane proteins) or the amino terminus (type ii membrane proteins) exposed on the cytoplasmic side. membrane insertion of type i membrane proteins most likely proceeds in a manner very similar to that of secretory proteins (lingappa et al., ) . type i membrane proteins are usually synthesized with a cleavable signal sequence and, in contrast to secretory proteins, are held in the membrane by a "stop transfer" sequence. examples of type i membrane proteins are the vesicular stomatitis virus g protein and class i and class ii histocompatibility antigens (lingappa et al., ; dobberstein et al., ) . of the type ii membrane proteins so far investigated, all are synthesized without a cleavable signal sequence. the neuraminidase of influenzavirus ( s et al., ) , the invariant chain (ii or ly) of class ii histocompatibility antigens (claesson et al., ; strubin et al., ; long, ; lipp and dobberstein, ) , the transferrin receptor (schneider et al., ) , and the asialoglycoprotein receptor (chiacchia and drickamer, ; holland et al., ; spiess and lodish, ) all belong to this class of membrane proteins. some steps in their membrane insertion must be similar to that of secretory and type i membrane proteins, as an srp-and docking protein-dependent membrane insertion has been demonstrated for some of them (spiess and lodish, ; lipp and dobberstein, ) . membrane insertion might occur in a loop-like fashion as this scheme can most easily explain how the different membrane topologies of membrane proteins are achieved (engelman and steitz, ) . as type ii membrane proteins contain only a single stretch of hydrophobic amino acid residues, this might function as a signal for membrane insertion as well as a membrane anchor (markoff et al., ; spiess and lodish, ) . to identify and characterize this sequence, we tested membrane insertion of the human invariant chain (ly) and several deletion and fusion proteins derived from it in a cell-free membrane insertion system. ly is a typical type ii membrane protein (claesson et al., ; strubin et al., ; lipp and dobberstein, ) . it exposes amino-terminal residues on the cytoplasmic side, spans the membrane between residues and , and exposes a large carboxy-terminal domain on the exoplasmic side. this domain has two sites for the addition of n-linked carbohydrate units. membrane insertion of ly requires srp and docking protein (lipp and dobberstein, ) . as the amino-terminal, cytoplasmic domain is hydrophilic and shows no resemblance to a signal sequence, it has been proposed that the membrane-spanning region, or part of it, functions as an internal, uncleavable signal sequence (dobberstein et al., ; claesson et al., ; lipp and dobberstein, ) . we demonstrate here that the membrane-spanning region of ly is composed of a potentially cleavable signal sequence fused to part of a membrane anchor, which together with the cytoplasmic domain determine the orientation of ly in the er membrane. deletion of the cytoplasmic domain exposes the signal sequence at the amino terminus of the membrane-spanning region, resulting in cleavage of this otherwise uncleaved signal. claesson et al., ) . ply. the complete ly coding and all of its ' noncoding sequence was cloned behind the t promoter (p) in the pds expression vector. plycat, the portion downstream of the pstl site in ply was replaced by the chloramphenicol acetyltransferase (cat) gene resulting in an in-frame fusion protein. pan-lycat, the segment between the sau a and sstll sites of plycat coding for the cytoplasmic domain was deleted. a new atg initiation codon right in front of the membrane-spanning segment is provided by the vector (see figure a ). the regions coding for protein are boxed. the membrane-spanning region of ly is indicated by loops; the hydrophilic domains by dots. cat-derived sequences are indicated by slanted lines. the position of n-linked glycosylation sites in ly and the potential n-linked glycosylation site in cat protein are indicated by an asterisk. relevant cleavage sites for restriction endonucleases are also indicated. protein segments that perform a particular function can be identified by their deletion or addition to unrelated proteins. we used this approach to localize and characterize the region in ly that is responsible for membrane insertion. deletions and fusions were made at the dna level after cloning of ly cdna into an expression vector. messenger rna was transcribed from these plasmids and translated in a cell-free system. the resulting proteins were tested for their ability to insert into microsomal membranes (blobel and dobberstein, ; stueber et al., ) . plasmids ply, plycat, and pan-lycat we have shown previously that cdna sequences cloned behind the strong t promoter in pds can be transcribed very efficiently by e. coli rna polymerase (stueber et al., ) . when transcription is performed in the presence of the cap analog mgpppa, the resulting mrna can be translated efficiently in eukaryotic cell-free systems. we have observed, however, that a stretch of gc residues at the ' end of a cdna negatively affects expression of the resulting rna (unpublished observation). the ly cdna construct (py- ) had been gc tailed and was inserted into the pstl site of pbr (claesson et al., ) . we deleted the ' gc tail and cloned ly cdna, or part of it, into the polylinker site of pds or p / r (see experimental procedures for details). ply contains the entire l-y coding region behind the t promoter ( figure ). plycat is an in-frame fusion between the ' region of ly encoding the cytoplas, mic, membrane-spanning segment plus amino acids of the exoplasmic portion of ly and the gene encoding the cytoplasmic protein chloramphenicol acetyltransferase (cat). the cat protein contains one potential site for the addition of n-linked oligosaccharide amino acid residues downstream of its original initiator methionine. in an-iycat, the entire hydrophilic, cytoplasmic segment from ly was deleted. the new initiator methionine is provided by the vector and is located in front of the hydrophobic segment. in vitro translation and membrane insertion of ly when ply was transcribed by e. coli rna polymerase and the resulting mrna translated in the wheat germ cell-free system, a single polypeptide species of kd was obtained ( figure , lane ). this is the expected molecular weight for nonglycosylated ly (claesson et al., ) . when rough microsomes (rm), derived from dog pancreas, were added to the translation system, a higher molecular weight species of kd appeared. this increase of kd in molecular weight is consistent with the addition of two oligosaccharides to the two n-glycosylation sites. the kd form ly* was reduced in molecular weight by about kd when proteinase k was used to remove the cytoplasmically exposed domain (figure , lanes and ). when protease digestion was performed in the presence of the detergent np , ly' was digested. these data suggest that ly' is integrated into the membrane and exposes - amino acid residues on the cytoplasmic side and a kd domain on the exoplasmic side of the membrane. the identity of ly and its glycosylated form was confirmed by immunoprecipitation with antibodies raised against the amino-terminal (anti-iyn) or against the carboxy-terminal (anti-iyc) residues of ly. as shown in figure , lanes , , , and , these antibodies recognize glycosylated and nonglycosylated forms of ly. no protein could be precipitated with anti-iyn antibody when the cytoplasmic domain was removed from membrane-integrated ly' by protease digestion (figure , lane ) . as the antibody is directed against the amino-terminal portion of ly, the data directly demonstrate that the amino terminus is located on the cytoplasmic side and is accessible to the protease. with anti-iyc antibody, the processed form of ly is readily detectable, demonstrating an exoplasmic location of the carboxy-terminal portion of ly ( figure , lane ). membrane insertion of iycat an analysis of membrane insertion was performed for ly-cat and cat as described above for ly. cat was expressed from pds . iycat was synthesized in the absence of microsomal membranes as a kd protein ( figure , lane ) and in the presence of microsomal membranes as a kd protein called lycat* ( figure , lane ). in vitro translation and membrane insertion of ly ply was transcribed in the presence of the cap analog mgpppa by e. coli rna polymerase. the resulting mrna was translated in the wheat germ cell-free system in the absence (lanes , . and ) or presence (lanes , , , , , . and ) of rm. the membrane topology of ly was determined by treatment with proteinase k (pk) (lanes , , and ) or pk and the detergent np (lane ). proteins were separated by sds-page and visualized by autoradiography. lanes - show total protein synthesized. samples characterized in lanes - were immunoprecipitated with an antibody raised against the amino-terminal amino acid residues of ly (anti-iyn); in lanes - , with an antibody against the carboxy-terminal portion of ly (anti-iyc). the increase in molecular weight is consistent with the addition of one n-linked oligosaccharide to the cat-derived portion. there is one potential site for n-linked glycosylation in the cat protein. after protease digestion in the presence of microsomal membranes, lycat* is reduced in molecular weight by about kd, suggesting that it exposes - amino acid residues on the cytoplasmic side ( figure , lanes and ). cat protein obtained after transcription-translation from pds is not modified by the added microsomes. as expected, no shift in molecular weight can be seen (figure , lanes and ). cat protein was very resistant to protease digestion even in the presence of np (figure , lanes and ). i-&at, in contrast, was very sensitive to added protease. this might reflect a difference in conformation between the free cat protein and the cat-derived portion in iycat. the location of cat outside of the membrane vesicles can be demonstrated by sedimenting the membranes by centrifugation. cat protein is then found in the supernatant (data not shown). we conclude from the data obtained with ircat and cat that the signal for membrane insertion must be located within the first amino acid residues of ly. to localize this signal more precisely, we deleted the first residues of iycat. cat- insertion of k&at and cat protein rna derived from plycat or pds was translated in the wheat germ cell-free system in the absence or the presence of rm. membrane insertion was tested by treatment with proteinase k (pk) and np . addition of rm, pk, and np is indicated at the bottom of each lane. of an+cat in all secretory proteins the cleavable signal for membrane translocation is located at the amino-terminal end of the precursor polypeptide. the main feature of this signal appears to be its hydrophobicity. in ly the only hydrophobic stretch of amino acid residues that resembles a signal sequence is located in the membrane spanning region about amino acid residues away from the amino-terminal initiator methionine. we asked whether removal of the amino-terminal residues in iycat would affect its membrane insertion and topology. the cytoplasmic domain of iycat was deleted and the initiator methionine was placed in front of the membranespanning segment. the amino-terminal sequences of ly-cat and an-i$at as deduced from the dna sequences are shown in figure a . when rna derived from panlycat was translated in the wheat germ cell-free system, a single polypeptide of kd was synthesized, an-iycat ( figure , lane ). this was, as expected, about kd smaller than the iycat protein ( figure , lane ). in the presence of microsomes, two new protein bands appeared, one about kd smaller and one kd larger than an-ircat both of these forms were resistant to proteinase k, indicating that they were inserted into or translocated across microsomal membranes (figure , lanes and ). we suspected that the smaller molecular weight form was generated by signal peptidase cleavage without concomitant glycosylation and that the larger molecular weight form was glycosylated and cleaved by signal peptidase. these possibilities were tested. from pan-lycat was translated in the wheat germ cell-free system in the absence or presence of rm. membrane insertion and topology was tested by treatment with proteinase k (pk) and np . components were added as indicated below the lanes. iycat translated in the wheat germ cell-free system is shown for comparison. processed and glycoeylated to detect the signal peptide cleavage of a glycosylated protein on a polyacrylamide gel it is necessary to block its glycosylation, but still allow membrane insertion to occur. addition of n-linked oligosaccharides onto nascent polypeptides can be blocked by including synthetic acceptor peptides in an in vitro membrane insertion assay (bause, ; lau et al., ) . iycat and an-iycat were translated in the presence of microsomes with and without the acceptor peptide asn-leu-thr. the size of iycat synthesized in the presence of rm and acceptor peptide was indistinguishable from that made in the absence of rm. when proteinase k was used to digest its cytoplasmically exposed domain, the size was reduced by about - kd ( figure a ). we can conclude that nonglycosylated iycat synthesized in the presence of rm and acceptor peptide is inserted into the membrane in the same way as its glycosylated form and that no signal sequence is cleaved during membrane translocation ( figure a figure , lanes and ) . an-iycat'was also found to be protected against exogenous proteinase k ( figure , lane ) . this suggested to us that the larger form was glycosylated and proteolytically processed and that an-iycat' was generated by a proteolytic cleavage, most likely by signal peptidase. to determine the site of cleavage in the proteolytically processed forms of an-ircat, the positions of leucine in the amino-terminal regions of an-itcat and membraneinserted an-lrcat*' were determined. an-i$at was translated in the absence or presence of rm with [sh]leutine as label. as an-iycat is essentially the only protein synthesized from pan-lycat-derived mrna, the complete translation mixture was subjected to automated edman degradation. as seen in figure a , leucine residues are found at the positions , , , , and , as predicted from the sequence deduced from py- cdna (claesson et al., ) . the initiator methionine is probably removed during or shortly after translation (kozak, ) . the positions of leucine residues in the membranetranslocated forms of an-i$at were similarly determined. as rm in the in vitro assay do not translocate all chains, some cytoplasmic forms remained (see inserts in figures a and b ). leucine residues were found at positions , , , and ( figure ) . larger peaks at positions and are consistent with the presence of some unprocessed an-iycat (see insert in figure b ). taking into account the size reduction of about kd by the processing , , and in authentic an-i$at, we conclude that processing has occurred between amino acid residues and ( figures b and c ). proteolytically processed an-lycat is translocated into the lumen of microsomal vesicles with the proteolytic removal of of the hydrophobic amino acid residues in the membrane-spanning region of an-iycat, the question arose as to whether the processed protein was still anchored in the membrane or whether it was now released into the lumen of the microsomal vesicles as is the case for secretory proteins. we used the ex- tractability with carbonate as a criterion for membrane integration. treatment of rm with carbonate at ph releases proteins that are not integrated into the lipid bilayer as well as proteins present in the lumen of microsomal vesicles. an+cat was translated in the presence of rm. membranes were isolated by centrifugation through a sucrose cushion and resuspended in carbonate buffer. solubilized components were then separated from membranes by centrifugation. proteins in the membrane pellet and supernatant were analyzed by sds-page and autoradiography. membrane-spanning proteins, ly and iycat, and the secretory protein, mouse granulocyte-macrophage colony stimulating factor (gm-csf), were used as control (gough et al., ) . as is shown in figure , ly" and i-&at*, as expected for membrane-spanning proteins, were found in the membrane fraction. both an-i$at' and the gm-csf' were found essentially in the soluble, carbonatereleased fraction. thus an-iycat' is released after the proteolytic processing into the lumen of the microsomal vesicles. proteolytic processing, as described above for an-iycat, was also obtained for an+, a protein that lacks the amino-terminal residues of ly (data not shown). our results show that the membrane-spanning segment of the type ii membrane protein ly contains a potentially cleavable signal sequence. this signal sequence is located in the amino-terminal half of the membrane-spanning segment, and it is cleaved when the preceding cytoplasmic domain is removed. all properties known to identify a signal sequence and a cleavage by signal peptidase can be demonstrated. to restrict vertical mobility of the membranespanning segment. ( ) an-itcat, during its initial stage of membrane insertion, also spans the membrane with its hydrophobic segment. however, as no charged amino acid residues are present at the extreme amino-terminal end, the hydrophobic segment has some freedom to change its topology across the membrane. part of the hydrophobic segment might now be pulled into the lumen of the er membrane, and a former cryptic site for signal peptidase cleavage might become accessible to the active center of signal peptidase. first, the cleavage occurs concomitant with insertion into the er membrane as is typical for cleavable signal sequences of presecretory proteins (blobel and dobberstein, ) . second, the cleaved segment is located at the aminoterminal end of the deletion protein an-iycat. it is amino acid residues long and composed entirely of hydrophobic or uncharged residues. signal sequences can vary in length from about to over residues. the only structural element identified so far for a signal sequence is its hydrophobic core, usually - residues long. it is followed by a more polar region - residues long, which is thought to define the cleavage site for signal peptidase. thus, a "minimal" signal sequence would be composed of an residue hydrophobic core followed by a residue region conferring cleavage specificity (von heijne, (von heijne, , . the segment cleaved from protein an-iycat would be consistent with such a minimal length signal sequence. finally, the amino acid residues around the cleavage site in membrane-translocated an-lycat*' are consistent with cleavage by signal peptidase. based on a sequence comparison of eukaryotic signal sequences, von heijne found that only small neutral residues are found at the site of cleavage (- position) and that only small neutral and uncharged ones are found at the - position, that is amino acid residues in front of the signal peptidase cleavage site (von heijne, ) . in the segment cleaved from an-i$at, threonine, a small neutral amino acid, is found at the - position, and leucine, an uncharged amino acid, at the - position. both of these residues fulfill the above described criteria for a signal peptidase cleavage site. thus, place (rm) and time of cleavage (cotranslational), hydrophobic character of the cleaved segment, and property of the cleavage site demonstrate that an-i$at contains a signal sequence at its amino terminus which is cleaved upon membrane insertion by signal peptidase. how can we possibly explain how the deletion of the cytoplasmic, hydrophilic segment from iycat reveals a cleavable signal sequence in a formerly membranespanning region? to us the most plausible explanation is that the position of the hydrophobic segment in the membrane is different in iycat and an-i$at signal peptidase is known to be an integral membrane protein not exposed on the cytoplasmic side of rm (jackson and blobel, ; lively and walsh, ; evans et al., ) . as in many secretory proteins, the cleavage site for signal peptidase is surrounded on either side by or even charged amino acid residues. it is reasonable to assume that the active center of this enzyme is located close to the exoplasmic side of the er membrane, not within the membrane. we propose that the removal of the cytoplasmic, hydrophilic segment from iycat allows the hydrophobic segment to shift its position within the er membrane. most likely it positions itself more toward the exoplasmic side. hence, a potential signal peptidase cleavage site becomes accessible to the active center of signal peptidase (see figure b ). it has been noted previously in type i membrane proteins that a deletion of the charged amino acid residues flanking the membrane-spanning region does not affect the overall topology (zuninga and hood, ; cutler et al., ) . in the case of es glycoprotein of semliki forest virus, it has been shown that mutation of the basic amino acid residues at the cytoplasmic side of the membranespanning segment reduces the stability of the mutant protein in the membrane (cutler et al., ) . when the membrane-spanning regions of type i and type ii membrane proteins are compared, no obvious structural difference can be found. in both types of membrane proteins these regions comprise a stretch of to hydrophobic amino acid residues that is flanked on the cytoplasmic side by positively charged amino acid residues. in type i membrane proteins the segment spanning the membrane does not appear to participate in the initial stage of membrane insertion. type i membrane proteins usually have cleavable signal sequences that initiate the membrane translocation of the amino-terminal half of the protein. the membrane-spanning region, in its position close to the carboxy-terminal end, seems only to function in anchoring the protein in the membrane. yost et al. placed the membrane-spanning segment of the murine surface immunoglobulin heavy chain close to the amino-terminal end of a fusion protein (yost et al., ) . in this position the segment did not provide the signal function for membrane insertion. as, however, a hydrophilic segment of about amino acid residues precedes the membrane-spanning segment, the question still remains as to whether a membrane-spanning region from a type i membrane protein, when placed into the appropriate surrounding, can also initiate translocation across the er membrane. it is well conceivable that certain hydrophilic sequences preceding a hydrophobic segment play a crucial role in exposing a potential signal for membrane insertion. up to now no special structural features, besides hydrophobicity, are known to be crucial for the function of a signal sequence. a common step has been proposed for the early stage of membrane insertion of secretory and membrane proteins (dobberstein et al., ; spiess and lodish, ; lipp and dobberstein, ) . this was based largely on the finding that both of these types of proteins require srp and docking protein for their membrane insertion. here, we show that a type ii membrane protein can be converted into a secretory protein by removal of the cytoplasmic segment. this directly demonstrates that the signal for membrane insertion of these two types of proteins can be the same. further deletion into the carboxy-terminal half of the ly hydrophobic segment is required to elucidate whether the cleaved signal sequence contains all the information for membrane insertion. it is conceivable that the functional signal sequence extends over the cleaved signal sequence into the adjacent hydrophobic part. for some secretory protein it has been observed that the cleavable signal sequence is not sufficient for membrane insertion. in the case of staphylococcal protein a, sequences of the amino-terminal part of the mature protein are required for membrane insertion and correct processing (abrahmsen et al., ) . srp can arrest elongation of presecretory and type ii membrane proteins after or even more amino acid residues have been polymerized (walter and blobel, a; meyer et al., ; lipp and dobberstein, ; lipp et al., unpublished data) . these domains are then inserted into the er membrane by a yet unknown mechanism. as the amino terminus of a type ii membrane protein has to remain on the cytoplasmic side, the formation of a loop during membrane insertion has been proposed. in the case of a secretory protein, signal peptidase would be able to act as soon as the loop appears on the exoplasmic side. an initial interaction of basic residues in a signal sequence with the phosphates of the membrane lipids was originally proposed by lnouye for the lipoprotein of e. coli (inouye et al., ) . our results rule out an essential role of these basic residues in er membrane insertion. the an-iycat protein does not contain any charged amino acid residues preceding the hydrophobic segment. it is nevertheless translocated across the er membrane and processed. the rules that define the cleavage site for signal peptidase in presecretory proteins are not yet fully understood. von heijne points out that the type of amino acids at the - and - position in front of the site of cleavage are im-portant in assigning a cleavage site. here we show that sequences at the very beginning of a signal sequence can also influence cleavage by signal peptidase. in the case of ly, these charged residues can prevent cleavage by signal peptidase. the variability in the length and in the amount of charged amino acid residues at the amino terminus of asignal sequence has not as yet been explained. mutation and deletion experiments have clearly shown that charged residues are not essential for membrane insertion. in the light of our findings, we propose that the charged amino acids at the amino terminus of signal sequences function in the alignment of signal sequences in the er membrane such that signal peptidase can cleave at a very specific site with high fidelity. our prediction is that removal of charged residues from the amino-terminal end of the signal sequences can lead to an altered or less specific signal peptidase cleavage. wheat germ was obtained from general mills, california. the acceptor peptide benzoyl-asn-leuthr-n-methylamide was a generous gift from e. bause, cologne. standard molecular cloning techniques, as described by maniatis et al. ( ) were used. the cdna clone py- , containing the entire coding region of the human invariant chain cloned into the pstl site of pbr , was obtained from p. a. peterson's laboratory, uppsala, sweden (claesson et al., ) . the expression plasmids pds , pds , and pds / have been described previously (stueber et al., ) . they allow efficient transcription by e. coli rna polymerase of cdnas cloned behind the strong t promoter p . figures a and b summarize the construction of the fusion and deletion plasmids described below. plycat py- was digested with pstl, and the bp fragment containing the ' end and the bp fragment containing the ' end of the ly coding region were isolated. the bp fragment coding for the ly cytoplasmic domain, the membrane-spanning segment and amino acid residues of the exoplasmic domain, was cleaved by sauda to remove the sgc tail. the bp sau a-pstl fragment was isolated and cloned into bamhiipstl-cut pds . this results in an in-frame fusion of the ' end of ly to the cat gene. ph initial attempts to clone the completely coding region into pds failed. when expressed, this region is probably lethal to the bacterium. to repress transcription from the t promoter/operator (p/o) in bacteria, we cloned the lac i repressor between the b/a gene and the t p/o. this plasmid is called pfllycat. for the construction of ply, prlycat was linearized by pstl and the bp pstl fragment, coding for the carboxy-terminal domain of it, was ligated into this site. transformants containing the bp fragment were screened for expression of immunoprecipitable ly chain after in vitro transcription-translation. to delete the cytoplasmic domain from ircat, the bp sstll-xbal fragment from plycat was isolated and ligated at the xbal site of bamhllxbal cut p / r. the protruding ends at the bamhl and the sstll sites were blunted with sl nuclease and ligated. as a result, a new atg initiation codon is placed just in front of the membrane-spanning segment of ly. the construction was confirmed by dna and amino acid sequence analyses (see figure a ). p lsr to repress transcription from the t promoter the lac i gene was inserted between the b/a gene and the t p/o region of pds / (stueber et al., ) . against ly domains to raise antibodies against the amino-and the carboxy-terminal domains of ly, fusion proteins of b-galactosidase and parts of ly were produced in bacteria and used as antigens to raise antibodies in rabbits. from a pstl digest of w- , the bp fragment coding for the aminoterminal amino acids of ly and the bp fragment coding for the exoplasmic carboxy-terminal domain of ly were isolated. each of the fragments was inserted into the pstl site of the bacterial expression vector pex (stanley and luzio, ) . fusion proteins expressed in nfl bacteria were separated on preparative sds-polyacrylamide gels ( % acrylamide; laemmli, ) . protein bands were visualized by koac precipitation, and fusion proteins were eluted from gel slices. two rabbits were immunized with each of the two fusion proteins. antibodies against the amino terminus of ly (anti-iyn) and its carboxyl terminus (anti-iyc) were obtained. they reacted with authentic ly chains synthesized by human raji cells (data not shown). lmmunoprecipitations after translation and posttranslational assays, antigens in a vi aliquot were solubilized by adding nonidet-p (np ) to . %. then ~ of either anti-iyn or anti+c antiserum was added and the mixtures incubated for min at oc. forty microliters of a :l slurry of protein a-sepharose (equilibrated in . % np ,lo mm tris-hci [ph . , mm naci, and mm edta) was added to each sample, and incubation continued for min at °c. beads were sedimented by centrifugation and washed three times with . % np , mm tris-hci (ph . ), mm naci, and mm edta, twice with . % np ,lo mm tris-hci (ph . ), mm naci, and mm edta, and once with mm tris-hci (ph . ). sample buffer for sds-page was added to the sedimented beads, and antigens were analyzed by sds-page and fluorography. in vitro transcription and translation plasmids were transcribed in vitro by e. coli rna polymerase, and the resulting mrna was translated in a wheat germ cell-free system as described by stueber et al. ( ) . to test for membrane translocation, rough microsomes from dog pancreas were included in the translation (blobel and dobberstein, ) . glycosylation onto asparagine residues was blocked by the addition of the acceptor peptide benzoylasn-leuthr-n-methylamide to a final concentration of pm (lau et al., ; bause, ) . assays to test translocation of in vitro-synthesized proteins across, or their insertion into, the er membrane, accessibility to proteinase k was used. a pl aliquot of a translation mixture containing rough microsomes was incubated for min at oc with either . mg/ml of proteinase k or . mg/ml of proteinase k and . % np . further proteolysis was stopped by the addition of phenylmethylsulfonyl fluoride (pmsf) to . mglml, and the sample was further characterized by sds-page (laemmli, ) and fluorography or, where indicated in the figure, by immunoprecipitation. to remove secretory and peripheral membrane proteins, rough microsomes were subjected to a carbonate wash with . m na&os, ph (fujiki et al., ) . peptide; h. gausepohl for performing automated amino acid analysis: m. t. haeuptle, i. ibrahimi, and d. meyer for critical reading of the manuscript, and annie steiner for expert typing. this work was supported by grant do / -z from the deutsche forschungsgemeinschaft. the costs of publication of this article were defrayed in part by the payment of page charges. this article must therefore be hereby marked "advertisement" in accordance with usc. section solely to indicate this fact. received april , ; revised july , . multiple mechanisms of protein insertion into and across membranes a stop transfer sequence confers predictable transmembrane orientation to a previously secreted protein in cell-free systems clonal variation in cell surface display of an h- protein lacking a cytoplasmic tail we thank p a. peterson and l. claesson, uppsala, for plasmid py- ; e. bause, cologne, for the acceptor abrahamsen, l., moks, t., nilsson, b., hellman, u., and uhlen, m. ( ) . analysis of signals for secretion in the staphylococcal protein a gene. embo j. , - . adams, g. a., and rose, j. k. ( ) . structural requirements for a membrane-spanning domain for protein anchoring and cell surface transport.cell , looi- .anderson, d. j., mostov, k. e., and blob& g. ( ) . mechanisms of integration of de novo-synthesized polypeptides into membranes: signal recognition particle is required for integration into microsomal membranes of calcium atpase and of lens mp but not of cytochrome be. proc. natl. acad. sci. usa , - . bause, e. ( ) . structural requirements of n-glycosylation of proteins. biochem. j. , - . blobel, g., and dobberstein, b. ( ) lingappa, v. r., katz, f. n., lodish, h. f., and blobel, g. ( ) . a signal sequence for the insertion of a transmembrane glycoprotein. similarities to the signals of secretory proteins in primary structure and function. j. biol. chem. , - .lipp, j., and dobberstein, b. ( ). signal recognition particle-dependent membrane insertion of mouse invariant chain: a membrane spanning protein with a cytoplasmically exposed amino-terminus. j. cell biol. , - . long, e. . ( . in search of a function for the invariant chain associated with la antigens. surv. immunol. res. , - . lively, m. o., and walsh, k. a. ( ) spiess. m., and lodish. h. f. ( ) . an internal signal sequence: the asialoglycoprotein receptor membrane anchor. cell , - . stanley, k. k., and luzio, j. p ( ) . construction of a new family of high efficiency bacterial expression vectors: identification of cdna clones coding for human liver proteins. embo j. , - . strubin, m., mach, b., and long, e. . ( . the complete sequence of the mrna for the hla-dr associated invariant chain reveals a polypeptide with an unusual transmembrane polarity. embo j. , ~ . key: cord- -pqxkyg z authors: reggiori, fulvio title: membrane origin for autophagy date: - - journal: curr top dev biol doi: . /s - ( ) - sha: doc_id: cord_uid: pqxkyg z autophagy is a degradative transport route conserved among all eukaryotic organisms. during starvation, cytoplasmic components are randomly sequestered into large double‐membrane vesicles called autophagosomes and delivered into the lysosome/vacuole where they are destroyed. cells are able to modulate autophagy in response to their needs, and under certain circumstances, cargoes, such as aberrant protein aggregates, organelles, and bacteria can be selectively and exclusively incorporated into autophagosomes. as a result, this pathway plays an active role in many physiological processes, and it is induced in numerous pathological situations because of its ability to rapidly eliminate unwanted structures. despite the advances in understanding the functions of autophagy and the identification of several factors, named atg proteins that mediate it, the mechanism that leads to autophagosome formation is still a mystery. a major challenge in unveiling this process arises from the fact that the origin and the transport mode of the lipids employed to compose these structures is unknown. this compendium will review and analyze the current data about the possible membrane source(s) with a particular emphasis on the yeast saccharomyces cerevisiae, the leading model organism for the study of autophagosome biogenesis, and on mammalian cells. the information acquired investigating the pathogens that subvert autophagy in order to replicate in the host cells will also be discussed because it could provide important hints for solving this mystery. in eukaryotic cells, the principal locations where protein catabolism occurs are the proteasome and the lysosome. the proteasome mostly recognizes and degrades cytosolic factors that have been specifically marked with polyubiquitin chains (roos-mattjus and sistonen, ) . the lysosome in contrast, requires active transport in order for the diverent substrates destined for elimination to reach its interior where the proteases are located. four diverent pathways can deliver intracellular proteins into the lysosome lumen: endosomal transport routes, chaperone-mediated autophagy (cma), microautophagy, and macroautophagy, the latter generally referred to as autophagy katzmann et al., ; klionsky, ; majeski and dice, ) . the endosomal transport routes and cma are mostly devoted to the transport of polypeptides, whereas microautophagy and autophagy deliver other cellular constituents because these pathways are the only ones able to internalize entire organelles and bacteria. eukaryotes, in particular fungi, can use microautophagy to eliminate peroxisomes and is the only cellular function that has indisputably been assigned to this pathway . autophagy, on the other hand, can deliver various cargoes to the lysosome interior and has multiple physiological roles. the hallmark of this catabolic pathway is the sequestration of cargoes by large cytosolic double-membrane vesicles called autophagosomes (reggiori and klionsky, ) . the autophagosomes successively dock and fuse with mammalian lysosomes or the yeast and plant vacuoles releasing the inner vesicles into the lumen of these organelles (reggiori and klionsky, ) . the biogenesis and consumption of these structures can be divided into six discrete steps: induction, expansion, vesicle completion, docking, fusion, and breakdown ( fig. ). autophagosomes are generated by the elongation of a small template membrane, termed the isolation membrane or phagophore (fengsrud et al., ; mizushima et al., ; noda et al., ; reggiori and klionsky, ) . there are several of these structures per cell but it still remains unknown where they are derived from. the surface of this small compartment is decorated with atg and atg , and its formation requires phosphatidylinositol (ptdins)- -kinase activity (mizushima et al., , . there are two ways of triggering the expansion of the isolation membrane, and they diver depending if the process of autophagy is selective or nonselective (section i.b) (reggiori and klionsky, ) . when this figure conceptual model for autophagy. the basic mechanism of autophagy is the sequestration of the cargo material (bulk cytoplasm, protein aggregates, organelles, or pathogens) by a cytosolic double-membrane vesicle named an autophagosome. extracellular stimuli or the recognition of a specific intracellular cargo induce the expansion of the isolation membrane. upon vesicle completion, the autophagosome docks with the lysosome/vacuole and successively fuses with it. in this way the inner vesicle is liberated inside the vacuole where it is finally consumed together with the cargo by resident hydrolases. this schematic represents nonspecific autophagy and does not show specific types of autophagy including the cvt pathway. pathway is selective, the binding to the isolation membrane of the cargo that has to be specifically eliminated (or in the case of resident hydrolases, activated) leads to the expansion of this structure (ogawa et al., ; shintani and klionsky, b) . in contrast to selective autophagy, which is induced by intracellular components, the nonselective process is governed by extracellular stimuli such as nutrients or cytokines (gutierrez et al., ; lum et al., ; shintani and klionsky, a) . in both cases, covalent conjugation of the ubiquitin-like atg -atg seems to be the step that initiates the expansion of the isolation membrane . the expansion of the isolation membrane is basically the simultaneous elongation and nucleation of this little cisterna (fig. ) . it is not known how the atg -atg complex recruits additional membranes, but the crescent autophagosome acquires more atg -atg and atg along with a second ubiquitin-like molecule, atg /lc , that is unconventionally linked to phosphatidylethanolamine (pe), and probably the rest of the atg proteins (mizushima et al., , . two expansion mechanisms are possible, one that relies on delivery of lipid bilayer by vesicular trayc (vesicular expansion) and one based on the fusion of small compartments (cisternal expansion) (reggiori and klionsky, ) . in addition, it has been suggested that retrograde trayc balances double-membrane vesicle biogenesis by recycling some atg proteins, such as atg , and also recovering from the forming autophagosome membrane components specific to the compartment(s) of origin (meiling-wesse et al., ; nazarko et al., ; nice et al., ; reggiori et al., reggiori et al., , a . when the two extremities of the forming autophagosomes reach each other, they fuse together sealing the vesicle (fig. ). this fusion event, at least in yeast, appears to be snare-independent and triggers an uncoating reaction where the externally localized components dissociate from the vesicle surface (ishihara et al., ; reggiori and klionsky, ; reggiori et al., b) . in particular, the ubiquitin-like protein atg -pe is proteolytically released from its lipid moiety by the atg protease, whereas the transmembrane protein atg is completely retrieved (kirisako et al., ; reggiori et al., a) . it is still unknown which factor senses completion of the double-membrane vesicle and initiates this disassembly. once uncoated, the double-membrane vesicle docks with the lysosomes/ vacuoles (fig. ) . in mammalian cells, this association is facilitated by microtubules and seems to require dynein whereas in yeast it is independent of these structures (aplin et al., ; fengsrud et al., ; kirisako et al., ; punnonen and reunanen, ; ravikumar et al., ; webb et al., ) . the fusion between the autophagosome and the lysosome/ vacuole occurs as soon as these organelles dock and it is mediated by a set of proteins also used for other fusion reactions with the lysosome/vacuole (section i.c). during this event, the external membrane of the autophagosome becomes part of the lysosome/vacuole surface whereas the inner autophagosomal vesicle is liberated in the interior of this organelle and now called an autophagic body (fig. ) . the limiting membrane of the autophagic body is immediately attacked and consumed by resident lysosomal/vacuolar hydrolases allowing these enzymes to gain access to the content of this vesicle. as a result, the cargoes are also degraded into their basic constituents (or in the case of certain resident hydrolases, processed to their active form; fig. ). autophagy has been known for a long time as an adaptation response to starvation and as the major factor in the turnover of long-lived proteins. but in recent years, it has become evident that autophagy plays an active role in several other physiological tasks highlighting its versatility and adaptability. we now know that this catabolic pathway participates in cellular processes such as development, cellular diverentiation and rearrangement, elimination of aberrant structures, lifespan extension, mhc class ii presentation of cytoplasmic antigens, and type ii programmed cell death, as well as protecting against pathogens (both viruses and bacteria) and tumors (cuervo et al., ; debnath et al., ; deretic, ; thompson, , ; kirkegaard et al., ; komatsu et al., ; kondo et al., ; levine and klionsky, ; paludan et al., ; rubinsztein et al., ; shintani and klionsky, a) . as a result, this degradative transport route plays a relevant role in the pathophysiology of neurodegenerative, cardiovascular, muscular, and autoimmune diseases, and some malignancies thompson, , ; kondo et al., ; rubinsztein et al., ; shintani and klionsky, a; towns et al., ). autophagy provides one evective way to adjust and cope with these various situations by rapidly delivering large fractions of the cytoplasm, aberrant protein aggregates, superfluous or damaged organelles, and invading pathogens into the lysosome/vacuole interior where they are destroyed by resident hydrolases (reggiori and klionsky, ) . the adaptability of this pathway is due to its ability to select specific cargoes when forced by circumstances. it has been believed for a long time that autophagy was a nonspecific process because when induced by starvation, cytoplasmic components and organelles were randomly sequestered into autophagosomes; however, this pathway can also be selective (table i) (reggiori and klionsky, ) . in the yeast saccharomyces cerevisiae, for example, aminopeptidase i (ape ) and -mannosidase (ams ) form a large oligomer that is unconventionally delivered from the cytoplasm directly to the vacuole interior through a process known as the cytoplasm to vacuole targeting (cvt) pathway (kim et al., ; shintani et al., ) . this transport route is specific and biosynthetic. precursor ape (prape ) is packed into double-membrane vesicles called cvt vesicles, which are four to eight times smaller in surface area than autophagosomes scott et al., ) . in the same organism, dysfunctional mitochondria are preferentially eliminated by autophagy (mitophagy) as well as superfluous peroxisomes (pexophagy) ( table i) (hutchins et al., ; priault et al., ) . the specific sequestration of peroxisomes into double-membrane vesicles and their subsequent degradation has also been very well described in other fungi such as pichia pastoris, hansenula polymorpha, and yarrowia lipolytica . mammalian cells on the other hand, seem not to possess a transport route similar to the cvt pathway, but there are indications that mitophagy could occur (bota and davies, ; elmore et al., ; rodriguez-enriquez et al., ) . pexophagy has also been report ed (lui ken et al. , ; yokota , ; yoko ta et al. , ) . it has lately been shown that autophagy can be selective in mammalian cells as the diverent types of selective autophagy, their specific cargoes, and the organisms that have been described are indicated. fulvio reggiori well, as evidenced by the specific recognition and disposal of invading bacteria and potentially also of intracellular viruses (table i) (deretic, ; kirkegaard et al., ; levine, ) . in addition, a study analyzing conditional knock-out mice defective for autophagy has revealed that the mutant animal accumulates numerous ubiquitinated aggregates in the cytosol, suggesting that this covalent protein modification could serve to specifically target to autophagosomes large structures that have to be eliminated (komatsu et al., ) . the process of autophagy has been known for at least years, but because none of the specific components involved in this pathway were known, the studies about this degradative transport route were limited to morphological and phenomenological observations. in the last years, genetic screens, mostly in the yeast s. cerevisiae and fungi such as p. pastoris and h. polymorpha, have lead to the isolation of genes termed autophagyrelated (atg) genes whose products are specifically involved in this catabolic pathway (table ii) (klionsky, ; klionsky et al., ) . the extent of the conservation of this pathway between eukaryotes was first revealed by comparing the genomes once various sequencing projects were completed (reggiori and klionsky, ) . it became immediately evident that most of the atg genes had one or more homologs in higher eukaryotic organisms. the cellular role of some of them has now been explored and in all the analyzed cases, it has been demonstrated that the homologs function as orthologs (table ii) (levine and klionsky, ; reggiori and klionsky, ) . the same genetic approaches have also led to the discovery of nine atg genes dispensable for bulk autophagy but essential for the cvt pathway and/ or pexophagy (table iii) . their products are mostly involved in cargo selection and the final sealing of the double-membrane vesicle, indicating that additional components are required for the autophagosomes to be able to enwrap specific cargoes. it is important to note that these genes involved in specific types of autophagy do not have clear homologs in higher eukaryotes sustaining the idea that the cvt pathway and pexophagy are probably only present in fungi (reggiori and klionsky, ) . in addition to the atg proteins, the genetic screens in yeast have also permitted the identification of additional components required for the normal progression of autophagy that are shared with other intracellular transport routes (table iv) . the function of several of these factors in the other pathways was already known and that has helped in clarifying the mechanism of autophagy. for example, yeast vacuoles can fuse with late . membrane origin for autophagy . in all these cases, cells use an identical fusion machinery, which consists of snare proteins, sec (nsf), sec (-snap), a rab-gtpase, and the class c vps protein complex also known as the hops complex. the same components have also been found to be exploited for the fusion of double-membrane vesicles (table iv) (reggiori and klionsky, ; wang et al., ) . similarly, it is also now evident that the dissolution of autophagic bodies is mediated by the same hydrolases that degrades the a in s. cerevisiae, these proteins also catalyze the retrieval transport from early endosomes. b atg is not required for pexophagy in s. cerevisiae but is essential for the same process in h. polymorpha. c these factors have no counterparts in s. cerevisiae or the homologs do not have a role in pexophagy. ? one report has indicated that atg is essential for pexophagy, another affirms that atg is not required for this process. mvb internal vesicles once these are released into the vacuole lumen (table iv) (epple et al., ; reggiori and klionsky, ) . most of the atg components are peripheral membrane proteins that transiently associate with the nascent autophagosomes. in contrast to mammalian cells where several isolation membranes can be simultaneously activated, a single perivacuolar site of organization for double-membrane vesicle formation (named the pre-autophagosomal structure, pas) is observed in the yeast s. cerevisiae (kim et al., ; suzuki et al., ) . the pas is believed to be the yeast counterpart of a mammalian isolation membrane and in this unicellular eukaryote, most of the atg proteins appear to be primarily restricted to this location. this unique site seems also to be present in h. polymorpha (monastyrska et al., a,b) . in p. pastoris, however, several atg components are distributed to more than one punctate structure (ano et al., ; chang et al., ; kim et al., b; mukaiyama et al., ; stromhaug et al., ) . it is unclear if this represents a diverence between organisms or is due to diverent growth conditions. p. pastoris is mostly used for the study of pexophagy and therefore grown in special media containing carbon sources that induce peroxisome proliferation. it is unclear where the pas is derived from and at which point it becomes membranous. the study of the cvt pathway has provided insights into how this structure is generated. after synthesis, prape forms a large oligomer that first associates with the atg cargo receptor and then with the atg adaptor to form the cvt complex (shintani et al., ) . this large cytosolic protein aggregate then moves in close proximity to the vacuole surface, where it induces the recruitment of the rest of the atg factors, triggering the formation of the cvt vesicle yorimitsu and klionsky, ) . neither the pas nor the vesicles are eyciently formed in the absence of any of the cvt complex components, indicating that the cargo stimulates the biogenesis of these structures . this requirement is overcome when cells are nitrogen-starved (kim et al., b; shintani and klionsky, b) . because of its dynamic properties, the pas should not be seen as a static or defined organelle but more as a structure in constant remodeling. it remains unclear at which stage and how membranes are transported at the pas, but because of their association with lipid bilayers, two proteins, atg and atg , could be important for dissecting this event. atg is a soluble ubiquitin-like protein and its carboxy-terminal arginine is removed by the atg cysteine protease leaving a glycine residue at the new carboxy terminus (kim et al., a; kirisako et al., ) . atg is activated by the e enzyme atg through a thioester bond between its carboxyterminal glycine and cysteine of atg (kim et al., ; kirisako et al., ; komatsu et al., ) . atg is subsequently transferred to the e enzyme atg via a new thioester bond between these two proteins kim et al., a) . atg is finally covalently conjugated to a pe molecule, becoming tightly membrane associated . this linkage is reversible because atg can be proteolitically released from its lipid moiety by atg , an event that takes place once the double-membrane vesicles are completed (section i.a. ) (kirisako et al., ; reggiori et al., a) . it is unclear where the atg conjugation to pe occurs. this protein is normally lipidated in mutants unable to form the pas indicating that this modification takes place at a diverent subcellular location (suzuki et al., . membrane origin for autophagy ) . this is supported by the fact that atg -pe localizes to the pas but also to tiny cytosolic vesicles (kirisako et al., ) . these data, however, do not exclude the possibility that atg -pe conjugates are formed at the pas as well. the association of atg -pe with membranes prior to getting concentrated at the pas suggests that the atg -pe structures could be at least in part the source of autophagosome lipid bilayers. this idea is supported by the observation that in the absence of atg , membranes fail to be delivered to the pas and therefore the size of autophagosomes is strongly reduced (abeliovich et al., ; kirisako et al., ; lang et al., ) . it remains a mystery where the tiny atg -pe containing vesicles are derived from, but one possibility is that that they originate from early compartments of the secretory pathway, for example, the endoplasmic reticulum (er) and/or the golgi apparatus. this hypothesis is based on two experimental findings. first, atg binds two vsnares required for both anterograde and retrograde transport between the er and golgi apparatus . second, this ubiquitin homolog has been detected on autophagosome-like structures derived from the golgi complex and/or er (section ii.d. ) (reggiori et al., b) . atg is the only integral membrane protein essential for double-membrane vesicle formation . this protein is probably transported to the pas with at least part of the lipids or lipid bilayers required to create this structure. this notion is corroborated by the fact that the totality of atg is associated with membranes reggiori et al., b) . atg cycles between the pas and several unknown punctate structures dispersed in the cytosol supporting the idea that it could partially supply the forming autophagosomes with membranes (reggiori et al., a) . a fraction of these punctate structures are atg aggregates residing on the mitochondria surface (reggiori et al., b) . this suggests that this organelle could provide the nascent autophagosomes with at least part of its lipid bilayers. however, it cannot be excluded that atg traycking carries out other functions. under certain conditions, autophagy becomes one of the principal sources of energy for the cell (kuma et al., ; lum et al., ) . because the mitochondria provide the other primary supply of energy, one could imagine that atg is used to coordinate the two sources. the sorting mechanism for atg transit from mitochondria to the pas is unknown, but under growing conditions this event is induced by cvt complex assembly and requires actin (reggiori et al., a; shintani and klionsky, b) . in contrast, the retrieval transport of this transmembrane protein from the pas has been characterized in more detail and shown to be regula ted by the atg -at g signal ing complex and requ ires atg , atg , and the ptdins - -phosp hate generat ed by the atg con taining ptd ins -kinase complex ( reggiori et al. , a) . this recycl ing eve nt, howeve r, seems to be to some extent di verentl y organiz ed in p. pasto ris dur ing micro pexoph agy, possibly because other membr ano us struc tures a re used an d assem bled in a di verent way during this invaginati on pro cess ( chang et al. , ) . an initial analys is concerning the role of yeast early secretion (sec ) mutant s in autoph agy has reveal ed that several of them are essent ial for autop hagosome form ation (ishiha ra et al. , ) . this class of genes is involved in trans port out of the er (kais er an d schekman, ). successi ve studi es, howeve r, have shown that these mutant s ha ve an indir ect negati ve e vect on both the cvt pa thway and au tophagy ( hama saki et al. , ; reggiori et al. , b ) . one possibl e explanation of their phe notype is that they alter the er morpho logy and con sequently impair severa l function s of this organel le, includi ng the put ative one to supp ly membra nes for doublemembr ane vesic le formati on. for example, the er is structural ly connected wi th the mitoc hondria and the disruption of the er organ ization in the early sec mutant s causes the fragmenta tion of the mitocho ndrial reti culum ( prinz et al. , ) . as mention ed, atg partiall y local izes to mito chondria, and in this class of mutants its tray cking out of this compartmen t is severe ly impai red (re ggiori and klio nsky, sub mitted). atg , atg , tlg , tl g , trs , vps , and the subunit s of the vpsfiftythree (vft) complex are part of retrieval transp ort routes from en dosomal compart ments back to the golgi app aratus, and consequ ently they are important in maintaining certain functions of this organelle (hettema et al., ; holthuis et al., ; sacher et al., sacher et al., , siniossoglou and pelham, ) . these proteins have also been shown to be required for the cvt pathway and some of them also play an important role in doublemembr ane vesicle biog enesis during pe xophagy and autophagy (tables iii and iv reggiori et al., ) . it is unclear, however, why these pathways are impaired in the absence of these factors. one possibility is that retrograde trayc from the forming double-membrane vesicles is . membrane origin for autophagy essential for the expansion and/or completion of these structures (meiling-wesse et al., ; reggiori et al., a,b) . a second hypothesis is that similarly to what was predicted for early sec mutants, an alteration of the golgi apparatus functions could interfere with the lipid bilayer delivery essential for the creation of these large vesicles. the major diyculty in investigating the contribution to autophagy of both the er and the golgi apparatus is that these two organelles depend on each other for their proper function. mutations that avect one of these two compartments indirectly perturb the other one. along these lines, the interpretation of the block of both the cvt pathway and autophagy in the sec mutant is not simple (reggiori et al., b) . nevertheless, the analysis of this strain has led to important information. sec is a gdp/gtp exchange factor required for traycking through the golgi complex (franzusov and schekman, ; jackson and casanova, ) . the inactivation of this protein provokes the accumulation of unsealed, autophagosome-like structures that are decorated with atg (reggiori et al., b) . these membranous arrangements enwrap ribosomes and cytosol and have been previously named berkeley bodies (esmon et al., ; novick et al., ) . this surprising result indicates that potentially, double-membrane vesicles can be created in large part by altering the activity of a single enzyme; however, it cannot be excluded that this is an indirect phenomenon. vps is a protein essential for the invagination of the late endosome limiting membranes and therefore mvb biogenesis (babst et al., ; katzmann et al., ) . a unique vps allele was isolated in a screen for mutations that result in autophagy induction even in the presence of nutrients (shirahama et al., ) . this led to an initial interpretation that endosomes play a relevant role in autophagosomes biogenesis. however, reports where the functions of these compartments have been severely impaired by specific gene deletions have revealed that the integrity of the endosomal system is not essential for either the cvt pathway or autophagy (epple et al., ; reggiori et al., b) . in contrast to the late stages of the autophagosome biogenesis where lipid bilayers are derived from endosomal compartments, the origin of the mammalian isolation membrane or phagophore remains uncertain. it is still unknown if this small sequestering cisterna is formed de novo or derived from a preexisting organelle (fengsrud et al., ) . a major problem in trying to investigate its origin is that these structures and autophagosomes are mostly composed of lipids and depleted in transmembrane proteins making particularly diycult the detection of specific organelle markers (fengsrud et al., ; hirsimaki et al., ; punnonen et al., ; reunanen et al., ; stromhaug et al., ) . this unique characteristic is one line of evidence that the isolation membranes and autophagosomes diver structurally from the other subcellular organelles. this observation also implies that whatever the origin of the lipid bilayers used to form autophagosomes, integral membrane components are segregated away from them. two models could explain how protein-depleted membranes are obtained. in the first, isolation membranes are derived from a specialized organelle subdomain where autochthonous proteins are gradually excluded. a similar process has been shown to occur during peroxisome biogenesis from the er tabak et al., ; tam et al., ) . in the second model, the same cisterna is progressively emptied of integral membrane factors by retrieval transport-a phenomenon hypothesized to occur during double-membrane vesicle formation in yeast (reggiori et al., a,b) . it is also possible that both mechanisms coexist. numerous studies have been published investigating the source for autophagosome lipid bilayers in mammalian cells but their conclusions often contrast. thus various organelles, such as the er, the golgi complex, and the plasma membrane, have been suggested to be the origin of doublemembrane vesicles. because of the heterogeneity in the results, no unanimous agreement in the field has been reached. for example, several studies have reported the presence of er-marker proteins in the isolation membranes and autophagosomes but others have shown that these structures lack er-resident factors (arstila and trump, ; dunn, a; furuno et al., ; reunanen et al., ; stromhaug et al., ; yamamoto et al., ; yokota et al., ) . as with the er, the role of the golgi complex as a lipid donor for the early autophagosome intermediates remains ambiguous. some studies have shown the presence of golgi protein markers in these structures whereas others have failed to detect them (arstila and trump, ; dunn, a; frank and christensen, ; locke and sykes, ; yang and chiang, ; yokota et al., ) . the membranes of the cis-golgi network have been shown to possess the same compositional characteristics of the isolation membrane (fengsrud et al., ; locke and sykes, ; reunanen et al., ; yamamoto et al., ) . only a few reports have indicated that the autophagic cisternae are derived from the plasma membrane and their conclusions have been challenged when other investigators have failed to detect plasma membrane protein markers in these structures (araki et al., ; arstila and trump, ; bosabalidis, ; ericsson, ; fengsrud et al., ; oledzka-slotwinska and desmet, ; reunanen et al., ) . importantly, autophagosomes have a low cholesterol content validating the idea that their membranes are not derived from the plasma membrane (reunanen et al., ) . the discrepancy between all these analyses could be due, in part, to diverent experimental approaches and techniques used in the various laboratories. but one possibility that should not be discarded a priori is that autophagosomes could be a mosaic of membranes derived from more than one organelle. for example, the isolation membrane could originate from one compartment and the additional lipid bilayers required for its expansion be acquired from other sources. in addition, the diverent contributions could vary depending on the tissues with cells able to derive the membranes from the most suitable reservoirs. the atg conjugation system is highly conserved in higher eukaryotic cells (table ii) tanida et al., ) . in mammals, there are at least three atg homologs: the microtubule-associated protein (map ) light chain (lc ), the golgi-associated atpase enhancer of kda (gate- ), and the -aminobutyric acid (gaba) a -receptorassociated protein (gabarap) (mann and hammarback, ; sagiv et al., ; wang et al., ) . it should be noted that these three proteins were first isolated because of their involvement in other traycking pathways. the mammalian counterparts of atg process these three atg homologs by exposing their conserved c-terminal glycine which then interacts with mammalian atg and atg homologs before being covalently linked to a lipid (hemelaar et al., ; scherz-shouval et al., ; tanida et al., tanida et al., , . the target phospholipid has not yet been unequivocally identified, but strong evidence suggests that it is pe (kabeya et al., ; tanida et al., ) . of the three homologs, lc has been best characterized as an autophagosomal marker in mammalian autophagy. the newly synthesized lc precursor is processed cotranslationally to generate a soluble lc form (lc -i) that, upon starvation, becomes membrane-bound and has greater mobility than lc -i when resolved by sds-page . the lipidated protein, called lc -ii, localizes on both autophagosomes and autolysosomes . these in vitro results have been confirmed using transgenic mice expressing gfp-lc . unfortunately, the small amount of lc -ii generated prior to induction of autophagy is already associated with the double-membrane vesicles formed by the basal activity of this pathway and lc -i is not clearly associated with a distinct membranous structure (kabeya et al., ) . therefore, the subcellular localization of these molecules has not furnished insights about the lipid bilayer source. both gate- and gabarap possess a form ii and localize to lc positive autophagosomes that are induced by starvation (kabeya et al., ) . thus, it remains a possibility that they participate in autophagy in addition to, or instead of, their originally described functions. because the three mammalian atg homologs are diverently expressed in various tissues (tanida et al., ) , another intriguing option is that these proteins are involved in supplying the autophagosome with membranes derived from diverent compartments depending on the cell type; for example, gate- from the golgi complex and gabarap from the same organelle as well as the synaptic cisternae (kittler et al., ; kneussel et al., ; sagiv et al., ) . a report has demonstrated that the two human proteins with high homology to atg , hsatg l and hsatg l , are its orthologs (yamada et al., ) . in human adult tissues, hsatg l is ubiquitously expressed, whereas hsatg l is highly expressed in placenta and pituitary gland. importantly, the authors have also shown that these two factors are not distributed on mitochondria. instead they localize to a perinuclear region, suggesting that in higher eukaryotes atg could supply autophagy with membranes by deriving lipid bilayers from a diverent reservoir. this observation could also explain why hsatg l and hsatg l cannot substitute for the yeast atg (reggiori et al., b; yamada et al., ) . however, hsatg l possesses a nonfunctional mitochondrial targeting sequence that is also present in its closest higher eukaryote homologs (yamada et al., ) . this characteristic raises the possibility that this is an ancient localization signal. because the subcellular distribution of hsatg l and hsatg l have not been carefully examined and the preliminary localization analysis was performed with overexpressed proteins, the identification of the precise localization of these two proteins could provide insights into membrane dynamics during autophagosome biogenesis in mammals. in mammalian cells, autophagosomes, also called initial autophagic vacuoles (avi), undergo a stepwise maturation process that can be followed ultrastructurally by monitoring the disintegration status of their internal lipid . membrane origin for autophagy bilayer and cargoes (fig. ) (dunn, b; eskelinen, ; fengsrud et al., ; rabouille et al., ) . these morphological changes correlate with the increasing acquisition of lysosomal makers dunn, b; liou et al., ; tanaka et al., ) . autophagosomes, which contain intact cytosol and organelles, fuse first with endosomal vesicles and mvb turning into early degradative autophagic vacuoles (avd) or amphisomes. these structures successively fuse together or with lysosomes becoming late avd or autolysosomes. the degradation of the internal material starts in the early avd and continues in the late avd until completion. in contrast to yeast, the endosomal system plays an essential role in mammalian autophagy (see section ii.d. ). this divergence between species has been highlighted by the discovery that skd is necessary for autophagosome maturation in mouse cells (nara et al., ) . skd is the mouse homolog of yeast vps and, as its counterpart, it is also essential to maintain endosome morphology and endosomal transport . as mentioned, vps is not required for autophagy in yeast (section ii.d. ) (reggiori et al., b; shirahama et al., ) . it is unclear why mammalian autophagosomes need the additional maturation step characterized by their fusion with endosome-and/or trans-golgi network (tgn)-derived transport vesicles and mvb. in yeast, doublemembrane vesicles fuse with a much larger vacuole one after the other. therefore, their cargoes do not influence the hydrolytic capacity of this compartment by altering, for example, the ph because the volume of their contents is just a fraction of that of the entire vacuole. lysosomes, in contrast, are much smaller than vacuoles and their size is comparable to that of autophagosomes. figure autophagosome maturation in mammalian cells. once sealed, the autophagosome (or avi) fuses with endosome-and/or tgn-derived transport vesicles and the mvb becoming an amphisome (or early avd). this event leads to the acquisition of hydrolytic enzymes that initiate the consumption of the autophagosome cargo. the amphisome then fuses with a lysosome generating a new organelle termed autolysosome (or late avd) where the degradation of the content of the initial autophagosome is completed. consequently, if these two structures would immediately fuse together, an important dilution of the lysosome content could occur impairing its internal enzymatic activity. addition of extra hydrolytic enzymes prior to autolysosome formation could help to compensate for this dilution phenomenon. autophagy provides a cellular defense against invading pathogens but unfortunately, some of them have developed systems to avoid the sequestration and elimination by double-membrane vesicles (deretic, ; kirkegaard et al., ; levine, ) . in addition, there are virus and bacteria that exploit this transport route to enter and replicate inside the host cell (kirkegaard et al., ) . the study of this latter class of pathogens has furnished some indications about the possible origin of autophagosome membranes even if it should be kept in mind that these invading microorganisms are also altering other cellular pathways, and therefore autophagy could progress in part diverently in the infected cells. upon infection, positive-strand viruses disassemble and release their genomic rna into the cytoplasm of the host cell. the genomic rna is subsequently translated to produce the replicase proteins that induce the formation of the rna replication complexes. these complexes are assembled and anchored on membrane surfaces and this is an essential requisite for their virulence. some of the positive-strand viruses, such as the poliovirus, the mouse hepatitis virus (mhv), the equine arterivirus (eav), and the severe acute respiratory syndrome (sars) coronavirus, seen to use autophagosomes as a membrane platform (kirkegaard et al., ) . factors of the poliovirus rna-replication complex localize to doublemembrane vesicles that are derived from the er by the action of viral proteins bc and a by a mechanism that excludes resident host proteins (schlegel et al., ; suhy et al., ) . importantly, these structures contain lc /atg and are highlighted with the fluorophore monodansylcadaverine, a dye that specifically stains autophagosomes (jackson et al., ) . the idea that poliovirus subverts components of the cellular autophagy machinery to promote its replication is also supported by the fact that inhibition of this pathway by -methyladenine or by rna interference against mrnas that encode two diverent atg proteins (lc /atg and atg ) decrease the poliovirus yield (jackson et al., ) . coronaviruses (mhv and sars) and arteriviruses (eav) are the two families within the order nidovirales. cells infected by these viruses accumulate double-membrane vesicles and the viral rna-replication complexes are associated with them (goldsmith et al., ; gosert et al., ; pedersen et al., ; shi et al., ; van der meer et al., ) . in the case of the mhv and sars coronaviruses, it has also been shown that these structures are decorated with lc /atg , revealing that they are autophagosomes (prentice et al., a,b) . for the mhv in addition, it has been demonstrated that the autophagy machinery is required to generate these compartments and in its absence the virus replication is severely blocked (prentice et al., a) . importantly, studies about the origin of these doublemembrane vesicles generated in cells infected by nidoviruses indicate that they are derived from the er (pedersen et al., ; prentice et al., a; shi et al., ; van der meer et al., ) . after endosomal uptake of porphyromonas gingivalis and brucella abortus, by the host cell, the endosomes that contain these bacteria immediately fuse with structures resembling autophagosomes (dorn et al., ; pizarro-cerda et al., a,b; progulske-fox et al., ) . this event prevents their delivery to the lysosome where they would be eliminated. in addition containing endosomal factors, the double membranes surrounding these two pathogens are decorated with er protein markers and their formation is blocked by autophagy inhibitors such as -methyladenine and wortmannin (rich et al., ) . legionella pneumophila is a gram-negative bacterium that can replicate within human macrophages. after being taken up by phagosomes, this pathogen becomes enwrapped within a double-membrane compartment that contains the er resident chaperone bip through an unknown mechanism, and starts to replicate (coers et al., ; joshi et al., ; sturgill-koszycki and swanson, ; swanson and isberg, ) . it has been shown that this compartment also progressively becomes decorated with typical autophagosome markers such as atg and atg (amer and swanson, ) . however, it remains unclear if these structures are autophagosomes or similar conformations derived from the er that at successive stage acquire autophagosomal membranes or subvert the autophagy machinery to complete their biogenesis (kagan and roy, ; tilney et al., ) . in dictyostelium discoideum, a natural host for l. pneumophila, deletion of atg genes leads to a defect in autophagy without avecting the formation of the doublemembrane compartment and therefore the replication of this invading microorganism is unavected (otto et al., ) . but this could just reflect host-specific diverences. listeria monocytogenes is another gram-negative bacterium that after entering into host cells destroys the phagosome membrane using hemolysin to gain access to the cytoplasm where it starts to multiply. however, when infected cells are treated with chloramphenicol, an inhibitor of bacterial protein synthesis, or lack the acta gene, the bacteria become trapped into double-membrane compartments shortly after phagosome lysis (rich et al., ) . these structures are autophagosomes because l. monocytogenes sequestration is enhanced by autophagic induction through serum withdrawal and blocked by autophagy inhibitors such as -methyladenine and wortmannin (rich et al., ) . the formation of these autophagosomes seems to be mediated by the assembly of small vesicles and cisternae with variable morphology, which contain the er protein marker protein disulfide isomerase (pdi). importantly, pdi-positive vesicular structures are accumulated around the cytoplasmic bacteria during the early stages of autophagosome biogenesis but not at later stages when these structures begin to acquire endosomal makers (rich et al., ) . our knowledge about the physiological roles of autophagy has enormously increased and we have realized how important this pathway is for cell survival in several extreme situations. despite the identification and partial characterization of the atg proteins, however, the molecular mechanism of this catabolic transport route remains largely unknown. a major challenge in studying this process arises from the fact that the origin and the transport mode of the lipids employed to compose these structures is unknown. investigations on this topic seem to indicate that the er and possibly the golgi complex are involved in supplying the nascent autophagosomes with membranes. endosomal compartments, in contrast, play a relevant role only in mammalian cells and at a later stage during autophagosome 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polymorpha atg gene encodes a novel coiled-coil protein that is required for macropexophagy atg is essential for macropexophagy in hansenula polymorpha modification of a ubiquitin-like protein paz conducted micropexophagy through formation of a novel membrane structure skd aaa atpase-dependent endosomal transport is involved in autolysosome formation early secretory pathway gene trs is required for selective macroautophagy of peroxisomes in yarrowia lipolytica cooperative binding of the cytoplasm to vacuole targeting pathway proteins, cvt and cvt , to ptdins ( )p at the pre-autophagosomal structure is required for selective autophagy fulvio reggiori apg p/cvt p is an integral membrane protein required for transport vesicle formation in the cvt and autophagy pathways yeast autophagosomes: de novo formation of a membrane structure identification of complementation groups required for post-translational events in the yeast secretory pathway escape of intracellular shigella from autophagy 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triggers mitophagy in yeast mutants avecting the structure of the cortical endoplasmic reticulum in saccharomyces cerevisiae porphyromonas gingivalis virulence factors and invasion of cells of the cardiovascular system evects of vinblastine, leucine, and histidine, and -methyladenine on autophagy in ehrlich ascites cells intramembrane particles and filipin labelling on the membranes of autophagic vacuoles and lysosomes in mouse liver membrane origin for autophagy the diverential degradation of two cytosolic proteins as a tool to monitor autophagy in hepatocytes by immunocytochemistry dynein mutations impair autophagic clearance of aggregate-prone proteins autophagy in the eukaryotic cell vps is part of the yeast vps fifty-three tethering complex essential for retrograde trayc from the early endosome and cvt vesicle completion the atg -atg complex regulates atg and atg retrieval transport from the pre-autophagosomal structure early stages of the secretory pathway, but not endosomes, are required for cvt vesicle and autophagosome assembly in saccharomyces cerevisiae autophagosomes: biogenesis from scratch? the actin cytoskeleton is required for selective types of autophagy, but not nonspecific autophagy, in the yeast saccharomyces cerevisiae atg cycles between mitochondria and the pre-autophagosomal structure in yeasts studies on vinblastine-induced autophagocytosis in mouse liver. v. a cytochemical study on the origin of membranes cytochemical studies on induced autophagocytosis in mouse exocrine pancreas cytoplasmic bacteria can be targets for autophagy tracker dyes to probe mitochondrial autophagy (mitophagy) in rat hepatocytes the ubiquitin-proteasome pathway autophagy and its possible roles in nervous system diseases, damage and repair identification and characterization of five new subunits of trapp trapp i implicated in the specificity of tethering in er-to-golgi transport gate- , a membrane transport modulator, interacts with nsf and the golgi v-snare gos- the cooh terminus of gate- , an intra-golgi transport modulator, is cleaved by the human cysteine protease hsapg a cellular origin and ultrastructure of membranes induced during poliovirus infection fulvio reggiori aminopeptidase i is targeted to the vacuole by a nonclassical vesicular mechanism colocalization and membrane association of murine hepatitis virus gene products and de novo-synthesized viral rna in infected cells autophagy in health and disease: a double-edged sword cargo proteins facilitate the formation of transport vesicles in the cytoplasm to vacuole targeting pathway mechanism of cargo selection in the cytoplasm to vacuole targeting pathway mutational analysis of csc /vps p: involvement of endosome in regulation of autophagy in yeast an evector of ypt p binds the snare tlg p and mediates selective fusion of vesicles with late golgi membranes purification and characterization of autophagosomes from rat hepatocytes gsa encodes a unique -kda protein required for pexophagy and autophagy in pichia pastoris legionella pneumophila replication vacuoles mature into acidic, endocytic organelles remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles the pre-autophagosomal structure organized by concerted functions of apg genes is essential for autophagosome formation association of legionella pneumophila with the macrophage endoplasmic reticulum peroxisomes start their life in the endoplasmic reticulum. trayc pex p initiates the formation of a preperoxisomal compartment from a subdomain of the endoplasmic reticulum in saccharomyces cerevisiae accumulation of autophagic vacuoles and cardiomyopathy in lamp- -deficient mice the human homolog of saccharomyces cerevisiae apg p is a protein-activating enzyme for multiple substrates including human apg p, gate- , gabarap, and map-lc lc conjugation system in mammalian autophagy human apg p/aut p homologue is an authentic e enzyme for multiple substrates, gate- , gabarap, and map-lc , and facilitates the conjugation of hapg p to hapg p how the parasitic bacterium legionella pneumophila modifies its phagosome and transforms it into membrane origin for autophagy rough er: implications for conversion of plasma membrane to the er membrane sera from patients with type diabetes and neuropathy induce autophagy and colocalization with mitochondria in sy y cells orf a-encoded replicase subunits are involved in the membrane association of the arterivirus replication complex yeast homotypic vacuole fusion requires the ccz -mon complex during the tethering/ docking stage gaba areceptor-associated protein links gaba a receptors and the cytoskeleton microtubule disruption inhibits autophagosome-lysosome fusion: implications for studying the roles of aggresomes in polyglutamine diseases endothelial nitric-oxide synthase antisense (nos as) gene encodes an autophagy-related protein (apg -like ) highly expressed in trophoblast characterization of the isolation membranes and the limiting membranes of autophagosomes in rat hepatocytes by lectin cytochemistry formation of a whorl-like autophagosome by golgi apparatus engulfing a ribosome-containing vacuole in corpora allata of the cockroach diploptera punctata formation of autophagosomes during degradation of excess peroxisomes induced by administration of dioctyl phthalate degradation of excess peroxisomes by cellular autophagy: immuno-cytochemical and biochemical analysis atg links cargo to the vesicle-forming machinery in the cytoplasm to vacuole targeting pathway the mouse skd , a homologue of yeast vps p, is required for normal endosomal traycking and morphology in mammalian cells the author thanks daniel klionsky, judith klumperman, catherine rabouille, and ger strous for critically reading the chapter. the author also wishes to thank marc van peski and rené scriwanek for figs. and . key: cord- -oa hh ky authors: collins, r.n.; holz, r.w.; zimmerberg, j. title: . the biophysics of membrane fusion date: - - journal: comprehensive biophysics doi: . /b - - - - . - sha: doc_id: cord_uid: oa hh ky a crucial interplay between protein conformations and lipid membrane energetics emerges as the guiding principle for the regulation and mechanism of membrane fusion in biological systems. as some of the basics of fusion become clear, a myriad of compelling questions come to the fore. is the interior of the fusion pore protein or lipid? why is synaptic release so fast? why is pip( ) needed for exocytosis? how does fusion peptide insertion lead to fusion of viruses to cell membranes? what role does the tmd play? how can studies on membrane fission contribute to our understanding of membrane fusion? what exactly are snare proteins doing? the biological membrane is ancient, and crucial to the emergence of life itself. while a single topology of membrane was sufficient for bacteria and archebacteria, eukaryotic cells contain specialized subcellular systems of more topologies, enclosing many discontinuous volumes. in order to mix these intracellular volumes, and to secrete such volumes to the extracellular space, or to add a volume to the cytoplasmic space, the membranes enclosing one of these volumes must either rupture or merge with another membrane, a process termed membrane fusion. topological membrane rearrangements, then, such as fusion, its inverse -fission, or formation of a membrane pore, are the most essential ingredients of the complex membrane dynamics of living cells. these membrane transformations are key elements of dynamic intracellular trafficking networks; they are also intimately linked to important pathological processes including cellular entry and egression of enveloped viruses and various parasites, membrane rupture and apoptosis. the topological membrane remodeling generally converges to highly bent intermediates. the characteristic length scales associated with key structural intermediates, such as fusion or fission pores, are typically of the order of tens of nanometers. it has become increasingly clear that critical properties of biological material at this nanoscale cannot be readily extrapolated from bulk measurements. neither can they be obtained from experiments with individual molecules, as in many cases, especially in most membrane processes, the functional unit is not a single protein (e.g., channel), but a selectively self-assembled cluster of proteins and lipids acting in a highly co-operative manner. therefore, for intuition and information about the nature of these transformations, we must create and study them where possible. since the biological membrane is thin ( nm in thickness, that of two lipid molecules) we cannot see the process of membrane fusion under the microscope; we can only see the sequellae of fusion as organellar or cellular contents mix or are secreted. the simplest natural occurring instance of fusion is the coalescence of two miscible liquid droplets in air, whose two outer molecular layers merge into one. but these layers can dive into the interior, any given molecule is an ephemeral rather than integral part of the droplet's surface. films made of soap or protein (e.g., bubbles made by children from detergent or blowing bubbles in their milk) provide a better model, and we have all seen them fuse and lyse before our eyes. however, they also have surfaces that can exchange with a bulky interior, and their width can vary. these films are unlike the biological membrane in that their apolar moities face the low dielectric air rather than the high dielectric watery interior of the film. the biological membrane is composed of lipids, proteins, and carbohydrates of varying chemical structure. it exists within the context of an aqueous cellular environment that prefers to avoid the interior of the membrane fusion. this entropically-driven hydrophobic effect leads to two important constraints on topological transformations, ( ) a tension at the interface of the polar head groups of the lipids to resist any stretching, and ( ) a uniform thickness which is primarily determined by the lipid constituents. bilayers made from phospholipids have been used extensively to study the fusion process, and more recently the fission process. since the number of phospholipids molecules far outnumber the other constituents, membrane entropy is dominated by the thermodynamics of the lipids themselves, and thus it is likely that whatever is learned from investigations of lipid bilayer fusion will inform us about biological membrane fusion. indeed, it seems that many of the key intermediates of membrane fusion are the same in the fusion of synthetic and natural membranes. [ ] [ ] [ ] we will not cover this ground, as many excellent reviews serve this purpose. [ ] [ ] [ ] [ ] [ ] [ ] [ ] rather, in this chapter we will introduce a number of controversies that the reader may be stimulated to solve. in general there are a number of energy barriers that prevent fusion from proceeding spontaneously. first and foremost, there is the fact that in distilled water and dilute solutions of monovalent salt, all lipid bilayers resist close approach with a force that rises exponentially from an equilibrium distance of about nm. with divalent cations in the solution bathing the membranes, close approach is possible, depending upon the lipid composition. this is readily seen as exchange of components in the contacting leaflets. , presumably, this occurs through minute and transient contact sites in which the contacting leaflets are joined (hemifusion). however, this is not generally sufficient either for membrane fusion or the formation of an extended diaphragm composed of the outer, or non-contacting leaflets of the joining bilayers (termed hemifusion diaphragm), for there are energetic restrictions to the widening of the hemifusion intermediate that joins the two leaflets (termed a stalk, [ ] [ ] [ ] . the main way to facilitate the formation of a stable hemifusion diaphragm is to add hydrocarbon solvent to the membrane, so as to lower the energy of the three-way junction between the two bilayers and the joined diaphragm. , once there is a sufficient junction, another way to complete fusion is via the formation of a lipidic pore within the hemifusion diaphragm. this can be facilitated by either increasing membrane tension, or by adding lipids whose composition favors spontaneous pore formation. , (formally, such lipids would be defined as having positive monolayer spontaneous curvature. ) thus it is possible to set up ionic and membrane compositional situations which allow the demonstration of membrane fusion and its intermediates without adding energy, thus spontaneous fusion is possible under a set of restricted conditions. what is not at all clear is the favorable effect of tension on the formation kinetics of hemifusion contacts, and how the huge hydration repulsion between contacting monolayers is breached during the tension-driven fusion of liposomes to bilayers. the fusion pore links the interior of a vesicle to the external space, or the interior of a virus to the cytoplasm. a relatively long-lived fusion pore is universal in biological fusion, regardless of whether exocytosis, viral fusion, or cell-cell fusion is studied. since its discovery as a sub-ultrastructural entity, its architecture and composition has been in debate. in part, this is because of two very different views of the mechanism of membrane fusion: lipid centric and protein centric. in the lipid centric view, proteins surround a fusion site, and the conformational energy of the protein (as it transitions from a pre-fusion to a post-fusion form) is harnessed into stressing the encircled lipids to the point of a spontaneous transition in topology along a well-studied set of molecular intermediates, first a hemifusion intermediate -the stalk -and then the fusion pore ( figure ) . , , the prediction of this hypothesis is clear: the proteins should be outside of an hourglass-shaped pore scaffolded by proteins. in the protein-centric view, proteins link up the two membranes that are to fuse and make a solid proteinaceous connection, a potential channel that is first closed, then opens to form the initial fusion pore. the prediction of this hypothesis is also clear: the proteins should be at the center of the pore, surrounded and later infiltrated by lipids as the proteins dissociate to guide the topological change of the lipids. a direct method is needed to determine which of these predictions is met during different instances of biological fusion. one of the abiding mysteries in biology is the great speed that synaptic transmission is capable of, with fusion of some vesicles beginning some tens of microseconds after ca þ floods the presynaptic intracellular release site. , this finding leads one to wonder if there is a physical state to a small population of vesicles whose fusion machinery is beyond the stages of priming and conformational change. , there are two main proposals: . that synaptotagmin provides positive curvature stress that translates into hemifusion at the center of a ring of protein at the base of a dimple, and . that ring assemblies of snare (soluble n-ethylmaleimidesensitive factor attachment protein (snap) receptor) and synaptotagmin complexes form to appropriately concentrate and orient c b domains of synaptotagmin. this ring of ordered domains effectively creates a tube-like scaffold of positively charged protein residues that span the two membranes that are to fuse, a favorable location for dimples of membrane to approach each other. in other words, it would be an electrostatic tunnel for membrane fusion that is extended by the polybasic linker regions of syntaxin and synaptobrevin. variations of this model can account for many physiological pathways, including a small fraction of the vesicles that may already interacting at the level of the hemifusion prior to the entry of calcium. what is the role of calcium once it enters? first, ca þ turns on an 'electrostatic switch' initially proposed for synaptotagmin-syntaxin interaction, but better suited to instantaneously stressing the phospholipid bilayers of the presynaptic membrane and the synaptic vesicle for the ultra-rapid exocytosis seen in the nervous system. second, even without synaptotagmin, ca þ speeds up fusion of snare-reconstituted membranes considerably. perhaps divalent ions play a direct role, electrostatically complexing ps headgroups to promote fusion between negatively charged phospholipid bilayers. is this effect specific for calcium over magnesium? there are indications that the spontaneous curvature of ps in the presence of calcium, but not magnesium, is significantly more negative with calcium than with magnesium. ultimately, synaptotagmin, snares, and the other proteins that comprise the exocytotic fusion machine must cajole lipids to move through a pathway that culminates in fusion pore opening. the snare proteins and synaptotagmin are the guides that walk and pull the membrane through a bumpy stalk-pore path, with electrostatic interactions playing a larger role than hitherto realized. . . why is ptdins- , -p needed for exocytotic fusion? experiments on the permeabilized chromaffin cell established a requirement for atp in membrane fusion in exocytotic secretion. , further work revealed the product of atp in exocytosis to be ptdins- , -p , which stands out as a key player amongst bilayer lipids. it has been almost years since this minor plasma membrane constituent was directly implicated in exocytosis. the early studies used biochemical approaches in permeabilized cells that directly implicated the polyphosphoinositides and subsequently pip kinase and ptdins- , -p as key components late in the fusion pathway. imaging of ptdins- , -p in secreting cells demonstrates that the lipid is located on the plasma membrane and not on the secretory granule. , ptdins- , -p associates with syntaxin puncta in plasma membrane lawns from pc cells. the concentration of ptdins- , -p in the cytoplasmic leaflet of puncta is surprisingly high,b mole%. an initial fusion pore of nm diameter and nm , would have room for approximately phospholipid molecules (area ¼ Å ) facing the cytosol including nine ptdins- , -p molecules at mole%. clearly, ptdins- , -p is absolutely required for exocytosis. ptdins- , -p can have two, potentially interrelated functions: . as a scaffold for proteins of the exocytotic and endocytotic pathways, such as the exocyst and indirect effects through the actin cytoskeleton. - . as a lipid involved in the topological rearrangement of the plasma membrane during fusion and fission. a distinguishing feature of ptdins- , -p is its high negative charge of b À at ph . the highly charged lipid creates a highly dynamic electrostatic scaffold that interacts with unstructured (e.g., in marcks, gap ) and structured (e.g., in ph and c domains) basic moieties on a variety of proteins (for a review see ref. ) . unstructured basic peptides cause lateral sequestration of ptdins- , -p on membranes in vitro containing mole% ptdins- , -p with as much as mole % ps , an effect that is explained by electrostatic considerations. several proteins that play important roles in exocytosis have structured basic moieties in c domains that interact with ptdins- , -p including synaptotagmin and rabphilin. stop-flow techniques demonstrate that ptdins- , -p greatly speeds the ca þ -dependent interaction of synaptotagmin with membranes in vitro , suggesting that this interaction may have important physiological consequences. in addition, fret has been used to show that ptdins- , -p directly interacts with syntaxin in vitro. there are numerous proteins involved in exocytosis and endocytosis that contain a structured basic sequences in pleckstrin homology (ph) domains that interact with ptdins- , -p . these include caps in the exocytosis pathway and dynamin in the endocytosis pathway. ptdins- , -p also plays an important regulatory role in conjunction with small gtpases and proteins that regulate the actin cytoskeleton. these pathways also influence fusion and fission. while there is strong evidence for ptdins- , -p interacting with proteins that are important for fusion and fission, there is little direct evidence at this time for a direct role of the lipid in the fusion or fission reactions. for example, both long term and acute modulation of ptdins- , -p in chromaffin cells alter the size of the releasable granule pools but not the fusion kinetics, consistent with a role prior to but not during fusion. nevertheless, it seems likely that ptdins- , -p molecules with high charge and relatively high concentrations at fusion sites (as many as nine molecules in the cytoplasmic leaflet of the fusion pore) would directly influence lipid rearrangements. this is an important area for future investigation. it will be challenging to distinguish between the lipid simply being a scaffold for a multitude of proteins involved with trafficking at the plasma membrane, and having a direct function in the bilayer rearrangements of fusion and fission. in fact, these two roles may sometimes be indistinguishable, since one or more of the interacting proteins may have as its primary task the regulation of ptdins- , -p function in fusion or fission. the superficially shared structural features of ha with the snare proteins essential for internal cellular fusion have stimulated the hope that there is a universal mechanism for protein-mediated fusion. as the prototype of class i fusion proteins, and the epitope for flu serotyping the influenza virus ha has been studied extensively. while the speed of lipid mixing of ha-mediated fusion is rapid in vitro relative to infection speeds ( figure ), fusion pore formation has not been measured and thus we do not know the kinetics of complete fusion for ha in an intact virus. however, the accessibility of ha-mediated cell-cell fusion, and the large body of investigation into ha make this the best studied fusion protein that is sufficient for fusion. we learn the importance of guided conformational transitions together with protein-protein interactions in conjunction with clear phenotypic discrimination between intermediates of hemifusion and the fusion pore opening and subsequent widening. clearly there is much more work to be done, because we do not know why changing a single amino acid residue at the tip of the fusion peptide gives hemifusion instead of fusion. ha is the first viral glycoprotein for which the structures of both pre-and post-fusion forms were solved at atomic resolution. , ha is synthesized as a single-chain precursor protein ha , which then oligomerizes into a trimer during protein transport through the secretory pathway . the precursor ha ( amino acids in a/hong kong/ /h n ) then needs to be cleaved into the ha and the ha polypeptides after the conserved arginine at residue to be primed for the subsequent low ph-triggered conformational changes ( figure ) . although the crystal structures of both pre-and post-fusion forms of ha have been available for more than a decade ( figure ) , we still do not know exactly how conformational rearrangements occur step by step, how low ph environment triggers the rearrangements of the ha ectodomain, and which structural elements are crucial for ha fusion activity. it has been suggested that the low ph might lead to an enhanced protonation of the ha domain, and generate enough electrostatic repulsion force to partially dissociate the ha domain from the ha to allow the loop region in the ha to contact with water; this would cause the loop to transition into a helix and extend the pre-existing central coiled coil ( figure ). [ ] [ ] [ ] in this model, it seems that the only step requiring a low ph trigger is the exposure of the loop region connecting the two alpha helices in ha to water by partial disorientation of ha from ha . consistent with this idea, spontaneous formation of extended coiled coils are observed in bacterially expressed ha polypeptide at neutral ph, indicating that the low ph trigger is not necessary for the extension of coiled coils. the prevailing hypothesis for the mechanism of how conformational changes lead to the fusion of two membranes is that extension of coiled coils triggered by the low ph directs the fusion peptide to insert into the target membrane, and then a helix-to-loop conformational change reorients the protein to pull the fusion peptide toward the transmembrane domain. these molecular transitions result in a tight packing of the cooh-terminus of ha against grooves of the nh -terminal coiled coil, proceeding to the fusion of the two bilayers. [ ] [ ] [ ] this hypothesis emphasizes that both the extension of the coiled coil and the bending of the protein resulting from the helix-to-loop conformational change are important for fusion. the experimental results favoring this hypothesis demonstrate that a double proline substitution mutant (f p/f p) at the region supposedly undergoing loop-to-helix conformational change upon low ph failed to induce fusion, although the mutant still presented and inserted the fusion peptide to the target membrane. the block to fusion was demonstrated to occur in the tight packing of cooh-terminal extended regions into the grooves between the helices of the nh -terminal half of the coiled coil due to the splayed nh -terminal half of ha , suggesting that the insertion of fusion peptide into target membrane alone is not sufficient for fusion, and the tension caused by the packing of cooh-terminus against the nh -terminus of ha may be the driving force for membrane merging. another piece of evidence for the role of the packing of the cooh-terminus to the nh -terminus in viral fusion is that the alanine substitution mutants at five apolar residues after the short cooh-terminus of ha fails to cause both lipid and content mixing. as a result of conformational changes of ha upon low ph exposure, the fusion peptide located at the tip of the ha molecule is exposed to the close proximity of the target membrane. when the target membrane is available, the fusion peptide inserts into the lipid bilayer to induce lipid mixing between the target cell membrane and the viral membrane. it has been experimentally determined that the free energy associated with the insertion of full-length fusion peptide into the lipid membrane is . kbt, and as many as fusion peptides binding to the target membrane might generate enough energy to stabilize a stalk-like fusion intermediate, which has high membrane curvature. while necessary for fusion, the packing of fusion peptides does not appear to be sufficient for fusion. lipids present in the membrane act together with ha to cause fusion. changing membrane lipid physical properties or composition in ways that well defined also blocks fusion, despite conformational-specific antibody binding indicating that ha is in the post-fusion state. in other words the protein conformational change, while essential, is not sufficient, as there are post-protein conformational changes in lipid conformation that are needed for fusion to continue along its path. thus the pathway of ha-mediated membrane fusion involves conformational changes to induce lipids to undergo the more general pathway outlined above for lipid membrane fusion. the role of the fusion peptide domain for ha-mediated membrane fusion has been the subject of many biophysical studies. the fusion peptide of ha is rich in glycine; for example, influenza a/x /h n contains glycine residues in residues of fusion peptide (glfgaiagfiengwegmidg). extensive mutagenesis studies of fusion peptides have revealed that both the primary sequence and the length of fusion peptide are crucial for ha fusogenic activity. for example, ha g e substitution abolishes cell-cell and rbc-cell fusion activity of expressed ha, while g e substitution decreases fusion efficiency and elevated the ph threshold for activation. alanine can substitute for glycine at positions and without impacting ha-induced cell-cell fusion; however, the polar amino acid serine substitution for glycine at position causes a hemifusion phenotype. the requirements for specific amino acids at certain positions and for a defined length in the fusion peptide have been further supported by the nmr-solved structure of fusion peptide in detergent micelles and in model lipid membranes , stabilized by a charge-dipole interaction between the n-terminal gly and the dipole moment of helix (figure ). at acidic ph, the residues of fusion peptide adopt a v-shaped 'boomerang' structure with an oblique nh -terminal amphipathic helix spanning residues - and a turn formed by residues , and followed by a short cooh-terminal helix , stabilized by a charge-dipole interaction between the nterminal gly and the dipole moment of helix ( figure ). the bent fusion peptide then may insert b - Å into the outer leaflet of the target membrane, almost to the mid-plane of the lipid bilayer with the residue leu and phe penetrating deepest into the membrane. , in the solution structure, the hemifusion phenotype mutant g s has a similar structure to that of wild type, but the glycine ridge on the outer surface of the nh -terminal helical arm is disrupted. in contrast, the mutant g v, where fusion is completely abolished, has a very irregular linear amphipathic helix instead of the fixed angled boomerang structure ( figure ). , another fusion-defective mutant, w a, has a more flexible kink than that of the wild type, in contrast, the alanine substitution at phenylalanine residue (f a) has a similar structure to that of the wild-type and has no defect in fusion ( figure ). these studies suggest that both the angled and deeply inserted structure as well as the glycine ridge make a contribution to the fusion activity. despite the fact that we have gained a large amount of information on the structure of the influenza fusion peptide over the past several years, there are still open questions surrounding how the insertion of fusion peptide leads to fusion of viral membrane and target membrane and also with regard to the thermodynamic profile during the mixing of two bilayers. it has been generally accepted that the transmembrane domain (tmd) of ha and cooperation of multiple ha molecules are required in the fusion pore initiation and fusion pore enlargement. replacing the tmd of ha with a glycerylphosphatidylinositol (gpi) anchor or the deletion mutants with less than residues in length cause a hemifusion phenotype, but a tmd with polar amino acids at the coohterminus still allows full fusion. , a synthetic peptide representing the transmembrane segment of x /ha spans an artificial dmpc/dmpg bilayer as an a-helix that aligns roughly perpendicular to the bilayer membrane. the consequences of ha fusion peptide insertion are not well understood, although it is clear that the proposed boomerang structure avoids placing the polar amino acids within the hydrophobic phase, which would be disruptive or structure-forming. insertion of a charged amphipathic helix per se would tend to promote positive curvature, and might aid in ''nippling'' the membranes towards each other to contact prior to fusion, as in the proposed mechanism for synaptotagmin. wt-ph . one suggestion is that the membrane helices of the fusion peptide and the tmd could interact with each other, so providing a driving force for the mixing of two membranes. other open questions include the study of ha fusion (and other enveloped viruses) in the context of negative membrane curvatures found in the multi-vesicular bodies of endosomal compartments, and lipids known to be enriched in endosomes. another open question is whether the ha conformational changes are strictly irreversible in the absence of a target membrane. the prevailing model that spring-loaded conformational changes are uni-directional is based in part on the fact that pre-treatment of virus particles from typical laboratory strains (e.g., the h strain x- ) with low ph effectively neutralizes infection. analysis of other viral subtypes (e.g., h ) show features consistent with reversible conformational changes and many natural isolates of influenza do not show the irreversible conformational changes associated with x- in the absence of a target membrane (g. whittaker personal communication). tatulian and tamm have demonstrated that the conformational change of the entire ha is reversible in the absence of bound target membranes. the formation of a six-helix bundle ( hb) is the structural feature that characterizes class i viral fusion proteins. because fusion peptides insert into target membranes and tmds span the viral envelope, the folding of fusion proteins into hairpins brings the viral envelope and host cell membrane into proximity. even for the two other classes of viral fusion proteins, which do not exhibit hbs and contain much b-sheet, in the final, post-fusion state, it is likely that fusion peptides and tmds are proximal. it is not experimentally certain that this proximity occurs, because the hydrophobic tmds and fusion peptides themselves are not present in the crystallized proteins. this apposition of the membrane imbedded tmd and fusion peptides of viral fusion proteins is similar to the proximity between tmds of v-and t-snares in their tetrameric coiledcoil. when the ubiquity of hbs was first demonstrated, it was assumed that bundle formation merely brought membranes into close contact, and fusion then occurred. we now know this is incorrect. the correlations between bundle formation and steps in the fusion process have been investigated for hiv- env, influenza ha, and aslv env. the precise steps of bundle formation depend on the precise protein. it appears that the longer the amino acid sequence that intervenes between tmds and the coiled-coil, the earlier the bundle can form. for hiv- env, which has a relatively short intervening sequence, not only does hemifusion occur prior to completion of bundle formation, but so does the creation of the initial fusion pore. bundle formation releases considerable energy and the late occurrence of formation indicates that considerable energy is required for pore enlargement -the last step in fusion. in fact, several lines of evidence lead to the picture that hemifusion is energetically easy to achieve, pore formation more difficult, and pore enlargement even more difficult. from the theoretical point of view of membrane mechanics, considerable work must be expended to enlarge a fusion pore because additional membrane must be bent from its relaxed state as the pore enlarges. the requirements for hemifusion are reasonably well understood: the standard helfrich concepts of spontaneous membrane curvature and bending energy are sufficient to account for experimental observation. the mechanisms by which fusion proteins create the pore formation is less understood and how fusion proteins contribute to pore enlargement remains a mystery. for topological reasons, tmds cannot span hemifusion diaphragms and can only enter the diaphragm from the junction between the diaphragm and the two original membranes. common sense suggests that this entry of tmds should destabilize the junction and thereby generate pores. this is consistent with demonstrations that tmds are directly involved in pore formation and with theory that predicts that pores form at the junction. but why tmds are forced through the junction has not been addressed and the determinants of pore properties are virtually uncharacterized. for example, some viral fusion proteins, such as hiv- env, generate large initial pores than readily open whereas others, such as influenza ha, create small pores that generally flicker open and closed before they enlarge. it seems reasonable that differences in pores are conferred by the proteins (rather than by the lipids), but the field has not formulated guiding principles as to which structural features of a protein control pore properties. because viral nucleocapsids can only enter cytosol if a pore enlarges, these questions are potentially of practical, in addition to biophysical, importance. the snare complex formed between two fusing membranes are the principal fusogens of the eukaryotic molecular machinery that mediates membrane fusion in intracellular trafficking pathways. the snare complex is a coiled bundle of four parallel helices provided by three or four individual snare protein molecules (figure ) . perhaps the best studied is the snare complex mediating neuronal exocytosis containing four helices provided by three snare proteins: snap- (synaptosome-associated protein of kd), synaptobrevin- , and syntaxin- . the free energy generated during the assembly of a single ternary snare is estimated to be - k b t. [ ] [ ] [ ] a key question is what proportion of the energy of snare complex formation is directed at membrane fusion and what the contribution of the energetics of this reaction, if any, contribute to membrane docking and tethering. several of the proteins engaged in and/or responsible for fusion have been studied at atomic resolution with biophysical structural approaches. these studies have greatly illuminated our understanding of the protein machines driving membrane fusion reviewed in refs. and . the minimal domains of snare proteins that can spontaneously engage in a stable -helix snare complex revealed a characteristic packing at the core of the snare complex. the majority of the packing interactions are hydrophobic, however there is an ionic layer typically consisting of a single arginine and three glutamine residues. , this ionic layer is found at the midpoint of the coiled-coiled bundle and hence is referred to as the zero ionic layer. the zero ionic layer is an evolutionarily conserved feature of all snare complexes examined to date, however the functional role of this feature is not known. biophysical characterization of snare complexes that perturb the zero ionic layer suggest that it may be important for the stability of snare complexes. one idea was that this layer could provide an intervention point for snare complex disassembly, however perturbation of the layer in an in vitro disassembly assay had no discernable impact on nsf/snap catalyzed snare disassembly. current hypotheses for how polar zero layer residues might impact snare function favor potential role(s) in snare complex assembly, such as the suggestion that a polar zero layer helps align assembly of the snare helices in register, or in other downstream functions of the snare complex. mutagenesis studies of the zero ionic layer in different systems suggest different effects, but they are all relatively subtle. , clearly, the influence of particular residues may vary according to the individual snare complex in question and the local parameters governing the assembly of a particular cognate snare complex. structures of the individual snare proteins have been tremendously stimulating in posing novel questions. the coiled-coil a-helix of synaptobrevin extends to the most membrane proximal residue, lysine and this residue is also part of the extended transmembrane helix. the energetics and topology of snare complex formation may influence local bending of the a-helix at the interfacial region, which in turn could generate local membrane destabilization to aid fusion ( figure ) . it is not known how the membrane itself may locally influence the structure of cytoplasmic portions of synpatobrevin or other proteins involved in fusion. a recent structure of lipid-bound synpatobrevin suggests that the amphipathic helix of synpatobrevin may lie on the surface of the membrane, providing a molecular explanation of the observation that the membrane may influence the cytoplasmic portion of synaptobrevin to adopt conformations not observed in the absence of lipid. some snares contain independently folded nh -terminal domains together with additional unstructured linker regions of significant length. the presence of such domains and their ability to interact inter-and intra-molecularly significantly increases the complexity of snare complex formation and the ability of the snare proteins to drive membrane fusion. syntaxin contains a linker region connecting the snare domain with the h abc nh -terminal domain. fully extended, this region may be up to approximately Å in length ( figure ). it is currently unknown whether there are proteins that selectively bind to or regulate this region. in contrast to synaptobrevin, syntaxin has an slightly extended membrane proximal region that may not be part of the initial core snare complex structure (residues - ). the post-fusion snare complex shows this region adopts an a-helical structure that directly links the snare complex a-helix to the transmembrane a-helix. the local secondary structure adopted by these residues prior to fusion is unknown; secondary structural predictions suggest the region is unstructured and could conceivably act as a hinge facilitating the molecule to sample up to a Å radius of the membrane proximal area ( figure ). there is evidence for an initial nh -terminal interaction between the snare proteins, but the final low energy -helical bundle may not be an intermediate in fusion, but rather either a dead-end conformation or a post-fusion conformation. an experiment missing from the field is a demonstration of helical bundle formation before, or simultaneous with fusion pore formation, as described above for viral fusion. there is strong evidence that prior interaction between the plasma membrane snares syntaxin and snap increases rate of interaction with vamp. the relationship of the post-fusion snare complex ( figure ) to lipid rearrangements that occur during fusion and content mixing are currently not known. one study has placed syntaxin as a pore-forming molecule with - syntaxin molecules making up a fusion pore. assembly of - snare complexes as a minimum number required to drive fusion events is in good agreement with theoretical considerations of the energetics of snare complex assembly and membrane fusion. b snare complexes were required to promote fast fusion in supported bilayer experiments, and fewer in other lipid mixtures including pe, presumably because pe promotes fusion by curvature. what is enormously fascinating is how the protein fusogens form such a pore, how the pore forms initially and how the pore dilates as fusion proceeds to completion. these pathways may have different kinetic and thermodynamic parameters for different types of biological fusion, depending on the physiological requirements of the particular fusion event. the snare machinery certainly appears to have the potential for adaptability, mediating types of fusion as diverse as ''kiss and run'', complete fusion during constitutive events of exocytosis and homotypic fusion events such as those between endosomes. the snare complexes for which the structures have been solved all contain the four helix bundle in a parallel orientation. perhaps not surprisingly, given the versatility of coiledcoil structures, the snare domains are also capable of associating in anti-parallel bundles, which are also stable, although not as stable as the parallel bundles. this is superficially reminiscent of the anti-parallel and parallel coiled-coil transitions experienced by different conformations of ha (figures and ) although caution should be exercised in extending these analogies given the topological constraints of the fusion machinery. it is not known what such associations may represent physiologically; they could possibly represent a means of tethering membranes independently of fusion, or may be unproductive molecules requiring reconditioning by accessory factors in order to participate in multiple rounds of membrane fusion. the profile of the energy landscape traversed by the fusion reaction will be influenced by a multitude of exogenous factors. known factors include the membrane lipid composition, the availability of snare proteins in suitable pre-fusion states and the specific activity of snare accessory factors ( table ) . these factors can have multiple influences, for example, membrane composition can play a role in providing molecular determinants for protein assembly and will also determine membrane elasticity. how conformational changes amongst snare proteins and their accessory factors control the thermodynamics and kinetics of docking, lipid mixing and content mixing after membrane fusion remain open questions. our understanding of the conformational plasticity of protein machineries and the hysteresis properties of biological fusion machines lead to an appreciation for the energetic complexities of the fusion reaction. the use of single molecule studies to unravel the complex energy landscape of membrane fusion will be an important biophysical approach with tremendous potential to relate the topography of the energy landscape to the mechanism and regulation of fusion. although the application of this approach to studying biological fusion machines is relatively new, , [ ] [ ] [ ] [ ] [ ] [ ] such experiments have some additional advantages -such as being able to distinguish fusion events from vesicle aggregation or vesicle rupture. single molecule approaches of fluorescently-labeled virus in living cells has allowed visualization of influenza fusion with intracellular compartments/endosomes, and the use of solid supported lipid bilayers, facilitates a more detailed analysis of the single-particle kinetics of ha-mediated fusion as well as snare-mediated fusion. in combination with assays that reflect lipid and content mixing, together with pore formation and expansion, such approaches are expected to contribute substantially towards providing missing information regarding the intermediates and pathways involved in fusion. membrane fusion? recent work has shown that two rungs of a dynamin spiral is the minimal structural unit responsible for the formation of the fission neck and hemifission intermediate in model membrane nanotubes (figure ) . requires the same hemifusion event, but in a cylindrically symmetrical way, as the inner leaflet of the tube must touch itself in the center to allow for the hemifission intermediate. once again, it is relatively straightforward to calculate the ring conditions that will lead to constriction and narrowing of the tube towards the center, as long as the distance between the two rings is not too short. but once again, we are faced with the hydration force resisting any further constriction of the neck, since the surfaces of the inner leaflet lipids would have to come closer than the nm equilibrium distance between attractive dispersion forces and repulsive hydration forces. but lo, there is a new modality for minimizing energy in this system; it is the tilt-like movement of lipid head groups away from each other at the very center of the hourglass constriction of the neck (figure in formation of a narrow separation of heads exposing the hydrophobic interior of the bilayers, termed a hydrophobic ''belt'' to indicate its presence as a ring (you can visualize this ring by rotating the figures of figure (a) around the axis of the horizontal dashed line under each bilayers). now the repulsive hydration force stabilizing inner aqueous diameters of nm gives way to a newly developed hydrophobic attractive force, which is effectively the desire of water to desolvate the space between the tilting headgroups in the center. this water ejection leads to the close approximation and finally merger of the neck with itself at the midpoint; that is, its closure. figure (d) shows the calculated energy of a -mm-long segment of neck, depending upon the width (h) and radius (rmid) of the hydrophobic belt discussed above. like the stalk, the belt width becomes that of a single monolayer at the point of merger. the energy barrier of k b t is also similar to those calculated for membrane fusion, so it is energetically feasible. the lipid bending and tilting needed to catalyze membrane merger are mainly motivated by high curvature stress in the neck inner leaflet. accordingly, the energy barrier depends sharply on the minimal radius of the thinned neck, rmin, which should approach - nm for the fission to occur, as seen in other estimations of hemifission. thus the rings of protein acting on the outside of a nm tube lead to boundary conditions whose effects propagate through the bilayer to influence and stress lipids facing each other across water, leading to their head groups parting and their oils merging in energetically feasible hemifission. the proteins act at length scales consistent with what we know about protein structural lengths, and the lipids respond fluidly to the protein's influence. mechanistic studies concerning dynamin offer not only a static view of the energetics of non-leaky fission/fusion reactions but also insights into the dynamics of the transition structures. dynamin assembly shapes bilayers into highly curved structures ( figure ). assembly also greatly enhances the rate of gtp hydrolysis ( -fold), which in turn leads to dynamin disassembly. thus, assembly is self-limited in the presence of gtp. recent experiments suggest that assembly-induced gtpase activity reduces the interaction of dynamin with the highly curved lipid membrane even before disassembly. the result is the unstable, highly curved lipid neck described above that resolves either in membrane fission (endocytosis, described above), or reversal of high membrane curvature. from these studies, performed in model membranes, membrane fission may therefore be considered to be a stochastic result of gtp hydrolysis. , studies in adrenal chromaffin cells suggest that ectopic expression can reverse the otherwise lethal neurodegeneration of cysteine string protein-a knockout mice. ( , ) dynamin functions in a similar way to control the fate of a recently fused secretory granule. increasing the dynamin gtpase activity increases the rate of fusion pore expansion and the likelihood of rapid endocytosis. the results are consistent with a function for dynamin in restricting fusion pore expansion. increased gtp activity catalyzes a more rapid stochastic decision that results in either fusion pore expansion or (less frequently) membrane fission and endocytosis. recent theoretical and experimental studies of membrane fission by dynamin and viral matrix protein reveal how protein complexes are arranged to effectively apply localized curvature stress to membranes without perturbing lateral membrane integrity; that is, without leakage. one conceptual approach is that sites of membrane remodeling are organized as membrane domains, both through membrane composition and membrane curvature; thus membrane remodeling is a collaborative effort accomplished by the entire domain, involving protein complexes and multiple lipids. we emphasize that despite decades of studies we still know only a little about fundamental physical principles underlying the spatial and temporal organization of membrane domains specialized in membrane remodeling. for example, the structure and composition of the fusion pore are unknown. new synergistic experimental and theoretical approaches are needed to resolve how proteins merge and separate membranes. for example, the ability to detect submicron deformations of the plasma membrane with the combination of polarization and tirfm techniques , permits detection of the expanding fusion pore and may enable investigations of the molecular basis for membrane curvature changes in living cells. the key is to study membrane remodeling in the context of concrete biological processes, taking into account corresponding length scales for the key membrane intermediates, dynamic cooperation between protein machineries and lipids, component segregation and sorting and, importantly, longrange interactions which are, ultimately, of critical importance for highly localized membrane rearrangements leading to membrane fusion or fission. functional determinants of a synthetic vesicle fusion system influence of lipid composition on physical properties and peg-mediated fusion of curved and uncurved model membrane vesicles: ''nature' s own'' fusogenic lipid bilayer fusion pores and fusion machines in ca + -triggered exocytosis the molecular mechanism of mitochondrial fusion mitochondrial fusion and fission in mammals protein-driven membrane stresses in fusion and fission protein-lipid interplay in fusion and fission of biological membranes mechanisms of membrane fusion: disparate players and common principles conflicting views on the membrane fusion machinery and the fusion pore how does synaptotagmin trigger neurotransmitter release? flickering fusion pores comparable with initial exocytotic pores occur in protein-free phospholipid bilayers gpi-anchored influenza hemagglutinin induces hemifusion to both red blood cell and planar bilayer membranes the pathway of membrane fusion catalyzed by influenza hemagglutinin: restriction of lipids, hemifusion, and lipidic fusion pore formation possible mechanism of membrane fusion phospholipid surface bilayers at the air-water interface. ii. water permeability of dimyristoylphosphatidylcholine surface bilayers on the theory of membrane fusion. the stalk mechanism asymmetric membranes resulting from the fusion of two black lipid bilayers short-chain alcohols promote an early stage of membrane hemifusion the mechanisms of lipid-protein rearrangements during viral infection how proteins produce cellular membrane curvature lipids in biological membrane fusion intrinsic bending force in anisotropic membranes made of chiral molecules parameters affecting the fusion of unilamellar phospholipid vesicles with planar bilayer membranes a quantitative model for membrane fusion based on low-energy intermediates the exocytotic fusion pore modeled as a lipidic pore calcium in synaptic transmission timing of neurotransmission at fast synapses in the mammalian brain emerging roles of presynaptic proteins in ca þ þtriggered exocytosis how synaptotagmin promotes membrane fusion evidence that the inositol phospholipids are necessary for exocytosis. loss of inositol phospholipids and inhibition of secretion in permeabilized cells caused by a bacterial phospholipase c and removal of atp catecholamine secretion from digitonin-treated pc cells. effects of ca þ , atp, and protein kinase c activators arf regulates a plasma membrane pool of phosphatidylinositol( , )bisphosphate required for regulated exocytosis a pleckstrin homology domain specific for ptdins- - -p and fused to green fluorescent protein identifies plasma membrane ptdins- - -p as being important in exocytosis nonclassical pitps activate pld via the stt p ptdins- -kinase and modulate function of late stages of exocytosis in vegetative yeast exo interacts with phospholipids and mediates the targeting of the exocyst to the plasma membrane phosphoinositides in cell regulation and membrane dynamics clustering of syntaxin- a in model membranes is modulated by phosphatidylinositol , -bisphosphate and cholesterol a specific point mutant at position of the influenza hemagglutinin fusion peptide displays a hemifusion phenotype structure of the haemagglutinin membrane glycoprotein of influenza virus at a resolution structure of influenza haemagglutinin at the ph of membrane fusion helenius, a. folding, trimerization, and transport are sequential events in the biogenesis of influenza virus hemagglutinin receptor binding and membrane fusion in virus entry: the influenza hemagglutinin membrane structure and fusion-triggering conformational change of the fusion domain from influenza hemagglutinin protonation and stability of the globular domain of influenza virus hemagglutinin early steps of the conformational change of influenza virus hemagglutinin to a fusion active state: stability and energetics of the hemagglutinin the relevance of salt bridges for the stability of the influenza virus hemagglutinin a soluble domain of the membrane-anchoring chain of influenza virus hemagglutinin (ha ) folds in escherichia coli into the low-ph-induced conformation cdna cloning of component a of rab geranylgeranyl transferase and demonstration of its role as a rab escort protein specific single or double proline substitutions in the ''spring-loaded'' coiled-coil region of the influenza hemagglutinin impair or abolish membrane fusion activity new insight into the spring-loaded conformational change of influenza virus hemagglutinin leash in the groove mechanism of membrane fusion a host-guest system to study structure-function relationships of membrane fusion peptides structural intermediates in influenza haemagglutinin-mediated fusion composition and functions of the influenza fusion peptide membrane fusion by influenza hemagglutinin studies on the mechanism of membrane fusion: site-specific mutagenesis of the hemagglutinin of influenza virus hypothesis: spring-loaded boomerang mechanism of influenza hemagglutinin-mediated membrane fusion early endosomal snares form a structurally conserved snare complex and fuse liposomes with multiple topologies the cytoplasmic tail slows the folding of human immunodeficiency virus type env from a late prebundle configuration into the six-helix bundle fusion peptide of influenza hemagglutinin requires a fixed angle boomerang structure for activity locking the kink in the influenza hemagglutinin fusion domain structure membrane fusion mediated by the influenza virus hemagglutinin requires the concerted action of at least three hemagglutinin trimers multiple local contact sites are induced by gpi-linked influenza hemagglutinin during hemifusion and flickering pore formation secondary structure, orientation, oligomerization, and lipid interactions of the transmembrane domain of influenza hemagglutinin endosome-to-cytosol transport of viral nucleocapsids conformational changes and fusion activity of influenza virus hemagglutinin of the h and h subtypes: effects of acid pretreatment reversible ph-dependent conformational change of reconstituted influenza hemagglutinin amino acid sequence requirements of the transmembrane and cytoplasmic domains of influenza virus hemagglutinin for viable membrane fusion snares-energies for membrane fusion crystal structure of a snare complex involved in synaptic exocytosis at . a resolution energetics and dynamics of snarepin folding across lipid bilayers single molecule probing of snare proteins by atomic force microscopy is assembly of the snare complex enough to fuel membrane fusion crystal structure of the endosomal snare complex reveals common structural principles of all snares structure and function of snare and snare-interacting proteins structure of proteins involved in synaptic vesicle fusion in neurons exocytosis requires asymmetry in the central layer of the snare complex snare complex zero layer residues are not critical for n-ethylmaleimide-sensitive factor-mediated disassembly testing the q: r ''rule'': mutational analysis of the ionic ''zero'' layer in the yeast exocytic snare complex reveals no requirement for arginine exocytotic mechanism studied by truncated and zero layer mutants of the c-terminus of snap- dynamic structure of lipid-bound synaptobrevin suggests a nucleationpropagation mechanism for trans-snare complex formation the snare complex from yeast is partially unstructured on the membrane structural basis for the inhibitory role of tomosyn in exocytosis transmembrane segments of syntaxin line the fusion pore of ca þ -triggered exocytosis single vesicle millisecond fusion kinetics reveals number of snare complexes optimal for fast snare-mediated membrane fusion docking and fast fusion of synaptobrevin vesicles depends on the lipid compositions of the vesicle and the acceptor snare complex-containing target membrane reconstitution of rab-and snare-dependent membrane fusion by synthetic endosomes single-molecule studies of snare complex assembly reveal parallel and antiparallel configurations structural transitions in the synaptic snare complex during ca þ -triggered exocytosis novel targets and catalytic activities of bacterial protein toxins receptor and substrate interactions of clostridial neurotoxins x-ray structure of a neuronal complexin-snare complex from squid three-dimensional structure of the complexin/snare complex the synaptic vesicle protein csp alpha prevents presynaptic degeneration ddi , a eukaryotic protein with the retroviral protease fold different domains of the ubl-uba ubiquitin receptor, ddi /vsm , are involved in its multiple cellular roles a structure-based mechanism for vesicle capture by the multisubunit tethering complex dsl the rab effector eea interacts directly with syntaxin- involvement of lma and gate- family members in intracellular membrane dynamics structure of gate- , membrane transport modulator and mammalian ortholog of autophagocytosis factor aut p intracellular bacteria encode inhibitory snare-like proteins a phorbol ester/diacylglycerol-binding protein encoded by the unc- gene of caenorhabditis elegans an open form of syntaxin bypasses the requirement for unc- in vesicle priming modular architecture of munc /calmodulin complexes: dual regulation by ca þ and possibility function in short-term synaptic plasticity structural basis for a munc - homodimer to munc - /rim heterodimer switch cellular functions of nsf: not just snaps and snares isolation and characterization of a dual prenylated rab and vamp receptor structural basis of rab effector specificity: crystal structure of the small g protein rab a complexed with the effector domain of rabphilin- a a novel syntaxin -interacting protein, ship , regulates syntaxin -dependent sorting from early endosomes synaptic vesicle fusion complex contains unc- homologue bound to syntaxin specificity and regulation of a synaptic vesicle docking complex sec p binds to snare complexes and concentrates at sites of secretion tomosyn: a syntaxin- -binding protein that forms a novel complex in the neurotransmitter release process structure of the yeast polarity protein sro reveals a snare regulatory mechanism amisyn, a novel syntaxin-binding protein that may regulate snare complex assembly snap family of nsf attachment proteins includes a brain-specific isoform the yeast sec gene product is functionally equivalent to mammalian alpha-snap protein doc b is a high affinity ca þ sensor for spontaneous neurotransmitter release synaptotagmin-ca + triggers two sequential steps in regulated exocytosis in rat pc cells: fusion pore opening and fusion pore dilation synaptotagmin modulation of fusion pore kinetics in regulated exocytosis of dense-core vesicles synaptobrevin binding to synaptophysin: a potential mechanism for controlling the exocytotic fusion machine vesicle-associated membrane protein and synaptophysin are associated on the synaptic vesicle alpha-synuclein promotes snare-complex assembly in vivo and in vitro alpha-synuclein cooperates with cspalpha in preventing neurodegeneration a single-vesicle content mixing assay for snare-mediated membrane fusion discrimination between docking and fusion of liposomes reconstituted with neuronal snare-proteins using fcs single-molecule studies of the neuronal snare fusion machinery single molecule measurements of mechanical interactions within ternary snare complexes and dynamics of their disassembly: snap vs. snap single molecule mechanical probing of the snare protein interactions single molecule observation of liposome-bilayer fusion thermally induced by soluble n-ethyl maleimide sensitive-factor attachment protein receptors (snares) visualizing infection of individual influenza viruses single-particle kinetics of influenza virus membrane fusion gtpase cycle of dynamin is coupled to membrane squeeze and release, leading to spontaneous fission real-time visualization of dynamin-catalyzed membrane fission and vesicle release membrane fission: model for intermediate structures dynamin self-assembly stimulates its gtpase activity real-time detection reveals that effectors couple dynamin's gtp-dependent conformational changes to the membrane a new role for the dynamin gtpase in the regulation of fusion pore expansion localized topological changes of the plasma membrane upon exocytosis visualized by polarized tirfm this work was supported by nih grant no. r gm to r. collins, r -ns to rwh, and the intramural program of the nichd, nih. many thanks to fabio rinaldi for help with figure preparation, fred cohen for his discussion of the viral helical bundle timing experiments, and gary whittaker for helpful discussions. key: cord- - mertwj authors: netherton, christopher l; wileman, tom title: virus factories, double membrane vesicles and viroplasm generated in animal cells date: - - journal: curr opin virol doi: . /j.coviro. . . sha: doc_id: cord_uid: mertwj many viruses reorganise cellular membrane compartments and the cytoskeleton to generate subcellular microenvironments called virus factories or ‘viroplasm’. these create a platform to concentrate replicase proteins, virus genomes and host proteins required for replication and also protect against antiviral defences. there is growing interest in understanding how viruses induce such large changes in cellular organisation, and recent studies are beginning to reveal the relationship between virus factories and viroplasm and the cellular structures that house them. in this review, we discuss how three supergroups of (+)rna viruses generate replication sites from membrane-bound organelles and highlight research on perinuclear factories induced by the nucleocytoplasmic large dna viruses. many viruses replicate within subcellular microenvironments or 'mini-organelles' called virus factories or 'viroplasm'. formation of these structures involves rearrangement of host cell membranes and cytoskeleton and induces a 'cytopathic effect' indicative of virus infection. it is generally believed that factories and viroplasm create a platform to concentrate replicase proteins, virus genomes and host proteins required for replication, and at the same time physically separate replication sites from a myriad of cellular antiviral defences. the subversion of membrane trafficking pathways during the formation of replication sites may add further benefit by slowing the transport of immunomodulatory proteins to the surface of infected cells to protect against immune responses, while disruption of the cytoskeleton may enhance release of viruses from cells. there is growing interest in understanding how viruses induce such large changes in cellular organisation, and recent advances in electron microscopy coupled with tomography and live cell imaging are beginning to reveal the relationship between virus factories and viroplasm and the cellular structures that house them. in this review, we discuss how three supergroups of (+)strand rna viruses generate replication sites from membrane-bound organelles and highlight research on perinuclear factories induced by the nucleocytoplasmic large dna viruses (ncldv). the rna-dependent rna polymerases (rdrp) of the (+)strand rna viruses are targeted to the cytoplasmic face of membrane-bound organelles and subsequent assembly of the replicase complex induces membrane curvature and the formation of densely packed membrane vesicles (reviewed in [ , ] ) ( figure ). the alphaviruses generate membrane invaginations called spherules [ ] , while the flaviviruses and coronaviruses generate networks of double membrane vesicles (dmvs) connected to a complex of convoluted membranes (cm) derived from the endoplasmic reticulum (er). the picornaviruses also generate dmvs and a heterogeneous series of membrane vesicles and membrane rosettes [ , ] . the formation of spherules, and possibly dmvs, parallels mechanisms of capsid assembly where ordered assembly of replicase proteins induces membrane curvature and invaginations of uniform diameter [ ] . the more complex membrane rearrangements involving er networks and cm may involve additional recruitment of host proteins that modulate snap (soluble nsf [n-ethylmaleimide sensitive factor] attachment protein) receptor (snare) proteins and the rab and arf gtpases that control the secretory pathway (reviewed in [ ] ). rdrp supergroup viruses such as the animal alphaviruses, semliki forest virus (sfv) and sindbis virus produce nm diameter invaginations called spherules that are aligned along the inside face of the limiting membrane of endosomes and lysosomes [ ] . flock house virus (fhv) is a member of the nodaviridae family (rdrp supergroup ), which generates spherules in the outer membrane of mitochondria [ ] . each spherule contains approximately copies of the replicase protein packed along the inner membrane surface [ ] . tomographic models for formation of sperules and double membrane vesicles during replication of (+)strand rna viruses. panel : spherule produced by alphaviruses. replicase proteins (red spheres) are recruited to the cytoplasmic face of membrane-bound organelles. assembly of replicase proteins induces membrane curvature and invagination forming a spherule. the spherule remains connected to the limiting membrane of the organelle and a pore allows new genomes to enter the cytosol (adapted from [ ] ). panel : virus-induced vesicles and double membrane vesicles generated by denv flavivirus. replicase proteins (red spheres) are recruited to the cytoplasmic face of membrane-bound organelles. assembly of replicase proteins induces membrane curvature and invagination into the er forming a large spherule. the invagination remains connected to the limiting membrane of the organelle and a pore allows new genomes to enter the cytosol. close apposition of er membranes leads to the formation of dmvs connected to the er by convoluted membranes (cm) (adapted from [ ] ). this may close the pore leading to the cytosol. panel : virus-induced vesicles and double membrane vesicles generated by sars-cov coronavirus. replicase proteins (red spheres) are recruited to the cytoplasmic face of membrane-bound organelles. assembly of replicase proteins induces membrane curvature and invagination into the er forming a large spherule. it has been difficult to find evidence for a pore connecting invaginations to cytosol. close apposition of er membranes leads to the formation of dmvs connected to the er by cm. these may exclude replicase proteins and become sites for storage of viral rna. in some cases the close apposition of er membranes is lost and single membraned vesicles arranged in vesicle packets (vp) appear within membrane networks connected to the er (adapted from [ ] ). reconstructions show that spherule membranes are continuous with the outer mitochondrial membrane and that a membrane neck connecting the lumen of the spherule to the cytosol surrounds a channel wide enough to allow passage of (+)rna into the cytosol [ ] . rubella virus is the only member of the rubivirus genus of the togaviridae, which are distantly related to the alphaviruses. assembly of the rubella rdrp on the cytoplasmic face of endosomes and lysosomes generates spherules containing replicase proteins and double-stranded rna. in some cases, the spherules grow into large vacuoles and rigid membrane rods and sheets possibly coated with rdrp. freeze fracture studies and tomographic reconstitution have identified inter-connections between the vacuoles [ ] and protein bridges connecting vacuoles to er. the flavivirus genus are part of rdrp supergroup . yellow fever virus, west nile virus and dengue virus (denv) generate - nm diameter invaginations into the er. tomographic reconstructions of membranes induced by denv show a continuous network of er membrane connected to spherical vesicles and cm [ ] . virus-induced vesicles contain replicase proteins and dsrna and are found within the lumen of the er. most have double membranes suggesting that they are formed from invaginations into er cisternae [ ] . each spherical vesicle is connected to the er membrane by a neck with a pore opening to the cytosol that could allow transit of viral rna. in some images, the pores lie adjacent to sites of virus assembly making it possible that viral rna passes directly from the spherical vesicles to budding viruses [ ] . arterivirus and coronaviruses also generate densely packed membrane vesicles. three-dimensional reconstructions of vesicles induced by severe acute respiratory syndrome coronavirus (sars-cov) show dmvs between and nm in size bounded by two tightly apposed membranes connected to er [ ] . sars-cov also induces cm containing small tubular and reticular membranes connected to the er. later during infection cells contain 'vesicular packets' where single membraned vesicles are surrounded by a common outer er membrane [ ] . the interior of the dmvs contains dsrna but surprisingly, unlike dmvs generated by flaviviruses, the replicase proteins are absent from the dmvs but locate to the cm that lie between dmvs. this suggests that virus replication occurs in the cm rather than dmvs. neck-like structures extending from dmvs to the outer er membrane are visible, but evidence for a pore connected to the cytosol is lacking. enveloped sars-cov can be detected in the vesicular packets. this suggests that replication may take place on cm and that genomes are transferred to vesicular packets for envelopment and budding, while excess viral rna may be stored in dmvs. picornaviruses generate densely packed dmvs between and nm in diameter, a series of single membraned vesicles resulting from fragmentation of the golgi, and autophagosomes possibly generated as a bystander response to infection [ , - ] . the nature and relative numbers of vesicles vary greatly depending on the picornavirus family and it is not clear which population of vesicles house the replication complex. the dmvs generated during picornavirus replication lack an obvious opening to the cytosol making it possible that, as suggested for coronaviruses, they are a by-product of replicase assembly and are used as a storage site for viral rna. lipid biosynthesis plays an important role in both alphavirus replication and nodavirus replication as cerulenin treatment inhibits alphavirus and nodavirus replication [ , ] and both fhv and sfv appear to upregulate phosphatidylcholine synthesis [ ] . similarly, hepatitis c virus (hcv) replication requires fatty acid synthesis and geranylgeranylation [ ] and cerulenin also inhibits poliovirus (pv) viral rna synthesis [ ] and coxsackievirus b replication [ ] . this suggests that lipid synthesis is required for efficient replication of many different (+)strand rna viruses. viral proteins that generate dmvs are beginning to be identified. when expressed separately the picornavirus bc, c and a proteins generate er vesicles and tubules but these differ from the vesicles produced during infection. for pv, coexpression of bc and a can generate dmvs similar to those seen in infected cells [ ] . the arterivirus nsp and the coronavirus equivalent nsp proteins are multispanning er membrane proteins. coexpression of equine arterivirus nsp and [ ] can generate dmvs from the er, and protein interactions involving cysteine residues in the first loop domain are required for generating correct curvature [ ] . dmv formation may also require host proteins that regulate the formation of vesicles within the er and golgi. inhibition of sar and arf gtpases inhibits rna replication, and inhibition of the arf gtp exchange factor, gbf , by brefeldin a reduces the number of dmvs in infected cells [ ] . the downstream effector of arf required by coronaviruses remains unknown but may be the phosphatidylinositol- -kinase-iiib (pi kiiib) shown to play a role in picornavirus and hcv replication (see below). for enteroviruses such as pv and coxsackieviruses, the nonstructural protein a may play a crucial role in the recruitment of the rdrp, d pol , to the cytoplasmic face of membrane-bound organelles by increasing the recruitment of pi kiiib [ ] . the mechanism hinges on the ability of the enterovirus a protein to modulate the activity of the arf gtpase and its guanine nucleotide exchange factor gbf . arf and gbf play a central role in regulating membrane traffic between the er and golgi. activation of arf by gbf catalyses recruitment of cop proteins to the golgi to facilitate formation of the cop -coated vesicles that carry proteins from the golgi to the er. binding of enterovirus a protein to gbf activates arf and increases recruitment of an alternative arf effector protein, pi kiiib, to membranes [ ] . since d pol binds to phosphatidylinositol- -phosphate (pi -p), the localised production of pi -p increases recruitment of the d pol at the expense of cop . this results in disassembly of the golgi providing membrane vesicles enriched in pi -p for replication. this role for a may not hold for other picornaviruses where replication is not dependent on an active gbf , and for picornaviruses that express a proteins unable to disrupt er to golgi transport [ ] [ ] [ ] [ ] . hcv also promotes recruitment of pi k to replication sites [ , ] . hcv generates a membranous web with many similarities to the network of spherical vesicles and cm produced by denv. pi -p lipids are colocalised with hcv replicase protein ns a, and replication is reduced following pi kiiib knockdown or overexpression of the sac phosphatase that removes phosphate from pi -p. the hepatitis ns a protein also binds pi kiiia and stimulates enzyme activity and knockdown of pi kiiia prevents formation of the membranous web associated with virus replication [ , ] . the ns a protein is anchored to the cytoplasmic face of the membranous web and recruits ns b and a series of cellular proteins that regulate membrane vesicle formation. these include vesicle-associated membrane protein-associated proteins [ ] which bind snares involved in er to golgi transport, and a rab -gap protein, tbc d . ns b can recruit rab and rab effectors eea , rabaptin and rab [ ] suggesting the web may fuse with endosomes [ ] . double-membraned vesicles are usually rare in cells, but dmvs are induced during autophagy. this makes it possible that autophagosomes may provide a source for dmvs associated with virus replication. a role for autophagosomes in supporting replication of the coronavirus mouse hepatitis virus (mhv) was first provided by studies where mouse embryonic stem cells lacking crucial autophagy protein atg showed a -fold reduction in mhv replication and reduced numbers of dmvs [ ] . less clear-cut results come from studies of primary fibroblasts or macrophages where loss of atg has little impact on virus replication [ ] . vesicles labelled with autophagy marker lc are, however, produced during coronavirus infection suggesting that the virus activates autophagy. autophagy may be activated by the nsp proteins of cornaviruses, or in the case of the arteriviruses, the equivalent nsp - protein. the nsp (nsp - ) proteins locate to the er where they generate small vesicles enriched in phosphatidylinositol- -phosphate and early autophagy marker atg [ ] . these vesicles closely resemble cellular organelles called omegasomes that are formed from the er during the initial stages of autophagy [ ] , and mature into autophagosomes labelled with autophagy marker atg /lc . autophagy is activated during cornavirus infection but this does not mean that all the dmvs generated in the cytoplasm are autophagosomes. most of the dmvs are smaller than autophagosomes and may be formed from invaginations into the er (figure ). vesicles formed from the er during coronavirus infection can also recruit a non-lipidated autophagy marker lc (lc i) by a pathway, that is, paradoxically, independent of autophagy, and linked to the export of er chaperones from the er to endosomes [ ] . autophagosomes may not therefore play a direct role in the formation of virus-induced dmvs, but may represent a defence against infection. picornaviruses activate autophagy in cell culture models [ ] and in some cases inhibition of autophagy reduces replication while activation increases virus yields. the autophagosome marker lc colocalises with pv replicase proteins suggesting replication on autophagosomes. translocation of lc to vesicles can be induced by expression of pv bc alone, but formation of dmvs resembling autophagosomes requires coexpression of bc with a [ , ] . as with the coronaviruses, the picornavirus dmvs are approximately one third the diameter of cellular autophagosomes making it difficult to determine if assembly of the replicase complex results in the formation of dmvs directly, or if the dmvs represent modified autophagosomes. virus assembly and replication can also occur in virus factories close to the microtubule organising centre (mtoc) [ ] . these inclusions lack cellular membranes and resemble inclusions called aggresomes that form at the mtoc in response to protein aggregation. aggresome inclusions such as lewy and mallory bodies are a pathological hallmark of protein misfolding diseases, and protect cells from the damage associated with protein aggregation. aggresomes and factories share many features in common including recruitment of mitochondria, cellular chaperones and confinement within cages of rearranged vimentin filaments (reviewed in [ , , ] ). many viruses are delivered to the mtoc after entering cells, and in common with protein aggregates, this involves recognition by the microtubule motor protein, dynein. it is possible that viruses may appear foreign or misfolded to cells and stimulate an aggresome response. for the ncldv this may be beneficial and provide a site for replication, for other viruses it may lead to confinement at the mtoc and degradation. delivery of incoming african swine fever virus (asfv) to the mtoc is important for the initiation of replication [ ] and replication of both the asfv and the iridovirus frog virus appears to require rearrangement of vimentin [ , ] . vimentin may provide a scaffold to prevent diffusion of viral components into the cytoplasm. dna and rna are spatially separated within vaccinia virus (vacv) and asfv factories [ , ] and individual factories appear to be distinct entities within the cell. although virus factories recruit many host-cell proteins to facilitate their replication, factories also represent effector sites for antiviral activity as vacv and asfv replication sites are targeted by stress granule components and mx proteins respectively [ , ] . a number of clear similarities between the membrane rearrangements generated by the positive strand rna viruses are beginning to emerge [ , ] and these are shared between animal and plant viruses. spherules and dmvs differ morphologically but may be formed by similar mechanisms involving ordered assembly of replicase proteins on membrane-bound organelles leading to membrane invagination (figure ). dmvs may be modified spherules where close apposition of er-derived membranes follows initial invagination into the er lumen. for cornaviruses, and possibly picornaviruses, this may result in loss of the pore connecting the spherule to the cytosol and conversion of the spherule into a site for storage of viral rna to suppress innate immune responses to double-stranded rna. the site of spherule formation differs between viruses ( figure ) and is determined by membrane targeting sequences in the nonstructural proteins that recruit the rdrp. this has been demonstrated by the work of miller et al. who could retarget fhv replication complexes from the mitochondria to the er in yeast [ ] . recent work has shown that recruitment of rdrp may also involve modification of membrane lipids by arf -dependent recruitment of pi kiiib to membranes to generate pi -p. this mechanism is shared between the enteroviruses of the picornavirus supergroup and the flavivirus hcv [ , ] . the membrane vesicles generated by picornaviruses are most heterogeneous and varied between subgroups and may be derived from membrane compartments that fragment during infection, rather than be formed for virus replication. it is uncertain which sites of replication generated by plant and animal viruses. the diagram is in two halves. the left represents a plant cell and is surrounded by a cell wall (green). viral replication complexes (vrc) are generated by plant viruses at the er, chloroplast and peroxisomes. tobamovirus vrcs are initiated at the tonoplast. vrcs of many plant viruses can be transported within the cell along the er and between cells using the plasmodesmata. this movement is directed by plant movement proteins (mp); see accompanying article by jeanmarie verchot for details. the right represents an animal cell. alphavirus and nodaviruses form spherules at the endosomal-lysosomal system and mitochondria respectively. coronavirus, arterivirus and flaviviruses form double membrane vesicles (dmvs) from the er and coronaviruses and arteriviruses also form vesicle packets (vps). the membranes used to generate sites of picornavirus replication are unclear but may involve the golgi, er and/or autophagosomes. nucleo-cytoplasmic large dna viruses (ncldv) factories are formed after microtubule-mediated delivery of incoming viruses to the microtubule organising centre next to the nucleus. thereafter they recruit host chaperones, mitochondria and intermediate filaments. membranes house the virus replicase and replication may occur on the cytoplasmic face of the er rather than in vesicles [ ] . parallels between plant and animal picornaviruses are therefore difficult to define. the large dna viruses of animals such as poxviruses and other members of the ncldv generate perinuclear inclusions called virus factories that assemble at the mtoc and are maintained by dynein microtubule motor proteins. replication complexes generated in plants often move through cells onboard microtubule motor proteins (figure ), but perinuclear inclusions are not found for plant viruses. as pointed out in the accompanying review by jeanmarie verchot, plants lack a mtoc to concentrate motor cargoes, and replication sites are therefore dispersed throughout the cell. origins of membrane vesicles generated during replication of positive-strand rna viruses organelle-like membrane compartmentalization of positive-strand rna virus replication factories cytoplasmic viral replication complexes cellular origin and ultrastructure of membranes induced during poliovirus infection formation of the poliovirus replication complex requires coupled viral translation, vesicle production, and viral rna synthesis modulation of membrane traffic between endoplasmic reticulum, ergic and golgi to generate compartments for the replication of bacteria and viruses three-dimensional structure of rubella virus factories flock house virus rna replicates on outer mitochondrial membranes in drosophila cells em tomography provides the first d reconstructions of membrane invaginations housing the replication complex of (+)strand rna virus composition and three-dimensional architecture of the dengue virus replication and assembly sites em tomography provides a d reconstruction of membrane rearrangments induced by dengue virus. the vesicles are connected to a continuous network of endoplasmic reticulum and a pore connects the vesicles to the cytoplasm. some pores lie opposites sites of virus envelopment sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum em tomography provides a d reconstruction of membrane rearrangemnts induced by sars-coronavirus. the double-membraned vesicles are connected to a continuous network of endoplasmic reticulum poliovirus infection blocks ergic-to-golgi trafficking and induces microtubule-dependent disruption of the golgi complex subversion of cellular autophagosomal machinery by rna viruses the ultrastructure of the developing replication site in foot-andmouth disease virus-infected bhk- cells fragmentation of the golgi apparatus provides replication membranes for human rhinovirus a remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virusinduced vesicles synthesis of semliki forest virus rna requires continuous lipid synthesis complementary transcriptomic, lipidomic, and targeted functional genetic analyses in cultured drosophila cells highlight the role of glycerophospholipid metabolism in flock house virus rna replication hepatitis c virus rna replication is regulated by host geranylgeranylation and fatty acids phospholipid biosynthesis and poliovirus genome replication, two coupled phenomena the human fatty acid synthase: a new therapeutic target for coxsackievirus b -induced diseases? non-structural proteins and interact to modify host cell membranes during the formation of the arterivirus replication complex formation of the arterivirus replication/transcription complex: a key role for nonstructural protein in the remodeling of intracellular membranes mouse hepatitis coronavirus rna replication depends on gbf -mediated arf activation viral reorganization of the secretory pathway generates distinct organelles for rna replication studies on enterovirus replication show that the a nonstructural proten modulates the activity of the arf gtpase and its guanine nucleotide exchange factor gbf . this increases recrutment of srf effector protein, phosphatidylinositol- -kinase-iiib (pi kiiib) to the cytoplsmic face of the endoplasmic reticulum. formation of phosphatidylinositol- -phosphate provides a binding site for the viral rna polymerase and inhibition of the pi kiiib kinase slows replication inhibition of cellular protein secretion by picornaviral a proteins differential requirements for copi coats in formation of replication complexes among three genera of picornaviridae effects of foot-and-mouth disease virus nonstructural proteins on the structure and function of the early secretory pathway: bc but not a blocks endoplasmic reticulum-to-golgi transport inhibition of the secretory pathway by footand-mouth disease virus bc protein is reproduced by coexpression of b with c, and the site of inhibition is determined by the subcellular location of c recruitment and activation of a lipid kinase by hepatitis c virus ns a is essential for integrity of the membranous replication compartment the membrane web generated from the endoplsmic reticulum (er) during hepatitis c replication contains eleveated levels of phosphatidylinositol- -phosphate. the ns a protein of hepatitis c locates to er and binds phosphatidylinositol- -kinase-iii and stimulates kinase activity. knockdown of phosphatidylinositol- -kinase-iii reduces replication. hepatitus c replication is therefore facilitated by production of phosphatidylinositol- -phosphate on the cytoplsmic face of the er and this may facilitate recruitment of the rna polymerase roles for endocytic trafficking and phosphatidylinositol -kinase iii alpha in hepatitis c virus replication the study shows that the lipid kinase phosphatidylinositol- -kinase iii alpha is important for hepatitis c replication human vap-b is involved in hepatitis c virus replication through interaction with ns a and ns b participation of rab , an early endosome protein, in hepatitis c virus rna replication machinery coronavirus replication complex formation utilizes components of cellular autophagy virgin hw: coronavirus replication does not require the autophagy gene atg coronavirus nsp proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate identification of cornavirus proteins able to stimulate starvation independent formation of autophagosomes directly from the endoplasmic reticulum via an omegosome intermediate autophagosome formation from membrane compartments enriched in phosphatidylinositol -phosphate and dynamically connected to the endoplasmic reticulum coronaviruses hijack the lc -i-positive edemosomes, er-derived vesicles exporting short-lived erad regulators, for replication edemosomes are er-derived vesicles that transport er chaperones to endosomes. double-membraned vesicles induced by the murine cornaviruses, mouse hepatitis virus (mhv), recruit edemosome marker edem and a non-lipidated lc by a pathaway independent of autophagy modification of cellular autophagy protein lc by poliovirus a guide to viral inclusions, membrane rearrangements, factories, and viroplasm produced during virus replication aggresomes resemble sites specialized for virus assembly aggresomes and pericentriolar sites of virus assembly: cellular defense or viral design? aggresomes and autophagy generate sites for viral infection vimentin rearrangement during african swine fever virus infection involves retrograde transport along microtubules and phosphorylation of vimentin by calcium calmodulin kinase ii interaction of frog virus with the cytomatrix. iv. phosphorylation of vimentin precedes the reorganization of intermediate filaments around the virus assembly sites colocalization of transcription and translation within cytoplasmic poxvirus factories coordinates viral expression and subjugates host functions demonstrated that individual ncldv factories are compartmentalised and represent separate functional units within the cell regulation of host translational machinery by african swine fever virus formation of antiviral cytoplasmic granules during orthopoxvirus infection inhibition of a large double-stranded dna virus by mxa protein engineered retargeting of viral rna replication complexes to an alternative intracellular membrane the authors would like to thank jeanmarie verchot for helpful discussions during the preparation of this manuscript. tw is supported by bb/e and bb/f grants from bbsrc. cn is supported by bbsrc and defra (se ). papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest key: cord- -vm nh lc authors: perez espitia, paula judith; de fátima ferreira soares, nilda; dos reis coimbra, jane sélia; de andrade, nélio josé; souza cruz, renato; alves medeiros, eber antonio title: bioactive peptides: synthesis, properties, and applications in the packaging and preservation of food date: - - journal: compr rev food sci food saf doi: . /j. - . . .x sha: doc_id: cord_uid: vm nh lc abstract: bioactive peptides are protein fragments which have a positive impact on the functions and conditions of living beings. peptides have shown several useful properties for human health, including antimicrobial, antifungal, antiviral, and antitumor activities. these compounds are produced by almost all species of life. however, they are produced in limited quantities in nature. as a result, researchers have tried to synthesize bioactive peptides to study their properties and applications in various areas. among their applications in food preservation, peptides have been incorporated into packaging materials. this review begins with a brief description of the methods used for the synthesis, purification, and characterization of peptides. also, the main bioproperties and mechanisms of action of peptides are discussed. finally, some applications of peptides are presented, especially their use in active packaging, their effects on the polymeric matrix, and peptide migration. food safety is a growing concern of great importance worldwide. recently, the estimated costs of diseases caused by foodborne pathogens was about $ billion in the united states (scharff ) , and it is estimated that in the united states alone about . million illness cases, hospitalizations and deaths will be caused by foodborne pathogens in . the consumption of processed foods with chemical preservatives has led to increased consumer concern and the demand for more natural and minimally processed foods. as a result, researchers have shown a growing interest in natural antimicrobial agents such as certain peptides. bioactive peptides are defined as specific protein fragments that have a positive impact on the functioning or conditions of living beings, thereby improving their health (korhonen and pihlanto ) . the beneficial effects are attributed to different properties found in peptides such as antimicrobial (reddy and others ; rajanbabu and chen ) , antioxidant (sarmadi and ismail ) , antithrombotic (wang and ng ) , anti-hypertensive (erdmann and others ) , and immunomodulatory activities (st georgiev ; gauthier and others ) , among others. ms submitted / / , accepted / / . authors espitia, soares, coimbra, de andrade, and medeiros are with food technology dept., federal univ. of viçosa, av. p. h. rolfs, s/n, campus univ., - . viçosa, minas gerais, brazil. author cruz is with food technology dept., state univ. of feira de santana, av. transnordestina, s/n, campus univ., - . feira de santana, bahía, brazil. direct inquiries to author soares (e-mail: nfsoares @gmail.com) . peptides with antimicrobial properties are used as the first chemical barrier against microbial attack, being synthesized in response to bacterial infections. they are produced by almost all species of life, from microorganisms, plants and animals, to humans (st georgiev ; hancock and diamond ) . in animals, antimicrobial peptides are produced mainly in those tissues exposed to adverse conditions such as skin, eyes, and lungs, which are more likely to be in contact with microorganisms (zasloff ; papo and shai ) . more than antimicrobial peptides have been reported, showing significant variations with respect to their sequence, length, and structure (papo and shai ) . antimicrobial peptides have found many applications, including those in biomedical devices, food processing equipment, and food preservation. in food preservation, peptides can be incorporated into materials to create antimicrobial packaging (appendini and hotchkiss ) . in this way, antimicrobial packaging plays an important role in maintaining the safety and quality of food, since the aim is to prolong food shelf life and to reduce bacterial growth on the product surface (soares and others a) . this type of active packaging interacts with the product and/or the headspace inside to reduce, inhibit, or retard the growth of microorganisms that may be present (soares and others b) . this review highlights the main methods of peptide synthesis and noteworthy peptide bioproperties. also, specific peptide applications in food preservation are reviewed, focusing on their incorporation in polymeric matrices. finally, the effects of peptide incorporation on packaging characteristics as well as their migration into food are discussed. borgia and fields ( ) . copyright ( ) , elsevier. peptides are biomolecules that contain between to several dozen of amino acid residues joined by peptide bonds. the discovery of the different peptide activities has generated enormous interest in this class of compounds and in the methods of isolation, analysis, purification, identification, and quantification. these methods have been systematically studied and improved. however, most sources of natural peptides are poor in these compounds, thus preventing their isolation in sufficient quantities for research. as a result, there was a growing need to synthesize peptides for application in physiological, chemical, physical, pharmacological, biochemical, and clinical studies. total of methods of peptide synthesis have been developed and improved: chemical synthesis, which uses chemical reagents to mediate peptide bond formation (andreu and rivas ) , enzymatic synthesis, in which the peptide bond formation is catalyzed by enzymes (bongers and heimer ; boeriu and others ) , and the dna recombinant technology synthesis, based on the use of cloning and ribosomal techniques from biological systems for peptide formation (sewald and jakubke ) . research on this synthesis method was first initiated more than y ago. however, the construction of peptides has recently become more accessible due to advances in process efficiency, including the development and use of fast coupling reagents, as well as the minimization of side reactions (borgia and fields ) . the main aspects of chemical synthesis are protection and activation. protection strategies are intended to provide chemical selectivity necessary for the construction of a particular peptide sequence. activation refers to the chemical coupling necessary to ensure quantitative formation of each peptide bond in the sequence (andreu and rivas ) . in chemical synthesis, chemical reagents are used to activate the carboxylic acid (rcooh) of the amino acid, which will donate the acyl group (r-co-) to form the peptide bond. the peptide bond presents a nucleophilic attack of the α-amino group by another amino acid (h n-r). in this synthesis, the reactive functional groups that are not directly involved in peptide bond formation receive prior protection (machado and others ) . there are types of chemical peptide synthesis, synthesis in solution (classical synthesis) and solid-phase synthesis. chemical synthesis in solution is performed with all reagents and reaction products dissolved in the medium (kent ) . in comparison, solid-phase synthesis (sps) is a simple procedure to produce peptides in large quantities on a solid support which remains insoluble in the reaction medium (shigeri and others ) . the solid support is a polymeric resin that has a functional group on its surface (linker) that allows it to form stable bonds in the peptide sequence to the reagent used for the de-protection of the n-amino group. peptide synthesis in the solid phase generally consists on the acylation of an amino acid to be linked to an insoluble support (resin) via a linker ( figure ). after that, the protecting group of the n-terminal is removed (the unprotecting step) to allow the next amino acid of the sequence to be attached to the complex "peptide-linker-resin." the unprotecting-coupling cycle is repeated until the desired sequence is complete. finally, the cleavage reagent is used to separate the complex "peptide-linker-resin." this reagent should also remove the protecting groups of side chains that are stable to unprotecting conditions of the n-terminal group (borgia and fields ) . peptide chemical synthesis can use protocols, boc (tertbutyloxycarbonyl) and fmoc ( -fluorenylmethyloxycarbonyl), named according to the type of protector of the reactive group of the amino acids (n-terminal) involved in the synthesis. the first protocol employs the tert-butyloxycarbonyl (boc) group for n-amino protection. this protocol is based on gradual differences in their sensitivity to acids. thus, the boc group is typically removed with trifluoroacetic acid (tfa), while the protecting groups of the lateral chains (ester, ether, and urethane derivatives based on benzyl alcohol) are specifically designed to be stable to repeated cycles of boc removal and are removed only with a specific reagent, a relatively stronger acid, usually hydrofluoric acid (borgia and fields ) . the second protocol uses a -fluorenylmethyloxycarbonyl (fmoc) as the n-amino protecting group. this protocol provides a greater degree of chemoselectivity than the boc protocol, since the fmoc group is removed under basic conditions (piperidine in n, n-methylpyrrolidone or dimethylformamide), without alteration of the acid-sensitive lateral chains (andreu and rivas ) . protection groups of lateral chains are compatible with the fmoc protection group; these are mainly ether, ester, and urethane derivatives based on t-butanol. protection groups of lateral chains are removed by the end of the synthesis using tfa (borgia and fields ) . in this method, the peptide bond formation is mediated by an enzyme (protease) in free or immobilized form. the enzymatic method is especially useful in the synthesis of very short peptides ( - oligomers) and in the condensation of large peptide fragments (so and others ) . proteolytic enzymes such as chymotrypsin, papain, pepsin, subtilisin, termolisin, trypsin, among others, have been used in the presence of organic solvents as catalysts for the synthesis of peptide bonds (ogino and others ) . the enzymatic synthesis of peptides has several advantages over chemical methods, including good stereoselectivity and regioselectivity. however, it has certain shortcomings, such as peptide synthesis being thermodynamically unfavorable in water, as well as the secondary hydrolysis of synthesized peptide chains, which hinders their use in peptide synthesis with long sequences (so and others ) . thus, the main practical obstacle to employment of a protease for peptide bond formation is finding suitable conditions to allow bond formation without mediating secondary hydrolysis of the peptide or peptide fragments used as reagents (bongers and heimer ) . the formation of a peptide bond by enzyme catalysis can occur through several mechanisms, including the reverse hydrolysis reaction of amides and transpeptidation (machado and others ; boeriu and others ) . the mechanism of the reverse hydrolysis reaction is based on the microscopic reversibility principle. this indicates that the peptide bond formation and hydrolysis reaction come from the same intermediate ( figure ). thus, the reaction conditions are manipulated to shift the equilibrium towards peptide bond formation. the transpeptidation mechanism occurs as a result of the break of a peptide bond, with the formation of an active acyl-enzyme intermediate ( figure ). this intermediate is attacked in the presence of a nucleophile (peptide or amino acid blocked in the α-carboxyl group) and consequently causes the formation of a new peptide bond. for both mechanisms, the equilibrium should shift to the synthesis reaction direction, requiring the use of protective groups of α-amino and carboxyl substrates, the addition of organic solvents to the media reaction, excess substrates, and the removal of products from the reaction medium (machado and others ) . this synthesis uses modern methods of cloning and gene expression in microorganisms, allowing the production of a recombinant peptide or several peptides simultaneously. bacteria are the expression system generally used, with e. coli being the most widely used host. since antimicrobial peptides present a natural destructive activity against the host and relative sensitivity to proteolytic degradation, peptides are often expressed as fusion proteins to neutralize their innate toxic activity and increase their expression levels (wang and others ) . compared with isolation from natural sources and the other synthesis methods, the recombinant approach offers the most costeffective alternative for large-scale peptide production (li ) . peptides are increasingly being produced for various purposes, and these may contain closely related impurities resulting from incomplete reactions or from several side reactions. peptides synthesized for therapeutic and clinical research, as well as for biological and structural studies to explore the structure-activity relationships must have % purity or greater (ridge and hettiarachchi ) . however, there are other applications where low values of purity, between % and %, are tolerable (table ) . peptide purification depends on a series of separation techniques. peptides made on a preparative scale (in gram amounts) can be obtained from a separation process, to isolate one or more individual components from a peptide mixture for future research, or on an analytical scale (about mg of peptide) to identify and determine the relative amounts of some or all components in the mixture. studies on an analytical scale are the first steps for improving separation conditions, which are developed prior to the execution of any preparative separation process (sewald and jakubke ) . after the synthesis process, peptides are submitted to a separation procedure consisting of centrifugation and washing to remove residues of the reagents used, as well as products of side reactions. subsequently, peptides are cleaved and subjected to filtration, as well as lyophilization (dagan and others ) . the most widely used methods used for the purification of peptides are reverse-phase high-performance liquid chromatography (rp-hplc), ion-exchange chromatography, size exclusion chromatography, affinity chromatography, and capillary electrophoresis ( table ) . the purity of a peptide must be verified by a method different from that used for purification, since the results of homogeneity derived from such a system can lead to misinformation and be misleading (ridge and hettiarachchi ) . thus, the characterization should be analyzed by different methods of mass spectrometry. mass spectrometry has different ionization methods, such as electrospray ionization mass spectrometry (esi-ms), fast atom bombardment mass spectrometry (fab-ms) or matrix-assisted laser desorption/ionization mass spectrome-try (maldi-ms) that can be used for peptide characterization ( table ) . the different mass spectrometry techniques are based on the accurate determination of the molecular mass-charge ratio of the peptide, as well as on a chemical structure determination, with high sensitivity and resolution (sewald and jakubke ) . the growing resistance of pathogens against many commonly used antibiotics has led to research of new compounds with the same functions. an interesting approach is the study of molecules of natural origin to replace antibiotics (bechinger and lohner ) . several studies in recent decades have shown that peptides have certain bioactive properties (agyei and danquah ). short peptides ( - amino acids) with cationic and hydrophobic properties are known to be potent defenses of the host organism, providing activity against a wide variety of pathogenic microorganisms such as gram-negative and gram-positive bacteria, fungi, viruses, and parasites (hancock and sahl ) . studies have shown remarkable results of peptide antitumor activity, observed mainly in cancer therapy (korhonen and pihlanto ) . although several peptides have biological activity, antimicrobial activity is one of the most studied. one of the most-used analytical techniques to determine peptide antimicrobial activity is the broth microdilution test. in this test, the microorganisms are cultured in titration microplates and the peptide to be tested is added to each well at different concentrations. the microorganism growth causes turbidity in the wells. however, when a certain concentration of the peptide tested inhibits bacterial growth no turbidity is observed. turbidity is usually read by spectrophotometry, with the greatest frequency at nm, but it can also be seen through visual inspection of the wells (otvos and cudic ) . the standard methods developed by the clinical and laboratory standards inst. (clsi) have been used to test the activity of antimicrobial peptides. among them are the standards for antimicrobial disk susceptibility tests (clsi ) , the method for dilution antimicrobial susceptibility tests for bacteria that grow aerobically m -a (clsi ) , and the method for broth dilution antifungal susceptibility testing of yeast m -a (clsi ) , all of which have been widely used (jang and others ; rubinchik and others ; hwang and others ) . the most studied peptides are those with antimicrobial activity, characterized by their interaction with the cytoplasmic membrane of the microorganism regardless of the final target (powers and hancock ) . factors influencing the antibacterial activity are the electrostatic interactions between the peptide and positively charged and anionic lipids on the surface of the target microorganism. also, the hydrophobicity of the peptide (factor required for insertion into the membrane) and peptide flexibility allow peptide interaction with the microbial membrane (jenssen and others ) . although these characteristics are variable according to each peptide, all of them are essential to the function of peptides as antimicrobials. the exact mechanism of action of antibacterial peptides is not yet fully understood. however, there is a consensus among researchers regarding the first step in the initial interaction between peptide and the target cell (reddy and others ) . exclusion liquid chromatography based on separation process according to the size of the peptide relative to pore sizes in the stationary phase. used primarily in the early stages of purification of the peptide, when performed in multiple steps used to separate low-molecular-weight impurities from a mixture of peptides. however, the separation of the peptide of interest with other closely related peptides is virtually impossible affinity chromatography based on the biological specificity of the peptide. consists of a ligand (small specific biomolecule such as an antibody) that is immobilized in the column. the separation occurs because of highly specific biochemical interactions between the peptide and the ligand used when a high degree of specificity is required, for example, isolation of a target protein present in low concentration in a biological fluid or a cell extract capillary electrophoresis based on the migration of the peptide according to its charge in solution, depending on the application of an electric field. complementary technique to reversed-phase chromatography used for peptides and proteins table -ionization methods used in mass spectrometry. fundamental principle the ions are produced from a peptide contained in a solvent (for example, an organic compound such as methanol or acetonitrile) that is scattered in a fine aerosol fab-ms the peptide analyzed is mixed with a matrix, which is a non volatile reagent of protection (glycerol, diethanolamine, and triethanolamine, among others), and is bombarded with a beam of high-energy atoms ( to ev) in a vacuum. atoms are of an inert gas such as argon or xenon maldi-ms the peptide analyzed is bombarded by a laser beam (nitrogen), while a matrix (sinapinic acid) is used to protect the peptide. the matrix allows avoiding direct contact of the peptide with the beam, facilitating its vaporization, and ionization the initial attraction between the peptide and the target cell occurs via electrostatic binding between the cationic peptide and the components of the negatively-charged outer cell membrane, such as lipopolysaccharides in gram-negative bacteria or lipoteichoic acid on the surface of gram-positive bacteria (jenssen and others ) . this electrostatic interaction removes the native divalent cations (mg + , ca + ) from the cell surface, thus destabilizing the outer membrane and facilitating the entry of the peptide and subsequent peptide contact with the cytoplasmic membrane, a process known as autopromoted uptake (powers and hancock ) . after the peptide is bound to the target cell, an arrangement of the peptide occurs on the surface of the cytoplasmic membrane. this fact is of considerable debate, since several arrangement models have been proposed, such as the barrel-stave or the carpet model among others. depending on the model, the peptide can permeabilize the cytoplasmic membrane and/or translocate through it. thus, antimicrobial peptides can be classified into major groups, the first consisting of those peptides which act on the cytoplasmic membrane, and the second consisting of those which have no action on the cytoplasmic membrane of the target microorganism. this means that the peptide just moves into the cell without causing major disturbances in the membrane (powers and hancock ; jenssen and others ) . peptides acting on the bacterial membrane. several models have been proposed to explain how, after initial attachment, antibacterial peptides are distributed on the surface of the bacterial cytoplasmic membrane to form pores. pore formation results in membrane permeabilization, thereby affecting cellular respiration. it also deprives the microorganisms of their source of energy by interrupting the electrochemical gradient and causing an increase in the flow of water and ions across the membrane, thus leading to cell swelling followed by cellular lysis (bechinger and lohner ) . to explain the formation of pores, the aggregate toroidal pore, barrel-stave, and the carpet models have been proposed. the last models, the barrel-stave and carpet model, have been the most widely studied. barrel-stave model. this model describes the formation of a transmembrane channel (pore) through the binding of amphipathic α-helices. the hydrophobic surface of the peptide interacts with the lipid core of the membrane, while the hydrophilic surface of the peptide is oriented inside, producing an aqueous pore (figure ) . the progressive recruitment of additional peptides to the membrane surface increases the size of the pores, causing the loss of cell content and thus cell death (reddy and others ) . this model has been proposed to explain the activity of antimicrobial peptides, such as magainins (matsuzaki and others ) . carpet model. in this model, the peptide in high concentration is in contact with phospholipids located on the outer surface of the bacterial membrane, a fact that allows the peptide to permeate the membrane ( figure ). the peptides bind to the surface of the target membrane and cover it like a carpet. according to this model, the peptides exhibit a preferential binding for the phospholipid groups. the binding step is followed by the alignment of the peptide on the membrane surface so the hydrophilic surface is in contact with phospholipid or water molecules, causing a reorientation of hydrophilic residues and creating a hydrophobic core. finally, the peptide disintegrates the membrane by deformation of the membrane curvature (reddy and others ) . this model has been proposed to explain the action mechanism of dermaseptins (dagan and others ) . toroidal pore model. this model is considered a variant of the barrel-stave model. it is suggested that a perpendicular inclusion of the peptides to the membrane with their hydrophilic regions is associated with phospholipids, whereas their hydrophobic regions are associated with the lipid core. in this process, the membrane is bent inward so the pores are formed (jenssen and others ) . the main difference between this model and the barrel-stave model is the intercalation of the peptide with phospholipids to form the pore ( figure ). this model has been used to explain the mechanism of action of the peptide melittin (yang and others ) . aggregate model. this model, proposed by wu and others ( ) , has some similarity to the toroidal pore model. this model consists mainly of the arrangement of the peptide in the membrane forming an extension and developing micelle-like aggregate of peptides and lipids, but without adopting a particular orientation (jenssen and others ) . peptides with no activity on the bacterial membrane. these antimicrobial peptides have the ability to translocate into bacterial cells without causing membrane permeabilization. the peptide is accumulated within the cell where it reaches a variety of essential cellular processes that result in bacterial cell death (jenssen and others ) . the target process includes inhibitions of nucleic acid synthesis, protein synthesis, enzyme activity, and cell wall synthesis. peptides such as buforin ii and pleurocidin have shown this mechanism of action (park and others ; patrzykat and others ) . there is a growing need for new antifungal agents due to the increased resistance of molds to therapies with regularly used compounds (de lucca and walsh ) . peptides have emerged as alternative antifungal agents. initially, the antifungal mechanism of action was described as a result of fungal cell lysis or as a result of interferences in fungal cell wall synthesis. however, the discovery of new antifungal peptides in the last decade has led to the identification of new mechanisms of action, including membrane permeabilization, binding to ergosterol/cholesterol in the fungal membrane, the attack of mitochondria or other intracellular organelles, and the deformation of cell membrane structure (jenssen and others ) . according to de lucca and walsh ( ) , fungal peptides can be classified with respect to their mode of action into groups: peptides that act through cellular lysis; peptides that cross the fungal membrane and interact with the intracellular target; and peptides that act by forming pores. peptides acting by cellular lysis. these peptides are characterized by their amphipathic nature, being molecules with faces, one positively charged and the other neutral and hydrophobic. some of these peptides bind only to the membrane surface, damaging the membrane structure, and they may or may not pass through it. peptide smap- (a synthetic peptide derived from the sequence of cathelicidins) has shown antimicrobial activity against the fungus trichosporon beigelii by interaction, penetration, and subsequent damage to the cell membrane (lee and others ) . this result suggests that the main target of smap- peptide is the fungal plasmatic membrane. a similar mechanism was observed for the synthetic peptide ib-amps (an analogue sequence to peptides isolated from seeds of impatiens balsamina), showing antifungal activity by bonding the peptide to the fungal cell membrane and subsequent penetration (thevissen and others ) . peptides that pass into the membrane and interact with intracellular targets. these peptides interfere with cell wall synthesis or the synthesis of essential cellular components such as chitin or glucan. as such, the synthetic peptide omiganan (an indolicin analogue peptide, isolated from bovine neutrophils) has shown antifungal activity against candida albicans, and the main mechanism of action of this peptide is related to its activity in the cytoplasmic membrane, resulting in macromolecules synthesis inhibition of macromolecules and finally cell death (rubinchik and others ) . pore-forming peptides. these peptides are aggregated in a selective way to form pores of varying sizes, which then allow the passage of ions and other solutes. the synthetic peptide di-k hc (a halocidin analogue peptide, isolated from the invertebrate marine animal halocynthia aurantium known as sea peach), has shown antifungal activity against several strains of aspergillus and candida (jang and others ) . the activity of di-k hc results in the formation of pores on the surface of fungal membranes. moreover, these researchers pointed out the specific binding of di-k hc with b- , -glucan, a component of the cell wall of fungi. this mechanism has also been observed with the antifungal peptide psacotheasin, isolated from the yellow-spotted long-horned beetle (psacothea hilaris), which has shown activity against c. albicans (hwang and others ) . the researchers indicated that there was damage to the cell wall, membrane depolarization with the formation of pores ( . - . nm), as well as an increase in membrane permeability, all being responsible for the antifungal activity of this peptide. several studies have shown the ability of cationic peptides to inhibit viral infections. the peptide cecropin a has shown antiviral activity against junin virus (jv-which causes argentine hemorrhagic fever). the peptide melittin inhibited jv and herpes simplex virus (hsv- ) multiplication, as well as magainin i and ii, and has shown inhibitory activity against hsv- and hsv- (albiol-matanic and castilla ). antimicrobial peptides isolated from fish, such as tilapia hepcidin - , have shown activity against the nervous necrosis virus (nn virus), an infectious agent that causes mass mortality of several species of marine fish in the larval stage (chia and others ) . in addition, synthetic peptides consisting of arginine and tryptophan repetitions have shown activity against vaccinia virus (the cause of cowpox) (mohan and others ). the antiviral activity of peptides is often related to virus adsorption and its entry into the host cell or, in other cases, is the result of a direct effect on the viral envelope. thus, the antiviral activity of peptides may result from multiple mechanisms of action, the most important being blocking virus entry through interaction with the host cell and blocking viral entry through interaction with the virus. blocking viral entry through interaction with the host cell. peptides can interact directly with specific viral receptors on the host cell, thus preventing the virus from binding to the cell membrane or binding intracellularly (jenssen and others ) . proteoglycans are proteins found in all types of tissue, in intracellular granule secretions as well as in the extracellular matrix and cell surface. proteoglycans are covalently linked to one or more chains of glycosaminoglycans (gag), long polysaccharide unbranched structures, which have a sugar that contains nitrogen and are usually sulfated. gag chains are present on the surface of mammalian cells and their degree of sulfation makes these compounds more anionic. this network of strong negative charges allows gag to attract and bind to small cations, such as enzymes and proteins, and also pathogens such as viruses (spillmann ) . heparan sulfate, one type of gag chain, is one of the most important molecules related to viral binding (spillmann ) . thus, by blocking heparan sulfate molecules can be inhibited viral infection. jenssen and others ( ) have suggested that antimicrobial peptides which interact with heparan sulfate have the ability to block a number of viral infections. due to the large number of amino acid residues positively charged peptides can interact electrostatically with negatively charged heparan sulfate molecules on the cell surface. studies on lactoferrin (lf) have shown that this peptide prevents infection of the host cell rather than inhibiting virus replication after infection of the target cell. the interaction of lf with heparan c institute of food technologists ® vol. , r comprehensive reviews in food science and food safety sulfate molecules has been proposed as the mechanism responsible for lf antiviral activity (van der strate and others ). similarly, jenssen and others ( ) showed the antiviral activity of synthetic peptides (consisting of arginine and lysine residues) against herpes simplex virus and (hsv- and hsv- ). the peptides presented higher affinity in binding to heparan sulfate with an increasing number of cationic residues, thereby blocking the entry of hsv (- or - ). in addition, luganini and others ( ) reported the inhibition of cytomegalovirus by binding synthetic peptide dendrimers with molecules of heparan sulfate on the surface of fibroblasts and endothelial cells. thus, cytomegalovirus infection was blocked by the interaction of synthetic peptide binding sites with heparan sulfate. blocking viral entry through interaction with the virus. the interactions of peptides with the glycoproteins (gp) in the viral envelope have been proposed as another mechanism that influences the process of viral entry and virus inactivation. in this way, peptides generated from chemical modification of milk proteins, such as α-lactalbumin, β-lactoglobulin, and lysozyme with -hydroxyphthalic anhydride ( -hp) inhibited infection of vero cells with hsv- (oevermann and others ) . according to those researchers, the antiviral activity of these peptides is based on their direct interaction with viral glycoproteins (gb, gc, gd), which are responsible for adsorption and penetration of the virus into the host cell. similarly, lf has shown the ability to bind to the gp glycoprotein (a protein present in the outermost layer of the hiv virus) with antiviral effects, since the gp glycoprotein plays an important role in the adsorption and entry of hiv into target cells (van der strate and others ; pan and others ) . on the other hand, other peptides, such as magainins, have shown antiviral effects through direct interaction with virus cells. egal and others ( ) have indicated that the effect of magainins is the result of the peptide acting on the viral envelope. a similar mechanism was suggested for the activity of mucroporin-m , a defense cationic peptide present in scorpion venom, which has shown activity against the measles virus, the coronavirus that causes severe accurate respiratory syndrome (sars), and flu virus h n (better known as the bird flu virus) (li and others ) . the researchers have suggested that the antiviral activity of the peptide is the result of direct interaction with the virus envelope, thereby reducing viral activity in the host cell. cancer, also known as malignant neoplasm, is a general term that refers to more than different diseases affecting various tissues and different types of cells. all forms of cancer are characterized by abnormal cell growth, that is, they lack the mechanisms that control normal cell division. this lack of regulatory mechanisms is the result of a multistep process involving genetic mutations induced by inheritance or environmental changes (hütter and sinha ) . despite major advances in cancer therapy, there is considerable interest in the development of antitumor agents with a novel mode of action, since the cells have shown carcinogenic development of resistance to current chemotherapy (hoskin and ramamoorthy ) . carcinogenic cells often become resistant to chemotherapy. this mainly occurs as a result of increased expression of intracellular enzymes for the detoxification of antitumor agents, the correction of dna damage, generation of intracellular organelles with the ability to eliminate and/or transport the drugs out of the tumor, and irreversible defects in the cellular machinery that mediates apoptosis (hütter and sinha ) . thus, recent studies have shown peptides as an alternative to conventional cancer treatments. however, not all peptides have selective activity against carcinogenic cells. according to hoskin and ramamoorthy ( ) peptides that have antitumor activity can be classified into major groups: peptides with selective activity, and peptides with non selective activity, that is, those that have activity against bacteria, carcinogenic cells, and healthy cells. peptides with selective activity toward carcinogenic cells. these peptides have activity against bacteria and carcinogenic cells, but not against normal cells. several peptides, such as the cecropins, buforins, and magainins have shown antitumor activity without affecting normal eukaryotic cells (cruciani and others ; cho and others ) . studies with magainin ii have shown to inhibit the proliferation of carcinogenic cells (in bladder cancer) without any effect on normal cells (lehmann and others ) . similar results were observed by chen and others ( ) in the study of the synthetic peptide th - (isolated from tilapia and analogous to the peptide hepcidin), with antitumor activity shown primarily by direct interaction and lysis of target carcinogenic cells (human fibrosarcoma cells). these researchers indicated that the lytic activity of the peptide and proliferative cells were restricted mainly to carcinogenic cells, since normal cells showed no significant effects. likewise, the synthetic peptide th - (isolated from tilapia and an analogue to the peptide hepcidin) has shown antitumor activity against carcinogenic cells, due to interaction with and penetration of the membrane. this peptide has less toxicity toward normal cells supposedly because it can discriminate between healthy cells and carcinogenic ones (chang and others ) . researchers have also indicated that the interaction with the cell membrane and its subsequent damage is caused by the formation of pores on its surface. it has been suggested that the internalization of the peptide and the subsequent damage to the mitochondrial membrane activates apoptotic pathways (chang and others ) . according to hoskin and ramamoorthy ( ) there are fundamental differences between the membranes of malignant cells and normal cells which allow the selectivity of certain peptides to attack carcinogenic cells without affecting healthy cells. electrostatic interactions between cationic peptides and anionic components of the cell membrane have also been considered an important factor. carcinogenic cells typically have a negative charge due to a higher expression than normal of anionic molecules such as phosphatidylserine (ps) and mucin (glycoprotein) (oren and shai ) . however, normal cells are not affected, since these cells have a neutral surface charge, conferred by the zwitterionic nature of most membrane components such as phosphatidylethanolamine (also known as cephalin), phosphatidylcholine, and sphingomyelin (sok and others ) . membrane fluidity and the surface area of the cell are also considered factors that contribute to the selectivity of peptides for carcinogenic cells. the fluidity of carcinogenic cells is greater than that of normal cells, which may increase the activity of lytic peptides through the easy destabilization of the membrane. in addition, the carcinogenic cells have a higher surface area than healthy cells due to the presence of greater numbers of microvilli, which are small projections of the cell membrane, irregular in size and shape. the microvilli may allow the bonding between peptide and carcinogenic cells (hoskin and ramamoorthy ) . peptides with nonselective activity. this group is comprised of peptides with activity against bacteria, carcinogenic cells, and against normal eukaryotic cells (hoskin and ramamoorthy ) . according to papo and shai ( ) non selective activity of these antimicrobial peptides results from their ability to interact with and cause damage to negatively charged membranes and those of a zwitterionic nature. dathe and others ( ) have indicated that the hydrophobic moment of antimicrobial peptides exerts a substantial influence on the neutral lipidic membranes, although it has a small role in the permeabilization of highly charged lipid membranes. peptides of this group include melittin, isolated from bee venom; taquiplesin ii, isolated from the horseshoe crab; defensins, isolated from insects; and plantaricin, a bacteriocin isolated from lactobacillus plantarum (schweizer ). plantaricin has shown activity against carcinogenic cells and against normal lymphocytes and neuronal cells (sand and others ) . the mechanism of action of antitumor peptides consists of permeabilization of the cell membrane mediated by electrostatic interaction. the electrostatic interaction is generated by the negatively charged phospholipids in the cell membrane and the positively charged peptide (schweizer ). unlike carcinogenic cells, eukaryotic cells have most of their negatively charged phospholipids, particularly ps, in the inner membrane, while neutral lipids are positioned on the outside (zhao and others ) . however, the result obtained by sand and others ( ) suggests that in addition to the mechanism of action related to the electrostatic interaction, there is another mechanism which explains the sensitivity of normal eukaryotic cells to plactaricin. probably another negatively charged macromolecule present on the membrane surface of healthy cells is also involved in plantaricin activity. similar results were observed by nan and others ( ) in the study of synthetic peptides consisting of lysine or arginine enriched with tryptophan. the peptide with arginine residues showed higher toxicity against human erythrocytes and mammalian cells. the hydrophobicity of the peptides has been suggested as an important factor in the increase of hemolytic activity and cytotoxicity in mammalian cells, as hydrophobic regions are required for direct interaction between peptide with membrane lipid components. the peptide with arginine residues was slightly more hydrophobic than the peptide with lysine residues. thus, these researchers suggested that small differences in hydrophobicity of these peptides may be responsible for the cytotoxic activity of this peptide in mammalian cells. the growing problem of microorganism resistance to conventional antibiotics, as well as the need for new agents with antibiotic properties has stimulated interest in developing antimicrobial peptides aiming for their application in the medical field (zasloff ) . most of the studies are devoted to the development of topical agents with antibacterial and antifungal activities. also, due to their antiviral activity, antimicrobial peptides have also been proposed as chemical preservatives. in the food industry, antimicrobial peptides, especially those produced by bacteria, have been widely researched in recent years due to their potential use as natural preservatives (papagianni ; coma ; settanni and corsetti ) . the direct application of antimicrobial peptides in food preservation can be achieved by methods: the direct addition of peptide to the food matrix, or the inoculation of the food matrix with the bacteriocin producer strain under the conditions favorable for the in situ production of the antimicrobial peptide. bacteriocins can be obtained ex situ by the cultivation of the producer strain at an industrial scale in a food-grade substrate, followed by a series of separation and purification techniques. these ex situ bacteriocins are commercially available in concentrated form, such as alta tm or microgard tm , and can be added directly to the food matrix. the production of bacteriocins in the food matrix offers several legal and cost advantages. the use of bacteriocin producer strain requires careful selection depending on the particular food intended for inoculation to ensure the producer strains will produce bacteriocins in the necessary amounts to inhibit the target microorganism. in addition to the peptides being studied as antimicrobial agents for direct addition to foods, they also have shown potential for being incorporated into food preparation surfaces (such as cutting surfaces) and processing equipment, as well as in food packaging (appendini and hotchkiss ) . active packaging includes the incorporation of antimicrobial agents in the packaging material to control and extend the shelflife of food (soares and others a). these types of packaging are considered an innovative technology in food preservation, since they allow better antimicrobial efficiency on food surfaces, thus improving stability. the development of active packaging by incorporating antimicrobial peptides in food packaging material can be done either to prolong the life of the product or to reduce the microbial load of the packing before use (steven and hotchkiss ) . the development of active packaging with antimicrobial peptides can be accomplished by main methods of incorporation: direct peptide incorporation in the polymer; peptide coating on the polymeric surface; and peptide immobilization in the polymer. numerous studies have reported the incorporation of antimicrobial peptides directly in the polymeric material, especially bacteriocins. the peptides are relatively resistant to heat (appendini and hotchkiss ) . however, their antimicrobial activity may be greater when heat is not used in the incorporation process. moreover, bioactive peptides incorporated in polymer films must be able to diffuse to the package surface over time to be effective. thus, polymers such as cellulose acetate, alginate, chitosan, and soy protein, among others, have been widely used to develop films with direct incorporation of these antimicrobials (marcos and others ; pires and others ; sivarooban and others ; santiago-silva and others ). researchers have studied the antimicrobial activity of bacteriocins incorporated into polymeric materials in synergy with other antimicrobial agents. synergistic activity against staphylococcus aureus, listeria monocytogenes, and bacillus cereus has been observed for nisin with potassium sorbate and garlic oil when incorporated into chitosan films (pranoto and others ) . in addition, soy protein films incorporated with nisin, grape seed extract, and ethylenediaminetetraacetic acid (edta) have shown inhibitory synergistic activity against pathogenic microorganisms such as l. monocytogenes, e. coli o : h , and salmonella typhimurium (sivarooban and others ) . the activity of bacteriocins incorporated into polymeric materials in synergy with other conservation technologies has also been reported. films incorporated with enterocins a and b (bacteriocins produced by enterococcus faecium) have shown synergistic activity when used together with high-pressure processing. thus, the use of antimicrobial packaging developed in conjunction with the high-pressure process allowed the control of l. monocytogenes at below detectable levels after d of storage at • c (marcos and others ) . this is an alternative method when the polymer requires extreme processing conditions during packaging material manufacture, such as high pressure and temperature, which can result in inactivation of the antimicrobial agent (appendini and hotchkiss ) . in some cases, the antimicrobial coating is done by contacting the film with or immersing it in the peptide solution. in this way, linear low-density polyethylene (lldpe) has been coated with lactocin and lactocin al (both bacteriocins produced by lactobacillus curvatus crl ), by direct contact of the film with a bacteriocin solution, showing antimicrobial activity in vitro against lactobacillus plantarum crl and listeria innocua (massani and others ) . similarly, scannell and others ( ) used alternatively lacticin and nisin adsorbed on the surface of plastic bags (polyethylene/polyamide) through direct contact of the polymeric material with bacteriocin solution. the film coated with nisin showed inhibitory activity against l. innocua and s. aureus, maintaining its activity for mo either at room temperature or under refrigeration. however, the film coated with lacticin did not show antimicrobial activity. the researchers suggested that lacticin was not retained by the polymer (scannell and others ) . proper handling of solvents and polymeric structures has been suggested to increase the adsorption of the peptide into the polymer matrix (appendini and hotchkiss ) . for example, the polymeric surface can be coated by applying a filmogenic solution that can be deposited on the film surface by the casting method. accordingly, chollet and others ( ) developed a laminated film of polyethylene (pe) and polyamide (pa), with the structure pe/pa/pe, coated with a filmogenic solution of hydroxypropyl methyl cellulose (hpmc) and adsorbed with nisin. the developed film presented antimicrobial activity in vitro against kocuria rhizophila (chollet and others ) . peptides can be immobilized or attached to solid supports by physical methods, such as layer-by-layer assembly, or by chemical methods, such as covalent bonding (onaizi and leong ) . layer-by-layer assembly. in this process, the peptide is sandwiched between polyionic polymers and the number of peptides and polymers is flexible (figure ) . the effectiveness of the peptide depends on its relative mobility. the advantage of this method is that it allows the slow release of the peptide embedded in the surface of the polymer. however, a key drawback of this method is that the peptide immobilized in the layers closest to the solid support will not be in direct contact with the target surface, thus reducing peptide activity. this peptide must be able to diffuse through the different layers of the assembly to the interface (onaizi and leong ) , to ensure efficient release and consequent bioactivity. the diffusion process of the peptide in the different layers is more complex than its diffusion in solution, since additional factors such as tortuosity of the diffusion path, the number of layers, and the polymer-peptide interactions can affect the diffusion process (appendini and hotchkiss ; sukhishvili ) . covalent bonding. in this process, the antimicrobial peptide will react chemically with a given surface to form a stable bond, which results in the formation of an antimicrobial coating on the polymeric surface (haynie and others ) . covalent bonding offers several advantages, including a more stable attachment between the peptide and the polymer surface (goddard and hotchkiss ) . covalent bonding reduces attached peptide ability to destabilize and improves its bioactivity, by protecting it from denaturation. due to the inert nature of most polymers, they must be subjected to a functionalization process on the surface before bonding with the peptide. the polymers can be functionalized with different spacers, which are reactive functional groups that allow peptide attachment on the spacer surface (humblot and others ) . the quantity of reactive functional groups generated in the functionalization process results in a restricted number of covalent bondings, which limits the amount of peptide that can be attached to the packaging. according to goddard and hotchkiss ( ) , direct bioactive compound applications require small amounts to be effective. however, for the applications of peptides in polymeric matrixes it is necessary to maximize the amount of peptides per unit area. to accomplish this, the functionalization technique must be optimized with the objective of linking the desired type and quantity of reactive functional groups. stiff or flexible spacers have been used as reactive groups for the functionalization of polymers (figure ). stiff spacers, such as polymethyl-methacrylate (pmma) and polyvinyl chloride (pvc), restrict the lateral mobility of the peptide bond, keeping the peptide firmly in a specific orientation. on the other hand, flexible spacers, such as polyethylene glycol (sand and others ) allow lateral mobility of the peptide bound, which can result in different orientations of the peptide molecules at the interface (onaizi and leong ) . among the different types of spacers, polyethylene glycol (peg) is widely used in the immobilization of peptides. the use of peg has several advantages, such as rapid and free peptide orientation, promoting peptide-bacteria interactions (costa and others ) . although the potential penetration and translocation of peptides through the microorganism cytoplasmic membrane is low due to the covalent bond that attaches the peptide to the polymer, it has been reported that peptides have sustained their bioactivity after attachment to polymeric matrixes. the synthetic peptide k l (a peptide sequence derived from magainin) was covalently bound to polystyrene (ps) resin by functionalization with peg and showed antimicrobial activity against foodborne pathogenic microorganisms, including e. coli o : h , l. monocytogenes, and pseudomonas fluorescens (appendini and hotchkiss ) . similarly, the synthetic peptide e lkk was covalently immobilized in ldpe film after chromium oxidation and functionalization with peg. this active packaging had antimicrobial activity against e. coli showing a reduction of log cycles when compared with the control (steven and hotchkiss ) . the sustained bioactivity of attached peptides is caused by the presence of spacers which allow sufficient freedom of motion for the active portion of the peptide to contact microorganisms on the food surface (appendini and hotchkiss ) . haynie and others ( ) have previously demonstrated that peptide-bacteria interactions are sufficient for peptide bioactivity. moreover, costa and others ( ) have indicated that the efficacy of attached peptides could possibly result from a higher peptide-relative surface availability, contrary to the other methods of peptide applications in which peptide aggregation can occur, producing uneven distribution. on the other hand, the diffusion of attached peptides into the food surface is restricted due to the covalent bonding. however, diffusion to the food product can occur in extreme conditions, such as high temperatures, which can promote hydrolysis reactions. the characterization of active packaging involves processes: structural analysis and measurement of their properties (table ) . according to goddard and hotchkiss ( ) , the type of analytical tool used in the structural characterization of polymers depends on the kind of modification, the specificity required, and available resources. some of the techniques used in structural analysis of active packaging with antimicrobial peptides are the contact angle, x-ray photoelectron spectroscopy (xps), fourier transform infrared spectroscopy (ftir), and scanning electron microscopy (sem). steven and hotchkiss ( ) used the techniques of contact angle and xps to assess changes in the surface of ldpe films after treatment with chromic oxide and functionalization with peg as spacer, subsequently covalently binding a synthetic peptide. the contact angle for the film before being subjected to any process of functionalization showed values of • . however, after chromic oxidation and peg bonding, films presented values of • and • , respectively; which indicated that the film surface became hydrophilic. these researchers concluded that the decrease in the contact angle is the result of an increased ionization at the film surface after oxidation, due to the presence of functional groups, such as carboxylic acid (cooh); and a value even lower after functionalization was observed due to solubility of peg. the xps technique showed changes in chemical composition on the film surface, resulting in the detection of nitrogen ( . %) and an increased percentage of oxygen (initially from . % to . % after oxidation and . % after functionalization). the oxygen increase was due to the presence of carboxylic acid after chromic oxidation. also, this increase was the result of functionalization with peg due to the main presence of o in the peg chain backbone. in addition, the functionalization with peg also introduced nitrogen originating from its amino-terminal functions (nh -peg-nh ). the ftir spectroscopic technique has been used by pranoto and others ( ) to study the interactions between chitosan films and nisin. they observed an increase in the band of the amide i corresponding to the wave number cm − related to the increased concentration of nisin incorporated in the film. according to the researchers, this is probably due to the interaction between the amine functional groups of chitosan and functional groups of nisin, which resulted in covalent bonds, and consequently in a larger peak. microscopic techniques have also been widely used to evaluate morphological changes in the surface of films that have been incorporated with antimicrobial peptides. pires and others ( ) used sem and observed that cellulose-based films incorporated with nisin, or a mixture of nisin and nantamicin, showed crystals deposited on the surface. these results indicated a heterogeneous distribution of the peptide in cellulosic films, while the control film presented a homogeneous structure. similarly, santiago-silva and others ( ) used sem to observe changes in surface morphology of cellulose acetate film incorporated with pediocin. when the concentration of peptide was increased, the films incorporated with pediocin had a rough surface c institute of food technologists ® vol. , r comprehensive reviews in food science and food safety table -characterization techniques of packaging incorporated with antimicrobial peptides. technique factor studied structural analysis contact angle quantifies surface hydrophobicity by measuring how far a droplet of water spreads on a surface x-ray photoelectron spectroscopy (xps) determines the atomic composition of the top several nanometers of a solid. this technique can be used to quantify the percent atomic composition and stoichiometric ratios fourier transform infrared spectroscopy (ftir) detects and identifies the chemical functional groups present in the polymer scanning electron microscopy (sem) allows the characterization of the polymer surface morphology and the observation of the dispersion quality of the peptide in the polymeric matrix property measurements mechanical properties measurement of the mechanical performance of the polymer. generally according to the standard method astm d (astm a) barrier properties measurement of water vapor permeability. generally according to the standard method astm e /e m (astm b); astm f (astm ) due to large amounts of pediocin granules dispersed in the matrix. this resulted from the lack of peptide solubility. on the other hand, the control film showed a homogeneous and transparent surface. in addition to the analysis of structural changes and interactions between the peptide and the polymeric matrix, the study of packaging properties is important as well. these properties show the performance of the developed material and how it will relate to the primary functions of food packaging, such as physical integrity. thus, mechanical and barrier properties have become increasingly relevant and are more frequently studied. the mechanical properties of films incorporated with antimicrobial peptides serve as the basis for assessing the effects on the mechanical performance resulting from the modification made to the polymer (table ) . guiga and others ( ) investigated the effect of nisaplin ® ( . % purity of nisin) coating on the mechanical properties of laminated films (pe/pa/pe), studying the mechanical properties of elongation at break and young's modulus. their results showed a significant difference in mechanical properties between the films incorporated with the peptide and the control treatment; peptideincorporated films showed an increase of young's modulus and a decrease in elongation at break. the polymer-based coating (hpmc) applied in laminated film, as well as the interaction between proteins and salts present in nisaplin, may have modified the mechanical behavior of the manufactured packaging, thereby increasing the rigidity of the film with the consequent decrease in its elongation. a similar result was observed by santiago-silva and others ( ) in their study of cellulosic films incorporated with pediocin. the researchers indicated that the addition of % of the peptide increased the maximum load required for film rupture when compared to the control. the researchers pointed out that a possible interaction between the pediocin and the polymeric matrix allowed the development of a more resistant film. however, a significant drop in the maximum load value was observed at a % concentration. according to the researchers, there was an excessive amount of the peptide incorporated, which weakened the cellulose chains of the film and resulted in a reduction of film resistance. a decrease in the mechanical strength of films incorporated with antimicrobial peptides has also been observed. sivarooban and others ( ) reported a decrease in puncture resistance and tensile strength values of soy protein films incorporated with nisin. similarly, pires and others ( ) indicated that nisin incorporated into cellulose-based films affected the film structure, reducing maximum load and elongation values. these resulted from the heterogeneous distribution of the peptide in the polymeric matrix, which consequently lead to the formation of stress points and reduced film resistance. although several studies have indicated changes in film properties, in some cases peptide incorporation into polymeric matrices had no significant effects. massani and others ( ) reported no significant difference in tensile strength, elongation, and water vapor permeability of ldpe films coated with lac-tocin and lactocin al . similar results were observed by chollet and others ( ) who indicated that the incorporation of nisin into pe/pa/pe laminated films, by coating with hpmc, showed neither changes in tensile strength at break nor in water vapor permeability. similarly, guiga and others ( ) reported that the direct incorporation of nisin into multilayer films of ethyl cellulose (ec) and hpmc (ec/hpmc/ec) did not alter the properties of tensile strength, young's modulus, or elongation at break. barrier properties of packaging include resistance to water vapor or gases (o and co ). the water vapor barrier property of the packaging can be determined by calculating the water vapor permeability (wvp) or the water vapor transmission rate (wvtr). both parameters cover the determination of the passage of water vapor through a polymeric material. however, the wvp considers vapor pressure difference between specific surfaces (internal and external) of the analyzed packaging (astm b). both parameters, wvp as well as wvtr, have been used to indicate that the addition of nisin in different polymeric matrixes, such as ldpe film coated with a cellulose-based solution (grower and others ; massani and others ) , sodium caseinate films (kristo and others ) and pe-film coated with hpmc (chollet and others ), causes no significant changes in the water vapor barrier property. on the other hand, studies regarding gas permeability in active packaging have been limited due to their applications as films or coatings for food products. during the evaluation and characterization of antimicrobial packaging it is important to research the transference of antimicrobial substances from the packaging material into the food, since this information allows the determination of how the antimicrobial agent is released from the active packaging. the mass transfer can occur by the diffusion mass transfer mechanism or by the convective mass transfer mechanism. the convective mass transfer mechanism occurs in a moving fluid, known as natural convection, if the movement is caused by differences in the density, or as forced convection, if the movement is caused by external agents or when a fluid is flowing on the solid surface by forced movement. on the other hand, diffusion mass transfer consists of a random motion of individual molecules as a result of a concentration gradient (crank ; geankoplis ) . the migration of substances from packaging materials takes place through the diffusion mass transfer mechanism, since the active packaging and the food contain a concentration gradient for the antimicrobial agent incorporated in the packaging. unlike the research on the release of active substances from drugs or the release of solvents from polymers, the study of antimicrobial release from active packaging is still limited (buonocore and others ; bastarrachea and others ) . the knowledge of diffusion parameters allows an efficient design of active packaging. several factors must be considered when studying the migration from antimicrobial packaging, including the release rate of the antimicrobial molecules from the packaging. if this rate is high, the active packaging would release the antimicrobial rapidly, resulting in a large concentration at a determined time. however, the large concentration would not be maintained over time, depending on the solubility of the antimicrobial in the selected food. if the solubility is very high, the antimicrobial will migrate rapidly to the food matrix, and therefore result in a decreased concentration of the antimicrobial on the food's surface along time. on the other hand, if the release rate is low, the antimicrobial agent will be slowly released in a desired concentration and if it presents a low solubility in the selected food, the antimicrobial can accumulate on the food surface and slowly migrate into the food matrix. in this situation the release rate should not be slower than the microbial growth (bastarrachea and others ) . in either case, the release of the antimicrobial agent from the packing material is indicated by the diffusion coefficient (d). thus, the diffusion characteristics of the antimicrobial agent can be used to determine the amount needed to maintain the proper concentration on the food surface (buonocore and others ) . the literature reports a few migration studies of antimicrobial peptides incorporated in active packaging. most evaluate the migration of nisin, probably due to the fact that this is the only antimicrobial peptide substance indicated as generally recognized as safe (gras) for direct contact with food in the united states (fda ). nisin is also widely accepted as a food preservative in the european community where it is classified as a safe preservative for food contact, coded as e (fsa ), as well as in brazil where the use of nisin is permitted by brazilian law as a natural preservative for biological products (anvisa ) . diffusion of several antimicrobial agents, such as potassium sorbate or lysozyme incorporated in active packaging, has been explained by fick's second law (han and floros ; gemili and others ) : where, c is the diffusing substance concentration; d is the apparent diffusion coefficient; t is the diffusion time; and x the distance. depending on the conditions of the migration test, different analytical solutions have been applied to solve fick's second law and to calculate the d-value in the migration of the antimicrobial peptide nisin incorporated in different types of packaging materials (table ) . different analytical solutions of fick's second law have been used in previous studies to calculate the d-value of nisin at a specific temperature ( table ) . some of these studies were conducted at different temperatures to characterize the d-value as a function of this parameter. protein films (corn zein or wheat gluten) and poly(butylene adipate-coterephthalate) (pbta) films incorporated with nisin showed an increase in d-value with increasing temperature, indicating that the peptide concentration was higher in the simulant at equilibrium state with increasing temperature (teerakarn and others ; bastarrachea and others ) . similarly, at low temperatures, lower d-values of nisin diffusivity indicate that the film retains larger amounts of the peptide in the polymer matrix while in contact with the simulant. the arrhenius activation energy model (eq. ) has been shown to confirm the dependence of the diffusivity with respect to temperature. where, d is a constant; e a is the activation energy for the diffusion process (j/mol); r is the universal gas constant ( . j/mol k); and t abs is the absolute temperature (k (− ) n ierfc nh √ d t m , is the initial amount of nisin in the film; m t , released amount of nisin at time t; h, film thickness; d, diffusion coefficient; ierfc, associated function of the mathematical error function teerakarn and others ( ) paper coated with acrylic polymer and ethylene-vinyl acetate co-polymer (eva) is the amount of nisin released at time t; m ∞ , is the migration in a state of equilibrium; l p coating layer thickness, d, diffusion coefficient kim and others ( ) hydroxypropyl methyl cellulose (hpmc) films is the amount of nisin in the simulant at time t; m f, , amount of nisin in the film when t = ; α, mass ratio between the amount of nisin in the simulant and in the film at equilibrium; q n , is the ''n" root of tanq n = −αq n ; l, is a half of the film's thickness bastarrachea and others ( ) teerakarn and others ( ) indicated e a values of . and . kj/mol for corn zein and wheat gluten films, respectively, and bastarrachea and others ( ) obtained an e a value of . kj/mole for pbta film incorporated with nisin. the value of e a represents the degree of molecular interactions between the antimicrobial substance incorporated and the polymeric matrix. thus, higher e a values represent stronger antimicrobial-polymer interactions, which is reflected in a lower d-value due to the greater energy level required for antimicrobial release (bastarrachea and others ) . the relationship between temperature and diffusivity of the antimicrobial agent is the result of structural changes in the polymer matrix, since above the glass transition temperature (t g ) the molecular mobility in the system increases along with temperature, which leads to an increase in the ability of the packaging material to transport substances through its polymeric matrix (teerakarn and others ) . in addition to the interactions between polymer and antimicrobial agent, the d-value is also influenced by interactions between the antimicrobial and the food matrix. thus, the food nh -r-w-c-f-r-v-c-y-r-g-i-c-y-r-k-c-r-conh miyata and others ( ) * x = means that the specific identity of an amino acid cannot be determined unambiguously. * * dhb = (z)- , -didehydrobutyrine. * * * abu = -aminobutyric acid. composition, as well as the solubility of the antimicrobial in these components also affects the d coefficient. in the study of paper coated with eva incorporated with nisin, the d-values varied according to the composition of the food in contact with the active packaging (kim and others ) . the highest d-value was observed when the film was in contact with a % citric acid solution, and the lowest value was observed when in contact with a % nacl solution. characteristic parameters of each solution, such as ph and ionic strength, have been shown to influence nisin solubility. nisin has a high solubility (up to mg·ml − at ph ) at low ph, but at high concentrations of nacl (above m) nisin solubility dependence on ph almost disappears and the solubility decreases to values below mg·ml − at any ph (rollema and others ) . chollet and others ( ) also investigated the influence of food composition on the migration of nisin incorporated in pe/pa/pe films coated with hpmc by changing the fat percentage. they found that increasing the fat content in the food resulted in an increased d-value and, therefore, in a greater diffusion of incorporated nisin. in their experiment, nisin diffusion mechanism was governed by the fat content. the increase in fat content resulted in microstructural changes, such as enlargement of pore size in the food matrix, which favored nisin diffusion into it. consumer demand for minimally processed foods and additivefree products has led to the development of antimicrobial packaging. peptides have shown various bioproperties, among them antimicrobial activity, leading to the application of these compounds in the food preservation area by either direct addition or incorporation into packaging materials (table ) . active packaging materials incorporated with antimicrobial peptides have shown effectiveness in inhibiting pathogenic microorganisms, an improvement in food safety. moreover, antimicrobial peptides incorporated into the polymeric matrix may af-fect the engineering characteristics of the packaging material, and lead to differentiated diffusion performance. this review highlights the characteristics of pure peptides, as well as their incorporation into polymeric matrices. several studies have indicated significant changes in mechanical properties and surface morphology of the films incorporated with antimicrobial peptides. however, research related to the study of barrier properties to gases and water vapor is still limited. more studies on the release of other peptides, different from nisin, from food packaging materials are needed to better understand the mechanism of dissemination of antimicrobial agents. finally, in the years ahead, the advent of nanotechnology will lead to research on the synergistic effects of antimicrobial peptides and nanoparticles, such as metals, metal oxides, and nanoclays, with the objective being to improve the mechanical and barrier properties of antimicrobial packaging. bioactive peptides: synthesis, properties, and applications shortened cecropin a-melittin hybrids significant size reduction retains potent antibiotic activity uso da nisina com a função de conservador para queijos pasteurizados. portaria deten/ms n • , de de janeiro de surface modification of poly(styrene) by the attachment of an antimicrobial peptide review of antimicrobial food packaging in: standard test method for water vapor transmission rate through plastic film and sheeting using a modulated infrared sensor astm d in: standard test method for tensile properties of thin plastic sheeting astm e /e m in: standard test methods for water vapor transmission of materials biochemical and genetic characterization of enterocin a from enterococcus faecium, a new antilisterial bacteriocin in the pediocin family of bacteriocins engineering properties of polymeric-based antimicrobial films for food packaging: a review release kinetics of nisin from biodegradable poly(butylene adipate-co-terephthalate) films into water detergent-like actions of linear amphipathic cationic antimicrobial peptides optimized enzymatic synthesis of c-terminal peptide amides using subtilisin a from bacillus licheniformis recent applications of enzymatic peptide synthesis chemical synthesis of proteins a general approach to describe the antimicrobial agent release from highly swellable films intended for food packaging applications tilapia (oreochromis mossambicus) antimicrobial peptide, hepcidin - , shows antitumor activity in cancer cells a fish antimicrobial peptide, tilapia hepcidin th - , shows potent antitumor activity against human fibrosarcoma cells antimicrobial peptides (amp) with antiviral activity against fish nodavirus buforins: histone h a-derived antimicrobial peptides from toad stomach monitoring nisin desorption from a multi-layer polyethylene-based film coated with nisin-loaded hpmc film and diffusion in agarose gel by an immunoassay (elisa) method and a numerical modeling reference method for broth dilution antifungal susceptibility testing of yeast performance standards for antimicrobial disk susceptibility tests methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically bioactive packaging technologies for extended shelf life of meat-based products covalent immobilization of antimicrobial peptides (amps) onto biomaterial surfaces the mathematics of diffusion antibiotic magainins exert cytolytic activity against transformed cell lines through channel formation in vitro antiplasmodium effects of dermaseptin s derivatives hydrophobicity, hydrophobic moment and angle subtended by charged residues modulate antibacterial and haemolytic activity of amphipathic helical peptides antifungal peptides: novel therapeutic compounds against emerging pathogens antiviral effects of synthetic membrane-active peptides on herpes simplex virus, type the possible roles of food-derived bioactive peptides in reducing the risk of cardiovascular disease direct food substances affirmed as gras -electronic code of the bactericidal activity of pediocin pa- is specifically inhibited by a -mer fragment that spans the bacteriocin from the center toward the c terminus food standards agency. current eu approved additives and their e numbers defensins: antimicrobial peptides of innate immunity immunomodulatory peptides obtained by the enzymatic hydrolysis of whey proteins principles of mass transfer development of cellulose acetate-based antimicrobial food packaging materials for controlled release of lysozyme polymer surface modification for the attachment of bioactive compounds development and characterization of an antimicrobial packaging film coating containing nisin for inhibition of listeria monocytogenes antimicrobial plastic film: physico-chemical characterization and nisin desorption modeling innovative multilayer antimicrobial films made with nisaplin ® or nisin and cellulosic ethers: physico-chemical characterization, bioactivity and nisin desorption kinetics simulating diffusion model and determining diffusivity of potassium sorbate through plastics to develop antimicrobial packaging films the role of cationic antimicrobial peptides in innate host defences antimicrobial and host-defense peptides as new anti-infective therapeutic strategies antimicrobial activities of amphiphilic peptides covalently bonded to a water-insoluble resin a coating for use as an antimicrobial and antioxidative packaging material incorporating nisin and [alpha]-tocopherol studies on anticancer activities of antimicrobial peptides the antibacterial activity of magainin i immobilized onto mixed thiols self-assembled monolayers proteomics for studying cancer cells and the development of chemoresistance antifungal properties and mode of action of psacotheasin, a novel knottin-type peptide derived from psacothea hilaris antifungal activity of synthetic peptide derived from halocidin, antimicrobial peptide from the tunicate, halocynthia aurantium peptide antimicrobial agents chemical synthesis of peptides and proteins properties of nisin-incorporated polymer coatings as antimicrobial packaging materials bioactive peptides: production and functionality thermal, mechanical and water vapor barrier properties of sodium caseinate films containing antimicrobials and their inhibitory action on listeria monocytogenes antifungal mechanism of smap- ( - ) isolated from sheep myeloid mrna against trichosporon beigelii antitumor activity of the antimicrobial peptide magainin ii against bladder cancer cell lines virucidal activity of a scorpion venom peptide variant mucroporin-m against measles, sars-cov and influenza h n viruses recombinant production of antimicrobial peptides in escherichia coli: a review peptide-derivatized dendrimers inhibit human cytomegalovirus infection by blocking virus binding to cell surface heparan sulfate sínteses química e enzimática de peptídeos: princípios básicos e aplicações high-pressure processing and antimicrobial biodegradable packaging to control listeria monocytogenes during storage of cooked ham development and characterization of an active polyethylene film containing lactobacillus curvatus crl bacteriocins relationship of membrane curvature to the formation of pores by magainin antimicrobial peptides, isolated from horseshoe crab hemocytes, tachyplesin ii, and polyphemusins i and ii: chemical structures and biological activity antiviral activity of selected antimicrobial peptides against vaccinia virus mammalian cell toxicity and candidacidal mechanism of arg-or lys-containing trp-rich model antimicrobial peptides and their d-enantiomeric peptides purification and characterization of plantaricin a, a lactobacillus plantarum bacteriocin whose activity depends on the action of two peptides the antiviral activity of naturally occurring proteins and their peptide fragments after chemical modification peptide synthesis catalyzed by organic solvent-stable protease from pseudomonas aeruginosa pst- in monophasic aqueous-organic solvent systems tethering antimicrobial peptides: current status and potential challenges selective lysis of bacteria but not mammalian cells by diastereomers of melittin: structure-function study broth microdilution antibacterial assay of peptides purification and amino acid sequence of lactocin , a bacteriocin produced by lactobacillus casei crl antiviral properties of milk proteins and peptides ribosomally synthesized peptides with antimicrobial properties: biosynthesis, structure, function, and applications can we predict biological activity of antimicrobial peptides from their interactions with model phospholipid membranes mechanism of action of the antimicrobial peptide buforin ii: buforin ii kills microorganisms by penetrating the cell membrane and inhibiting cellular functions a novel antimicrobial peptide from bufo bufo gargarizans sublethal concentrations of pleurocidin-derived antimicrobial peptides inhibit macromolecular synthesis in escherichia coli development and evaluation of active packaging for sliced mozzarella preservation the relationship between peptide structure and antibacterial activity enhancing antimicrobial activity of chitosan films by incorporating garlic oil, potassium sorbate and nisin applications of antimicrobial peptides from fish and perspectives for the future antimicrobial peptides: premises and promises peptide purity and counter ion determination of bradykinin by high-performance liquid chromatography and capillary electrophoresis improvement of solubility and stability of the antimicrobial peptide nisin by protein engineering antimicrobial and antifungal activities of a novel cationic antimicrobial peptide, omiganan, in experimental skin colonisation models plantaricin a, a peptide pheromone produced by lactobacillus plantarum, permeabilizes the cell membrane of both normal and cancerous lymphocytes and neuronal cells antimicrobial efficiency of film incorporated with pediocin (alta ® ) on preservation of sliced ham antioxidative peptides from food proteins: a review development of bioactive food packaging materials using immobilised bacteriocins lacticin and nisaplin ® bioactive peptides: synthesis, properties, and applications health-related costs: from foodborne illness in the united states. produce safety project. available from: www.producesafetyproject.org. accessed cationic amphiphilic peptides with cancer-selective toxicity controlled diffusion of an antimicrobial peptide from a biopolymer film application of bacteriocins in vegetable food biopreservation synthesis and application of caged peptides and proteins physical and antimicrobial properties of grape seed extract, nisin, and edta incorporated soy protein edible films lipase-catalyzed synthesis of peptides containing d-amino acid: facts and artifacts recent patents on active packaging for food application active and intelligent packaging for milk and milk products membrane fluidity characteristics of human lung cancer heparan sulfate: anchor for viral intruders? immunomodulatory activity of small peptides covalent immobilization of an antimicrobial peptide on poly(ethylene) film responsive polymer films and capsules via layer-by-layer assembly nisin diffusion in protein films: effects of film type and temperature influence of amino acid substitutions in the nisin leader peptide on biosynthesis and secretion of nisin by lactococcus lactis antiviral activities of lactoferrin natural products with hypoglycemic, hypotensive, hypocholesterolemic, antiatherosclerotic and antithrombotic activities expression and purification of antimicrobial peptide buforin iib in escherichia coli lantibiotics: peptides of diverse structure and function mechanism of interaction of different classes of cationic antimicrobial peptides with planar bilayers and with the cytoplasmic membrane of escherichia coli barrel-stave model or toroidal model? a case study on melittin pores antimicrobial peptides of multicellular organisms interaction of the antimicrobial peptide pheromone plantaricin a with model membranes: implications for a novel mechanism of action the authors would like to thank to nicholas j. walker for providing language help and writing assistance. financial support for this research was provided by coordenação de aperfeiçoamento de pessoal de nível superior (capes) and the conselho nacional de desenvolvimento científico e tecnológico (cnpq). key: cord- -a vcapl authors: gottardi, cara j.; caplan, michael j. title: signals and mechanisms of sorting in epithelial polarity date: - - journal: nan doi: . /s - ( ) -x sha: doc_id: cord_uid: a vcapl this chapter discusses epithelial-membrane polarity, sorting pathways in polarized cells, and the sorting-signal paradigm. polarized epithelial cells have long captured the attention of cell biologists and cell physiologists. at the electron-microscopic level, one of the most apparent and fundamental features of this cell type is its polarized organization of intracellular organelles and its structurally and compositionally distinct lumenal (apical) and serosal (basolateral) plasma-membrane domains. the polarized epithelial phenotype is an absolute necessity for organ-system function. in the most general sense, these cells organize to form a continuous, single layer of cells, or epithelium, which serves as a semi-permeable barrier between apposing and biologically distinct compartments. within the tubules of the nephron, these cells orchestrate complex ion-transporting processes that ultimately control the overall fluid balance of the organism. at the surface of the gastrointestinal tract, specialized versions of this cell type control the digestion, absorption, and immuno-protection of the organism. polarized epithelial cells have long captured the attention of cell biologists and cell physiologists. this is largely because the architecture of these cells so tellingly bespeaks their function. at the electron microscopic level, one of the most apparent and fundamental features of this cell type is its polarized organization of intracellular organelles and its structually and compositionally distinct lumenal (apical) and serosal (basolateral) plasma membrane domains (figures a, b) . through the eyes of the physiologist, the polarized epithelial phenotype is an absolute necessity for organ system function. in the most general sense, these cells organize to form a continuous, single layer of cells, or epithelium, which serves as a semi-permeable barrier between apposing and biologically distinct compartments. within the tubules of the nephron, these cells orchestrate complex ion-transporting processes that ultimately control the overall fluid balance of the organism. at the surface of the gastrointestinal tract, specialized versions of this cell type control the digestion, absorption and immuno-protection of the organism. thus while polarized epithelial cells can carry out myriad functions, they share one defining feature: a structural polarity which serves their underlying functional polarity. the differential distribution of membrane proteins between the plasmalemmal surfaces of polarized epithelial cells enables these cells to both respond to and effect changes upon their environment in a directed fashion. the gastric parietal cell of the stomach, for example, contains a population of h,k-atpase-rich vesicles. upon stimulation, these vesicles fuse selectively with the lumenal membrane, resulting in the massive apical secretion of hcl which initiates digestion. without two important elements of the polarized phenotype, that is, junctional integrity and the precision of this membrane insertion, proton pumps might be delivered to a compartment which would be adversely affected by the secretion of acid. another illustration of the utility of the polarized phenotype is provided by the principal cells of the kidney, which carry out net sodium absorption through a mechanism which is entirely dependent upon the polarized distribution of two membrane proteins. sodium absorption is stimulated by the hormone aldosterone, which increases the amount or activity of na,k-atpase at the basolateral surface, while increasing the number or activity of apical sodium channels and thus the sodium conductance of the lumenal membrane (doucet and barlet-bas, ) . because the na,k-atpase generates low intracellular { na+}, sodium is these morphologically distinct apical and basolateral membrane domains are separated by a unique ultrastructure known as the tight junction (tj). this structure is just visible as an area of close, uniform membrane apposition located at the apices between adjacent epithelial cells. (photo courtesyof dr. marian neutra, children's hospital, boston, ma). able to pass from the lumen of the kidney tubule through apical sodium channels and into the cytoplasm down its electrochemical gradient. the na+ is then pumped across the basolateral membrane and into the interstitum by the sodium pump and is ultimately prevented from leaking back into the lumen by impermeable tight junctions. therefore, it is the differential assignment of na' channels to the apical surface and na,k-atpase molecules to the basolateral domain that ensures the vectoriality of this transport process. how the polarized cell assigns these two proteins (and apical and basolateral membrane proteins in general) to their respective surface domains has been the subject of much investigation and is the general focus of this review. it is perhaps important to point out that the fundamental questions of plasma membrane protein aniosotropy are not unique to surface membrane proteins or even to the study of epithelial polarity. the golgi apparatus, for example, is a polarized organelle whose cis-and trans-most cisternae are structurally and biochemically distinct. this organization is thought to enable the ordered addition and trimming of glycoprotein sugar residues as they traverse the stacked cisternae. as is clearly represented in the breadth of topics covered in this book, numerous cell types adopt a polarized state for some functional purpose. the propagation of a nervous impulse from dendrite to axon requires compositionally different membrane proteins in each of these domains, while the localization of determinants to specific parts of an egg's cytoplasm gives rise to cells with different growth potentials and the necessary assymetries required for embryo development . what we hope will become clear in this chapter and related chapters in this book is that we are beginning to appreciate the universality of polarity. the mechanisms involved in establishing and maintaining the polarized state appear to be so fundamental that some of the schemes through which a cell is able to localize a particular protein to a given cellular domain are turning out to be conserved between epithelia and neurons, and even between epithelia and yeast. while the need for protein asymmetries in development, or membrane polarity in epithelial transport is clear, the means through which it is achieved are only beginning to be elucidated. before we embark upon our review of the field, we first introduce the conceptual framework onto which the results in this field are organized and interpreted. first, a protein destined to accumulate with a polarized distribution needs to be recognized as different from other proteins. we presume that what is recognized is some structural aspect of the protein itself. we refer to that part of the protein that is recognized for polarized localization as a sorting signal or localization determinant. these two terms are often used interchangeably, but in fact there is a subtle difference between the two. "sorting signal" is often taken to imply a signal that is recognized and acted upon before the protein is delivered to its ultimate residence. sorting signals are thought to be those signals that enable a cohort of similar proteins with similar destinations to be sorted and sifted away from all of the other molecules traversing the biosynthetic pathway at the same time. a "localization determinant" is perhaps a more general term that carries fewer mechanistic implications. it is defined here as the determinant that specifies a protein's polarized distribution, but it does not make a distinction between recognition that takes place before the protein has reached its final destination or after (e.g., through a selective retention mechanism). the proteins which serve to recognize a particular signal and act upon it are generally referred to as sorting machinery. often, a distinction is made in the literature between "sorting" and "targeting machinery." in these cases, the sorting machinery is exclusively those elements which recognize the sorting signal. any downstream effectors of this sorter that orchestrate the vectorial directing of a vesicle to its final destination are referred to as targetting machinery. a simple schematic of these elements is presented in figure . as is discussed in the second half of this review, we know much more about general targeting machinery than the sorters themselves. one of many possible ways to think about how a secretory or membrane protein could be sorted into a vesicle. it is presumed that the "sorters" will recognize a sorting signal (" embedded within the protein structure. it seems likely that this recognition event would need to take place in the lumen of the golgi for a secretory protein, but this might not be necessary for a membrane protein, which could interact with a sorter from either a lumenal-or cytoplasmic-facing signal domain. ultimately, the sorted protein(s) could be contained within a "domain-specific vesicle," which would then be targetted (with the help of protein targetting machinery x, y, and z) to the appropriate apical or basolateral surface domain. it is thought that proteins destined for either the apical or basolateral domain of a polarized cell occupy the same golgi cisternae during their biosynthesis ( m a t h and simons, ; misek et al., ; rindler et al., ; fuller et al., ; pfeffer et al., ) . immunoelectron microscopic studies performed on nonpolarized endocrine cells which manifest two biochemically and kinetically different secretory pathways suggested that the process of sorting components away from one another takes place at the tgn (orci et al., ; tooze et al., ) . however, recent studies have demonstrated that sorting may not take place exclusively at the tgn. sorting mechanisms have been suggested to take effect as early along the biosynthetic pathway as the er (balch et al., ) as well as at the recycling endosome (matter et al., ; . in hepatocytes, sorting appears to occur after all newly synthesized membrane proteins are delivered to the basolateral plasmamembrane (bartles et al., ) . similar delivery routes have been detected in polarized intestinal epithelial cell lines (matter et al., ) . finally, in at least one subclone of the canine renal mdck cell line, sorting may take place both at the golgi as well as at the level of the plasma membrane. while most proteins in this cell line are sorted in the tgn, the na,k-atpase can be preferentially localized to the basolateral membrane through domain-specific stabilization mechanisms after random insertion into both plasmamembrane domains (hammerton et al., ; siemers et al., ) . apically and basolaterally sorted proteins have been shown to be packaged into distinct classes of golgi-derived vesicles which are ultimately targeted to their appropriate domains. recently it has been shown that membrane and secretory proteins are segregated into distinct vesicular carriers upon transport from the golgi to the basolateral surface of hepatocytes (saucan and palade, ) the extent to whch distinct basolateral (or apical) proteins are cosorted and incorporated within the same vesicle either due to common localization signals or the ability to co-aggregate has not yet been determined. after proteins are sorted, the targeting of a vesicle to a particular surface domain can occur directly (vectorially) from the tgn to the apical domain (matlin and simons, ; rindler et al., ; fuller et al., ) , basolateral domain (caplan et al., ) or indirectly as has been shown for the poly-immunoglobulin receptor (pigr) (mostov and deitcher, ) . in the latter case, the protein is first targeted to the basolateral surface where the receptor can bind its ligand and is then transported to the apical surface via a process known as transcytosis (reviewed in mostov and simister, ) . as noted above, in hepatocytes all apical proteins studied to date make use of this indirect pathway for apical delivery (bartles et al., ) , while cell lines derived from intestine and kidney can employ both routes for surface delivery (matter et al., ; casanovaet al., ; low et al., ) while the details of the routes have been determined for a number of sorting pathways, the molecular signals and recognition components which control each of them are not well understood. the search for these molecular signals and recognition components has been the focus of much study over the last years. during this period, the subjects of protein sorting and epithelial polarity have been extensively reviewed. several of these reviews are listed here for those seeking more background on specific aspects of this field: for general reviews on protein sorting pathways (burgess and kelly, ) ; general concepts of sorting and targeting (caplan and matlin, ); a discussion of the mechanisms required for the establishment and maintenance of epithelial polarity (rodriguez-boulan and nelson, ) ; polarized transport of surface porteins and lipids in epithelial cells ; comparative epithelial and neuronal polarity (rodriguez-boulan and powell, ) ; the generality of the polarized phenotype (nelson, ) ; cytoskeleton as a component of the protein sorting machinery (mays et al., ) ; summary of the few known sorting signals in polarized epithelial cells ; common signals involved in sorting from the tgn and endosomes . perhaps now more than ever before, it is becoming a rather daunting task to provide a synthesis of the observations relevant to the study of epithelial polarity. this is in part due to the fact that important insights into the mechanisms of sorting are being contributed by fields that are not exclusively focussed on epithelial biology. as we discussed in this review, some important contributions are emerging from studies of endocytosis, secretion in yeast and neurons, and the sorting of yeast lyso-soma enzymes (see chapter i of this volume), in addition to more "classical" approaches to epithelial polarity. in this review, we explore the current paradigm that the generation and maintainance of distinct membraneous compartments requires "sorting signals," the recognition domains embedded within the amino acid sequence or polypeptide structure of the protein, and "sorting machinery," the proteins which interpret and act upon these signals. in the first half, we review and categorize the signals that have begun to be elucidated, as well as discuss the approaches and difficulties associated with finding and interpreting sorting signals. while the polarity field itself has not yet succeeded in characterizing the definitive sorting machinery, numerous components of the membrane budding and fusion apparatus are rapidly being elucidated. we have chosen to review some of the important findings in the field of membrane transport, and in particular examine the potential roles that gtp-binding proteins of the rab, arf and heterotrimeric classes may play. we also discuss a class of proteins referred to as adaptins as well as the implications that the snare hypothesis may have for epithelial polarity. although these components have not been shown to be directly involved in sorting per se, it is becoming increasingly clear that in a general sense, the composition of the membrane vesicle budding and fusion machinery may be part of the overall apparatus which "acts upon" the sorted species and contributes to domain specific surface targeting. as stated above, the paradigm for conceptualizing the mechanisms responsible for biosynthetic sorting requires that each protein contains signal information embedded within its polypeptide sequence/structure (sorting signal) which is interpreted and acted upon by components referred to as sorting machinery. this scheme takes its cue from the process through which ribosomes translating secretory and membrane proteins are targeted to the endoplasmic reticulum to initiate cotranslational protein translocation (blobel, ) . prior to the elucidation of this process, it was suggested that protein targeting might require cellular sorting machinery to recognize certain signals which would be shared by proteins with common destinations (blobel, ) . shortly after this suggestion, it became clear that targeting to the rer, mitochondria and chloroplasts required short, contiguous, n-terminal signal peptides (reviewed in burgess and kelly, ) . in the case of the former, the signal was recognized by a receptor, srp (lingappa et al., ; von heijne, ; kurzchalia et al., ; walter and lingappa, ). subsequently, a number of short, contiguous amino acid domains have been shown to play a role in later stages of post-synthetic targeting. these include: ( ) the kdel and adenovirus e l signals which ensure the retention or recapture of resident er proteins (munro and pelham, ; nilsson et al., ); ( ) a transmembrane domain signal responsible for golgi retention (swift & machamer, ; machamer, ) ; ( ) the cluster of positively charged lysine residues (sv -nls) sufficient for nuclear targeting (richardson et al., ) ; ( ) the critical tyrosine/ "tight-turn'' structural motif which can mediate localization to clathrin coated-pits (goldstein et al., ; pearse and robinson, ; collawn et al., ) ; and ( ) the discovery that lysosomal hydrolases were targeted to lysosomes through the recognition of a phosphorylated sugar residue (mannose- -phosphate; reviewed by kornfeld and mellman, ) . in several of these cases receptors for these signals have been well-characterized: the signal recognition particle (srp) for secretory and membrane proteins (walter and lingappa, ) , the mannose- -phosphate receptor (m pr) for the targeting of lysosomal hydrolases to the lysosome (sly and fischer, ; vonfiguraandhasilik, ) , the kdelreceptor (tanget al., ) and the adaptins which couple coated pit localization sequences to clathrin cages (pearse and robinson, ; robinson, ) . the search for definitive signals which mediate the delivery of proteins to a particular epithelial surface domain has proven to be quite difficult. this is due in part to general limitations imposed by certain molecular biological approaches, as well as to some inherent difficulties specific to the investigation of epithelial polarity. our goal in this section is to outline reasonable criteria for the identification of a sorting signal. the observation that the influenza and vesicular stomatitis viruses bud from opposite surface domains of polarized mdck cells (madin darby canine kidney) (rodriguez-boulan and sabatini, ) spawned an extensive search in which chimeric and deletion analyses were applied to the problem of identifying the underlying apical and basolateral sorting signals (reviewed in caplan and matlin, ) . these efforts to characterize sorting signals have generally involved the generation of chimeric or truncated contructs prepared from portions of apical and basolateral membrane proteins. through analysis of the subcellular distributions of the resulting proteins, sorting information can, at least in theory, be assigned to particular portions of the parent molecules. while a large number of chimeric and truncated viral glycoproteins have been generated and analyzed, it has been difficult to interpret many of the resultant observations. with the benefit of hindsight, we now know that these difficulties can be attributed to a number issues that we discuss in more detail below (including the tertiary stuctures of the experimental constructs, the confounding possibilities introduced by uncharacterized default pathways, and the potential for multiple and hierarchical signals to be embodied within the structures of the studied proteins). until recently (thomas and roth, ) , the analysis of viral spike glycoproteins did not produce a definitive sorting signal. much of the uncertainty associated with this work is likely attributable to the fact that these studies engineered chimeras from portions of structurally dissimilar molecules. the tertiary structures of the resultant chimeras may thus differ substantially from those of either parent molecule, which may in turn exert unpredictable effects upon sorting behavior. clearly, if sorting signals are formed from domains arising from noncontiguous regions of a polypeptide, for example, in much the same manner that heterotrimeric g proteins are thought to "see" their effectors (berlot and bourne, ) , or in the way that the human growth hormone receptor (hghbp) is thought to interact with its ligand (cunningham and wells, ) , it is easy to imagine how the structural integrity of the putative sorting signal could become compromised in a chimeric construct. while producing a rough map of the signal-bearing domain of a protein can be relatively straight forward, determining the exact residues which constitute the signal is turning out to require a collaboration between many different types of mutagenesis approaches. often, contradicting results can arise from alanine scanning, truncation and point mutation/deletion mutagenesis, since a mutated protein can manifest impaired sorting behavior even though the altered residues are not part of the actual sorting signal (aroeti et al., ) . it is becoming clear that a judicious and thorough comparison of many different types of mutagenesis approaches may be necessary to determine definitively the key residues necessary for sorting. perhaps another difficulty in looking for apical or basolateral sorting signals is that the default pathway for "signal-less" membrane proteins is still not known. a protein that is sorted "by default" is, by definition, unable to interact with and be acted upon by any sorting machinery whatsoever. in theory, at least, such "unsorted'' proteins may be distributed with polarity, depending on the nature and characteristics of the membrane vesicular traffic arising from the golgi complex in a particular cell type. obviously, if the localization of a protein construct under study is identical to that produced by the cell's default pathway, elucidation of a signal will be difficult, since elimination of the signal will not alter the protein's distribution. thus, one can appreciate the difficulty in assigning localization information to a particular domain in the context of an undefined default pathway. this caveat accounts for at least some of the reasons which explain why a definitive basolateral sorting signal in the c-terminal domain of vsv-g protein took so long to discern. in the following example we summarize the ha-vsvg spike glycoprotein chimera literature as a means to illustrate the difficulties in interpretating these types of studies. when acdna encoding the influenza ha was expressed in mdck cells, the encoded protein localized to the apical membrane (roth et al., ) , while a cdna encoding the vsvg polypeptide produced a protein that is localized to the basolateral domain (gottlieb et al., b; stephens and compans, ) . when truncation mutants were expressed in which soluble ectodomain versions of these proteins were synthesized, the vsvg ectodomain was secreted from both apical and basolateral domains gonzalez et al., ) while the ha ectodomain was predominantly secreted from the apical domain (gonzalez, et al., ; roth et al., b) . based on evidence that the default pathway for secreted proteins leads to nonpolarized secretion from both surface domains (kondor-koch et al., ; gottlieb et al., a; caplan et al., ) , it was reasoned that the ectodomain of ha encodes an apical sorting signal while the vsvg ectodomain lacks signal information. this was further confirmed by the observation that a hybrid ha-vsvg protein comprising the ha ectodomain fused to the vsvg transmembrane and cytoplasmic tail region was targeted to the apical membrane (mcqueen et al., ; roth et al., a) . but if the vsvg ectodomain is randomly secreted and the vsvg tail domain fused to ha is apical, which domain of vsvg encodes basolateral sorting information? the complementary hybrid comprised of the ectodomain of vsvg (presumably signal-less) tethered to the ha transmembrane and tail region (perhaps also signal-less) was targeted either to the basolateral membrane or to both surface domains (mcqueen et al., ; puddington et a] ., ; roth et a]., a; compton et al., ) . the interpretation of the behavior of this chimera was clearly complicated; it was suggested that this protein could be pursuing its distribution by default. (as discussed above, the default pathway for membrane proteins is still not defined in polarized cells). an alternative interpretation was that the vsvg ectodomain indeed contains basolateral sorting information, but that perhaps this domain needs to be tethered to the plasmamembrane with a transmembrane anchor in order to interact with its presumptive sorting machinery. this interpretation, however, was proved incorrect by the observation that the anchoring of this ectodomain to the membrane through a lipid-linkage resulted in apical targeting (brown et al., ) . interestingly, when the ectodomain of the normally apical placental alkaline phosphatase (plap) was attached to the vsvg transmembrane and cytosolic tail domains (which were though to lack a dominant signal), the resulting chimeric protein was targeted basolaterally. it is difficult to reconcile the ha-vsvg and plap-vsvg chimeras without invoking hierarchical and competing signals. recently, a basolateral targeting signal has been precisely localized to the cytoplasmic domain of the vsvg protein (thomas and roth, ) . in light of the vicissitudes which attended the interpretation of each round of chimeric constructs discussed above, it was certainly unexpected that definitive sorting information would be localized to the cytoplasmic tail of vsvg. the nature and function of this signal will be discussed in depth below. the preceding discussion was presented simply to reinforce the caveat that the default pathway, protein structural considerations and the possible interactions between "dominant" and "recessive" sorting signals can considerably cloud the interpretation of chimera experiments. recent studies of the polymeric immunoglobulin receptor (pigr), the low density lipoprotein receptor (ldlr) and polytopic hetero-oligomeric proteins (h,k-atpase and na,k-atpase) suggest that individual proteins can interact in multiple and complex fashions with the machinery responsible for surface targeting. it is becoming increasingly clear that there can be an array of signals encoded within an individual protein, and the sorting problem is becoming evermore complicated by the apparent redundancy, multiplicity and hierarchical nature of these signals (matter et al., ; mostov et al., ) . for example, brewer and roth's ( ) demonstration that they could completely overwhelm the apical signal present in the ha ectodomain and redirect it to the basolateral surface by changing a single amino acid in this protein's cytoplasmic tail strongly suggests that multiple signals present in a single protein can interact in a heirarchical fashion. the newly created cytoplasmic signal is dominant over the presumed apical sorting signal present in the ectodomain of ha. as discussed below, the ldl receptor has been shown to encode redundant, basolateral sorting information, since either of two cytoplasmic determinants could independently mediate basolateral delivery (matter et a]., ) . moreover, the protein may also contain acryptic apical sorting signal in its ectodomain, since a cytoplasmic tail-minus construct of this protein (ct ) is sorted with great efficiency to the apical membrane in mdck cells (matter et al., ). an ectodomain apical localization signal has also been found within the pigr, whose initial surface delivery is to the basolateral plasmalemma. why do these proteins need multiple signals? what does the ldlr gain by expressing two basolateral localization signals? recent studies (discussed in greater detail in the following section) have more finely decoded these two signals and are revealing functional differences. for ex-ample, the "membrane proximal determinant" encodes coated-pit internalization information, while the "membrane distal determinant" appears to ensure efficient sorting from a basolateral endosome back to the basolateral surface (matter et al., ) . analysis of the sorting behavior of multisubunit ion pumps provides further insight into the possible utility of multiple signals (reviewed in the gastric h,k-atpase and the na,k-atpase are close cousins in the large family of p-type ion transporting atpases. both are composed of kda a-subunits and heavily glycosylated kda p-subunits. they share similar reaction mechanisms and catalytic properties and, not surprisingly, are highly homologous at the amino acid sequence level. the a-subunits are - % identical, whereas the p-polypeptides manifest roughly % identity. while the na,k-atpase is a basolateral protein in most polarized epithelial cell types (with the exception of neural epithelia such as choroid plexus and retinal pigment epithelium), the h,k-atpase occupies the apical membrane and a pre-apical storage compartment in gastric parietal cells. hormonal stimulation of gastric acid secretion induces fusion of the membrane vesicles which comprise the intracellular reservoir with the plasma membrane, resulting in delivery of the h,k-atpase to the apical cell surface. during the interdigestive period, the h,k-atpase is re-endocytosed and returned to its storage compartment. chimera studies reveal that each subunit of the h,k-atpase possesses a sorting signal which participates in regulating this complex traffic . the a-subunit is endowed with a dominant apical targeting signal, which can drive the apical sorting of chimeric pumps expressed in both mdck and llc-pkl renal epithelial cells. the p-subunit of the h,k-atpase possesses a tyrosine-based endocytosis signal (roush et al., manuscript submitted). this signal causes the protein to be sorted basolaterally when it is expressed in mdck cells and apically when it is expressed in llc-pk cells. the na,k-atpase p-subunit does not possess a similar sequence domain. it seems likely that the two h,k-atpase signals participate in distinct stages of pump sorting in the gastric parietal cells. the apical signal in the a-subunit probably mediates the sorting of the entire complex to the apical membrane or the pre-apical storage compartment, whereas the p-subunit signal is responsible for ensuring the re-internalization of the pump following the cessation of secretagogue stimulation (courtois-coutry et al., ) . it remains to be determined why the p-subunit's tyrosine-based signal is differentially interpreted by mdck and llc-pk cells. investigation of this phenomenon may shed light on the nature and function of the epithelial sorting machinery. this apparent trend towards a multiplicity of signals is not entirely surprising, since many proteins are required to perform highly sophisticated feats of membrane targeting during the course of their transits throughout the endomembranous networks of the cell. for example, the pigr receptor expressed in its native hepatocytes or by transfection in mdck cells travels first to the basolateral membrane to pick up ligand and is then transported to the apical surface domain. it appears that an apical sorting signal in this protein's ectodomain might be required for basolateral to apical transcytosis, while a basolateral signal in the cytoplasmic domain ensures the initial basolateral delivery. unlike proteins that are constitutively expressed at one surface domain, a number of distinct and individually acting signals are necessary to orchestrate the more complicated surface targeting events displayed by pigr receptor, and other molecules like it. obviously, the hierarchical (both temporal and spatial) regulation of each signal will be of utmost importance in ensuring that a protein follows a physiologically relevent trafficking pathway. recent evidence, for example, demonstrates that the pig receptor undergoes phosphorylation on a cytosolic serine residue around the time that it is delivered to the basolateral surface (larkin et al., ) . this phosphorylation event appears to inactivate the protein's basolateral signal and thus permit its transcytosis to the apical membrane (casanova et al., ). perhaps not surprisingly, the greatest advances in the elucidation of sorting signals have been made with single membrane-spanning monomeric or homooligomeric proteins (e.g., pigr, ldl-r, tfr). with these molecules the requirements for surface expression are easily met and the effects of mutagenesis on tertiary structure can be assessed through well-characterized functional assays, such as receptor-ligand or antibody binding. through deletion analysis and heterologous expression in mdck cells, it was determined that the pigr (casanova et al., ) and the ldlr (hunziker et al., ) each contained basolateral targeting determinants which mapped to short, contiguous regions of their cytoplasmic domains (table ). both signals could be grafted onto heterologous proteins and cause them to be targeted to the basolateral surface, supporting the idea that each determinant was truly an autonomous basolateral sorting signal. exhaustive mutagenesis studies have more finely mapped each of these determinants. the ldlr possesses two distinct basolateral targeting determinants, one that is "coated-pit related" (proximal determinant) and another which is tyrosine-dependent but not capable of mediating localization into coated-pits (distal determinant) (matter et al., ; . interestingly, the polymeric immunoglobulin receptor (pigr) signal may constitute yet another class of basolateral targetting determinant, since it shares little in the way of sequence homology with either determinant of the ldlr and shows weak tyrosine dependence (aroeti et al., ) . the general characteristics of these three determinants and the degree to which they are related are only beginning to be eluciated thomas and roth, ). an attempt to categorize these basolateral sorting determinants has been made by and is summarized in table . before discussing the nature of the "coated-pit related" basolateral targeting determinant, it is necessary to be familiar with the signals that are known to mediate the accumulation of plasma membrane receptors into clathrin-coated pits (goldstein et al., ) . it is now generally accepted that tyrosine-and dileucine- containing sequence motifs present in the cytoplasmic tails of a number of coated-pit clustering proteins serve as the critical recognition elements for the adaptor components of clathrin coats (pearse and robinson, ; trowbridge, ) . more recently, numerous studies have demonstrated a strong relationship between the signals which mediate localization into coated pits and a subset of those involved in basolateral targeting (brewer and roth, ; hunziker et al., ; lebivic et al., ) . for example, brewer and roth ( ) found that the apically targeted ha molecule could be completely rerouted to the basolateral membrane by replacing a strategically localized cysteine residue (cys ) with a tyrosine in the cytoplasmic domain. this tyrosine was also sufficient to localize this protein into coated-pits and direct the protein's incorporation into endosomes. this observation that an endocytosis signal might also double as a basolateral targeting signal led to the suggestion that the recognition determinants for endocytosis and for tgn-to-basolateral targeting might be similar or identical to one another. thorough mutagenesis studies on the coated-pit localization and basolateral sorting determinants of ha-y (thomas and roth, ; lin et al., ) , vsvg protein (thomas et al., ) , and the ldlr , however, have led to a revision of this initial interpretation. it turns out that the "endocytosis signal" of both the ha-ys and the ldlr (proximal signal) can be resolved into two overlapping but distinct signal components. in other words, there is information recognized for endocytosis that is distinct from that recognized for basolateral sorting, even though the sequences are in part superimposed and share marked similarity. table shows the systematic mutagenesis that ultimately unraveled this relationship. brewer and roth ( ) found that ha-y is capable of both basolateral sorting and endocytosis. the second generation mutant ha-y ,rs , however, behaved as aprotein that was capable of endocytosis, but whose basolateral localization was inhibited (lin et al., ) . similar results were found with the ldlr proximal determinant. matter and colleagues ( ) showed that the truncation mutant ct was basolaterally targetted and rapidly endocytosed, while the removal of terminal acidic residues in ct pro- duced a protein that was not capable of basolateral targetting, but could nonetheless be endocytosed. thus, the initial correlation between endocytosis signals and basolateral targeting has now resolved into two distinct but overlapping signals that can share common residues for their respective activities. the implications of this result are very exciting for the field of epithelial polarity. first, they suggest that the signals for basolateral sortingkargetting may be structurally similar to signals for clathrin-coated pit localization and endocytosis. the involvement of similar signals suggests that the sortinghecognition molecules themselves may be related. at least for endocytosis signals, there is evidence in favor of clathrin "adaptins" (of the ap plasma membrane class) playing a role in recognizing these sequences (pearse, ; glickman et al., ; beltzer and spiess, ; sorkin and carpenter, ; sosa et al., ) . in light of the recent characterization of adaptin related molecules (cops, discussed in section , below), it has been suggested that a family of structurally and functionally similar sorting adaptors may serve as the sorting machinery which interacts with these basolateral sorting signals . the findings support the more general contention that sorting at the level of the tgn may be mechanistically similar to that at the level of the endosome (matter et al., . taken altogether, there now appear to be two general classes of basolateral targeting determinants. one of these is biochemically related to the signals that mediate sorting into coated pits. this type of signal can be colinear with an endocytosis determinant and may share the critical tyrosine residue required for the activity of both, but it is nonetheless distinct and dissociable from an endocytosis signal. the second class of basolateral targeting determinants appears to be unrelated to clathrin-coated pit localization signals, although it may also strongly depend on a tyrosine for activity. this second type of determinant appears to be unique to the ldlr, pigr (casanovaet al., ) and the tw (dargement et al., ) , although these signals share no primary sequence homology with one another. it is possible however, that this second determinant present in these three proteins may be mutually similar in three-dimensional structure but not in primary sequence. in this context it is important to note that adaptor proteins are thought to recognize tyrosine residues in the context of a tight turn, which can be achieved by many different primary sequences (glickman et al., ; collawn et al., collawn et al., , bansal and gierasch, ) . more detailed analyses are revealing that while the dependency on tyrosine is crucial, other residues which are acidic and c-terminal to the tyrosine are also important. demonstrated that the clusters of two or more acidic amino acids downstream from a tyrosine, phenylalanine or di-leucine are important for signal function (see table ). while the authors of this study have argued that it is premature to propose a common motif characteristic of all basolateral targeting determinants, they have found that this critical aromatic amino acid followed by acidic residues can be discerned in the cytoplasmic domains of many known proteins which are targeted to the basolateral membrane of mdck cells, including ecadherin, transferrin receptor, cation-independent and dependent mannose- -phosphate receptors, lap, pigr and fcriib . (see discussion of . as these authors have suggested, it will be exciting to define mutations that will prevent the recognition of these sequences so that the identification and characterization of the molecules which serve to interact with and interpret these signals can be facilitated. an ever growing list of proteins are anchored to membranes through a covalent attachment to glycosylphosphatidylinositol or gpi. proteins of this class are initially synthesized on bound polysomes as transmembrane polypeptides and, while still resident within the er, are cleaved from their transmembrane portions and transferred covalently to lumenally facing glycosyl-phosphatidylinositol molecules (cross, ) . gpi-anchored proteins are widely distributed with respect to both cell type and function. members of this class of proteins include protozoal surface coat proteins (e.g, the variant surface glycoproteins of trypanosomes), differentiation antigens (e.g., thy-i ) , adhesion molecules (e.g., the gpi-linked isoform of n-cam), hydrolases (e.g., alkaline phosphatase and snucleotidase), and receptors (folate receptor). the functional advantages that this membrane linkage confers upon a particular protein is presently unclear, and has been the focus of a great deal of attention (reviewed in brown, ) . in general, the gpi-linkage has been suggested to be important for enabling proteins to "c uster"at a surface density much higher than is possible for single-pass transmembrane proteins (hooper, ) . studies have also shown that these clusters of gpi-anchored proteins may be important for certain cell surface signal transducing events (reviewed in anderson, ) . gpi-linked proteins captured the attention of epithelial biologists because of their polarized distribution in mdck cells (lisanti et al., ) and other cultured epithelial cell lines (lisanti et al., ) . the nearly exclusive correlation of membrane anchoring via gpi with apical localization raised the question as to whether or not the gpi membrane anchor was itself a signal for apical targeting. chimeric analyses showed clearly that the gpi-linkage is sufficient for apical targeting in mdck cells (brown et al., ; lisanti et al., a,b) . of course, in the absence of a known default pathway for membrane proteins, it remains formally possible that the gpi-anchor prevents a protein from gaining entry into the basolateral sorting pathway. moreover, the fact that the cytoplasmic tail-minus versions of the ldl and pig receptors are directly targetted to the apical membrane is consistent with the possibility that apical sorting occurs by default (discussed in . nonetheless, the gpi-linkage is the field's best accepted apical localization signal characterized to date. interestingly, glycosphingolipids (gsls) share the apical preference of gpi-linked proteins and are generally found exclusively in the outer leaflets of the apical membranes of mdck cells. the means through which gpi-anchored proteins and glycosphingolipids (gsls) are sorted and subsequently targetted to the apical membrane are poorly un-derstood. it has been shown that gsls manifest biophysical properties which enable them to self-associate or form clusters in the plane of the membrane (thompson and tillack, ) . these properties have been invoked to support the proposal that gsl clustering occurs at the level of the tgn, and that newly synthesized gpi-linked proteins might co-cluster with these lipids (simons and van meer, ) . it has been further suggested that apically-destined transmembrane proteins could similarly be sorted through an ability to co-cluster with gsls and gpi-linked proteins . according to this model, apical sorting could take place through selective inclusion within these gsl microdomains, while certain basolateral membrane protein components would be sorted by selective exclusion. however, it should be pointed out that there is still no experimental evidence showing that the gsl clusters are important for apical sorting. one cell line in particular suggests that the role of gsls in sorting of gpi-anchored proteins may be more complex. a rat thyroid epithelial cell line (frt) distributes its gsls and gpi-anchored proteins to the basolateral surface while the polarized distribution of a number of transmembrane proteins is identical to that of mdckcells (zurzolo et al., ) . this suggests that at least some of the apical proteins analyzed (e.g., ha) do not partition with basolaterally directed gsls. the frt cell line will serve as an excellent tool for furthering our understanding about the role of glycolipid clustering in the sorting of proteins and lipids in polarized epithelial cells. most of the early studies in epithelial polarity used the kidney-derived mdck cell line as their workbench. however, the last six years has seen the introduction of a number of new cell culture models into the field: caco (pinto et al., ; matter et al., ; costa de beauregard et al., ) ; ht- and t- (human intestinal epithelial), (madara et al., ; polak-charcon et al., ; mikogami et al., ) ; llc-pk (pig kidney proximal tubule) (hull et al., ; gstrauthaler et al., ; gottardi et al., ) ; mdbk (madin-darby bovine kidney) (furuse et al., ) , frt (fischer rat thyroid) (zurzolo et al., ) , as well as primary cultures of choroid plexus and retinal pigmented epithelium (marrs et al., ) . as we have discussed in the first half of this review, we arejust beginning to elucidate the nature of certain "apical" and "basolateral" sorting signals. however, the "nonstandard" sorting of gpi-link proteins in frt cells mentioned above, and the fact that a number of proteins display tissue and cell-type specific membrane localizations (see table ), calls into question the ways in which we think about polarized sorting signals and the mechanisms of sorting. as shown in table , there are notable differences in the localization of certain membrane proteins expressed in different tissue cell-types. the na,k-atpase, nearly ubiquitously expressed at the basolateral domain of most polarized cell types, is localized to the lumenal (apical) domain of both retinal pigmented epithelial and chorid plexus cells (wright, ; steinberg and miller, ; spector and shiel and caplan, a,b; (m) schwartz et al., ; (n) brown et al., . johanson, gundersen et al., ) . when the cdna encoding the ldl receptor was placed under the control of a metallothionein promoter and employed in the generation of a transgenic mouse, the receptor was expressed at the basolateral domains of liver and intestinal epithelial cells, but unexpectedly localized to the apical domains of proximal kidney tubule cells (pathak et al., ) . the polarized budding of certain viruses and the localization of their respective spike glycoproteins was shown to vary considerably between kidney derived mdck and thyroidderived frt cells (zurzolo et al., a) . in some instances, ashift in the type of targeting pathway used by a protein can depend on the differentiated state of the cell culture (zurzolo et al., b) . furthermore, the polarized localization of a particular gpi-linked protein was found to be developmentally regulated in drosophila embryos (shiel and caplan, a) . finally, a remarkable flexibility and "plastic-ity" of protein sorting has been suggested to be present in kidney intercalated cells, which appear to direct the vacuolar proton pump to either surface domain, depending on particular environmental cues (schwartz et al., ; brown et al., a) at the present time, we have little understanding of the signals or sorting mechanisms that mediate the differential sorting of the same protein in distinct cellular types. are different signals recognized by the different epithelial cells or is the same signal interpreted differently? is the sorting machinery itself different between polarized cells, or is the sorting machinery basically conserved between different cell-types while its regulation, adaptation, or wiring to the targeting machinery is different? evidence discussed in the second half of this review on the rab family of proteins suggests that elements of the targeting machinery are in fact highly conserved between different cell types, and it is the cell-type specific adaptation of this machinery which accounts for differences. nonetheless, it is becoming clear that the sorting of a particular protein can be a highly idiosyncratic feature of each polarized cell model. the observation that different epithelial cell lines may handle the same protein (or the same signal) differently has to reflect more than a mere capriciousness of epithelial cells in culture. each of the cultured cell models employed in polarity studies derive from and reflect some of the differentiated features of a tissue or organ system. accordingly, the sorting behavior observed in a particular cell type needs to be evaluated in the context of this cell's functional history. for example, is this cell derived from a tissue specialized for apical secretion or apical endocytosis? studies of the sorting of ion-transporting atpase molecules expressed in distal tubule-derived mdck and proximal tubule derived-llc-pk kidney cells suggest that the distinct cell surface distributions which an atpase subunit achieve in these two lines are consistent with established physiologic differences between the distal and proximal tubule epithelial cells (roush et al., manuscript submitted). these observations have led to the suggestion that sorting mediates delivery to functionally defined rather than topographically defined domains (gottardi and caplan, a) . it is becoming quite clear that the findings in the field of intracellular protein transport (reviewed by rothman, and by mellman, ) will prove to be extremely valuable to the discipline of epithelial polarity. in this field, the convergence of studies on synaptic vesicle (regulated) secretion in neurons, constitutive secretion in yeast, and intra-golgi transport have led to the rapid identifcation and characterization of the basic components necessary for vesicle formation, docking and fusion. clearly, the general components of the bud-ding and docking machinery lie at the heart of any transport process, whether we are considering the transport of a membrane protein from er to golgi, or a secretory protein from the tgn to a particular cell surface. in the following sections we touch upon some of the key discoveries in the field of intracellular transport and focus on the relevant molecules that may contribute to polarized sorting and delivery processes. one of the recent paradigms in intracellularprotein transport is based on the concept that vesicle shuttling between different organellar compartments is regulated through the coordinated efforts of different gtp-binding proteins. there are two broad classes of gtp-binding proteins which have been shown to regulate membrane trafficking events; the small g proteins (rabs and arf) reviewed by (donaldson and klausner, ; pfeffer, pfeffer, , and the trimeric g proteins (reviewed by bomsel and mostov, ) . the role of a gtp-binding protein in regulating vesicular transport was first realized with the analysis of one of the temperature sensitive sec (secretory) mutants in yeast (salminen and novick, ) . sec mutants display a rather striking accumulation of secretory vesicles when cultured at the restrictive temperature. the cloning, sequencing, and characterization of the sec gene revealed that it encoded a ras-like or 'small' gtp-binding protein which was present on the surfaces of the vesicles and could bind and hydrolyze gtp (salminen and novick, ; goud et al., ; kabcenell et al., ) . since the phenotype of cells bearing mutant sec is the accumulation of transport vesicles, it was apparent that sec is necessary for the targeting and/or fusion of secretory vesicles with the plasma membrane. similar results were found with another yeast protein yptl( % identical to sec ), which in its mutant form inhibited vesicular transport between the er and golgi complex (gallwitz et al., ; segev et al., ) . the suggestion that two small gtpases were important in the regulation of two different vesicular transport events in yeast led to the hypothesis that each step in vesicular traffic was regulated by a specific gtpase (bourne et al., ) . these ras-like gtpases are known to zdopt either of two distinct conformations, depending upon whether or not they are complexed with gtp or gdp. consequently, these gtpases have been postulated to serve as key regulators or "molecular switches" for membrane fission and fusion events. the apparent generality of ras-like gtpase in yeast, as revealed by sec and yptl, inspired asearch for these proteins' mammalian counterparts. to date, yptl/seccrelated proteins have been identified and are often referred to as rab proteins ("ras-like" proteins from rat brain) reviewed in (balch, ; hall, ; goud and mccaffrey, ; zerial and stenmark, ) . a number of rabs have been localized to specific organelles within the cell and through the combined efforts of in vitro and in vivo approaches have been shown to regulate membrane traffic between these organelles (reviewed in zerial and stenmark, ) . how this class of molecules contributes to the overall fidelity of membrane trafficking events is still unclear (rothman, ) . the idea that specific rab proteins regulate distinct steps along the transport pathway (e.g., rabl always regulates er to golgi traffic, whether in a kidney cell or neuron) led to the hypothesis that cells which contain unique, cell-type specific transport processes might be regulated by disinct rabs. indeed, the best example of this is the family of rab isofoms which have been found to be localized within cells which are well-adapted for regulated secretory events. rab a has been suggested to be important in the regulation of caz+ dependent secretion in neuronal (fischer von mollard et al., ) , neuroendocrine (darchen et al., ) and endocrine cell types (mizoguchi et al., ) . interestingly, an isoform of rab a, rab d, has been localized to the glucose transporter-containing vesicles of adipocytes, which are known to undergo regulated exocytosis after insulin stimulation (baldini et al., ) . thus, despite cell-specific differences, or vesicle-content differences, these regulated pathways rely on similar rabs (rab ). thus, distinct regulated exocytic events in a variety of cell types make use of similar molecular machinery (lutcke et al., ) . in this context, it has been speculated that polarized epithelial cells, with their distinct apical and basolateral targeting pathways, may employ epithelia-specific rab molecules. recent data suggest that this may be true. there are four rabs which have been implicated in polarized epithelial-specific functions: rab , rab b, rabl , and rab . of the four, only rabl is truly specific to polarized epitheiial cells. in the developing kidney, rabl mrna is detected only after mesenchyme is induced to differentiate into polarized epithelial structures (lutcke et al., ) . interestingly, rabl induction was shown to occur just prior to the appearance of apical markers and has therefore been suggested to be involved in the generation of apicalhasolateral polarity in these cells. rab localizes to the basolateral membrane and to electron dense tubules near the apical membrane. since rab proteins have been shown to regulate transport between the subcellular compartments with which they associate, it has been suggested that rabl regulates epithelial transcytosis. as we stated previously, two isoforms of rab ( a and d) have been implicated in the regulated exocytosis events shared by neuronal, endocrine and adipocyte cell types. interestingly, another isoform of rab , b, has been shown to be specific for polarized epithelial cells and is exclusively localized to the apical pole of cells, near the tight junctions (weber et al., ) . rabl , like rab b, also accumulates at the apical poles of polarized cells and co-localizes with the tight junction associated protein, zo- (zahraoui et al., ) . it has been suggested that these two rabs could regulate events necessary for the establishment of polarity. for example, since the localization of both rabs are completely dependent on the presence the of cell-cell contacts, it is possible that these mole-cules control the recruitment of membrane protein-containing vesicles required for establishing the tight junction "fence," a structure thought to maintain the distinct protein and lipid compositions of apical and basolateral membranes (dragsten et al., ) . it has also been proposed that these rabs control general vesicle targetting to the apical membrane (zahraoui et al., ) . this hypothesis was based on two independent observations. it has been shown that an apical membrane protein (aminopeptidase) inserts preferentially into the apical membrane at regions of cell-cell contact in mdck cells (louvard, ) . furthermore, under conditions in which mdck cells are denied intercellular contacts, apical proteins appear to be sorted and retained within a large subapical vacuolar compartment (vacuolar apical compartment, or vac) which, after initiation of cell-cell contact, is inserted preferentially at regions of cell-cell contact (vega-salas et al., ) . taken together, the localization of r a b l and rab b at this region of cell contact places these monomeric gtpases in a position to regulate the delivery of apical proteins to the cell surface (zahraoui et al., ) . moreover, the localization of a regulated, exocytic compartmentspecific rab (rab ) to a subdomain of the apical membrane of polarized cells is intriguing and suggests possible functional relationships between these subcellular compartments. the last rab worth exploring in the context of epithelial polarity is rab . while rab is not solely expressed in polarized cells, it is the only rab that has been functionally implicated in vectorial targeting. a peptide derived from the c-terminal region of rab can inhibit basolateral but not apical transport of membrane proteins in a permeabilized-mdck cell assay (huber et al., a) . interestingly, rab can also regulate membrane transport to the dendritic plasma membranes of neurons in culture; antisense rab oligonucleotides decrease the level of viral glycoprotein transported to this domain (huber et al., b) . this observation is consistent with the model which suggests that the mechanisms which produce axoddendrite polarity in neurons may be similar to those involved in apicallbasolateral polarity in epithelia (simons et al., ) . taken together, the identification of a polarized epithelia-specific rab (rab ), and the localization of other rabs to specific polarized epithelial domains (rab and rab b, apical; rab , basolateral) suggests that rabs may regulate specific pathways in polarized epithelial cells. for the epithelial cell biologist, the obvious question is, "what brings about the pathway-specific localizations of rab proteins in polarized epithelial cells?" it has been demonstrated that the carboxy-terminal regions of rab proteins are responsible for their unique cellular localizations (chavrier et al., ) . it has been suggested that organelle-specific receptors exist which recognize the c-terminal domains of these molecules. at least in terms of polarized cells, it would be tempting to speculate that identification of such receptors for rabl , b and rab will bring us one step closer to an understanding of the overall machinery that orchestrates domain-specific vesicle formation and targeting. recent evidence, however, suggests that rabs may not provide the primary level of specificity in membrane targeting events (brennwald and novick, ; reviewed by rothman and warren, ) . as we discuss below, a new class of proteins, the snares, may provide the necessary specificity for vesicle-membrane targeting events throughout the cell. the snare hypothesis for vesicle targeting arose from research in three related fields: synaptic vesicle release in neurons, transport between cisternae of the golgi, and secretion in yeast. briefly, a number of synaptic proteins were discovered to be important for the regulated fusion of synaptic vesicles with their targets on the pre-synaptic plasma membranes (reviewed by pevsner and scheller, ) . homologues of these proteins were found in yeast and shown to be required for constitutive vesicle transport (aalto et al., ) . at the same time, key elements of the general machinery for intracellular membrane fusion were being elucidated. in all three .cases, membrane fusion requires an nem-sensitive factor (nsf), adaptors that link nsf to membrane proteins (snaps: soluble nsf attachment proteins) and the membrane receptors for the nsf-snap complexes (snares: snap receptors) (reviewed in rothman and warren, ) . distinct snare proteins are present in the membranes of the vesicle and the target. the snare hypothesis stipulates that each transport vesicle is endowed with its own vesicle-(v-) snare (or vamp-like molecule) that can specifically interact with its cognate target-( t -) snare (or syntaxin/snap -like protein). this 'pairing' could ensure vesicleharget membrane specificity, while a general fusion apparatus consisting of nsf and snaps could be used throught the cell (sollner et al., ) . in the context of epithelial polarity, this hypothesis suggests that vectorial targeting of apical and basolateral proteins will require distinct v-snares. interesting recent data suggest that the situation in at least one epithelial cell type may be somewhat more complicated. when the surface delivery of membrane proteins is examined in mdck cells permeabilized at their apical or basolateral surfaces with streptolysin , it appears that basolateral transport involves all of the machinery discussed above. toxins which cleave snares inhibit basolateral delivery, as do antibodies directed against snaps. in contrast, apical protein insertion is unaffected by these reagents. isolation of apically-bound vesicles from mdck cells reveals the presence of high concentrations of an adducin homologue in their surface membranes. adducins are calcium-dependent phospholipid binding proteins thought to be involved in a number of membrane fusion events (ilkonen et al., ) . it would appear, therefore, that completely distinct classes of vesicular targeting and fusion machinery may operate in the two membrane delivery pathways present in polarized epithelial cells. in the absence of a readily available genetic system with which to identify the genes and gene products necessary for such higher eukarotic functions as transcytosis or polarized targeting, epithelial cell biologists have been resigned to the prospect of "poking" at the epithelial cell with various reagents and watching how it responds. reagents which prevent the polymerization of actin (gottlieb et al., ; jackmon et al., ) and tubulin (achler et al., ; parczyk et al., ) , toxins which modify a particular class of g proteins (stow et al., ; pimplikar and simons, b) , or toxins that inactivate the vamp, syntaxin and snap- molecules described above, second messanger stimulators, analogues of the messangers themselves (apodaca et al., ; cardone et al., ; hansen and casanova, ) and the remarkable fungal metabolite brefeldin a (bfa) are all being incorporated into the repetoire of tools which we hope will enable us to gleen more information from a particular transport pathway. those interested in polarized and nonpolarized cell functions alike have made use of such cell-perturbing reagents. since the focus of this review is epithelial polarity, we have chosen to summarize some of the studies which are providing insights about the mechanisms of polarized sorting and targeting. brefeldin a is a fungal metabolite that endeared itself to cell biologists because of its dramatic effect on the protein secretory pathway (reviewed in . protein secretion is inhibited by bfa: membrane trafficking out of the er is blocked and the golgi appears to breakdown and become redistributed into the er (lippincott-schwartz et a]., ). before golgi redistribution, bfa causes this organelle to form tubular extensions which are devoid of any cytoplasmic (nonclathrin) "coat" material (lippincott- schwartz et al., ) . it has been shown that these morphological changes are not restricted to the golgi but rather are observed in a number of organelles of the endomembranous network such as endosomes, lysosomes and the tgn (hunziker et al., ; lippincott-schwartz et al., ; wood et al., ) , suggesting that the bfa "effector" might play a role in membrane transport events all over the cell. perhaps surprisingly, while membrane transport phenomena are remarkably altered in the presence of bfa, several processes are clearly unaffected, including receptor mediated endocytosis and endocytic recycling . from the standpoint of sorting and polarized delivery, bfa's most interesting property is its ability to differentially affect polarized cell surface targeting events. for example, low and colleagues ( i , ) determined a concentration of bfa where er-golgi trafficking was not inhibited, so that delivery from the tgn to the surface could be assayed for bfa sensitivity. interestingly, bfa inhibited the apical delivery of both endogenous, mdck secretory proteins ( ) and the membrane protein dppiv ( ) while also enhancing their mis-delivery to the basolateral surface. basolateral targeting of the endogenous mdck protein, uvomorulin, was not affected under these conditions. taken together, it would seem that a target molecule for bfa action exists that is exclusively involved in directing apical vesicles or which is simply more sensitive to the effects of bfa than similar molecules participating in the basolateral pathway. either way, these results provide a hint that there are indeed molecular differences between these two targeting pathways. it is important to add that in addition to inhibiting the exocytic apical pathway in mdck cells, basolateral to apical transcytosis is also inhibited by this drug (hunziker et al., ; low et al., ) . these findings have led to the suggestion that sorting mechanisms for apically destined proteins, whether along the exocytic or the transcytotic pathway may be functionally and biochemically similar (hunziker et al., ) . the fact that the loss of the structural integrity of the golgi induced by bfa correlates with a striking absence of its characteristic "coat" (observed at the em level) led to the idea that coat proteins might be rendered non-functional due to bfa action. through a number of studies (reviewed by donaldson et al., ; helms and rothman, ; rothman & orci, ) molecules which make up this "coat" were identified and characterized (e.g., pcop and a m ) . an "order of events" necessary for vesicle budding emerged from these studies and is outlined below. arf is a gtp-binding protein loosely related to ras and distinct from the family of rabs. in its gtp-bound state, it is capable of associating with the membrane by virtue of its myristoyl group, while its gdp-bound form is soluble and not membrane bound. arf binding to membranes appears to be the signal for coatomer binding, that is, the binding of pcop in addition to other as yet uncharacterized coat proteins. coatomer binding is believed to be absolutely necessary for vesicle budding. therefore proper coatomer assembly would be required for any event downstream of budding, such as targeting. recently, it has been determined that bfa inhibits coatomer assembly and vesicle formation through arf, by essentially allowing it to remain in its gdp-bound or inactive form. there exists a class of proteins which are able to catalyze the exchange of gdp for gtp called guanine nucleotide exchange factors (gne). bfa has been proposed to antagonize the action of a gne on arf, thus preventing coatomer assembly and membrane budding helms and rothman, ) . with the recent identification of an ever-growing family of new arf-related genes (kahn et al., ) and the speculation that different cops may exist in the control of membrane budding events from different organelles , there is growing excitement that arfs and cops will turn-out to be essential components for regulating a particular level of specificity inherent to membrane targetting events. in the context of bfas affect on apical sorting and targeting in polarized mdck cells (low et al., (low et al., , , it is likely that distinct arfkoatomer complexes regulate the budding of apical and basolaterallydestined vesicles from the tgn. moreover, the fact that significant missorting into the basolateral pathway was observed in the presence of bfa (low et al., ) suggests that coatomer assembly may be inextricably linked to proper secretory and membrane protein sorting. it has been known for some time that members of the heterotrimeric family of g proteins are associated not only with the plasma membrane but also with intracellular membranes (reviewed by bomsel and mostov, ) . a number of toxins (cholera, pertussis and mastoparan) known to activate or inhibit various classes of g proteins have been applied to studies of polarized sorting and targeting. stow et al. ( ) found that overexpression of gai- in polarized llc-pk cells significantly reduced the level of constitutive basolateral secretion of an extracellular matrix component, heparan sulfate proteoglycan. pertussis toxin, which adp-ribosylates and inactivates the a-subunits of the g a i/o class of heterotrimeric g proteins, relieved this inhibition. similarly, pimplikar and simons ( ) suggested that gi and gs may differentially regulate the trafficking of apical and basolateral vesicles in slo-permeabilized mdck cells, while leyte et al. ( ) found that gi/o and gs associated with the tgn could oppositely regulate constitutive secretory vesicle formation. it should be noted that in no case did the g protein related inhibition or stimulation appear to affect the actual sorting or missorting of apical or basolaterally destined proteins (in contrast to the bfa results discussed above (low et al., ) , but rather may only affect the rate or "efficiency" of sortingkargetting . a possible link between heterotrimeric g proteins and coatomer formatiodvesicle budding was provided by ktistakis et al., ( ) . this group found that activation of a g a protein with mastoparan promoted pcop binding and prevented bfainduced effects. pretreatment of cells with pertussis toxin, which is known to specifically affect g a i subclass of heterotrimerics, prevented mastoparan's antagonizing effects on bfa. stated more simply, these results showed that activation of a pertussis-toxin-sensitive ga promotes the binding of pcop to golgi membranes and thus antagonizes the action of bfa. the authors of this study suggest further that different subclasses or isoforms of ga could be responsible for some of the differences in bfa-sensitivities observed between cell types and organellar membranes. these key observations have led to the idea that heterotrimeric g proteins, by virtue of their membrane topology would be ideal candidates for coordinating the transfer of sorting information to the cytoplasmic surface of the tgn necessary for vesicle budding (bomsel and mostov, ; ktistakis et al., ) . the outer surface of a fruit fly embryo is composed of a monolayer of polarized epithelial cells. the apical membranes of these epithelial cells face the outer shell, or chorion, while their basolateral surfaces face the embryonic interior and yolk space. invaginations of this surface epithelium give rise rise to all of the embryo's internal tissue structures (for review see shiel and caplan, b) . recent investigations have examined the mechanisms through which proteins are sorted in these epithelial cells. human placental alkaline phosphatase (plap) is a gpi-linked protein which has been shown to be sorted to the apical plasma membrane when it is expressed by transfection in mdck cells. a chimeric construct of plap, in which the gpilinkage domain is replaced by the transmembrane and cytoplasmic domains of the vsv g protein (plapg), is sorted to the basolateral surfaces of mdck cells (brown et al., ) . these two proteins have been expressed under the control of heat shock promoters in transgenic flies and their distributions have been examined in embryonic epithelia throughout embryogenesis (shiel and caplan, a) . as would be expected, the plapg protein is restricted to basolateral surfaces throughout ontogeny in the surface epithelial cells as well as in the internal epithelia which derive from invaginations of the surface cells. surprisingly, plap was also restricted to a basolateral distribution in the surface epithelial cells in both early and late stage embryos. biochemical experiments demonstrated that this mis-sorting of the plap protein can not be attributed to problems with the addition of the gpi-linkage, since at all embryonic stages plap is correctly glipiated. internal epithelial cells sorted plap exclusively to their apical surfaces. since in many cases internal epithelia form from surface epithelia without undergoing any mitosis (e.g., salivary gland), essentially the same epithelial cell is capable of differentially sorting plap depending on that cell's physical position within the embryo. examination of epithelia undergoing invagination (e.g., ventral furrow, tracheal placode) demonstrate that the transition in plap sorting occurs in the early stages of the invagination process. while the mechanism responsible for this switch remains unclear, the power of drosophilu genetics will hopefully allow the cellular components responsible for this transition to be readily identified. it is likely that the isolation of the proteins responsible for this phenomenon will shed light on the drosophilu as well as on the mammalian epithelial sorting machinery. a drosophilu mutation whose phenotype includes peturbations of the polarized organization of the surface epithelial cells has recently been identified and characterized at the molecular level wodarz et al., ) . the crumbs gene encodes a transmembrane protein which is normally expressed in the apical membranes of surface and internal epithelial cells. mutation of the crumbs gene results in a loss of crumbs polarity and markedly alters embryonic morphology. genetic studies have demonstrated that the crumbs gene product is necessary not only for its own apical sorting, but for the apical delivery of other proteins as well. furthermore, the product of the stardust gene appears to interact with the crumbs protein and also appears to participate in apical sorting. understanding these proteins' biochemical functions and their intermolecular associations will undoubtedly provide enormous insight into the cellular components responsible for generating and maintaining the polarized phenotype. hopefully, the development of genetic approaches such as these, in concert with the continuing refinement of in vitro and model systems, will allow us to develop a clear and fundamental understanding of how epithelial cells produce their remarkable asymmetry. yeast syntaxins ssolp and sso p belong to a family of related membrane proteins that function in vesicular transport role ofmicrotubules in polarized delivery of apical membrane proteins to the brush border ofthe intestinal epithelium distribution of transport proteins over animal cell membranes podocytosis of small molecules and ions by caveolae the calmodulin antagonist, w- , alters transcytosis, recyclingand morphology ofthe endocyticpathway in madine-darby canine kidney cells mutational and secondary structural analysis of the basolateral sorting signal of the polymeric immunoglobulin receptor vesicular stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic reticulum cloning of a rab isotype predominately expressed in adipocytes the npxy internalization signal of the ldl receptor adopts a reverse turn conformation biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation. in vifro binding oftheasiatoglycoproteinreceptor to the betaadaptin of plasma membrane coated vesicles identificationofeffector-activatingresidues ofgsa lntracellular protein topogenesis. proc. natl. acad. sci. usa role of heterotrimeric g proteins in membrane traffic the gtpase superfamily: aconserved switch sorting of gpi-anchored proteins to glycolipid-enriched membrane an h+-atpase in opposite plasma membrane - . for diverse cell functions . subdomains during transport to the cell surface interactions between gpi-anchored proteins and membrane lipids mechanism of membrane anchoring affects polarized expression oftwo proteins in mdck cells constitutive and regulated secretion of proteins intracellular sorting and polarized cell surface delivery of nqk-atpase, an endogenous component of mdck cell basolateral plasma membranes dependence on ph of polarized sorting of secreted proteins sortingofmembrane and secretoryproteins in polarizedepithelial cells phorbol myristate acetate-mediated stimulation of transcytosis and apical recycling in mdck cells phosphorylation of the polymeric immunoglobulin receptor required for its efficient transcytosis an autonomous signal for basolateral sorting in the cytoplasmic domain of the polymeric immunoglobulin receptor hypervariable c-terminal domain of rab proteins acts as a targeting signal transferrin receptor internalization sequence yxrf implicates a tight turn as the structural recognition motif for endocytosis transplantedldl and mannose- -phosphate receptor internalization signals promote high-efficiency endocytosis of the transferrin receptor asortingsignal for the basolateral delivery of the vesicular stomatitis virus (vsv) g protein lies in its luminal domain: analysisofthe targetingofvsv g-influenzahemagglutininchimeras suppression of villin expression by antisense rna impairs brush border assembly in polarized epithelial intestinal cells a tyrosine-based signal targets h,k-atpase to a regulated compartment and is required for the cessation of gastric acid secretion high-resolution epitope mapping of hgh-receptor interactions by alanine-scanning mutagenesis association of the gtp-binding protein rab a with bovine adrenal chromaffin granules the internalization signal and the phosphorylation site of transferrin receptor are distinct from the main basolateral sorting information arf: a key regulatory switch in membrane traffic and organelle structure brefeldin a inhibits golgi membrane-catalysed exchange of guanine nucleotide onto arf protein polarized sorting ofglypiated proteins in hippocampal neurons involvement of na,k-atpase in antinatriuretic action of mineralocorticoids in mammalian kidney membrane assymetry in epithelia: is the tight junction a barrier to diffusion in the plasma membrane? a small gtp-binding protein dissociates from synaptic vesicles during exocytosis an enzymatic assay reveals that proteins destined for the apical or basolateral domains of an epithelial cell line share the same late golgi compartments direct association of occludin with zo- and its possible involvement in the localization of occludin at tightjunctions a yeast gene encoding a protein homologous to the human c-hashas proto-oncogene product specificity of binding of clathrin adaptors to signals on the mannose- -phosphate/insulin-like growth factor receptor receptor-mediated endocytosis: concepts emerging h m the ldl receptor system nonpolarized secretion of truncated forms of the influenza hemagglutinin and the vesicular stomatitis virus g protein from mdck cells an ion transporting atpase encodes multiple localization signals biotinylation and assessment of membrane polarity: caveats and methodological conerns sorting of ion transport proteins in polarized cells secretion of endogenous and exogenous proteins from polarized mdck monolayers sorting and endocytosis of viral glycoproteins in transfected polarized epithelial cells actin microfilaments play a critical role in endocytosis at the apical but not the basolateral surface ofpolarized epithelial cells small gtp-binding proteins and their role in transport a gtp-binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast biochemical characterization ofrenal epithelial cell cultures (llc-pk and mdck) apical polarity ofna,k-atpase in retinal pigment epithelium is linked to a reversal of the ankyrin-fodrin submembrane cytoskeleton mechanism for regulatingcell surfacedistributionofna,k-atpase inpolarizedepithelial cells gs alphastimulates transcytosis and apical secretion in mdck cells through camp and protein kinase a inhibition by brefeldin a of a golgi membrane enzyme that catalyzes exchange of guanine nucleotide bound onto arf protein more than just a membrane anchor rab , a small gtpase involved in vesicular traffic between the tgn and the basolateral plasma membrane protein transport to the dentritic plasma membrane of cultured neurons is regulated by rab p the origin and characteristics of a pig cell strain, llc-pk basolateral sorting in mdck cells requires a distinct cytoplasmic domain determinant different requirements for nsf, snap, andrab proteins in apical and basolateral transport inmdck cells inhibition of apical but not basolateral endocytosisofricinandfolateincaco- cells by cytochalasind binding and hydrolysis of guanine nucleotides by sec p, ayeast protein involved in the regulation of vesiculartraffic human adp-ribosylationfactors: a functionally conserved family of gtp-binding proteins crumbs and stardust, two genes of drosophila required for the development of epithelial cell polarity. development suppl exocytotic pathways exist to both the apical and the basolateral cell surface of the polarized epithelial cell mdck the biogenesis of lysosomes action of brefeldin a blocked by activation of a pertussis-toxin-sensitive g protein the signal sequence of nascent preprolactin interacts with the k polypeptide of the signal recognition particle phosphorylationofthe rat hepaticpolymericiga receptor an internal deletion in the cytoplasmic tail reverses the apical localization of human ngf receptor in transfected mdck cells multiple trimeric g-proteins on the trans-golgi network exert stimulatory and inhibitory effects on secretory vesicle formation tyrosine-dependent basolateral sortingsignals are distinct from tyrosine-dependent internalization signals a signal sequence for the insertion of a transmembrane glycoprotein rapid redistribution of golgi proteins into the er in cells treated with brefeldin a: evidence for membrane cycling from golgi to er microtubule-dependentretrograde transport of proteins into the er in the presence of brefeldin a suggests an er recycling pathway brefeldin a's effects on endosomes, lysosomes, and the tgn suggest a general mechanism for regulating organelle structure and membrane traffic polarized apical distribution of glycosyl-phoshatidylinositol-anchoredproteins in a renal epithelial cell line steady-state distribution and biogenesis of endogenous mdck-glycoproteins: evidence for intracellular sorting and polarized cell surface delivery preferred apical distribution of glycosyl-phosphatidylinositol (gpi) anchored proteins: a highly conserved feature of the polarized epithelial cell phenotype apical membrane aminopeptidase appears at site of cell-cell contact in cultured kidney epithelial cells selective inhibition ofprotein targeting to the apical domain of mdck cells by brefeldin a inhibition by brefeldina ofprotein secretion from the apical cell surfaceofmadin-darby caninekidney cells rabl , a novel small gtpase, is specific for epithelial cells and is induced during cell polarization targeting and retentioon ofgolgi membrane proteins structural analysis o f a human intestinal epithelial cell line distinguishing roles of the membrane-cytoskeleton and cadherin mediated cell-cell adhesion in generating different na,k-atpase distributions in polarized epithelia sortingofan apical plasmamembraneglycoproteinoccurs before it reaches the cell surface in cultured epithelial cells sortingofendogenous plasmamembrane proteins occurs from two sites in cultured human intestinal epithelial cells (caco- ) basolateral sorting of ldl receptor in mdck cells: the cytoplasmic domain contains two tyrosine-dependent targeting determinants mechanisms ofcell polarity: sorting and transport in epithelial cells structural requirements and sequence motifs for polarized sorting and endocytosis of ldl and fc receptors in mdck cells polarizedexpressionofa chimeric protein in which the transmembrane and cytoplasmic doamins of influenza hemagglutinin have beenreplaced by those of the vesicular stomatitis g protein membranes and sorting. c u r apical-to-basal transepithelial transport biogenesis of epithelial cell polarity: of human lactoferrin in the intestinal cell line ht- c . a intracellular sorting and vectorial exocytosis of an apical plasmamembrane glycoprotein tissue distribution of smg p a, a ras p -like gtp-binding protein, studied by use of a specific monoclonal antibody polymeric immunoglobulin receptor expressed in mdck cells transcytoses iga plasma membrane protein sorting in polarized epithelial short cytoplasmic sequences serve as retention signals for transmembrane proteins in the endoplasmic reticulum the trans-most cisternae of the golgi complex: a compartment for sorting of secretory and plasma membrane proteins microtubules are involved in the secretion of proteins at the apical cell surface of the polarized epithelial cell, madin-darby canine kidney tissue-specific sorting of the human ldl receptor in polarized epithelia of transgenic mice clathrin, adaptors, andsorting receptors compete for adaptors found in plasmamembranecoated pits mechanisms of vesicle docking and fusion: insights from the gtp-bindingproteins inintracellulartransport. trendsincell biology intracellular sorting and basolateral appearance of the g j. , - . nervous system isoformsofthena,k-atpase are presentin both axons and dendrites of hippocampal neurons in culture regulation of apical transport in epithelial cells by a gs class of heterotrimeric g protein role of heterotrimeric g proteins in polarized membrane transport the effect ofmodifying the culture medium on cell polarity in a human colon cell line replacement of the cytoplasmic domain alters sorting of a viral glycoprotein in polarized cells localization of sodium pumps in the choroid plexus epithelium nuclear localization signals in polyomavirus large-t viral glycoproteins destined for apical or basolateral plasma membrane domains traverse the same golgi apparatus during their intracellular transport in doubly infected madin-darby canine kidney cells the distribution of na,k-atpase in the retinal pigmented epithelium from chicken embryo is polarized in vivo but not in primary cell culture the roleofclathrin, adaptors and dynamin inendocytosis morphogenesis of the polarized epithelial cell phenotype polarity of epithelial and neuronal cells asymmetric budding of viruses in epithelial monolayers: a model system for study of epithelial polarity influenza virus hemagglutinin expression is polarized in cells infected with recombinant sv viruses carrying cloned hemagglutinin dna the large extracellular domain is sufficient for the correct sorting of secreted or chimeric influenza virus hemagglutinins in polarized monkey kidney cells the large external domain is sufficient for the correct sorting of secreted or chimeric influenza virus hemagglutinins in polarized monkey kidney cells mechanisms of intracellular protein transport molecular dissectionofthesecretory pathway implications ofthe snare hypothesis for intracellularmembrane topology and dynamics a ras-like protein is required for a postgolgi event in yeast secretion membrane and secretory proteins are transported from the golgi complex to the sinusoidal plasmalemmaofhepatocytes by distinct vesicular carriers plasticity of functional epithelial polarity developmental regulation of membrane protein sorting in the generation of epithelial polarity in mammalian and lipid sorting in epithelia polarized sorting in epithelia biogenesis of cell-surface polarity in epithelial cells and neurons the phosphomannosyl recognition system for intracellular transport of lysosomal enzymes snap receptors implicated in vesicle targeting and fusion interaction of activated egf receptor with coated pit adaptins in vitro binding of plasma-coated vesicle adaptors to the cytoplasmic domain of lysosomal acid phosphatase ), c < . drosophila embryos atpase in polarized epithelial cells the mammalian choroid plexus transport and membrane properties of the retinal pigment epithelium nonpolarized expression of a secreted murine leukemia virus glycoprotein in polarized epithelial cells a heterotrimeric g protein, gai- , on golgi membranes regulates the secretion o f a heparan sulfate proteoglycan in llc-pki epithelial cells a golgi retention signal in a membrane-spanning domain of coronavirus el protein molecularcloning,characterization, subcellular localization and dynamics of p , the mammalian kdel receptor vesicular stomatitis virus glycoprotein contains a dominant cytoplasmic basolateral sorting signal critically dependent on tyrosine the basolateral targetingsignal inthe cytoplasmicdomainofvsv g protein resembles a variety of intracellular targeting motifs related by primary sequence but having diverse targeting activities organization of glycosphingolipids in bilayers and plasmamembranes of mammalian cells sorting of progeny coronavirus from condensed secretory proteins at the exit from the trans golgi network of att cells endocytosis and signals for internalization exocytosis of vacuolar apical compartment (vac): a c e l k e l l contact controlled mechanism for the establishment ofthe apical plasma membrane domain in epithelial cells analysisofthedistributionofchargedresidues inthen-terminal regionofsignal sequences: implications for protein export in prokaryotic and eukaryotic cells mechanisms of protein translocation across the endoplasmic reticulum distinct transport vesicles mediate the delivery of plasmamembrane proteins to the apical and basolateral domains of mdck cells expression and polarized targeting of a rab isoform in epithelial cells crumbs is involved in the control of apical protein targeting during drosophrlu epithelial development brefeldin a causes amicrotubule-mediatedfusionofthe mechanismsofiontransportacrossthe choroidplexus a small rab gtpase is distributed in cytoplasmic vesicles in non-polarized cells but colocalizes with the tightjunction marker - in polarized epithelial cells opposite polarity of virus budding and of viral envelope glycoprotein distribution in epithelial cells derived from different tissues modulation of transcytotic and direct targeting pathways in a polarized thyroid cell line glycosylphosphatidylinositol-anchored proteins are preferentially targeted to the basolateral surface in fischer rat thyroid epithelial cells key: cord- -qdbgkm authors: kinnun, jacob j.; bolmatov, dima; lavrentovich, maxim o.; katsaras, john title: lateral heterogeneity and domain formation in cellular membranes date: - - journal: chem phys lipids doi: . /j.chemphyslip. . sha: doc_id: cord_uid: qdbgkm as early as the development of the fluid mosaic model for cellular membranes, researchers began observing the telltale signs of lateral heterogeneity. over the decades this has led to the development of the lipid raft hypothesis and the ensuing controversy that has unfolded. here, we review the physical concepts behind domain formation in lipid membranes, both of their structural and dynamic origins. this, then leads into a discussion of coarse-grained, phenomenological approaches that describe the wide range of phases associated with lipid lateral heterogeneity. we use these physical concepts to describe the interaction between raft-lipid species, such as long-chain saturated lipids, sphingomyelin, and cholesterol with non-raft forming lipids, such as those with short acyl chains or unsaturated fatty acids. while debate has persisted on the biological relevance of lipid domains, recent research, described here, continues to identify biological roles for rafts and new experimental approaches have revealed the existence of lipid domains in living systems. given the recent progress on both the biological and structural aspects of raft formation, the research area of membrane lateral heterogeneity will not only expand, but will continue to produce exciting results. the fluid mosaic model of membranes was proposed by singer and nicolson in , and almost immediately, there were reports showing the existence of membrane lateral heterogeneity . by the late 's, it was suggested that lipids could segregate into liquid disordered and liquid ordered domains. over the next decade, research showed that proteins could also co-localize , and in some cases, preferentially associate with lipids, such as sphingomyelin. in the early 's, experiments focusing on cholesterol in model membranes showed the sterol's ability to increase lateral heterogeneity and other studies, such as those using detergents to extract biomolecules from natural membranes, soon followed demonstrating protein co-localization with sphingomyelin and cholesterol . eventually, these data led to the hypothesis that sphingomyelin and cholesterol formed liquid ordered domains, called "rafts", in which proteins could associated with . although it was shown that some proteins had a preference for certain lipids, the idea that these would form large domains of functional significance remained controversial. however, recent research has revealed evidence for domain formation, consistent with lipid rafts, in fully functional, living cells . in this review, we discuss the concepts behind lipid domain formation in membranes, the biomolecules that they are made of, and their biological significance. j o u r n a l p r e -p r o o f the propensity for domain formation results from the interaction energy between chemically distinct lipids and proteins. for example, unfavorable interaction energies can result from lipids with different length fatty acid chains, forming for example, different bilayer thicknesses residing near each other. some of these unfavorable interactions can be eliminated or minimized by sequestering lipids of similar length within a domain. to model this, an interaction energy, i.e., the so-called called line tension, λ, is used, and which is defined as: where e b is the total interaction energy at the domain boundary and l b is the length of the domain boundary. note, that the total interaction energy is proportional to the domain boundary length, but the line tension, is not. in general, the greater the line tension, the greater the propensity for domain formation. line tension can be thought of as a string surrounding the domain perimeter that tightens as a function of an increasing unfavorable interaction energy between the the domain and its surroundings. note, that line tension should be considered in relation to the repulsive electrostatic forces that exist between the different lipids. points in phase separation studies. , further, experiments have revealed that domain formation is to some extent, dependent on the collective membrane material properties and long-range fluctuations, which will be discussed later. thus, line tension, itself, can vary as a function of system size, under certain circumstances. in addition to domain size having an effect on line tension, it is known that lipid-lipid repulsive interactions -which limit domain formation -also affect line tension. one way to account for lipid-lipid repulsive forces that counteract line tension, is to treat each lipid phase as a density of dipoles. what is important here, is not the absolute dipolar density, but the dipole density difference, ∆m, where lipids of one phase redistribute into the other phase in order to reduce repulsive dipolar interactions. the energy per molecule, e/n , for a circular domain can be written in terms of the opposing line tension and dipolar density difference as: where a is the area per molecule, r is the domain radius, ε is the dielectric constant of the interfacial water, ε is the permittivity of free space, e is euler's number, and δ is the molecular cut-off distance ∼ . nm lipids and membrane proteins have varying intrinsic hydrophobic thicknesses. coexistence of these species within a membrane results in local deformations at boundaries, where lipids splay and tilt to accommodate different thicknesses (see fig. ). however, these deformations have an associated energy cost that results in line tension. energetics of local deformation can be discussed in terms of material properties, such as the splay elastic modulus (b), tilt modulus (k), and the intrinsic curvature (j) between two domains. if we consider two domains with thickness difference, δ, and an average thickness, h , the line tension at the domain boundary can be written as: a feature of increasing importance is the packing defects between membrane lipids. these are introduced when the structure of a lipid is unable to conform to its neighbor. defects can be therefore be introduced as a result of the presence of unsaturated acyl chains or methylated segments that do not pack well with rigid moieties, such as the hydrocarbon rings in sterols, as shown in fig. c . akin to material rigidities -as discussed in the previous section -increasing biomolecule rigidity enhances packing mismatch, which increases line tension. in terms of specific theoretical energetics, this contribution to line tension is less j o u r n a l p r e -p r o o f figure : (a) the registration of domains across membrane leaflets maximizes dynamics, is entropically favorable, and is one mechanism for domain coalescence (adapted from haataja et al. where a is the characteristic length of the monolayer that lies between the lipid headgroup diameter and monolayer thickness. an increasing bending rigid difference between ordered and disordered lipid phases increases the value of the logarithm in eq. . this repulsive energy increase can result in co-alignment of rigid domains across bilayer leaflets or domains aggregating to more rigid areas of the membrane, such as those with proteins. for the case of domain registration across bilayer leaflets, it has been determined experimentally that domains with similar bending rigidiities can coalesce. it has also been observed that domains are able to coalesce across adjacent bilayers, and membrane undulations are thought to play a similar role . moreover, effects of acyl chain packing across bilayer leaflets should not be discounted, as there is evidence that leaflets can influence each other's molecular order. thus the packing between leaflets may influence domain registration and ultimately, domain formation. evidence for lipid-driven lateral heterogeneity in membranes began to appear in lipid mixtures, such as dimyristoylphosphatidylcholine ( carbon acyl chains) mixed with distearoylphosphatidylcholine ( carbon acyl chains) . in general, longer-chained, unsaturated lipids tend to be more ordered, the entropic difference between these two states results in a tension at domain boundaries (see fig. b ) and adds another possible mechanism for cholesterol increasing domain size through registration. although specific bonding between cholesterol and sphingomyelin may play a major role in cholesterol's ability to increase the size of domains, the effect of non-raft domains should not be discounted. there is research showing that increasing the unsaturated lipid presence of non-raft lipid constituents drives increasing domain size, implying that acyl packing effects may be a big contributor to domain size. it has also been suggested that the "push" mechanism that drives cholesterol from non-raft domains is equally important as the "pull" mechanism, where cholesterol is incorporated into rafts many proteins are relatively rigid and require a hydrophobic surface of sufficient thickness for them to incorporate "properly" into the membrane. differences between the protein's hydrophobic portions and those of the surrounding lipid membrane can result in hydrophobic mismatch, i.e., increased line tension (see fig. c early on, some research indicated that certain proteins preferential interact with sphin-gomyelin . since proteins can increase membrane thickness, it is likely that the ordering nature of sphingomyelin reduces membrane tension by deforming the membrane around proteins. also, it has previously been discussed that the nature of sphingomyelin's acyl chains allows for them to be compatible with the rigid cholesterol. since proteins can also be rigid, it is then possible that sphingoymelin is able to interact with proteins in a manner similar to that with cholesterol. it should also be pointed out that proteins, themselves, can form domains on the membrane surface, e.g., scaffolding proteins . often their organization is guided by electrostatics through specific charged amino acids, leading to oligomerization. although some do not penetrate into the bilayer, they do introduce a region of increased local rigidity, which may be sufficient to cause ordered domain registration of apposing bilayer leaflets . where the units are defined such that the coefficients of the quartic terms are unitless. this simple model has three parameters: i.e., κ, r, and µ, with the latter two parameters derived from the thermodynamic potential v [φ] and where κ is the rescaled surface tension. often, the fourth-order derivative term is ignored. this is acceptable as long as κ > in eq. ( ). as a result, the fourth-order derivative term becomes irrelevant with respect to the renormalization group and the dominant term becomes the second-order derivative. r is a tunable parameter across the mixing/demixing phase transition, which, in the mean-field, would occur at r = µ / or where phase separation takes place. conversely, for r > µ / , the potential v [φ] is minimized around φ = and we would expect no phase separation, but rather a fluctuating, disordered mixture of the liquid-ordered and liquid-disordered regions with a characteristic correlation length ξ ∼ κ/r. we may also be a somewhat more specific and identify the constant values φ o,d for which the potential is at a minimum in the phase separated regime with r < µ / . for < r < µ / , the potential v [φ] has two minima at constant values φ = φ d = and respectively. note that when µ = , this transition would have a first-order character and the potential will have a local maximum between the two minima (i.e., a thermodynamic barrier to transitioning between the liquid-disordered and liquid-ordered phases). if µ = , the two minima in the free energy become degenerate at φ = at the transition (r = ) so that there is no thermodynamic barrier and the transition is second-order (continuous). thermal fluctuations change this picture, somewhat, as the distinction between an unstable and a metastable phase becomes ambiguous when thermal fluctuations are taken into account. in lipid vesicles, it is easy to tune across the mixing/demixing transition by varying the temperature. in biological cells, however, the situation is more complicated as it is unclear whether or not the cell membrane is at equilibrium, whereby these phases may not be meaningful. nevertheless, the free energy in eq. ( ) is useful as a conceptual tool. we may use it as a basis for constructing the dynamics of the lipid phases by employing an appropriate dynamical model. for instance, it has been recently argued that natural cells tune their membranes such that, they have compositions near criticality (µ = and r ≈ ). , through dynamical processes, such as lipid diffusion, the free energy in eq. ( ) trends towards minimization. as the lipid membrane is fluid, the most appropriate model would include the hydrodynamic coupling of the lipids across the membrane. another crucial aspect of the dynamics is the conservation of total lipids, which means that the integrated order parameter, dx φ(x), is a constant (typically zero for equal proportions of liquid-ordered and liquid-disordered regions). excluding the hydrodynamics and thermal fluctuations, the time-dependence of the order parameter is given by: with Γ representing mobility of the phase. eq. may be evolved for various initial conditions, for example, to study the evolution of the phase separation of domains in the r < µ / region. with such conserved dynamics, we would typically expect lifshitz-slyosov domain evolution, where domains grow as ∼ t / . however, hydrodynamic effects may modify this scaling in more realistic scenarios. the different phase-separated regions in a lipid vesicle may exhibit different preferred curvatures of the membrane, due to the particular geometry of the constituent lipid molecules. here, the shape of the membrane couples to the dynamics of φ, and we have to combine the free energy in eq. ( ) with elastic terms for the membrane and a coupling between φ and membrane curvature. leibler and andelman showed that in the presence of such couplings, the line tension term, κ|∇φ| / , gets contributions from membrane curvature and can even change signs. when κ is driven to negative values, the quartic derivative term, |∇ φ| / , must be included in the theory and the free energy, f, is now minimized by spatially modulated configurations. microscopic models also reveal that a coupling to membrane curvature is sufficient to drive the surface tension term negative. by including sign changes in κ, this makes the phase space of the theory in eq. ( ) much richer, and the various possibilities present a unified way of thinking about lipid membrane heterogeneity. in the mean-field (ignoring thermal fluctuations), the phase portrait of the model is shown in fig. . figure : schematic of the various phases described by the free energy in eq. ( ) for µ = . the phase boundaries are given in the single-mode mean-field approximation. we see that when the gradient term κ < , it is possible to form an ordered modulated (patterned) phase and a disordered "microemulsion", with a characteristic wave number q . here we have shown possibilities for the phases on the surface of a vesicle, which introduces its own complications due to the finite spherical geometry. for example, the striped modulated phase shown has stripes terminating at the poles. moreover, any of the modulated phases will have defects induced by the spherical topology. when κ < , the free energy develops minima with configurations of φ(x) with non-zero fourier modes. in particular, using a simple single-mode approximation, one can show that in the mean-field, the preferred wave number is |q| = q = −κ/ (see eq. ( ) have nanoscopic domains and are more likely to be in the disordered "microemulsion" phase instead of these highly ordered patterned phases. note that we may incorporate dynamics by using an equation such as eq. ( ). in the case of a free energy with parameters corresponding to a modulated phase, the dynamics reduce to the so-called "phase field crystal". the dynamics of the modulated phases include interesting cases such as, the formation of a foam after a rapid quench into the modulated phase. such foamy states appear to be relevant for pollen grain patterns, as well. foamy structures may also be observed in synthetic lipid vesicles with specific compositions. if κ is large and negative, then we will be deep in the modulated phase/microemulsion regime and we can expand our free energy around the dominant fourier modes with |q| = q . for a spherical vesicle, fourier modes are inappropriate and one has to expand the field figure : (a) mean-field phase diagram for the modulated phases with a fixed κ < in eq. ( ) and a flat and infinite two-dimensional substrate. we see that the cubic term µ tunes between the striped phase (middle), the hexagonal phase (top), and the inverted hexagonal phase (bottom). when r > , we also find a microemulsion region, denoted by the yellow. (b) modulated phases on a sphere have a more complex structure, since the pattern has to wrap the sphere = q r times, and has defects. we show the various possible shapes for small values of . note the wide range of shapes, including continuously varying "intermediate" states. in general, on the sphere the free energy landscape becomes much more complicated than for a flat, infinite substrate. the phase diagram is adapted from radja et al. here, we have a rescaled cubic termμ ∝ µ. r is fixed to a negative value ensuring that we are always in a modulated phase. is the location on the spherical membrane in spherical coordinates. we also have the usual spherical harmonics y m (θ), with = , , , . . . the "total angular momentum" mode number and j o u r n a l p r e -p r o o f m = − , − + , . . . , the azimuthal mode number. then, we expect that the field has contributions primarily around the spherical harmonic modes with ≈ ≈ q r, with r the vesicle radius. this parameter will strongly influence the kinds of patterned phases that can form, as shown in fig. . we can rewrite the free energy in eq. ( ) on the sphere in the following (rescaled) landau-brazovskii , form: where Υ , , m ,m ,m are the so-called gaunt coefficients coming from integrations of products of three spherical harmonics. we see here that the cubic and quartic terms in the thermodynamic potential will couple different spherical harmonic modes. minimizing eq. ( ) over the set of coefficients c m yields a rich set of possibilities for modulated phases, some of which are illustrated in fig. (b) . a free energy of this type is not only useful for understanding lipid vesicles and pollen grains, but also viral capsid formation. we have spent some time considering the modulated phases (κ, r < in eq. ( )). however, as mentioned previously, lipid heterogeneities in living cells and in many synthetic lipid mixtures are best thought of as disordered, microemulsion phases. the main difficulty of this hypothesis is explaining the origin of the sign change of κ. however, one possibility is that differences in composition between lipid leaflets can induce such a sign change via a mechanism similar to spontaneous curvature. this is plausible as living cells are known to maintain asymmetric lipid compositions on their inner and outer membrane leaflets. in this phase, the order parameter remains, on average, zero ( φ = ), but exhibits particular fluctuations with dominant contributions at the characteristic wavevectors with |q| = q (or modes = on the sphere), resulting in a structure factor s(q). the structure factor, s(q), for the lipid membrane can be measured via a scattering (neutron or x-ray) experiment. small angle neutron scattering is particularly valuable as it provides better contrast to probe the lateral membrane heterogeneity, even when these heterogeneities are nanoscopic. , to get good contrast on the lateral heterogeneity within the membrane, deuterated lipid mixtures may be used to mask one of the liquid phases, for example. a schematic of the idea is shown in fig. (a) . here, lipid vesicles are prepared with particular deuteration levels such that, at high temperatures when the lipids remain mixed the sld of the acyl tails match the sld of the surrounding fluid. thus, the lipid vesicles become "invisible" to neutrons, as shown by the flat curves at high temperatures in fig. (b) . at lower temperatures, we have the liquid-liquid phase separation (or possibly microemulsion or modulated phase formation) and the slds of the liquid-ordered and liquid-disordered regions will be different, creating contrast for the neutron scattering as shown in fig. (a) . in this case, the neutron scattering will come from fluctuations in the lipid composition. therefore, it is a direct probe of our order parameter, φ(x). for scattering from lipid vesicles, the scattering intensity, i, is a probe of the fluctuations |c m | of the spherical harmonic modes of φ(x). for a microemulsion phase, we expect that where r is the vesicle radius and ξ the correlation length. such a phenomenological approach was used successfully to interpret scattering data of lipid vesicles in the presence of melatonin -although a true microemulsion phase could not be established as the scattering data was also consistent with = (a regular phase-separated phase). fits using the microemulsion theory are shown in fig. (b) . note, that this theory works well even when compared to a more microscopic model of domain configurations shown in fig. (c) . in other words, the simple phenomenological free energy approach presented here provides a conceptual framework for understanding the scattering data and interpreting the wide range of phase behaviors observed for lateral lipid organization in cell membranes. being near a phase transition allows for small changes in the environment to have a large effect on the appearance and size of rafts. one proposed scenario is that rafts may provide a buffering role in stabilizing membrane physiological properties across a range of temperatures. here, high-melting temperature raft lipids diffuse into the disordered phase as temperature increases, maintaining membrane bending rigidity and viscosity. it has also been theorized that cells vary their lipid composition to control the formation of rafts. the fluid mosaic model of the structure of cell membranes lateral phase separation in phospholipid membranes clusters in lipid bilayers and the interpretation of thermal effects in biological 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membrane in different cell lines target membrane cholesterol modulates single influenza virus membrane fusion efficiency but not rate influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion sphingolipid and cholesterol dependence of alphavirus membrane fusion lack of correlation with lipid raft formation in target liposomes tuning membrane phase separation using nonlipid amphiphiles • review article describing recent experimental and theoretical membrane lateral heterogeneity research • describes the static and dynamic physical concepts behind domain formation in model membranes • describes coarse-grained, phenomenological approaches that result in phases associated with lipid lateral heterogeneity key: cord- -fwbz t authors: lemieux, m. joanne; overduin, michael title: structure and function of proteins in membranes and nanodiscs date: - - journal: biochim biophys acta biomembr doi: . /j.bbamem. . sha: doc_id: cord_uid: fwbz t abstract the field of membrane structural biology represents a fast-moving field with exciting developments including native nanodiscs that allow preparation of complexes of post-translationally modified proteins bound to biological lipids. this has led to conceptual advances including biological membrane:protein assemblies or “memteins” as the fundamental functional units of biological membranes. tools including cryo-electron microscopy and x-ray crystallography are maturing such that it is becoming increasingly feasible to solve structures of large, multicomponent complexes, while complementary methods including nuclear magnetic resonance spectroscopy yield unique insights into interactions and dynamics. challenges remain, including elucidating exactly how lipids and ligands are recognized at atomic resolution and transduce signals across asymmetric bilayers. in this special volume some of the latest thinking and methods are gathered through the analysis of a range of transmembrane targets. ongoing work on areas including polymer design, protein labelling and microfluidic technologies will ensure continued progress on improving resolution and throughput, providing deeper understanding of this most important group of targets. with a short sma( : ) copolymer with a : ratio of s:ma subunits shows optimum copolymer concentrations of . - % under temperatures in the - °c range with incubation times of - hours. lower temperatures warrant longer incubations, while excessive polymer concentrations should be avoided to minimize aggregates. optimal ionic strength ( - mm nacl), divalent cations (< mm) and ph ( ) ( ) are evident for kcsa solubilization but can be protein and membrane dependent as they are subject to respective charge distributions. comparisons with the performance of conventional detergents suggests that the development of neutral or basic copolymers related to sma could offer advantages, providing avenues for solubilization and analysis of a broader array of biological membrane:protein assemblies (memteins). the utility of sma lipid particle (smalp) technology for analysis of memteins by mass spectrometry (ms) and x-ray diffraction methods remains limited. this problem stems from the requirements of the respective gas phase or crystalline samples and the heterogeneity of currently used sma copolymers. in order to address this, muench and colleagues have developed a method to transfer membrane proteins from smalps into amphipols or detergents for downstream analysis [ ] . the homotrimeric e. coli multidrug transporter acrb was used as a test case. the vulnerability of sma to precipitation in the presence of divalent cations was exploited by using stepwise addition of mgcl from . to mm to cause the solubilizing copolymer to precipitate in the presence of a rescue solution composed of amphipol a - or % n-dodecyl-β-d-maltoside (ddm) detergent. the resulting amphipol j o u r n a l p r e -p r o o f journal pre-proof and detergent complexes are more homogeneous, tolerate higher divalent cations levels, and yield observable signals by native mass spectrometry, including of phosphatidylglycerol in the amphipol-stabilized protein trimer. new methods are being applied to analyze the process of forming nanodiscs from membranes in greater detail. in particular, a microfluidic diffusional sizing (msd) system which was recently developed to detect protein hydrodynamic radii between . to nm has been adapted to probe nanodisc formation [ ] . this microfluidics technology requires small sample volumes (< μl) which are injected into a microfluidic chip. the complexes diffuse across from one side of the chamber to the other during a minute time course and are then sensed via primary amines of, for example, phosphatidylethanolamine. several stages can be reliably seen from comparison of mds and dynamic light scattering (dls) data collected with increasing ratios of sma( : ) to lipid. first the polymer inserts into bilayers until it reaches saturation. this is followed by solubilization into particles with - nm diameters. as excess polymer is applied these become progressively smaller until reaching a limiting diameter of nm. the lipid-specific association of an aggregation peptide derived from the tau protein is also demonstrated, indicating utility for detecting how fibers are nucleated on membranes. two longstanding challenges are the characterization of endogenous heteromultimeric membrane proteins and the development of conformation-specific antibodies for memteins. a study by mouro-chanteloup and coworkers of red blood cell ghosts addresses these confounding issues [ ] . the individual components of erythrocyte membranes including rh proteins are well known, but their multimeric lipid complexes are dissociated in detergent-based preparations and hence no longer recognizable or extractable using conformation-specific antibodies. in contrast using sma( : ) copolymer for solubilization preserves the native states of rh complexes, and membrane-associated cytoskeletal proteins can be gently removed by washing in very low ionic strength buffer. the biological relevant states of detergent-free rh complexes can then be isolated using gel filtration and monoclonal antibodies. the native states are preserved based on detection by conformation-dependent antibodies, paving the way for antibody screening and structure-function analysis. the extraction and purification of low abundance drug targets from membranes in stable, functional forms is a key goal for the pharmaceutical industry. the g-protein coupled receptor (gpcr) superfamily is critical, being targeted by many therapeutic agents. they respond to a diversity of signals and mediate cellular responses through interactions with heterotrimeric g proteins, gpcr kinases and arrestins. the complexity and conservation of the interactions of the phosphorylated and palmitoylated vasopressin receptor type , its peptide agonist vasopressin and arrestin is modeled by fanelli and coworkers, revealing coupled motions between key gpcr sites [ ] . conformational changes that are induced by ligands including full agonists, partial agonist, antagonists and inverse agonists are j o u r n a l p r e -p r o o f journal pre-proof membrane-dependent. the adenosine a (a a) receptor is a well-characterized gpcr from a structural perspective, although the roles of dynamics and lipids remain unclear. after expression in pichia pastoris, the wild-type a a receptor and a pair of mutants with trp to tyr substitutions were solubilized into nanodiscs using sma( : ) copolymer by wheatley and colleagues [ ] . a fluorescent reporter (iaedans) was also attached to an introduced cys residue by the sixth helix, providing an additional probe. binding of specific ligands induces concentration-dependent changes in the fluorescence signals of trp residues, which exhibit greater steric hindrance based on fluorescence anisotropy measurements. the association with phospholipids from the plasma membrane is apparent by ms/ms analysis of the smalps. watts et al. show that the dopamine d receptor can be purified from human embryonic kidney (hek) cells with a yield of g/l using sma( : ) copolymer [ ] . as with the a a receptor, the purification of this gpcr was carried out at °c to preserve the native state. binding studies of the protein in smalps provides estimated native-like affinities for a neurotensin peptide ligand and a specific antagonist. the transport of materials including drug molecules out of cells is carried out by the atp binding cassette (abc) family of membrane transporters. the structures and functions of these proteins are reviewed by kanelis and coworkers, with an emphasis on the intrinsically disordered regions that mediate key interactions in a phosphorylation-dependent fashion [ ] . a set of abcg protein constructs were designed with n-terminal gfp or snap and his tags and solubilized from hek cells using sma( : ) copolymer, as were cd and cd antibiotics include lipid-specific peptides that self-associate into pores, which permeabilize bacterial membranes. however, the intact pores are difficult to resolve due to their dissociation in detergents. palmer's lab studies daptomycin, a cyclic lipopeptide consisting of amino acids and a decanoic acid group that is a critical line of defense against infections caused by gram-positive bacteria. it works by binding phosphatidylglycerol-containing membranes in a calcium-dependent manner to form octameric pores. the design of alternating sma copolymers with methylamine sidechains allows octamers and tetramers to be obtained by varying on the amount of added polymer, illuminating the assembly of membrane complexes [ ] . this paves the way for structure-determination of the native pore state and design of improved antibiotics to combat multi-drug resistant bacteria. aquaglyceroporin proteins allow the selective permeation of molecules across membranes. this family includes the e. coli glycerol facilitator, which mediates passive diffusion of glycerol across the inner membrane of the bacterium. although prone to aggregation, careful optimization of its expression and solubilization in various media showed that this helical bundle protein is most monodispersed in lauryl maltose neopentyl glycol [ ] . this detergent j o u r n a l p r e -p r o o f journal pre-proof stabilizes the physiological tetramer, although a non-native octamer persists due to interactions between the disordered termini. the dynamics of lipids in nanodiscs formed by sma( : ) and diisobutylene/maleic acid copolymer (dibma) copolymers, which differ in their hydrophobic groups, are compared by steinhoff and coworkers [ ] . the application of electron paramagnetic resonance (epr) spectroscopy and a set of phosphatidylcholine lipids with nitroxide groups located at the th , th or th carbon atom positions reveals that the lipids are more constrained in the more stable smalp discs. this can be explained by coarse-grain molecular dynamics simulations, which indicate that in the case of dibma a single belt of polymer contains lipids within the nanodisc with dynamics more closely resembling their mobility in a liposome. in the overduin lab, new amphipathic copolymers are being developed to broaden the utility of native nanodiscs. a series of alkylamine derivatives of sma with alternating sidechains were designed to reduce polymer sequence heterogeneity and improve resolution [ ] . in the major facilitator superfamily, various solute carrier (slc) families exist. one of the challenges is to functionally characterize these transporters. in particular, a different transporter function may be present for the same transporter when it resides in different cell types. the cordat lab determined that the slc a protein is a chloride/bicarbonate exchanger in kidney cells, which is in contrast to its role as a chloride channel in oocytes [ ] . furthermore, they show that the abundance of this protein at the cell surface is dependent on the osmolarity of the cell culture medium, and ph of the cells. this is important considering the slc a is expressed in outer medullary collecting duct cells near acid secreting cells that can render the local environment hyperosmotic compared to plasma. in human cells, both concentrative and equilibrative nucleoside transporters (cnt and ent) are found. with multiple transporters in this family having overlapping function it is important to characterize each individually. radioactive hplc was used by the young lab to determine the metabolism of nucleosides in oocytes, for which little previous study has been conducted [ ] . this study revealed little metabolism occurred in oocytes and helped develop this novel means to examine nucleoside levels. next, in mice, the contribution of cnt and ents, which have overlapping specificities, were examined using radioactive j o u r n a l p r e -p r o o f hplc. nucleotide metabolites were assessed from plasma of mice with cnt or ent knockouts, illuminating the specificities of these transporters in vivo. the seca protein works to facilitate the transport of proteins destined for secretion through the secyeg translocon. despite structural information that reveals helical nucleotide binding domains, how seca interacts with diverse secretory proteins remain a question in the field. to address this, the bondar group conducted a sequence analysis of bacterial seca proteins ( ), which was guided by sequence alignment, structural analysis and phylogeny [ ] . they identified that seca proteins have varied length as a result of insertions and deletions, with clustering revealing three main groups. the first residues housing the nucleotide binding region (nbd ) is highly conserved in the family, with preferred residues at the n-terminus, which include a conserved phe at position , followed by hydrophobic sequences, and an arg/lys, are important for lipid anchoring. in addition they highlight the tyr residue located at the end of a helix finger structural motif, and other charged residues that may play a role in recognition of the positively charged pre-sequence. they further suggest diversity in sequence length contributes to increased conformational dynamics that may influence target interactions. the development of new tools to produce and analyze of integral membrane proteins is covered in a timely review by danmaliki and hwang [ ] . these targets remain challenging to characterize at atomic resolution by any method. their analysis by nuclear magnetic j o u r n a l p r e -p r o o f journal pre-proof resonance (nmr) spectroscopy is complicated by the requirement for many milligrams of pure protein containing h, c and n isotope labels to ensure high sensitivity and resolution, as well as the complex dynamics within the bilayer that broaden signals from transmembrane regions. advanced isotope labelling protocols allow chemical groups such as methyl and methylene groups and f nuclei on specific residue types to be resolved, yielding structural information from such moieties. successes include an array of  barrel structures, which have been solved in various detergent micelles, as well as -helical structures that are inherently more challenging as they exhibit greater chemical shift degeneracy and fewer observable distances between secondary structure elements. hence nmr is well-positioned to provide unique insights into dynamics and interactions, as well as structures of proteins that are small or difficult to crystallize. single pass transmembrane proteins pose challenges for structural biology, being particularly dynamic, prone to aggregation and dependent on lipid microenvironments. consequently, very few have been characterized at high resolution. ramamoorthy and colleagues solubilized the full length form of the microsomal protein cytb , which includes a heme-containing soluble domain, transmembrane helix and flexible linker, into nanodiscs using an -residue amphipathic helical peptide and various synthetic lipids [ ] . through expression in e. coli the four tryptophan residues could be labelled with fluorine- for detection by f-nmr spectroscopy. due to the rapid tumbling of the ~ nm diameter nanodiscs, the multi-domain proteins that reversibly bind membrane surfaces provide technical challenges for unravelling biological pathways. the phafin protein plays a critical role in inducing autophagy, but its membrane recognition mechanism is unclear. this modular protein contains a pleckstrin homology (ph) domain and a (fab , yotb, vac , and eea ) fyve domain, both of which bind phosphoinositol -phosphate (pi p), a key signaling lipid that is found in endocytic membranes. the capelluto group showed that its fyve domain is indispensable for constitutive and specific pi p recognition [ ] . in contrast, its ph domain binds acidic lipids such as phosphatidylserine or pi p when they are present in bilayers (but not as soluble lipids). the ph domain is autoinhibited by interactions with a conserved acidic c-terminal motif. thus the multivalent and tightly regulated binding of proteins to membrane surfaces can deciphered via the individual interactions. in the future soluble and homogeneous nanodiscs may offer opportunities to reveal the native binding mechanisms via concerted recognition of multiple lipids and protein elements that are heavily phosphorylated. together, these studies illustrate the challenges and potential of the growing field of membrane structural biology. membranes have traditionally been the least well understood components of the cellular ecosystem, and new tools have been sorely needed. the coming years promise further gains as the interfaces between proteins and lipids become better understood, and are certain to yield many more secrets into memtein formation and function. our ability to probe such mechanisms in native states and recombinant forms will in turn lead to improved understanding and exploitation of membrane targets for applications including synthetic biology and drug discovery. isolation of intramembrane proteases in membrane-like environments factors influencing the solubilization of membrane proteins from escherichia coli membranes by styrene-maleic acid copolymers styrene maleic-acid lipid particles (smalps) into detergent or amphipols: an exchange protocol for membrane protein characterisation microfluidic diffusional sizing probes lipid nanodiscs formation detergent-free isolation of native red blood cell membrane complexes dynamics and structural communication in the ternary complex of fully phosphorylated v vasopressin receptor, vasopressin, and beta-arrestin ligand-induced conformational changes in a smalp-encapsulated gpcr detergent-free extraction of a functional low-expressing gpcr from a human cell line intrinsically disordered regions regulate the activities of atp binding cassette transporters application of fluorescence correlation spectroscopy to study substrate binding in styrene maleic acid lipid copolymer encapsulated abcg expression and detergent free purification and reconstitution of the plant plasma membrane na(+)/h(+) antiporter sos solution structure of the cytoplasmic domain of nhap a k(+)/h(+) antiporter from vibrio cholera characterization of multimeric daptomycin bound to lipid nanodiscs formed by calcium-tolerant styrene-maleic acid co-polymer solution structure and oligomeric state of the e. coliglycerol facilitator lipid dynamics in nanoparticles formed by maleic acid-containing copolymers: epr spectroscopy and molecular dynamics simulations the effect of hydrophobic alkyl sidechains on size and solution behaviors of nanodiscs formed by alternating styrene maleamic copolymer slc a protein is a chloride/bicarbonate exchanger and its abundance is osmolarity-and ph-dependent in renal epithelial cells hplc reveals novel features of nucleoside and nucleobase homeostasis, nucleoside metabolism and nucleoside transport diversity and sequence motifs of the bacterial seca protein motor solution nmr spectroscopy of membrane proteins expression, purification, and functional reconstitution of ( )f-labeled cytochrome b in peptide nanodiscs for nmr studies where he serves as director of nanuc, canada's national nmr centre. his research focuses on membrane structural biology and the discovery of ligands of proteins involved in cell adhesion, signaling and endocytosis. he studies desmosomal protein interactions, phosphoinositide recognition by signaling proteins, and ligand binding by novel drug targets including calcium/calmodulin dependent ser/thr kinases and gtpase regulators. he co-developed the moda program to identify membrane binding domains and the smalp system for detergent-free purification of native membrane proteins into stable as director of the membrane protein disease research group she leads a multidisciplinary research program focused on membrane protease structure and function. she is internationally recognized as a leader in membrane protein crystallography having solved two distinct membrane protein crystal structures key: cord- -ing qma authors: roy, nimisha; kashyap, jyoti; verma, deepti; tyagi, rakesh k.; prabhakar, amit title: prototype of a smart microfluidic platform for the evaluation of sars-cov- pathogenesis, along with estimation of the effectiveness of potential drug candidates and antigen–antibody interactions in convalescent plasma therapy date: - - journal: trans indian natl doi: . /s - - - sha: doc_id: cord_uid: ing qma originating in china during december , the novel corona-virus, sars-cov- , has created mayhem worldwide in a very short time. the outbreak has been so rapid and widespread that the only option to treat the patients was administering drugs already available in the market like chloroquine/hydroxychloroquine (an antimalarial drug) and remedesivir. a large number of patients have been cured but the attribution to survival by these drugs has been controversial. till date, we do not have any specific drug or vaccine available for covid- and the pandemic seems to be far from over. to handle the current challenges posed by the outbreak effectively, we need to employ innovative interdisciplinary approaches. organ-on-chip (ooc), particularly lung-on-chip, is one such approach which combines the potential of microfluidics, cell culture and molecular biology into a single miniaturised platform. the device is realized to be capable of simulating in-vivo physiological responses of an organ. in the current study, an ooc, which is a multichannel d cell culture microfluidic device, is made via soft lithography technique, using polydimethylsiloxane-polymer and diverse polymeric porous/semipermeable membranes. several polymer membranes i.e. pdms, polyvinylidene fluoride (pvdf), nitrocellulose, polyester etc., integrated into the microdevices, were efficiently explored to realize their better cell-adhesion and viability property. we also propose for the application of a simple, smart and cost-effective lung-on-chip platform to study the sars-cov- pathogenesis in humans, drug toxicity testing and provide insights into antigen–antibody interactions. this platform will enable us to study multiple phenomena at a micro-level generating more reliable data and a better understanding of the underlying mechanisms of sars-cov- infection and pathogenesis. on march , , the who declared covid- a pandemic considering the global apocalyptic effects of this disease. although the scourge of this pandemic did not spare any nation, the top ten among most severely affected countries includes usa, brazil, russia, united kingdom, italy, spain, india, germany, france and peru. as per the may , situation report of who, the total number of cases and deaths worldwide are , , and , , respectively. presently, no drug or vaccine for this disease is available. however, several potential drug and vaccine candidates are in various stages of clinical trials. nevertheless, considering the lengthy procedures of conventional drug discovery pipeline it would take substantial time before these drugs reach the market. the rate at which covid- spread across the globe has been alarming, and based on the initial information about the pathogenesis the only option was to administer existing drugs approved for other infectious diseases; however, their usage for covid- has been controversial because of the associated side effects. as the pandemic continues to spread, some important queries are imminent. firstly, can existing drugs be repurposed for effective treatment of covid- ? if so, how their efficacy can be evaluated rapidly? secondly, what alternative options can be employed to reduce the time of clinical trials by rapid drug testing and approval? the current need is a screening platform that can provide reliable results in less time. the organ-on-chip technology (ooc) is one such alternative. though the field is still in its infancy, it has tremendous potential to revamp the conventional drug discovery pipeline. the ooc technology emerged with the pioneering work by huh et al. at the wyss institute at the harvard university in (huh et al. ) in which they described a fabrication protocol for organ-on-chip using polydimethylsiloxane (pdms). since the inception of the concept, enormous efforts have gone into converting this proof-of-concept into actual working devices which are currently being globally used by researchers with some of the breakthrough results (zhu et al. ; neuzil ) . various startups have been set up as spin-offs from various research institutions across the globe with huge market potential and substantial revenue-earning. in india, however, this promising platform has not been harnessed. in the current pandemic situation, this technology may prove to be a valuable tool for not only for performing drug toxicity testing but may also be used for analysing the sars-cov- pathogenesis in human. oocs are a designed network of microfluidic channels intended for mimicking the smallest physiological and biochemical functional units of various organs. the chip dimensions are in the range of a few centimetres with microchannels network in the range of micrometres. these oocs provide significant advantages over the traditional d cell culture (fig. ) in petri-plates like: (i) closely mimicking the cell's natural microenvironment or niche, (ii) minimal requirements of reagents, (iii) options for flexible design to suit experiment needs and (iv) high throughput efficiency. initially, the primary goal for developing the ooc was to expedite the conventional drug discovery pipeline (wikswo ) . in the current drug discovery and development paradigm, it takes about years for a single drug to reach the market. about % of the drugs fail at various stages of clinical trial (kola and landis ) . this culminates into severe loss in terms of money and time, and sometimes volunteer's life. in addition, the present animal testing is deemed inhumane with organisations such as peta protesting against it. there have also been many incidents where a novel drug performed excellently in animal models but subsequently failed to produce the same expected response in humans. sometimes, this too resulted in life-threatening repercussions to the human clinical trial candidates (suntharalingam et al. ; strooper ) . opposed to all the above-mentioned drawbacks, ooc being an enclosed d cell culture platform mimics the smallest functional unit of an organ hence it provides reliable results as they are capable of generating in-vivo like environment on a chip in a comparatively lesser amount of time. one of the most important advantages is in its ability to recreate the cell microenvironment by precisely controlling the flow rate of fluids using pumping devices, such as syringe pumps. some of the other pertinent advantages include reduced risk of contamination, resolution up to the single-cell level to study cellular morphology, mechanisms with the scope of parallelisation to study cellular reactions simultaneously. figure represents the comparison between and d cell culture platforms (halldorsson et al. ) . although it would be too early to say that these chips would replace animal testing, they will surely provide a platform to study diseases at the molecular level and for rapid drug screening. this fig. comparative presentation of a d, and b d cell culture platfroms would be a boon to the society in urgent situations such as in current pandemic (fukumoto and narasaiah ) . our group has been working on organ-on-chip technology that is primarily focussing on lung/liver-on-chip devices and searching for alternate ways of fabrication and application of versatile materials to lower the cost. in the liver-on-chip device, we tried to mimic the hepatic sinusoids which are the site for mixing of the oxygen-rich blood coming from the hepatic artery and the nutrient-rich blood from the portal vein. further, we performed experiments in the search for the most optimal materials, by replacing the commonly used porous pdms membrane that will be cell life-supporting, economical and easily accessible membrane to serve as an integral part of oocs. we conducted a comparative study of cell adhesion on various easily available membranes to examine parameters of cell adhesion and viability properties. we also performed static cultures in the chip to assess for cell viability inside the chip. for this purpose, we conducted studies with human liver cell line hepg as they are a good representative model of hepatocytes. although the ultimate aim of developing this platform is to assess the drug toxicity on the hepatocytes the protocol can be easily adapted to fabricate other organs too. we illustrate the application of 'lung-on-chip', a microengineered biomimetic platform to get a deeper insight into the infectious mechanism of the virus on the lung epithelial cells along with the evaluation of the efficacy of drug/ vaccine and their toxicity testing. our fabrication protocol takes inspiration from the protocol mentioned by huh et al. but we have introduced some major changes in fabrication and materials to lower the cost of developing the chip. in addition, the bonding of membranes with upper and lower channel layers has been customized with a novel membraneprocessing method. this 'lung-on-chip' platform is basically a -dimensional alveolar-capillary interface (fig. ) , which is the site of gaseous exchange between blood and alveolus (the functional unit of lungs). under normal conditions, the alveolar membrane and the capillary boundary are close and hence optimal exchange of gases will occur to support life. patients with a severe infection of covid- develop acute respiratory distress syndrome (ards). in this syndrome, the alveolus sacs are filled with fluid and hence the interface between oxygen and blood capillary wall thickens. as a result of the fluid accumulation, the amount of oxygen in the alveoli decreases leading to less oxygen being diffused into the blood-stream. since there is a decline in the amount of oxygen reaching the organs, the patient dies slowly due to multiple organ failure. studies have shown that such a microfluidic platform can be effectively utilised to study ards (viola et al. ) . recent studies (chen et al. ; shen et al. ) have suggested that the plasma from the patients, who have recovered from covid- , can be used to treat patients based on successful results of this therapy during sars, mers and ebola outbreak. the plasma of recovered patients develops antibody against the virus and these can be transfused to other patient's body to help fight the infection. however, the fda has only allowed it to be used as an experimental therapy for only a few critically ill patients (tanne ). one of the major issue associated with this therapy is the risk of mild to severe allergic responses that could even be life-threatening (roback and guarner ) . utilising this platform we can also study the mechanism of convalescent plasma therapy on a micro-scale by introducing antibody-rich plasma into a virally infested chip. the results so obtained may be of immense importance for designing future therapies. in addition, there is immense flexibility in fig. illustration showing a lung alveoli and blood capillary interface, and b the proposed lung-on-chip platform aiming to recapitulate this alveoli-capillary interface, as it is the place where the exchange of gases takes place the design of ooc devices, it can be easily tailored to the needs of a single cell type culture or multiple cell type coculture on the same chip (yeo et al. ). while designing the 'lung-on-chip' device, the critical consideration is that the device assembly must resemble the alveolar-capillary interface as is found in the human lung. besides, a mechanism to induce the stretching of the membrane should be possible as is the case with the alveolar membranes during breathing. the device consists of three layers: an upper channel layer, lower channel and the side vacuum channels. a d microfluidic device replicating the alveolar-capillary interface has been described in fig. . it consists of two channels separated by a porous membrane. in the original practice, a pdms membrane was used; however, we have also explored the possibilities of other membranes easily available in the laboratory i.e. polyvinylidene fluoride (pvdf), nitrocellulose, polyester etc. the width of channels is selected after considering the average diameter of the human lung-alveolus. initially, the device uv-mask were designed using auto-cad and printed on transparency-sheet using the high-resolution printer. further, the master-moulds of su- polymer were fabricated using the photolithography technique. later, the upper and the lower channels were fabricated separately using pdms polymer, via the general soft-lithography technique. afterwards, the upper and lower channels were bonded together using oxygen plasma, after proper alignment under the microscope, keeping the membrane sandwiched in between the two channels. the microchannel network (fig. ) and its respective geometry and purpose are be defined below: (i) the upper channel: it is lined with the alveolar lung cells, and oxygen/air-flow is introduced through the channel. this channel has a greater depth as compared to the lower channel. (ii) the porous membrane: it is the membranous-interface, from where the diffusion of nutrients and waste material takes place, as well as, it is also the site for the cell-cell interactions. in general, this membrane needs to be very thin and stretchable. the porous membrane is present between the upper and lower cell culture channels. (iii) the lower channel: it represents the endothelial lining of the blood vessels that exchanges oxygen from alveolus in return of carbon dioxide. (iv) side vacuum channels are for inducing relaxation and contraction as experienced by the human lung. the above-mentioned fabrication process has resulted in the development of an efficient device with appropriate geometry and dimension, having ideal microchannel bonding strength. both upper and lower channel is made of pdms (polydimethylsiloxane) which is a transparent biocompatible polymer, along with polymer membrane, which is observed to be permeable to life-supporting gases. once the device was fabricated after soft-lithography and bonding; the cells were seeded and immobilised inside the channels at % confluence. the cells were regularly observed under the phase-contrast microscope to assess for their normal morphology and health (fig. ) . initial results of staining and microscopy observations have shown excellent cell-viability inside the channels through live-dead cell staining. in this section, we describe the overall methods used and intermediate results we obtained till now. we describe our work plan and timeline under the sub-heading 'timeline envisaged' for the future work through an illustrative diagram. human hepatocellular carcinoma (hepg ) cells were procured from the national cell repository (ncss, pune, india) and were cultured and maintained according to the atcc guidelines. cells were maintained in dmem (sigma-aldrich, germany) supplemented with % fetal bovine serum (fbs) (pan biotech, germany), µg/ml of penicillin, streptomycin, and . μg/ml amphotericin. cells were grown and maintained under conditions of % co with % atmospheric air at °c temperature in a humidified incubator. for propidium iodide (pi) and hoechst staining cells were seeded in mm plate on both membranes (nitrocellulose and polyester) and control plate and allowed to grow to - % of confluency. after h, the cells were stained with a blue fluorescent nuclear dye hoechst (sigma-aldrich, . µg/ml) and red fluorescent dye pi (thermo fisher scientific, µm) for dead cells and incubated for a minimum of h at °c. subsequently, the cells were rinsed with pbs, and fresh media was added. cells were observed for live-dead cells under a nikon upright fluorescence microscope with water immersion objectives (model evolution fig. fluorescence images of hepg cells cultured on both nitrocellulose and polyester membrane and stained via pi/ hoechst dual-staining process performed for h at °c. both nitrocellulose and polyester membranes exhibit cell adhesivity and viability similar to control. cells were visualized for live/dead cells using live-cell imaging. hoechst fluorescence images indicate that all cells have a normal healthy and intact nuclei; pi fluorescence images represent dead cells vf, media cybernetics, usa) microscope, and images were taken at × magnification. in our experimental study, we envisage examining some of the cost-effective and commonly available synthetic biopolymer membranes as a substitute to the porous/semipermeable pdms polymer membrane that works similar to it in our ooc device and exhibits better cell-adhesion and viability. these membranes are also promising because they are chemically and mechanically stable, and biologically inert. in addition, its porous structure allows it for the exchange of biomolecules. in our study, we analysed the effect of nitrocellulose and the polyester membrane. these membranes were tested for its ability to support cell viability and adhesivity through dual staining with pi and hoechst for live/dead cell nuclei using hepg cells as a cell model for hepatocytes followed by live-cell microscopy. strikingly, we observed that both the membranes were efficient for adhesion of cells, and cells remained viable even after h. in both membranes, high cell viability was maintained similarly to the control as shown in fig. . hoechst staining revealed that all cells have intact and evenly shaped nuclei. in terms of the pi staining, images displayed non-apoptotic changes in cells. this outcome confirms that both membranes are suitable for cell adhesivity and viability and might serve as promising candidates for the porous/semi-permeable polymeric membranes in our occ device. based on the results obtained during this study and based on available literature we can assert that these membranes will also serve as excellent substrates for the cells lines used for the lung-on-chip device. li et al. ( ) performed a study concluding that nitrocellulose membrane displays excellent cytocompatibility of different cell lines on nitrocellulose membrane for tissue culture. due to its affinity towards membrane proteins, nitrocellulose membrane provides essential support for cell adhesion. apart from being porous, this membrane can be rendered transparent enabling easy visibility of cells under microscopes. hanke et al. ( ) in their elegant study demonstrated that excellent and immediate cell adhesion on both sides of the permeable polyester membrane by simple surface enhancement techniques of dielectrophoresis and electrostatic forces enabling a quick cell adhesion, even against gravity. therefore, both these membrane can effectively be utilised for our intended lung-on-chip platform. the technology innovation supports advanced in-depth insights concerning the covid- and lung-cell interactions, requisite biomarker and transcription of various factors that need to be studied including enzyme-linked immunosorbent assay (elisa) for il- and il- , mucus secretion, cell surface p-gp expression, sodium fluorescein permeation etc. the major goals of our study have been illustrated in fig. . there is immense flexibility in the plan of the proposed microfluidic device, as it can be customized, according to the specific needs of individual cell types and cell cocultures, and can be implemented on the same chip. the reported 'lung-on-chip' devices closely mimic the cell's natural microenvironment; for example, by continuous culture perfusion or by creating chemical gradients. further, there are possibilities of analysing low numbers of cells or single cells in high temporal and/or spatial resolution via automation, parallelization, on-chip analysis, as well as schematic for our objective of the proposed study. the major goals include: a studying antigen-antibody interaction, and b screening of the potential drug candidates for toxicity, which will aid in c speeding of drug development. d prediction of any outcomes that may arise as a result of drug treatment. further, it will be possible to e study disease mechanisms at micro levels which would give us useful insights in fighting the disease. f other drugs that are available in the market and show potential to fight covid- can be tested and repurposed accordingly direct coupling to downstream analytical chemistry platforms. one of the major issue associated with drugs is hepatotoxicity, and hence by combining this platform with the liver-on-chip platform already developed by us, we can get a deeper insight into the pharmacokinetics of the drug. this research documented herein requires an interdisciplinary approach as it requires the expertise of researchers working in the domain of microfluidics, mammalian cell culture and virology. to fabricate the chip using photolithography, appropriate consumable fabrication-polymer materials i.e. biocompatible polymer poly-dimethyl siloxane (pdms) and su- photoresist; however, other low-cost biocompatible polymers may also be explored. the basic laminar hood is essential to ensure the contamination-free fabrication of the cell-impregnated chip. the essential equipment set-up exclusively used for producing such contaminationfree micro-devices, includes desiccator, centrifugal mixer, spin-coater, uv-curer, hot plate, hot air oven, plasma system, etc. in addition, while performing dynamic cell culturing, syringe-pumps and peristaltic-pumps would be required for continuous controlled flow of fluids at precise and very low flow rate. the entire chip can be moved from one place to another without any concern, ensuring that the surface remains contamination-free. for this purpose, the device needs to be packed suitably and kept in an appropriate thermally controlled atmosphere to maintain the cell viability. to mimic the alveolar-capillary interface, calu- (cultured in dulbecco's modified eagle's medium: f- supplemented with % fbs, % l-glutamine, % penicillin and % non-essential amino acids) and egm- mv human lung microvascular endothelial cell line will be required, along with other reagents such as the specific culture media, buffers, antibiotics, antimycotic solutions, pbs, fbs, trypsin. the types of equipment present in a standard mammalian cell culture lab are sufficient to transfer and incubate cells inside the device. to study the disease model and pathophysiology of covid- , the cells would have to be infested with the isolated virus. as this is potentially hazardous, therefore, bsl- and bsl- plus labs would be required to perform these studies. the government of india has approved labs, with bsl- and bsl- plus, across the country for covid- testing and conducting research. because the devices are portable, these can easily be transferred to distant laboratories for carrying out the requisite tests. figure explains the entire work plan in a nutshell. the proposed study can be divided into three main stages: . design, fabrication and rapid prototyping of the device ( - days): the devices are proposed to be fabricated via the soft-lithography process. to begin with, the channels would be designed using the autocad software. the mask would then be printed on transparent sheets with the help of high-resolution inkjet printer and would be left to dry in sterile conditions and cut in the appropriate size. device fabrication will be done using optimised cost-effective photolithography process which does not necessarily require a clean-room facility. all the steps are performed under a laminar hood. (i) su- master fabrication: the first step in the fabrication is developing a master using su- polymer, which is an epoxy-based negative photoresist. substrate surface preparation: the glass slides ( × mm) would be used as the substrate for developing the su- master. initially, the glass slides will first be cleaned through acetone boiling using sonicator followed with detergent and di water. further, wiped with ipa to ensure perfect cleaning. thermal treatment will be given via heating the slide at °c for min on a hotplate spin coating: since our device consists of two channels of varying depths, hence for each channel su- mask will be required. as the thickness of the su- layer varies with the rpm, hence, the final thickness of the microstructure will be defined by adjusting the rotational speed of spin coater chuck. pre-bake: this is done on a hotplate at two different temperatures. first, at °c to create slow evaporation of the solvent resulting in a uniform coating and enhanced adhesion to the substrate. the second cycle consists of gradually heating the substrate with the su- layer up to °c, to densify the su- . exposure using uv-aligner: placing the su- polymer-coated glass-slide, beneath a predesigned uv-mask, and exposed with uv-aligner, for a defined period to suitably cross-link the polymer. post-bake: this step is accountable for increasing the degree of cross-linking in the su- irradiated areas (due to the photoacid generated in the uv exposure step) making it resistant to the action of solvents in the developing step. the temperature is applied gradually to reach two specific temperatures: °c followed by °c. development of the su- microstructure: for the final su- microstructure, it is necessary to remove the non-polymerized su- . to achieve this, the microstructures are immersed in a container with su- developer from microchem while continuously shaking the container to give a rigorous wash to the master mould. rinse and dry: once all the non-polymerised su- is dissolved in the developer, it is wiped with a clean wipe and rinsed with a stream of di water. passivation of the su- master: to make the su- master more durable and resistant to peel off, the silanization process is necessary. to do this, the su- mould is placed in the vacuum desiccator and few drops of aptes (silanizing agent) is placed, in a small open container using a disposable dropper. further, a vacuum environment is quickly created and the lid of the desiccator was sealed. the vacuum pump is switched off and the setup kept stand like this for an hour so that a passivation monolayer is formed on the su- mould. the process is always done inside the fume hood. (ii) pdms polymer casting and rapid prototyping: for making the upper and lower channel, first the pdms elastomer was mixed with few drops of curing agent in the ratio : (wt/wt) or : (v/v) and vigorous mixing is done manually or via mixers for around min. once the mixing is completed, it is kept inside desiccator to remove all the bubble formed as a result of vigorous mixing. the duration of this degasification may range from min to h fig. schematic diagram for the overall plan of the proposed work, illustrating the major planned steps in the entire study. cells from petri dish is seeded inside the device channels (both upper and lower) at specified cell density (cells/ml). the device is then incubated overnight. to check cell viability, cells are stained with fluorescent dyes like hoechst and pi. further to perform studies under dynamic conditions, the platform is connected with pumps keepin the floe rates very precise and slow. the media is changed routinely and all assays are performed under controlled flow conditioned depending upon the number of bubbles. while degasification is in progress, the hot plate is maintained at °c. the temporary boundary is made around the su- master using aluminium foil, ensuring for no leakage and the bubble-free pdms prepolymer mixture were poured/cast in it. pouring/ casting should not start from the region where the microstructures are present. the complete pdms casting arrangement is placed on the hotplate, which is maintained at °c for min and the mould is removed from the hotplate. the foil boundary is removed and the pdms layer is gently peeled off, and inlets and outlets holes were created at preplanned places using . mm hole puncher. the membrane to be used in between the channels is incised precisely by observing under a microscope. the width of the membrane should be slightly greater than the channel width and length equal to the length of the channel. the membrane is treated with corona treater for min by doing sweeping motions and it is placed on top of the bottom pdms layer under a microscope. the top pdms layer is treated with corona treater for min and it is placed on top of the membrane under a microscope to ensure proper alignment. the whole assembly is kept inside the oven at °c overnight ( - h). further, the assembly is taken out and brought at room temperature, and with a sharp scalpel, the edges of the device are trimmed and cleaned. finally, it is kept in a clean closed box. . cell seeding and culturing inside the channels ( - days): the cells will be cultured following the standard protocol given by the manufacturer following all measures to avoid contamination. a confluency of - % is required before seeding cells inside the device. the cell seeding density (cells/ml) is always done according to the volume of the channels and the days of continuous incubation of cells in the device. the counting of cells before seeding is done using a haemocytometer. before seeding the cells inside the channels, sterility of the channels should be ensured to avoid contamination. to do this, % ethanol (v/v) is perfused for min followed by washing with autoclaved di water. further, the device is placed under uv light for - min. once the device is sterilised completely, the cells are seeded as done routinely. the pipet tip the placed on the inlet of the channel and the suspension is dispensed swiftly. the device with the seeded cells is placed inside the incubator for h. the cells are visualized under the microscope for cell adhesion. if the cells have adhered properly, the inlet/outlet ports of the top channel are closed and the device is reversed to seed cells inside the lower channel via repeating the same step. to ensure a long term contamination-free health cell culture, the media may be changed every/alternate day depending on confluency. . immunological staining for cell viability and functionality testing ( - days): (i) staining with hoechst and pi: to observe live/dead cells, the cells are stained with hoescht and pi dye using the abovementioned process applied in the intermediate result section and subsequently observed under the microscope. (ii) enzyme-linked immunosorbent assay (elisa) for il- and il- : the media collected from the chips will be stored at − °c until experimenting. after thawing them, the levels of secreted il- and il- will be analyzed according to the manufacturer's instructions, using commercial human il- and il- elisa kits to find the corresponding absorbance (shrestha et al. ). (iii) immunofluorescence staining of intercellular junctions: at room temperature, the cells are fixed with % volume/volume (v/v) paraformaldehyde in pbs after washing three times with pbs. it is incubated for min and washed with pbs twice. follow permeabilization of the cells with . % (v/v) triton x- for min and blocking with % (w/v) bovine serum albumin (bsa) in pbs for h. for staining of endothelial junctions, primary ve-cadherin antibody is diluted in bsa solution at a dilution of : , and introduced into the lower channel. the mixture is then incubated at °c overnight. antibody solution is removed by flushing the channels with d-pbs three times. fluorescently labelled goat anti-rabbit antibody is diluted in : bsa solution and introduced into the lower channel. it is incubated at room temperature in the dark for h. antibody solution is removed by flushing the channels with d-pbs three times. the device is now ready for visualization of endothelial adherens junctions using fluorescence microscopy. the whole process is repeated with fluorescently labelled anti-occludin antibody in the upper microchannel to visualize tight-junction formation in the epithelial cells (huh et al. ). . drug efficacy and toxicity testing ( - days): to study the drug efficacy/toxicity of various drug candidates, the primary requirement is to infest the chip with the isolated virus under bsl- laboratory. once the chip is infested, the appropriate dosage of the drug would be given and drug toxicity study would be performed using mts assay kit according to the manufacturer's protocol. . studying the mechanism of convalescent plasma therapy: to study the underlying mechanism and the associated immune responses-like variations in cytokine activity at the cellular level, we will introduce plasma containing the antibody inside the infested channels. the proposed 'lung-on-chip' platform is a simple yet innovative device containing a defined network of microfluidic channels lined by living human cells on the microchannel wall. these may replicate the smallest functional units of organs and organ-level physiology. the protocol followed here can be customised for other tissues or organs, as well, to study the effect of the drug on multiple organs. it is a rapid and low-cost alternative to the lengthy conventional drug testing pipeline that involves animal testing which would undoubtedly be an advantageous study tool in pandemic situations calling for urgent measures. we are in the initial experimental stages with very promising results. with further experimentation and validation, this platform would help tackle a resolve several issues associated with various aspects of covid- resulting in designing therapies that are effective and elicit negligible side effects. convalescent plasma as a potential therapy for covid- lessons from a failed γ-secretase alzheimer trial human lung on a chip: innovative approach for understanding disease processes and effective drug testing advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices generating cell co-cultures by rapid cell adhesion on opposite sides of polyester membranes can the pharmaceutical industry reduce attrition rates? use of nitrocellulose membranes as a scaffold in cell culture revisiting lab-on-a-chip technology for drug discovery convalescent plasma to treat covid- : possibilities and challenges treatment of critically ill patients with covid- with convalescent plasma a rapidly prototyped lung-on-a-chip model using d-printed molds cytokine storm in a phase trial of the anti-cd monoclonal antibody tgn covid- : fda approves use of convalescent plasma to treat critically ill patients microphysiological systems modeling acute respiratory distress syndrome that capture mechanical forceinduced injury-inflammation-repair the relevance and potential roles of microphysiological systems in biology and medicine world health organization ( ) coronavirus disease (covid- ): situation report, . world health organization cultured human airway epithelial cells (calu- ): a model of human respiratory function, structure, and inflammatory responses publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -us dybue authors: kanjanahaluethai, amornrat; chen, zhongbin; jukneliene, dalia; baker, susan c. title: membrane topology of murine coronavirus replicase nonstructural protein date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: us dybue mouse hepatitis virus (mhv) is a member of the family coronaviridae. these positive strand rna viruses encode a replicase polyprotein that is processed into nonstructural proteins (nsps). the nsps assemble with membranes to generate double membrane vesicles, which are the sites of viral rna synthesis. mhv nsp contains multiple domains including two papain-like protease domains, plp and plp , and a predicted transmembrane (tm) domain. in this study, we determined the membrane topology of nsp -tm and showed that tm-mediated tethering of plp is important for processing at cleavage site . biochemical analysis revealed that nsp is an integral membrane protein that is inserted into endoplasmic reticulum (er) membranes co-translationally and glycosylated at asparagine- . proteinase k digestion experiments indicate that the tm domain of nsp has membrane-spanning helices. we show that nsp -tm is sufficient to mediate er membrane association of a cytosolic protein. this study is the first detailed analysis of the topology and function of the coronavirus nsp tm domain. proteolytic processing of a replicase polyprotein and the generation of a membrane-associated replication complex are common themes in studies analyzing the replication of positive strand rna viruses. for the majority of coronaviruses, the proteolytic processing of the viral replicase polyprotein is mediated by three distinct viral proteinases to generate replicase products [reviewed in (ziebuhr, ) ]. both coronavirus and the related arterivirus replicase products have been shown to assemble with cellular membranes to generate double-membrane vesicles that are the sites of viral rna synthesis (goldsmith et al., ; gosert et al., ; pedersen et al., ; snijder et al., ) . the goal of our research is to characterize the coronavirus replicase proteolytic processing cascade and identify factors that mediate membrane-association of the replication complex. our model system is the replication of mhv, one of the prototype coronaviruses. mhv is a ∼ . -kilobase (kb) positivestrand rnavirus that replicates in the cytoplasm of infected cells. the ′-most kb of the mhv genomic rna contains two large open reading frames (orfs), termed orf a and orf b (lee et al., ) . during translation of the genomic rna, the two orfs are joined via a ribosomal frameshifting mechanism to produce a polyprotein of ∼ kilodaltons (kda) in size (brierley et al., ; lee et al., ) . this polyprotein is the viral rnadependent rna polymerase, termed the replicase. the mhv replicase is processed by three distinct proteases contained within orf a of the replicase polyprotein. two of the proteases are papain-like cysteine proteases, termed plp and plp . the third protease domain is distantly related to the picornavirus family of c-like proteases, and is termed clpro. these three proteases process the replicase polyprotein to produce intermediates and products that function in the replication of genomic rna and the synthesis of a nested set of subgenomic mrnas [reviewed in (brian and baric, ; sawicki and sawicki, ) ]. studies have shown that the replicase products alone are sufficient to mediate viral rna synthesis , and to generate the double-membrane structures that serve as the sites for viral rna synthesis (pedersen et al., ) . however, the role of proteolytic processing in regulating the assembly and function of the mhv replication complex is not yet understood. therefore, we wanted to identify regions of the replicase polyprotein that may direct or regulate proteolytic processing and/or membrane association. the focus of this study was to identify the regions of mhv nsp that direct membrane association and plp activity. previously, we showed the mhv plp can act in trans to process cleavage site (cs ) at the nsp /nsp junction (kanjanahaluethai and baker, ; kanjanahaluethai et al., ) . other coronaviruses, such as the human coronavirus e, have been shown to utilize plp (also termed pl pro) to process sites both upstream and downstream of the catalytic domain (ziebuhr et al., ) . the papain-like protease (plpro) encoded by the coronavirus that causes severe acute respiratory syndrome (sars-cov) processes three sites in the replicase polyprotein (harcourt et al., ) , and has recently been shown to have de-ubiquitinating activity (barretto et al., ; lindner et al., ) . the crystal structure of this enzyme has been resolved and is currently being targeted for anti-viral drug development (ratia et al., ) . a better understanding of the coronavirus papain-like proteases may facilitate anti-viral drug development for sars and also other recently identified human coronavirus infections caused by nl (van der hoek et al., ) and hku (woo et al., ) , which can cause pneumonia and respiratory tract infections in children and the elderly. the aims of this study were to determine the topology of mhv nsp and to identify the regions in nsp required for plp activity. we used a trans-cleavage assay to determine if an expressed plp domain was sufficient for the recognition and processing of a substrate containing cs . we found that constructs containing plp and the downstream putative tm domain were able to efficiently process the substrate. bioinformatic analysis of the nsp -tm domain indicated the presence of putative membrane-spanning helices and consensus sites for n-linked glycosylation. site-directed mutagenesis of the asparagine residues and analysis of endoglycosidase h (endo h) sensitivity revealed that asparagine- in nsp -tm is glycosylated. to investigate the topology of nsp -tm, we tested for sensitivity to digestion with proteinase k and found that at least two lumenal domains are protected, consistent with membranespanning regions in nsp -tm. we showed that nsp -tm alone is sufficient to confer membrane association to a normally cytosolic protein, enhanced green fluorescent protein (egfp), and that a predicted multi-spanning tm domain is conserved in nsp of all coronaviruses. thus, the nsp -tm domain is important for membrane association of the replicase and tethering the plp domain for viral polyprotein processing activity. the mhv nsp -tm domain is important for plp processing at cleavage site analysis of sars-cov plpro activity showed that the nsp hydrophobic domain downstream of plpro is essential for processing at cs (harcourt et al., ) . to determine if a similar domain is important for mhv plp activity, we generated a series of plp c-terminal deletion constructs and tested each construct for expression and processing activity. plp expression constructs were generated by polymerase chain reaction (pcr) amplification and cloning of the amplified region into pcdna . /v -his, as described in materials and methods. to determine if each clone was expressing a protein of the expected size, we analyzed the products after t -mediated expression and immunoprecipitation (fuerst et al., ; kanjanahaluethai and baker, ) . we detected the expected series of truncated plp proteins that ranged in size from ∼ kda to ∼ kda (fig. b, lanes - ) . to determine if these plp products were sufficient to mediate processing of cs , we tested each construct in the trans-cleavage assay by co-transfection with the substrate (fig. c, lanes - ) . we found that only two of the plp expression products, pplp - and pplp - , were able to efficiently process the substrate and produce the -kda cleavage product, nsp (fig. c, lanes and ). these two constructs encompass all or a major part of the predicted nsp -tm domain indicating that membrane tethering of plp is important for recognition and processing at cs . thus, both sars-cov plpro (harcourt et al., ) and mhv plp require the downstream tm domain for recognition and processing at the nsp /nsp junction. bioinformatic analysis of nsp -tm predicts a series of putative membrane-spanning sequences previously, we showed that mhv nsp is indeed an integral membrane protein, but the role of the tm in mediating this membrane association was not investigated (gosert et al., ) . initial bioinformatic analysis indicated two transmembrane helices in nsp (ziebuhr et al., ) . to extend these studies of membrane association of coronavirus replicase products, we analyzed the amino acid sequence of mhv-jhm nsp (from glycine- to glycine- ) for probability of transmembrane helices using the five different programs designed to search for putative membrane-spanning sequences: phobius, tmhmm, hmmtop, sosui and tmpred (fig. ) . interestingly, each program generated a unique prediction for the topology of nsp (fig. c ). the number of predicted membrane-spanning domains varied from three (phobius) to seven (tmpred). however, since both the n-and c-termini of nsp are cleaved in the cytosol, the number of membrane-spanning helices must be either two [as previously predicted (ziebuhr et al., ) ], four or six. to better understand the topology of the nsp -tm domain, we performed membrane-association, fractionation and proteinase k protection experiments. to determine if the nsp -tm is indeed required for membrane association, we performed in vitro transcription and translation of the plp expression constructs in the absence or presence of canine microsomal membrane (cmm) and assayed for membrane association. the newly translated proteins were metabolically radiolabeled with [ s]-translabel, subjected to centrifugation to separate the membrane-associated pelleted fraction from the soluble fraction. protein products of both fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page), and visualized by autoradiography (fig. ) . the percentage of total proteins detected in soluble and pelleted fractions was quantitated by phosphoimaging analysis. in the absence of cmm, the translated protein products were detected predominantly in the soluble fraction (fig. a) . in contrast, when cmms were added to the mixture, protein products that included all or part of nsp -tm (plp - , - and - ) were detected predominantly in the pelleted fraction, consistent with membrane association (fig. b ). to determine if the membrane association occurred co-translationally or post-translationally, we added the cmms after termination of translation, and assessed membrane association. previous studies with hepatitis c virus revealed post-translational insertion of the ns a replicase protein (brass et al., ) . we found that the translation product of plp - is inserted co-translationally, with no detectable post-translational insertion into membranes (fig. c ). these results indicate that mhv nsp membrane insertion likely occurs via the normal, signal recognition particle (srp)-dependent er translocation mechanism, and not via a tail-anchoring or post-translational mechanism. to distinguish between membrane association versus integral membrane insertion, membrane extraction experiments were performed. protein products expressed from pplp - , pplp - and pplp - constructs were translated in vitro in the presence of cmm and the pelleted fraction was subsequently subjected to differential extraction methods as indicated in fig. . as expected, treatment with nte buffer alone had no effect and the proteins pelleted with membranes, whereas treatment with detergent triton x- disrupted the membranes, and the proteins were detected in the soluble fraction (fig. a) . treatments with urea, nacl or sodium carbonate, ph . have been shown to disrupt the association between peripheral, membrane-associated proteins, but such treatments do not disrupt integral membrane proteins (bordier, ) . we tested for disruption of plp -tm proteins and found that majority of the proteins remained in the pelleted membrane fraction, consistent with integral membrane proteins (fig. b ). we noted that the protein product expressed from pplp - , which contains only one or two predicted membrane spanning helices, was dissociated by treatment with high ph, but that the protein products expressed from constructs that extended further into the tm were not disrupted from the membranes by these treatments, consistent with the characteristics of integral membrane proteins. bioinformatic analysis also revealed two consensus sequences for n-linked glycosylation [the consensus being nx(t/s), where x is any amino acid (helenius and aebi, ) ] in nsp -tm. if one or more of the predicted sequences was indeed a transmembrane helix, then the sites for n-linked glycosylation could be lumenal, and subject to modification. to determine if either of the putative sites was modified by glycosylation, we the amino acid numbering is according to ncbi accession number nc . conserved domains previously identified by comparative sequence analysis (ziebuhr et al., ) and the recently described adp-ribose- ʺ-phosphatase (adrp) domain (saikatendu et al., ) are indicated. two putative sites for n-linked glycosylation were predicted at asparagine residues and . (b) the diagram shows the results of an analysis of the nsp for predicted transmembrane helices using tmhmm program. the predicted tm domain designates the boundaries of the putative membrane-spanning regions a, b, c, d, and e. (c) sequence of the plp -tm (amino acid residues to ) were analyzed for the prediction of transmembrane domains using phobius, tmhmm, hmmtop, sosui and tmpred programs. subjected the proteins expressed from wild-type pplp -cen (kanjanahaluethai and baker, ) and two asparagine-toalanine substitution mutants to endo h treatment (fig. ) . expressed proteins were radiolabeled with [ s]-translabel, lysates were prepared, and subjected to immunoprecipitation with anti-v antibody as described in materials and methods. the plp -cen protein was either untreated, or treated with endo h for h, then analyzed for mobility by electrophoresis through a % polyacrylamide gel. we found that the untreated wild-type protein migrated at approximately kda (lane ). however, after digestion with endo h, the protein migrated at kda (lane ), consistent with the loss of one n-linked modification. the plp -cen-n a protein with an asparagine-to-alanine substitution migrated more quickly than the wild type or n a protein, indicating that n is the site modified by n-linked glycosylation (compare lanes - ). furthermore, proteins generated by endo h treatment of the pplp -cen-n a and pplp -cen-n a migrated more quickly than wild type plp -cen (lanes - ). these results indicate that nsp is glycosylated at asparagine- . overall, these experiments demonstrate that nsp -tm does have transmembrane and lumen sequences that can tether the plp domain to intracellular membranes. to further investigate the topology of nsp -tm, we tested for sensitivity to proteinase k digestion. cytosolic domains are sensitive to proteinase k, whereas transmembrane and lumenal domains are protected from digestion with proteinase k. analysis of plp - revealed two major fragments of kda and kda protected from proteinase k digestion (fig. , lane ) . we noted the predicted size of the tm domain from k -y is . kda, and the predicted size of the k -i (a and b helices) is . kda. analysis of plp - revealed two protected fragments of kda and kda, respectively (fig. , lane ) . the predicted size of the k -s fragment is . kda. by combining the results of the integral membrane assays (figs. and ) , the glycosylation assay (fig. ) , and the proteinase k sensitivity assay (fig. a ), we were able to generate a topology model for nsp -tm that is consistent with all the data (figs. b and c). our results indicate that nsp -tm has four membrane-spanning sequences and two lumenal domains (fig. b ) and that the kda fragment likely represents the proteinase k resistant fragment generated by the first two membrane-spanning sequences. our model is most similar to the predicted models generated by the tmhmm and sosui programs with the exception that the fourth predicted membrane-spanning sequence (which is the only domain not consistent in these two predictions) is not a membrane-spanning sequence, but instead remains lumenal, and the final membranespanning domain is in the reverse orientation. our results, and the results of others (hugle et al., ; miller et al., ) , demonstrate the importance of experimental validation of bioinformatics predictions of membrane-spanning sequences. for example, in the case of dengue virus type nonstructural protein b, miller and co-workers found that two computerpredicted transmembrane helices were in fact lumenal and one was glycosylated (miller et al., ) . in addition, we note the value of using multiple programs to better estimate the complexity of the bioinformatic prediction. the differences in the predicted models could then be tested experimentally. here, we provide an initial model of nsp -tm that should be refined by further experimentation. similar studies should also be performed to determine the topology of nsp and nsp , the other coronavirus replicase products with multiple predicted transmembrane helices. finally, to determine if the tm domain of mhv nsp was sufficient to confer membrane association to a cytosolic protein, we appended nsp -tm to egfp and determined the localization of the fusion protein using confocal microscopy (fig. ) . egfp normally is distributed throughout the cell (fig. a) . we found that appending the mhv nsp -tm sequence to egfp fig. . membrane extraction experiments of the plp expressed protein products treated with triton x- , urea, nacl or sodium carbonate solution ph . . in vitro transcription and translation reactions of pplp - , pplp - or pplp - were performed in the presence of cmm. subsequently, reaction mixtures were centrifuged to sediment cmm containing associated plp protein. the supernatant were removed and the pellets were resuspended in nte buffer or . % triton x- (a), m urea, m nacl or mm sodium carbonate solution ph . (b) and incubated for min at °c. subsequently, membrane sedimentation analyses were performed as described under materials and methods. soluble (s) and pellet (p) fractions were applied in equivalent amounts and subjected to % sds-page analysis. quantitation was performed by phosphoimaging and values expressed in % were given at the bottom and depicted as bars. was sufficient to tether it to membranes, as shown by the intense, perinuclear localized signal (fig. b ). to determine if the nsp -tm domain is retained in the er membranes or is transported through the medial golgi, we radiolabeled the efgp-nsp tm protein in transfected cells, immunoprecipitated the protein and subjected the immunoprecipitated products to endo h. we found that efgp-nsp tm is sensitive to treatment with endo h, indicating that the protein is retained in the er and does not pass through the medial golgi. thus, the nsp -tm domain is sufficient to confer membrane-localization and retention in the er. these studies are in agreement with our previous findings showing that the tm domain of sars-cov nsp (previously termed the hd) confers membrane association of egfp (harcourt et al., ) . in summary, using biochemical fractionation and proteinase k protection assays, we show that nsp -tm likely has four membrane-spanning domains, and that lumenal residue asparagine- is modified by glycosylation. furthermore, we found the region nsp -tm domain is required for efficient mhv plp process activity at cleavage site in the polyprotein. why does mhv plp require membrane association for proteolytic processing of the plp cleavage site? one possible explanation is that membrane-tethering brings plp into close proximity with a membrane-associated substrate. it is also possible that the nsp -tm membrane tether is important for anchoring the replicase complex to intracellular membranes. bioinformatic analysis of the nsp of other coronaviruses revealed that the membrane-spanning features of nsp -tm are conserved in all viruses (fig. , analysis using the tmhmm program is shown as an example), even though the amino acid identity is relatively low ( - % identity within nsp ). therefore, the tm domain is likely to be important for both plp activity and assembly of the replication complex. further studies will be required to determine the precise topology of the tm domain in other coronaviruses, and if the lumenal sequences in nsp -tm play any role in interacting with host factors during viral replication. hela cells expressing the mhv receptor, hela-mhvr cells (gallagher, ) were used for all transfection experiments. the cells were grown in dulbecco's modified eagle's medium supplemented with % fetal bovine serum (invitrogen, carlsbad, ca), . % penicillin/streptomycin, % glutamine, and mm sodium n- -hydroxyethylpiperazine-n′- ethanesulfonic acid, ph . . recombinant plasmid dna constructs expressing the plp coding region were generated using specific primers (listed in table ) to amplify the designated region from the parental plasmid pplp -cen (kanjanahaluethai and baker, ) . the region of interest was generated by pcr amplification using la-taq polymerase according to the manufacturer's instructions (clontech, palo alto, ca). the amplified region was then digested with restriction enzymes bamhi and xhoi and ligated into the corresponding sites in the pcdna . /v -his expression vector (stratagene, la jolla, ca) using t ligase (new england biolabs). the ligated dna product was transformed into xl- blue competent cells according to the manufacturer's instructions (stratagene), except that the bacteria were grown at °c. hela-mhvr cells were infected with a recombinant vaccinia virus expressing the bacteriophage t polymerase (vtf - ) at a multiplicity of infection of . then, infected cells were co-transfected with recombinant plasmid dnas encoding the mhv-jhm indicated protease domain and the substrate using lipofectamine according to manufacturer's instruction as previously described (fuerst et al., ; kanjanahaluethai and baker, ) . newly synthesized proteins were metabolically labeled with μci/ml [ s]-translabel (icn, costa mesa, ca) from . to . h post-infection (hpi). to harvest the cells, radioactive labeled cells were washed with phosphate buffered saline (pbs), and cell lysates were prepared by scraping the cells in lysis buffer a [ % sds, % dtt, % glycerol and . m tris, ph . (schiller et al., ) ]. the lysates were either used directly for immunoprecipitation assays or stored at − °c for future studies. radiolabeled cell lysate was diluted in . ml ripa buffer [ . % triton x- , . % sds, mm nacl, mm edta, and mm tris-hcl, ph . (schiller et al., ) ] and subjected to immunoprecipitation with anti-v monoclonal antibody (invitrogen) and protein-a sepharose beads (amersham biosciences, piscataway, nj). the immunoprecipitated products were eluted with × laemmli sample buffer, incubated at °c for min, and analyzed by electrophoresis on a . - . % gradient polyacrylamide gel containing . % sds. following electrophoresis, the gel was fixed in % methanol- % acetic acid, enhanced with amplify (amersham biosciences) for min, dried, and exposed to kodak x-ray film at − °c. site-directed mutagenesis of putative glycosylation sites in mhv-jhm nsp -tm domain plasmid dna pplp -cen which encompasses mhv-jhm gene amino acid residues - (kanjanahaluethai and baker, ) was subjected to site-directed mutagenesis at positions and for pplp -n a and positions and for pplp -n a using synthetic oligonucleotides with mismatches encoding specific nucleotides changes as shown in table . mutagenesis was performed according to the manufacturer's instructions (quickchange site-directed mutagenesis; stratagene), and as previously described (kanjanahaluethai and baker, ) . mutations were confirmed by dna sequence analysis. the tnt t -coupled reticulocyte lysate system (promega, madison, wi) was used according to the manufacturer's instructions. the recombinant plasmid dna encoding the designated plp region was linearized by digestion with pmei. in vitro transcription and translation was performed for min at °c in the presence of . μci of [ s]-translabel per ml in a volume of μl. where indicated, . μl of cmm (promega) was added prior to the incubation. for analysis of membrane association, the products of in vitro transcription and translation were centrifuged at , rpm for min. the supernatant was removed, the pellet that may contain aggregated or membrane-associated protein was suspended in × laemmli sample buffer, heated at °c for min, and both fractions were analyzed by sds-page and subjected to autoradiography. protection of translation products by microsomal membranes was examined by digestion with proteinase k (hugle et al., ) . following translation, reaction mixtures (after incubation with rnase a) were adjusted to . mg/ml of proteinase k (roche, indianapolis, in) and incubated for min on ice. proteinase k digestion was terminated by addition of phenylmethylsulfonyl fluoride to mg/ml and incubation was continued for min on ice. a portion of each reaction mixture (generally μl) was mixed with μl of × laemmli sample buffer, heated at °c for min, analyzed by % sds-page and subjected to autoradiography. for endo h treatment, lysates from vtf . -infected and pplp -tm transfected cells were prepared and subjected to immunoprecipitation as described above. protein-a sepharoseantibody-antigen complexes were washed once in ripa buffer and endo h treatment was performed as suggested by the manufacturer (roche). briefly, the complexes were resuspended in μl of mm sodium phosphate buffer, ph . , and incubated in the presence or absence of a final concentration of unit/μl of endo h for h at °c. following the incubation, μl of × laemmli sample buffer was added to each sample, mixed, and incubated for min at °c. the sepharose beads were pelleted by a brief, high-speed spin in a microfuge, and the supernatant loaded directly for analysis by % sds-page and subjected to autoradiography. for membrane extraction experiments, the pellets from μl in vitro transcription-translation reactions performed in the presence of cmm were resuspended in μl nte buffer, . % triton x- , m urea, m nacl, or mm sodium carbonate (ph . ) and incubated for min at °c (hugle et al., ) . subsequently, supernatant and pellet fractions were separated by centrifugation at , rpm for min, and analyzed by sds-page and autoradiography. quantitation was performed by using phosphoimaging analysis. the putative transmembrane domain region of mhv-jhm nsp (nt to ) was pcr amplified from the pplp -cen (kanjanahaluethai and baker, ) with primers b and b (table ) , cloned into the mammalian expression vector for egfp, pegfp-c (bd biosciences), and designated pegfp-nsp tm. the plasmid dna encoding egfp or egfp-nsp tm was transfected into hela-mhvr cells in well chamber culture slides with lipofectamine according to manufacturer's instructions, for h. expression of egfp and egfp-nsp tm fusion protein products was detected by confocal microscopy (zeiss lsm lazer-scanning confocal microscope). egfp-nsp tm was amplified by primers zcp and zcp (table ) using pegfp-nsp tm as template, and then cloned into bamhi and xbai sites of pcdna . /v -hisb (invitrogen) to generate the construct of pcdna . -egfp-nsp tm. the plasmid dna was expressed via the vaccinia virus-t expression system, proteins radiolabeled with stranslabel, cell lysates were subjected to immunoprecipitation with anti-v antibody, and products were either incubated with endo h or buffer alone, and analyzed by electrophoresis on % sds-page. the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity phase separation of integral membrane proteins in triton x- solution an amino-terminal amphipathic alpha-helix mediates membrane association of the hepatitis c virus nonstructural protein a coronavirus genome structure and replication an efficient ribosomal frame-shifting signal in the polymerase-encoding region of the coronavirus ibv eukaryotic transientexpression system based on recombinant vaccinia virus that synthesizes bacteriophage t rna polymerase murine coronavirus membrane fusion is blocked by modification of thiols buried within the spike protein rna replication of mouse hepatitis virus takes place at double-membrane vesicles identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity intracellular functions of n-linked glycans the hepatitis c virus nonstructural protein b is an integral endoplasmic reticulum membrane protein identification of mouse hepatitis virus papain-like proteinase activity identification of the murine coronavirus mp cleavage site recognized by papain-like proteinase the complete sequence ( kilobases) of murine coronavirus gene encoding the putative proteases and rna polymerase the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme subcellular localization and membrane topology of the dengue virus type non-structural protein b open reading frame a-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme structural basis of severe acute respiratory syndrome coronavirus adp-ribose- ʺ-phosphate dephosphorylation by a conserved domain of nsp coronavirus transcription: a perspective processing of the coronavirus mhv-jhm polymerase polyprotein: identification of precursors and proteolytic products spanning kilodaltons of orf a ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex viral replicase gene products suffice for coronavirus discontinuous transcription identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku , from patients with pneumonia the coronavirus replicase the autocatalytic release of a putative rna virus transcription factor from its polyprotein precursor involves two paralogous papain-like proteases that cleave the same peptide bond we thank nicole kreuziger, kari severson and ami knop ullrich for excellent technical assistance, and dr. alexander gorbalenya, leiden university medical center for useful suggestions during the course of this work. this research was supported by public health service research grants ai and hhsn c. key: cord- - g kmpdi authors: makino, hisao; emi, hitoshi; yamaguchi, akimasa; iritani, eiji; namiki, norikazu; myojo, toshihiko; yamamoto, kenji title: environmental and safety issues with nanoparticles date: - - journal: nanoparticle technology handbook doi: . /b - - . - sha: doc_id: cord_uid: g kmpdi this chapter evaluates the relationship between nanoparticles and the environment, and describes the trouble caused by nanoparticles as well as the safety issues. the relationship between nanoparticles and the environment is clarified from the viewpoint of the kind of influence nanoparticles generated either artificially or naturally have on the environment, such as in atmosphere, groundwater, wastewaters, and exhaust gases. indoor nanoparticles originate from the several sources such as products of chemical reactions, nonvolatile residues (nvrs) of liquid droplets, printers/photocopiers, combustion, bioaerosols, and infiltration of outdoor air. the influence of nanoparticles on the indoor environment is discussed in the chapter. it describes the sources of nanoparticle generation in general industrial processes such as grinding processes, and in cleanroom or controlled environment industrial processes, such as exhaled air, ionizers, and haze by chemical reaction on solid surfaces. the chapter discusses safety issues related to nanoparticles such as possibility of dust explosion, health risks and biological effects of nanoparticle materials such as carbon nanotubes, fullerenes, nanosized metal oxides, and carbon black. the chapter also discusses methods for removing nanoparticles from gas and liquid as technology to control the influence of nanoparticles on the environment. since nanoparticles have superior surface activity and can be applied to the production of particles with various functions, they are extremely important for the future development of sophisticated material technologies. on the other hand, this superior activity of nanoparticles is a cause of trouble from the perspective of safety, and does not always have a positive influence on the environment. attention must also be paid to impact on health. nevertheless, all technologies have negative aspects, and overcoming these kinds of problems, we will be able to utilize the superior characteristics of nanoparticles for practical purposes. to achieve this goal, it is necessary to fully understand the influence of nanoparticles on the environment and the relevant safety issues. this chapter evaluates the relationship between nanoparticles and the environment, and also describes the trouble caused by nanoparticles as well as the safety issues. the relationship between nanoparticles and the environment will be clarified from the viewpoint of what kind of influence nanoparticles generated either artificially or naturally have on the environment. the influence on the indoor environment, where nanoparticles are produced, will also be clarified. the safety of nanoparticles will be clearly described from the perspective of the trouble caused by the superior surface activity of nanoparticles; the effect of the compositional characteristics of nanoparticles, and also the influence on health. a method for assessing the influence of nanoparticles using quantum dots is also explained. in the final section, methods for removing nanoparticles from gas and liquid are described as technology to control the influence of nanoparticles on the environment. in our atmospheric environment, particles ranging from several nanometers to several tenth micron orders are suspended. they are emitted into the atmosphere at the rate of . billion tons every year. emission sources are classified as either natural or artificial. natural particles occupy % of total particles, consisting mainly of salt particles (ϳ billion ton) from the sea and soil particles (ϳ . billion ton) from the land. on the other hand, the latter particles are brought about by human activities. although occupying only % of the total emitted particles, their size is mostly of submicron order and because they contain hazardous chemical components such as nitrates, sulfates, hydrocarbons, heavy metals, etc. in high concentration, their effects on the ecosystem are serious. fig. . . shows an overview of the size and concentration ranges of various aerosol particles. as it can be seen, the number concentration of atmospheric aerosol which we inhale every day ranges from several thousand particles per cm in clean area to several hundred thousands in dusty areas, and the size range lies between nm and several tens of micrometer. fig. . . shows mass-based size distribution of atmospheric aerosol particles. since the size distribution in the nanosize range appears only when the sources of particle generation exist, the size distribution is usually bimodal with peaks in the size range of a few to micron and submicron. the former peak consists of naturally generated coarse particles such as soil dust, sea salt spray, and so on. on the contrary, the latter contains plenty of artificially generated particles, some of which grow from molecules (in most cases vapor state) exhausted by human activities through chemical reaction, condensation, and coagulation. particle growth rarely leads to particles larger than m unless high concentration of vapors or particulate matters which cause the above-mentioned growth mechanisms exist in the atmosphere. as it can be seen from the differences in the particle generation process, fine particles generated from molecules or nanoparticles are much more complicated in their chemical component than the coarse particles, and sometimes have serious adverse health effects. such fine particles are called pm . , which is defined for particles less than . m including nanosized particles. recent epidemiologic investigation reports that the concentration of pm . showed a positive correlation to the mortality due to pulmonary diseases [ ] . various research techniques are used in order to understand the process of particle growth and to trace back to the source of pollution. an example is shown in fig. . . where a characteristic function of sulfur dioxide is shown taking into account all possible factors related to particle growth. where f is the characteristic function that expresses particle size, particle concentration, particle composition, and so on [ , ] . particulate materials in water are present in the form of colloids. these colloid particles are classified into inorganic colloids. examples of the former are oxides of aluminum, silicon and other substances, and typical examples of the latter are substances such as humic acid and fulvic acid. while the structure and molecular weight of particles vary depending on the area of water, it is known that what are usually present in water are comparatively small colloids (particles smaller than nm). the number concentration of colloid particles in ground water, or a typical water area environment, ranges from to (number / m ) and varies significantly depending on the geochemical conditions of the aquifer. it is known that in moving water, colloid particles sometimes act as a medium in conjunction with water and in some cases move faster than water. homogeneous porous layer such as a sand layer, and most of the colloid particles are trapped. the mechanism of partical trap in this layer is explained by the sand filtration theory. c and d are a gravel layer and a rock bed, respectively, and both have high water permeability with large gaps and cracks. particles can also pass through easily. safety and movement characteristics of colloid particles have a significant influence on the movement of materials such as ionized molecules in aquatic environments. since fine particles such as nanoparticles in particular are highly stable as colloid particles, it will be very important in the future to understand their influence. at the same time, these characteristics are considered to have a high potential to be developed for further application of nanoparticles. in most cases, nanoparticles in exhaust gases are studied from the viewpoint of the influence of total particulate matters on the environment. the term "nanoparticles" is used only in a few cases, "fine particles" is usually used for investigation. since nanoparticles are part of fine particles, this section will be described from this perspective. major sources of combustion exhaust gases are stationary large-scale combustors and diesel engines for stationary and portable use. for stationary combustors, fuels such as coal, oil, and gas are used. lighter fuels have a lower rate of particulate emission, but have a higher fine particle content including nanoparticles. fig. . . [ ] shows the frequency distribution in combustion of coal and heavy oil. fig. . . a and b are the distributions on a number and mass basis, respectively. as these figures clearly show, the total weight of particles of a size of m or smaller is extremely low, while their total number is, on the contrary, very large. it is clear that, while the total quantity of particulate material is far larger in coal combustion than in oil combustion, the difference is less when it comes to particles m or less in diameter, including nanoparticles. most of the particles contained in pulverized coal combustion exhaust gases are considered to be formed as particulate materials directly from ash content, which is originally contained in coal and also includes some unburned carbon. particularly, almost all large-size particles are considered to be this type of particle. on the other hand, fine particles include two types. one type is formed in the process by which low boiling point metal contained in coal ash is evaporated and vaporized in a high-temperature combustion field and then becomes particles in the exhaust gas cooling process. the other includes carbon particles formed in the gas phase, or so-called soot, which is generated due to the delay in oxygen supply for combustion of evaporated volatile matter in the initial stage. fig. . . [ ] shows the relationship between the trace metal content in coal ash and the particle diameter. aluminum with a high boiling point has a constant concentration regardless of the particle diameter. however it is obvious that in the case of metals with a lower boiling point, the smaller the particle diameter, the larger the content. with regard to particles with sizes m or smaller in the nano domain, it has been clarified that the generated amount is increased rapidly by reducing combustion air supply or by weakening the oxidation atmosphere in the volatile matter combustion area, for example, when air supply from a burner is reduced in twostage combustion. this also demonstrates the significant contribution of carbon particles formed in the gas phase. also in the case of ash from heavy oil combustion, there are large particles of a carbon residue type generated from sprayed liquid particles and particles formed in the gas phase as well. as in the case of coal, trace metal contained in heavy oil with a low boiling point is concentrated into fine particles and discharged. also in the case of the combustion of liquefied natural gas, carbon particles formed in the gas phase are generated, albeit in trace amounts. in contrast, only in the case of diesel engines, fuel is injected into the high-temperature and highpressure atmosphere produced by compressing only air to induce spontaneous ignition, and combustion continues with a heterogeneous mixture of fuel and air in the combustion chamber. therefore, particulate materials mainly consisting of unburnt carbon are generated due to incomplete combustion. fig. . . [ ] shows changes in the diameter of particles according to changes in the diesel engine load. it is obvious that the overall concentration of particles increases with the increase in the load rate of the engine. according to observations using sem, fine particles in diesel engine exhaust gases have also been found to comprise fine primary particles of a size several tens of nanometers, and coarse particles with carbon hydride condensed on the surface of secondary aggregates of primary particles. influence of particle diameter on trace element contents. the volume of industrial and domestic wastewater is increasing significantly year by year with the change in the lifestyle based on mass consumption and mass disposal brought about by the dramatic development of the economy and industry. effective advanced wastewater treatment is required because wastewater contains a variety of constituents such as particles, organic materials, and emulsion depending on the resource. inorganic nanoparticles are not generally stabilized in the liquid because they form aggregates of some sort more or less. for wastewater reclamation and reuse, these nanoparticles can be removed from the liquid by the advanced treatment processes such as membrane filtration following biological treatment processes. organic materials such as macromolecules are regarded as soft nanoparticles judging from their sizes, in contrast with hard inorganic particles. chemical mechanical polishing (cmp) is one of the fastest growing processes in semiconductor industry, and it has become an integral part of the state-of-the-art fabrication line for the multilayer wiring board of large-scale integrated circuit (lsi). besides the semiconductor devices, cmp is widely applied to the magnetic head, the magnetic memory, and the imaging devices. the process is primarily used for polishing the device side of a semiconductor wafer through the mechanical downforce of slurry abrasive in combination with chemical oxidation of wafer surface. in general, colloidal silica is used as abrasive slurry to planarize the oxide wafer surface. particles in slurry are highly charged to avoid aggregations between particles or between particles and wafer surfaces. during the process, large volumes of ultrapure water are consumed to clean the surface of the wafer, which generates large quantity of cmp wastewater typically having high solid content resulting from slurry abrasive particles of sio , al o , or ceo , depending on the nature of the cmp application. the quantity of cmp wastewater generated is expected to increase proportionally with the growing needs of the cmp processes. as a result, the treatment and reuse of cmp wastewater has become increasingly necessary. the cmp wastewater has been generally treated with the conventional chemical coagulation-sedimentation process, producing large quantity of sludge. currently, a membrane filtration process coupled with chemical pretreatment is used to separate the nanoscale particles from the cmp wastewater to reclaim the water [ , ] . wastewater of nanosized metal colloid, which is hard to be removed by coagulation-sedimentation process, is discharged in such diverse fields as metalworking factory, electronic components factory, and pigment-manufacturing factory [ ] . it is reported that various trace elements of heavy metal are contained in wastewater discharged from a pulp production plant [ ] . a spent emulsion, which contains nanosized copper colloid, is discharged from plants manufacturing copper cables for electrical industry, and the treatment for purification of effluents is examined by the integrated membrane system based on ultrafiltration (uf) and nanofiltration (nf) [ ] . a glass company generates the wastewater containing fine clay and glass particles from the grinding process of glass surfaces during production of crt glass used for tvs and monitors. separation of fine clay and glass particles by microfiltration (mf)/uf is examined in order to treat glass industry wastewater for reuse in the manufacturing process [ ] . the colored substances are free from regulatory constraint of water quality so far because they are not considered hazardous substances. however, water color is being recently used as a standard for the judgment of the purity in water because the removal of color becomes important for wastewater reclamation and reuse. dye works are scattered across the country as the industry with local tradition. the dyehouse effluent is discharged in large quantity, and it has extremely complex composition because it contains not only dye but also dyeing aid and finishing agent. in general, dye cannot be removed by standard biological treatment because of its low environmental biodegradability. dye wastewater is treated by coagulation-sedimentation and activated sludge processes, and nanoparticles produced in the course of the treatment are released into the environment [ ] . the color is often imparted by organic substances, predominantly humic substances. aquatic humic substances including humic and fulvic acids are a term referring to a broad class of naturally occurring mixture of organic compounds, ubiquitous in surface waters, ground waters, and soil pore waters. they are a complex mixture of heterogeneous organic materials in terms of elemental composition, chemical functionality, and molecular size distribution since humic substances can be derived from any organic materials, including plant and animal debris, microfauna, biowaste, pesticide, and others. the molecular weights of humic acids range from several thousands to several tens of thousands daltons, and those of fulvic acids range from several tens of thousands to several hundred thousands daltons. because of this versatility, humic substances are known to significantly affect the behavior of some pollutants in natural environments, such as trace metal speciation and toxicity, solubilization and adsorption of hydrophobic organic compounds, and disinfection by-product formation [ ] . melanoidins are natural polymeric compounds of dark brown color, and they are closely related to humic substances. they are produced by a set of consecutive and parallel nonenzymatic reactions taking place between amino compounds and carbohydrates during a maillard reaction [ ] . they are contained in the molasses wastewater from alcohol distillery, sugar processing and refinery industry, and glutamate processing industry. such wastewater containing melanoidins has frequently caused a coloration problem of water environment, and thus the suitable decolorization treatment is required in many fermentation and sugar industries using molasses. treatments by flocculation, ozonation, and electrolysis are promising in color removal [ ] . food-processing wastewater usually contains a variety of organic materials in varying degree of concentration. in cheese-making in the dairy products industry, only ϳ % of the initial milk volume becomes product, cheese, and the other % becomes by-product, liquid cheese whey. since cheese whey is a protein-and lactose-rich by-product of cheese production, its cost-effective utilization is becoming increasingly important. recent developments in membrane technology have provided exciting new opportunities for large-scale protein and lactose fractionation in whey treatment [ ] . in textile industry, typically it takes over l of water to process just kg of textile material. not only the washing water must be treated to recover important by-products such as lanolin, but bleaching and dyeing chemicals must also be removed before discharge back to the rivers [ ] . surfactants are a primary constituent of the detergent used in the household routinely, and also they are widely used in industry and agriculture because they have several functions such as washing, emulsification, and dispersion. the surfactants are usually present in the solution in the form of the micelle, and large amounts of surfactant wastewater are discharged in the rivers [ ] . pesticides whose molecular weight ranged from to da (ϳ nm) have been used in great quantities not only for agricultural use but also in golf links and resort. therefore, the wastewater and effluent treatments have become an important issue, and pesticide separation by nf membranes is found to be very efficient [ ] . the potential reclamation of high-quality water produced by the advanced treatment of the secondary effluent of the municipal sewage has come a long way in recent years. the sewage contains various components such as virus [ ] , pharmaceutical substances [ ] , and endocrine disrupting compounds derived from zoonotic excretory substances [ ] . the advanced treatment of such chemical contaminants at low level becomes increasingly important. as mentioned above, the removal of nanoparticles contained in wastewater is stringently required to recycle the reclaimed wastewater in a wide variety of industries such as chemical industry, textile industry, pulp and papermaking industry, food-processing industry, dairy products industry, and pharmaceutical industry. also for domestic wastewater, the reuse of the reclaimed wastewater for nonpotable purposes is becoming more and more important, and this is expected to raise awareness of the behaviors of nanoparticles contained in wastewater in order to upgrade the water treatment processes. in recent urbanized lifestyles people tend to spend more time in enclosed buildings or residences than outdoors. therefore, it is of great importance to characterize indoor particles and correlate between indoor and outdoor ones from the viewpoint of evaluating the influence of indoor air quality (iaq) on human health. as shown in table . . , indoor nanoparticles originate from the several sources such as products of chemical reactions, nonvolatile residues (nvrs) of liquid droplets, printers/photocopiers, combustion, bioaerosols, and infiltration of outdoor air. . . . secondary particle formation by gas phase ozonolysis for particle formation resulting from chemical reaction via ozone, the reaction of terpenes is very common in indoor environments as well as atmospheric ones. terpenes are emitted from fragrance-containing vegetable oils such as pine oil and citrus oil, and wooden materials including woody furniture [ ] . meanwhile, sources of emission of ozone are air cleaners, air-conditioners, laser printers using corona discharge, and infiltration of outdoor air. terpenes are generic terms of unsaturated organic compounds that are composed of isoprene as unit (e.g. -pinene and limonene). these compounds used for household applications readily react with ozone because they have one or more double bonds. it has been proposed that, as shown in fig. . . , the reaction mainly proceeds to form less volatile pinonic acid via pinonaldehyde of intermediate [ ] . furthermore, the acid-catalyzed reaction allows the products to convert into higher molecular weight compounds by the polymerization via carbonyl groups in the aldehydes and the aldol condensation [ ] . it has been reported that the resultant generated particles have a size distribution with a peak diameter of about nm, and that the products by terpenes ozonolysis irritate human airways [ ] . nanoparticles are also generated from air humidifiers or negative air-ion generators in which water is atomized. in general, humidifiers are mainly categorized into vaporization type and atomization type [ ] . the former does not entrain impurities in water when it is fed into indoor spaces of interest. meanwhile, the latter has the drawback that nvrs are suspended in spaces to be humidified by feeding water via spraying and sonication. the nvrs in tap water include colloidal particles and soluble fraction such as silicates, sulfates, carbonates, and chlorides. the size of nvr particles, d p _ r can be estimated from the following equation: where dp_ m is the droplet size, c the mass fraction of nvr in the droplets, m the droplet density, and r the nvr particle density, respectively. assuming that m-sized droplets ( m ϭ , kg/m , r ϭ , kg/m ) are formed by an ultrasonic nebulizer, and the mass fraction of nvr in city water is ppm ( Ϫ ), the nvr particle size, d p_r is estimated to be nm. recently, a wide variety of negative ion generators using the lenard effect, corona discharge, uv/photoelectron emission and electrospray have been commercialized and attention has been focused on features such as air purification and physiological activation [ ] . among them there are the ion generators that atomize water based on the lenard effect and electrospray form the nvr particles as by-product in addition to ion products if the supplied water contains nonvolatile impurities. fig. . . shows an example of electrical mobility distribution for ions generated by the electrospray method (positive in this case) [ ] . this method atomizes liquid fed to a tip of a capillary electrode to form fine droplets with large amounts of charge by applying high voltage between the tip and the downstream counter electrode. when water in the generated droplets evaporates and their surface charge density attains the charge limit called "rayleigh limit", this phenomenon induces their self-fragmentation followed by the formation of a high concentration of cluster ions. in this figure, the high-mobility peak on the right side corresponds to the cluster ions. these ions are nm in size assuming that they are singly charged. meanwhile, another peak on the figure results from nvr in water and then its height increases with the increase in the fraction of tap water in the fed liquid. the electrical-mobility-equivalent size of nvr particles measured by differential mobility analyzer (dma) -condensation nucleus counter (cnc) method ranges from to nm and their concentration is on the order of particles/cm . comparing the forementioned electrical mobility distribution with the particle size one, the nvr nanoparticles are estimated to hold about charges. accompanying the recent proliferation of computers, the use of inkjet printers and electrophotographic machines such as laser printer and photocopier is becoming common in homes as well as offices. it has been reported that these devices emit various sorts of pollutants. the eco-friendlinessoriented standards such as blue angle standard [ ] regulate the maximum permissible limits of benzene, styrene, total volatile organic compounds (tvoc), ozone and particles. as the regulation of particles is based on the emitted mass per hour, mainly the relatively coarser particles such as toner and dust adhering to paper have been targeted. however, some reports have revealed that nanoparticles are emitted from inkjet printers or laser printers [ ] . fig. . . depicts the size distribution of particles emitted from a laser printer measured by a scanning mobility particle sizer (smps). as seen in the figure, nanoparticles with a peak diameter of around nm are generated in printing mode, whereas the emission in the case of feeding paper without printing is about one third of the normal printing mode. furthermore, these particles were dried by passing them through a diffusion dryer because they are thought to originate from the nucleation of water vapor emitted from papers in the fixation process. as a result, it was found that most particles formed in the paper feed mode evaporated and then vanished, while particles in the printing mode contained nonvolatile components as well as water. from these results it is anticipated that the particles are derived from styrene remaining in the toner even though their composition has still not been identified. meanwhile, it is thought that during ink discharge ink-jet printers emit not only the main ink droplets but also their satellites (about m) to result in nanometer-sized nvrs during printing. one of the most significant source of indoor nanoparticles relevant to combustion is cooking such as frying and sautéing [ ] . some reports said that over % of the particles by number were in the ultrafine fraction range during cooking with bimodal peaks at and nm, attaining the number concentration on the order of particles / m and the emission rate of particles / h. owing to lifestyles in asian countries, cigarette smoke, incense, and mosquito coils also contribute to indoor nanoparticle levels [ ] . it was reported that especially in the indian subcontinent the combustion of biofuels such as straw and dried cattle manure used for cooking could have a significant impact on climate change in the south asian region [ ] . sidestream cigarette smoke also contains nanoparticles, having a concentration distribution with the main peak between . and . m [ ] . in addition, it was found that nanoparticles a peak size of nm formed by the nucleation of vapor fraction in filtered sidestream smoke immediately after burning when the dilution of smoke by air was insufficient [ ] . attention should be paid to air cleaners when a high concentration of cigarette smoke has to be treated by the cleaners using a single unit air filter. airborne virus particles or virions are typically in the - nm size range, and are a good example of nanoparticle bioaerosols. smaller viruses typically contain one subunit, which consists of an outer protein capsid, internal nucleic acid (e.g. dna and rna), and other internal proteins. corona virus that causes sars and influenza are good examples of them. the viruses most often are transmitted through direct contact with an infected person, such as by shaking hands, hugging or kissing, while sometimes it is spread by nasal droplets. however, it is still unknown how these virus particles behave in the case of airborne infection. recently, the studies that attempt to elucidate the behavior have been progressing [ ] . this section describes the sources of nanoparticle generation in industrial processes by categorizing them into specific processes where a cleanroom is used and other general ones. the sources of emission of unwanted nanoparticles in general workplaces are categorized as fumes from hot processes (e.g., smelting, refining, and welding) and from (incomplete) combustion processes. favorable conditions required for the generation of nanoparticles are found in workplaces where there is ( ) presence of vaporizable material, ( ) sufficiently high temperature to produce enough vapor, followed by condensation to form an independent aerosol, and ( ) rapid cooling and a large temperature gradient. there have been so many studies on occupational exposure to fine particles in the field of public health. in general, high spikes of nanoparticle concentration are observed during active operations, followed by a gradual decay after the operation, primarily because of coagulation, evaporation, dilution, and/or deposition. the fraction of the total number of nanoparticles generally decreases, whereas that of the number of submicrometer particles increases with time and distance from the point of emission. in order to accurately estimate exposure, the effects of spatial and temporal changes will need to be evaluated. therefore, it is important to identify the time required for the concentration to decline to the normal or background levels. as an example of reports on grinding processes, fig. . . shows the case where a steel substrate was ground upon using a high-speed grinder [ ] . from the figure the distribution of concentration of generated particles has a distinct bimodality, one with the finer peak at around nm and the coarser one at around m. the former results from within the grinder motor and the volatilization or combustion of amenable ground substrate and/or grinding materials, the latter from the mechanical abrasion and attrition. however, the resultant total concentration on the order of particles/cm is not so high. cleanrooms and associated controlled environments (e.g., in the case of an iso class cleanroom, the maximum permissible airborne particle concentration is less than particles/m for particles with the size of . m or larger, while the airborne particle concentration in ordinary indoor environments is on the order of particles/m or higher) are usually adopted to avoid particle contamination in industrial processes where precision products such as engineered nanoparticles, semiconductors, and other electronic or optical devices are fabricated because the deposition of particles onto product surfaces causes their yield reduction and quality deterioration. the emission sources in cleanroom environments are tabulated in table . . . since some of the listed emission sources emit trace amounts of nanoparticles, these nanoparticles are not regarded as particulate contaminant but as chemical or molecular one. in this section, these nanometer-sized solid substances formed on solid surfaces by chemical reaction are also included. size distribution of nanoparticles generated when a steel plate was ground with a high-speed grinder. ( ) air exhaled by humans emissions from human bodies are a minor contribution in ordinary indoor situations because airborne particle concentration in such places is quite high, whereas the emission cannot be seen as negligible in cleanroom environments. the major human emissions are thought to be atmospheric dust deposited on clothes and skin fragments, and most of these particles are submicrometer in size. meanwhile, particles in exhaled air are composed of fine liquid droplets from spittle ( . % of water), and then evaporate to form nanoparticles of nvr. in fig. . . an example of size distributions of particles in exhaled air before and after smoking is shown [ ] . when measuring particles in exhaled air, the air was introduced into a measuring device after drying them by passing them through a diffusion dryer. the size distribution of particles in exhaled air before smoking (n db ϭ ) has a bimodality, one with the peak size of . m, and the other of nm or smaller. the former peak comes from atmospheric aerosols as it decreases with the increase in number of deep breaths in a clean booth (n db ). the latter originates from nvr particles of spittle droplets. incidentally, since smoking induces the rapid increase in number concentration of particles . m or larger by times or more and for nanoparticles by about double, special attention should be paid to the management of personnel's clothes such as face mask when they enter a cleanroom after smoking. ( ) emission from ionizers ionizers are commonly used in cleanrooms to eliminate electrostatic charge on substrates for precision electronic devices. the most popular ionizer is a corona-discharge type. corona-discharge type ionizers are categorized into the following three groups; ac, dc and pulsed-dc types. the issues of emission of contaminants such as ozone, no x and particles have been pointed out [ ] . these issues are also applicable to air cleaners using a corona discharger. among these problems is that the particle emission has a potential for particle contamination onto product surfaces and eventually decline in product yield. the particle emission, which has been studied since the s, is caused by foreign particle deposition onto electrodes, electrode erosion, and gas-to-particle conversion. the issue of electrode erosion can be solved by the improvement of electrode materials, whereas for the issue of gas-to-particle conversion, the airborne molecular contamination (amc) control to be ionized has to be made. it was reported that exhaled air of human corona-discharge ionizer (e.g. gas-to-particle conversion of low-molecular-weight cyclosiloxane) boron-containing particles from borosilicate glass fibers of hepa filter haze by chemical reaction on solid surfaces (precipitation of ammonium salt and silica) watermark on wafer surfaces at drying leakage from thin film and nanoparticles processing equipment silicon-containing compounds that precipitated on the electrodes result from the gas-to-particle conversion of low-molecular-weight cyclosiloxane (lmcs) from silicone sealant via corona discharge [ ] . ( ) boron-containing particles from hepa filter with borosilicate glass fibers the use of hepa and ulpa filters made of borosilicate glass fibers prevails in the most cleanrooms. it has been known that owing to chemical reaction in equation ( . . ) bf vapor is formed from glass fiber filters by passing hf gas leaking from wet cleaning equipment through the filters. boron, which is a dopant element for semiconductors, has been thought to be a contaminant that might cause failure in semiconductor devices if it comes from the surroundings. in addition, it has been revealed that trace amounts of boron in the form of boric acid (h bo ) are also formed from the fibers via the reaction with moisture in the surrounding air (equation ( . . )). fig. . . depicts the change in volatilized boron mass from various filters in terms of airborne boron concentration. especially, at the initial stage just after the initiation of ventilation, the volatilized boron mass increases with increase in relative humidity [ ] . boric acid, which is solid at room temperature, is surmised to form in the particulate form. however, its existence was identified only by chemical analysis because it is present only in trace amounts. haze might form on glass surfaces of lenses and mirrors for optical instruments if they are exposed for a long time to cleanroom environments where amcs are not controlled properly. the haze is more likely to bring about the insufficient light delivery onto a surface to be exposed in photolithographic processes. one of the reasons is that ammonium sulfate ((nh ) so ) is formed, which then precipitates on the glass surfaces via chemical reaction of sulfur dioxide (so ) with ammonia or amines. for another reason, hexamethyldisilazane (hmds) used as additive in resist coating or lmcs from silicone sealant is adsorbed, and then decomposed to form silica precipitates on glass surfaces by photochemical reaction during laser irradiation, followed by the unwanted decline in laser penetration [ ] . as another example a report said that tiny projections, which are also known as "haze", with a size of . m or smaller were formed on silicon wafer surfaces owing to the adsorption of organosilicate compounds in thin film formation processes with cvd. it is similarly caused by the precipitation of sio [ ] . ( ) watermarks on solid surfaces during drying when a silicon wafer surface is cleaned with deionized water and then dried in air, a watermark is formed on it via the mechanisms demonstrated in fig. . . . oxygen in air is dissolved and diffused into water droplets or adsorbed water on a wafer surface, followed by the formation of silicate compounds via silanol reaction. the watermark on a wafer surface is detected in the form of nanometer-sized particles by an electron microscope [ ] . ( ) leakage from nanoparticle production processes in regard to the risks to processing equipments by nanoparticle leakage from production processes, the vdi report in germany [ ] has been described in detail. the production of engineered nanoparticles can be generally categorized into two approaches. one is a "top-down" approach that is initiated with a bulk material and then breaks it into finer pieces using some form of energy such as etching, ball milling, sputtering, and laser ablation. the other approach is to synthesize materials from the atomic or molecular level by growth and assembly to form the desired nanoparticles. processes included in this "bottom-up" category are sol-gel, chemical vapor deposition, flame synthesis laser pyrolysis, and so on. most of these processes are performed in a closed reaction chamber installed in a cleanroom or associated controlled environment. human exposure to these engineered particles does not take place during synthesis unless there is an unexpected system failure (e.g. rupture of a seal). human exposure is more likely to occur after the manufacturing when opening the reaction chamber, drying the products, or in the post-process handling of the products. the release of nanoparticles during production chamber cleaning operations is another critical point. cleaning typically involves using water or some solvent. brushes, sponges, or tissues used in the cleaning will carry nanoparticles into the waste stream. disposal of the waste and wastewater may become a source of nanoparticle release into the environment. further, conditioning of nanoparticles such as compression, coating, and composition to form final products may also result in the release to the environment and resultant exposure although very few studies have been carried out on this subject. recent studies [ ] to evaluate the aerosol discharge during the handling of carbon nanotubes showed that the generation of nanoparticles occurred under vacuum to remove spilled nanotube materials or vigorous mechanical agitation. however, they reported that the concentrations were very low. in addition, measurements of nanoparticle levels during final packaging of carbon black, which is a typical engineered nanoparticle material, showed that there was no increase in nearby air [ ] . study on the safety of nanoparticles has started only recently, and no sufficiently systemized results have been obtained. what should be noted in particular is that the possibility of radial troubles caused by particulate matters are considered to increase by the decrease of particle diameter in nanoparticles. one typical example is the problem of dust explosion, caused by the high surface reactivity of fine particles. in other words, since nanoparticles are extremely fine particles, dust explosion is more likely to occur. explosion is more likely to occur because fine particles are different in their composition, in that low boiling point metal can be easily condensed, as described in section . . however, of particular note here is that, since all particles do not necessarily exist independently in the form of a single particle, the possibility of dust explosion does not simply increase as particles become finer. fine particles with sizes of m or smaller such as nanoparticles have an extremely high agglomeration propensity and secondary particles can be easily generated. therefore, in some cases they conversely behave like large particles. these are the points to be taken into consideration when studying the problems caused by nanoparticles. as shown in fig. . . [ ] , the effect of the particle diameter on dust explosion tends to be that the smaller the particle diameter of the dust, the lower the minimum explosion concentration. in other words, explosion can be induced under conditions of lower concentration of particles in air as the particle diameter becomes smaller. due to the difficulty of conducting experiments to suspend particles with the same size in a uniform concentration, this result was obtained from particles far larger than nanoparticles; however, it has been clarified qualitatively that the smaller the particle diameter, the higher the possibility of dust explosion. from the perspective of composition, the possibility of explosion increase if materials that react easily with oxygen at low temperature are condensed into particles of small diameter. therefore, with regard to the effect of particle diameter on dust explosion, more careful attention needs to be paid in the case of combined materials than in the case of uniform materials, as assumed in fig. . . . as described before, however, since nanoparticles are considered to exist often as agglomerates, it is necessary from the perspective of the particle diameter to take into consideration the diameter of not only primary particles but also of particles after agglomeration. to address the safety of nanoparticles, it will be important in the future to elucidate their behavior in detail including these factors. the terms 'nanoparticles' and 'nanomaterial' have been used for particles of which one representative dimension, for example, diameter of particles on cross-sectional diameter of fibers has at least nm or less. some people hold that the majority of such fine particles are exhaled without depositing in the respirator tract, and that therefore the particles may not cause pulmonary diseases. however, the properties of nanoparticles are known to be different from the bulk material they are derived from. in cases where the biological effects of bulk materials have been reported, nanosized particles of these materials may be expected to have stronger dose response for the health effects. every effort must be made to clarify the uncertainty on the risks of these nanomaterials [ ] . at the present time, there is no regulation or standard for assessing the biological effects of nanomaterials, and therefore there is a paucity of toxicological data concerning nanomaterials. much more systematic and strategic studies are needed to enable risk assessments for human health [ ] [ ] [ ] [ ] [ ] . as regards risk assessment and risk management of nanomaterials, the characterization and identification of anticipated risks should be first determined for chemical substances or foods. conventional assessment methods are applicable for water-soluble particles. for insoluble nanoparticles, the assessment of potential health hazards should be made based on their properties or toxicity and dose-response relationship. the risk is a product of hazard and exposure; even if a nanoparticle has a hazard, the risk is lower when the possibility of exposure to the nanoparticle is small [ ] . influence of particle diameter on lower limit of particle concentration for explosion. ( ) exposure routes for nanoparticles nanoparticles can either be deliberately introduced into the body for medical purposes (drug delivery systems) or absorbed involuntarily from the environment (inhalation of nanoparticle-containing dust in the air). a distinction should also be drawn between nanoparticles manufactured for industrial application and those unintentionally generated and released in the environment, such as welding fumes or diesel exhaust particles (dep). in the fields of environmental science and toxicology, numerous studies on the potential health hazards caused by ultrafine particles have been conducted. practically, there are several definitions of nanoparticles or ultrafine particles, however, findings regarding biological effects of the ultrafine particles are useful as a starting point for estimating the effects of nanoparticles on human health. human and animals contact with nanoparticles through various routes: nanoparticles can be inhaled in the air, swallowed in the water, ingested in food, and absorbed via the skin in cosmetics. for successful risk assessment, it is important to determine how nanomaterials or nanoparticles are used, such as composites, surface coating, or powders. coatings or powders have the potential to release a part of their nanomaterials into the environment. workers who come into contact with nanomaterials have the possibility of exposure to nanoparticles at the workplace. consumers of products using nanotechnology can also be exposed to them. attention needs to be paid to the environments and ecosystems in which nanoparticles and nanomaterials are released. nanoparticles in the products may change their size, quantity, and composition during their life-cycle of manufacturing, use, transportation, and disposal. ( ) respiratory uptake of nanoparticles inhalation is the main route of exposure to nanoparticles. particles inhaled with the air through the mouth and nose pass through the throat (nasopharynx and oropharynx) and tracheobronchial tree before reaching the alveolar region where oxygen moves from the alveoli to the blood and carbon dioxide moves from the blood to the alveoli. how deeply particles can penetrate and where they become deposited on each respiratory airway such as the nasal cavity, tracheobronchial tree, and the alveoli depend on their size under the various deposition mechanisms: inertial impaction, gravitational sedimentation and diffusion, etc. the respiratory airway includes the anterior nasal passage, posterior nasal passage, pharynx, larynx, trachea, main bronchi, bronchi, bronchioles, terminal bronchioles, alveolar duct, and alveoli, as shown in fig. . . . in the human lungs, the trachea divides asymmetrically into the right main bronchus that enters the right lung where it divides into three lobes, that is, an upper, a middle, and a lower, and the left main bronchus that enters the left lung where it divides into two lobes, that is, an upper and a lower. the trachea divides into two branches, dividing progressively to the terminal alveolus. to quantitatively assess the pulmonary particle deposition needs human lung morphology models, respiratory physiology based models of the entire lung airway system and aerosol deposition models based on many experimental findings. in , the icrp task group on lung dynamic (icrp: the international commission of radiological protection) published their revised lung model [ ] . the deposition, clearance, and translocation of particles in each of the compartments were described. while the model has been widely used in the nuclear field, it is applicable to conventional aerosols as well as radioactive aerosol. in the nuclear field, aerosols including radon progeny that used to be nanoparticles have been studied. fig. . . shows the deposition fractions of inhaled particles per adult nasal respiration of . m /h in each region re-calculated for the nasopharynx, tracheobronchial, and the pulmonary (alveolar) region based on the model. inhaled aerosol particles deposit on different regions depending on their size; for example, nanoparticles larger than nm deposit mostly in the alveoli and those less than nm deposit in the nasal cavity. how deeply particles penetrate into the lung depends on their size. nanoparticles can reach pulmonary region in the lung and deposit more intensively and this, therefore, has become one of the reasons for concern about the effects of nanoparticles on human health. however, deposition of nanoparticle chain-like agglomeration and fibrous particles such as carbon nanotubes cannot be estimated by this model. most inhaled nanoparticles are deposited on the surface of the respiratory tract. generally, if insoluble particles are deposited in the ciliated airspaces which are lined with a mucous layer, they are transported to the digestive tract with the mucous flow by mucociliary movements. particles deposited on nonciliated bronchioles and alveoli are phagocytosed by macrophages, which is a kind of white blood cell. as a result, their residence time is longer, however, they are usually transported to the ciliated upper part of the respiratory tract. removal of deposited particles described above is called clearance. when the amount of deposited particles is below a certain value, no health effect is produced. in relation to a macrophage response to particles, crystalline silica is hazardous, whereas titanium dioxide is not. pneumoconiosis is a well-known lung disease that is caused by exposure to dust particles of several micrometers in diameter. silicosis is a typical form of pneumoconiosis resulting from exposure to crystalline silica dust and characterized by a progressive fibrosis of the lungs. the macrophage-mediated clearance (phagocytes) was effective for micron and submicron particles. it has been reported that only % of deposited nanoparticles were removed by the clearance mechanism [ ] . it has been suggested that the remaining nanoparticles may pass through the alveolar walls, penetrate into the blood or lymphatic circulation, and be transported to other organs. many studies have shown that the smaller the particle size the greater the mobility or they pass easily through the alveolar wall and enter into the bloodstream. it is further presumed that the mechanism of health effects of nanoparticles on cardiovascular system other than the respiratory tract is similar to that in the airborne ultrafine particles from dep. in japan, two reference values, the 'administrative control levels' (acl) and the 'occupational exposure limits' (oels), are used for the regulation of hazardous chemicals as well as dust (particle matters). oels are recommended and revised every year by the japan society for occupational health. oel (oel-mean) for mean concentration of a chemical substance is defined as the reference value to the mean exposure concentration at or below which adverse health effects caused by the substance do not appear in most workers working for h a day, h a week under a moderate workload [ ] . the 'threshold limit value' (tlv) has the same definition (but may not be the same values as oel of the same substance) provided by the american conference of governmental industrial hygienists (acgih). acl is an index to determine the control class to judge the propriety of the working environment control based on the results for working environment measurement, which have been implemented for the unit work area in accordance with the working environment measurement standard. the results of working environment measurement are evaluated by classifying the working environments concerned into three control classes (control class i, ii, and iii). these classes are used as the standards to classify the level of the working environment concerned. among those subject to working environment measurement, these standards apply to workplace where dust, lead, organic solvent, and specified chemical substances are used. article of the industrial safety and health law stipulates that certain workplaces in which harmful substances are involved or harmful work operations are performed shall be the subject to working environment measurement. a % cut-off size of the dust particle is set at m for both standards and is far larger than nanosized particles. oel or acl [ ] for particulate matters are usually based on mass concentration, that is, milligram per cubic meter. therefore, if the particulate matters have broad size distribution, the contribution of nanoparticles is not large to in terms of mass of particles. evidence from a number of toxicological studies on insoluble particles indicates that the primary determinant of the health effect of particles depends on the surface area of particles deposited [ , ] . on the basis of the results from a number of in vitro studies of insoluble nanoparticles, a hypothetical cellular interaction has been proposed [ ] : inflammation and oxidative stress can be mediated by several primary pathways: ( ) the particle surface causes oxidative stress resulting in increased intracellular calcium and gene activation; ( ) transition metals released from particles result in oxidative stress, increased intracellular calcium, and gene activation; ( ) cell surface receptors are activated by transition metals released from particles, resulting in subsequent gene activation; or ( ) intracellular distribution of insoluble nanoparticles to mitochondria generates oxidative stress. in the workplace, the concentration of nanoparticles may be at a high level and most of the nanoparticles become agglomerates, while nanoparticles will form single nanoparticle at low levels in the general environment. it is a matter of debate whether agglomerates of nanoparticles react as a larger particle or a single nanoparticle in the lung or other organs. if insoluble particles are retained in the lung for a longer time without enough clearance mechanisms, they can cause pulmonary inflammation or pneumoconiosis. it is of interest that nanoparticles deposited in the lung can move into the blood vessel through alveolar epithelium and they can damage vessels or produce blood clots [ , ] . in a recent study, nanoparticles deposited in the nose may move directly to the brain via the olfactory bulb [ ] . ( ) biological effects of fullerene the biological effects of fullerene have being investigated intensively. in rats dosed orally with radioisotope-labeled c fullerenes, most were excreted in the feces and some were found in the urine. a small amount of them can be absorbed via the gastrointestinal tract. in contrast, in the same study, % of the same labeled fullerenes administered intravenously were retained after week, with most found in the liver [ ] . ld s (acute toxicity) by intraperitoneal injection in mice and rats were . and . g/kg, respectively. the dose of . g/kg orally in rats did not result in death. the reproductive translocation of fullerenes was also observed in mice. fullerenes have shown mutagenic activity in ames tests. fullerenes have shown no skin irritation or allergic reactions [ ] . on the other hand, fullerenes are being tested for possible medical use. fullerenes are basically hydrophobic but water-soluble derivatives have been synthesized to be used as drugs or its carrier. the derivative can be anticipated as drugs, for example, anti-aids drug. it has been stated that the toxicity of fullerenes changes due to slight structural changes including chemical modification [ ] . ( ) biological effects of carbon nanotubes carbon nanotubes are chemically stable and are similar in form and size to asbestos; these characteristics have given rise to concern that carbon nanotubes may have the potential to cause pulmonary diseases such as lung cancer and mesothelioma similar to asbestos. a few data are available concerning the biological effects of carbon nanotubes. the biological effects of carbon nanotubes are being researched. epithelioid granulomas and interstitial inflammation are induced in mice and rats following exposure to single-walled carbon nanotubes [ ] [ ] [ ] . untreated carbon nanotubes contain the nanoparticles of transition metals such as iron and nickel, which are used as catalysts in forming carbon nanotubes. these nickel-containing carbon nanotubes have been reported to be toxic [ ] . the concentration of airborne asbestos fibers is expressed as a number concentration, that is, fibres per cubic centimeter or fiber per liter. when fiber concentrations are determined by phase contrast light microscopy, the fibers with a diameter of less than m, a length longer than m, and a lengthto-diameter ratio (aspect ratio) greater than are counted [ ] . asbestos fibers having nanosized diameter were often observed in analyses of environmental samples using electron microscopy. international agency for research on cancer (iarc) rated asbestos as a known human carcinogen (group ) [ ] and the concentration of chrysotile asbestos is expressed as a risk level of . fiber/cm [ ] . health effects of vitreous fibers and other asbestos substitutes have been assessed to determine their oels or their carcinogenicity in humans. the health effects of carbon nanotubes are being intensively investigated now. the oel for carbon black respirable dust is mg/m and these for activated charcoal and graphite are . mg/m in each [ ] . in the ref. [ ] , while rats and mice inhaled carbon black with a particle diameter of ϳ nm at a concentration of - mg/m did not produce any specific changes, particles (agglomerate of small particles) of ϳ nm at a concentration of - mg/m produced early pulmonary changes. micronsized titanium dioxide particles are thought to have almost no toxicity and often used as a negative control substance. the oel for titanium dioxide is mg/m for respirable fraction [ ] . however, the results of a series of studies by oberdörster et al. [ , , , ] on submicron-and nanosized titanium dioxide suggested that as size decreases, inflammatory effects are intensified, and normally nontoxic substances may assume hazardous characteristics. fig. . . shows a part of the results by oberdörster et al. in which rats and mice were exposed to anatase titanium dioxide particles [ ] . their results have been frequently cited in the discussion of whether the health effects of fine particles should be based on its mass or its surface area. in fig. . . , percentages of neutrophils in lung lavage of rats are shown as indicators of inflammation after intratracheal instillation of different mass doses of and nm tio particles. the steeper dose response of nanosized tio particles is observed than for submicron tio particles when the dose is expressed as mass (fig. . . a ). if the same dose-response relationship as in fig. . . a is indicated as particle surface area (fig. . . b), the particle surface area seems to be a more appropriate dosimetric for comparing effects of different-sized particles of the same chemical structure. zinc oxide is a white powder and used in pigments. the oel is mg/m for its respirable fraction [ ] , and the value for zinc oxide fume which causes metal fume fever is under consideration. nanoparticles of transition metals and rare earth elements and their oxides will be used widely. since many of these metals and their oxides have biological effects, particular attention should be given to them. nickel compounds are rated as a human carcinogen (group ) by iarc. in particular, nickel oxide is particularly insoluble among the nickel compounds and remains longer in the lung. nanosized nickel oxide particles have greater toxicity to the lung than larger particles [ , ] . pulmonary inflammatory responses induced by nanosized cobalt particles have been reported [ ] [ ] [ ] . biological effects of nanosized particles of other transient metals such as iron and manganese have received attention [ ] . rare earth elements are a general term of chemical elements consisting of scandium (sc), yttrium (y), and a lanthanide series of elements from lanthanum (la) to lutetium (lu). these elements have been used in magnetic alloy, fluorescent and hydrogen storage alloy. particularly cerium oxide nanoparticles are frequently used as a fuel additive and are incorporated in cosmetics formulation. the potential biological and environmental effects of these elements have not sufficiently been investigated. it has been demonstrated that ld s for these elements in oral and intravenous administration are in a range from several dozens to several thousands milligrams per kilograms, indicating that none of these elements has high toxicity. in the results of studies in which the biopersistence and the distribution of rare earth compounds in the body were investigated, for example, the compounds deposited in bones and teeth, and organs including lung, liver, spleen and kidney following intratracheal, oral, intravenous, and intraperitoneal administration. although the compounds deposited predominantly in the liver other than bones, it has also been reported that the distribution of the compounds in the lung and spleen increased when the dose was increased [ ] . the demand for indium compounds has been sharply increasing. the compounds have been used in the materials for transparent electrodes for flat panel displays. in japan, the cases of pulmonary interstitial pneumonia and pulmonary fibrosis have been reported in workers engaged in cutting and grinding of sintered indium-tin oxide (ito) and potentially having inhaled the dusts released from ito [ , ] . the biological effects of indium arsenic compounds and indium phosphorus compounds also have been investigated. the acgih has proposed a value of . mg / m for their tlv, and the value has been applied tentatively in japan. we have been experiencing amazing progress of the technology on the processing for the nanometer-sized materials. the applications cover even biomedical engineering in addition to information technology, material, environmental science, and energy production [ ] [ ] [ ] [ ] [ ] . as the result, many kinds of new materials have been designed, fabricated, and discarded. from now on, this movement will be accelerated and even more new functional materials will be distributed in the world. here, we should not forget the safety of those materials in the process of the production, usage, and discard. without this safety assessment, we will go into the same problems as that of asbestos just we are facing now. first of all, we have to conduct experiments to reveal the minimal concentration for emergence of the toxicity. in other words, we have to fix the standard value for the threshold concentration for each material first of all. if we do not fix it, we should not use the material at any concentration, which means any engineering process could not be carried out. the applications in the various fields have started all over the world, and the safety assessment is urgently needed. in this article, we introduce the methods of the safety assessment of the semiconductor nanoparticles and describe the safety and the threshold depending on the surface treatment. the production process and the surface treatment play one of the most important roles for the safety of the nanoparticles. cd and se semiconductor nanoparticles coated with zns are one of the most widely used for the strong intensity of the fluorescent activity. cd oxide and se compounds once dispersed in the tri-octhil-phosphine oxide (topo) heated up to Њc generate nanoparticles by self-assembly. then zns enhances the stability of the structure and raises up the fluorescent intensity than without the coated one. nanoparticles, thus manufactured, as materials for a novel memory in the field of intelligence technology (it), and as super-micro devices for laser in the field of optics, have been developed all over the world [ ] [ ] [ ] [ ] [ ] . these nanoparticles cannot be dissolved into water, but dissolved into organic solvents like toluene. therefore, for biological and medical applications, various technologies for surface-conjugations to make them hydrophilic [ , ] have been developed. for example, nanoparticles covered with topo are hydrophobic because an alkyl group on them is hydrophobic. therefore, a technology for replacing this alkyl group with hydrophilic carbonic acid (making the whole particles soluble in water) has been developed [ ] . nanoparticles, thus surface-treated, can be dissolved into water, like sodium salt or potassium salt. with this method, various kinds of materials have been surface-conjugated. the mtt assay method is a way to evaluate the hazard assessment of nanoparticles, in which the activation metabolism in a mitochondrium in a cell is measured and the influence of nanoparticles on the prolification of the cell is qualitified. the mtt is a kind of tetrazolium, whose molecular formula is c h brn s. taken into a cell, it is decomposed by a dehydrogenase enzyme in a mitochondrium into a pigment called 'hormazan'. the measurement of the fluorescence intensity of the pigment shows the prolification of the cell [ ] [ ] [ ] . fig. . . . shows the hazard assessment of vero cells and kidney cells of the african green monkey against cdse/zns nanoparticles. the horizontal axis shows the concentrations of the nanoparticles and the vertical axis the fluorescence intensity of the hormazan at nm, that is, the metabolism of the cells. the figure indicates that cyto-toxicity is not observed for cells when the concentration is less than . m. this result means that this concentration is the threshold of the cell toxicity. likewise, cyto-toxicity is not observed for the hela cells and for the human primary cells. further, in order to find out how the sizes of nanoparticles influence the cyto-toxicity, the cyto-toxicities were evaluated with three kinds of quantum dots; one whose fluorescence wavelength is nm, red, one with nm, yellow, and the other with nm, green. the following results were obtained. the largest quantum dots whose fluorescence wavelength is nm show a tendency to give cyto-toxicity. cyto-toxicity is observed at concentrations more than m [ ] . another method to evaluate cyto-toxicity is the flow cytometry [ ] . the mtt assay alone cannot tell whether the toxicity observed is lethal to the cells or just restrains the prolification of them. in the flow cytometry, the nuclei of dead cells are dyed with propidium iodide (pi) after the nanoparticles are taken in and the ratios of the dead cells are measured. fig. . . shows the lethal cyto-toxicity of mua conjugated nanoparticles ( nm, green) against vero cells. the vertical axis indicates the numbers of the cells, and the horizontal axes show the fluorescence intensities and the cyto-toxicities. these experiments also show that dead cells cannot be observed at concentrations less than . m even though nanoparticles are taken in, as was shown in the mtt assay. however, at concentrations more than m, the nanoparticles taken in cause damage to more than the half of the cells. that is, the cyto-toxicity of mua quantum dots against cells is lethal [ ] . nanoparticles have been surface-conjugated for applications for various uses. some surface-conjugations cause more grave cyto-toxicity than others. therefore, relations between surface-conjugations and their safety for cells have to be considered. in order to find out the relations, the safety evaluations of nanoparticles surface-conjugated with two materials were made; one is with mua (quantum dots-cooh) and the other is with glycerol (quantum dots-oh), and their purified and unpurified particles. fig. . . shows that the purification reduces the cyto-toxicity for the quantum dots-oh, and that the toxicity remains the same after the purification for the quantum dots-cooh. mua itself, a material with which particles are conjugated, has cyto-toxicity. this experiment shows that toxicity against cells is connected not only with particles themselves but also with kinds of surfaceconjugations and degrees of purification. it is shown in the above experiments that the toxicity of nanoparticles is lethal to cells. next, we found out by the comet assay method whether the toxicity is derived from damaged dna. the method is a way to evaluate quantitative damage of dna by electrophoresis. the fragmented dna seeps out of their cells by treating cells, whose dna has been fragmented, with agarose-gel to break their cell membrane, and then electrophoresing them. it looks like the "tales of comets". cells, whose dna has not been fragmented, have their nuclei keeping their spherical shape after electrophoresis, and the tales of comets cannot be observed [ ] [ ] [ ] [ ] . fig. . . shows the results of the experiments with quantum dots conjugated with cooh (both purified and unpurified) to wtk- cells, a human lymphoblast mutation strain [ ] . the vertical axis shows the lengths of the tails, and the horizontal axis the concentrations of the quantum dots. the concentration of the nanoparticles is m. the results are as follows: the unpurified quantum dots with cooh caused damage to dna. on the other hand, the electrophoresis with the purified quantum dots does not show dna damage. this is probably because the dna damage has been repaired during the longer cultivation time. conducted with quantum dots conjugated with mua, and quantum dots-nh with an amino group. dna damage was not observed in either case. the results indicate that as far as particles themselves do not break down, the cyto-toxicity of quantum dots is derived from the chemical properties of the materials covering the quantum dots. the safety evaluation of nanoparticles has not been conducted sufficiently. as indicated above, the procedure for surface-conjugation could apply not only to cd/se nanoparticles but also to other nanoparticles. today, various kinds of techniques for surfaceconjugations have been available in academic papers, proceedings, and on the internet. some of them are widely known and others are patented. those techniques are all shared among the human race. even at this moment, the human beings are making breakthroughs in various fields and developing different kinds of technologies. sharing these technologies will lead to still more speedy developments of yet more advanced technologies. to achieve it, these technologies should be so structured that different fields, for example, bioimaging and biotechnology, structured on their own, can be linked to each other. such structured knowledge will play an essential part in merging different fields. in order to prevent nanoparticles release from a system so as to maintain environmental safety, the removal technique of nanoparticles must be established. in this section, separation techniques of particles from exhausted or suspended gas and liquid are described focusing on particles less than nm. generally, as shown in fig. . . , all particle separators for a dispersed system employ either one of three basic forms of particle separation. on the left hand side of the figure lie the separation methods in which particles are collected only by force field (electrostatic force, centrifugal force, gravity force, etc.), and the representative separator is electrostatic precipitator (esp). if some obstacles (collectors) are placed into the particle laden stream, particle separation is facilitated because particles are collected on obstacles with a smaller deviation from the fluid flow by the force exerting on the particles compared to the case without obstacles. typical collectors of this form are air filter, deep bed filter, etc. on the right hand side of the figure lie separators that collect particles utilizing only sieving effect of obstacles without any force field. in this case, geometrical size of channel between the obstacles must be smaller than that of particles. membrane filter, fabric filter, etc. belong to this group. when we apply the above collection forms to nanoparticles, the major collection mechanisms are brownian diffusion and electrostatic force for particles in gas, while sieving effect and interception/adhesion forces for those in liquid. as mentioned above, most airborne particles are collected by separators utilizing various kinds of forces such as gravity, centrifugal force, electrostatic forces, inertia, brownian diffusion force, and so on. therefore, the migration velocities or displacement of a particle per second due to the individual forces gives the basis for the comparison of removal efficiencies due to each force. in fig. . . , migration velocities of particles due to various forces are depicted against particle diameter at normal temperature and pressure for particle density of g/cm [ ] . as seen from the figure, the velocities due to gravity, centrifugal force, and inertia monotonically decrease with decreasing particle diameter, suggesting that the removal of nanoparticles with these forces is difficult. on the contrary, the velocities due to brownian diffusion and electrostatic forces increase with decreasing particle diameter for particles less than nm. this suggests that brownian diffusion and electrostatic forces are most effective in collecting nanoparticles. fig. . . summarizes typical conventional dust collectors. among them, the effective collectors for nanoparticles are esp and fabric/air filter. however, for the case of esp, which relies on only electrostatic force, nanoparticles (Ͻ nm) fail to carry even one electron resulting in low collection efficiency. in this case electrically charged filters are effective because we can expect the combined effects of electrostatic forces and brownian diffusion. among charged filters, so-called electret filter, which consists of permanently polarized fibers, is the most favorable filter because of its charge stability. particle penetration data of electret filter are plotted against particle diameter in fig. . . and compared with that of uncharged filter with the same structure. for the three combinations of charged states of fiber and particle there exist the most penetrating particle diameters. for the uncharged fiber, collection efficiency of uncharged particle has a minimum at nm and increases with decreasing particle diameter, showing that nm is the most penetrating particle size. for the charged fiber, particle collection efficiency is very high even for uncharged particle, and the efficiency for charged nanoparticles is extremely high because of strong coulombic force between fiber and particle. the experimental data plotted in fig. . . are qualitatively in good agreement with the theoretical prediction following the particle size dependency on particle migration velocity (shown in fig. . . ). however, as particle size becomes smaller and comparable with the size of a molecule, particles may rebound on a collector surface, and the adhesion probability of particles drops, resulting in a decrease in collection efficiency. fig. . . is an example of experimental data that confirm the particle rebound [ ] . the figure shows the penetration of nanoparticles through a grounded circular tube. the solid curve is the theoretical line derived by assuming that particles are deposited from a laminar flow in a tube by brownian diffusion. it is evident that experimental penetration deviates from the theoretical line for particles less than nm. this means that molecular behavior begins to appear when the particle size becomes as small as nm, and as a result, the collection efficiency is reduced. it should be noted that considerable amounts of nanosized particles are contained in diesel exhaust particles (dep), possibly penetrating through the honeycomb type (tubular channel) diesel particulate filters (dpf). there are two types of methods that differ in the way the nanoparticles in liquid are collected. the first group, called membrane filtration, constrains the particles by a membrane, and the liquid is allowed to flow freely through the membrane. in the second group of ultracentrifugation, the liquid is constrained in a rotating vessel, and the particles move freely within the liquid by an external field of acceleration caused by ultracentrifugal field. these methods have been quite extensively used in separation of macromolecules and molecules from liquid, and they are recently becoming important also in separation of nanoparticles from liquid. in pressure-driven membrane filtration processes, the pressure gradient across the membrane would force solvent and smaller species through the pores of the membrane, while the larger molecules/particles would be retained. membrane filtration processes are usually classified into three general categories according to the size of separating components, as shown in fig. . . . microfiltration (mf) is designed to retain suspended particles in the range of nm- m. ultrafiltration (uf), on the other hand, retains macromolecules or nanoparticles in the range of - nm (nominal molecular weight cut-off (nmwco) ranging from , to , , da). nanofiltration (nf) is a relatively new process that uses charged membranes, and it covers molecular sizes ranging from . to nm (nmwco ranging from to , ). it is useful in that it can separate dissociated forms of a compound from the undissociated form. one of the major factors limiting the use of membrane filtration is membrane fouling, resulting in a dramatic decline in flux with time of operation. to account for the membrane fouling, the resistancein-series model is frequently employed. the resistance model becomes ( . . ) where u is the permeate flux, v the filtrate volume per unit membrane area, the filtration time, p the applied transmembrane pressure, the viscosity of the permeate, r t the total resistance, r bm the resistance of the membrane per se plus the clogging of the membrane pores, r c the resistance of the filter cake, and r cp the resistance of the concentration polarization layer. significance of each resistance in membrane filtration is as follows. the membrane, even in the absence of any suspended particle, has a natural flow resistance. during membrane filtration, particles become attached to the pore channel of the membrane thereby reducing the flow channel dimension, or pores become blocked off completely. the last two effects lead to resistances that are due to adsorption and pore blocking. the blocking filtration model introduced by hermans and bredée [ ] , and grace [ ] is most commonly used as an interpretation of such phenomena. the clogging of the membrane pores is strongly influenced by such solution environment as ph and the ionic strength. the permeate flux of bovine serum albumin (bsa) (pi . , molecular weight , , stokes-einstein diameter . nm) solution by permeable mf membrane (nominal pore size . m) is lowest at around the isoelectric point [ ] . as the bsa molecule carries no net charge at the isoelectric point, the molecule is in its most compact state at that point. the bsa molecules deposit themselves rather densely onto the pore walls of the membrane to form a compact configuration with a smaller lateral electrical interaction between the molecules. as a result of this, the flow resistance increases markedly at around the isoelectric point. in dead-end membrane filtration, which has a feed and permeate stream, each with the same mass flow rate, the resistance of the filter cake plays a major part in the filtration resistance. therefore, the cake filtration theory can be applied, and thus the permeate flux u is described by ( . . ) where m is the ratio of wet to dry cake masses, s the mass fraction of solids in the solution, av the average specific filtration resistance, the density of the permeate, and v m the fictitious filtrate volume per unit membrane area, equivalent to the flow resistance of the membrane [ ] . for fine particle suspensions, colloidal forces which arise from interaction between the suspended particles control the nature of the filter cake. the average specific filtration resistance av and the average porosity av of the filter cake are strongly affected by the solution properties, including ph and electrolyte strength. for instance, in mf of suspensions of the titanium dioxide (pi . , the original mean specific surface area size nm), av goes through a minimum, and av is much larger near the isoelectric point [ ] , as shown in fig. . . . the titanium dioxide particles are destabilized around the isoelectric point where the van der waals attraction is more dominant. consequently, the particle tends to come together, that is, to flocculate, and the very porous flocs are then formed. thus, it is speculated that the filter cake formed from such porous flocs has often loose and wet structures. on the other hand, the filter cake becomes compact and dry when the particle carries the charge. since the most loose filter cake forms around the isoelectric ph, the filter cake is most permeable. it is interesting to note that the results in protein uf had a distinctly different behavior. in protein uf of bsa solution, the filter cake is in its most compact state around the isoelectric point [ ] , as shown in fig. . . . since the bsa molecules are hydrophilic colloids, their stability in the solution would appear to be influenced not only by the presence of a surface charge on the protein but also by hydration of the surface layers of the protein. the bsa molecules, because of hydrated layers surrounding them, are not destabilized by such consideration as depression of the electrical double layer. thus, the bsa molecules have water bound to them even around the isoelectric point. the hydrophilic bsa molecules maintain a dispersed state in the solution due to hydration of the surface layers of the protein even around the isoelectric point. when a bsa molecule acquires a charge, the filter cake becomes loose and wet due to electrostatic repulsion between the charged bsa molecules. this contrasts to the compact filter cake around the isoelectric point. the average specific filtration resistance av has a definite maximum around the isoelectric point since a compact filter cake provides a large hydraulic flow resistance. most membrane filtration processes are operated in the cross-flow mode, in which the feed is moved tangentially to the membrane surface so that the filter cake is continuously sheared off. during membrane filtration, particles in the feed are brought to the upstream surface of the membrane by convective transport, and this results in a higher local concentration of the rejected particles at the membrane surface as compared to the bulk solution which is referred to as concentration polarization [ ] . the particle concentration in the solution adjacent to the membrane varies from the value at the membrane surface, c m , to that in the bulk feed solution, c b , over a distance equal to the concentration boundary layer thickness . the resulting concentration gradient causes the particles to be transported back into the bulk solution due to diffusional effects. at steady state, the rate of convective transport of particle toward the membrane is balanced by the rate of particle transport through the membrane plus the rate of the diffusive back transport of particle. thus, the permeate flux u is given by ( . . ) where cp is the particle concentration in the permeate, k (ϭd/ ) the mass transfer coefficient, and d the diffusion coefficient. the osmotic pressure model assumes that the deviation from pure water flux occurs solely due to the osmotic pressure difference across the membrane, and thus the permeate flux u is given by ( . . ) where is the osmotic pressure, which is a function of the concentration. equation ( . . ) means that the effective driving force across the clean membrane is pϪ{ (c m )Ϫ (c p )}. replacing pϪ{ (c m )Ϫ (c p )} by the hydraulic pressure at the membrane surface, p m , equation ( . . ) reduces to the cake filtration equation. to minimize the effects of cake buildup and concentration polarization, membrane filtration is usually conducted using the cross-flow geometry in which the feed flow is parallel to the membrane and perpendicular to the filtrate flow [ ] . however, especially in mf the energy requirements associated with pumping the feed (plus any recirculation flow) along the membrane surface are typically very high. thus, there have been some innovations in recent years with cakeless filtration. the rotating disk module in which the membrane disk is stationary is suited for large-scale operation [ ] . it is possible to enhance the permeate flux by using the vibrating modules [ , ] . in the rotating cylinder device with the membrane on the inner rotating cylinder, counter-rotating taylor vortices within the annular gap are available [ , ] . dean vortices that twist and spiral in the direction of flow inside a highly curved channel, similar to vortices in rotary modules can result in enhanced flux [ ] . these vortices, or flow instabilities, induce turbulence into the system. periodic removal of the formed filter cake is also effective for enhancing the permeate flux. recently, several methods have been investigated: back washing using the filtrate or air pressurization [ ] , periodic rotation of the cylindrical membrane [ ] , pulsatile flow [ ] , high-frequency transmembrane pressure pulsing with a frequency around . - hz [ ] . dead-end upward filtration, where the filtrate flow is in the opposite direction to gravity, and dead-end inclined filtration, where the membrane is inclined, can reduce the cake formation onto the membrane in uf of nanoparticulate suspension and protein solutions. in upward uf of silica sol (mean diameter . nm) and bsa solution, a sustained permeate flux is achieved, as shown in fig. . . [ , ] , because the filter cake overlying the membrane is exfoliated continuously under the gravitational force acting on the particles comprising the filter cake. another approach for enhancing the permeate flux is to employ external force fields. electrofiltration, in which an applied electric field is used to drive charged particles away from the membrane surface, has been developed. in electrofiltration, the accumulation of the particles on the membrane surface is limited by the imposed electrophoretic force. in addition, the permeate flux through the filter cake is dramatically enhanced due to electroosmosis as a secondary electrokinetic phenomenon. this method can be applied to a broad combination ranging from mf of particulate suspension such as bentonite [ ] to uf of protein solution. fig. . . shows the reciprocal permeate flux (d /dv) versus the permeate volume per unit membrane area, v, for various values of the strength of the dc electric field, e [ ] . the steady permeate flux increases noticeably with the magnitude of the imposed field strength. also, a higher electric field strength causes the permeate flux to equilibrate more rapidly. a method has been developed for removing humic substances by hybrid uf combined with both flocculation and adsorption treatments, as shown in fig. . . [ ] . flocculation by use of polyaluminum chloride (pacl) is particularly effective for the removal of humic acids, which constitute the relatively high molecular weight fractions of humic substances, whereas adsorption by use of powdered activated carbon (pac) is able to remove fulvic acids of relatively low molecular weight effectively, which cannot be fully flocculated by pacl. hybrid uf in combination with flocculation and adsorption treatments exhibits high permeate quality because the flocs and pac are easily retained by the uf membrane. in ultracentrifugal sedimentation, ultracentrifugal force field of several tens of thousands of revolutions per minute is applied to a rotor. in recent years, ultracentrifugal sedimentation is employed for concentrating dilute protein solutions and for separating proteins and other large biological molecules from low-molecular-weight solutes or from much larger particles. fig. . . shows the results for ultracentrifugal sedimentation of an aqueous solution of the mixtures of bsa and egg white lysozyme (pi . , mw , ) measured using schlieren optics in an analytical ultracentrifuge [ ] . the angular acceleration of the rotor is , rad/s. the symbol r i and r i in the figure represent the distances from the center of rotation to the sedimentation boundary at time and , respectively. the electrical nature of macromolecules plays a significant role in determining the sedimentation behavior in ultracentrifugation of binary protein mixtures. in the ph range where both protein molecules were electropositive, the molecules sediment independently due to the electrostatic repulsive force acting between bsa and lysozyme molecules. denpun kagaku filtr. sep institute for quality assurance and certification: basic criteria for the award of the environmental label (printer ral-uz ) proc. air cleaning contam proc. air cleaning contam proc. air cleaning contam chemical contamination in semiconductor processing environments and its countermeasures the japan society of industrial machinery manufactures: report on behavior control of individual sort of contaminants - report on introduction of advanced technologies to environmental equipment industry industrial application of nanomaterials -chance and risks. future technologies, division of vdi technologiezentrum nanoscience and nanotechnologies: isbn recommendation of occupational exposure limits who: environmental health series . world health organization summaries & evaluations -nickel and nickel compounds rapid colorimetric assay for cellular growth and survival ouyou earozorugaku crossflow filtration: theory and practice encyclopedia of fluid mechanics: slurry flow technology filtr. sep key: cord- -g zhoyo authors: mukherjee, shruti; bhattacharyya, dipita; bhunia, anirban title: host-membrane interacting interface of the sars coronavirus envelope protein: immense functional potential of c-terminal domain date: - - journal: biophys chem doi: . /j.bpc. . sha: doc_id: cord_uid: g zhoyo the envelope (e) protein in sars coronavirus (cov) is a small structural protein, incorporated as part of the envelope. a major fraction of the protein has been known to be associated with the host membranes, particularly organelles related to intracellular trafficking, prompting cov packaging and propagation. studies have elucidated the central hydrophobic transmembrane domain of the e protein being responsible for much of the viroporin activity in favor of the virus. however, newer insights into the organizational principles at the membranous compartments within the host cells suggest further complexity of the system. the lesser hydrophobic carboxylic-terminal of the protein harbors interesting amino acid sequences- suggesting at the prevalence of membrane-directed amyloidogenic properties that remains mostly elusive. these highly conserved segments indicate at several potential membrane-associated functional roles that can redefine our comprehensive understanding of the protein. this should prompt further studies in designing and characterizing of effective targeted therapeutic measures. induce global metabolic changes on infected cells, leading to the rearrangement of the lipid dynamics to facilitate viral multiplication. ( ) in some cases, these alterations produce the reorganization of intracellular membranes of the host cell, building an adequate microenvironment for viral replication. all these findings highlight the intimate connections between viruses and host lipid interactions. typical of the genus, the initial steps of sars-covs entry include the attachment of the virus particle to a cellular receptor, angiotensin-converting enzyme (ace -specific for sars-cov) located on the host plasma membrane surface. ( ) the viral genome has to take entry into the host cell to reach the replication sites. different lipids found either on the plasma membrane and/or the endosomal membranes can contribute to these processes by enabling receptor clustering, virus internalization, or membrane fusion. the host spectrum/tropism of individual covs have been known to be primarily determined by the s protein. ( ) the functional aspects of the s protein have been known to mediate receptor binding and virus-cell fusion that occur at either the interface of the plasma membrane or within the endosomal vesicles. ( ) the mechanism of membrane fusion of sars-cov- mostly belongs to the type i fusion system, where s protein of sars-cov- comprises of a number of membrane binding regions in the s domain identified as fusogenic peptide or fps by several researchers. ( ) ( ) ( ) recently bhattacharjya et. al. have shown that one of these fusion peptides adopts β-sheet conformation either in the presence of phosphatidylcholine or phosphatidylcholine/cholesterol comprising membrane models with the help of circular dichroism spectroscopy. ( ) moreover, it has also been shown that the peptide undergoes oligomerization with increasing concentration of cholesterol in the membrane. in addition to receptor binding, the action of host cell-specific proteases can likely cleave the s protein, in turn, regulating the cov infection and host-or tissue-tropism. ( ) once inside the host cellular environment, positive-stranded rna viruses such as cov compartmentalize their replication machinery efficiently within the host membranous compartments to evade detection by host pattern recognition receptors. ( ) such compartments are formed for the benefit of the virus by taking advantage of cellular pathways and lipid-modifying enzymes. ( ) after transcription within the rough endoplasmic reticulum (er), the viral membrane proteins associate with the endoplasmic reticulum-golgi intermediate compartment (or the ergic) before being folded and promoting the budding off of the viral particle by hijacking the cellular secretory mechanism.( ) a major portion of the viral e protein has been known to remain localized at the site of intracellular trafficking, viz. the ergic system, participating in cov assembly and budding. ( , ) thus, much of the lipid components of the virus particles are comparable to these host organellar membranes being rich in phosphatidylcholines, phosphatidylethanolamines and a small fraction of phosphatidylinositols. ( ) this has provided direct evidence that the host membranes can serve as crucial functional domains that are indispensable for viral propagation. thus, mechanistic insight into the functional interface of host-membranes is particularly essential to obtain a comprehensive understanding of the target conformers for effective therapeutic strategies. e protein of cov is an integral membrane protein about - residues long and has been known to be most dynamic at the membranous functional interface. ( ) the primary structural elements of the protein make it particularly interesting that delve into its possible role in viral propagation. interestingly, despite the everevolving nature of the virion particles, the primary sequence of the protein remains mostly conserved. the sars-cov- e protein bears about % sequence similarity with that of the sars-cov. this alternatively directs into understanding the uniqueness of the sequence that is conserved to mediate some crucial functional roles of the protein (figure ). the primary amino acid sequence reveals a very short n-terminal hydrophilic sequence of about ten amino acids. ( , ) this is followed by a stretch of hydrophobic sequence that serves as the transmembrane domain (tmd). this twenty-eight residue long stretch has been known for the membrane-induced topologies of the protein that have been extensively defined and studied. ( ) the absence of a canonical cleaved signal sequence suggests equal likelihood for the protein to be a type ii (with its c-terminal targeted to the er lumen) or type iii (with its n-terminal targeted to the er lumen) membrane protein. therefore, the topology of the cov e protein is highly debated in the literature and remains largely inconclusive. table summarizes the contradictory topology predicted for some of the widely studied cov strains. the predicted topologies of the sars cov- e protein using prediction servers have been represented in table . however, further complicating matters, the topology models devised using prediction programs are mostly inconsistent with the previous experimental observations. two membrane-associated topologies have been demonstrated for e protein (figure )-hairpin or transmembrane. ( , ) in a report by arbely et al. ( ) , the protein was shown to adopt a highly unusual topology, comprising of a short transmembrane helical hairpin conformation that inverses about a previously unidentified pseudo-center of symmetry. ( ) this hairpin structure could deform lipid bilayers and cause fragmentation. ( , ) it has been suggested that post-translational modifications and several intermediate stages of maturation in the er and golgi environment can modulate the overall conformation of the molecule in response to the physiochemical properties of the membrane. ( ) ( ) ( ) the specific lipid composition of the particular lipid bilayers of the er-golgi protein synthesis-packing machinery of the cell defines divergent physicochemical properties at the membranous interface. the lipid packing density has been known to underlie significant features of the resulting membrane-inducing characteristic local curvatures, surface charge, phase behavior, elasticity, hydrophobicity, degree of hydration, etc.( ) membrane phase behavior in a cell has been known to permit the transient concentration of specific proteins and lipids into dynamic nanoscopic domains. ( ) the level of palmitoylation may moderate the relative proportion of the membrane-associated conformers of the protein that again, in turn, can modulate the local membrane curvatures, facilitating further favorable association of the protein conformers. ( ) substitutions of the hydrophobic amino acid residues in the tmd of the e protein with charged amino acids have been shown to alter the migrating properties of the e protein in sds-page. ( ) this, therefore, suggested changes in the overall conformation and membrane association of the mutant forms in comparison to the wild type e protein. the tmd is followed by a lesser hydrophobic c-terminus with a fraction incorporated as part of the envelope in the virion particle ( figure ). the c-terminus, in particular, has been known to be associated with the redirection to the host er and golgi complex membranes that underlies some of the major functional domains of the protein. ( , ) comprising of more than % of the entire protein sequence, this segment has been known to influence the topology in the host cell. there are several conflicting theories that either suggests a luminal localization of the c-terminal when associated with the ergic system or directed towards the cytoplasm. ( , ) the several experimental and in-silico prediction studies have been mostly inconclusive with respect to its localization and hence functional domain in the different strains of cov. nevertheless, the amino acid sequence of the c-terminal harbors some of the answers to the many known functional attribute of the protein sequence. interestingly, it comprises of the conserved "fyxy" peptide sequence (where x= any amino acid) that can be correlated to its high amyloidogenic propensity. ( , ) also, another highly conserved cysteine containing motif-"cxxc" can enable disulfide isomerization to prompt membrane-directed conformational changes. ( ) apart from these highly conserved sequences throughout the genus, there are distinct potent glycosylation sites along the stretch that can serve as chaperone interacting motifs to help in the protein folding and/or aid in j o u r n a l p r e -p r o o f journal pre-proof trafficking along with the cellular machinery.( ) glycosylation of particular asparagine residues (asn , asn , asn , and asn ) in the sars-cov has been shown to be crucial in maintaining the proteinoligomerization events associated with the host membranes. ( ) conversely, maintaining the glycosylation events have been suggested to be critical to maintaining the broad functional roles of the protein. ( , ) further terminally, there is a distinct phosphorylation domain comprising of the "s sr " motif. additionally, a pdzbinding domain (pbm) in the c-terminal plays an essential role in host cell modifications necessary for viral infection and pathogenesis (figure ). ( , ) mutation studies with the pbm have established its importance for viral infection, making it a significant factor of research for therapeutic and vaccine designing. ( , ) . the e protein as a functional viroporin. the primary structural features of the e protein have striking similarities with the viroporin class of proteins. ( ) ( ) ( ) viroporins ideally comprise of about - amino acids, with a distinct hydrophobic transmembrane domain. they are known for their interactions with membrane surfaces, often resulting in the expansion of the lipid bilayer. ( ) viroporins participate in several viral functions, including the prompting of viral particle release from host cells. ( ) although not essential for viral replication, viroporins modulate the host cellular machinery to make it favorable for viral propagation. they affect host cellular functions, including membrane vesicularization, glycoprotein tracking, and membrane permeability. the transmembrane domain could form hydrophilic pores in the membranes of virus-infected cells by oligomerization. ( , ) these hydrophilic channels would allow low molecular weight hydrophilic molecules to cross the membrane barrier, leading to the disruption of membrane potential, collapse of ionic gradients, and release of essential compounds from the cell several studies with sars cov e protein have demonstrated the structural and functional similarities with viroporins. it has been demonstrated that the sars-cov e protein could enhance the membrane permeability of bacterial cells to o-nitrophenyl-β-d-galactopyranoside and hygromycin b, suggesting that the protein may function as a viroporin. ( ) similar observations were reported on mouse hepatitis virus e protein. ( ) the e protein, as a viroporin, is present in low copy numbers in the virus particle, but has been implicated in membrane scission being mostly associated with the ergic and cis golgi-consistent with a predominant role as a mediator of virus assembly and release at this location. ( ) the lipid content at these locations may also enhance virus budding. ( ) compartments are formed by manipulating the protein-synthesis, packaging, and distribution system of the cell involving the er-ergic-golgi complex. this helps the viral replication machinery to evade detection by host pattern recognition receptors ( figure ). both viral and hijacked host proteins are used in this process, taking advantage of cellular pathways and lipid-modifying enzymes to the benefit of the virus, sequestering newly formed rnas away from host immune sensors. ( , ) this eventually allows the virus to manipulate the normal secretory pathway of the host cellular machinery to transport and deliver the virus protein cargo at the site of final packaging, and eventual budding. ( ) the virulence of the strain is, in fact, determined by the efficiency in their mechanism to concentrate the rna synthesis machinery and subsequent packaging within these specialized compartments that are, in turn, modulated by the associated structural proteins.( ) among the structural proteins, the e protein has been particularly intriguing either alone or in collaboration with the m protein. ( ) interestingly, studies have shown that overexpression of the e protein is sufficient to induce the packaging and assembly of the cov particles upon efficient manipulation of the host cellular machinery. ( ) rottier et al. suggested that the e protein has no genuine structural function in the virion envelope itself where it occupies frequent, regular positions within the lattice built by m protein. ( ) however, its primary role lies in the functional interface of the host packaging and assembling membranes ( figure ). the frequent but strategic positions within the lattice hint at a possible morphogenetic function to generate the required membrane curvature for the final viral particle formation ( figure , inset). studies have endowed the c-terminal to be associated with the redirection of the e protein to the er, and golgi complex for assembly, budding, and intracellular trafficking of infectious virions.( ) interestingly, sequence analyses of the c-terminal residues suggest the prevalence of some degenerate er export signal sequences. these are closely comparable to the most common fxxxfxxxf( ) and the [r/k](x)[r/k]( ) dibasic motifs that have been known to be responsible for the export of glycosyltransferases. the last -residue "r dklys " stretch from the ibv e protein terminal has been defined to serve as the er retention signal. ( ) the site-directed mutagenesis of this composite lysine residue (k ) to glutamine resulted in the accumulation of e in the golgi apparatus. [ ] maturing through the golgi complex, the e protein is expected to impart some very crucial functions with respect to vesicularization events, by inducing local curvatures at the site of membrane-directed accumulation ( figure ). two principle mechanisms have been described for moving and delivering cargo proteins through the secretory pathway of the golgi complex, the cisternal maturation, and the formation of mega-vesicles. ( , ) despite the extensive studies performed, experimental evidence for both the systems remains inadequate and far from being elusive. during cov infection, virions have been observed in large vesicle depots resembling megavesicles derived from golgi/ergic membranes, indicating that remodeling of the golgi complex may be crucial for virion trafficking. ( ) understanding the structural transitions that the e protein undergoes, from after its synthesis to its association with the ergic membrane complex, would greatly facilitate the characterization of potential targets. we expect the protein to undergo stepwise dynamic structural transitions with specific roles bound to the membranous component. this gradual transition is dependent equally on both the respective membrane composition along with the conformational changes in the protein as it is translated, modified, and matures along with the er-golgi network system. previous studies performed by knoops as membrane fusion and fission to prompt viral propagation by hijacking the entire cellular machinery. however, progress in our in-depth understanding of this whole machinery has been stymied by the inability to track the functional interface of the protein at the atomic-level resolution in real-time. identifying these intermediate functional conformers could broaden our chances of finding the best target motif for therapeutic design. the cov e protein has been known for its possible pro-apoptotic viroporin activity majorly by mobilizing calcium ions, prompting an overall change in the ionic environment of the homeostatic cell. ( , ) the virus particles, generally, hijack the cellular homeostatic machinery of the host cell by compromising the membrane integrity that facilitates its regulatory mechanisms within the environment of the host cells. it has been suggested that the e protein can directly disrupt membranes through the formation of ion channels upon membrane-directed oligomerization events mediated upon the insertion of the tmd. in this context, the tmd has received much interest owing to their membrane-spanning attribute. molecular dynamic simulation studies with synthetic peptides have revealed that the tmd could form ion channels by homo-oligomerization into stable dimers, trimers, and pentamers. ) additionally, in silico prediction studies based on high-resolution nmr spectroscopic studies provided useful insight into understanding the probable conformers that define the tmd induced ion channels. hence, these studies directed the defining of therapeutics targeting the resultant ion channel pores. ( ) the hollow structure of the oligomers allows the unregulated efflux of ions from the cellular compartments across the hydrophilic interior of the pore, modulating the membrane potential, altering the ionic environment of the lumen. alterations in ion concentration would promote translation of viral mrnas, given the translation of mrnas from many cytolytic animal viruses is reasonably resistant to high sodium concentrations. ( , ) since the secretory pathway of the host cellular machinery is highly sensitive to perturbations in ionic strength, viral propagation can trigger a detrimental apoptotic cascade. unregulated ion channel formation upon e protein oligomerization in the host membrane is believed to be one of the primary processes underlying the viral pathophysiology.( ) as discussed previously, much of the studies have been focusing on the tmd for the high molecular weight conformers (multimers) of the protein upon host membrane interactions. ( , ) however, interestingly, the immediate adjacent segment (towards the c-terminal) hint, at least in part, at a possible role in the membrane directed oligomerization propensity of the molecule. upon inspection into the c-terminal segment of the protein, we can see that it is marked by the presence of unique motifs that can prompt much of the membrane-directed conformational changes of the protein molecule. the primary sequence of the c-terminal region reveals possible functional characteristics of the protein that are lesser known in literature. interestingly, the sequence is distinctive of amyloidogenic proteins and peptides that have been studied in the literature for their membrane-directed indispensable functions. highresolution structural models of amyloid oligomers have been obtained in the context of membrane interactions in neurodegenerative diseases.( ) membrane-induced oligomerization prompts membrane compromise, often leading to vesicularization into smaller lipid aggregates. ( ) ( ) ( ) studies with amyloid proteins have hinted at the possibilities of a change in the local curvature being induced by the membrane-associated high molecular weight conformers of the proteins (figure , inset).( ) several reports have suggested specific lipid domains to serve as templates for protein anchorage. this is followed by rearrangement of the protein conformers resulting in aggregates that form "wedges" within the bilayer to facilitate curvature change. ( , ) such protein aggregates are driven mostly by the hydrophobic interactions that relocate within the acyl chain region of the lipid bilayer. these interactions result in a change in the lipid packing density and hence the overall membrane integrity. ( ) the structural similarity of the c-terminal segment of the e protein to amyloid proteomics indicates at analogous purposes that can be complementary to or function in parallel to its viroporin activities. the oligomerization properties of e protein have been defined previously.( ) e protein expressed in bacterial and mammalian cell systems under reducing conditions was shown to exist as monomers. however, upon change to non-reducing conditions, they formed homodimers and homotrimers. ( ) the existing literature has focused on the phenomenon of tmd multimerization associated with the ion channel formation that lacks mechanistic details on the initiation. however, site-directed mutagenesis studies revealed that the two-cysteine residues, immediately adjacent to the tmd in the c-terminal, were essential for the oligomerization, leading to the induction of membrane permeability. the tmd is immediately followed by a "cxxc" redox motif, which is highly conserved among the coronaviridae family of viruses. this motif is very crucial for several reasons. the oxidized cxxc motif can maintain the structural topology of the transmembrane region as well as that of its contiguous cytoplasmic domain, inclusive of glycosylation sites, involved in signaling and protein-protein interaction.( ) a probable activation of a thiol in this motif can trigger the participation of the other cys residues in the formation of the inter-subunit disulfide bond. the activated thiol will then attack the disulfide and cause its isomerization into a disulfide isomer within the motif. this may lead to the refolding of the transmembrane region and may activate its fusogenic potential.( , ) interestingly, a similar cxxc motif is present as a part of the c-terminal of s protein sequence. bioinformatic studies have suggested at the possibilities of forming inter-protein disulphide bridges that might indicate at a crossover or cooperation between these two structural proteins in the viral membrane interactions. ( ) the formation of a disulfide bond may also play a crucial role in the oligomerization of the e protein, forming stable dimers, trimers, and pentamers depending on its functional requirement.( ) thus even though the tmd spans the lipid bilayer, the cxxc motif could serve as an essential key to defining the membrane-associated oligomerization events-providing newer targets for preemptive therapeutic intervention. apart from the consequential involvement of this segment in the viroporin function, the amyloidogenic segment of the e proteins can function independently at the membranous interface. further downstream, the cterminal harbors the "fyxy" composite peptide sequence ( figure ) that has been known for a high propensity of amyloid formation. ( , ) interestingly, a self-assembling short peptide segment with the composite sequence has been studied to have a membrane-mediated function. bhunia and co-workers studied a -residue long peptide stretch (tk , t vyvysrvk ) in sars cov e protein (figure ) for its physical changes in the presence of both zwitterionic and negatively charged model membrane micelles.( ) the tk peptide segment was shown to adopt amyloid fibrillary structures in solution ( figure ). ( ) further studies with tk provided evidence for its aggregation propensity that closely resembles amyloid proteins. intriguingly, the composite -residue peptide motif-t vyv in tk was found to serve as the critical sequence motif that may be critical for the beta-sheet formation. ( ) however, in vitro measures demonstrated the differential orientation of the tk peptide with respect to the membranous environment: correlating well with the structure and function at the membrane-protein interactions interface. this atomic-resolution nmr based structural elucidation in the presence of membrane mimics have improved understanding of the molecular mechanism of cov infection under physiological conditions. studies demonstrated the helical propensity of this particular segment from the c-terminal region (figure ) of the protein to impart host specificity to the composite virus. the peptide's affinity was further j o u r n a l p r e -p r o o f journal pre-proof manifested by its pronounced membrane-integrity disruption ability towards the mammalian compared to the bacterial membrane mimic-implicated in the viral pathogenesis. tk penetrated deeper into the acyl region of the neutral micelles mimicking the mammalian membrane and its intracellular organelles as opposed to the superficial conformation when in association with the bacterial model membrane mimics (figure ). this provided evidence for the host membrane-associated conformational change in the protein segments that is specific to a particular lipid composition (species specificity). this study alternatively offered useful insight into the fact that the composite lipid molecules play particularly important roles in the interaction dynamics and conformational uptake. these segment-based studies highlighted the exclusive function of the amyloidogenic segment of the e protein that acts independently of the tmd-associated oligomerization events. similarly, another short -residue peptide, nk (n ivnvslvk ) further upstream, was identified and characterized, being marked by sheet breaker-hydrophobic residues, like ile, val, and leu. ( ) the experimental intervention into these segments can provide useful insight into the topology of the protein, its membrane-bound orientation, and eventual functional conformation uptake. these preliminary studies have provided useful links to the immense possibilities of the functional membrane-directed conformers of the e protein, which remains largely unexplored. nevertheless, epitope mapping for these interfaces can serve as critical targets for effective therapeutic intervention. preliminary sequence homology analyses performed to compare the e protein primary sequence of sars cov to that of the novel cov- , revealed a minimal difference with the "fyxy" motif remaining conserved (figure ). the first threonine and the second valine from the tk sequence have only been substituted by a serine and phenylalanine residues, respectively ( figure ). this might indicate at an unperturbed oligomerization potency of the segment. this conserved amyloidogenic sequence indicates a very crucial functional role of the segment that contributes significantly to the virulence. the fact that hydrophobic interactions can provide stability in localization within the acyl-chain layer of the lipid bilayers leaves us with some obvious open questions. does the c-terminal induce the initial docking of the protein to the host membranes? do these hydrophobic interactions prompt the downstream oligomerization events to the formation of the membrane-associated multimers? alternatively, does the lipid-composition specificity of the c-terminal determine the conformational uptake and the eventual functional topology? these questions are vital for a comprehensive understanding of viral propagation. interestingly, hydrophobic protection of these segments within the acyl-chain region can serve as open templates for downstream aggregation. this can result in membrane-associated aggregates that, in analogy to the amyloidogenic proteins, can modulate the overall membrane integrity. thus, it is essential to study the interface of interaction between the e protein and the host membrane, either as whole or directed to particular functional peptide segments. one of the major functional aspects of cov e protein has been associated with virus assembly and its subsequent release for propagation in the host physiology. indirect immunofluorescence microscopy showed that e protein is localized to the golgi complex in cells transiently expressing ibv e.( ) when co-expressed with ibv m, both from cdna and in ibv infection, the two proteins are colocalized in golgi membranes, near the cov budding site.( ) thus, even though ibv e is present at low levels in virions, it is apparently expressed at high levels in infected cells near the site of virus assembly. the subcellular localization of the sars cov e protein was analyzed using the sera from immunized mice by elisa and immunofluorescence using cells j o u r n a l p r e -p r o o f infected with recombinant (rsars-cov) and viruses lacking the e gene (rsars-cov-∆e) as a negative control. ( ) several parallel studies have endowed the c-terminal to be associated with the redirection of the e protein to the er, and golgi complex for the intracellular trafficking of infectious virions. ( ) experimental studies have shown that the c-terminal domain of the e protein, in fact, plays a crucial role in virus budding. ( , ) the deletion of the domain resulted in the free distribution of the mutant protein and a dysfunctional viral assembly. alternatively, mutations introduced into the cytoplasmic tail of mhv e protein by targeted rna recombination resulted in elongated virions, further indicating at the critical involvement of the domain in the budding mechanism. ( ) the difference in the lipid composition along the secretory pathway might, in fact, have a direct role on the e protein topology. moving from the er to the golgi and finally the plasma membrane, there is a definite gradient in the concentration of specific lipid components that induce a unique physical property of the lipidbilayers in terms of thickness and rigidity. ( ) the er presents a lower fraction of cholesterol or sphingolipids that allows maintenance of a relatively fluid lipid compartments for the remodeling associated with the protein association, sorting and accumulation. ( ) this fluidity can have a direct role in the gradual compartmentalization of the er-associated membranes prompting viral particle formations. the host membrane-mediated oligomerization of the e protein may be responsible for much of the membrane compromise in favor of the viral propagation. the membrane-associated aggregates can compromise the overall lipid bilayer integrity. ( , ) the hydrophobic interactions involving the lipid acyl regions can result in increased fluidity of the membrane surface, prompting phase separation of the lipid domains. biomolecular phase separation is suggested to drive the organization of cellular organelles involved in cellular packaging and compartmentalization.( ) differential distribution of the membrane proteins and their site of interaction with specific lipid rafts, help determine the local curvature and hence characterize propensity to bud off from the surface. ( , ) the membranous compartments of er and the golgi complex presents with a fair fraction of zwitterionic and neutral lipids-i.e., phosphatidylcholine and phosphatidylethanolamines. ( ) in vitro studies by khattari et al have demonstrated that the membrane-directed topology of the sars cov e protein imposes a direct constraint on the lipid-bilayer thickness and the acyl-chain ordering. ( ) their studies suggested that the increasing protein concentrations in the organellar membrane along the secretory pathway (greater protein/lipid concentrations), the tmd of the sars cov e protein induces bilayer lipid arrangements. specific inter-molecular interactions with the lipid molecules and intra-chain hydrogen bonds and salt-bridge interactions allow the uptake of a topology that traverses across the membrane bilayer. however, the exact transmembrane topology of the protein was highly debated and remains elusive.( ) nevertheless, these intra-and intermolecular interactions can be considered to have the ability to rearrange and induce direct morphological changes in the host membranes. the final viral packaging requires assembling of the replicated rna strands and the other viral proteins before the final membrane budding. the putative transmembrane domains of the e protein can also serve a 'catalytic' function in the membrane packaging. ( ) cross-talk between distant protein molecules can, in fact, serve as a "zipper" to close the 'neck' of the viral particle as it pinches off from the membrane in the terminal phase of budding. ( , ) despite the immense possibilities, much of the progress in our understanding of the actual mechanism of action is still hypothetical and far from being elusive. it is, therefore, essential to study the interface of interaction between the e protein and the replication complex. particularly, the membrane-mediated aggregation, that compromises the local curvature, inducing the final scission. cov gained particular notoriety when the sars cov outbreak shook the world around - . the aftermath leads to the identification of many newer family members. soon after that, studies were in full swing all around the globe to determine the mechanism of viral propagation. several parallel studies provided useful insight into the understanding of the structural and functional uniqueness of the viral proteins and rna in their search for finding the ideal target for therapeutic intervention. almost immediately after sars, the endemic spread of the mers-cov in reignited the urge to gain in-depth insights into the system all over the world for designing effective therapeutic measures. the recent outbreak of the novel covid- soon turned into a pandemic, threatening health standards globally. lu et al. first published the genomic characterization of covid- and also reported it to be significantly divergent from sars-cov, claiming it to be a new human-infecting βcoronavirus. ( ) a comparative analysis of small membrane/ envelope protein of different coronaviruses could provide an interpretation of the molecular events that could advance these viruses from developing acute infections to one producing a pandemic. an analysis of the sequence conservation can also help in identifying the regions required in the typical functions of membrane interaction, oligomerization, localization in the host cell, and viral infection. such information is of immediate significance and would contribute to vaccine designing and facilitate the evaluation of vaccine candidate immunogenicity. additionally, this would help in reflecting the potential effects of mutational events as the virus is transmitted through the human population. recent studies have revealed that the homology with a cov strain isolated from pangolin was ~ %. this indicated that while sars-cov- have been known to evolve from the bat cov, pangolins might have helped as an intermediate host. ( ) nevertheless, further research is required to justify these theories. we performed a blast (https://blast.ncbi.nlm.nih.gov/blast.cgi) search for the e protein and selected the sequences from the different genus of the coronaviridae family. homology analyses and sequence alignment were conducted using the mega software. the muscle program of the mega software was used to perform multiple sequence alignment, and a phylogenetic tree was created by using a maximum likelihood approach ( figure ). ( ) predictive tools such as tmhmm ( ), memsat( ), phobius ( ) , and the hydrophobic moment plot method( ) of eisenberg and co-workers allowed the calculation of the transmembrane regions. much has been defined to correlate the structural conformation of the tmd with its ion channel formation. however, sequence analyses of the c-terminal have revealed crucial information that has been out of the limelight for several years. despite the fact that the c-terminal has shown the potential to underlie much of the protein's dynamic functionality associated with membrane budding and scission, it has received much lesser attention. several secondary structure prediction programs predicted two β-strands within the sars-cov e protein tail sequence, including the n ivnvslvk and the t vyvysrvk . the predicted β-strands fit the criteria for forming a β-hairpin, which is a simple structural motif with two β-strands linked by a short loop of two to five amino acid residues. interestingly, a highly conserved proline residue, pro , resides between the predicted βstrands-responsible for the β-coil-β motif. ( ) the antiparallel β-strands form hydrogen bonds to stabilize the hairpin structure. previous reports with the synthetic peptide fragment of e (i.e., i -s ), encompassing the predicted β-hairpin segment, was found to produce ∼ % β-structure and was completely resistant to hydrogen-deuterium exchange in fourier transform experiments. alternatively, the study showed that titration with the drug molecule, hma ( -(n, n-hexamethylene) amiloride) resulted in large perturbations of the val and leu residues, which are far apart in the sequence. the structural model proposed by jaume torres's group, therefore, suggested that these two residues were coming spatially close in the membrane-associated pentamer and might belong to different monomers. comparison with mutation-based data hypothesized this β structure to be in dynamic equilibrium with an α-helical intermediate form. in fact, a delicate balance between j o u r n a l p r e -p r o o f these two forms may alter the membrane-dynamics and subsequent processes in the infected cell (e.g., membrane scission, binding to protein partners, or e protein localization). disturbing this predicted β-strand region along with the substitution of the conserved proline residues had resulted in the disruption of the cytoplasmic golgi complex signal, consequently changing the subcellular localization of e protein. ( ) taken together, the results support the hypothesis that the three-dimensional structure of e protein and not the primary sequence dictates much of its function. the sum of amino acid substitutions per site from between sequences is presented in figure . analyses were performed using the poisson correction model. the estimated value of the shape parameter for the discrete gamma distribution is ~ . , suggesting that the occurrence of the conserved amino acid residues among the coronavirus family e protein is not entirely random. however, upon close inspection with other e protein sequences of different species of the same genus, the value decreased to ~ . . substitution patterns and rates were estimated under the jones-taylor-thornton ( ) model (+g). ( , ) the model evolutionary rate differences among sites were obtained using a discrete gamma distribution ( categories, [+g]). all the ambiguous positions were eliminated for each of the sequence pairs (pairwise deletion option). evolutionary analyses were conducted in mega x. ( ) collectively, the data reveals that sites observed to be constant among sequences might have crucial biological significance correlated to their function. besides, deleterious mutations are much more likely to be found in population-level data. this indicates that sites that would predictably be considered as 'invariable' at the phylogenetic level might have transient polymorphisms at the population level. in the analyses of the e protein sequences from the cornaviridae family, altering the number of gamma categories did not have any noticeable effect on the estimate of the substitution rate. cov e protein has received lesser attention when compared to the other structural proteins (like s and m) majorly owing to a very low copy number in the viral envelope. ( , , ) it is the smallest and yet has been the most puzzling of the major structural proteins. earlier deletion based studies had proven that the viral life cycle endures the absence of e protein, implying that other viral genes could counterbalance for its loss. ( ) however, recent evidence collected with recombinant covs missing e protein, exhibit considerably lowered viral titers, crippled viral development, or produce progeny incapable of further propagation. ( , ) several parallel studies have highlighted the crucial role played by the e and m proteins in the propagation of the viral genome into the host cell and virulence. remarkably, studies have reported that viruses produced by the deletion of the sars-cov e gene can be attenuated in at least three different animal models that conferred protection against sars-cov pathogenesis. ( , ) but, the lack of complete information and a limited number of findings have prevented an understanding of the exact mechanism underlying the definite functional role of e protein in viral infection. nevertheless, these have provided the basis of understanding that the cov e might be involved in several aspects of the viral replication cycle, including host cell responses such as apoptosis, inflammation, and even autophagy in association with other nonstructural viral proteins.( ) soon after the identification, studies have been in full swing all around the globe to determine the mechanism of the viroporin action. several parallel studies from all over provided useful insight into understanding the structural and functional uniqueness of the viral protein in their search for finding the absolute target for therapeutic intervention. extensive studies performed in prof. torres's laboratory provide useful insight into the structural conformation of the ion channel pores created by e protein that seems to be a very attractive target for therapeutic intervention. ( ) their studies have prompted the designing and characterization of specific inhibitors for these channels that would perturb the primary functional attribute of the protein. hexamethylene amiloride has been studied to have blocked this e protein-associated ion channel activity in the mammalian cells expressing sars-j o u r n a l p r e -p r o o f cov e protein. ( ) however, viroporin proteins, like the sars-cov e protein, can exhibit a multitude of diverse functions unrelated to their ion-channel properties that are yet to be defined. the dynamic functional interfaces of the protein have been underestimated for its functionality in the propagation. this study can be correlated with earlier docking screening by gupta et al., where more than phytochemicals were evaluated on the same protein, and three of those, i.e., belachinal, macaflavanone e, and vibsanol b were found to be particularly promising. ( ) based on the experimental evidences, it is worth mentioning that the two hits, i.e., glycyrrhizic acid and cepharanthine, are supporting their significant activity against the sars viruses. ( ) more recently, anatoly chernyshev has established the potential of the sars-cov- e protein as a pharmaceutical target. ( , ) these studies have defined approved drugs, which theoretically could block the e protein-induced ion channel and eventually hinder the virus's life cycle. ( ) however, these studies have been based on in silico approaches, and hence much of the drug candidates have to be subjected to further experimental investigations. considering the lengthy procedure of new drug development, the existing strategy of drug repurposing has turned into one of the preferred solutions for the immediate treatment of sars-cov- affected individuals. ( ) long-term drug development objectives for the pharmaceutical industry incorporate the identification of inhibitors targeted at the replication or infection routes associated with sars-cov- or other allied coronavirus infections. ( ) knowledge of the coronavirus structural proteins and their mechanisms of viral action can be crucial in defining alternative therapeutic targets. despite the extensive research efforts worldwide, a definite cure to target cov propagation is still elusive. effective therapeutic intervention into viral systems without compromising the host cell integrity requires an in-depth understanding of the specific functional interface. especially, with the ever-evolving viral components, as seen in the covid- specimen, immense multidimensional research is the need for the hour. among the structural proteins, the e protein have not been a convincing choice for therapeutic targeting, despite the evidence for a crucial viroporin activity. much of the knowledge has remained restricted to only its ion channel formation ability. although this might be serving as a significant functionality, it is definitely not the comprehensive representation of the immense potential that the protein is possibly capable of undertaking. we believe, in-depth understanding of its functional interface with the host-membranes can undermine newer possibilities and functional attributes that have not received the deserved attention so far. attempts to determine the crucial protein domains associated with the host membranes are innovative in terms of targeted therapeutic designing. several studies have been gradually exemplifying the protein's essential role in viral packaging and propagation. the conserved sequences in the c-terminal region directly prompt the understanding of evolutionarily crucial functional roles of this segment that is yet to be conclusively defined. the knowledge is incomplete, essential insight into the protein's functional interface should provide newer insight into understanding and structure-function correlation. this should enable the effective designing of targeted therapeutics. the entire portrayals of biologics with characters suitable for inhibiting several key cov proteins could serve as a basis for drug development. j o u r n a l p r e -p r o o f infected cell table : list of predicted topologies of sars cov- envelope protein using in silico prediction servers. alternatively, the segment has been studied for the differential membrane-directed functioning. the peptide undergoes a helical conformation when in association with membranous environments. comparison between bacterial and mammalian model membrane mimicking systems showed the different orientation of the peptide. this suggested the specific functional role of this peptide segment and its membrane-directed structural change. preliminary studies with the ty peptide segment (underlined) had also shown the significance of the "fyxy" sequence, characteristic of amyloid proteins, and peptides. ( , ) the figure was prepared with https://biorender.com. j o u r n a l p r e -p r o o f highlights:  the sars cov envelope protein is a small structural protein of the virus that has been suggested to have significant viroporin like activity.  majority of its function is mediated at the interface of host-membrane interactions j o u r n a l p r e -p r o o f  focus at the membrane-directed features of the protein provide useful insight into gaining mechanistic insight into its viroporin functions.  studies have elaborated the central hydrophobic transmembrane domain of e protein, known to affect ion-channel formation.  the c-terminal region of the protein show further potential host-membrane directed functional roles.  the highly conserved amyloidogenic amino acid stretches of the c-terminal suggest at significant contribution to cov propagation. sars-cov- : an emerging coronavirus that causes a global threat world health organization declares global emergency: a review of the 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hawes, philippa c.; mantell, judith; verkade, paul; alrashedi, hasan; maier, helena j. title: infectious bronchitis virus nonstructural protein alone induces membrane pairing date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: uidpr positive-strand rna viruses, such as coronaviruses, induce cellular membrane rearrangements during replication to form replication organelles allowing for efficient viral rna synthesis. infectious bronchitis virus (ibv), a pathogenic avian gammacoronavirus of significant importance to the global poultry industry, has been shown to induce the formation of double membrane vesicles (dmvs), zippered endoplasmic reticulum (zer) and tethered vesicles, known as spherules. these membrane rearrangements are virally induced; however, it remains unclear which viral proteins are responsible. in this study, membrane rearrangements induced when expressing viral non-structural proteins (nsps) from two different strains of ibv were compared. three non-structural transmembrane proteins, nsp , nsp , and nsp , were expressed in cells singularly or in combination and the effects on cellular membranes investigated using electron microscopy and electron tomography. in contrast to previously studied coronaviruses, ibv nsp alone is necessary and sufficient to induce membrane pairing; however, expression of the transmembrane proteins together was not sufficient to fully recapitulate dmvs. this indicates that although nsp is able to singularly induce membrane pairing, further viral or host factors are required in order to fully assemble ibv replicative structures. this study highlights further differences in the mechanism of membrane rearrangements between members of the coronavirus family. viruses rely on their host cell to provide most of what they need to replicate and in order to do this, they hijack many cellular processes. a well-studied example is the ability of positive-sense single-stranded rna viruses (+rna) to induce cellular membrane rearrangements upon expression of viral proteins [ , ] . this reorganization of cellular membranes is a critical step in the viral replication cycle since these areas of restructured membranes act as a site for assembly of all components required for viral rna synthesis as well as offer protection from detection by the host antiviral defenses [ , ] . although the structures of these membranes are relatively well-understood, the mechanisms behind their formation, and particularly the viral and host proteins involved, are often not. the precise structure of virally induced membrane rearrangements varies between viruses [ , ] , but viruses generally cause proliferation of membranes, forming structures, such as convoluted membranes (cm), as well as distinct types of vesicles. most common are double membrane vesicles (dmvs), which are discrete from the cytoplasm and are produced by viruses, such as poliovirus [ , ] , hepatitis c virus [ , ] , human norovirus [ ] , and recently the equine torovirus, berne virus [ ] . spherules, which are invaginated vesicles with a channel connecting them to the cytoplasm, have been found in semliki forest virus [ ] , some flaviviruses [ ] [ ] [ ] [ ] , as well as brome mosaic virus (bmv), which is able to induce their formation with the expression of just one viral protein [ ] . an important +rna virus family, the coronaviruses, include pathogens of both animal and human importance, such as severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov), mouse hepatitis virus (mhv), porcine epidemic diarrhea virus (pedv), and infectious bronchitis virus (ibv). within this subfamily of viruses, we see variations in membrane rearrangements formed. dmvs and cm are found in cells infected with the alphaand betacoronaviruses, such as sars-cov, mers-cov, and mhv [ ] [ ] [ ] [ ] [ ] [ ] . in the case of the gammacoronavirus ibv, although dmvs are found, the virus induces little cm and instead induces membrane zippering to form zippered endoplasmic reticulum (zer) as well as double membrane spherules, which are found tethered to the zer [ ] , producing a much more defined structure when compared to cm. subsequent to this discovery, mers-cov infection has also been shown to produce small circular structures similar in appearance to the spherules seen in ibv infection but less distinct [ ] . the coronaviral proteins involved in the production of membrane rearrangements have been recently investigated with the three transmembrane non-structural proteins (nsps) nsp , , and , which are the focus of these studies. nsps , , and from different coronaviruses are accepted as functional homologues, although amino acid sequence conservation is low (ranging from . to . % amino acid homology for nsps , , and between ibv strain beaur and mhv strain a ). these proteins do, however, have conserved secondary structure and conserved domains, including enzymatic domains in nsp , transmembrane domains in all three proteins, and cytoplasmic endo-domains in nsps and . for a detailed review of the domain organization and known functions of nsps , , and , see [ ] . nsp of mhv has been shown to be important for the normal function and stability of dmvs, where mutations in nsp resulted in attenuated virus and impairment of dmv formation [ ] [ ] [ ] . in addition, nsp has been shown to localize to dmvs and cm in sars-cov-infected cells [ ] . in a related group of viruses, the arteriviruses, expression of two nsps (nsps and ) was able to produce dmvs [ ] [ ] [ ] . these nsps of the arterivirus are considered functional homologs to coronavirus nsp and [ ] . upon co-expression of nsp and from mhv, both proteins located to areas of curved membranes from where they were shown to be able to recruit nsp and ; however, nsp and alone were not able to induce the formation of dmvs [ , ] . following on from this, it was shown that co-expression of sars-cov nsp and induced membrane pairing and with the addition of nsp the formation of dmv-like structures [ ] . in a subsequent study by others, it was shown that expression of only nsp and from either mers-cov or sars-cov was able to induce dmv formation, and furthermore, addition of nsp made no difference to their shape or size, and did not induce the spherule-like structures seen following infection with whole virus [ ] . interestingly, however, a small molecule inhibitor, k , has been shown to inhibit the replication of several coronaviruses in vitro. in hcov- e, k impaired dmv formation, while k resistance was associated with mutations in nsp , emphasizing a role for nsp in dmv formation [ ] . ibv is a pathogen of poultry, causing significant economic losses to the poultry industry worldwide as well as animal welfare problems. various strains of ibv cause disease that varies in severity from mild respiratory problems to virulent strains that can cause nephropathology and reproductive organ pathology. in this study, we compared the membrane rearrangements induced by viral proteins from two different strains of ibv, the pathogenic m and the apathogenic beaur. these strains were chosen because beaur and other strains of ibv induce dmv, zer, and spherule formation; however, m produces a low spherule phenotype when compared with other strains of the virus [ ] . as the role in membrane rearrangements for nsp and is well-established for several nidoviruses and considering that nsp may also play some role, here we investigated the role that these three nsps play in the formation of ibv membrane rearrangements. avian df cells were maintained in dmem (sigma aldrich, gillingham, uk) supplemented with % fcs (sigma aldrich, gillingham, uk). ibv strains beaur and m -ck (here referred to as m ) have been described previously [ , ] . plasmids expressing tagged nsps derived from either the apathogenic strain beaur or the pathogenic strain m were generated to produce pegfp-n -m nsp , pmcherry-n -beaur nsp , pmcherry-n -m nsp , pcdna . (-)-beaur nsp - xflag, and pcdna . (-)-m nsp - xflag. rna was extracted from virus-infected cells using an rnaeasy kit (qiagen, hilden, germany) following the manufacturer's protocol. rna was reverse transcribed using superscript iii (fisher scientific, loughborough, uk) and a random primer following the manufacturer's protocol. pcr was carried out on cdna using primers specific for each gene, including flanking restriction sites. pcr products were digested and ligated into pegfp-n (takara bio europe, saint-germain-en-laye, france) or pmcherry-n (takara bio) using xhoi and bamhi restriction sites. plasmid pcdna . (-) was modified by insertion of a xflag motif between the kpni and hindiii sites to generate pcdna . (-)- xflag. the pcr products were then ligated into this backbone using the xhoi and bamhi restriction sites. plasmid sequences were verified using sanger sequencing. the er marker plasmid pyfp-er was kindly provided by dalan bailey. df cells seeded into six-well plates were transfected with pegfp-n -m nsp , pmcherry-n -beaur nsp , pmcherry-n -m nsp , pcdna . (-)-beaur nsp - xflag or pcdna . (-)-m nsp - xflag, pegfp-c , pmchery-n , or pcdna . (-)-beaur nsp - xflag using lipofectamine (fisher scientific). cells were transfected with a total of ng plasmid with a dna:lipofectamine ratio of : following the manufacturer's instructions. after h, cells were lysed in cell lysis buffer ( mm tris-hcl (ph . ), mm nacl, mm edta, % v/v triton-x , % v/v glycerol, × halt protease inhibitor complex (fisher scientific). cell lysates were heated with × sample buffer (bio-rad laboratories, watford, uk) and separated on - % tgx gels (bio-rad). proteins were transferred to a nitrocellulose membrane and blocked in % milk in pbs-t. membranes were incubated with primary antibodies to detect gfp (biolegend, london, uk), mcherry (abcam, cambridge, uk), or flag (m ; sigma aldrich, gillingham, uk). after h, membranes were washed with pbs-t and incubated with irdye conjugated secondary antibodies (li-cor, cambridge, uk). membranes were imaged using an odyssey clx infrared imaging system (li-cor). df cells seeded onto glass coverslips were transfected with pegfp-n -m nsp , pmcherry-n -beaur nsp , pmcherry-n -m nsp , pcdna . (-)-beaur nsp - xflag, and pcdna . (-)-m nsp - xflag alone or in combination using lipofectamine . cells were transfected with a total of ng plasmid with a dna:lipofectamine ratio of : following the manufacturer's instructions. after h, cells were fixed for min in % paraformaldehyde in pbs at room temperature. cells were then permeabilized in . % triton x- in pbs for min and blocked in . % bsa in pbs for h. primary anti-flag m antibody (sigma aldrich) and anti-pdi antibody (enzo life sciences, exeter, uk) were diluted in blocking buffer and cells incubated for h. after three washes in pbs, alexa fluor conjugated secondary antibodies (fisher scientific) were diluted / and cells incubated for h. after a further three washes in pbs, nuclei were strained using topro (fisher scientific) or dapi (sigma aldrich) and coverslips mounted with vectashield (vector laboratories, peterborough, uk). cells were visualized using a leica sp confocal microscope (leica microsystems, milton keynes, uk). quantitation of transfected cells was performed manually on three randomly selected fields of view. df cells in six-well plates were either infected with beaur and incubated for h at • c when fresh × bes medium (mem, . % tryptose phosphate broth, . % bovine serum albumin, mm n,n-bis( -hydroxyethyl)- -aminoethanesulfonic acid (bes), . % sodium bicarbonate, mm l-glutamine, u/ml nystatin, u/ml penicillin, and u/ml streptomycin) was added, or were transfected with plasmids as described above. at hpi, cells were washed once in . % saline and scraped into the saline buffer. cells were pelleted at × g for min at • c and µl % glutaraldehyde in . m sodium cacodylate was added to the pellet. df cells were then rinsed three times in . m sodium cacodylate and incubated in % osmium tetroxide for h. after three washes in water, cells were incubated in % uranium acetate aqueous for h at • c. cells were dehydrated in increasing concentrations of acetone and then embedded in agar resin (agar scientific, stansted, uk). sections approximately to nm in thickness were cut and stained with % uranyl acetate to enhance contrast. data was recorded at kv on a phillips cm (amsterdam, netherlands) with a charge-coupled device (ccd) camera. cell sections used here each contained a single visible nucleus, with intact nuclear and plasma membranes. alternatively, df cells were seeded onto thermanox coverslips (fisher scientific) and either infected with beaur and incubated for h at • c, after which time fresh × bes medium was added, or cells were transfected with plasmids as described above. after h, cells were fixed in % glutaraldehyde for h, incubated in % aqueous osmium tetroxide solution for h, then dehydrated in increasing concentrations of ethanol. cells were embedded into agar resin and sections of nm were cut, collected on hexagonal thin bar copper grids, and stained with % uranyl acetate and lead citrate. data was recorded on a fei tecnai tem (fei, cambridge, uk) used at kv with a tvips f digital camera. df cells seeded onto thermanox coverslips were transfected and processed as before. sections or nm thick were cut from the resin-embedded blocks and collected on mesh copper hexagonal grids coated in formvar or pioloform-coated copper slot grids. ten or nm gold particles were applied to the grids to serve as fiducial markers for subsequent alignments. data was recorded on a jeol f tem (jeol, welwyn garden city, uk) used at kv with a tvips f digital camera, or on a tecnai tem (fei) used at kv with a fei k × k eagle ccd camera. samples were mounted in a jeol high angle tilt holder or a fischione double tilt tomography holder, respectively. a single axis tilt series was collected using serial em or fei software. each single axis tilt series was collected over • to • in increments of between • and . • and subsequently aligned and reconstructed in imod [ ] . our previous studies have shown that ibv is able to induce diverse membrane rearrangements in vero cells, primary chicken kidney cells (ckcs) and tracheal organ cultures (tocs). these membrane rearrangements include dmvs, zer, and spherules [ , ] . in order to further characterize membrane rearrangements induced by ibv, we analyzed the membrane rearrangements induced by beaur in df s. unlike primary ckcs, df s are a continuous avian cell line that are more easily transfected and are therefore used throughout this study. although the spike protein of beaur has increased tropism to allow for virus entry into additional cell lines, including df cells, m is not adapted to infect these cells [ , ] . df cells were infected with beaur, fixed after h, processed for em, and imaged. consistent with previous work, dmvs, zer, and spherules were all seen in ibv-infected df cells ( figure ). beaur. df cells were infected with beaur for h, fixed, and processed for electron microscopy (em). viral particles are indicated with arrowheads, double membrane vesicles (dmvs) with asterisks, and zippered endoplasmic reticulum (zer) and associated spherules with arrows. scale bar represents nm. other viruses in the nidovirales order have been shown to require expression of only two or three nsps to induce membrane rearrangements similar to those seen under virus infection conditions [ , , ] . to begin to understand the roles of ibv nsps in rearranging cellular membranes, nsps and from apathogenic beaur and nsps , , and from pathogenic m were tagged with fluorescent or epitope tags. it was not possible to generate a plasmid expressing nsp from beaur due to presumed toxic sequences, as has been found for this region in other coronaviruses [ ] [ ] [ ] . df cells were transfected with these plasmids and after h cells were lysed and proteins separated by sds-page and detected by western blot. all fusions proteins were found to be intact with bands detectable at the predicted molecular weights (figure a) , although an additional kda band was present in nsp -gfp expressing cells, presumably due to a cleavage event within nsp . it was also noted that nsp - xflag from m migrated at a higher molecular weight than nsp - xflag from beaur, most likely due to differences in post-translational modification. subsequently, df cells were transfected with these plasmids and after h cells were fixed, labelled with an anti-flag antibody, and visualized by confocal microscopy. all three nsps showed reticular cytoplasmic labelling consistent with localization to the er (figure b ), as has been observed previously [ , , [ ] [ ] [ ] [ ] [ ] . in addition to er localization, nsp was found in both small and large puncta in cells where the level of nsp expression was higher (comparison shown in figure b ). nsp was also found in small cytoplasmic puncta when expressed alone (figure b ). to confirm er localization, df cells were transfected with either the plasmid expressing nsp -gfp alone or plasmids expressing nsp or together with pyfp-er, as indicated. after h, cells were fixed and labelled with anti-flag-and nsp -expressing cells with anti-protein disulphide isomerase (pdi), a resident er protein. colocalization between yfp-er or pdi and nsp , , and was observed, confirming that these proteins localize to the er (figure c ). cells were transfected with plasmids expressing tagged nsps, as indicated, or empty vectors or nsp - xflag as controls. cell lysates were separated by sds-page and proteins detected by western blot. from left to right, nsp -gfp detected using anti-gfp, nsp -mcherry detected using anti-mcherry, and nsp - xflag detected using anti-flag, as labelled. molecular weight markers are shown on the left and asterisks indicate the nsp bands on each blot. (b) df cells were transfected with plasmids expressing nsp -mcherry and nsp - xflag from beaur, and nsp -egfp, nsp -mcherry, and nsp - xflag from m . after h, cells were fixed with % paraformaldehyde and imaged. nsp (green), nsp (red), and nsp (blue) were imaged as labelled. nuclei were stained with topro (grey) and scale bars indicate µm. (c) df cells were transfected with plasmids expressing nsp -mcherry and nsp - xflag from beaur, and nsp -egfp, nsp -mcherry, and nsp - xflag from m together with yfp-er. after h, cells were fixed with % paraformaldehyde and imaged. nsp (green) and nsp and nsp (red) were imaged along with markers for the er; pdi (red) or yfp-er (yellow) as indicated. nuclei were stained with dapi (grey) and scale bar represents µm. next, to understand whether co-expression of these proteins results in changes in their localization, df cells were transfected with combinations of the plasmids. after h, cells were fixed and labelled with an anti-flag antibody. upon co-expression of some combinations of these viral proteins, this staining pattern changed. expression of nsp with nsp resulted in both proteins localizing to cytoplasmic puncta, although some signals for both proteins also remained in the er (figure ). co-expression of nsp with nsp , or nsp with nsp , did not result in relocalization of either protein, with nsp remaining er-associated, nsp remaining both er-associated and localized in cytoplasmic puncta, and nsp remaining both er-localized and in cytoplasmic puncta (figure ) . interestingly, co-expression of nsps , , and resulted in relocalization of all three proteins to cytoplasmic puncta, some containing nsp and , some nsp only, and some puncta containing nsp , , and ( figure ). nsps and derived from either beaur or m exhibited the same pattern of localization. this demonstrates that co-expression of ibv nsps in the absence of any other viral components can result in their relocalization within the cell, presumably as a result of protein-protein interactions and potentially associated with rearrangement of cellular membranes. figure . co-expression of ibv non-structural proteins results in their relocalization from the er to cytoplasmic foci. df cells were transfected with plasmids expressing nsp -mcherry and nsp - xflag from beaur, and nsps -egfp, nsp -mcherry, and nsp - xflag from m in pairs or in a combination of three, as indicated. solid arrows indicate areas of nsp and colocalization, open arrows indicate areas of nsp , , and colocalization. nuclei were strained with topro (grey) and scale bar represents µm. to further understand the ability of ibv nsps , , and to rearrange cellular membranes, proteins were expressed in cells and analyzed by electron microscopy (em). initially, to assist with subsequent analysis by em, the percentage of total cells in figures b and that were expressing the nsps of interest, as well as the percentage of cells expressing other combinations of nsps, was quantified (table s ). df cells were transfected with tagged nsp , , and derived from beaur or m alone and in combination. after h, cells were chemically fixed, embedded in resin, and visualized using an electron microscope. a phenotype common to all transfected cells was small, tight whorl-like structures which stained more strongly than other structures (figure a ). these were considered an artefact of transfection. transfection of cells with empty pegfp-n , pmcherry-n , or pcdna . (-)- xflag did not result in changes to cellular membranes (figure a ). different types of membrane structures were observed in the transfected cell samples that were absent from mock treated cells, including paired membranes, disordered or piled membranes, and dmv-like structures. nsp in other coronaviruses has been shown to be important in membrane modifications, particularly in the formation of conventional dmvs [ , ] . initially, the effect of expression of nsp in df cells was investigated. interestingly, it was observed that expression of beaur nsp alone was capable of forming paired membranes. this the first time this has been observed for any coronavirus nsp . these paired membranes were observed both as very large areas of extensive accumulations or as small regions of shorter sections of paired membranes. the paired membranes were tightly apposed, often connected to the er, were largely free of ribosomes, and strongly resembled ibv-induced zer (figure b) , although the electron density often surrounding ibv-induced zer was missing here and no spherules were present. transfection of m nsp also induced membrane pairing (figure b) with an appearance comparable to that of beaur nsp -induced paired membranes. for cells transfected with the beaur nsp expression vector, out of cell sections ( %, percentage of total cells not transfected cells) contained piled membranes, and out of ( %) sections for m , significant to p < . by a fisher's exact test. it has previously been shown for other coronaviruses that membrane pairing requires co-expression of nsps and or that co-expression of these proteins results in dmv accumulation [ ] . therefore, the effect of co-expression of nsp with nsp was investigated. firstly, the effect of expression of nsp alone on cellular membranes was determined. although over cells were examined from multiple experiments, expression of nsp was found to have no striking phenotype with cellular membranes appearing unchanged in the presence of nsp derived from m when compared with untransfected cells. furthermore, surprisingly, expression of nsp with nsp had no effect on the membrane pairing ability of nsp (figure c) , with membrane rearrangements appearing comparable to cells expressing nsp alone, i.e., paired membranes connected to the er and lacking ribosomes, found covering both large and smaller areas of the cytoplasm. specifically, the numerous dmv-like structures observed in cells expressing nsp and from either mers-cov or sars-cov were not observed here [ , ] . overall, this data confirms that ibv nsp alone is the main driving factor in membrane pairing and co-expression of nsp does not alter this function. coronavirus nsp has previously been linked to autophagy induction when expressed alone [ ] . nsp derived from sars-cov has also been shown to induce single membrane vesicle accumulation and microtubule organizing center vesiculation [ ] . therefore, the cellular membrane rearrangements induced by expression of ibv nsp were analyzed. in cells expressing nsp alone from either beaur or m , large areas of tangled single membranes, which appear to be derived from the er, were observed ( figure ). these piled, disordered membranes strongly resemble the disordered membrane bodies seen previously upon expression of sars-cov nsp [ ] . to determine the effect of expression of nsp with other nsps on the formation of disordered membranes or any other structures, samples transfected with plasmids expressing nsp and either nsp or nsp were analyzed. in cells co-expressing nsp and , it was observed that cells expressing beaur nsp formed disordered membranes while those expressing m nsp did not. co-expression of nsp and nsp produced the paired membranes associated with nsp expression (for both beaur and m nsp ). disordered membranes were only found in cells co-expressing beaur nsp but none when co-expressing m nsp . this indicates that while nsp from either beaur or m can induce the formation of disordered membranes when expressed singly, co-expression of nsp with either nsp or disrupts this mechanism and to a greater extent in m . finally, the membrane rearrangements induced by co-expression of ibv nsps , , and were investigated by electron microscopy to determine whether co-expression of all three transmembrane nsps could result in the formation of structures comparable to replication organelles in ibv-infected cells. the major phenotype observed following co-expression of all three nsps was the paired membranes induced by expression of nsp alone ( figure ). when nsp and nsp derived from beaur were expressed with m nsp , a very limited number of dmv-like structures was observed ( in cell sections). in cells co-expressing nsp , , and derived from m , no dmv-like vesicles were found in cell sections with only nsp -associated paired membranes being detected. in neither combination were the spherules usually found during virus infection observed. therefore, although co-expression of ibv nsps , , and may be sufficient for formation of dmvs, this does not seem to be a very efficient process compared with dmv formation by nsp and from the betacoronaviruses studied previously [ , ] and nsp is unlikely to be the additional nsp required for ibv dmv formation. in order to further understand the paired membranes induced by expression of ibv nsp , electron tomography (et) was used to visualize membrane rearrangements in three dimensions. in addition, et was used to confirm that, unlike for other coronaviruses [ , ] , co-expression of ibv nsp and does not result in the formation of dmvs. df cells were transfected with plasmids expressing either beaur nsp or beaur nsp with m nsp . after h, cells were fixed and processed for et. the paired membranes produced by nsp expression (indicated by arrows) were found to form sheet-like structures with sections of paired membranes dilating in several places (arrowheads) (figure a , video s ). a comparison with cells expressing nsp and showed there is no noticeable difference between the areas of paired membranes induced upon expression of these nsps (figure b, video s ) . therefore, expression of ibv nsp alone results in the formation of paired er membranes. addition of nsp does not alter the membrane structures induced with no formation of either dmvs, as seen for other covs or spherules. induction of host cell membrane rearrangements is a tool used by many +rna viruses, such as coronaviruses [ , ] . these membrane rearrangements vary between the different members of the family, with the alpha and betacoronaviruses inducing convoluted membranes and dmvs and the gammacoronavirus ibv inducing zippered er, spherules, and dmvs [ ] [ ] [ ] [ ] [ ] , ] . the formation of these membrane rearrangements is, however, a well-conserved mechanism used by these viruses in order to provide a site for viral rna synthesis. although the pool of knowledge about these structures has been growing, the mechanisms behind their formation remain largely unclear. some light has been shed in recent years on the specific viral proteins involved in the formation of these structures; however, these studies were lacking in ibv. in this study, we looked at the involvement of nsps , , and , which have all been implicated in the formation of membrane rearrangements. as transmembrane proteins, these are likely candidates in reordering the host cell membranes to the advantage of the virus. we showed firstly that df cells are a suitable cell type to use for studying ibv membrane rearrangements in addition to those already tested [ ] . in order to assess the involvement of nsps , , and in virus-induced membrane rearrangements, plasmids expressing gfp, mcherry, or xflag fusion proteins were generated. to confirm expression of full-length fusion proteins, western blots were performed using antibodies against the tags. for all the constructs, full-length nsp fusion proteins were detected. however, in cells expressing nsp -gfp, an additional kda band was seen indicating that as well as full-length protein, a cleavage product corresponding to the c-terminus of nsp plus gfp was also being produced. next, we expressed nsps alone or in combination in df cells to assess their ability to rearrange cellular membranes. when expressed alone, all three nsps had a reticular, cytoplasmic localization consistent with previous observations that these nsps localize to the er [ , , [ ] [ ] [ ] [ ] [ ] , although nsp and nsp in addition had a punctate localization with nsp in particular forming large foci in some cells. er localization was subsequently confirmed by colocalization of the three nsps with er markers. when nsps and were co-expressed, both proteins localized to large and small cytoplasmic puncta with some protein also remaining in the er. this suggests that nsp and are able to interact with one another, again consistent with previous findings for other coronaviruses [ , ] , resulting in nsp moving into the nsp -containing puncta. co-expression of nsp and or nsp and did not result in alteration of their cellular localization. however, when nsp , , and were co-expressed, nsp and colocalized as seen before but some puncta now also contained nsp , although some puncta contained only nsp and or nsp alone. this suggests that, as seen in other coronaviruses, nsp and together, but not alone, are able to direct nsp into the nsp / puncta [ , ] . subsequently, em was used to identify changes to the structure of cellular membranes upon expression of these three proteins. surprisingly, expression of nsp did not induce any notable phenotype. expression of nsp from either sars-cov or mers-cov results in the production of disordered membrane bodies likely derived from the er [ , ] . it is not clear why nsp derived from ibv behaves so markedly differently from nsp s expressed by other coronaviruses. however, the previously studied nsp s have all been derived from betacoronaviruses so nsp from gammacoronaviruses, including ibv, may function somewhat differently. indeed, an amino acid sequence comparison between nsp sequences from beaur and the betacoronavirus mhv a shows only . % homology and . % similarity. therefore, although these are accepted as functional homologs, there is scope for these proteins to behave differently from one another. furthermore, given that we have previously demonstrated that ibv-induced membrane rearrangements are distinct from those induced by alphaand betacoronaviruses [ ] , differences in the mechanism of their formation might reasonably be expected. interestingly, expression of nsp alone induced membrane proliferation and the formation of disordered membranes similar to the disordered membrane bodies (dmbs) induced by sars-cov and mers-cov nsp [ , ] . expression of nsp alone did not appear to induce microtubule organizing center vesiculation as seen upon expression of sars-cov nsp [ ] and the presence of autophagosomes was also not apparent [ , ] , although this is likely due to differences in experimental approaches, namely the use of em in this study compared to immunofluorescence of whole cells used previously [ ] . therefore, ibv nsp also appears to function somewhat differently to nsp from sars-cov in its ability to rearrange membranes. the most striking phenotype came upon expression of nsp ; expression of nsp alone was sufficient to induce areas of paired membranes. furthermore, et demonstrated that these are sheet-like areas of paired er membranes, highly similar to zer in ibv-infected cells. it was noted that the paired membranes, although resembling zer in infected cells, lacked the electron density often surrounding the membranes [ ] . this reflects the lack of the other viral proteins making up the replication complex, which, presumably, accumulate on the cytoplasmic surface of the zer. nsp -induced paired membranes were observed as both small regions throughout the cytoplasm and also in extensive areas of paired membranes. these two phenotypes potentially reflect the different localizations observed by confocal microscopy with some cells containing nsp localized only to the er and some cells containing large cytoplasmic puncta corresponding to the large areas of paired membranes. use of correlative light electron microscopy (clem) in the future would confirm this. attempts were made to confirm the nsp homotypic interaction by co-immunoprecipitation; however, this was not successful. it has previously been shown for mhv that nsp can self-associate [ ] , although earlier attempts to demonstrate the interaction in sars-cov failed [ , ] , highlighting that detection of this interaction can be challenging. however, it is likely that self-interaction between nsp proteins located in both membranes of the er zippers the two er membranes together to generate the paired membranes seen, although it cannot be ruled out that instead an interaction with one or more cellular proteins is required. significantly, this is the first time for any coronavirus that, regardless of mechanism, a membrane pairing function for nsp alone has been described. surprisingly, addition of nsp did not alter the membrane rearrangements induced by nsp alone. previous work by others has shown that for other related coronaviruses and arteriviruses, membrane pairing requires the expression of nsp and (or their homologs) [ ] [ ] [ ] ] . in addition to this, however, co-expression of nsp and for other coronaviruses resulted in the formation of numerous dmv-like structures [ , ] . despite extensive searching and the use of electron tomography to gain three-dimensional information, we were not able to detect any dmvs in cells expressing nsp and . the reason for this difference is not clear. here, we used separate plasmids to express nsp and but this strategy was also used in previous work and when compared with a cleavable nsp - precursor did not yield different results [ ] . therefore, the protein expression strategy is unlikely to be the reason that dmvs were not formed. it is possible that the presence of the shorter nsp fragment detected by western blot prevented the formation of dmvs. however, full-length nsp was also present and therefore should have been capable of inducing dmvs in combination with nsp . in addition, dmvs were not detectable in cells expressing either nsp from m and nsp from beaur or cells expressing nsp and from m , indicating that the use of proteins from different virus strains was not the reason for the lack of dmvs. indeed, nsp relocalized to both beaur and m nsp -containing foci suggesting that m nsp is capable of interacting with both nsp proteins. again, attempts were made to confirm interaction between nsp and nsp by co-immunoprecipitation, but this was not successful. interactions between full-length or the c-terminus of nsp and nsp from other coronaviruses have been shown previously [ , ] . interestingly, sakai et al. showed that just two amino acid residues in nsp are necessary for the interaction with nsp ; however, these residues are only conserved in betacoronaviruses, not in alphaor gammacoronaviruses [ ] , so it is likely that the mechanism of any nsp /nsp interaction is different in ibv. overall, the data indicates that dmv formation by ibv requires the presence of additional viral protein(s), either to direct an interaction between nsp and nsp if it cannot occur directly or because dmv formation is via another mechanism. co-expression of nsps , , and did appear to result in the formation of a very small number of dmv-like structures. however, these were significantly less numerous and less easily identifiable than those observed by oudshoorn et al. [ ] . therefore, nsp does not appear to be the ibv protein required, in addition to nsp and , to induce dmvs and other viral proteins must play a role. throughout this study, we were unable to detect spherules associated with ibv infection, although we did identify membranes highly similar to zer. in our previous work, we demonstrated that m virus has a low spherule phenotype and the region of the genome from the end to nsp was responsible for this [ ] . unfortunately, we were unable to clone nsp from beaur due to toxicity problems in escherichia coli. it was also not possible to clone nsp from two further strains of ibv. as the nsp used in this study was derived from m , it is possible that this is the reason that spherules were not detected under any conditions. nsp from beaur and m are highly related with . % amino acid homology and . % similarity with the majority of the differences occurring within the non-functional papain-like protease domain. despite that fact, it cannot be ruled out that these differences are sufficient to prevent spherule formation. in future, cloning the c-terminal part of nsp from beaur, as other groups have done for mhv [ ] , may provide further insight into the role of nsp in membrane modifications. an alternative explanation for the lack of spherules could be that the precise molar ratio of nsps to one another, as well as the presence of cleavage intermediates, generated as a result of expression via a polyprotein during virus infection is critical for spherule formation. in that case, the expression approach taken here of transfecting multiple plasmids into cells would not result in the correct ratio of proteins or presence of cleavage intermediates, thereby preventing spherule formation. however, oudshoorn et al. were also unable to identify cms and spherule-like structures when combinations of nsps were expressed either from separate plasmids or as a polyprotein [ ] . instead, it is more likely that additional viral proteins are required for spherule formation. this is not necessarily surprising. for alphaviruses, spherules are only formed in the presence of all nsps and although they are able to form in the absence of rna, the length of rna present directly affects the size of the spherule produced [ , ] . furthermore, in the case of flock house virus, spherules only form when rna synthesis is actively occurring [ ] . therefore, spherule formation by ibv may require expression of additional nsps, including those required for rna synthesis, as well as an rna template. alternatively, it may require expression of additional nsps that direct interaction with cellular proteins that facilitate changes to the membrane. the mechanisms behind the formation of virus-induced membrane rearrangements required for replication organelle formation are doubtlessly complex. although we have identified a clear role for ibv nsp in membrane pairing and the formation of zippered er, numerous questions remain and further differences between ibv and members of the betacoronavirus sub-family have been highlighted. the identity of the ibv proteins required for both spherule and dmv formation remain unknown and further study is required to complete our understanding of the critical stage of the virus replication cycle. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , table s : cells expressing each nsp compared to the total number of cells counted, video s : electron tomographic reconstruction of paired membranes in an nsp -expressing cell, video s : electron tomographic reconstruction of paired membranes in an nsp and nsp -expressing cell. modification of intracellular membrane structures for virus replication virus factories, double membrane vesicles and viroplasm generated in animal cells organelle-like membrane compartmentalization of positive-strand rna virus replication factories the hepatitis c virus-induced membranous web and associated nuclear transport machinery limit access of pattern recognition receptors to viral replication sites ultrastructure of the replication sites of positive-strand rna viruses architecture and biogenesis of plus-strand rna virus replication factories structural and functional characterization of the poliovirus replication complex complex dynamic development of poliovirus membranous replication complexes identification of the hepatitis c virus rna replication complex in huh- cells harboring subgenomic replicons sequential biogenesis of host cell membrane rearrangements induced by hepatitis c virus infection membrane alterations induced by nonstructural proteins of human norovirus ultrastructural characterization of membranous torovirus replication factories biogenesis of the semliki forest virus rna replication complex composition and three-dimensional architecture of the dengue virus replication and assembly sites the endoplasmic reticulum provides the membrane platform for biogenesis of the flavivirus replication complex ultrastructure of kunjin virus-infected cells: colocalization of ns and ns with double-stranded rna, and of ns b with ns , in virus-induced membrane structures ultrastructural characterization of zika virus replication factories a positive-strand rna virus replication complex parallels form and function of retrovirus capsids ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex rna replication of mouse hepatitis virus takes place at double-membrane vesicles qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-α treatment ultrastructural characterization of membrane rearrangements induced by porcine epidemic diarrhea virus infection infectious bronchitis virus generates spherules from zippered endoplasmic reticulum membranes expression and cleavage of middle east respiratory syndrome coronavirus nsp - polyprotein induce the formation of double-membrane vesicles that mimic those associated with coronaviral rna replication bioinformatics and functional analyses of coronavirus nonstructural proteins involved in the formation of replicative organelles mutation in murine coronavirus replication protein nsp alters assembly of double membrane vesicles murine hepatitis virus nonstructural protein regulates virus-induced membrane modifications and replication complex function mutations across murine hepatitis virus nsp alter virus fitness and membrane modifications sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum formation of the arterivirus replication/transcription complex: a key role for nonstructural protein in the remodeling of intracellular membranes open reading frame a-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex non-structural proteins and interact to modify host cell membranes during the formation of the arterivirus replication complex evolving the largest rna virus genome membrane rearrangements mediated by coronavirus nonstructural proteins and . virology mobility and interactions of coronavirus nonstructural protein severe acute respiratory syndrome coronavirus nonstructural proteins , , and induce double-membrane vesicles targeting membrane-bound viral rna synthesis reveals potent inhibition of diverse coronaviruses including the middle east respiratory syndrome virus extensive coronavirus-induced membrane rearrangements are not a determinant of pathogenicity reverse genetics system for the avian coronavirus infectious bronchitis virus taxonomic studies on strains of avian infectious bronchitis virus using neutralisation tests in tracheal organ cultures computer visualization of three-dimensional image data using imod selection of and recombination between minor variants lead to the adaptation of an avian coronavirus to primate cells contributions of the s spike ectodomain to attachment and host range of infectious bronchitis virus engineering the largest rna virus genome as an infectious bacterial artificial chromosome strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model systematic assembly of a full-length infectious cdna of mouse hepatitis virus strain a coronavirus nsp proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate detection of nonstructural protein in murine coronavirus-infected cells and analysis of the transmembrane topology by using bioinformatics and molecular approaches membrane topology of murine coronavirus replicase nonstructural protein topology and membrane anchoring of the coronavirus replication complex: not all hydrophobic domains of nsp and nsp are membrane spanning localization and membrane topology of coronavirus nonstructural protein : involvement of the early secretory pathway in replication visualizing the autophagy pathway in avian cells and its application to studying infectious bronchitis virus analysis of protein-protein interactions and involvement of viral proteins in sars-cov replication analysis of intraviral protein-protein interactions of the sars coronavirus orfeome biogenesis and architecture of arterivirus replication organelles two-amino acids change in the nsp of sars coronavirus abolishes viral replication template rna length determines the size of replication complex spherules for semliki forest virus partially uncleaved alphavirus replicase forms spherule structures in the presence and absence of rna template nodavirus-induced membrane rearrangement in replication complex assembly requires replicase protein a, rna templates, and polymerase activity the authors would like to thank paul britton and pip beard for helpful discussions. the authors declare no conflict of interest. the funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. key: cord- -ls zgipi authors: norris, rachael p.; terasaki, mark title: gap junction internalization and processing in vivo: a d immuno-electron microscopy study date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: ls zgipi gap junctions have well-established roles in cell-cell communication by way of forming permeable intercellular channels. less is understood about their internalization, which forms double membrane vesicles containing cytosol and membranes from another cell, called connexosomes or annular gap junctions. here, we systematically studied the fate of connexosomes in intact ovarian follicles. high pressure frozen, serial sectioned tissue was immunogold labeled for connexin . within a volume of electron micrographs, every labeled structure was categorized and counted. surface area measurements indicate that large connexosomes undergo fission. subsequent modifications are separation of inner and outer membranes, loss of cx from the outer membrane, and outward budding of the modified membranes. we also documented several clear examples of organelle transfer from one cell to another by gap junction internalization. we discuss how connexosome formation and processing may be a novel means for gap junctions to mediate cell-cell communication. gap junctions are arrays of permeable channels between two cells that have well-established roles in intercellular signaling (nielsen et al., ) . the basic structural unit is the transmembrane protein connexin. six connexins assemble to form the connexon, which is a pore within the membrane. a connexon in one cell docks head-on with a connexon in a neighboring cell to form a channel between the two cells. large gap junctions may consist of hundreds or thousands of channels packed densely in a patch a few microns in diameter (larsen, ) . the current view is that connexons are added to the plasma membrane via small post-golgi vesicles, followed by docking between two cells, but how connexons are removed from the membrane and their subsequent fate are incompletely understood. to turn over the gap junction, cells could undock connexons and then endocytose them in small parcels. instead, connexons remain docked and the gap junction is taken up by one of the two cells (laird et al., ; falk et al., ) . this was first suggested by electron microscopists who interpreted circular gap junction profiles (first called annular gap junctions) as internalized gap junctions (espey and stutts, ; merk et al., ) . this interpretation was convincingly corroborated by live imaging of gfp-connexins, which showed formation of vesicles of comparable size (jordan et al., ; piehl et al., ) . the internalized gap junction structure is now often called a connexosome (laird, ) . gap junction internalization is therefore a type of endocytosis, in which the plasma membrane of the neighboring cell remains attached to the endocytosed plasma membrane (heck and devenport, ) . gap junction internalization can also be considered to be a form of trogocytosis (joly and hudriser, ) in which a portion of the plasma membrane and cytosol of the neighboring cell is transferred to the engulfing cell (see fig. a ). there is a possibility that further processing after the initial engulfment is involved in other modes of cell-cell communication (see discussion). internalized gap junctions could likewise play new roles in cell-cell communication. in a previous study (norris et al., ) , we addressed several methodological issues that have limited electron microscopic studies of gap junctions in the past. mouse ovarian follicles were high pressure frozen in order to preserve structure better than chemical fixation, which involves diffusion of aldehydes through the cell membrane(s) and cross linking of proteins, during which abnormal processes may occur (murk et al., a) . the frozen tissue was freeze-substituted, embedded in lowicryl, sectioned, then immunolabeled with an antibody to cx . this allowed us to unambiguously identify gap junctions. serial sections were then imaged to obtain threedimensional information; this distinguished between internalized connexosomes and gap junctions in the process of invagination. likewise, a round profile in a single section could be a vesicle or an invagination, or an apparently empty vesicle could contain intraluminal vesicles; serial sections distinguished these alternatives. here, we investigate the fate of internalized gap junctions by examining cx localization. as before, we high pressure froze ovarian follicles and immunolabeled serial sections. we found a surprisingly large number of cx labeled structures that we interpret as modifications of the connexosome. mouse ovarian antral follicles were high pressure frozen minutes after exposure to luteinizing hormone (lh). this tissue was used because it has large numbers of cx gap junctions (okuma et al, ; norris et al., norris et al., , baena et al., ) , which are caused to internalize in response to hormone (larsen et al., ) . as in our previous study, the follicles were embedded in lowicryl, serial sectioned, then labeled with primary antibody to cx and gold-labeled secondary antibody (norris et al., ) . we used a different scanning electron microscope in order to obtain higher resolution images from the sections labeled in our previous study. this provided clearer images of the classic double membrane with a gap structure ( figure b -d). we will use the term connexosome to mean a double membrane vesicle completely detached from the plasma membrane in which both limiting membranes were contacting each other and labeled with cx throughout the periphery of the vesicle. connexosomes were the most abundant cx labeled structure in the cytoplasm, but there were also many other membranous structures that contained cx (video ) which we interpreted to be modifications of the connexosome. we characterized these cx structures by classifying and counting them in a defined volume. by doing so, we gained information on the relative abundance of various forms, which could be useful in deducing dynamics. we analyzed two volumes of mural granulosa cells that were each μm x μm in the x/y plane (imaged at nm per pixel), in sections of nm thickness. thus each volume was , μm . in this volume, there were gap junctions. of these, were a planar patch. the others were present on highly infolded membranes, which we refer to as invaginating gap junctions. there were internalized structures. of the cx labeled structures, were connexosomes and appeared to be connexosomes that had undergone processing. direct imaging of living cultured cells has shown that connexosomes are formed by internalization of entire gap junctions, and that connexosomes can undergo fission (piehl et al., ; bell et al., ) . we examined the high pressure frozen and serial sectioned ovarian follicles for connexosome formation. the areas of structures are measureable in d data sets, so we also measured the surface areas of all of the connexin containing membranes because this information could be relevant to connexosome formation ( figure e ). the average surface area of the "flat" gap junction plaques in the volume was . ± . μm (mean ± sd), with a range of . - . μm . serial sections showed that most were disc shaped, corresponding to a disc of average diameter . μm. the invaginated gap junctions looked like they could form connexosomes. these were larger than the "flat" gap junctions, with an average surface area of . ± . μm with a range of . - . μm (n = ). connexosomes, on the other hand, had an average surface area of . ± . µm , and a range of . - . µm (n= ). the average connexosome area thus was % of the average gap junction area (figs. e, f). it seems likely that invaginated gap junctions were frozen in the process of internalization and are therefore the source of connexosomes. the surface areas of invaginated gap junctions are significantly larger than connexosomes. four of the invaginated gap junctions contained organelles or vesicles in addition to cytosol. one deeply invaginated gap junction contained a multivesicular endosome and a mitochondrion ( fig. a, video ). if such structures were to form connexosomes, it would result in transfer of organelles from one cell to another. we indeed found a connexosome containing a mitochondrion and an apparent endosome (fig. b , video ), and another connexosome enclosing a tubular organelle, possibly er (fig. c , video ). because the entire periphery of the connexosome is gap junction, these organelles must have come from other, previously gap junction-coupled cells. in total, out of connexosomes contained vesicles or organelles in addition to cytosol. of the cx labeled internalized structures, appeared to be modified connexosomes. to describe the modifications, we will use the following terms to refer to the membranes and compartments of the unmodified connexosomes. there are an outer and an inner membrane, which are closely apposed because the connexons are docked. the small space between the two membranes originally was the extracellular space. the compartment within the inner membrane came from the cytoplasm of the neighboring cell. the initial modification appears to be fusion of a connexosome with another vesicle. in of the modified connexosomes, a patch of unlabeled outer membrane bulged outward from a labeled inner membrane ( suggesting that different types of vesicles were involved. cx labels the inner membrane in areas where the inner and outer membranes have separated. in these regions, the connexons must have become undocked. the inner and outer membranes have become more separated in of the modified connexosomes and small vesicles were present in this enlarged space (fig. c ). the small vesicles were generally not labeled with cx , and the diameters averaged nm. this corresponds closely to the reported diameters of intraluminal vesicles ( - nm) found in multivesicular endosomes (murk et al., b; hanson and cashikar, ; scott et al., ) . the inner membrane is identifiable and labeled while the outer membrane is much less labeled ( fig. b -d, video ). there was a striking example in which the inner and outer membranes were completely separated, and no cx label was present in the outer membrane (fig. d ). if the connexons had merely become undocked, the amount of label in the outer membrane should be comparable to the amount in the inner membrane. therefore, this is evidence for connexon degradation in the outer membrane. in the remaining of the modified connexosomes, there was an outer membrane similar in diameter to that of unmodified connexosomes that had little to no cx labeling. also, instead of a single inner membrane labeled with cx , there were various different sized vesicles labeled with cx ( fig. a and b, video ). the diameters of the cx labeled vesicles in modified connexosomes averaged nm, and ranged from nm to nm (fig. c ). based on the range of diameters and the presence of cx , these seem likely to have formed by fission or outward budding (akin to cytokinesis) of the inner connexosome membrane. for comparison, we also measured small vesicles within structures that lacked cx labeling (fig. d ). unlabeled vesicles were smaller, averaging nm in diameter, with a range of to nm ( fig. c ). in addition to subdivision of the inner membrane, some of the modified connexosomes had outer membranes with short tubules extending into the cytosol, (see arrows in fig. a and b), as is often seen in endosomes. (klumperman and raposo, ). luteinizing hormone stimulates the internalization of gap junctions in ovarian granulosa cells (larsen et al., ) . we high pressure froze follicles minutes after stimulation and then labeled cx by immunogold staining in serial sections ( nm thick). every cx containing structure was categorized and counted in a defined volume of ~ , µm . this data allows us to make several novel observations and measurements on connexosome formation and modification. live cell imaging studies of several cultured mammalian cell lines expressing cx -gfp chimeras showed that either entire gap junctions are internalized, followed by fission (piehl et al., ; bell et al., ) , or that a small portion of the gap junction center is internalized (falk et al., ). the d data from high pressure frozen tissue allowed us to look for internalization intermediates and also to make the first systematic measurements of gap junction and connexosome areas. most gap junctions were disk shaped on a flattened piece of plasma membrane (n = ). there were invaginated gap junctions, and their average areas were larger than those of the flat gap junctions. it seems likely that the largest gap junctions begin to invaginate and are the source of connexosomes in this tissue. the internalization of whole invaginated gap junctions should produce correspondingly large connexosomes, but connexosomes as a group are smaller than gap junctions. this is consistent with fission occurring soon after internalization. the simplest connexosome modification was a partial separation of the inner and outer membrane while the rest of the gap junction is intact. the separated outer membrane bulges out, and lacks cx while the inner membrane retains it (figures b and c) . it seems likely that a vesicle has fused with the connexosome, perhaps at a bare patch left over from the internalization process (falk et al., (falk et al., , if this vesicle fusion leads to a lowering of ph, it could cause the connexons of the gap junction to undock (falk et al., ) . subsequent modifications seem to involve two different processes. one is complete separation of the two membranes with loss of cx in the outer membrane but retention in the inner membrane. the other process is the appearance of numerous smaller compartments within the boundary of the former connexosome. small cx -free vesicles seem likely to derive from outward budding of the outer membrane; they resemble intraluminal vesicles of multivesicular endosomes. outward budding or fission of the inner membrane appears to produce cx containing compartments that are somewhat larger than the cx free vesicles. there is evidence from previous studies of other tissues for connexosome or connexin degradation by autophagy. autophagosomes engulf internalized gap junctions in the equine hoof wall (leach and oliphant, ) , canine ventricular myocardium (hesketh et al., ) , hela cells and mouse embryo fibroblasts (lichtenstein et al., ; fong et al., ) . in mouse liver cells, connexins are degraded by autophagy (bejarano et al., ) . however, we did not observe intermediates resembling a phagophore or an autophagosome in our images. our data comes from minutes after application of luteinizing hormone, which might be too early for the final stages of cx degradation. another possibility is that in ovarian granulosa cells, connexosomes become something more related to multivesicular endosomes. this conclusion was made by leithe et al ( ) in cultured rat liver epithelial cells that were treated with phorbol ester. their evidence was based on immunolocalization of endosomal markers and cx is highly phosphorylated after lh treatment (norris et al., ) . if we had not immunogold labeled sections with cx , a structure as seen in figs. a or b would likely be identified as a multivesicular endosome. this suggests that in other tissues, some apparent multivesicular endosomes could be modified connexosomes. we propose a sequence of connexosome processing events ( figure ). the initial event is fusion with a vesicle (fig. , step ) . the vesicle fusion adds unlabeled membrane to the outer membrane, and triggers the undocking of connexons, perhaps by lowering the ph (falk et al., ) . the connexons of the outer membrane are degraded, perhaps because the cytoplasm of the host cell can recognize that they are undocked (fig. , step ) the uncoupled inner membrane undergoes either fission or outward budding to form various sizes of cx containing vesicles (fig , step ) . possible fates of the modified connexosomes are degradation by autophagosome formation or direct fusion with a lysosome (leithe et al., ; falk et al., ) . however, an alternative possibility is the fusion of the inner vesicles with the modified outer membrane that lacks cx (fig. , step ) . this would result in the mixing of membranes from two cells, and the release of cytoplasm from one cell into another. evidence for this happening in other cells is discussed in the next section. what vesicles initially fuse with the connexosomes? many appear to be clear vesicles, which are consistent with endosomes (murk et al., a) , but a significant fraction of vesicles were dark which is characteristic of lysosomes (klumperman and raposo, ) . there is evidence that lysosomes are not always degradative and can instead function in secretion and signaling (settembre et al., ; perera and zoncu, ) . while there is solid evidence for clathrin involvement in connexosome formation (piehl et al., ) , an escrt driven process could also help explain some of our observations. escrt machinery for outward budding could become attached and activated on the donor side of the gap junction. there is evidence that escrt machinery can bind to ubiquitinated connexins (auth et al., ). if the machinery were transferred within the connexosome, it could generate single membrane vesicles by outward budding or fission of the internal membrane after separation of the inner and outer connexosome membranes. the channel properties of connexins are well established for the exchange of small molecules between cells (nielsen et al., ) . we discuss here how connexins, by way of forming connexosomes, may also facilitate cytoplasmic and membrane transfer between cells. first of all, our serial section images provide conclusive evidence for transfer of mitochondria and other organelles via connexosomes. a transferred mitochondrion could affect physiological processes of the host cell. in a mouse model for acute lung injury, cx -dependent mitochondrial transfer from bone marrow derived stroma cells rescued injured lung alveolar epithelial cells (islam et al., ) . the authors suggested that mitochondria were transferred in some way by microvesicles; our observations provide a clear cx -dependent mechanism for transfer. our observations demonstrate only the transfer of an organelle in an enclosed double membrane. if the double membranes were to fuse (or the inner membrane vesicles were to fuse with the outer membrane), this would result in mixing of cytoplasm (e.g. release of mitochondrion in the above example) and membranes ( figure , step ). it is established that the endosome membrane can fuse with a vesicle within it (bissig and gruenberg, ) . this occurs with enveloped viruses such as vesicular stomatitis virus (le blanc et al., ) or coronavirus (grove and marsh, ) , resulting in the release of nucleocapsids into the cytoplasm. there is now direct evidence that this also occurs with extracellular vesicles (joshi et al., ) , resulting in release of cargo to the cytoplasm. membrane mixing involving connexosomes might explain a previous finding in immune cells. macrophages transfer mhc ii bound antigens to dendritic cells in the gut to establish oral tolerance, and this depends on the presence of cx (mazzini et al., ) . without specifying the role of cx , the authors raise the possibility that transfer occurs via trogocytosis. in trogocytosis, like connexosome formation, a part of the plasma membrane and cytoplasm of one cell is internalized into another (joly and hudriser, ) ; a fusion between outer and inner compartments, followed by budding of the combined membranes would be required to achieve antigen transfer and presentation. we suggest that the mhc ii-antigen complex is transferred from the macrophage via connexosomes followed by fusion of inner and outer membranes to deliver it to the dendritic cell's endosomal system. cell-cell communication by way of connexosome formation and processing may be widespread and warrants further investigation. from this point, we followed the procedure of rubio and wenthold ( ) , with some modifications. samples were freeze-substituted with . % uranyl acetate (electron microscopy sciences) in dry methanol for hours at - ° c in an afs freeze substitution unit (leica biosystems). the temperature was then raised ° c per hour to - ° c. samples were then rinsed in methanol, and infiltrated with monostep uv light as the temperature in the afs was increased by ° per hour to ° c, then held at ° c for hours more. when samples were removed from the afs , they were pink in color and left to polymerize at room temperature until the pink hue was gone two days later. ultrathin sections ( nm) of lowicryl hm embedded follicles were cut on a uc- ultramicrotome (leica biosystems) with a diamond knife (diatome, hatfield, pa). the sections were picked up by an automated tape collector on glow-discharged kapton tape (terasaki et al., ; kasthuri et al., ; baena et al., ) . for immunostaining, ribbons of follicle sections on kapton tape were cut to lengths of approximately three inches and attached to a sheet of parafilm with doublesided carbon tape (electron microscopy sciences # ). sections were rehydrated with x pbs (life technologies, grand island, ny) and blocked in % normal goat serum (invitrogen, frederick, md) in a solution of % bovine serum albumin in pbs. following an overnight incubation at °c in primary antibody, sections were rinsed three times for minutes each in pbs, then rinsed once in % bsa in pbs. next, secondary antibody diluted at : was applied to sections for one hour at room temperature. sections were then rinsed with x pbs followed by milli-q filtered water and dried overnight. sections were placed back in their original order and post-stained with % uranyl acetate in : methanol: water for minutes, then rinsed generously in water. imaging serial sections of tissue with scanning em immuno-labeled sections on tape were attached to a cm diameter silicon wafer (university wafer, south boston, ma) with double-sided carbon adhesive tape (electron microscopy sciences). wafers were carbon coated (denton, moorestown, nj) and first imaged on a sigma field emission scanning electron microscope (zeiss, thornwood, ny) using a backscatter detector as described in norris et al., . two volumes of mural granulosa cells were imaged at nm/pixel resolution, with a field of view of square micrometers. original low-resolution images obtained on the zeiss sigma were aligned with the register virtual stack slices macro (fiji), then larger files were aligned and diced for convenient viewing with a custom program (piet, provided by duncan mak and jeff lichtman, harvard university). these files were used to track all cx -labeled internalized structures. when internalized structures looked complex, they were reimaged using a higher resolution electron microscope as described below. high-resolution images on fei verios l higher a. five serial sections through a modified connexosome containing several internal vesicles labeled with cx . b. five serial sections through a different modified connexosome with internal vesicles labeled with cx . this vesicle has a darker interior than the vesicle in a. in a. and b., arrows indicate short tubules extending from the outer membranes, as is often seen in endosomes. related video shows all sections through structures in a and b. c. diameters of cx -labeled internal vesicles are more variable in size and larger than unlabeled internal vesicles. internal vesicles labeled with cx were measured within nine modified connexosomes, and unlabeled internal vesicles were measured within nine structures that had no cx labeling. d. two sections through a vesicle with unlabeled intraluminal vesicles, and no cx labeling, as measured for panel c. scale bars in a,b, and d are nm. proposed events after internalization and fission. as in figure , gap junction proteins are represented by orange rectangles. ( ). fusion of a connexosome with a vesicle from the host / recipient cell leads to a local separation of the two membranes ( ). the formation of intraluminal vesicles (ilvs) from the outer membrane is also possible. a further modification is the outward budding of the inner membrane that still contains cx ( ). note that cx is decreased or absent in the outer connexosome membrane. while degradation is possible, another possible fate of the modified connexosomes is fusion of the inner vesicles with the limiting membrane of the processed connexosome ( ). this would result in release of contents from the other cell and mixing of the plasma membrane from the donor cell with the membrane of the processed connexosome. if some of the donor membrane budded away from the processed connexosome, it could get incorporated into the receiving cell plasma membrane. video . multiple structures labeled with cx polyclonal antibody. serial sections through an ovarian granulosa cells showing a variety of double membrane vesicles labeled with an antibody to cx and nm gold. other types of vesicles are also labeled with cx (yellow arrows). the scale bar is nm. video . organelle transfer by gap junction internalization. serial sections through ovarian granulosa cells labeled with anti-cx and nm gold. corresponding to figure re a, the cytoplasm of a cell protruding into another cell is shaded in blue. there is a multivesicular endosome and a mitochondrion protruding into the cell as well. scale bar is nm, as in figure a . corresponding to figure b , a connexosome contains a multivesicular endosome and a mitochondrion. corresponding to figure c , another connexosome contains smaller vesicles and other membranes. scale bars are nm, as in figures c and c . video . connexosome modifications of the outer and inner membranes. serial sections through ovarian granulosa cells labeled with anti-cx and nm gold. corresponding to figures and , the full structures of the modified connexosomes in figure a -d and in figures a and b are shown. scale bars are nm. the tsg protein binds to connexins and is involved in connexin degradation cellular heterogeneity of the luteinizing hormone receptor and its significance for cyclic gmp signaling in mouse preovulatory follicles. endocrinology serial-section electron microscopy using automated tape-collecting ultramicrotome (atum) autophagy modulates dynamics of connexins at the plasma membrane in a ubiquitin-dependent manner visualization of annular gap junction vesicle processing: the interplay between annular gap junctions and mitochondria alix and the multivesicular endosome: alix in wonderland exchange of cytoplasm between cells of the membrane granulosa in rabbit ovarian follicles gap junction turnover is achieved by the internalization of small endocytic doublemembrane vesicles degradation of endocytosed gap junctions by autophagosomal and endo/lysosomal pathways: a perspective degradation of connexins and gap junctions molecular mechanisms regulating formation, trafficking and processing of annular gap junctions internalized gap junctions are degraded by autophagy the cell biology of receptor-mediated virus entry multivesicular body morphogenesis trans-endocytosis of planar cell polarity complexes during cell division ultrastructure and regulation of lateralized connexin in the failing heart mitochondrial transfer from bone-marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury what is trogocytosis and what is its purpose? the origin of annular junctions: a mechanism of gap junction internalization endocytosis of extracellular vesicles and release of their cargo from endosomes saturated reconstruction of a volume of neocortex the complex ultrastructure of the endolysosomal system life cycle of connexins in health and disease structural diversity of gap junctions. a review differential modulation of rat follicle cell gap junction populations at ovulation degradation of annular gap junctions of the equine hoof wall endosome-tocytosol transport of viral nucleocapsids endocytic processing of connexin gap junctions: a morphological study autophagy: a pathway that contributes to connexin degradation oral tolerance can be established via gap junction transfer of fed antigens from cxcr + macrophages to cd + dendritic cells the fine structure of granulosa cell nexuses in rat ovarian follicles influence of aldehyde fixation on the morphology of endosomes and lysosomes: quantitative analysis and electron tomography endosomal compartmentalization in three dimensions: implications for membrane fusion gap junctions luteinizing hormone causes map kinase-dependent phosphorylation and closure of connexin gap junctions in mouse ovarian follicles: one of two paths to meiotic resumption localization of phosphorylated connexin using serial section immunogold electron microscopy colocalization of connexin and connexin but absence of connexin in granulosa cell gap junctions of rat ovary the lysosome as a regulatory hub internalization of large double-membrane intercellular vesicles by a clathrindependent endocytic process glutamate receptors are selectively targeted to postsynaptic sites in neurons endosome maturation, transport and functions signals from the lysosome: a control centre for cellular clearance and energy metabolism stacked endoplasmic reticulum sheets are connected by helicoidal membrane motifs we thank rindy jaffe and matthias falk for critical review of the manuscript and useful discussions. we thank art hand, maya yankova, and maria rubio for technical advice. we thank valentina baena, tracy uliasz, and other members of the jaffe and terasaki labs for technical assistance and help with collecting samples. this work was funded by the fund for science. the authors declare no conflicts of interest. rachael norris contributed to conceptualization; data collection, curation and analysis; visualization, and writing and editing of the original draft. mark terasaki contributed to conceptualization, supervision of the work, funding acquisition, software and resources for data collection; and writing and editing of the original draft. key: cord- -oawrnmhy authors: fahimirad, shohreh; fahimirad, zahra; sillanpää, mika title: efficient removal of water bacteria and viruses using electrospun nanofibers date: - - journal: sci total environ doi: . /j.scitotenv. . sha: doc_id: cord_uid: oawrnmhy abstract pathogenic contamination has been considered as a significant worldwide water quality concern. due to providing promising opportunities for the production of nanocomposite membranes with tailored porosity, adjustable pore size, and scaled-up ability of biomolecules incorporation, electrospinning has become the center of attention. this review intends to provide a detailed summary of the recent advances in the fabrication of antibacterial and antiviral electrospun nanofibers and discuss their application efficiency as a water filtration membrane. the current review attempts to give a functionalist perspective of the fundamental progress in construction strategies of antibacterial and antiviral electrospun nanofibers. the review provides a list of antibacterial and antiviral agents commonly used as water membrane filters and discusses the challenges in the incorporation process. we have thoroughly studied the recent application of functionalized electrospun nanofibers in the water disinfection process, with an emphasis on their efficiency. moreover, different antibacterial and antiviral assay techniques for membranes are discussed, the gaps and limitations are highlighted and promising strategies to overcome barriers are studies. removing bacteria from water supplies which meet the requirement of effective antimicrobial activity, superior filtration flux with acceptable retention potentials (zodrow et al., ) . nanofiber membranes, because of their high surface area to volume ratio, nano-sized pores, and high porosity, have been illustrated to improve the efficiency of conventional materials employed for the filtration and separation of particulate materials (aussawasathien et al., ) . a number of processing techniques including melt-blown, self-assembly, phase separation, template synthesis, and electrospinning have been employed to prepare nanofibers in recent years. among them, electrospinning is the most promising, efficient method to produce web-like non-woven ultrafine fibers including microfibers (> μm) or nanofibers (< nm) from different kinds of polymers. moreover, incorporation of bioactive, antimicrobial and antiviral agents into nanofiber structure is easily possible through the electrospinning process fahimirad and hatami, ; faccini et al., ) . the present work reviews previous studies on the production and application of electrospun nanofibers as antimicrobial water filtration membranes. the merits and demerits of these novel water microfiltration tools are discussed. moreover, their antibacterial efficiency and disinfection activities are compared with commercial water membrane filters comprehensively. finally, some points are recommended to be noticed as the subsequent future research plans. the objectives of this review were to: (i) introduce the different procedures, which have been applied for incorporation of the various antimicrobial agents into electrospun nanofibers (ii) discuss the different antimicrobial tests used for proving antimicrobial activity of the fabricated electrospun water filters (iii) study the efficiency of the produced antimicrobial electrospun application in the water treatment industry. j o u r n a l p r e -p r o o f the electrospinning approach was invented by cooley in (cooley, . this method is easy, cost-effective, uncomplicated, and has the potential for scale-up production. the flexibility in material selection and additive incorporation to obtain appropriate functionality, as well as its considerable capability to produce fibers in the sub-micron range with the high surface-area (up to m g - based on the fiber diameter), are prominent privileges of electrospinning process for fabrication of nanofibers. in addition, effective porosity of electrospun nanofibers (almost about % with no upper limit) with many small pores, interconnected pore structure directly promote both infiltration rate and contaminant rejection ratio in comparison with conventional materials being used for mf applications (nasreen et al., ; wang et al., ) . in this process, a prepared solution of polymer is loaded into a syringe and fed at a set flow rate to the spinneret. due to the needle connected to a high voltage power supply under the electric field with a specific voltage, a taylor cone is constructed by elongation of the polymer droplet at the end of the syringe into a characteristic conical shape. enhancing the electrical field causes the formation of a steady jet elongated and whipped consecutively by electrostatic repulsion. the solvent evaporates when electrostatic forces prevail over surface tension and the jet gets finer, so electrospun nanofiber in mf application, such as uniform fiber morphology with controllable pore size, interconnected open pore structure, high porosity, and membrane thickness, turn them to a superior substitute to replace the conventional mf membrane such as the millipore gswp mf membrane with an average pore size of . mm (wang et al., ; barhate and ramakrishna, ) . another novel application of electrospun nanofiber in water purification and bacterial rejection is thin-film nanocomposite membrane (tfnc), a major type of reverse osmosis (ro) and nanofiltration (nf) membranes, which compromises of three layers including the first barrier layer of interfacial polymerization, a polyacrylonitrile or poly (vinylidene fluoride) electrospun membrane as the second layer and nonwoven polyethylene terephthalate (pet) as the third layer. the third layer employed as a substructure layer to provide the whole membrane adequate mechanical strength (subramanian and seeram, ; yin et al., ; fig ) . high water flux, great solute rejection, minimum membrane fouling, and perfect mechanical persistence are main properties of an ideal tfc membrane and turn it into an excellent candidate for microfiltration and ultrafiltration applications (li and wang, ) . sato et al., ( ) fabricated a novel composite fibrous membranes, consisting of an ultra-fine cellulose nanofibrous infused into electrospun polyacrylonitrile (pan, with an average diameter of . μm a mean diameter of about μm as the barrier layer ( - μm in thickness) to provide filtration attributes) nanofibrous scaffold on a melt-blown polyethylene terephthalate (pet, with a mean diameter of μm as the support layer (about μm thick) to sustain mechanical strength) non-woven substrate for water purification. the nanostructure showed a retention rate of . % for e. coli filtering and the high percent of the ms virus, with nm sizes, captured in the electrospun pan scaffold infused with m-ufcns (sato et al., ) . recently, taheran et al. ( ) j o u r n a l p r e -p r o o f fabricated a methodical portable water purification instrument using electrospun nanofiber. the device contained three distinct electrospun membranes. the first membrane was made by electrospinning of polyacrylonitrile/chitosan solution at : mass ratio as an antibacterial membrane, the second membrane was produced from laccase ( unit g - ) immobilized onto pan/biochar : % electrospun mat for removal of micro-pollutants and the third layer was fabricated by electrospinning of pan/biochar at : ratio as an adsorptive membrane. the applied technology led to approximately % removal of microorganisms, % of micropollutant removal, and more than % of turbidity decline during less than min contact time (taheran et al., ) . the important characteristics of a nanofiber mat membrane for application as filters for the separation of contaminations and pathogens from a continuous fluid phase are wetting properties, permeability, porosity, fiber size distribution, and fiber structure. for water filtration, a membrane must be wet-table and surface wetting properties are generally specified by the contact angle. a surface with a low contact angle (below degrees) is considered a hydrophilic surface, while a surface illustrating a high contact angle (over degrees) is referred to as a hydrophobic surface. sessile drop and the captive bubble method are two common techniques used for measuring the nanofibers' contact angle (nuraje et al., ) . one of the key parameters in filter design and its performance is porosity. generally, porosity is calculated from the apparent density and bulk density of the membrane. however, other alternative procedures inclusive of image analysis and mercury porosimeter are frequent methods j o u r n a l p r e -p r o o f applied for the evaluation of porosity in the nanofiber membrane (ghasemi-mobarakeh et al., ) . electrospun nanofibers are highly porous with interconnected pores in the size range of just a few times the fiber diameter. the small pore size of the nanofibrous membrane introduces a higher retention rate, the interconnected pores leads to better tolerance against fouling and the high porosity defined a higher permeability capability (homaeigohar et al., ) . clean water permeability (cwp (l/m •h•bar)) illustrates the highest amount of attainable flux dependent on the membrane condition. it can be assayed by calculating the flux at various trans membrane pressures (tmp). the slope of the eventuated curve is regarded as the cwp (bjorge et al., ) . the high cwp grants high flux operation to the membranes, introduces the nanofiber mat as an energy-saving membrane, and means that if fouling does not happen, enormous volumes can be treated (daels et al., ; he et al., ) . the surface charge on membranes is related to affinity corresponding interactions and considered as a significant parameter influencing the disinfection capabilities of the membrane. surface charges can qualify the strength of biomolecular or even pathogen affinity on a material surface. in virus removal, surface charged nanofibers adsorb virus via electrostatic interactivity between the nanofibers and the counter-charges of virus and signify virus remediation improvement (cho et al., ) . a series of studies have confirmed that electrostatic attraction between the cationic membrane and the anionic surface of bacteria may lead to morphological defects in consequence of ros generation and cell membrane destruction. indeed, anionic membranes act as powerful non-adhesive site of bacteria attributable to electrostatic repulsion (mukherjee and de, ; kolewe et al., ) . operating conditions influence the antibacterial activity performance of the nanofibrous membrane. as proved by several experiments, bacterial cells are able to decline their size at higher operating pressure, hence resulting in enhancing permeation through the filter. therefore, less trans membrane pressure (tmp) is usually desired, to retain antibacterial activity during long term application of the membrane. the tmp is described as the mean feed pressure minus the permeate pressure that is essential to push down water through a membrane (mukherjee and de, ; . different factors including surface area, surface roughness, pore diameter, zeta potential, and inclusion of biocides or antibacterial agents determine the antimicrobial performance of membrane (rahaman et al., ; mukherjee and de, ) . accordingly, the employing of electrospun polymeric membranes in bacterial and virus removal from water is performed in two procedures including size exclusion and adsorption (lee et al., ) . in most cases, the diameter of water-borne bacteria is more than . μm. for example, the e. coli size is . - . μm and brevundimonas dimimuta dimension is . - . μm. previous studies have confirmed that using a . μm pore sized mf leads to a log- log bacteria reduction (gómez et al., ; ghayeni et al., ) . thus, based on the degree of exclusion, the electrospun membrane should have an average pore size of fewer than . μm. in addition, the narrow pore size distribution is requisite for achieving a high retention rate (ma et al., ) . there is a direct relationship between the pore size and the fiber diameter of a porous nonwoven structure. the relationship has been confirmed as the average pore size was approximately ± times the mean fiber diameter, and j o u r n a l p r e -p r o o f the greatest pore size was about ± times the mean fiber diameter. thus pore size of electrospun fiber generally grows with increasing fiber diameters (ma et al., ) . various conventionally employed membranes for the application as micro-filters have . μm theoretical pore sizes. the advantage of electrospun nanofiber membranes in comparison to conventionally used membranes are the simplicity of manufacture, adjustable size of the pores and high porosity (saleem et al., ) . in view of the fact that the membrane pore sizes can be controlled by adjusting the electrospinning parameters and besides the fact that the most aquatic bacteria dimensions are more than . mm, electrospun nanofibers can be designed efficiently with smaller pore dimensions suitable for mf applications (wang and hsiao, ) . for instance, accelerating the flow rate raises the pore diameter by enhancing the fiber diameter. moreover, increasing polymer solution concentration and using higher molecular weight polymer increases fiber diameter. employing a secondary ring electrode circling the nozzle cause reducing the fiber deposition and consequently decrease the density of the membrane, the parameter which reduces the pore size. in addition, controlling fiber distribution, post electrospinning modification and using temporary spacers can be utilized for controlling pore size (dong et al., ; haider et al., ) . as discussed above and based on the size exclusion process, microfiltration larger sized bacteria are substantially seized by the membrane but it is not efficient in separating small sized viruses within . - . µm range size (mi et al., a; barhate and ramakrishna, ) . so, rejection of bacteria smaller than membrane pores or viruses needs the incorporation of antiviral or antibacterial agents into the membrane. also, after size-exclusion microbial removal of the membrane, intercepted bacteria can be released and induce membrane biofouling during subsequent filtration. therefore, antimicrobial agents are commonly used to prohibit bacterial j o u r n a l p r e -p r o o f growth and biofoul formation that would decline filter efficiencies (botes and cloete, ; wen et al., ) . various bioactive agents with different fundamental properties may have consequential impacts on bacteria removal. plus, nanofiltration membranes or ultrafiltration membranes with a positive charge on the surface are able to remove viruses selectively (mukherjee and de, ). moreover, incorporating antimicrobial agents into electrospun nanofibers enhance the antimicrobial activity of fabricated nanofibrous membrane (nasreen et al., ; park and kim, ). an ideal bioactive agent incorporated into the functionalized membrane should be nontoxic, water insoluble with no or slight leaching property. also, the functionalization process should not cause adverse influences on the quality and overall performance of the membrane. based on the majority of researches studied in this review, blending and post-modification strategies are two commonly used techniques to incorporate biocide agents into nanofibers aiming for water disinfection application (shalaby et al., ; he et al., ; makaremi et al., ) . blend electrospinning is an easy one-step procedure, mostly used for agents' incorporation into nanofibers (shabafrooz et al., ) . using the same solvent, the bioactive agent is dissolved directly into the polymer solution and a homogeneous blended solution of the incorporating agents in the polymer solution is prepared for the electrospinning step (pillay et al., ; fahimirad and ajalloueian, ). j o u r n a l p r e -p r o o f the agents incorporation into electrospun fibers can be performed after the electrospinning process by physical or chemical treatments. covalent and non-covalent immobilizations are fundamental methods for molecules attached to the fiber surface. non-covalent immobilization is performed by immersion of electrospun mats in a solution compromising the bioactive molecules. by treating with plasma the surface gets activated for subsequent modification using specified ligands like active amine groups. the affinity of incorporated agents to the electrospun nanofiber surface improves by covalent immobilization (wang and windbergs, ; kurusu and demarquette, ) . some commonly used antimicrobial or antiviral agents in electrospun nanofibers are discussed in this section. silver nanoparticles (agnps) are considered the most efficient nanoparticles for biological applications and the most extensively applied antibacterial agent for water disinfection mukherjee and de, ) . agnps are capable to puncture the microorganisms' cell walls, interact with their nucleic acids and attach to their enzymes, which cause the cell membrane destruction and finally growth inhibition. different feasible interactions of ag + ions with various bacterial biomolecules are documented. furthermore, the extended range of antibacterial activities and virulence effects of ag + ions toward several microorganisms (e.g. bacteria, viruses, and fungi) at only a few mg ml - are confirmed in previous studies. thus, silver nanoparticles are recognized as potent disinfection agents (lópez-heras et al., ) . in water purification, nanosilver materials have been mainly applied to prevent the formation of there are three main methods for agnps incorporation into electrospun nanofibers including ) blending of prepared synthesized agnps solutions to the polymer solution, ) agnp synthesis in the polymer solution by employing a precursor, and ) post-treatments of the electrospun nanofibers for agnp synthesis by reduction of the precursor that has been spun along with the electrospinning solution (fahimirad and ajalloueian, ). there are three main approaches to produce iron oxide nanoparticle-nanofiber composites, including ( ) electrospinning of solution containing prepared ionps, ( ) in-situ synthesizing of ionps during the electrospinning process or in the solution to be electrospun and ( ) it has been indicated that due to electrostatic interaction, cunps illustrate antibacterial functions on the bacterial cell through different mechanisms, such as adhesion to the bacterial cell wall, lead to detrimental impacts on protein structure within the cell membrane, denaturation of j o u r n a l p r e -p r o o f proteins in inertial parts of the cell , and adverse effects on phosphorus-and sulfur-containing compounds like dna (raffi et al., ) .recently, cunps have gained considerable interest because of their broad-spectrum and acutely effective antibacterial activity with comparatively low cost and high scalability (taner et al., ) . recently, zinc oxide (zno) has received much attention due to its non-toxic profile, effective antibacterial activity, adsorptive properties, mechanical, chemical, and thermal stability while encountering diverse environmental conditions (tiwari et al., ) . zno particles have illustrated antimicrobial activity against both gram-positive, gram-negative bacteria and even against spores wagner et al., ) . zno nps are considered bio-safe, nontoxic, and biocompatible (hameed et al., ; farrokhi et al., ) . in comparison with bulksized particles, nanoparticles can pass through bacterial cell walls more simply. the release of zn + ions from nps destroy the cell membrane and subsequently enhance cellular internalization of the nanoparticles. it is also confirmed that the antimicrobial function of zno can be ascribed to photocatalytic activity. by receiving uv light which promotes its interaction with bacteria, ros, which has a phototoxic effect on bacteria, will be produced (dimapilis et al., ) . journal pre-proof tio is a biocompatible chemical thermally stable compound with high photocatalytic activity and has shown good antimicrobial activities with wide spectrum function against microorganisms (gram-negative and gram-positive bacteria, fungi, and virus). the generation of reactive oxygen species (ros) is the major mechanism of tio . due to its photocatalytic nature, antimicrobial activity of tio nps enhances by exposing uv light on its surface (de dicastillo et al., ; levchuk et al., b; levchuk and sillanpää, ) . it is proved that lanthanum compounds, such as lanthanum hydroxide (la(oh) ), lanthanum carbonate (la co ), and lanthanum hydroxide (la(oh) ) can attach to phosphate so firmly that they can generate lapo and remove redundant phosphate in a bacterial cell. according to the very significant band to phosphate, nano-lanthanum (la) species represent high effectiveness adsorption and suppress microbial growth by inhibition of the microorganism growth liu et al., ) . carbon is the chemical element with atomic number and six electrons situate s , s , and p atomic orbital. graphene is a one-atom-thick hexagonal structure consisting of a -dimensional bhatnagar et al., ) . furthermore, the existence of these functional groups advances the interactions with biomolecules and leads to bacterial death with no intracellular process. go nano-sheets with sharpe edges hurt the bacterial cell membranes, lead to leakage of the intracellular matrix and eventually cause inactivation of bacteria. plus, go generate oxidative stress by producing ros and lead to dna damage and mitochondrial dysfunction (kumar et al., ) . in addition, the antiviral activity of go is confirmed by several experiments (ye et al., ) . single-walled carbon nanotubes (swnts) are nanometer diameter cylinders fabricated of rolled up graphene sheet in the form of a tube. generally, swcnt length is in the micrometer range and their diameters vary from . to to nm (eatemadi et al., ) . swnts have presented strong and board spectrum antimicrobial activities. the antimicrobial activity of swcnts has been confirmed to be varied by several factors. for instance, longer length nanotubes exhibited superior antimicrobial activity, swcnts having surface groups of -oh and -cooh illustrate more strong antimicrobial activity in comparison with swcnts-nh , also the diameter of nanotubes is an important factor governing their antibacterial effects (dong et al., ) . chitosan [poly-(b- / )- -amino- -deoxy-d-glucopyranose] is quaternary ammonium cations are positively charged polyatomic ions. these ions contain a positively charged nitrogen "head" binding four bonds r including an alkyl group or an aryl group. quaternary ammonium compounds are salts of quaternary ammonium cations (tezel and pavlostathis, ) . because of their positively charged sites, they are able to generate electrostatic bonds with the negatively charged sites on bacterial cell walls, resulting in disruption of a cell wall, defect cell membrane permeability and consequently sever leakage of intracellular low-molecular-weight materials (chen et al., ) . qacs target bacterial cell membranes. therefore, they illustrate extended-spectrum antimicrobial activity and have been widely employed to construct an antibacterial surface (jennings et al., ) . quaternized poly benzalkonium chloride (bac) are some important kinds of qacs (zhu et al., ) . easy release of biocides from the membrane improves their exposure rate to bacterial cell. there is a challenging point since the leaching profile of incorporated biocides determines long term bactericidal efficiency of the membrane. leaching of bactericidal agents resulted in the diminution of the membrane antimicrobial performance over time. gradual leaching of the blended biocides during the filtration process not only declines the antibacterial activity, but may j o u r n a l p r e -p r o o f also lead to secondary pollution (fu et al., ) . besides chemical contamination and cytotoxicity issues, the continuous release of bactericidal agents causes the development of bacterial resistance due to being exposed to sub-inhibitory concentrations of biocides (sile-yuksel et al., ; mukherjee and de, ) . thus, there is a challenge to provide process eluding leaching of toxic materials while illustrating rapid pathogens killing ability. nanofiber coatings based methods which promote contact pathogen-killing capacity are promising and can be obtained by chemical modification with tethered biocides functionalities. these strategies may be successful by regarding the right control over the binding quality between the active agent and the underlying biomaterial surface (zhang et al., ; bazaka et al., ; hilpert et al., ) . despite there are numerous researches on application of antibacterial electrospun nanofiber membrane in water filtration, the leaching pattern and durable bactericidal efficiency of the membranes have not been studied comprehensively. this method is generally used for testing the inherent antibacterial performance of fabricated electrospun nanofibers as a membrane. this assay generally consists of qualitative detection and quantitative measurement techniques (zhu et al., ) . the inhibitory activity of electrospun nanofibers is assayed by the inhibition zone diameter or agar diffusion method toward the considered bacterial sample, based on the clinical and laboratory standards institute (clsi document m -a ) (clsi, ) . for this reason, μl overnight culture of the tested bacteria ( cfu/ml) is spread across the surface of an appropriate agar plate, the electrospun nanofiber is cut to disk with about mm diameters, sterilized under uv light for min and then incubated on the plates for - h at °c. then, the area of bacteria growth is detected, and the diameter of the inhibition zone around the electrospun nanofiber is measured. this procedure modifications are also used (santos et al., ; jatoi and al mamun, ; fig. a) . the antibacterial function of the membrane will lead to changes in the bacteria cell morphology. this method is a shaking flask method. briefly, an appropriate amount of sample sterilized nanofiber is weighted, dipped into a flask containing pbs buffer with a cell concentration of - × cfu ml - . the flask is incubated with continuous shaking at • c for a determined time. after serial dilutions by the phosphate buffer, the bacterial suspensions are plated in the agar plate. the inoculated plates were incubated at • c for h and the viable bacterial cells are counted by a colony counter (kleyi et al., ) . also, the number of bacteria after incubation for a determined time can be indirectly measured by spectrometric optical density at nm . then, the reduction rate is calculated with the following equation: where r is the reduction rate, a is the number of bacteria isolated from the inoculated electrospun nanofibers after defined time contact time, and b is the number of bacteria isolated from the inoculated electrospun nanofibers at zero contact time (yao et al., ) . j o u r n a l p r e -p r o o f the aatcc test method quantitatively evaluates the bacteriostatic (growth inhibition) or bactericidal (killing of bacteria) ability of textiles over a hours contact. for this test, firstly a defined weighted of nanofiber is cut (about mg), get sterilized by uv light, then inoculated with . ml microbial suspension ( - / × cfu ml - ) and finally overnight incubated at • c. over determined contact period, ml pbs buffer is added to the falcon tubes containing the inoculated treated electrospun nanofiber. after min shaking, µl of the solution is cultured on nutrient agar plates and incubated for h (ardekani et al., ). astm e is another antimicrobial standard method used for evaluating the antibacterial function of immobilized antimicrobial nanofibers under dynamic contact conditions. the antibacterial efficiency is evaluated depending on the contact time from several mints to h between the bacterial solution and the sample (ungur and hrůza, ) . for both methods, the percentage of growth reduction is calculated with the r equation, mentioned above. in this method, some known dyes are been used to probe if the entrapped bacteria are inactivated by membranes and quantify surviving bacteria, representing an operative, visual and precise antibacterial assay (zhu et al., ) . for example, a common dye-based method is detecting the optimal analytical parameters for fluorescence measurements from the dyes syto and propidium iodide (pi). the basis of this approach is the attachment of syto to live-cell and propidium iodide (pi) to dead cells or cells with defected membranes. the optimal analytical parameters are used for measurement fluorescence by evaluating the intensity of emissions at - nm for syto and - nm for pi which interpret to quantify of the live cells minimum inhibitory concentration (mic) represents the minimum amount of antibacterial membrane, which could inhibit bacterial growth. in this method, the defined weighted of nanofiber is dissolved in water (or proper dissolvent), µl of this solution is added to the first well and serially diluted by transferring µl of the well pipetted content to the next well containing µl media. thereafter, µl of bacterial cultures ( × cfu ml - ) is poured to each well and plate is incubated at ºc for h. to detect the bacterial growth, resazurin or piodonitrotetrazolium chloride is added to wells. the wells that turned pink (if resazurin used) or purple (if p-iodonitrotetrazolium chloride used) represents the surviving of bacteria, hence no growth inhibition. the nanofiber concentration in the last growth inhibited well is considered as the mic value (nthunya et al., ). the bacteria retention test can also be performed with a dead-end filtration module using a vacuum filtration cell, a syringe filter holder mm, millipore and a dead-end filtration cell j o u r n a l p r e -p r o o f system (jabur et al., ; daels et al., ; son et al., ) . before the experiments, the membrane cut diameter and sterilized. all pieces of filtration equipment are sterilized with an autoclave method for min at °c. the membrane is fitted into the device. after passing sterile water from the filter, the bacterial suspension is filtered through the membranes using a pressure. in this step there are two different techniques for evaluation of bacterial retention: ) the filtrate is serially diluted with sterile distilled water and viable counts are assayed by plate counts. the colony count can be facilitated by staining bacterial cells with syto fluorescent dye and using a fluorescence microscope . then the bacterial retention ratio is calculated in terms of lrv (log reduction value) by the equation: lvr= log (c f /c p ) where r= ( -(c p /c f )) × electrochemical disinfection can destroy bacteria and viruses by electroporation and reactive oxygen species (ros) during a short time. electrochemical treatment devices electrochemical disinfection regarded as an effective portable water disinfectant. fabrication of electrospun j o u r n a l p r e -p r o o f porous membrane filter using agents to provide a conducting bed and a strong electric field, facilitate electroporation and production of ros, which signifies the disinfection process (hong et al., ; huo et al., ) . for testing this ability, an electrochemical filtration device with electrospun nanofiber as a filtration membrane is used. then, a saline solution containing bacterial suspension flows through the nanofiber filter using low voltages at a defined flow rate. the bacterial removal efficiency is calculated by the lvr equation (wen et al., ; tan et al., ; xie et al., ) . in order to determine the deposition of bacteria on a filtrated membrane and the possibility of biofouling, instantly after filtration, the membrane is transferred to an autoclaved beaker filled with pbs buffer and sonicated. the bacteria in the suspended membranes are measured by counting the number of colony cells. moreover, the morphology of trapped bacteria is investigated using sem (xie et al., ; makaremi et al., ; wen et al., ) . different kinds of electrospun nanofibers have been recently fabricated for bacterial removal from water are illustrated in tables . usually, evaluation of the antiviral function of nano-filters is carried out using bacteriophage and polypropylene nonwoven textile as support layer, the structure shows excellent retention of bacteria and fine solids, with - nm pore size and operating pressure < mbar. naked filter is anther novel commercially application of nanofiber in household/bottled water filter with ability to remove . % of the micro-organic contaminants. nanotrap is another commercial household water filter produced by coway company. astrapool, fluidra has introduced nanofiber based product applied in filtration system for residential pools (http://electrospintech.com/products.html#.xvs_nm zbiu). liquidity nanotech corporation has created electrospun nanofiber membrane made water purification cartridge with superior flow rate, about a cup per minute, good microbiological retention, -log bacteria reduction, -log virus reduction and -log cyst reduction and simple usage process (https://product.statnano.com/product/ /liquidity-water-purification-cartridge). pentair company has produced polyethersulfone nanofiber-based cartridge for industrial water purification applications. the cartridge is an absolute barrier to bacteria and viruses: with more than -log reduction rate (https://www.directindustry.com/prod/pentair-x-flow/product- - .html). the researcher's and industry's attention to research and development of electrospun nanofibrous membranes has been growing because of its simplicity, low-cost, scalable molecules incorporation process on the fabricated non-woven mats, production of membranes with the high surface area.. high surface area to volume ratio, uniform pore size, and high pore interconnectivity and adequate antibacterial property improve the performance of the nanofibrous membrane in water disinfection application (subramanian and seeram, ) . however, there are several major concerns to be noticed for the application of electrospun nanofiber in water disinfection. although high surface area and porosity of the electrospun nanofiber are significant advantages, which enhance permeability and selectivity, they also lead to higher mechanical stresses. consequently, the membrane might be compacted or deformed through the filtration process, which causes loss the porosity and subsequently decreases the also, further experimental studies needed to conduct proper control of biomolecules release rate from nanofiber, to ensure a balance between successfully deactivate the bacteria strains and lengthen the period of the function, and minimize contamination. therefore, fabrication of membrane representing inherent self-cleaning, antiviral, and the antibacterial and anti-biofouling feature has gained immense attention for industrial application. recently, focusing on the production of smart antibacterial surfaces has led to a promising "kill−release" strategy. this approach proposed the fabrication of dual-functional antibacterial surfaces by incorporating biocides into non-fouling materials. these membranes are able to maintain their long-term antibacterial activity by killing bacteria attached to their surface and subsequently are potent to release the dead bacteria to reveal a clean surface (wei et al., ) . although these smart membranes are applied for biomedical applications, the strategy can be promising for further j o u r n a l p r e -p r o o f designing of novel electrospun nanofiber with these dual functions and strong long-term functional ability in water disinfection. as it is illustrated in tables and , despite the significant results obtained from the application of electrospinning in water filtration membrane designing, there are some gaps in this research area. for instance, there are no unanimous standard methods for evaluating the antibacterial or antiviral potential of fabricated electrospun water disinfecting filters. moreover, most of the researchers have used static antibacterial assay approaches that are unable to represent the membrane antibacterial performance under the dynamic water filtration process. moreover, recent related studies have not investigated comprehensibly the long-term antibacterial or antiviral performance of produced nano-membrane in water disinfection. due to extensive endeavors aiming to produce novel smart antibacterial and antiviral membranes and, electrospun nanofibers should be developed rapidly as great candidates for a high effective anti-biofouling membrane for water treatment. the water-stable nanofibers was able to bind to two different viruses and achieved a . lrv for ppv 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membranes using a controlled-release platform key: cord- - moy hr authors: stillwell, william title: membrane transport date: - - journal: an introduction to biological membranes doi: . /b - - - - . - sha: doc_id: cord_uid: moy hr life depends on a membrane's ability to precisely control the level of solutes in the aqueous compartments, inside and outside, bathing the membrane. the membrane determines what solutes enter and leave a cell. transmembrane transport is controlled by complex interactions between membrane lipids, proteins, and carbohydrates. how the membrane accomplishes these tasks is the topic of this chapter. life depends on a membrane's ability to precisely control the level of solutes in the aqueous compartments, inside and outside, bathing the membrane. the membrane determines what solutes enter and leave a cell. transmembrane transport is controlled by complex interactions between membrane lipids, proteins, and carbohydrates. how the membrane accomplishes these tasks is the topic of chapter . a biological membrane is semipermeable, meaning it is permeable to some molecules, most notably water, while being very impermeable to most solutes (various biochemicals and salts) found in the bathing solution. this very important concept of unequal transmembrane distribution and, hence, permeability between water and other solutes came out of the pioneering work of charles overton in the s (see chapter ) . how does a biological membrane accomplish semipermeability? the barrier to solute movement is largely provided by the membrane's hydrophobic core, a very thin (w Å thick), oily layer. the inherent permeability of this core varies from membrane to membrane. generally, the more tightly packed the lipids comprising the bilayer, the lower its permeability will be. lipid bilayers are very impermeable to most solutes because of their tight packing. fig. . depicts the membrane permeability of a variety of common solutes [ ] . note the data are presented as a log scale of solute permeability (p in cm/s) and ranges from na þ ¼ À cm/s to water ¼ .  À cm/s, spanning almost orders of magnitude! lipid bilayer permeability is not a constant but instead is affected by environmental factors. for example, luvs (large unilamellar veicles) made from dppc ( : , : pc) have a sharp phase transition temperature, t m , of . c. at temperatures well below t m , the luvs are in the tightly packed gel state and permeability is extremely low. at temperatures well above t m , the luvs are in the loosely packed liquid disordered state (l d , also called the liquid crystalline state) and permeability is high. however, maximum permeability is not found in the l d state, but rather at the t m [ ] . as the luvs are heated from the gel state and approach the t m , domains of l d start to form in the gel state. solutes can then pass more readily through the newly formed l d domains than the gel domains resulting in an increase in permeability. at t m there is a maximum amount of coexisting gel and l d state domains that exhibit extremely porous domain boundaries. it is through these boundaries that most permeability occurs. as the temperature is further increased, the luvs pass into the l d state and the interface boundaries disappear, reducing permeability to that observed for the single-component l d state. thus, maximum permeability is observed at the t m . the tendency for solutes to move from a region of higher concentration to one of lower concentration was first defined in by the physiologist adolf fick (fig. . ). his work is summarized in what is now the very well-known fick's laws of diffusion [ ] . the laws apply to both free solution and diffusion across membranes. fick developed his laws by measuring concentrations and fluxes of salt diffusing between two reservoirs through connecting tubes of water. fick's first law describes diffusion as: diffusion rate ¼ Àda dc dx where d ¼ diffusion coefficient (bigger molecules have lower ds); a ¼ cross-sectional area over which diffusion occurs; dc/dx is the solute concentration gradient (diffusion occurs from a region of higher concentration to one of lower concentration). the relationship between a solute's molecular weight and its diffusion coefficient is shown in table . . large solutes have low diffusion coefficients and therefore diffuse more slowly than small solutes. the diffusion rate for a particular solute under physiological conditions is a constant and cannot be increased. this defines the theoretical limit for an enzymatic reaction rate and also limits the size of a cell. if a solute starts at the center of a bacterial cell, it takes about À s to diffuse to the plasma membrane. for this reason, typical cells are microscopic (see chapter ) . at about . pounds and the size of a cantaloupe, the largest cell on earth today is the ostrich egg. however a fossilized dinosaur egg in the american museum of natural history in new york is about figure . log of the permeability (p in cm/s) across lipid bilayer membranes for common solutes ranging from na þ ( À cm/s) to water ( .  À cm/s). this range spans almost orders of magnitude [ ] . the size of basketball. since an egg's only function is to store nutrients for a developing embryo, its size is many orders of magnitude larger than a normal cell. osmosis is a special type of diffusion, namely the diffusion of water across a semipermeable membrane. water readily crosses a membrane down its potential gradient from high to low potential ( fig. . ) [ ] . osmotic pressure is the force required to prevent water movement across the semipermeable membrane. net water movement continues until its potential reaches zero. an early application of the basic principles of osmosis came from the pioneering work on hemolysis of red blood cells by william hewson in the s (see chapter ) . it has also been discussed that mlvs (multilamellar vesicles, liposomes) behave as almost perfect osmometers, swelling in hypotonic solutions and shrinking in hypertonic solutions (see chapter ) [ , ] . liposome swelling and shrinking can be easily followed by changes in absorbance due to light scattering using a simple spectrophotometer. therefore, osmosis has been investigated for many years using common and inexpensive methodologies and a lot is known about the process. membranes are rarely, if ever, perfectly semipermeable. deviation from ideality is defined by a reflection coefficient (s). for an ideal semipermeable membrane where a solute is totally movement of solutes across membranes can be divided into two basic types: passive diffusion and active transport [ ] . passive diffusion requires no additional energy source other than what is found in the solute's electrochemical (concentration) gradient and results in the solute reaching equilibrium across the membrane. passive diffusion can be either simple passive diffusion where the solute crosses the membrane anywhere by simply dissolving into and diffusing through the lipid bilayer, or facilitated passive diffusion where the solute crosses the membrane at specific locations where diffusion is assisted by solute-specific facilitators or carriers. active transport requires additional energy, often in the form of atp, and results in a nonequilibrium, net accumulation (uptake) of the solute on one side of the membrane. the basic types of membrane transport, simple passive diffusion, facilitated diffusion (by channels and carriers) and active transport are summarized in fig. . [ ] . there are countless different examples of each type of membrane transport process [ ] . only a few representative examples will be discussed here. even simple passive diffusion requires energy to cross a bilayer membrane. in order to cross a membrane, the solute must first lose its waters of hydration, diffuse across the membrane, and then regain its waters on the opposite side. the limiting step involves the energy required to lose the waters of hydration. table . shows the relationship between the waters of hydration (proportional to the number of eoh groups on a homologous series figure . osmosis and osmotic pressure. water is placed in a u-shaped tube where each of the tube arms is separated by a semipermeable membrane with pores of a size that water can easily pass through but a solute cannot. upon addition of the solute to the tube's right arm, water diffuses from left to right (high water potential to low). the column of water in the tube's right arm (the one containing the solute) rises until the extra weight of the column equals the osmotic pressure caused by the solute. a pump could then be used to counter the osmotic pressure whereupon the solution columns in the right and left arms of the tube are made the same. the pump pressure required to equalize the height of the two columns is the osmotic pressure [ ] . note a small amount of the solute leaks from right to left since no filter is perfect. of solutes) and the activation energy for transmembrane diffusion. as the number of waters of hydration increases from glycol < glycerol < erythritol, the activation energy for diffusion also increases. the activation energy compares very well with the energy of hydration. however, water diffusion does not fit this model. water permeability is just too high. several possibilities have been suggested to account for the abnormally high membrane permeability of water: . water is very small and so it just dissolves in bilayers better than larger solutes. . due to its size, water can readily enter very small statistical pores (w . Å in diameter). statistical pores result from the simultaneous lateral movement of adjacent membrane phospholipids in opposite directions. statistical pores have only a fleeting existence and cannot be isolated or imaged. . passage down water chains. . water can be carried down kinks in acyl chains that result from acyl chain melting (see lipid melting in chapter ). . water may rapidly cross membranes through nonlamellar regions (eg, micelles, cubic or h ii phasedsee chapter ). . high water permeability will occur at regions of packing defect (eg, surface of integral membrane proteins, boundary between membrane domains). . through pores or channels used to conduct ions. . through specific water channels known as aquaporins (see below, chapter , section . ). the only molecules that can cross a membrane by simple passive diffusion are water, small noncharged solutes, and gasses. charged or large solutes are virtually excluded from membranes and so require more than just simple passive diffusion to cross a membrane. facilitated diffusion (also known as carrier-mediated diffusion) is, like simple passive diffusion, dependent on the inherent energy in a solute gradient. no additional energy is required to transport the solute and the final solute distribution reaches equilibrium across the membrane. facilitated diffusion, unlike simple passive diffusion, requires a highly specific transmembrane integral protein or carrier to assist in the solute's membrane passage. facilitators come in two basic types: carriers and gated channels. facilitated diffusion exhibits michaelis-menton saturation kinetics ( fig. . , part a, right), indicating the carrier has an enzyme-like active site. like enzymes, facilitated diffusion carriers exhibit saturation kinetics and recognize their solute with exquisite precision, easily distinguishing chemically similar isomers like d-glucose from l-glucose. fig. . (part a) compares simple passive diffusion to facilitated diffusion. the figure is not to scale, however, as facilitated diffusion is orders of magnitude faster than simple passive diffusion. a well-studied example of a facilitated diffusion carrier is the glucose transporter, or glut [ ] . from the activation energies for transmembrane simple passive diffusion of glycol, glycerol and erythritol presented in table . , it can be estimated that the activation energy for glucose should be well over kj/mol, but instead it is only kj/mol. this large discrepancy is attributed to the presence of a glucose-facilitated diffusion carrier. fig. . demonstrates the mode of action of one of these transporters, glut- , from the erythrocyte [ ] . gluts occur in nearly all cells and are particularly abundant in cells lining the small intestine. gluts are but one example in a superfamily of transport facilitators. gluts are integral membrane proteins whose membrane-spanning region is composed of a-helices. gluts function through a typical membrane transport mechanism [ ] . glucose binds to the membrane outer surface site causing a conformational change associated with transport across the membrane. at the inner side of the membrane, glucose is released into the internal aqueous solution ( fig. in virtually all organisms there exists a wide variety of ion channels, the most widely distributed being potassium channels [ ] . there are four basic classes of potassium channels, all of which provide essential membrane-associated functions including setting and shaping action potentials and hormone secretion: . calcium-activated potassium channel . inwardly rectifying potassium channel . tandem pore domain potassium channel . voltage-gated potassium channel potassium channels are composed of four protein subunits that can be the same (homotetramer) or closely related (heterotetramer). all potassium channel subunits have a distinctive pore-loop structure that sits at the top of the channel and is responsible for potassium selectivity [ ] . this is often referred to as a selectivity or filter loop. the selectivity filter strips the waters of hydration from the potassium ion, allowing it into the channel. farther down the structure is a -Å-diameter, transmembrane, water-filled central channel that conducts potassium across the membrane. elucidating the three-dimensional structure of this important integral membrane protein by x-ray crystallography ( fig. . ) [ ] was a seminal accomplishment in the field of membrane biophysics. for this figure . three-dimensional structure of the potassium channel [ ] . the channel itself is the clear opening in the center of the structure and a single k þ is shown in the center of the channel. work from , rod mackinnon ( fig. . ) of rockefeller university was awarded the nobel prize in chemistry. until the potassium channel work, just obtaining the structure of nonewater-soluble proteins was next to impossible. mackinnon's work elucidated not only the structure of the potassium channel but also its molecular mechanism. it has served as a blueprint for determining the structure of other membrane proteins and has greatly stimulated interest in the field. in some ways, na þ channels [ ] parallel the action of k þ channels. they are both facilitated diffusion carriers that conduct the cation down the ion's electrochemical gradient. in excitable cells such as neurons, myocytes, and some glia, na þ channels are responsible for the rising phase of action potentials (see chapter ) . therefore agents that block na þ channels also block nerve conduction and so are deadly neurotoxins. there are two basic types of na þ channels: voltage-gated and ligand-gated. the opening of a na þ channel has a selectivity filter that attracts na þ . from there the na þ ions flow into a constricted part of the channel that is about e Å wide. this is just large enough to allow the passage of a single na þ with one attached water. since the larger k þ cannot squeeze through, the channel is selective for na þ . of particular interest are two extremely potent biological toxins, tetrodotoxin (ttx) and saxitoxin (stx) (fig. . , [ ] ), that, in seafood, have killed and injured many humans. both toxins shut down na þ channels by binding from the extracellular side. ttx is encountered primarily in puffer fish but also in porcupine fish, ocean sunfish, and triggerfish. ttx (fig. . , left) is a potent neurotoxin that blocks na þ channels while having no effect on k þ channels. puffer fish is the second most poisonous vertebrate in the world, trailing only the golden poison frog that is endemic to the rain forests on the pacific coast of colombia. in some parts of the world puffer fish are considered to be a delicacy but . membrane transport must be prepared by chefs who really know their business, as a slight error can be fatal. puffer poisoning usually results from consumption of incorrectly prepared puffer soup, and ttx has no known antidote! saxitoxin (stx, fig. . , right) is a na þ channeleblocking neurotoxin produced by some marine dinoflagellates that can accumulate in shellfish during toxic algal blooms known as red tide. saxitoxin is one of the most potent natural toxins, and it has been estimated that a single contaminated mussel has enough stx to kill humans! stx's toxicity has not escaped the keen eye of the united states military, which has weaponized the toxin and given it the designation tz. the driving force for transmembrane solute movement by simple or passive diffusion is determined by the free energy change, dg. is the solute concentration on the right side of a membrane; [s o ] is the solute concentration on the left side of a membrane; r is the gas constant; t is the temperature in k; z is the charge of the solute; f is the faraday; dj is the transmembrane electrical potential. solute movement will continue until dg ¼ . if dg is negative, solute movement is left to right (it is favorable as drawn). if dg is positive, solute movement is right to left (it is unfavorable in the left-to-right direction) or energy must be added for the solute to go from left to right. the equation has two parts; a transmembrane chemical gradient and a transmembrane electrical gradient (dj). the net movement of a solute is therefore determined by a combination of the solute's chemical gradient and an electrical gradient inherent to the cell. if the solute has no charge, z ¼ (as is the case for glucose) and the right hand part of the equation (zfdj) drops out. therefore, the final equilibrium distribution of glucose across the membrane will have the internal glucose concentration equal to the external glucose concentration and is independent of dj, the electrical potential. at equilibrium for a noncharged solute, the situation for a charged solute like k þ is more complicated. the net dg is determined by both the chemical gradient À s o à ½s o Á and electrical gradient (dj). the dj results from the sum of all charged solutes on both sides of the membrane, not just k þ . therefore even if the k þ concentration is higher inside the cell than outside (the chemical gradient is unfavorable for k þ uptake), the dj may be in the correct direction (negative interior) and of sufficient magnitude to drive k þ uptake against its chemical gradient. aquaporins are also known as water channels and are considered to be "the plumbing system for cells" [ , ] . for decades it was assumed that water simply leaked through biological membranes by numerous processes described above (chapter , section ). however, these methods of water permeability could not come close to explaining the rapid movement of water across some cells. although it had been predicted that water pores must exist in very leaky cells, it was not until that peter agre (fig. . ) at johns hopkins university identified a specific transmembrane water pore that was later called aquaporin- . for this accomplishment agre shared the nobel prize in chemistry with rod mackinnon for his work on the potassium channel. aquaporins are usually specific for water permeability and exclude the passage of other solutes. a type of aquaporin known as aqua-glyceroporins can also conduct some very small uncharged solutes such as glycerol, co , ammonia, and urea across the membrane. however, all aquaporins are impermeable to charged solutes. water molecules traverse the aquaporin channel in single file (fig. . ) [ ] . a characteristic of all living membranes is the formation and maintenance of transmembrane gradients of all solutes including salts, biochemicals, macromolecules, and even water. in living cells, large gradients of na þ and k þ are particularly important. typical cell concentrations are: cell interior: mmol/l k þ , mmol/l na þ cell exterior: mmol/l k þ , mmol/l na þ figure . peter agre, e. living cells will also have a dj from À to À mv (negative interior) resulting from the uneven distribution of all ionic solutes including na þ and k þ . the chemical and electrical gradients are maintained far from equilibrium by a multitude of active transport systems. active transport requires a form of energy (often atp) to drive the movement of solutes against their electrochemical gradient, resulting in a nonequilibrium distribution of the solute across the membrane. a number of nonexclusive and overlapping terms are commonly used to describe the different types of active transport. some of these are depicted in fig. . [ ] . figure . basic types of active transport [ ] . cell membrane cell membrane figure . aquaporin. water molecules pass through the aquaporin channel in single file. primary active transport is also called direct active transport or uniport. it involves using energy (usually atp) to directly pump a solute across a membrane against its electrochemical gradient. the most studied example of primary active transport is the plasma membrane na þ ,k þ -atpase discussed below (chapter , section . ). other familiar examples of primary active transport are the redox h þ -gradient generating system of mitochondria (see chapter ), the light-driven h þ -gradient generating system of photosynthetic thylakoid membranes, and the atp-driven acid (h þ ) pump found in the epithelial lining of the stomach. there are four basic types of atp-utilizing primary active transport systems (table . ). arguably the most important active transport protein is the plasma membrane-bound na þ ,k þ -atpase. this single enzyme accounts for one-third of human energy expenditure and is often referred to as the "pacemaker for metabolism." as a result the na þ ,k þ -atpase has been extensively studied for more than years. the enzyme was discovered in by jens skou (fig. . ) who, years later, was awarded the nobel prize in chemistry. as is often the case in biochemistry, a serendipitous discovery of a natural product from the jungles of africa has been instrumental in unraveling the enzyme's mechanism of action. the compound is ouabain (fig. . ) , a cardiac glycoside first discovered in a poison added to the tip of somali tribesmen's hunting arrows. in fact the name ouabain comes from the somali word waabaayo that means "arrow poison." the sources of ouabain are ripe seeds and bark of certain african plants and ouabain is potent enough to kill a hippopotamus with a single arrow. for decades after its discovery, ouabain was routinely used to treat atrial fibrillation and congestive heart failure in humans. more recently, ouabain has been replaced by digoxin, a structurally related, but more lipophilic cardiac glycoside. there are several important observations about na þ ,k þ -atpase that had to be factored in before a mechanism of action could be proposed. these include: . na þ ,k þ -atpase is an example of active antiport and primary active transport. . na þ ,k þ -atpase is inhibited by ouabain, a cardiac glycoside. . ouabain binds to the outer surface of na þ ,k þ -atpase and blocks k þ transport into the cell. . na þ binds better from the inside. . k þ binds better from the outside. . atp phosphorylates an aspartic acid on the enzyme from the inside. . phosphorylation is related to na þ transport. . dephosphorylation is related to k þ transport. . dephosphorylation is inhibited by ouabain. . three na þ ions are pumped out of the cell as two k þ ions are pumped in, driven by hydrolysis of one atp. . na þ ,k þ -atpase is electrogenic. mechanism of na þ ,k þ -atpase [ ] is based on toggling back and forth between two conformational states of the enzyme, enz- and enz- ( fig. . ). three na þ s bind from the inside to na þ ,k þ -atpase in one conformation (enz- ). this becomes phosphorylated by atp causing a conformation change producing enz- wp. enz- wp does not bind na þ , but does bind two k þ ions. therefore, three na þ ions are released to the outside and two k þ ions are bound from the outside, generating enz- wp ( k þ ). upon hydrolysis of wp, na þ ,k þ -atpase (enz ii) reverts back to the original enz- conformation that releases two k þ ions and binds three na þ ions from the inside. ouabain blocks the dephosphorylation step. secondary active transport (also known as cotransport) systems are composed of two separate functions. the energy-dependent movement of an ion (eg, h þ , na þ , or k þ ) generates an electrochemical gradient of the ion across the membrane. this ion gradient is coupled to the movement of a solute in either the same direction (symport) or in the opposite direction (antiport, see fig. . , [ ] ). movement of the pumped ion down its electrochemical gradient is by facilitated diffusion. the purpose of both types of co-transport is to use the energy in an electrochemical gradient to drive the movement of another solute against its gradient. an example of symport is the sglt (sodium-glucose transport protein- ) in the intestinal epithelium [ ] . sglt uses the energy in a downhill transmembrane movement of na þ to transport glucose across the apical membrane against an uphill glucose gradient so that the sugar can be transported into the bloodstream. the secondary active symport system for lactose uptake in escherichia coli is shown in fig. . [ ] . lactose uptake is driven through a channel by a h þ gradient generated by the bacterial electron transport system [ ] . the free energy equation for transport described above can be rearranges for cases employing h þ gradients (see chapter ) to: where dm h þ is the proton motive force; dj is the transmembrane electrical potential; r is the gas constant; t is the temperature in k; n is the solute charge (þ for protons); f is the faraday; dph is the transmembrane ph gradient. it is the force on an h þ (called the proton motive force) that drives lactose uptake. note that the ability to take up lactose is a combination of the electrical gradient and the ph gradient. although lactose uptake is directly coupled to h þ transmembrane movement, it is possible to take up lactose even if the ph gradient is zero (ie, if the dj is sufficiently large). over years ago, peter mitchell (see chapter , fig. . ) recognized the importance of what he termed "vectorial metabolism" [ , ] . water-soluble enzymes convert substrate to product without any directionality. mitchell proposed that many enzymes are integral membrane proteins that have a specific transmembrane orientation. when these enzymes convert substrate to product they do so in one direction only. this enzymatic conversion is therefore unidirectional, or "vectorial." mitchell expanded this basic concept into his now famous "chemiosmotic hypothesis" for atp synthesis in oxidative phosphorylation (chapter ) [ , ] . for this revolutionary idea mitchell was awarded the nobel prize in chemistry. vectorial metabolism has been used to describe the mechanism for several membrane transport systems. for example, it has been reported in some cases the uptake of glucose into a cell may be faster if the external source of glucose is sucrose rather than free glucose. through a vectorial transmembrane reaction, membrane-bound sucrase may convert external sucrose into internal glucose plus fructose more rapidly than the direct transport of free glucose through its transport system. mitchell defined one type of vectorial transport as group translocation, the best example being the pts (phosphotransferase system) discovered by saul roseman in . pts is a multicomponent active transport system that uses the energy of intracellular phosphoenol pyruvate (pep) to take up extracellular sugars in bacteria. transported sugars include glucose, mannose, fructose, and cellobiose. components of the system include both plasma membrane and cytosolic enzymes. pep is a high-energy phosphorylated compound (dg of hydrolysis is À . kj/mol) that drives the system. the high-energy phosphoryl group is transferred through an enzyme bucket brigade from pep to figure . lactose transport system in escherichia coli [ ] . uptake of lactose is coupled to the movement of an h þ down its electrochemical gradient. this is an example of active transport, co-transport, and active. glucose producing glucose- -phosphate in several steps (pep / ei / hpr / eiia / eiib / eiic / glucose- -phosphate). the sequence is depicted in more detail in fig. . [ ] . hpr stands for heat-stable protein that carries the high-energy wp from ei (enzyme-i) to eiia. eiia is specific for glucose and transfers wp to eiib that sits next to the membrane where it takes glucose from the transmembrane eiic and phosphorylates it producing glucose- -phosphate. although it is glucose that is being transported across the membrane, it never actually appears inside the cell as free glucose but rather as glucose- -phosphate. free glucose could leak back out of the cell via a glucose transporter, but glucose- -phosphate is trapped inside the cell where it can rapidly be metabolized through glycolysis. group translocation is defined by a transported solute appearing in a different form immediately after crossing the membrane. the term ionophore means "ion bearer." ionophores are small, lipid-soluble molecules, usually of microbial origin, whose function is to conduct ions across membranes [ , ] . they are facilitated diffusion carriers that transport ions down their electrochemical gradient. ionophores can be divided into two basic classes: channel formers and mobile carriers (fig. . ) [ ] . channel formers are long lasting, stationary structures that allow many ions at a time to rapidly flow across a membrane. mobile carriers bind to an ion on one side of a membrane, dissolve in and cross the membrane bilayer and release the ion on the other side. they can only carry one ion at a time. four representative ionophores will be discussed: the k þ ionophore valinomycin, the proton ionophore , -dinitrophenol, synthetic crown ethers, and the channel-forming ionophore nystatin (fig. . ). superficially valinomycin resembles a cyclic peptide (fig. . ) . however, upon closer examination the ionophore is actually a -unit (dodeca) depsipeptide where amino acid peptide bonds are alternated with amino alcohol ester bonds. therefore the linkages that figure . the bacterial pts system for glucose transport [ ] . . membrane transport hold the molecule together alternate between nitrogen esters (peptide bonds) and oxygen esters. the units that comprise valinomycin are d-and l-valine (hence the name "valinomycin"), hydroxyvaleric acid and l-lactic acid. the circular structure is a macrocyclic molecule with the carbonyl oxygens facing the inside of the ring where they chelate a single k þ . the outside surface of valinomycin is coated with nine hydrophobic side chains of d-and l-valine and l-hydroxyvaleric acid. the polar interior of valinomycin precisely fits one k þ . the binding constant for k þ -valinomycin is while na þ -valinomycin is only . this emphasizes the high selectivity valinomycin has for k þ over na þ . valinomycin, figure . representative examples of ionophores: the k þ ionophore valinomycin, the proton ionophore , -dinitrophenol, the synthetic crown ether -crown- , and the channel forming ionophore nystatin. therefore, has an oily surface that readily dissolves in a membrane lipid bilayer, carrying k þ across the membrane down its electrochemical gradient. valinomycin was first recognized as a potassium ionophore by bernard pressman in the early 's [ , ] . he reported that valinomycin, a known antibiotic, stimulated k þ uptake and h þ efflux from mitochondria. many studies showed that valinomycin dissipates essential transmembrane electrochemical gradients causing tremendous metabolic upheaval in many organisms including microorganisms. it is for this reason that valinomycin was recognized as an antibiotic long before it was identified as an ionophore. currently several ionophores are added to animal feed as antibiotics and growth enhancing additives [ ] . recently valinomycin has been reported to be the most potent agent against sars-cov (severe acute respiratory-syndrome coronavirus), a severe form of pneumonia first identified in [ ] . fig. . ) is considered to be the classic uncoupler of oxidative phosphorylation (see chapter ) . it is a synthetic lipid-soluble proton ionophore that dissipates proton gradients across bioenergetic membranes (mitochondrial inner, thylakoid, bacterial plasma). an uncoupler is therefore an h þ -facilitated diffusion carrier. elucidating the role of dnp in uncoupling oxidative phosphorylation was an essential component in support of peter mitchell's chemiosmotic hypothesis [ ] . electron movement from nadh or fadh to o via the mitochondrial electron transport system generates a considerable amount of electrical energy that is partially captured as a transmembrane ph gradient (see chapter ) . the movement of h þ s back across the membrane, driven by the electrochemical gradient, is through a channel in the f atpase (an f-type primary active transport system discussed above, (chapter , section . )) that is coupled to atp synthesis. dnp short-circuits the h þ gradient before it can pass through the f atpase, thus uncoupling electron transport, the energy source for the h þ gradient, from atp synthesis. therefore, in the presence of dnp, electron transport continues, even at an accelerated rate, but atp production is diminished. the energy that should have been converted to chemical energy in the form of atp is then released as excess heat. this combination of properties led to the medical application of dnp to treat obesity from to [ ] . upon addition of dnp: • the patient became weak due to low atp levels. • breathing increased due to increased electron transport to rescue atp production. • metabolic rate increased. • body temperature increased due to inability to trap electrical energy as chemical energy in the form of atp, releasing heat. • body weight decreased due to increased respiration burning more stored fat. dnp was indeed a successful weight loss drug. two of the early proponents of dnp use as a diet drug, cutting and tainter at stanford university, estimated that more than , people in the united states had tested the drug during its first year in use [ ] . dnp, however, did have one disturbing side effectddeath! fatality was not caused by a lack of atp, but rather by a dangerous increase in body temperature (hyperthermia). in humans, e mg/kg of dnp can be lethal. although general use of dnp in the united states was discontinued in , it is still employed in other countries and by bodybuilders to eliminate fat before competitions. crown ethers are a family of synthetic ionophores that are generally similar in function to the natural product valinomycin [ ] . the first crown ether was synthesized by charles pederson (fig. . ) while working at dupont in . for this work pedersen was co-awarded the nobel prize in chemistry. crown ethers are cyclic compounds composed of several ether groups. the most common crown ethers are oligomers of ethylene oxide with repeating units of (ech ch oe) n where n ¼ (tetramer), n ¼ (pentamer), or n ¼ (hexamer). crown ethers are given structural names, x-crown-y, where x is the total number of atoms in the ring and y is the number of these atoms that are oxygen. crown refers to the crown-like shape the molecule takes. crown ether oxygens form complexes with specific cations that depend on the number of atoms in the ring. for example, -crown- ( fig. . ) has high affinity for k þ , -crown- for na þ , and -crown- for li þ . like valinomycin, the exterior of the ring is hydrophobic, allowing crown ethers to dissolve in the membrane lipid bilayer while carrying the sequestered cation down its electrochemical gradient. it is now possible to tailor make crown ethers of different sizes that can encase a variety of catalysts for phase transfer into the bilayer hydrophobic interior where they can be used to catalyze reactions inside the membrane. nystatin (fig. . ) is a channel-forming ionophore that creates a hydrophobic pore across a membrane [ , ] . channel-forming ionophores allow for the rapid facilitated diffusion of various ions that depend on the dimensions of the pore. nystatin, like other channel-forming ionophores (eg, amphotericin b and natamycin), is a commonly used antifungal agent. finding medications that can selectively attack fungi in the presence of figure . charles pedersen, e . normal animal cells presents a difficult challenge since both cell types are eukaryotic. bacteria, being prokaryotes, are sufficiently different to present a variety of anti-bacterial approaches not amenable to fungi. however, fungi do have an achilles heel. fungal plasma membranes have as their dominant sterol ergosterol, not the animal sterol cholesterol (see chapter ) . nystatin binds preferentially to ergosterol, thus targeting fungi in the presence of animal cells. when present at sufficient levels, nystatin complexes with ergosterol and forms transmembrane channels that lead to k þ leakage and death of the fungus. nystatin is a polyene antifungal ionophore that is effective against many molds and yeast including candida. a major use of nystatin is as a prophylaxis for aids patients who are at risk for fungal infections. gap junctions are a common structural feature of many animal plasma membranes [ , ] . in plants similar structures are known as plasmodesmata. gap junctions were introduced earlier in chapter (see fig. . ). gap junctions represent a primitive type of intercellular communication that allows transmembrane passage of small solutes like ions, sugars, amino acids, and nucleotides while preventing migration of organelles and large polymers like proteins and nucleic acids. gap junctions connect the cytoplasms of two adjacent cells through nonselective channels. connections through adjacent cells are at locations where the gap between cells is only e nm. this small gap is where the term "gap junction" originated. gap junctions are normally clustered from a few to over a in select regions of a cell plasma membrane. early experiments involved injecting fluorescent dyes, initially fluorescein (molecular weight ), into a cell and observing the dye movement into adjacent cells with a fluorescence microscope [ , ] . currently lucifer yellow has become the fluorescent dye of choice for gap junction studies, replacing fluorescein. at first, the dye only appeared in the initially labeled cell. with time, however, the dye was observed to spread to adjacent cells through what appeared to be points on the plasma membrane. these points were later recognized as gap junctions. by varying the size of the fluorescent dye, it was shown that there was an upper size limit for dye diffusion. solutes had to have a molecular weight of less than w to cross from one cell to another [ ] . although gap junctions were obviously channels that connected the cytoplasms of adjacent cells, it was years before their structure, shown in fig. . , was determined [ , ] . each channel in a gap junction is made up of proteins called connexins. six hexagonally arranged connexins are associated with each of the adjacent cell plasma membranes that the gap junction spans. each set of six connexins is called a connexon and forms half of the gap junction channel. therefore, one gap junction channel is composed of aligned connexons and connexins. each connexin has a diameter of about nm and the hollow center formed between the connexins (the channel) is about nm in diameter. gap junctions allow adjacent cells to be in constant electrical and chemical communication with one another. of particular importance is the rapid transmission of small second messengers, such as inositol triphosphate (ip ) and ca þ . it appears that all cells in the liver are interconnected through gap junctions. this presents a possible dilemma. if even a single cell is damaged, deleterious effects may be rapidly spread throughout the entire liver. preventing this is one important function of ca þ . extracellular ca þ is w À mol/l while intracellular levels are maintained at w À mol/l. if a cell is damaged, ca þ rushes in, dramatically increasing intracellular ca þ . gap junction channels close if intracellular ca þ reaches À mol/l, thus preventing the spread of damage. gap junctions are particularly important in cardiac muscle as the electrical signals for contraction are passed efficiently through these channels [ ] . as would be expected, malfunctions of gap junctions lead to a number of human disorders including demyelinating neurodegenerative diseases, skin disorders, cataracts, and even some types of deafness. there are several other ways that solutes, including large macromolecules, can cross membranes. these methods include receptor-mediated endocytosis (rme, discussed in chapter ), phagocytosis, pinocytosis, exocytosis, and membrane blebbing. these methods involve large sections of a membrane containing many lipids and proteins. two similar transport processes that have been known for a long time are pinocytosis and phagocytosis [ ] . both involve nonspecific uptake (endocytosis) of many things from water and ions through to large macromolecules and, for phagocytosis, even whole cells. pinocytosis is greek for "cell drinking" and involves the plasma membrane invaginating a volume of extracellular fluid and anything it contains including water, salts, biochemicals and even soluble macromolecules. phagocytosis is greek for "cell eating" and involves the plasma membrane invaginating large insoluble solids. figure . gap junction [ ] . six connexins form a connexon and one connexon from each cell unite to form a gap junction. pinocytosis is a form of endocytosis involving fluids containing many solutes. in humans, this process occurs in cells lining the small intestine and is used primarily for absorption of fat droplets. in endocytosis the cell plasma membrane extends and folds around desired extracellular material, forming a pouch that pinches off creating an internalized vesicle ( fig. . , [ e ] ). the invaginated pinocytosis vesicles are much smaller than those generated by phagocytosis. the vesicles eventually fuse with the lysosome whereupon the vesicle contents are digested. pinocytosis involves a considerable investment of cellular energy in the form of atp and so is many times less efficient than rme (see chapter ) . also, in sharp contrast to rme, pinocytosis is nonspecific for the substances it accumulates. pinocytosis is not a recent discovery as it was first observed decades before the other transport systems discussed in chapter . its discovery is attributed to warren lewis in . phagocytosis is a type of endocytosis that involves uptake of large solid particles, often > . mm [ ] . the particles are aggregates of macromolecules, parts of other cells, and even whole microorganisms and, in contrast to pinocytosis (shown in fig. . ), phagocytosis has surface proteins that specifically recognize and bind to the solid particles. fig. . [ ] depicts events in phagacytosis. phagocytosis is a routine process that ameba and ciliated protozoa use to obtain food. in humans, phagocytosis is restricted to specialized cells called phagocytes that include white blood cell neutrophils and figure . pinocytosis, a type of endocytosis. an invagination of the plasma membrane encapsulates many water-soluble solutes ranging in size from salts to macromolecules. . membrane transport macrophages. as with pinocytosis, phagocytosis generates intracellular vesicles called phagosomes that have sequestered solid particles they transport to the lysosome for digestion. phagocytosis is a major mechanism used by the immune system to remove pathogens and cell debris. in fact, very early studies of the immune system led elie metchnikoff to discover phagocytosis in . for this work metchnikoff shared the nobel prize in medicine with paul ehrlich. exocytosis is the process by which cells excrete waste and other large molecules from the cytoplasm to the cell exterior [ ] and therefore is the opposite of endocytosis. exocytosis generates vesicles referred to as secretory or transport vesicles (chapter ). in exocytosis, intracellular (secretory) vesicles fuse with the plasma membrane and release their aqueous sequestered contents to the outside at the same time that the vesicular membrane hydrophobic components (mostly lipids and proteins) are added to the plasma membrane ( fig. . , [ ] ). steady state composition of the plasma membrane results from a balance between endocytosis and exocytosis. the resultant process of plasma membrane recycling is amazingly fast. for example, pancreatic secretory cells recycles an amount of membrane equal to the whole surface of the cell in w min. even faster are macrophages that can recycle contents of their plasma membrane in only min. before approaching the plasma membrane for fusion, exocytosis vesicles had a prior life that is considered in chapter . the vesicles must first dock with the plasma membrane, a process that keeps the two membranes separated by < e nm. during docking, complex molecular rearrangements occur to prepare the membranes for fusion. the process of vesicle fusion and release of aqueous compartment components is driven by snare proteins (see chapters and ) [ , ] . blebbing of the plasma membrane is a morphological feature of cells undergoing late stage apoptosis (programmed cell death, see chapter ) [ ] . a bleb is an irregular bulge in the plasma membrane of a cell caused by localized decoupling of the cytoskeleton from the plasma membrane. the bulge eventually blebs off from the parent plasma membrane taking part of the cytoplasm with it. it is clear in fig. . [ ] that the plasma membrane of an apoptotic cell is highly disintegrated and has lost the integrity required to maintain essential transmembrane gradients. blebbing is also involved in some normal cell processes, including cell locomotion and cell division. carefully controlled solute movement into and out of cells is an essential feature of life. there are many ways solutes are transported across the thin (w Å) membrane hydrophobic barrier. transport is divided into passive diffusion and active transport. a biological membrane is semipermeable, being permeable to some molecules, most notably water (osmosis), while being very impermeable to most solutes that require some form of transporter. passive diffusion (simple and facilitated) only requires the energy inherent in the solute's electrochemical gradient and results in its equilibrium across the membrane. in contrast, active transport requires additional energy (ie, atp), and results in a nonequilibrium, net accumulation of the solute. passive transport can involve simple diffusion or facilitated carriers including ionophores and channels. active transport comes in many, often complex forms. examples of active transport include primary active transport (uniport), secondary active transport (co-transport, antiport), and group translocation. besides the multitude of transport systems, transport can be accomplished by gap junctions, receptor mediated endocytosis, phagocytosis, pinocytosis, exocytosis, and apoptotic membrane blebbing. chapter will discuss bioactive lipids, highly specialized lipids that are functional at very low levels. discussed bioactive lipids include ceramides, diacylglycerol, eicosanoids, steroid hormones, and phosphatidic acid. principles of membrane transport effect of the gel to liquid crystalline phase transition on the osmotic behaviour of phosphatidylcholine liposomes fick's laws of diffusion water systems: aqua technology for the st century osmotic properties and water permeability of phospholipids liquid crystals osmotic behaviour and permeability properties of liposomes (review) membrane transport: a practical approach figure . . comparison of passive and active transport the glut glucose transporter transporte de glucosa: glut y sglt seminarios de biología celular y molecular e usmp filial norte potassium channels. methods and protocols the structure of the potassium channel: molecular basis of k þ conduction and selectivity resurgence of sodium channel research differences in saxitoxin and tetrodotoxin binding revealed by mutagenesis of the na þ channel outer vestibule aquaporins in kidney pathophysiology what are aquaporins for? the royal swedish academy of sciences. the nobel prize in chemistry: peter agre, roderick mackinnon. . nobelprize.org. the official web site of the nobel prize biological membranes and transport. solute transport across membranes. figure - atpase: structure, mechanism, and regulation renal na þ -glucose cotransporters chapter e signal transduction. a. energy transduction: uses of atp nutrient transport by ruminal bacteria: a review group-translocation: a consequence of enzyme-catalysed group-transfer coupling of metabolism and transport by enzymic translocation of substrates through membranes chemiosmotic hypothesis of oxidative phosphorylation proton current flow in mitochondrial systems enzyme i of the phosphoenolpyruvate: sugar phosphotransferase system. proteopedia, weizmann institute of science in israel biological applications of ionophores structural aspects of ionophore function principles of membrane transport. figure . mechanism of action of valinomycin on mitochondria induced active transport of ions in mitochondria the role of enteric antibiotics in livestock production. canberry (australia): avcare limited deciphering the biosynthetic codes for the potent anti-sars-cov cyclodepsipeptide valinomycin in streptomyces tsusimaensis atcc use of dinitrophenol in obesity and related conditions: a progress report synthesis and molecular recognition studies of crown ethers probing the structureÀfunction relationship of polyene macrolides: engineered biosynthesis of soluble nystatin analogues revealing the orientation of nystatin and amphotericin b in lipidic multilayers by uv-vis linear dichroism hexagonal array of subunits in intracellular junctions of the mouse heart and liver gap junctions: molecular basis of cell communication in health and disease. benos d, series editor. current topics in membranes and transport, series ed size limit of molecules permeating the junctional membrane channels cell-to-cell diffusion of fluorescent dyes in paired ventricular cells chapter: channels and transporters. molecular pathogenesis of cholestasis. madame curie bioscience database a quantitative analysis of connexin-specific permeability differences of gap junctions expressed in hela transfectants and xenopus oocytes gap junctions in cardiovascular disease mechanisms of phagocytosis in macrophages phagocytosis of microbes: complexity in action phagocytosis of bacteria and bacterial pathogenicity the molecular machinery of synaptic vesicle exocytosis cell and cell structure membrane fusion: grappling with snare and sm proteins synaptic vesicles really do kiss and run membrane blebbing during apoptosis results from caspase-mediated activation of rock human development, bio f key: cord- -og sg qw authors: howell, gareth j.; holloway, zoe g.; cobbold, christian; monaco, anthony p.; ponnambalam, sreenivasan title: cell biology of membrane trafficking in human disease date: - - journal: int rev cytol doi: . /s - ( ) - sha: doc_id: cord_uid: og sg qw understanding the molecular and cellular mechanisms underlying membrane traffic pathways is crucial to the treatment and cure of human disease. various human diseases caused by changes in cellular homeostasis arise through a single gene mutation(s) resulting in compromised membrane trafficking. many pathogenic agents such as viruses, bacteria, or parasites have evolved mechanisms to subvert the host cell response to infection, or have hijacked cellular mechanisms to proliferate and ensure pathogen survival. understanding the consequence of genetic mutations or pathogenic infection on membrane traffic has also enabled greater understanding of the interactions between organisms and the surrounding environment. this review focuses on human genetic defects and molecular mechanisms that underlie eukaryote exocytosis and endocytosis and current and future prospects for alleviation of a variety of human diseases. the human cell is a complex network of membranes and protein enclosed in a membrane lipid bilayer. the interactions within and associated with such biomembrane bilayers have profound consequences for the organism as a whole; a single defect in just of the potential - , gene products made by each cell can cause devastating, if not fatal, evects for the whole organism. in addition to this, humans pass genetic information onto their ovspring and, with it, any genetic mutations or polymorphisms. it is believed that at least in people have, or will eventually develop, a disease caused by mutation or variation at the gene level. understanding how genetic mutations increase risk for human disease is critical in our understanding and treatment of the majority of human ailments that are caused by interactions between the organism and the environment. this review focuses on the research undertaken in the past years relating to the molecular mechanisms that underlie membrane traycking within eukaryotic cells. we address mechanisms and factors that control protein progression through the secretory and internalization pathways and highlight key human diseases that illuminate mechanisms of membrane traycking. in addition, current and future strategies for therapeutic intervention in such genetic disorders are considered. common to all eukaryotic cells is the presence of multiple biomembrane lipid bilayer compartments, or organelles, which are maintained by specific protein-protein and protein-lipid interactions. such interactions are maintained within each compartment in spite of continuous traycking of membrane-bound and soluble components to diverent intracellular locations, and for secretion from the cell. in the majority of cases, this transfer of material occurs through vesicular movement: fission, docking, and fusion of membrane bilayer-enclosed intermediates occurs between donor and acceptor compartments (palade, ) . proteins, including membrane-bound receptors, secreted enzymes, and antibodies, begin their journey by entering the early secretory pathway at the endoplasmic reticulum (er). from here they are transported through the golgi apparatus and finally distributed to their final destination such as other intracellular organelles, the plasma membrane, or the extracellular environment. but how does a specific protein ''know'' how to reach a specific cellular destination when hundreds of newly synthesized, diverent molecules require specific transport and targeting? many of these transport intermediates or vesicles, whether derived from the er, other internal organelles, or the plasma membrane, are ''coated'' with unique protein complexes, tethering factors, and regulatory factors that ensure correct targeting to an acceptor compartment. vesicle coat proteins, such as the clathrin or coat protein (cop) complexes, are relatively well studied. such complexes are assembled onto the cytoplasmic face of donor compartments to facilitate the fission of transport intermediates. allied with these coat proteins are diverent molecules that mediate recognition of cytoplasmic motifs in cargo proteins either directly (e.g., transmembrane proteins) or indirectly (e.g., soluble secreted enzymes). the snare hypothesis is central to our understanding of vesicular targeting to intracellular compartments (rothman, ; sollner et al., ) . initially uncovered in a screen for intra-golgi transport docking and fusion regulators, the snare (soluble n-ethylmaleimide-sensitive fusion attachment protein receptor) proteins have been found to regulate diverent membrane interactions in all eukaryotes via a highly conserved mechanism for membrane traycking based on accessory docking and fusion regulators. snare proteins are present on both the vesicle (vesicular or v-snare) and the acceptor (target or t-snare) and comprise coiled-coil domains that assemble to facilitate vesicle docking and membrane fusion (bennett, ; pelham, ) . in conjunction with snare proteins, small ras-related rab gtpases are implicated in further ensuring the fidelity of vesicle docking and fusion (olkkonen and stenmark, ) . these -to -kda proteins are gtphydrolyzing enzymes that act to recruit diverent proteins or evectors to membranes in a gtp/gdp-regulated manner (collins, ) . rab gtpase activity and protein conformation are regulated by interaction with soluble and membrane-bound proteins; such regulators can also tether vesicles to acceptor membranes and mediate intracellular signaling. a. early secretory pathway the endoplasmic reticulum (er) is the first stage of quality control along the secretory pathway. proteins destined for secretion (e.g., hormones), the plasma membrane (e.g., membrane-bound receptors), or other intracellular membrane compartments such as the lysosome (e.g., lysosomal proteases) emphysema and liver cirrhosis (perlmutter, ) endophillin ii clathrin-coated pit formation leukemia (dreyling et al., ; jones et al., ; narita et al., ; tebar et al., ) alzheimer's disease presenilin presenilin -involved in cleavage and trafficking of amyloid precursor protein to plasma membrane neurodegenerative disorder (uemura et al., ) tau tau -microtubular stability through formation of aggregates autosomal dominant polycystic kidney disease (adpkd) polycystin- or causes a defect in e-cadherin assembly and basolateral trafficking renal cysts in kidney and other tissues leading to endstage renal failure (charron et al., ) (continued ) autosomal dominant retinitis pigmentosa rhodopsin inhibited interaction of rhodopsin and arf , leading to inhibited post-golgi delivery to rod outer segment narrowing of visual fields, night blindness (deretic et al., ) autosomal dominant ventricular tachycardia cardiac arrhythmia, hyperthermia (yano et al., ) autosomal recessive primary hyperoxaluria mistargeting of peroxisomal proteins to mitochondria kidney disease (danpure, ) ab-lipoproteinaemia mtp er retention thus preventing apob secretion vascular disease (sharp et al., ) batten's disease cln -cln group of gene products implicated in regulating the processing and targeting of lysosomal and synaptic proteins neurological disease (pearce, ) breast cancer caveolin- deletion or dominant negative mutation of caveolin- promotes tumor progression breast cancer (bouras et al., ; williams and lisanti, ) (hayasaka et al., ; matsuyama et al., ) chediak-higashi syndrome (chs) chs /lyst lyst involved in regulation of protein secretion from lysosomes -enlarged lysosomes partial albinism, recurrent bacterial infections, impaired chemotaxis and abnormal natural killer cell function (shiflett et al., ; ward et al., ) choroideremia (chm) rab escort protein (rep ) rab a remains cytosolic due to defective geranylgeranyl modification in chm lymphoblasts x-linked form of retinal degeneration combined factors v and viii deficiency ergic- /p c-type lectin er retention and defective secretion of factors v and viii blood disease congenital finnish nephritic syndrome nephrin (nphs ), podocin (nphs ) er retention kidney inflammation (kestila et al., ; kramer -zucker et al., ) pancreatic atp-sensitive potassium channel (k-atp) er or golgi retention of k-atp due to mutations in its sulfonylurea- (sur ) subunit excess insulin leading to hypoglycaemia (dunne et al., ; yan et al., ) congenital hypothyroid goiter thyroglobulin er storage disease. thyroglobulin is misfolded and accumulates in er constipation, large tongue, swelling around the eyes, failure to suckle, mental retardation (hishinuma et al., ; kim and arvan, ) (fan et al., ; garman and garboczi, ) familial hemophagocytic lymphoschistiocytosis (fhl) perforin -defective ctl (cytotoxic t lymphocytes) mediated killing immunodeficiency (feldmann et al., ; stepp et al., ) munc (hogg et al ., ; mathew et al., ) three forms of menkes disease can arise from diverent mutations in the atp a gene: premature stop codons, deletions, or splicing defects. these can prevent atp a function and/or traycking. classical menkes disease is the most common and fatality usually results by the age of years. in two other nonfatal forms of menkes, mild and occipital horn syndrome, atp a maintains the ability to transport copper ions across intracellular membranes, although traycking to the plasma membrane can be compromised (la fontaine et al., ) . atp a is ubiquitously expressed and is the major copper transporter in cells of the intestine, kidney, and brain. in the liver, however, the major copper transporter is the wilson's disease protein, atp b (bull et al., ) . this second p-type atpase shares strong similarity with atp a and also translocates copper ions across membranes. although these gene products share functional similarities, mutations in atp b result in copper accumulation in the liver and brain. familial hypercholesterolemia is an autosomal dominantly inherited disease caused by mutations in the low-density lipoprotein receptor (ldlr), leading to premature atherosclerosis and coronary heart disease. in healthy individuals, the ldlr is expressed on the surface of cells, where it binds circulating ldl particles and promotes uptake and cellular metabolism of its constituents, which includes cholesterol. in these patients, ldlr alleles display amino acid substitutions (cassanelli et al., ; jensen et al., ) , truncations (lehrm an et al ., ) , or mis sense mutat ions ( leiter sdorf et al ., ) , which can result in er retention and degradation. the point mutation at residue of the insulin receptor compromises the ability of the receptor to dimerize correctly within the er, therefore leading to er retention. decreased plasma membrane levels of insulin receptor cause inhibited insulin binding after stimulus by a meal, and subsequent elevations in plasma glucose levels. this then leads to type ii diabetes mellitus (kadowaki et al., ) . a number of human diseases can induce the er stress response. here, the mutant protein is retained within the er, resulting in either dilation of the organelle, such as in congenital hyperthyroidism (medeiros-neto et al., ) and hypofibrinogenemia (callea et al., ) , or chronic er stress as is the case for hereditary emphysema (perlmutter, ) . in pelizaeus-merzbacher disease, an x-linked leukodystrophy disease, er accumulation of proteolipid protein (plp) results in oligodendrocyte apoptosis (gow et al., ) and the subsequent disruption of white matter formation in the brain observed in humans and mouse models. plp is a central nervous system protein that is the major component of myelin and, when expressed in cultured fibroblasts, is localized to the plasma membrane (gow et al., ) . the link between plp and the er stress response provides a tool for elucidating the cellular response to misfolded protein accumulation . accumulation of proteins within the er, leading to blockage of protein secretion, is an unwanted cellular property and mechanisms have evolved to overcome such events. this disposal of unwanted proteins is termed er-associated degradation (erad) (fig. ) . as recently as the early s, it was still believed that aberrant proteins were degraded within the er (fra and sitia, ) ; however, current models suggest that aberrant er-retained proteins actually undergo retrotranslocation and subsequent degradation in the cytoplasm. retrotranslocation has been proposed to occur through the same ''pore'' used to translocate nascent proteins into the er lumen during translation, namely the sec translocon (biederer et al., ; rö misch, ) . various yeast and mammalian proteins have been shown to be retrotranslocated from the er and degraded within the cytoplasm in a proteasomedependent manner, including the budding yeast proteins carboxypeptidase y, and a mutant pro-a-factor. when a mammalian protein such as cftr is expressed in budding yeast, it matures relatively slowly within the yeast er, leading to retrotranslocation to the cytoplasm and degradation (ward et al., ) . a further example is that of a -antitrypsin deficiency. a -antitrypsin is responsible for inactivating the enzyme elastase produced by lung neutrophils. in this inherited disease, a mutated form of a -antitrypsin is retrotranslocated and degraded in proteasomes, leading to retention of active elastase in lung tissues and thus is a cause of lung emphysema (rutishauser and spiess, ) . however, retrotranslocation and proteasomal degradation may not be functionally coupled processes. pharmacological inhibitors that cause proteasome inactivation lead to egress of molecules such as mhc class i (wiertz et al., a,b) , ribophorin (de virgilio et al., ) , and carboxypeptidase y (biederer et al., ) from the er to the cell cytoplasm. in contrast, inhibition of protein ubiquitination results in the retention of such molecules within the er. schmitz et al. ( ) suggest that two distinct proteasome-regulated pathways mediate degradation of retrotranslocated b-amyloid precursor protein. interestingly, endocytosed toxins that target key cytosolic factors appear to use the erad pathway to move out of the er and into the cytosol (deeks et al., ; hazes and read, ) . cholera and ricin toxins are routed from the cell surface through the golgi apparatus and to the er before being retrotranslocated into the cell cytosol. it is believed that the unusually low lysine content of these protein toxins prevents subsequent er-associated ubiquitination for degradation by the cytosolic proteasome. protein cargo is shuttled between the er and golgi within vesicular intermediates or -nm-diameter spherical vesicles containing coat protein complexes fig. quality control of protein assembly within the endoplasmic reticulum. proteins destined for the secretory pathway (this example shows a transmembrane protein) are cotranslationally translocated from the ribosome into the lumen of the endoplasmic reticulum (er) through a portal referred to as the sec translocon. as the newly synthesized protein enters the er, quality control mechanisms in the form of protein chaperones bind to it and fold it to its correct conformation. further processing occurs through interactions with other chaperones before the successfully folded protein is loaded into copii-coated vesicles and shuttled from the er to the golgi apparatus. however, if the protein carries a mutation that causes it to take on an aberrant conformation the er chaperones will trigger a misfolded protein response. this has two outcomes: either the chaperones will remain bound to the misfolded protein, preventing its escape from the organelle (er retention), or the protein will be ubiquitinated and retrotranslocated through the sec complex for proteasomal degradation in the cell cytoplasm. a number of human genetic diseases are a result of key proteins failing to trayc through the secretory pathway and as a consequence are retained or degraded in this manner. such as copi or copii. initially discovered in mammals and yeast (kaiser and schekman, ; malhotra et al., ; novick et al., ; rothman and wieland, ) , cop complexes are required for the formation of vesicles at the er, er-golgi intermediate compartment (ergic), and golgi apparatus. cop recruitment to membranes facilitates the specific capture, packaging, transport, and delivery of membrane-bound and soluble protein cargo to an acceptor compartment. copii recruitment to sites on the smooth er initiates the formation of anterograde (forward) transport vesicles. these copii vesicles move from the er to the ergic, or vesicular tubular clusters (vtcs). from here, copi-coated vesicles are thought to mediate the continued anterograde movement from the ergic to the cis face of the golgi apparatus (scales et al., ) . the sar p gtpase regulates copii vesicle formation via interaction with the sec p guanine exchange factor (gef). sec p-mediated activation of sar p to a gtp-bound form leads to recruitment of the sec p-sec p heterodimer to membranes; this also initiates protein cargo selection within the er and recruitment of v-snares such as bet p and bos p. binding of sec p-sec p mediates further recruitment of the sec p-sec p complex. this copii complex then acts as a protein scavold that causes deformation of the membrane, resulting in vesicular fission, with anterograde movement of protein cargo-containing copii vesicles to the ergic. copii docking at an acceptor compartment is thought to trigger sec p function, causing a conformational change in sar p and gtp hydrolysis and dissociation or uncoating of the copii complex. thus copii vesicle docking and fusion with an acceptor compartment are mediated by cognate v-snare/ t-snare interactions (kirchhausen, ; kuehn et al., ; matsuoka et al., ; tang et al., ) . a severe hereditary bleeding disorder called combined deficiency of factor v factor viii (f f d) highlights the functional importance of traycking between the er and ergic. some f f d patients are deficient in the ergic-localized ergic- (lman ) protein and display defective secretion of the factor v and viii clotting factors. ergic- is a mannosebinding lectin that acts as a ''cargo receptor'' and recycles between the er and ergic (neerman-arbez et al., ; . however, % of f f d patients show normal levels of ergic- /lman , but are deficient in an associated protein, mcfd , another ergic resident that interacts with ergic- /lman in a calcium-dependent manner . small intestinal cells called enterocytes absorb fats and fat-soluble vitamins from food in the form of fatty acids and monoglycerides. the fats enter the lumenal surface of absorptive enterocytes by free divusion across their membranes, and emerge from the basolateral surface as particulate structures referred to as chylomicrons. formation of chylomicrons occurs within the er and golgi apparatus by vesicular transport before being traycked from the golgi to the plasma membrane. chylomicron retention disease (cmrd), anderson disease, and a neuromuscular disorder, cmrd associated with marinesco-sjö gren syndrome (cmrd-mss) , are examples of inherited diseases that result in compromised fat absorption, low blood cholesterol, and severely depleted blood chylomicron levels. jones et al. ( ) identified eight mutations in the sar p gene product and copii component associated with these lipid absorption diseases, thus strongly implicating a role for the copii vesicular transport system in the movement of dietary fats from the intestine to the circulating bloodstream. copii mediates anterograde trayc from the er to the golgi apparatus; however, copi vesicles appear to function primarily in the retrograde (backward) transfer of proteins from the golgi and ergic back to the er. this retrograde trayc is necessary for recovering escaped er resident proteins, coat and snare proteins that have arrived at the ergic and golgi from copii vesicles, or glycosylation enzymes that have been incorrectly modified (duden, ; lee et al., ) . the golgi-associated copi coatomer is a complex of seven polypeptides: a-, b-, b -, g-, d-, e-, and z-cop gene products, which interact with the donor membrane to form copi vesicles. vesicle formation is triggered by the gtpase adp-ribosylation factor (arf ), which recruits copi coatomer to the donor membrane. transmembrane proteins containing cytoplasmic lysine-based motifs such as kkxx or kxkxx, or soluble proteins containing the c-terminal kdel motif, are recycled by copi-coated vesicles from the golgi apparatus back to the er. the kdel motif, present in soluble er chaperones such as bip and protein disulfide isomerase, is recognized by the membrane-bound kdel receptor (majoul et al., ) . in both cases, cytoplasmic motifs in these transmembrane proteins are recognized and bound by copi coatomer, promoting inclusion into vesicles destined for the er. actin microfilaments are also involved in this retrograde transport step (valderrama et al., ) . this golgi-er step is regulated by the gtpase cdc and n-wasp protein (luna et al., ) , factors previously implicated in actin-linked processes at the plasma membrane. live imaging of cells expressing an engineered fluorescent and temperaturesensitive vesicular stomatitis virus g-glycoprotein (ts vsvg) demonstrated sequential action of copii-and copi-coated vesicles (scales et al., ) . vsvg accumulated in structures close to the er that contained intermediate compartment resident proteins. these structures then matured into vesicles that contained copi proteins. stephens et al. ( ) showed that this ''segregation'' between copii and copi vesicles occurred at a location in close proximity to exit sites on er membranes. a cop-independent mechanism has also been implicated in retrograde trayc between the golgi apparatus and the er. the rab gtpase is implicated in regulating the movement of bacterial shiga toxin b fragment (stb) via a retrograde step from the golgi apparatus to the er. expression of a dominant-negative gdp-bound form of rab inhibited stb retrograde movement, whereas copi transport was unavected (white et al., ) . the golgi apparatus is composed of flattened cisternae and membrane compartments that are closely juxtaposed in a stack-like appearance. in mammalian cells these stacks are positioned end-to-end, forming a ribbonlike structure near the nucleus (barr and warren, ) . the golgi apparatus is a highly dynamic organelle sited at the hub of the secretory pathway with key processing and sorting functions. the golgi is a polarized structure with proteins and lipids from the er received at the cis side, followed by the medial and trans subcompartments, where further glycosylation modifications occur; the trans-golgi network (tgn) is the final subcompartment where sorting and packaging events take place. the golgi apparatus also sorts proteins and lipids bound on a retrograde pathway from the cis-golgi back to the er. in addition, proteins can also return to the tgn from the endomembrane/lysosomal system (fig. ) . controversy exists regarding the mechanism for anterograde movement of cargo proteins within the golgi apparatus. the golgi apparatus contains secretory proteins that can vary in physical size, from relatively small polypeptides to large, bulky multisubunit complexes; all need to reach the tgn for final sorting into transport intermediates. there are also resident glycosylation enzymes that have spatially restricted functions within the golgi, that is, enzymes that function within specific subcompartments to ensure the correct addition or trimming of n-and o-linked sugars on secreted proteins as they progress through the pathway. this raises a key question: how do protein and lipid cargo move through the golgi apparatus while resident enzymes retain their localization? we know that many golgi enzymes contain transmembrane golgi localization signals that mediate targeting to a specific compartment (munro, ) . two models have been proposed: the cisternal maturation model and the vesicular transport model (elsner et al., ; storrie et al., ) . briefly, the cisternal maturation model suggests that large proteins or aggregates remain within a single golgi cisterna, which matures through the retrograde transfer of resident enzymes via copi vesicles. in contrast, the vesicular transport model proposes that newly synthesized protein is traycked from cisterna to cisterna via copi-coated vesicles that sequentially bud ov membranes and fuse with the next subcompartment. in either case, copi-coated vesicles play a central role in intra-golgi the secretory pathway and vesicular traycking. protein enters the secretory pathway at the endoplasmic reticulum (er) and is traycked in copii-coated vesicular structures to the intermediate compartment (ergic/vtc), from which copi-coated vesicles carry it to the cis face of the golgi. cargo protein (c) continues along the secretory pathway through the golgi apparatus to the trans-golgi network (tgn). retention signals in er resident proteins (r) ensure they undergo retrograde traycking from the golgi in copi vesicles. retrograde transport of transport. a number of snare proteins, such as membrin, rbet , gs , and syntaxin- , have also been localized to the golgi apparatus and are required for intra-golgi transport and homeostasis (nichols and pelham, ) . golgi-tethering molecules called golgins and golgi reassembly stacking proteins (grasps) belong to a family of regulatory factors involved in golgi maintenance and vesicular transport. the reader is pointed to an indepth review that covers golgins in more detail (short et al., ) . in brief, the golgins can be anchored to golgi membranes through various mechanisms and contain characteristic coiled-coil domains that extend from the membranes as a rod-like structure (burkhard et al., ) . golgins such as giantin and golgin- are securely anchored to the membrane via a transmembrane domain near their c terminus. electrostatic or ionic interactions mediate the attachment of other golgins to membranes. for example, proteins of the grasp family (grasp and grasp ) bind to gm and golgin- to recruit these factors to the cis and medial golgi membranes, respectively. moreover, a large number of golgins are recruited to membranes via interactions with the rab, arf, and arl (arf-like) gtpases. vesicular and cis-golgi membrane recruitment of golgin p is regulated by rab , whereas membrane attachment of yeast golgin rud p is regulated by arf p. golgin- binds to membranes by interaction with arl p, a member of a new class of arf-like gtpases termed arls (short et al., ) . interestingly, autoantibodies directed against giantin, golgin- , golgin- , gm , and golgin- golgins and grasps are present in patients with autoimmune conditions such as sjö gren's syndrome and systemic lupus erythematosus. in sjö gren's syndrome, moisture-producing glands are targeted by the autoimmune response, resulting in dry eyes and mouth (lichtenfeld et al., ) . systemic lupus erythematosus is a chronic rheumatic condition that avects joints and muscles, causing skin rash and kidney problems. sjö gren's syndrome patients can also simultaneously display both rheumatoid arthritis and systemic lupus erythematosus. golgi biogenesis requires golgin function at diverent stages during cell division. mammalian p is crucial for maintenance of the stacked nature of the golgi cisternae (puthenveedu and linstedt, ) . during mitosis, the golgi stack disperses into clustered vesicles. these vesicles then fuse in the daughter cells to form new cisternae, alignment and stacking of which result in the formation of a fully functional organelle. grasp tethers have been proposed to hold cisternae in close proximity through interactions with p and gm (shorter and warren, ) . the golgin p is also involved in tethering copi vesicles to golgi membranes (sonnichsen et al., ) and may be needed for snare complex assembly (shorter et al., ) . the budding yeast p homolog (uso p) tethers copii-coated vesicles to golgi membranes during anterograde transport from er exit sites to the cis-golgi (barlowe, ; cao et al., ; sapperstein et al., ) . mammalian p is also essential for the tethering of transport vesicles to the cis-golgi (alvarez et al., ) and during intra-golgi transport (seemann et al., ; waters et al., ) . golgins such as golgin- are implicated in the regulation of golgi structure and the formation of the golgi ribbon (diao et al., ) . golgin- may function as a tethering molecule in retrograde trayc from the endosome to the tgn (lu et al., ) . moreover golgins are also implicated as tethering components between the cytoskeleton and the golgi apparatus (short et al., ) . the trans-golgi network (tgn) is the final golgi subcompartment where secreted proteins are sorted, packaged, and directed to their final destination. traycking from the tgn can occur in either a constitutive or regulated manner. constitutive transport is the continuous release of protein from the trans-golgi network. regulated secretion occurs in response to extracellular stimuli such as secretagogues, metal ions, hormones, or growth factors, which trigger the docking and fusion of secretory granules or vesicles with the plasma membrane. various mechanisms control the traycking of proteins from the tgn by the formation and delivery of membrane-derived transport vesicles to the plasma membrane, endosomes, or lysosomal structures (ponnambalam and baldwin, ) . the expression of inactive (dominant-negative) protein kinase d isoforms in tumor lines (liljedahl et al., ) , polarized canine kidney cells (yeaman et al., ) , and mouse fibroblasts (prigozhina and waterman-storer, ) has been shown to inhibit vesicle fission (release) from the tgn. vesicle release is modulated by this family of kinases in response to cellular diacylglycerol (baron and malhotra, ) and binding to an as yet unknown evector protein on the cytoplasmic face of the tgn (van lint et al., ) . in addition, the cdc gtpase is linked to actin remodeling and has been shown to inhibit the exit of basolateral targeted proteins in polarized cells (kroschewski et al., ; musch et al., ) and copper-regulated protein transport (cobbold et al., ) . copper is an essential element and cofactor required for functionality of many secreted enzymes (cuproenzymes). at steady state, atp a (menkes howell et al. disease copper transporter; section iii.a. ) resides in the tgn, where it provides newly synthesized cuproenzymes such as lysyl oxidase with copper ions as they traverse the secretory pathway. when intracellular copper ion levels rise, atp a responds to this environmental danger by redistributing to the plasma membrane in a cdc -regulated manner (cobbold et al., ) . here, atp a acts as a copper ezux pump to remove copper ions from the cytoplasm to maintain homeostatic function and prevent toxicity. when copper levels are reduced, atp a recycles back to the tgn. this endocytic internalization and sorting event is independent of both clathrin and caveolae (cobbold et al., ) , although relying on a cytoplasmic dileucine motif present in the atp a c-terminus petris and mercer, ) . dent's disease, an x-linked kidney disorder that presents with hypercalciuria, nephrocalcinosis (kidney stone formation), and progressive renal failure, is caused by missense, nonsense, and deletion mutations within the endosomal clc- voltage-gated chloride channel. clc- is a member of a large family of voltage-gated chloride channels that have a diverse array of cellular functions including membrane excitability, transepithelial ion transport, and cell volume regulation (thakker, ) . when expressed in xenopus oocytes, a number of missense mutations in the clc- gene localized the channel to the golgi apparatus and showed reduced conductance and significantly reduced plasma membrane (pm) localization (ludwig et al., ) . similarly, expression of mutant clc- alleles in cultured cells revealed an approximate -fold increase in golgi retention (carr et al., ) . a. receptor-mediated endocytosis clathrin-coated vesicles (ccvs) are a route for protein internalization conserved from yeast to humans. roth and porter ( ) first observed this process in mosquito oocytes and these vesicles have subsequently become one of the best characterized membrane transport steps in eukaryotes. clathrin is one of the principal proteins involved in this transport step and, in combination with more than clathrin-associated factors, this unique structural component forms transport vesicles on the cytoplasmic face of the tgn, endosomes, and the plasma membrane. clathrin-coated vesicles bud from their donor membranes and are directed to target membranes by associated proteins and factors. this highly conserved -kda clathrin complex comprises heavy ( kda) and light ( kda) chain proteins that are assembled into a three-legged structure called a triskelion. triskelions can be polymerized by accessory factors into striking lattice-like ''cages'' comprising pentagons and hexagons, resembling a soccer ball structure or buckminsterfullerene. clathrin cages are - nm in diameter; significantly larger than copi or copii vesicles (ccvs). clathrin-coated vesicles are believed to assemble through a sequence of events that can be designated as activation, cargo capture, coat assembly, scission, movement, and vesicle uncoating (kirchhausen, ) . members of a class of clathrin-associated factor termed adaptor protein (ap) complexes are recruited to donor membranes through interactions with a docking complex, which then further interacts with motifs within the cytoplasmic tail of cargo proteins, resulting in ''cargo capture.'' this leads to clathrin cage assembly and the concomitant polymerization of the clathrin triskelion and resultant deformation of the donor membrane. scission, or vesicle release from the plasma membrane, is believed to occur through the action of the gtpase dynamin and other accessory proteins, such as amphiphysin (wigge et al., ) . in the fruit fly drosophila melanogaster, a dynamin gene mutation (shibire) causes temperature-sensitive paralysis. this is likely due to a block in the endocytic uptake of synaptic vesicle proteins at the plasma membrane, leading to a block in recycling and reformation of competent synaptic vesicles at nerve terminals (chen et al., ; koenig and ikeda, ; kosaka and ikeda, ; van der bliek and meyerowitz, ) . the expression of a dominant-negative gdp-bound dynamin mutant, k a, results in compromised ccv formation (herskovits et al., ; van der bliek et al., ) and inhibition of clathrin-mediated internalization of the glucose transporter glut (al-hasani et al., ) , human immunodeficiency virus (hiv) (daecke et al., ) , and influenza virus (roy et al., ) . the scission function of dynamin is assisted by specific lipid-modifying enzymes such as endophilin, synaptojanin, and phospholipase d (bi et al., ; havner et al., ; ringstad et al., ; schmidt et al., ; woscholski et al., ) . finally, ccv uncoating at the target membrane occurs through the actions of the heat shock protein hsc (schlossman et al., ) and auxilin (ungewickell et al., ) . sorting of proteins from donor to target membranes involves the recognition of cytoplasmic sequences in membrane proteins by clathrin-associated ap complexes. four adaptor protein complexes (ap -ap ), each comprising four diverent subunits, have been identified (robinson, ) . the ap complex is involved in clathrin-coated vesicle formation at the tgn for transport to late endosomes; evidence has also implicated a role for this complex in a tgn-to-plasma membrane step (folsch et al., ) . ap is the best-studied of the four complexes and mediates internalization of transmembrane howell et al. receptors at the plasma membrane via clathrin-coated vesicles. the ap complex is involved in traycking from early endosomes to either late endosomes or lysosome-related organelles such as melanosomes, platelet-dense bodies, and antigen-processing compartments. finally, the ap complex was the last to be cloned (dell'angelica et al., a; hirst et al., ; ) . in contrast to ap -ap , ap does not possess the b ''ear'' domain (see below), which allows interaction with clathrin and other cytosolic factors such as eps and auxilin (lundmark and carlsson, ) . by electron microscopy, ap has been localized to vesicles at the tgn, plasma membrane, and early endosomes, although there is debate as to whether these vesicles are clathrin-coated (barois and bakke, ; hirst et al., ) . interestingly, ap and ap may function independently of clathrin (hirst et al., ; vowels and payne, ) , suggesting the existence of another, as yet unidentified, coat protein that is analogous to clathrin. all four ap complexes comprise two large -kda subunits: a b subunit (b -b ) plus a g (ap ), a (ap ), d (ap ), or e (ap ) subunit. in addition, each ap complex contains a -kda subunit (m -m ) and a small -kda subunit (s -s ). ap , - , and - contain two carboxyl ''ear'' domains connected to the head of each large -kda subunit by a flexible hinge of approximately - residues. importantly, the ear domain of the b subunit and the hinge domains of the g and a subunits have been shown to bind clathrin (goodman and keen, ; morgan et al., ; owen et al., ) , and consensus sequences in the hinge domains of b and b have clathrin-binding properties (dell'angelica et al., ) . the b and m subunits of the ap complex interact with motifs present in the cytoplasmic domains of transmembrane proteins to mediate cargo recruitment into clathrin-coated vesicles. such motifs include npxy, yxxØ, and dileucine-based sequences (Ø represents a bulky hydrophobic amino acid). one such motif, npxy, is present in key cellular receptors such as low density lipoprotein receptor (ldlr), epidermal growth factor receptor (egfr or erb ), and insulin receptor, and mediates endocytosis and sorting. importantly, the jd mutation (y c) in ldlr lies within this key motif and causes familial hypercholesterolemia (knoblauch et al., ) . the second tyrosinebased motif, yxxØ, mediates plasma membrane internalization, lysosomal targeting, and basolateral targeting of cargo. this motif is found in lysosomal residents such as lamp- and - , cd , the recycling transferrin receptor (tfr), and tgn-associated recycling membrane proteins, furin and tgn . di-leucine motifs present on transmembrane transporters such as glut (glucose transporter), atp a, and mannose- -phosphate receptors (m pr) can fall into two categories: [de]xxx[li] and dxxll related motifs. the [de]xxx[li] motif is associated with proteins internalized from the plasma membrane and targeted to lysosomes, while dxxll motif is found in transmembrane proteins that shuttle between the tgn and endosomal system (bonifacino and traub, ) . another class of clathrin-associated factor is the golgi-localized, g-earcontaining, arf-binding proteins (ggas) found on the tgn and postulated to interact with ap to mediate transport of m pr (section v.b) to endosomes (doray et al., ) . ggas can act as multifunctional adaptors that link transmembrane proteins, arf gtpases, clathrin and accessory proteins at sites of ccv formation (robinson and bonifacino, ) . the disease oculocerebrorenal syndrome of lowe (ocrl) is an x-linked disorder caused by mutations in the ocrl gene (lowe, ) . the gene product is an inositol -phosphatase that catalyzes the removal of the phosphate from this position on the inositol moiety. the preferred ocrl substrate is pi( , )p , a phosphoinositide shown to be important in endocytosis because of its central role in recruiting accessory proteins to ccvs (padron et al., ) . ocrl has been localized to clathrin-coated vesicles associated with endosomal and tgn membranes (choudhury et al., ) . this is not surprising as ocrl interacts with clathrin and promotes its assembly into clathrin lattices and cages (choudhury et al., ; ungewickell et al., ) . ocrl also interacts with the rac gtpase that regulates actin dynamics, possibly via a gtpase activation domain to accelerate gtp hydrolysis (faucherre et al., ) . although the exact function of ocrl is still unclear, the disease phenotype hints to ocrl function in membrane traycking. ocrl mutations can cause loss of protein expression and phosphatase activity. rnai-mediated inhibition of ocrl expression in cultured human cells results in partial redistribution of a cation-independent mannose- -phosphate receptor and a tgn recycling protein (tgn ) to early endosomes (choudhury et al., ) . this suggests that loss of ocrl perturbs endosome-to-tgn vesicle transport, suggesting a functional requirement for this membrane trayc step. it is possible that ocrl plays a role in anterograde traycking from the tgn-to-endosomes as well, since ocrl is abundantly present on tgn-associated clathrin buds destined for the endocytic pathway. ocrl disease symptoms include congenital cataracts, mental retardation, and renal tubular dysfunction (lowe et al., ) . renal failure in ocrl patients is probably partly caused by defects in solute and protein readsorption in kidney proximal tubules. this is likely due to missorting of megalin and cubilin, cell surface receptors involved in kidney solute uptake. in ocrl patients plasma membrane shedding of these receptors is reduced (norden et al., ) , indicating ocrl regulation of either receptor traycking from the tgn-to-plasma membrane or recycling from plasma membrane-to-tgn. paraneoplastic stiv-person syndrome (sps) is a neurological autoimmune disease characterized by severe muscle stivness and spasms, and often has secondary symptoms including diabetes, epilepsy, and breast cancer. autoantibodies are produced against the clathrin-associated regulator, amphiphysin i (de camilli et al., ) , a protein shown to bind dynamin in nerve terminals (david et al., ) and which is implicated in regulating the endocytosis of neuronal synaptic vesicle components (burns, ) . in support of this hypothesis, sommer et al. ( ) showed that sps-like symptoms could be triggered in rats injected with anti-amphiphysin antibodies from a human sps patient. genetic translocations leading to the formation of hybrid clathrin-accessory proteins can lead to other forms of acute myeloid leukemia, lymphoblastic leukemia and acute megakaryoblastic leukemia (dreyling et al., ; jones et al., ; narita et al., ; tebar et al., ) . in these diseases, an aberrant hybrid protein consisting of the putative transcription factor af and the clathrin accessory protein calm (clathrin assembly lymphoid myeloid leukemia protein) is formed because of a partial inversion of the af gene on chromosome (salmon-nguyen et al., ) . finally, in hermansky-pudlak syndrome (hps) type , a condition that results in partial albinism and prolonged bleeding, mutations have been found in the b a gene that encodes a subunit of the ap adaptor complex (dell'angelica et al., b) . hps is discussed in more detail in section v.c. originally identified more than years ago (palade, ; yamada, ) , caveolae are flask-shaped invaginations of approximately - nm in diameter at the plasma membrane. these plasma membrane profiles are related to lipid rafts and contain unique mixtures of gpi-anchored proteins, transmembrane proteins, signaling factors and lipids, such as cholesterol. caveolae are believed to mediate the uptake of small solutes, regulate protein traycking (hommelgaard et al., ; tagawa et al., ) , transcytosis (transport across endothelial cells) (simionescu et al., ) , signal transduction (insel et al., ; lisanti et al., ; ostrom and insel, ) and cholesterol homeostasis (fielding and fielding, ) . however, their exact role in the internalization of membrane proteins and soluble protein ligands is controversial. caveolin- , also known as vip , is a structural component essential for the formation and stability of caveolae (kurzchalia et al., ; rothberg et al., ) . of the three members of the caveolin gene family (caveolin- , - , and - ) tang et al., ) , caveolin- and - are abundant in a wide variety of cell types including endothelial cells, adipocytes, alveolar type i pneumocytes, and smooth muscle cells (williams and lisanti, ) , whereas caveolin- is a muscle-specific isoform expressed in striated muscle cells such as cardiac and skeletal myocytes (cohen et al., ; tang et al., ) . caveolin- and - are both able to induce formation of caveolae at the plasma membrane (galbiati et al., ; li et al., ) . however, caveolin- requires the presence of caveolin- for expression, membrane localization, and formation of caveolae (razani et al., ) . caveolae are absent from cells that lack caveolin- but can be induced by ectopic expression of the gene (fra et al., ) . caveolins adopt a hairpinlike structure that inserts into the membrane such that the n and c termini are cytoplasmic. caveolins can polymerize to form a striated coat surrounding an invagination site . caveolin- can bind cholesterol (murata et al., ) , which is enriched within both caveolae and lipid rafts (sargiacomo et al., ) ; this may explain why caveolae have been considered a subset of lipid rafts. however, caveolae and lipid rafts are considered to be independent entities as some proteins can be found in one but not the other (liu et al., ) . certain ligands can internalize via a lipid raftdependent but clathrin-independent mechanism in cells that lack caveolae (lamaze et al., ) . a large pool of the plasma membrane caveolar vesicles cluster into dense grape-like structures where individual caveolae appear stacked on top of each another (thomsen et al., ) . these structures are intimately associated with the actin cytoskeleton (stahlhut and van deurs, ) ; caveolaassociated proteins are also implicated in regulating plasma membrane dynamics and cellular movement. a small pool of ''transport-competent'' caveolar vesicles may undergo short-range constitutive fusion and budding cycles just under the plasma membrane . caveolae and caveolins can also be detected at the tgn (dupree et al., ; kurzchalia et al., ) and may form stable ''platforms'' for the movement of proteins and lipids from the tgn to the plasma membrane (tagawa et al., ) . the caveolar pathway can be hijacked and used by pathogens or toxins to gain entry into the cell. viruses such as polyomavirus, echovirus , and simian virus (sv ) use caveolae to internalize viral particles. these viruses cluster lipid rafts and sequester them into caveolae through interactions with raft components such as integrins and glycosphingolipids ; in the case of sv , the virus binds to the raft component ganglioside gm (tsai et al., ) . tagawa et al. ( ) have shown that sv can trigger the long-range movement of transport-competent caveolar vesicles. moreover, cell infection with sv more than doubles the number of caveolae capable of undergoing viral internalization and long-range traycking. caveolae contain much of the molecular machinery required for ''classical'' vesicle fission, docking, and fusion, for example, snare proteins, monomeric and trimeric gtpases, annexins ii and vi, n-ethylmaleimide (nem)sensitive fusion protein (nsf), and atpases (schnitzer et al., ) . caveolae also contain the dynamin gtpases, which can be transiently recruited to sv -loaded caveolae and implicated in membrane scission (henley et al., ; oh et al., ; pelkmans and helenius, ) . internalized caveoladerived vesicles move to an endocytic compartment termed the ''caveosome'' and eventually arrive at the early endosome. after fusion with the target compartment, caveolae do not disassemble but maintain their integrity in the membrane, preserving their compartmentalization and retaining their lipid and protein components (pelkmans et al., ) . the fate of internalized sv viruses after reaching the caveosome eventually results in arrival at the smooth er (pelkmans et al., ) . interestingly, mutations in caveolin have been implicated in muscular dystrophy and cardiovascular disease, and mutations causing the downregulation of caveolin have been linked to the progression of various human carcinomas; it is therefore possible that caveolins may have a tumor suppressor role. the caveolin- and caveolin- genes are located on human q . near the microsatellite repeat marker d s . this region is commonly deleted in various cancers (engelman et al., ) , hinting that caveolin gene deletion may be advantageous for tumor progression. in one report, the caveolin- p l mutation was present in % of breast cancer patients studied (hayashi et al., ) . the p l mutation was also linked to the metastatic potential of tumors and disease prognosis. the caveolin- p l mutation also conferred increased cell migration and altered morphology. caveolin- protein levels can be reduced or absent from a number of human breast cancer cell lines compared with normal mammary cells (lee et al., ) . similarly, silent and missense mutations in caveolin- have also been associated with oral carcinomas (han et al., ) . caveolin- , and to a lesser extent caveolin- , gene expression is downregulated in some cases of thyroid carcinoma (aldred et al., ) . although it remains unclear as to why the loss of caveolin causes cell proliferation diseases such as cancer, one can speculate on the role of caveolin in regulating signaling pathways. in endothelial cells, which have a high abundance of caveolin, the key vascular endothelial growth factor receptor (vegfr ) has been shown to be inactive when localized to caveolae (labrecque et al., ) . this receptor tyrosine kinase modulates the endothelial response to the key vegf-a cytokine and controls angiogenesis and new blood vessel formation, thus regulating neovascularization and tumor growth (neufeld et al., ) . similarly, platelet-derived growth factor (pdgf) receptor tyrosine kinase activity is reduced when associated with caveolae (yamamoto et al., ) . in addition to vegfr and pdgfr, a number of g protein-coupled receptors (gpcrs) have been shown to interact with caveola-associated factors (insel et al., ) . gpcrs are a large family of transmembrane receptors involved in a variety of signal transduction events. these receptors are activated by a range of ligands, including hormones and peptides, and have been linked to a number of cancers such as cell biology of membrane trafficking in human disease thyroid, lung, and gastric. the presence of a number of gpcrs in caveolae suggests that these plasma membrane structures may interact with gpcrs and modulate their signaling potential. lisanti and others (li et al., a) have shown that caveolin- interacts solely with inactive forms of g-protein a subunits, lending credence to the negative regulation hypothesis caused by the association of caveolae with transmembrane signaling receptors. a number of mutations in muscle-specific caveolin- have been associated with four distinct but related autosomal dominant muscle disease phenotypes (woodman et al., ) : limb girdle muscular dystrophy type c (minetti et al., ) , rippling muscle disease, hyperckemia (persistently elevated levels of serum creatine kinase), and distal myopathy. some mutations cause aberrant retention of caveolin- in the golgi and subsequent degradation; other mutations may cause mutant caveolin- to act in a dominant-negative manner by forming unstable aggregates with wild-type caveolin- (galbiati et al., ; sotgia et al., a,b) . hypertrophic cardiomyopathy (hcm) patients have a caveolin- t s mutation that reduces plasma membrane levels (hayashi et al., ) . caveolin gene knockout mice are providing insights into protein function in diverent human diseases. for example, lack of caveolins can cause diabetes, atherosclerosis, and cardiomyopathies in mouse models (cohen et al., ; williams and lisanti, ) . however, such phenotypes have yet to be linked to caveolin dysfunction in humans. phagocytosis is a process used by white blood cells such as macrophages, neutrophils, and dendrites to ingest large particulate material into specialized vesicles called phagosomes. these professional phagocytes are paramount in the defense against infection as they engulf and ingest whole microorganisms such as bacteria. they also use this route for ''mopping up'' apoptotic debris or senescent cells from tissues. in contrast to constitutive pinocytic transport, phagocytosis is regulated by cell surface-localized fc receptor (fcr) contact or interaction with complement-or antibody-coated particles which results in clustering of fcr on the cell surface, a step important for subsequent intracellular signaling and cellular activation (daeron, ) . polymorphisms in leukocyte-specific fcg receptors may contribute to autoimmune diseases such as guillain-barré syndrome or rheumatoid arthritis, and enhanced susceptibility to infection (van sorge et al., ) . fc-mediated binding can trigger a complex signaling response involving extrusion of fine plasma membrane projections (pseudopodia) from the macrophage to surround and engulf the pathogen, forming a phagosome. the signaling response is reviewed in greater detail elsewhere (bokoch, ; chimini and chavrier, ; niedergang and chavrier, ) . in brief, the activation of tyrosine kinases and rho gtpases is triggered through fcr signaling. the rac and cdc gtpases, in conjunction with the downstream evector wasp, mediate remodeling of the actin cytoskeleton, leading to pseudopodium formation and phagosome closure (castellano et al., ; chimini and chavrier, ) . in contrast, the complement mediated-uptake of opsonized particles divers such that they appear to ''fall'' into the cell in a process that requires rho, but not rac or cdc (bokoch, ) . phagocytosis, although designed to destroy pathogens, can paradoxically be used as a route of entry by pathogens such as mycobacterium (m. leprae and m. tuberculosis) or leishmania (nguyen and pieters, ; scott et al., ) . normally, internalized pathogens are destroyed successfully through phagosome maturation into lysosomes and subsequent degradation. mycobacterium can evade host degradation by secreting a soluble serine/threonine protein kinase g molecule into the phagosome. this molecule initiates a signaling response that interferes with phagosome-lysosome fusion, and promotes intracellular pathogen survival (walburger et al., ) . furthermore, phagosome maturation is compromised by a pathogen induced block of p map (mitogen-activated protein) kinase recruitment to the tethering molecule early endosome antigen (fratti et al., ) . the leishmania protozoan parasite, which is transmitted to humans by sand flies, produces a membrane molecule called a lipophosphoglycan, which is inserted into the lipid bilayer of the phagosome in infected macrophages. this lipophosphoglycan is thought to modulate intracellular signaling pathways, resulting in a less fusogenic phagosome and preventing maturation; this would facilitate pathogen replication and disease progression (lodge and descoteaux, ) . molecules internalized from the cell surface by receptor-mediated endocytosis and clathrin-coated vesicles are delivered to the early endosome for sorting. molecules such as low-density lipoprotein receptor (ldlr) and transferrin receptor (tfr) are eyciently recycled between the early endosome and the plasma membrane. however, after ligand-mediated activation (fig. ) , receptor tyrosine kinases such as epidermal growth factor receptor (egfr) are sorted along the endocytic pathway for degradation. early endosomes are thought to be formed through the fusion of internalized vesicles and recruitment of specific proteins and lipids. one key protein traycking through the endosomal-lysosomal system. cell surface receptors are internalized through clathrin-coated vesicles (ccvs) at the plasma membrane. in the cell cytoplasm, ccvs shed their coat components and fuse to produce endosomes. internalized receptors are either recycled from sorting endosomes (housekeeping receptors, e.g., transferrin receptor) or targeted for degradation within the lysosome (signaling receptors, e.g., growth factor receptors) after movement through the late endosome and multivesicular body (mvb) compartments. endosomal regulator is the ubiquitously expressed rab a gtpase. rab a is present on the cytosolic face of the plasma membrane, vesicles, and tubular endosomal profiles (chavrier et al., ) . a number of rab a-associated evector proteins regulate endosomal fusion and mediate protein cargo movement and endosomal sorting (zerial and mcbride, ) . such evector proteins, including , are clustered on the cytosolic face of the early endosome and stabilize the gtp-bound rab a in an activated state (horiuchi et al., ) . gtp-bound rab a directly binds to early endosome antigen eea to regulate vesicular and endosomal tethering. eea contains a c-terminal rab a-binding domain, and a phosphatidylinositol -phosphate-binding zinc finger domain referred to as an fyve (conserved in fab , yotb, vac , and eea ) domain (gaullier et al., ; stenmark et al., ) . overexpression of wild-type rab a, or a constitutively active rab a mutant, causes endosome enlargement and defective traycking through this compartment, whereas expression of a constitutively inactive rab a mutant leads to formation of small endosomes and decreased endocytosis (bucci et al., ) . a family of evector proteins that accelerate gtpase hydrolysis (rabgaps) have been identified: rabgap- binds to rab a and regulates traycking through the endocytic pathway . the importance of rab a activity is further illustrated in the genetic disorder tuberous sclerosis (ts), a disease that causes tumors in the brain, eyes, heart, kidneys, lungs, and skin. ts arises when the tumor suppressor gene, tuberous sclerosis complex (tsc), is absent; introduction of the wild-type tsc gene into an animal model or cultured cells results in tumor suppression and reduced cellular proliferation (kobayashi et al., ; yeung et al., ) . interestingly, the tsc gene product (tuberin) is implicated in regulating gtp/gdp exchange on rab a, thus regulating traycking through this endosome system (xiao et al., ) . in chronic myelomonocytic leukemia (cmml) a genetic translocation causes fusion of rab a evector rabaptin- and the pdgfbr (magnusson et al., ) . this chromosomal translocation results in enhanced cellular proliferation by compromising endosomal fusion and traycking, and thus regulation of growth factor degradation. it is likely that this aberrant gene product is not degraded and triggers sustained intracellular signaling, leading to cell proliferation and tumor progression in a subset of lymphoid cells. recycling from endosomes back to the cell surface is often used by receptors that internalize nutrients such as lipoproteins and ions. receptor recycling rather than degradation conserves receptor functionality and nutrient uptake and reduces energy expenditure in the synthesis of new receptors (mukherjee et al., ) . genetic screens in the nematode caenorhabditis elegans identified rme- and delineated a new family of conserved class of eps homology (eh) domain proteins . both the worm and mouse homologs of rme- are associated with the endosomal compartment: a dominant-negative rme- g r mutant had little evect on receptor-mediated endocytosis but had a substantial evect on endosomal recycling, suggesting a functional role in this step . although information is currently limited, a number of neurological diseases are associated with dysfunction of early endosomal proteins. in some cases of demyelinating polyneuropathy, characterized by progressive weakening and sensory dysfunction of the legs and arms, eea autoantibodies have been detected (selak et al., ) . a number of disorders, from muscular dystrophy to rheumatoid arthritis, reveal the presence of circulating anti-eea antibodies. interestingly, eea epitopes recognized by such autoantibodies varied from patient to patient (selak et al., ) . autoantibodies against eea have also been detected in cases of subacute cutaneous systemic lupus erythematosus (scle), characterized by the appearance of an unsightly red rash, often occurring after sun exposure (mu et al., ) . lysosomes are terminal, membrane-enclosed degradative compartments that interact with other organelles through vesicular transport originating from the secretory, endocytic, and autophagic pathways. this organelle stores various proteases, lipases, hydrolases, and degradative enzymes within an acidic environment that maximizes enzymatic activity and degradation. resident lysosomal membrane proteins, integral proteins, and glycoproteins are targeted to the organelle via the endosome. lysosomal proteases such as cathepsin d are processed in the golgi apparatus to add a mannose -phosphate (m p) moiety to n-linked sugars. the m p moiety is recognized by plasma membrane or tgn-resident mannose -phosphate receptors (m prs) and sorted to the late endosome and eventually the lysosome. here, the acidic ph (ph < . ) results in receptor-ligand disassociation and recycling of the m pr to the tgn. fusion between the endosome and preexisting primary lysosomes allows the delivery of lysosomal resident proteins. the importance of m p-mediated targeting of lysosomal proteins is highlighted in the human neurological disorder, i-cell disease (mucolipidosis ii), where lysosomal enzymes are secreted from cells rather than targeted to the lysosome. the defect in i-cell disease involves lack of m p moiety addition as a result of mutations to the n-acetylglucosamine- -phosphotransferase enzyme usually present within the golgi apparatus (ben-yoseph et al., ) . how lysosomes are formed is still unclear (luzio et al., ) . three mechanisms have been proposed to explain lysosomal biogenesis: vesicular transport between late endosomes and preformed primary lysosomes (griyths and gruenberg, ), early endosomal ''maturation'' to lysosomes (murphy, ) , or the current favored model of ''kiss-and-run,'' in which transient interactions between endosomes and lysosomes transfer endosomal contents to the latter compartment (duclos et al., ; storrie and desjardins, ) . late endosome and lysosome interactions in the kiss-and-run model are thought to be regulated by the rab gtpase, which is present on late endosomes; a vps complex, homologous to budding yeast vacuole fusion regulators, is also implicated in sorting and delivery to lysosomes (seals et al., ) . the mammalian form of the vps complex interacts with syntaxin- , a t-snare that is concerned in regulating membrane dynamics along this route (kim et al., ) . danon disease is caused by point mutations in, or complete absence of lysosome-associated membrane protein (lamp ) or complete absence of this protein: changes which result in cardiomyopathy, myopathy, and mental retardation. in danon disease patients and lamp -deficient mice, autophagic vacuoles accumulate within the cytoplasm; these vacuoles arise via intracellular engulfment of old membranes to form an autophagosome, thus sequestering membranes and proteins for eventual degradation (shintani and klionsky, ) . autophagosomes fuse with lysosomes, leading to degradation for provision of molecules for cellular homeostasis. the accumulation of autophagic vacuoles in lamp -deficient cells suggests that lamp mediates interactions between autophagosomes and lysosomes. this pathway is commonly activated during conditions of cellular stress such as starvation or pathogenic infection (kirkegaard et al., ) . lysosomal storage diseases are caused through insufficient degradation of targeted components within lysosomes, leading to substrate accumulation and lysosome enlargement. more than lysosomal storage diseases have been documented and generally manifest themselves as neurological disorders; disease severity correlates with the levels of lysosomal enzyme activity. niemann-pick disease is a neurodegenerative condition caused by sphingomyelin accumulation in reticuloendothelial cells and ganglion neurons, leading to cell death. it is classified into five types (a-e), each distinguished by either clinical severity or age-related disease phenotype. niemann-pick type a (npa) is most common, with death occurring before years of age. npa patients have point mutations in the smpd gene that encodes a lysosomal sphingomyelinase (levran et al., ; takahashi et al., ) . interestingly, in npc patients, endocytosed ldl particles are not fully degraded in lysosomes, leading to defects in cholesterol metabolism (li et al., a) . the npc disease is caused by mutations in the npc gene, which encodes a lysosomal resident protein with similarity to sterol-sensing enzymes and proteins (scott and ioannou, ) . fabry disease is an x-linked condition caused by changes in lysosomal a-galactosidase activity resulting in glycosphingolipid accumulation within vascular endothelial lysosomes. this leads to angiokeratomas (a wart-like thickening of the skin), progressive renal impairment, cardiomyopathy, and cerebrovascular disease. mutations in the a-galactosidase a gene can also show reduced enzymatic activity of the encoded protein and retention within the endoplasmic reticulum (yasuda et al., ) . receptor tyrosine kinases such as epidermal growth factor receptor (egfr) are degraded by lysosomes after ligand binding and receptor activation. egfr lysosomal targeting is dependent on ligand-stimulated ubiquitination of the cytoplasmic domain. binding of egf to egfr causes downstream signaling, clathrin-mediated internalization, and traycking to endosomes. internalized receptor-ligand complexes are sorted to the late endosome or multivesicular bodies (mvbs), which eventually deliver their contents to the lysosome (katzmann et al., ) . whereas other receptors such as tfr are recycled to the plasma membrane, egfr is moved through the endosome-lysosome system by a ubiquitin-dependent sorting and recognition system. these include the hrs/stam heterodimer and tsg (bilodeau et al., ) present on endosomal membranes. the tsg tumor suppressor gene is mutated in nearly % of breast cancer patients and encodes a membrane-associated protein (lee and feinberg, ) . this factor participates in the sorting of ubiquitinated proteins on the endosome, but its exact function is not clear. in some specialized cells, such as cytotoxic t lymphocytes (ctls), platelets, and melanocytes, regulated secretion can be routed through compartments other than the tgn. such cells have evolved mechanisms whereby modified or secretory lysosomes release their contents at the plasma membrane in response to extracellular stimuli. these secretory lysosomes (sls) share lysosomal characteristics such as acid ph and lamp (lysosome-associated membrane proteins) residents but also contain unique markers such as tyrosinase, present in melanosomes. the ctl secretory lysosomes contain unique components such as perforin and granzymes required for triggering apoptosis in target cells. on ctl contact with a target cell, sls trayc toward the immunological synapse formed between the ctl and target cell. a signal then causes sl fusion with the ctl plasma membrane (stinchcombe et al., ) , and release of sl contents and subsequent target cell death. a number of autosomal genetic diseases causing immunodeficiency and albinism involve defects in regulated lysosomal secretion (stinchcombe et al., ) . in the rare, fatal disease familial hemophagocytic lymphohistiocytosis (fhl) sls congregate at the plasma membrane in ctls, where they can dock but cannot fuse with the membrane. in one group of fhl patients, this disease is due to a mutation in the gene encoding munc - ; this is closely related to the neuronal munc - gene product that is involved in snare complex formation in neuronal cells (feldmann et al., ) . assembly of this neuronal syntaxin- , snap- , and synaptobrevin complex is regulated by munc - , which binds and locks syntaxin- (t-snare) in a closed, inactive conformation, thus preventing it from interacting with snap- (yang et al., ) . however, munc - (sassa et al., ) and rim (rab a-interacting protein evector) (koushika et al., ) can compete with munc - and displace it from syntaxin- . this reinforces the syntaxin- open conformation and allows snare complex formation to occur. munc - may act as a conformational switch to promote t-snare into an ''open'' state, thus allowing formation of the snare complex that mediates synaptic vesicle docking and fusion. from observations of fhl patients, one speculation is that munc - has a role similar to that of munc - in regulating snare complex formation for sl docking and fusion in ctls (yang et al., ) . chediak-higashi syndrome (chs) is a key example of a disease avecting sl function in ctls, with patients displaying hypopigmentation (stinchcombe et al., ) . chs patients have genetic mutations in the lyst or chs gene (barbosa et al., ; perou et al., ) and produce ctls containing strikingly enlarged sls that are able to polarize to the immunological synapse but are unable to fuse with the pm. this suggests a role for the chs gene product in regulating membrane docking and fusion . overexpression of chs leads to the presence of small lysosomes, indicating increased lysosomal fission (ward et al., ) . in addition, chs interacts with snare proteins, further indicating a role in sl fusion (tchernev et al., ) . griscelli syndrome patients also display defects in sl dynamics within ctls and exhibit hypopigmentation and silvery hair. in melanocytes, cells responsible for pigment storage and production, rab a is required to recruit melanophilin to pigment granules called melanosomes (sls). melanophilin binds the myosin motor protein myosin va and regulates melanosome movement along actin cables to the plasma membrane strom et al., ; wu et al., ) . in type griscelli syndrome patients, rab a gtpase is missing or defective, whereas in type griscelli syndrome patients the myosin va motor protein is absent. these defects are also evident in mouse models such as ashen (rab a defective), dilate (myosin va defective), and leaden (melanophilin defective). in both the human griscelli syndromes and the mouse models, melanosomes are clustered in a perinuclear location, a defect attributed to rab a dysfunction (wilson et al., ; wu et al., ) . interestingly, ctls isolated from type griscelli syndrome patients and ashen mice (rab a deficient) are unable to kill target cells, whereas type griscelli syndrome patient and dilate mouse ctls are functional. this suggests that rab a interacts with diverent evectors to induce sl fusion with the plasma membrane in diverent cell types (haddad et al., ) . hermansky-pudlak syndrome (hps) is a fourth example of sl dysfunction and is characterized by oculocutaneous albinism, ceroid deposition, and excessive prolonged bleeding (hermansky and pudlak, ; swank et al., ) . however, hps cannot be viewed as a single disease but a group of at least seven autosomal genetic disorders. each of the seven subgroups (hps - ) is due to mutations in individual genes, most of which encode components of multisubunit protein complexes involved in vesicle traycking , whereas the function of others remains unclear. three of these complexes, termed blocs (biogenesis of lysosome-related organelle complexes), play a role in regulating traycking involved in platelet and melanosome secretion, but their exact functions are unclear (di pietro and dell'angelica, ) . in hps patients a nonsense mutation in the gene encoding the b a subunit of the ap adaptor protein prevents expression of this subunit (huizing et al., ) . as previously mentioned, ap is involved in the recruitment of transmembrane proteins into vesicles at the early endosome for delivery to the lysosome (peden et al., ) . in melanocytes derived from hps patients, the tyrosinase that catalyzes the formation of melanin is not transported to maturing melanosomes (huizing et al., ) . this leads to the characteristic pattern of albinism seen in patients with this condition. furthermore, patients with hps display an impaired ctl response and immune response. ctls from hps patients have lytic granules that cannot move in an oriented fashion toward the microtubule-organizing center; therefore, when ctls are stimulated by contact with target cells, the lytic granules are not targeted to the immunological synapse for cell killing . studies on the cell biology of hiv infection have suggested the existence of a viral secretory compartment. work by marsh and others pelchen-matthews et al., ) has localized viral envelope (gp ) and matrix proteins (p ) to tetraspanin-positive endosome-related organelles in infected macrophages and dendritic cells. these viral secretory compartments move from an intracellular localization to an infectious synapse when infected macrophages or dendritic cells form an immunological synapse with activated t cells. this may be one mechanism for subsequent viral infection of cd -positive t cells, thus causing the impaired immune response seen in patients with acquired immunodeficiency syndrome (aids). cells require a highly organized framework or cytoskeleton to station and move membrane organelles within three-dimensional space. components of the cytoskeleton can guide organelles or vesicles to specific destinations within the cell. the microtubule cytoskeleton is commonly associated with the directional movement of intracellular transport vesicles or intermediates. in contrast, actin has been envisioned to have a structural role in determining cell shape, plasma membrane dynamics, and cell locomotion. however, evidence points to a role for actin in regulated traycking from the tgn (allan et al., ; badizadegan et al., ; cobbold et al., ) and endocytosis (ascough, ; engqvist-goldstein and drubin, ) . the cytoskeleton is a dynamic structure likened to a collapsible scavold that can be rapidly disassembled and reconstituted depending on cellular requirements. actin or tubulin polymerization (elongation) and depolymerization (breakdown) rely on the controlled addition or removal of monomers in a polarized and energy-dependent manner. protofilaments in either structure are both polarized, with the plus end growing at a faster rate. actin cables are each composed of two parallel protofilaments that twist around each other, whereas microtubules are composed of a hollow cylindrical structure comprising parallel protofilaments. actin nucleation is an initial step required for elongation involving formation of a stable trimer subunit base for protofilament elongation. a heptameric complex termed arp / (actin-related protein) binds to the ends and sides of actin filaments to nucleate and further accelerate the growth of the actin network (millard et al., ) . the function of the arp / complex can be regulated by membrane-associated rho gtpases. these regulators, which include cdc and various rac isoforms, act as molecular switches that cycle between an active gtp-bound state and an inactive gdp-bound state. cdc regulates arp / indirectly through its downstream target wiskott-aldrich syndrome protein (wasp), which binds directly to the arp / complex (jave and hall, ) . patients with x-linked wiskott-aldrich syndrome display mutations in the wasp gene and have thrombocytopenia (reduced platelet count), eczema, recurrent infections, hematologic malignancy, and autoimmune disorders (lemahieu et al., ) . approximately disease mutations in wasp have been reported, which lead to defective control of wasp in actin polymerization and severe disease phenotypes (burns et al., ) . wasp expression is restricted to hematopoietic cells, although the ubiquitously expressed n-wasp is present in various cells and tissues (burns et al., ) . the actin network is important for the formation of immunological synapses between cytotoxic t lymphocytes (ctls) and their targets, as well as t lymphocytes and antigen-presenting cells such as macrophages. the formation of the immunological synapse in ctls is essential for the transport, docking, and fusion of sls and subsequent destruction of the target cell as described above. defective wasp inhibits the formation of the immunological synapse and t cell activation (notarangelo and ochs, ) , probably causing the immunological deficiencies observed in wiskott-aldrich syndrome patients. wasp deficiency in t lymphocytes also avects the regulation and composition of lipid rafts (dupre et al., ) , indicating that the cell biology of membrane trafficking in human disease formation of the immunological synapse is dependent on lipid rafts, wasp, and actin dynamics. motor proteins provide the physical force to move membrane vesicles along the polymerized cytoskeletal filaments via atp-dependent hydrolysis. actinbased motor proteins belong to the myosin superfamily. the myosin va gene is mutated in a small number of patients with griscelli syndrome (bahadoran et al., ; pastural et al., ) (other traycking mutations contributing to griscelli syndrome are discussed in section v.c). mutations in the myosin viia gene can cause usher's syndrome, resulting in blindness and deafness. intracellular transport is probably compromised in usher syndrome patients; the mouse shaker model has a mutant myosin viia gene, displaying defective melanosome transport in retinal pigment epithelial cells (liu et al., ) and altered distribution in photoreceptor cells (richardson et al., ) . microtubule motor proteins, which actively move vesicles along the microtubules, and microtubule-associated proteins (maps), serve as docking molecules to bind cargo to motor proteins (gerdes and katsanis, ) . microtubule motor proteins belong to either the kinesin or dynein families. with the exception of the c-terminal kinesins, kinesin-based motors generally transport cargo toward the plus end of the microtubule, whereas the dyneins are minus end-directed motors. long-range vesicular transport is particularly important in neurons, where axons can reach up to m in length. newly synthesized lipids, and secreted or membrane-associated proteins, are made in the cell body; long-range and directional transport is crucial for replenishing the constituents of the presynaptic cleft (at the terminal end of the axon) with synaptic vesicles and plasma membrane receptors (holzbaur, ) . a number of human neurological diseases are linked to mutations in microtubule motors and associated proteins. lissencephaly, a greek term meaning ''smooth brain,'' causes severe brain malformation resulting in mental retardation and epilepsy. one of the genes mutated in the disease is lis (originally identified in miller-dieker syndrome patients with lissencephaly) (reiner et al., ) . the lis protein regulates microtubule motor function by binding dynein and p glued , a component of the dynactin complex that binds to and activates dynein (smith et al., ) . it is proposed that lis regulates retrograde axonal transport. another gene mutated in some patients with lissencephaly is doublecortin, a microtubule-associated protein that binds tubulin and stabilizes microtubules (horesh et al., ; moores et al., ) . the kif b kinesin regulates transport of synaptic vesicle precursors along the neuronal axon. patients with charcot-marie-tooth disease type a display neuronal axonal degeneration due to a loss-of-function mutation in the motor domain of kif b . in alzheimer's disease, a classical sign is hyperphosphorylated aggregates of the microtubule-associated protein, tau, in neuronal cells and tissues. tau protein can influence vesicular transport (ebneth et al., ) by regulating the attachment of motors to microtubules (trinczek et al., ) . one theory is that the tau protein can interfere with kinesin-dependent transport by blocking motor access to microtubules, thus slowing or preventing vesicle movement along axons (mandelkow et al., ) . moreover, an early sign of alzheimer's disease is the loss of synapses and retrograde degeneration of neurons, complemented by a breakdown in intracellular transport. the disruption of microtubule-mediated vesicular traycking may also be a causative factor of the neurodegenerative phenotype of huntington's disease. this disease is caused by expansion of polyglutamine repeats occurring in the brain-enriched protein huntingtin (htt). it has been demonstrated that htt enhances vesicular transport of brain-derived neurotrophic factor (bdnf) along microtubules (gauthier et al., ) . htt is localized in the cytoplasm and is associated with vesicular and microtubule-based trayc through its ability to bind huntingtin-associated protein (hap ) (li et al., b) , a protein that has aynity for the dynactin p glued subunit (engelender et al., ) . dysfunctional polyq-htt associated with the disease state may disrupt the transport of bdnf by binding and blocking the hap /dynactinmediated delivery of bdnf vesicles along microtubules (gauthier et al., ) . this is further supported by the finding that bdnf levels are decreased in brains of huntington's disease patients (ferrer et al., ) . the current treatment of genetic disorders involves addressing the symptoms rather than the cause. to that end, many mild forms of disorders such as niemann-pick disease, and familial hypercholesterolemia, can be controlled by diet regimens and lifestyle changes. in contrast, a life-threatening disease such as cystic fibrosis requires extensive physiotherapy and pulmonary exercise to loosen and prevent mucus accumulation within the lungs. new antidementia drugs are increasingly successful in treating neurological disorders such as alzheimer's disease. drugs such as galantamine, donepezil, rivastigmine, and memantine target the posttranslational processing of bapp to reduce amyloid deposits (prasher, ) . in familial hypercholesterolemia, statin treatment is a common strategy for reducing plasma ldl and cholesterol levels by targeting hmg-coa reductase, the rate-limiting enzyme in cellular cholesterol biosynthesis. furthermore, less commonly used ldl-lowering drugs such as probucol have shown some success in (buckley et al., ) . the administration of adrenalin receptor antagonists (b-blockers) to patients with the cardiac condition long-qt syndrome reduces arrhythmia risk. enzyme replacement therapy (ert) has been carried out in patients with fabry's disease, a lysosomal storage disease. patients are given recombinant lysosomal a-galactosidase (mignani and cagnoli, ) to reduce the risks of strokes and kidney failure associated with the condition. finally, organ transplantation is occasionally carried out for some disease states: for example, bone marrow transplants for chediak-higashi syndrome patients (liang et al., ) and rab a-defective patients with griscelli syndrome (schuster et al., ) and wiskott-aldrich syndrome (filipovich et al., ) . however, although transplant operations can be successful in alleviating the immunological issues associated with these diseases, it does not address problems associated with the nervous system or pigmentation. b. gene therapy: the next generation of medical treatment? completion of the human genome sequencing project has given science the ability to track gene(s) responsible for potentially any genetic disorder and, as a consequence, to allow these genes to be corrected in patients. this is the goal of gene therapy research. of course, gene therapy has a fundamental limitation: it is only really suitable for single-gene defect diseases, and multigenic or chromosomal defects will be beyond the ability of the technique because of the complex nature of the disease. however, there are more than single gene defects that cause human disease, so there are many diseases requiring such approaches. the history of gene therapy is discussed in more detail by russell ( ) and scollay ( ) . much evort has been made in developing techniques that allow successful replacement or augmentation of defective genes. gene therapy is performed by introducing a gene vehicle directly into the patient (in vivo) or by removing cells from a patient, introducing the gene into these cells in culture, and replacing the cells back in the patient (ex vivo). most studies have focused on the use of viral vectors as delivery vehicles. retroviruses are potentially the best gene delivery system (kurian et al., ) . these rna viruses are able to infect a great many cell types and replicate by inserting their viral genes into the genome of the host. the host cellular machinery is then modulated to produce and assemble viral particles. in gene therapy, retroviruses could be used to express the target gene to be replaced but be modified to prevent viral disease (hiv, which is the causative agent of aids, is also a retrovirus). the principal drawback of a retrovirus vector is the possibility that genomic integration could elevate oncogene expression, thus causing cancer. therefore, the majority of clinical trials using retrovirus vectors have been performed ex vivo. a ''successful'' gene therapy experiment was exemplified in the case of a -year-old female patient lacking adenosine deaminase (ada), which results in severely compromised immunodeficiency (ada-scid) and dysfunctional t cells (blaese et al., ) . in this case, a retroviral vector was used to deliver the coding sequence for ada into cells, resulting in successful expression of this enzyme in hitherto defective cells. although successful, it is uncertain whether enzyme replacement treatment (recombinant ada injections) also influenced the patient outcome. adenovirus (adv) (mcconnell and imperiale, ) is a dna virus and key gene therapy vehicle that maintains the viral genome as a separate transmissible episome within the nuclei of infected host cells. the use of attenuated or inactivated adv for human gene delivery has attracted much interest. the advantages of adv gene transfer are that its genome can easily be manipulated and recombinant virus can be grown to high titers in vitro with eycient transduction of target cells in vitro or in vivo. as adv can evectively infect nondividing cells such as lung pulmonary tissues it is a popular vector of choice for gene therapy to treat cystic fibrosis (cf) patients. although there are promising studies (zabner et al., ) , failures have also been noted (knowles et al., ) . a major disadvantage of an adv-based approach is the triggering of a strong host immune response to the virus, which becomes a serious problem in subsequent long-term delivery of recombinant virus for disease alleviation. one approach to circumventing such an issue is to use a viral delivery system that produces a low host immune response such as the adeno-associated virus (aav) (flotte, ) . aav is a nonpathogenic virus that requires coinfection with a helper virus to replicate. however, aav has broad host cell specificity and is diycult to grow in large quantities, probably because of its reliance on a helper virus. finally, nonviral methods are increasingly available for the delivery of dna constructs directly into cells and tissues. these are often lipid-based reagents (e.g., liposomes) that bind to the plasmid dna and fuse with the plasma membrane, thus enabling cytosolic delivery of the gene. the plasmid dna would then be transported into the host nucleus by endogenous cellular machinery. this type of gene delivery can only be performed ex vivo and can be limited by the poor dna transfection eyciency of primary cells or tissues. this type of method, however, is a potentially useful method for delivering genes into progenitors or precursors (e.g., stem cells) before cellular cell biology of membrane trafficking in human disease diverentiation and tissue formation within a particular microenvironment in the body (mendell et al., ) . numerous disease states are caused by protein misfolding within the er, leading to degradat ion (table i ; section s iii. a. an d iii.a. ). one strategy would be to promote the correct protein conformation in a mutant gene product either chemically or pharmacologically. a number of membrane-divusible chemical and pharmacological ''chaperones'' have been identified that could avect protein folding in cells. chemical chaperones such as glycerol and trimethylamine n-oxide (tmao) can restore the wild-type traycking and activity of cftrÁf in cultured epithelial cells (brown et al., ) , and porcine kidney epithelial cells expressing cftrÁf and treated with dimethyl sulfoxide (dmso) increased plasma membrane levels of the channel protein (bebok et al., ) . loo et al. ( ) have demonstrated that a novel quinazoline derivative specific for cftr will rescue the defective traycking of cftrÁf in cultured cells. cell surface levels of a water channel, aquaporin- , can be enhanced with dmso (tamarappoo and verkman, ) . defects in this gene can result in x-linked nephrogenic diabetes insipidus, a condition in which patients are unable to concentrate their urine because of an inability to reabsorb water from the kidneys into the blood. although chemical chaperones are somewhat nonspecific in their action (the protein folding of the whole cell is avected and not just the target protein), pharmacological chaperones can be tailored to individual proteins. for example, the compound sr a is a nonpeptide vasopressin v receptor antagonist (morello et al., ; robert et al., ) . patients with a mutant vasopressin v receptor can also display nephrogenic diabetes insipidus. on treatment, the cell-permeant sr a compound would act as a chaperone and accompany the mutant v receptor to the cell surface to rescue correct functionality. geldanamycin, a naturally occurring antifungal agent, has potential as an anticancer drug (beliakov and whitesell, ; miyata, ) . geldanamycin interacts with and inhibits activity of the heat shock protein and chaperone hsp , a cytosolic cellular stress protein that supports the correct folding, stability, and function of ''client'' proteins. many hsp client proteins are implicated in cell cycle progression, proliferation, and angiogenesis (whitesell and lindquist, ) . the erbb tyrosine kinase complex is implicated in regulation and development of epithelial breast tumors and is an hsp client. inhibition of hsp action by geldanamycin results in degradation of both erbb and downstream signaling evectors, resulting in reduced cellular growth and tumor formation (citri et al., ) . a number of cell-permeable peptide sequences found in viruses and host proteins have been discovered that mediate the delivery of cargo (proteins, drugs, plasmid dna, oligonucleotides) directly into cells (brooks et al., ; gupta et al., ; schwartz and zhang, ) . such peptide sequences could be fused or attached to recombinant or engineered proteins and administered to patients to complement defects of a particular gene product. for example, the drosophila melanogaster antennapedia homeodomain (antp) transcription factor contains a short -residue sequence that mediates protein translocation across biological membrane bilayers in an energy-independent manner (derossi et al., ; joliot et al., ) . other sources of cell membrane-permeable proteins have been uncovered in viruses. the hiv- replication protein tat contains a basic, arginine-and lysine-rich peptide sequence (residues - ) that modulates the translocation of exogenous tat across the plasma membrane in a number of cell types, and is able to activate intracellular genes controlled by an hiv promoter (frankel and pabo, ; mann and frankel, ) . this basic -residue sequence can internalize conjugated b-galactosidase and horseradish peroxidase (fawell et al., ) as well as a fab antibody fragment (anderson et al., ) . the major structural protein of herpesvirus (hsv- ), vp , can trayc between cells (elliott and o'hare, ) , whereas the pres- domain of hepatitis b virus surface antigen acts as a shuttle for peptides and functional proteins (such as egfp) in hepatocytes and other cells (oess and hildt, ) , suggesting further the existence of naturally occurring peptide sequences that may act as drug delivery vectors. finally, a ''synthetic'' amphipathic peptide, fluos-klalklalkalkaalkla-nh , has been shown to be internalized in mast and endothelial cells (oehlke et al., ) . the employment of small molecular inhibitors as a method of treating human disease has moved at exponential pace. a number of compounds have been synthesized or isolated from nonhuman organisms that directly avect cellular function and have been used in research on a variety of diseases. plant-and microorganism-derived polyhydroxylated alkaloids referred to as iminosugars have been used in the treatment of patients with gaucher disease (cox et al., ) . gaucher disease type i and type ii is a lysosomal storage disorder caused by a mutation in the gene encoding the cell biology of membrane trafficking in human disease acid b-glucosidase (gba) enzyme and results in the accumulation of toxic glucosylceramide in a patient's spleen, liver, and bones; manifesting itself in enlargement of these organs, as well as heart and lung disease. iminosugars act on glycosylating enzymes present within the er and golgi and inhibit their ability to transfer sugar moieties onto proteins. one member of the iminosugar family, n-butyldeoxynojirimycin (nb-dnj; also called miglustat or zavesca) inhibits the enzyme important in the maturation of the gba substrate glucocerebroside, namely ceramide glucosyltransferase (cgt) (butters et al., ) . inhibition of cgt has resulted in significantly reduced levels of glucocerebroside in the liver and spleen of patient in clinical trials (cox et al., ) . however, nearly % of patients in the trials displayed osmotic diarrhea as a side evect of the treatment. in a mouse model for human tay-sachs disease, which is caused by a mutation in the gene encoding hexosamidase a, levels of the harmful glycophospholipid gm were significantly reduced on treatment with nb-dnj (platt et al., ) . in addition to the treatment of lysosomal storage diseases, an nb-dnj derivative called miglitol has been used in the treatment of diabetes mellitus, resulting in reduced activity of the sucrose-isomaltase enzyme complex and reduction of carbohydrate digestion (mitrakou et al., ) . a major aspect of human disease is the production and subsequent degradation of misfolded proteins, either by the proteasome or within the lysosome. lysosomotropic agents such as chloroquine cause an increase in the intralumenal ph of endosomes and lysosomes, reducing lysosomal protease activities and the traycking of proteins through the endosome-lysosome system. a number of proteasome inhibitors such as mg , lactacystin, and alln can specifically inhibit the activity of a range of serine and cysteine proteases and chymotrypsin-like enzymes (kisselev and goldberg, ) . proteasome inhibition has been linked with a number of aspects of human disease. treatment of endothelial cells with proteasome inhibitors resulted in apoptosis of proliferating cells (drexler et al., ) and inhibition of plasminogen activator levels; this factor promotes angiogenesis and new blood vessel sprouting (oikawa et al., ) . however, inhibiting proteasome function has broad cytotoxic and apoptotic evects in cells and tissues. chemotherapeutic agents targeting signaling pathways are currently of much interest in relation to cancer therapeutics. cellular proliferation can be regulated by growth factor binding to a cell surface receptor and signaling through either the mitogen-activated protein kinase (mapk) or phosphoinositide- -kinase (pi k) cascades. activation of these pathways induces the expression of oncogenes such as c-jun and c-fos and inhibits apoptosis through a sequence of protein phosphorylation events. shelton et al. ( ) demonstrated that inhibition of the mapk pathway with small molecule inhibitors specific for raf- or mek reduced cell proliferation and induced apoptosis in conditionally transformed hematopoietic cells. however, such pathways also regulate other cellular functions besides proliferation or apoptosis and there are likely to be consequences for cellular homeostasis. structural studies are important in the development of new small molecule inhibitors that target specific enzymes and regulators. c-akt (pkb) is a serine/ threonine protein kinase required for survival and proliferation in many human cancers and its structure has been elucidated (kumar and madison, ; yang et al ., ; and refer ences therei n). chem otherape utic agents have been consequently designed that inhibit c-akt activity; molecules such as h- target the atp-binding pocket in c-akt (kumar and madison, ) . compounds that bind specifically to c-akt isoforms or target specific domains within the kinase have been reported (barnett et al., ) , but there are no reports of clinical trials with such compounds (kumar and madison, ) . finally, small molecule inhibitors are being developed to target the posttranslational processing of proteins or peptides implicated in human disease. the enzyme that catalyzes the initial steps in b-amyloid synthesis, g-secretase, is an attractive target for prevention of amyloid deposits in alzheimer's disease patients (churcher and beher, ) . such small molecule inhibitors could also be used to treat pathogenic infections such as those caused by severe acute respiratory syndrome (sars), influenza, hiv, or hepatitis c viruses. attractive targets are virus-encoded or host proteases required for processing of viral proteins to generate infectious virus particles from the host cell. in the case of the sars virus, a viral chymotrypsin-like cysteine protease is responsible for processing sars viral proteins required for viral replication. inhibition of this protease would evectively inhibit viral replication. a molecule referred to as cs was found to inhibit the replication of human sars with no toxic evect on normal cells (dooley et al., ) . much work is also being carried out in targeting host proteases required for the processing of hiv envelope glycoproteins by the biosynthetic secretory pathway to generate viral gp and gp polypeptides. the completion of the human genome sequencing project has led to the prediction that a large number of diseases will be identified and understood at the gene level . as noted in this review, a number of examples exist in which a single gene mutation can have devastating evects on human function. at present, the symptoms of some mild forms of genetic diseases can be modulated through diet or drug regimens, and some success has been achieved with organ transplantation. gene therapy has attracted much attention but has suvered setbacks due to viral toxicity issues. an alternative strategy is the use of small molecule therapeutics, which cell biology of membrane trafficking in human disease may override specific defects or target specific pathways to compensate for gene defect(s). in addition, our understanding of how we respond at a genetic level to pathological infection will enable us to design evective drug strategies to presently chronic infections. in essence, understanding the cell biological basis for human diseases will enable us to design evective methods to deliver therapeutic strategies to patients. caveolin- and caveolin- , together with three bone morphogenetic protein-related genes, may encode novel tumor suppressors down-regulated in sporadic follicular thyroid carcinogenesis endocytosis of the glucose transporter glut is mediated by the gtpase dynamin motoring around the golgi the p -interactive proteins gm and giantin participate in endoplasmic reticulum-golgi trayc cftr and chaperones: processing and degradation tumor cell retention of antibody fab fragments is enhanced by an attached hiv tat protein-derived peptide endocytosis: actin in the driving seat traycking of cholera toxin-ganglioside gm complex into golgi and induction of toxicity depend on actin cytoskeleton comment on elejalde syndrome and relationship with griscelli syndrome identification of the homologous beige and chediak-higashi syndrome genes coupled er to golgi transport reconstituted with purified cytosolic proteins the akt/pkb family of protein kinases: a review of small molecule inhibitors and progress towards target validation the adaptor protein ap- as a component of the clathrin coat machinery: a morphological study role of diacylglycerol in pkd recruitment to the tgn and protein transport to the plasma membrane loss of function associated with novel mutations of the scn a gene in patients with brugada syndrome disassembly and reassembly of the golgi apparatus activation of deltaf cftr in an epithelial monolayer hsp : an emerging target for breast cancer therapy snares and the specificity of transport vesicle targeting mutations associated with neutropenia in dogs and humans disrupt intracellular transport of neutrophil elastase altered molecular size of n-acetylglucosamine -phosphotransferase in i-cell disease and pseudo-hurler polydystrophy congenital and acquired neutropenia the role of regulated cftr traycking in epithelial secretion phosphatidic acid formation by phospholipase d is required for transport from the endoplasmic reticulum to the golgi complex degradation of subunits of the sec p complex, an integral component of the er membrane, by the ubiquitin-proteasome pathway role of cue p in ubiquitination and degradation at the er surface the vps p hse p complex binds ubiquitin and mediates endosomal protein sorting t lymphocyte-directed gene therapy for ada-scid: initial trial results after years regulation of innate immunity by rho gtpases signals for sorting of transmembrane proteins to endosomes and lysosomes caveolin- in breast cancer tat peptide-mediated cellular delivery: back to basics chemical chaperones correct the mutant phenotype of the delta f cystic fibrosis transmembrane conductance regulator protein the small gtpase rab functions as a regulatory factor in the early endocytic pathway probucol. a reappraisal of its pharmacological properties and therapeutic use in hypercholesterolaemia the wilson disease gene is a putative copper transporting p-type atpase similar to the menkes gene coiled coils: a highly versatile protein folding motif mechanisms of waspmediated hematologic and immunologic disease a step forward for stiv-person syndrome small-molecule therapeutics for the treatment of glycolipid lysosomal storage disorders hepatic endoplasmic reticulum storage diseases initial docking of er-derived vesicles requires uso p and ypt p but is independent of snare proteins a role for cbs domain in traycking of chloride channel clc- a ''de novo'' point mutation of the low-density lipoprotein receptor gene in an italian subject with primary hypercholesterolemia actin dynamics during phagocytosis adpkd: a human disease altering golgi function and basolateral exocytosis in renal epithelia localization of low molecular weight gtp binding proteins to exocytic and endocytic compartments multiple forms of dynamin are encoded by shibire, a drosophila gene involved in endocytosis function of rho family proteins in actin dynamics during phagocytosis and engulfment lowe syndrome protein ocrl interacts with clathrin and regulates protein traycking between endosomes and the trans-golgi network gamma-secretase as a therapeutic target for the treatment of alzheimer's disease the achilles heel of erbb- /her : regulation by the hsp chaperone machine and potential for pharmacological intervention lytic granules, secretory lysosomes and disease adaptor protein -dependent microtubule-mediated movement of lytic granules to the immunological synapse novel membrane trayc steps regulate the exocytosis of the menkes disease atpase the menkes disease atpase (atp a) is internalized via a rac -regulated, clathrin-and caveolae-independent pathway actin and microtubule regulation of trans-golgi network architecture, and copper-dependent protein transport to the cell surface role of caveolae and caveolins in health and disease the human genome project: lessons from large-scale biology rab and arf gtpase regulation of exocytosis novel oral treatment of gaucher's disease with n-butyldeoxynojirimycin (ogt ) to decrease substrate biosynthesis copper transporting p-type atpases and human disease defective intracellular transport and processing of oa is a major cause of ocular albinism type involvement of clathrin-mediated endocytosis in human immunodeficiency virus type entry fc receptor biology the molecular basis of alanine: glyoxylate aminotransferase mistargeting: the most common single cause of primary hyperoxaluria type a role of amphiphysin in synaptic vesicle endocytosis suggested by its binding to dynamin in nerve terminals the synaptic vesicle-associated protein amphiphysin is the -kd autoantigen of stiv-man syndrome with breast cancer a novel form of hereditary myeloperoxidase deficiency linked to endoplasmic reticulum/proteasome degradation the low lysine content of ricin a chain reduces the risk of proteolytic degradation after translocation from the endoplasmic reticulum to the cytosol low-density lipoprotein receptor-its structure, function, and mutations association of the ap- adaptor complex with clathrin ap- , a novel protein complex related to clathrin adaptors altered traycking of lysosomal proteins in hermansky-pudlak syndrome due to mutations in the beta a subunit of the ap- adaptor rhodopsin c terminus, the site of mutations causing retinal disease, regulates traycking by binding to adp-ribosylation factor (arf ) the third helix of the antennapedia homeodomain translocates through biological membranes rab geranylgeranyl transferase alpha mutation in the gunmetal mouse reduces rab prenylation and platelet synthesis ubiquitination is required for the retrotranslocation of a short-lived luminal endoplasmic reticulum glycoprotein to the cytosol for degradation by the proteasome mutations in the mdr gene cause progressive familial intrahepatic cholestasis the coiled-coil membrane protein golgin- is a novel rab evector required for golgi ribbon formation the cell biology of hermansky-pudlak syndrome: recent advances from genome to drug lead: identification of a small-molecule inhibitor of the sars virus cooperation of ggas and ap- in packaging mprs at the trans-golgi network inhibition of proteasome function induces programmed cell death in proliferating endothelial cells the t( ; )(p ;q ) in the u cell line results in the fusion of the af gene and calm, encoding a new member of the ap- clathrin assembly protein family remodeling of endosomes during lysosome biogenesis involves ''kiss and run'' fusion events regulated by rab er-to-golgi transport: cop i and cop ii function (review) hyperinsulinism in infancy: from basic science to clinical disease wiskott-aldrich syndrome protein regulates lipid raft dynamics during immunological synapse formation caveolae and sorting in the trans-golgi network of epithelial cells overexpression of tau protein inhibits kinesin-dependent traycking of vesicles, mitochondria, and endoplasmic reticulum: implications for alzheimer's disease a novel gene for autosomal dominant stargardt-like macular dystrophy with homology to the sur protein family intercellular traycking and protein delivery by a herpesvirus structural protein cisternal maturation and vesicle transport: join the band wagon! (review) huntingtin-associated protein (hap ) interacts with the p glued subunit of dynactin genes encoding human caveolin- and - are co-localized to the d s locus ( q . ), a known fragile site (fra g) that is frequently deleted in human cancers actin assembly and endocytosis: from yeast to mammals accelerated transport and maturation of lysosomal alpha-galactosidase a in fabry lymphoblasts by an enzyme inhibitor lowe syndrome protein ocrl interacts with rac gtpase in the trans-golgi network tat-mediated delivery of heterologous proteins into cells munc - is essential for cytolytic granules fusion and is mutated in a form of familial hemophagocytic lymphohistiocytosis (fhl ) brain-derived neurotrophic factor in huntington disease caveolae and intracellular traycking of cholesterol impact of donor type on outcome of bone marrow transplantation for wiskott-aldrich syndrome: collaborative study of the international bone marrow transplant registry and the national marrow donor program adeno-associated virus-mediated gene transfer for lung diseases the ap- a and ap- b clathrin adaptor complexes define biochemically and functionally distinct membrane domains the endoplasmic reticulum as a site of protein degradation de novo formation of caveolae in lymphocytes by expression of vip -caveolin identification of a di-leucine motif within the c terminus domain of the menkes disease protein that mediates endocytosis from the plasma membrane cellular uptake of the tat protein from human immunodeficiency virus induction of p mitogen-activated protein kinase reduces early endosome autoantigen (eea ) recruitment to phagosomal membranes caveolin- null mice show a loss of caveolae, changes in the microdomain distribution of the dystrophin-glycoprotein complex, and t-tubule abnormalities phenotypic behavior of caveolin- mutations that cause autosomal dominant limb girdle muscular dystrophy (lgmd- c). retention of lgmd- c caveolin- mutants within the golgi complex hiv- traycking to the dendritic cell-t-cell infectious synapse uses a pathway of tetraspanin sorting to the immunological synapse structural basis of fabry disease fyve fingers bind ptdins huntingtin controls neurotrophic support and survival of neurons by enhancing bdnf vesicular transport along microtubules microtubule transport defects in neurological and ciliary disease wilson disease the alpha chain of the ap- adaptor is a clathrin binding subunit intracellular transport and sorting of the oligodendrocyte transmembrane proteolipid protein disrupted proteolipid protein traycking results in oligodendrocyte apoptosis in an animal model of pelizaeus-merzbacher disease evidence that rme- , a conserved c. elegans eh-domain protein, functions in endocytic recycling the arguments for pre-existing early and late endosomes intracellular delivery of large molecules and small particles by cell-penetrating proteins and peptides a gtpase-activating protein controls rab function in endocytic traycking defective granule exocytosis in rab a-deficient lymphocytes from ashen mice synaptojanin : localization on coated endocytic intermediates in nerve terminals and interaction of its kda isoform with eps mutation and aberrant expression of caveolin- in human oral squamous cell carcinomas and oral cancer cell lines traycking, turnover and membrane topology of prp charcot-marie-tooth neuropathy type b is associated with mutations of the myelin p gene invasion activating caveolin- mutation in human scirrhous breast cancers identification and functional analysis of a caveolin- mutation associated with familial hypertrophic cardiomyopathy accumulating evidence suggests that several ab-toxins subvert the endoplasmic reticulum-associated protein degradation pathway to enter target cells the delta f mutation shortens the biochemical half-life of plasma membrane cftr in polarized epithelial cells dynaminmediated internalization of caveolae albinism associated with hemorrhagic diathesis and unusual pigmented reticular cells in the bone marrow: report of two cases with histochemical studies evects of mutant rat dynamin on endocytosis characterization of a fourth adaptor-related protein complex missense mutation (c r) in the thyroglobulin gene causes congenital goiter with mild hypothyroidism by impaired intracellular transport a novel leukocyte adhesion deficiency caused by expressed but nonfunctional beta integrins mac- and lfa- caveolae: stable membrane domains with a potential for internalization doublecortin, a stabilizer of microtubules a novel rab gdp/gtp exchange factor complexed to rabaptin- links nucleotide exchange to evector recruitment and function hereditary neutropenia: dogs explain human neutrophil elastase mutations ap- mediates tyrosinase but not trp- traycking in human melanocytes nonsense mutations in adtb a cause complete deficiency of the {beta} a subunit of adaptor complex- and severe hermansky-pudlak syndrome type the leaden gene product is required with rab a to recruit myosin va to melanosomes in melanocytes caveolae and lipid rafts: g protein-coupled receptor signaling microdomains in cardiac myocytes rho gtpases: biochemistry and biology a common w s mutation in the ldl receptor gene of danish patients with familial hypercholesterolemia encodes a transport-defective protein antennapedia homeobox peptide regulates neural morphogenesis identification and molecular characterisation of a calm-af fusion in acute megakaryoblastic leukaemia mutations in a sar gtpase of copii vesicles are associated with lipid absorption disorders substitution of arginine for histidine at position in the alpha-subunit of the human insulin receptor. a mutation that impairs receptor dimerization and transport of receptors to the cell surface distinct sets of sec genes govern transport vesicle formation and fusion early in the secretory pathway metabolic and molecular bases of menkes disease and occipital horn syndrome receptor downregulation and multivesicular-body sorting stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls positionally cloned gene for a novel glomerular protein-nephrin-is mutated in congenital nephrotic syndrome endocrinopathies in the family of endoplasmic reticulum (er) storage diseases: disorders of protein traycking and the role of er molecular chaperones a conditional mutation avecting localization of the menkes disease copper atpase. suppression by copper supplementation molecular characterization of mammalian homologues of class c vps proteins that interact with syntaxin- three ways to make a vesicle cellular autophagy: surrender, avoidance and subversion by microorganisms proteasome inhibitors: from research tools to drug candidates a cholesterol-lowering gene maps to chromosome q a controlled study of adenoviral-vector-mediated gene transfer in the nasal epithelium of patients with cystic fibrosis a germline insertion in the tuberous sclerosis (tsc ) gene gives rise to the eker rat model of dominantly inherited cancer disappearance and reformation of synaptic vesicle membrane upon transmitter release observed under reversible blockage of membrane retrieval possible temperature-dependent blockage of synaptic vesicle recycling induced by a single gene mutation in drosophila a post-docking role for active zone protein rim hiv interaction with endosomes in macrophages and dendritic cells organization of the pronephric filtration apparatus in zebrafish requires nephrin, podocin and the ferm domain protein mosaic eyes cdc controls secretory and endocytic transport to the basolateral plasma membrane of mdck cells copii-cargo interactions direct protein sorting into er-derived transport vesicles akt crystal structure and akt-specific inhibitors defective human ether-a-go-go-related gene traycking linked to an endoplasmic reticulum retention signal in the c terminus retroviral vectors vip , a -kd membrane protein is an integral component of trans-golginetwork-derived transport vesicles regulation of vascular endothelial growth factor receptor- activity by caveolin- and plasma membrane cholesterol intracellular localization and loss of copper responsiveness of mnk, the murine homologue of the menkes protein, in cells from blotchy (mo blo) and brindled (mo br) mouse mutants interleukin receptors and detergent-resistant membrane domains define a clathrinindependent endocytic pathway aberrant splicing but not mutations of tsg in human breast cancer tumor cell growth inhibition by caveolin re-expression in human breast cancer cells bi-directional protein transport between the er and golgi a missense mutation in the low density lipoprotein receptor gene causes familial hypercholesterolemia in sephardic jews novel mutations in the wiskott-aldrich syndrome protein gene and their evects on transcriptional, translational, and clinical phenotypes niemann-pick disease: a frequent missense mutation in the acid sphingomyelinase gene of ashkenazi jewish type a and b patients evidence for a regulated interaction between heterotrimeric g proteins and caveolin a huntingtin-associated protein enriched in brain with implications for pathology expression and characterization of recombinant caveolin hermansky-pudlak syndrome type (hps- ) results from mutant dysbindin molecular, anatomical, and biochemical events associated with neurodegeneration in mice with niemann-pick type c disease the listeria protein internalin b mimics hepatocyte growth factor-induced receptor traycking bone marrow transplantation from an hla-matched unrelated donor for treatment of chediak-higashi syndrome familial sjogren's syndrome with associated primary salivary gland lymphoma protein kinase d regulates the fission of cell surface destined transport carriers from the trans-golgi network rme- regulates the distribution and function of the endocytic recycling compartment in mammalian cells caveolae, caveolin and caveolin-rich membrane domains: a signalling hypothesis organized endothelial cell surface signal transduction in caveolae distinct from glycosylphosphatidylinositol-anchored protein microdomains mutant myosin viia causes defective melanosome distribution in the rpe of shaker- mice rescue of deltaf and other misprocessed cftr mutants by a novel quinazoline compound organic-aciduria, decreased renal ammonia production, hydrophthalmos, and mental retardation; a clinical entity structure and function of the lowe syndrome protein ocrl autoantigen golgin- , an evector of arl gtpase, participates in trayc from the endosome to the trans-golgi network functional evaluation of dent's disease-causing mutations: implications for clc- channel traycking and internalization regulation of protein transport from the golgi complex to the endoplasmic reticulum by cdc and n-wasp the beta-appendages of the four adaptor-protein (ap) complexes: structure and binding properties, and identification of sorting nexin as an accessory protein to ap- function and regulation of the mammalian coppertransporting atpases: insights from biochemical and cell biological approaches membrane dynamics and the biogenesis of lysosomes rabaptin- is a novel fusion partner to platelet-derived growth factor beta receptor in chronic myelomonocytic leukemia kdel-cargo regulates interactions between proteins involved in copi vesicle trayc: measurements in living cells using fret purification of a novel class of coated vesicles mediating biosynthetic protein transport through the golgi stack clogging of axons by tau, inhibition of axonal trayc and starvation of synapses endocytosis and targeting of exogenous hiv- tat protein a novel point mutation in cd causing the expression of dysfunctional cd /cd leucocyte integrins in a patient with leucocyte adhesion deficiency (lad) copii-coated vesicle formation reconstituted with purified coat proteins and chemically defined liposomes altered traycking and adhesion function of mpz mutations and phenotypes of charcot-marie-tooth disease b biology of adenovirus and its use as a vector for gene therapy the putative tumor suppressors ext and ext form a stable complex that accumulates in the golgi apparatus and catalyzes the synthesis of heparan sulfate congenital hypothyroid goiter with deficient thyroglobulin. identification of an endoplasmic reticulum storage disease with induction of molecular chaperones mutations in rab a cause griscelli syndrome associated with haemophagocytic syndrome myoblast transfer in the treatment of duchenne's muscular dystrophy enzyme replacement therapy in fabry's disease: recent advances and clinical applications signalling to actin assembly via the wasp (wiskott-aldrich syndrome protein)-family proteins and the arp / complex mutations in the caveolin- gene cause autosomal dominant limb-girdle muscular dystrophy long-term evectiveness of a new alpha-glucosidase inhibitor (bay m -miglitol) in insulin-treated type diabetes mellitus genetic disorders avecting proteins of iron and copper metabolism: clinical implications hsp inhibitor geldanamycin and its derivatives as novel cancer chemotherapeutic agents mechanism of microtubule stabilization by doublecortin identification and functional analysis of two novel mutations in the multidrug resistance protein gene in israeli patients with dubin-johnson syndrome pharmacological chaperones: a new twist on receptor folding a conserved clathrin assembly motif essential for synaptic vesicle endocytosis eea , an early endosome-associated protein. eea is a conserved alpha-helical peripheral membrane protein flanked by cysteine ''fingers'' and contains a calmodulin-binding iq motif localization of proteins to the golgi apparatus vip /caveolin is a cholesterol-binding protein maturation models for endosome and lysosome biogenesis cdc regulates the exit of apical and basolateral proteins from the trans-golgi network consistent detection of calm-af chimaeric transcripts in haematological malignancies with t( ; )(p ;q ) and identification of novel transcripts molecular analysis of the ergic- gene in families with combined factor v-factor viii deficiency vascular endothelial growth factor (vegf) and its receptors the trojan horse: survival tactics of pathogenic mycobacteria in macrophages snares and membrane fusion in the golgi apparatus mutations in the er-golgi intermediate compartment protein ergic- cause combined deficiency of coagulation factors v and viii regulation of phagocytosis by rho gtpases urinary megalin deficiency implicates abnormal tubular endocytic function in fanconi syndrome wiskott-aldrich syndrome: a model for defective actin reorganization, cell traycking and synapse formation identification of complementation groups required for post-translational events in the yeast secretory pathway cellular uptake of an alpha-helical amphipathic model peptide with the potential to deliver polar compounds into the cell interior non-endocytically novel cell permeable motif derived from the pres -domain of hepatitis-b virus surface antigens dynamin at the neck of caveolae mediates their budding to form transport vesicles by gtp-driven fission from the plasma membrane of endothelium the proteasome is involved in angiogenesis role of rab gtpases in membrane trayc the evolving role of lipid rafts and caveolae in g proteincoupled receptor signaling: implications for molecular pharmacology the structure and function of the beta -adaptin appendage domain phosphatidylinositol phosphate -kinase i{beta} recruits ap- to the plasma membrane and regulates rates of constitutive endocytosis fine structure of blood capillaries intracellular aspects of the process of protein synthesis two genes are responsible for griscelli syndrome at the same q locus localization and processing of cln , the protein associated to batten disease: where is it and what does it do? localization of the ap- adaptor complex defines a novel endosomal exit site for lysosomal membrane proteins infectious hiv- assembles in late endosomes in primary macrophages snares and the specificity of transport vesicle targeting caveolar endocytosis of simian virus reveals a new two-step vesicular-transport pathway to the er caveolin-stabilized membrane domains as multifunctional transport and sorting devices in endocytic membrane trayc alpha -antitrypsin deficiency: liver disease associated with retention of a mutant secretory glycoprotein in the endoplasmic reticulum alpha- -antitrypsin deficiency: diagnosis and treatment identification of the murine beige gene by yac complementation and positional cloning the menkes protein (atp a; mnk) cycles via the plasma membrane both in basal and elevated extracellular copper using a c-terminal dileucine endocytic signal participation of the endoplasmic reticulum chaperone calnexin (p , ip ) in the biogenesis of the cystic fibrosis transmembrane conductance regulator prevention of lysosomal storage in tay-sachs mice treated with n-butyldeoxynojirimycin correction of a mineralization defect by overexpression of a wild-type cdna for col a in marrow stromal cells (mscs) from a patient with osteogenesis imperfecta: a strategy for rescuing mutations that produce dominant-negative protein defects constitutive protein secretion from the trans-golgi network to the plasma membrane review of donepezil, rivastigmine, galantamine and memantine for the treatment of dementia in alzheimer's disease in adults with down syndrome: implications for the intellectual disability population protein kinase d-mediated anterograde membrane traycking is required for fibroblast motility gene replacement reveals that p /snare interactions are essential for golgi biogenesis constitutive skipping of alternatively spliced exon in the atp a gene abolishes golgi localization of the menkes protein and produces the occipital horn syndrome traycking and folding defects in hereditary spherocytosis mutants of the human red cell anion exchanger caveolin- -deficient mice show evidence of severe pulmonary dysfunction without disruption of caveolae isolation of a miller-dicker lissencephaly gene containing g protein [beta]-subunit-like repeats myosin viia is required for aminoglycoside accumulation in cochlear hair cells endophilin/sh p is required for the transition from early to late stages in clathrinmediated synaptic vesicle endocytosis mechanisms of cell-surface rerouting of an endoplasmic reticulum-retained mutant of the vasopressin v b/v receptor by a pharmacological chaperone adaptor-related proteins adaptable adaptors for coated vesicles surfing the sec channel: bidirectional protein translocation across the er membrane yolk protein uptake in the oocyte of the mosquito aedes aegypti caveolin, a protein component of caveolae membrane coats mechanisms of intracellular protein transport protein sorting by transport vesicles early stages of influenza virus entry into mv- lung cells: involvement of dynamin science medicine, and the future. gene therapy endoplasmic reticulum storage diseases calm-af fusion gene in leukemias: simple and inversion-associated translocation ( ; ) assembly of the er to golgi snare complex requires uso p signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in mdck cells regulation of the unc- -caenorhabditis elegans syntaxin complex by unc- visualization of er-to-golgi transport in living cells reveals a sequential mode of action for copii and copi identification, sequence, and expression of caveolin- defines a caveolin gene family an enzyme that removes clathrin coats: purification of an uncoating atpase endophilin i mediates synaptic vesicle formation by transfer of arachidonate to lysophosphatidic acid endoplasmic reticulumlocalized amyloid beta-peptide is degraded in the cytosol by two distinct degradation pathways endothelial caveolae have the molecular transport machinery for vesicle budding, docking, and fusion including vamp, nsf, snap, annexins, and gtpases griscelli syndrome: report of the first peripheral blood stem cell transplant and the role of mutations in the rab a gene as an indication for bmt peptide-mediated cellular delivery gene therapy: a brief overview of the past, present, and future phagosome maturation: a few bugs in the system the npc protein: structure implies function rab gtpases, intracellular trayc and disease a ypt/rab evector complex containing the sec homolog vps p is required for homotypic vacuole fusion the role of the tethering proteins p and gm in transport through the golgi apparatus in vivo early endosome antigen. : an autoantigen associated with neurological diseases identification of the b-cell epitopes of the early endosome antigen (eea ) cloning and gene defects in microsomal triglyceride transfer protein associated with abetalipoproteinaemia diverential evects of kinase cascade inhibitors on neoplastic and cytokine-mediated cell proliferation chediak-higashi syndrome: a rare disorder of lysosomes and lysosome related organelles autophagy in health and disease: a double-edged sword golgins and gtpases, giving identity and structure to the golgi apparatus a role for the vesicle tethering protein, p , in the postmitotic stacking of reassembling golgi cisternae in a cell-free system sequential tethering of golgins and catalysis of snarepin assembly by the vesicle-tethering protein p transcytosis of plasma macromolecules in endothelial cells: a cell biological survey regulation of cytoplasmic dynein behaviour and microtubule organization by mammalian lis snap receptors implicated in vesicle targeting and fusion paraneoplastic stiv-person syndrome: passive transfer to rats by means of igg antibodies to amphiphysin a role for giantin in docking copi vesicles to golgi membranes phenotypic behavior of caveolin- r q, a mutant associated with hyperckemia, distal myopathy, and rippling muscle disease phosphofructokinase muscle-specific isoform requires caveolin- expression for plasma membrane recruitment and caveolar targeting: implications for the pathogenesis of caveolin-related muscle diseases identification of filamin as a novel ligand for caveolin- : evidence for the organization of caveolin- -associated membrane domains by the actin cytoskeleton endosomal localization of the autoantigen eea is mediated by a zinc-binding fyve finger perforin gene defects in familial hemophagocytic lymphohistiocytosis copicoated er-to-golgi transport complexes segregate from copii in close proximity to er exit sites the immunological synapse of ctl contains a secretory domain and membrane bridges linking albinism and immunity: the secrets of secretory lysosomes the biogenesis of lysosomes: is it a kiss and run, continuous fusion and fission process? breaking the copi monopoly on golgi recycling a family of rab -binding proteins. melanophilin links rab a and myosin va function in melanosome transport retinal stimulates atp hydrolysis by purified and reconstituted abcr, the photoreceptor-specific atp-binding cassette transporter responsible for stargardt disease abnormal vesicular traycking in mouse models of hermansky-pudlak syndrome protein folding and translocation across the endoplasmic reticulum membrane role of calnexin in the glycan-independent quality control of proteolipid protein assembly and traycking of caveolar domains in the cell: caveolae as stable, cargo-triggered, vesicular transporters identification and expression of five mutations in the human acid sphingomyelinase gene causing types a and b niemann-pick disease. molecular evidence for genetic heterogeneity in the neuronopathic and non-neuronopathic forms defective aquaporin- traycking in nephrogenic diabetes insipidus and correction by chemical chaperones misfolding of mutant aquaporin- water channels in nephrogenic diabetes insipidus molecular cloning of caveolin- , a novel member of the caveolin gene family expressed predominantly in muscle copii and exit from the endoplasmic reticulum identification of a familial hyperinsulinism-causing mutation in the sulfonylurea receptor that prevents normal traycking and function of katp channels mutations of the pds gene, encoding pendrin, are associated with protein mislocalization and loss of iodide ezux: implications for thyroid dysfunction in pendred syndrome the chediak-higashi protein interacts with snare complex and signal transduction proteins clathrin assembly lymphoid myeloid leukemia (calm) protein: localization in endocytic-coated pits, interactions with clathrin, and the impact of overexpression on clathrin-mediated trayc how peroxisomes arise chloride channels cough up caveolae are highly immobile plasma membrane microdomains, which are not involved in constitutive endocytic traycking tau regulates the attachment/detachment but not the speed of motors in microtubule-dependent transport of single vesicles and organelles gangliosides are receptors for murine polyoma virus and sv protein traycking and alzheimer's disease role of auxilin in uncoating clathrincoated vesicles the inositol polyphosphate -phosphatase ocrl associates with endosomes that are partially coated with clathrin actin microfilaments facilitate the retrograde transport from the golgi complex to the endoplasmic reticulum in mammalian cells dynamin-like protein encoded by the drosophila shibire gene associated with vesicular trayc protein kinase d: an intracellular trayc regulator on the move fcgammar polymorphisms: implications for function, disease susceptibility and immunotherapy a dileucine-like sorting signal directs transport into an ap- -dependent, clathrin-independent pathway to the yeast vacuole protein kinase g from pathogenic mycobacteria promotes survival within macrophages degradation of cftr by the ubiquitinproteasome pathway use of expression constructs to dissect the functional domains of the chs/beige protein: identification of multiple phenotypes a novel -kd peripheral membrane protein is required for intercisternal transport in the golgi stack rab coordinates a novel golgi to er retrograde transport pathway in live cells hsp and the chaperoning of cancer sec -mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction the human cytomegalovirus us gene product dislocates mhc class i heavy chains from the endoplasmic reticulum to the cytosol amphiphysin heterodimers: potential role in clathrin-mediated endocytosis the caveolin genes: from cell biology to medicine a mutation in rab a causes the vesicle transport defects observed in ashen mice caveolinopathies: mutations in caveolin- cause four distinct autosomal dominant muscle diseases four contiguous amino acid substitutions, identified in patients with laron syndrome, diverently avect the binding aynity and intracellular traycking of the growth hormone receptor synaptojanin is the major constitutively active phosphatidylinositol- , , -trisphosphate -phosphatase in rodent brain rab a enables myosin va-dependent melanosome capture by recruiting the myosin to the organelle identification of an organelle receptor for myosin-va the tuberous sclerosis gene product, tuberin, functions as a rab gtpase activating protein (gap) in modulating endocytosis the fine structure of the gall bladder epithelium of the mouse caveolin is an inhibitor of platelet-derived growth factor receptor signaling sulfonylureas correct traycking defects of atp-sensitive potassium channels caused by mutations in the sulfonylurea receptor nsec binds a closed conformation of syntaxin a abnormal ryanodine receptor function in heart failure fabry disease: characterization of alpha-galactosidase a double mutations and the d y plasma enzyme pseudodeficiency allele protein kinase d regulates basolateral membrane protein exit from trans-golgi network predisposition to renal carcinoma in the eker rat is determined by germ-line mutation of the tuberous sclerosis (tsc ) gene adenovirus-mediated gene transfer transiently corrects the chloride transport defect in nasal epithelia of patients with cystic fibrosis rab proteins as membrane organizers bleeding due to disruption of a cargo-specific er-to-golgi transport complex charcot-marie-tooth disease type a caused by mutation in a microtubule motor kif b the lebanese allele at the low density lipoprotein receptor locus. nonsense mutation produces truncated receptor that is retained in endoplasmic reticulum sucrase-isomaltase deficiency in humans. diverent mutations disrupt intracellular transport, processing, and function of an intestinal brush border enzyme surfing the sec channel: bidirectional protein translocation across the er membrane crystal structure of an activated akt/protein kinase b ternary complex with gsk -peptide and amp-pnp key: cord- -h aozg authors: modis, yorgo title: class ii fusion proteins date: - - journal: viral entry into host cells doi: . / - - - - _ sha: doc_id: cord_uid: h aozg enveloped viruses rely on fusion proteins in their envelope to fuse the viral membrane to the host-cell membrane. this key step in viral entry delivers the viral genome into the cytoplasm for replication. although class ii fusion proteins are genetically and structurally unrelated to class i fusion proteins, they use the same physical principles and topology as other fusion proteins to drive membrane fusion. exposure of a fusion loop first allows it to insert into the host-cell membrane. conserved hydrophobic residues in the fusion loop act as an anchor, which penetrates only partway into the outer bilayer leaflet of the host-cell membrane. subsequent folding back of the fusion protein on itself directs the c-terminal viral transmembrane anchor towards the fusion loop. this fold-back forces the host-cell membrane (held by the fusion loop) and the viral membrane (held by the c-terminal transmembrane anchor) against each other, resulting in membrane fusion. in class ii fusion proteins, the fold-back is triggered by the reduced ph of an endosome, and is accompanied by the assembly of fusion protein monomers into trimers. the fold-back occurs by domain rearrangement rather than by an extensive refolding of secondary structure, but this domain rearrangement and the assembly of monomers into trimers together bury a large surface area. the energy that is thus released exerts a bending force on the apposed viral and cellular membranes, causing them to bend towards each other and, eventually, to fuse. enveloped viruses acquire a lipid bilayer membrane when they bud across the plasma membrane or the membrane of the endoplasmic reticulum (er) during assembly of the virion. , during infection, the viral membrane must be fused to the host-cell membrane to deliver the viral genome into the cytoplasm for replication (fig. ) . the fusion of the viral and host-cell membranes is therefore the central molecular event during the entry of viral entry into host cells, edited by stefan pöhlmann and graham simmons. © landes bioscience and springer science+business media. enveloped viruses into cells. adjacent membranes do not fuse spontaneously; membrane fusion requires considerable energy (on the order of kj mol - or kt). , envelope proteins anchored in the viral membrane provide this energy in the form of a conformational rearrangement that bends the apposed membranes towards each other, inducing them to fuse. [ ] [ ] [ ] most 'fusion proteins' (or their cleavage products) also effect cellular attachment of the virus prior to the membrane fusion event by binding to a receptor on the cell surface, except the paramyxo-and alphaviruses, in which a second envelope protein binds the receptor. fusion proteins of enveloped viruses fall into two structural classes. the influenza virus haemagglutinin (ha) is the prototype of class i fusion proteins, which encompass those of other orthomyxo-and paramyxoviruses such as measles virus, retroviruses such as human immunodeficiency virus (hiv), filoviruses such as ebola virus, and coronaviruses such as sars (see . class ii fusion proteins are a structurally and evolutionarily distinct class of proteins found in flaviviridae, such as dengue, yellow fever, and west nile viruses, and on alphaviruses, such as semliki forest and sindbis viruses. hepatitis c has a similar genomic organization to the flaviviruses, and therefore most likely relies on a class ii fusion protein as well. crystal structures of several class i and class ii fusion proteins before [ ] [ ] [ ] [ ] [ ] [ ] [ ] and after , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] their fusogenic conformational rearrangements have provided us with a detailed molecular understanding of the fusion mechanism ( table ). the structures show that, despite the absence of similarities in the protein folds of the two classes, fusion proteins from both classes use the same physical principles and general topology to drive membrane fusion. first the fusion protein inserts a hydrophobic fusion anchor partway into the outer bilayer leaflet of the host-cell membrane. the fusion anchor is either an n-terminal peptide, as in influenza and hiv, or an internal loop, as in sars coronavirus, avian sarcoma leucosis virus and all class ii enveloped viruses. second, the fusion protein folds back on itself, directing the (c-terminal) viral transmembrane anchor towards the fusion anchor. this fold-back forces the host-cell membrane (held by the fusion anchor) and the viral membrane (held by the c-terminal transmembrane anchor) against each other, resulting in fusion of the two membranes. in this chapter, i describe our current picture of how class ii fusion proteins drive viral membrane fusion, based on the structural and biochemical data available to date. three-dimensional structures of eight class ii fusion proteins in their native, or prefusion states, , [ ] [ ] [ ] , [ ] [ ] [ ] that is, the conformation that they adopt on the surface of a mature virus particle, have been determined at near atomic resolution. figure shows the three-domain structures of e and e , the fusion proteins of dengue virus (a representative flavivirus) and of semliki forest virus (an alphavirus), respectively. the two proteins share a common molecular architecture, despite a lack of significant sequence similarity. domain i, an eight-stranded -barrel, organizes the structure. two long insertions between pairs of consecutive -strands in domain i form the elongated domain ii, which bears the fusion anchor, a fusion loop in class ii proteins, at its tip (figs. , ) . domain ii contains twelve -strands and two -helices. domain iii is an igc-like module, with ten -strands. domain iii contains most of the antigenic sites on e, as well as most of the structural determinants of virulence and tropism. this observation, and the widespread occurrence of immunoglobulin modules in cell-adhesion proteins, suggest that domain iii participates in attachment to a cellular receptor. indeed, positively charged patches on the surface of domain iii in dengue virus have been suggested to promote attachment by binding heparan sulfate on the cell surface. both e and e have one or more glycosylation sites. these glycans can aid viral attachment to the cell surface, in as found in the mature virus particle. the fusion loop in a-c is marked with an asterisk. a second subunit of e, forming the dimer found on the viral surface and in solution, is shown in light gray. d) view rotated ° relative to c, with the second subunit omitted for clarity. e) crystal structure of sfv e in the prefusion conformation, as found in the mature virus particle. the fusion loop is marked with an asterisk. f) view rotated ° relative to e. the case of dengue virus by binding to the lectin dc-sign. , as expected from their differ only in the length and structure of surface-exposed loops, some of which have been implicated in receptor binding. , , despite these hints on the basis of cellular attachment, however, a cellular receptor that specifically recognizes an envelope protein on a class ii enveloped virus has yet to be conclusively identified, although candidate receptors for dengue virus type and west nile virus were recently suggested. it is important to note that all the crystal structures of fusion proteins determined so far, from both classes and regardless of their conformational state, lack the c-terminal viral membrane anchor. this anchor consists of one or two transmembrane helices, and has been intentionally omitted in constructs targeted for crystallization to facilitate expression and handling, and to promote crystallization. the crystallized species are therefore referred to as soluble fragments of the ectodomains of the full-length fusion protein. furthermore, all available crystal structures of class ii fusion proteins lack the 'stem' region, a - amino acid linker between domain iii and the c-terminal transmembrane anchor (figs. a-b, ). as i will discuss below, the stem region plays a key role in the final stages of membrane fusion. its function is analogous to that of the 'outer helix' in class i fusion proteins. both class i and class ii fusion proteins rely on a proteolytic cleavage event to become primed to respond to the environmental conditions appropriate for fusion. these conditions are usually the acidic ph of an endosome ( fig. ), but for some class i enveloped viruses, such as hiv, coreceptor binding is required instead. in contrast to class i fusion proteins, however, class ii fusion proteins rely on a priming proteolytic cleavage that does not cleave the fusion protein itself. instead, class ii proteins associate with a second, 'protector' protein, called m (for membrane protein) in flaviviruses or e in alphaviruses. the protector protein is cleaved by furin when immature virus particles assembled in the er reach the trans-golgi network. the cleavage produces mature virus particles, which are then released from the host cell by exocytosis. the cleavage of the protector protein releases a conformational constraint on the fusion protein, which allows it to adopt its mature conformation (described above) in a large rearrangement on the viral surface. in the mature conformation, the fusion protein is primed to respond to acidic ph and induce membrane fusion with a further conformational rearrangement (described below). structures from electron cryomicroscopy of both immature , and mature - flavivirus and alphavirus particles, provide a detailed picture of the rearrangement that accompanies maturation in these viruses. alphaviruses retain the t = icosahedral packing of their envelope proteins, but domains that form spikes on the immature virion swing in towards the threefold symmetry axis, during maturation. , the rearrangement is more dramatic in flaviviruses, in which the fusion protein e breaks the t = icosahedral symmetry of the immature virion to adopt an unusual icosahedral herringbone pattern in the mature virion. , in both alphaviruses and flaviviruses, the fusion proteins form dimers in the mature virion albeit in different configurations. , the key feature of the maturation process in both genera, however, is that cleavage of the protector protein allows the fusion loop to reposition itself so that it is poised to insert into the host-cell membrane in response to acidification of the solute in the endosome. mature virus particles are sequence identities (≥ %), flaviviral e proteins have very similar overall structures, and therefore infectious, , unlike immature virions, , which are insensitive to ph. the fusion loop is shielded from the viral surface in mature virions by e-e dimer contacts in flaviviruses, or by protein e in alphaviruses (figs. a,b, a). the three-dimensional structures of four class ii fusion proteins in their postfusion states , , , reveal striking differences from the prefusion forms (fig. ) , and suggest a and b) two sfv e molecules in the prefusion conformation as found on the viral surface, viewed perpendicular to the viral membrane. the fusion loop is buried, either in the dimer interface (a), or under e (b). the outer (proximal) bilayer leaflets of the cellular and viral membranes are shown to scale as solid rectangles. the thin outer layer within each leaflet represents the polar headgroup layer, and the thicker inner layer represents the hydrocarbon layer. the stem-anchor segments are absent from the crystal structure, but are represented here schematically as rods in the viral membrane. c,d) upon acidification of the solute in the endosome, the domain ii rotates - ° about a hinge in the domain ii-domain i interface. this exposes the fusion loop, which then inserts into the host cell membrane. the postfusion, trimeric structures of den e (e) and sfv e (f). after insertion of their fusion loops into the target membrane, the fusion proteins form trimers and fold back on themselves, bringing the fusion loops close to the c-terminal transmembrane anchors. molecular mechanism for membrane fusion (see below and fig. ). like class i fusion proteins, flaviviral e proteins and alphaviral e proteins are both homotrimers in their postfusion conformations. class ii proteins form trimers from monomers on the viral surface, while class i proteins are trimeric in their prefusion state. however, a comparison of the pre-and postfusion states of influenza ha-the only example in its class where both structures are known for the same protein-shows that, as in class ii fusion proteins, nearly all of the trimer contacts in the postfusion state are formed during the fusogenic conformational rearrangement. unlike influenza ha, which undergoes extensive refolding during membrane fusion, the three domains of class ii fusion proteins retain most of their folded structures (fig. ) . instead, the domains undergo major rearrangements in their relative orientations, through flexion of the interdomain linkers. domain iii undergoes the most significant displacement in the fusion transition. it rotates by about °, and its center of mass shifts by - Å towards domain ii. this folding-over brings the c-terminus of domain iii about Å closer to the fusion loop, at tip of domain ii (fig. ) . domain ii rotates - ° with respect to domain i about a hinge region in which mutations affect the ph threshold of fusion in various flaviviruses. [ ] [ ] [ ] [ ] [ ] [ ] these conformational rearrangements position the end of domain iii-and the beginning of the stem region that links domain iii to the c-terminal viral transmembrane anchor-towards the fusion loop ( fig. e-f) . , a deep channel extends from the c-terminus of the crystallized fragment along the intersubunit contact between domains ii to the fusion loops, in both the dengue and semliki forest virus postfusion trimer structures. in the full-length fusion proteins, it is thought that the stem binds in this channel in an extended, but mainly -helical conformation. , this proposed stem conformation places the viral transmembrane anchor in the immediate vicinity of the fusion loop, just as in the postfusion conformation of class i viral fusion proteins. the fusion transition in class ii viral proteins is irreversible. the refoldings just described may impart irreversibility by contributing a high barrier to initiation of trimerization and an even higher barrier to dissociation of postfusion trimers once they have formed. moreover, many new polar and nonpolar contacts are formed during the fusion transition, in several different areas along the threefold axis of the trimer. the total surface buried is , - , Å , , nearly four times more than is buried in the prefusion dimer. the stem, which is missing from currently available crystal structures, most likely forms additional contacts with the core trimer structure. the stem does indeed promote trimer assembly even in the absence of liposomes. the process of viral membrane fusion in both class i and class ii enveloped viruses begins with the exposure of a fusion anchor, and its subsequent insertion into the host-cell membrane. fusion anchors from both viral classes vary in length but are in general rich in glycines and hydrophobic residues, particularly aromatic residues such as trp or phe. sequence conservation is poor between fusion proteins of both classes. the fusion anchor in class i fusion proteins-the 'fusion peptide'-is a region of approximately residues at or near the n-terminus of the envelope protein. the crystal structure of the parainfluenza virus fusion (f) protein in its prefusion form reveals the fusion peptide wedged between two subunits of the protein, in a partly extended, partly -sheet and partly -helical conformation. structural studies on influenza ha in its postfusion conformation using nmr and other spectroscopic techniques show that the fusion peptide is mostly -helical in character and that its structure changes only subtly as it inserts partway into the outer leaflet of the host-cell lipid bilayer. , none of the currently available postfusion class i protein crystal structures contain information on the fusion peptide. the recently determined crystal structures of class ii fusion proteins in pre , - and postfusion , , conformations offer the first direct views of fusion anchors-in this case, the fusion loops-as they insert into a target membrane (fig. ) . like the class i fusion peptide, the class ii fusion loop penetrates only partway into the hydrocarbon layer of the target membrane. exposed carbonyls and charged residues prevent the fusion loop from penetrating further than Å. , in flaviviruses, the fusion loop adopts a tightly folded conformation, which is stabilized by a disulfide bond (fig. a) . the structure of the fusion loop is essentially identical in the pre-and postfusion conformations of the protein, suggesting that membrane insertion has no effect on the structure of the fusion loop. during the fusion transition, three hydrophobic residues in the fusion loop (trp, leu, and phe) become exposed on the molecular surface. three fusion loops end up in close proximity at the tip of the trimer in the postfusion conformation, where they form a crater-like surface with a hydrophobic rim (fig. e) . electron cryomicroscopy and mutagenesis studies confirm that these hydrophobic, mostly aromatic residues on the crater rim insert into the host-cell membrane, acting as an 'aromatic anchor' for the fusion protein. the concave shape of the crater is thought to be important in generating distortions or perturbations in the host-cell membrane, which are required for fusion. in alphaviruses, the fusion loop is also rich in aromatic and other hydrophobic residues. unlike flaviviral fusion loops, however, alphaviviral fusion loops do not form trimer contacts (fig. f) . indeed, in the postfusion structure of the semliki forest virus e trimer, the fusion loops have high temperature factors and exhibit a high degree of flexibility despite the presence of two disulfide bonds. thus, the structures of the fusion loops are poorly defined, but each fusion loop seems to adopt a very different conformation (fig. b,c) . the fusion loops in the postfusion semliki forest virus e structure form quite polar surfaces, with many mainchain carbonyls and some polar or charged sidechains exposed on the surface. this suggests that, in contrast to flaviviral fusion loops, alphaviral fusion loops either change their conformation upon membrane insertion to shield polar groups from the membrane, or the fusion loops only interact with the polar headgroups of the lipids, and do not penetrate into the hydrocarbon layer. semliki forest virus e trimers form irregular clusters, or 'rosettes' of about - trimers through contacts between fusion loops in adjacent trimers. this is reminiscent of influenza virus ha, which aggregates into rosettes through interactions between the fusion peptide, at low ph and after proteolytic activation. this fusion loop/peptide clustering may provide a mechanism for the direct coupling of several e /ha trimers to work in concert around a single fusion site (see below). combined with previous knowledge, the structures of the fusion proteins from class ii viruses in their postfusion states , , have led to a much better understanding of how conformational changes in the proteins drive membrane fusion. the structures confirm two major principles of membrane fusion machineries: ( ) the fusion protein must insert an anchor into each of the two membranes to be fused, and ( ) the protein folds back on itself in a thermodynamically favorable conformational rearrangement that drives membrane fusion by forcing the two anchors into close proximity. in the current model, viral membrane fusion proceeds as follows (fig. ) . first, receptor binding by an envelope protein, which in flaviviruses is also the fusion protein, leads to clathrin-mediated endocytosis of the virus (figs. , a ). when the virus reaches endosomal compartments the low ph of the lumen (ph ) causes an initial conformational rearrangement that leads to the exposure of the previously buried fusion loop , at the tip of domain ii. in flaviviruses, domains i and ii flex relative to each other by °. this hinge motion causes domain ii, and therefore the fusion loop, to swing away from the viral surface and towards the host-cell membrane (fig. b) . indeed, mutations at the domain i-domain ii interface in various flaviviruses alter the ph threshold of fusion. , [ ] [ ] [ ] [ ] [ ] [ ] as domain ii swings away from the viral surface, constraints imposed by the tight packing of e on the viral surface are released, allowing e to diffuse freely in the plane of the viral membrane. the stem may also be able to extend away from the membrane at this stage. in alphaviruses, constraints are released in response to low ph by the dissociation of the protector (and receptor-binding) protein e . this exposes the fusion loop and allows domain ii of e to swing towards the nearest threefold symmetry axis in the virus particle in a ° hinge motion relative to domain i, leading to the formation of trimer contacts with adjacent e molecules. the second key step in the fusion process is insertion of the exposed fusion loop into the host-cell membrane (fig. c) . alphaviral e has already formed some trimer contacts at this stage, but flaviviral e proteins probably insert their fusion loops as monomers. membrane insertion probably catalyzes trimerization of the fusion loops, by lateral rearrangement of e monomers. this trimeric prefusion intermediate (fig. c ) bridges host-cell and viral membranes, anchored by its fusion loops in the former and by the figure . proposed fusion mechanism for fusion mediated by class ii fusion proteins. a) the virus binds to a receptor on the cell surface. in flaviviruses, the fusion protein e binds the receptor, while in alphaviruses, the 'protector' protein e binds the receptor. following attachment, the virus is internalized to an endosome. b) acidic ph in the endosome causes domain ii to hinge outward from the virion surface, exposing the fusion loop, and allowing e monomers to rearrange laterally in the plane of the membrane. c) the fusion loop inserts into the hydrocarbon layer of the host-cell membrane, promoting trimer formation. d) formation of trimer contacts spreads from the fusion loop at the tip of the trimer, to the base of the trimer. the protein folds back on itself, directing the fusion loop towards the c-terminal transmembrane anchor. energy release by this refolding bends the apposed membranes. e) creation of additional trimer contacts between the stem-anchor and domain ii leads first to hemifusion and then (f) to formation of a lipidic fusion pore. viral transmembrane anchors in the latter. this proposed intermediate is analogous to the 'prehairpin' intermediate postulated for class i viral fusion mechanisms. upon insertion of the fusion loops into the host-cell membrane, formation of trimer contacts spreads from the fusion loops at the trimer tip to domain i at the trimer base. domain ii shifts and rotates, folding the stem and c-terminal anchor back towards the fusion loop (fig. d) , and burying additional protein surfaces. free energy released by this refolding drives the two membranes to bend towards each other, - as the c-terminal anchor is forced closer to the fusion loop, forming apposing nipples in the membranes (fig. d) . fusion-loop insertion may induce positive bilayer curvature, which would stabilize the lateral surfaces of the nipples. the concave shape of the crater-like surface formed by the fusion loops at the trimer tip may also have a destabilizing effect on the membrane, as has been postulated for fusion peptides in class i fusion proteins. based on the energy required to deform lipid bilayers, it seems likely that a ring of trimers refolding in concert is needed to provide sufficient energy to form nipples in the membranes. , it is unclear exactly how many trimers are needed to drive membrane fusion in class ii viruses, nor how their conformational changes are coupled. in the case of influenza, fusion requires the concerted action of at least three ha trimers, and is more likely driven by rings of - trimers. the clustering of fusion loops may provide a mechanism for the direct coupling of several e trimers to work in concert around a single fusion site in alphaviruses, but such clustering has not been observed in flaviviruses. it is possible that coupling occurs via the membrane: only when several trimers fold back in concert can they overcome the resistance of the membrane to deformation and reach their final, most stable postfusion conformation (figs. d-f). as the fusion transition proceeds, the stem zippers up onto the core of the trimer, along a channel that spans domain ii, at the intersubunit contact regions (figs. , d-f) . the zippering up of the stem onto the domain ii forces the fusion loop and the viral transmembrane anchor closer and closer, until the proximal leaflets of the two membranes fuse to form a 'hemifusion stalk' (fig. e ). hemifusion is thought to be an essential intermediate of membrane fusion. , , (fig. e ) illustrates the need for shallow penetration of the viral fusion anchor into the host-cell membrane: assuming several trimers do in fact act in concert around a single fusion site, fusion anchors from different trimers would collide if they inserted beyond the outer (proximal) lipid bilayer leaflet. this constraint on the length of the fusion anchor holds true for both class i fusion peptides and class ii fusion loops. hemifusion stalks can 'flicker' open into narrow fusion pores. in order to prevent the transient fusion pores from closing, the stem must complete its zippering up onto the core of the trimer, and the c-terminal transmembrane anchor must migrate into the pore (fig. f) . indeed, the transition from hemifusion stalk to full fusion pore appears to require that the viral transmembrane anchor span the membrane completely, in all biological membrane fusion systems. thus, the replacement of the c-terminal transmembrane anchor of influenza ha with a glycosylphophatidylinositol (gpi) lipid anchor, [ ] [ ] [ ] or with a half-length protein -helical anchor, stalls the fusion reaction at the stage of hemifusion. other viral fusion proteins and cellular snare fusion proteins also require at least one transmembrane anchor. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] upon completion of fusion, the trimer has reached the conformation seen in the postfusion crystal structures. , , the stems (not present in the structures) are docked along the surface of domains ii, and the fusion loops and transmembrane anchors lie next to each other in the fused membrane (fig. f ). some class ii fusion proteins, including those of alphaviruses, can only fuse membranes containing cholesterol and sphingolipids. the structural basis for this requirement is still not well understood. several mutations in different regions of the semliki forest virus fusion protein e lower its dependence on cholesterol and/or sphingolipids for membrane fusion. , it is unclear, however, whether the lower dependence on cholesterol of these mutants is due to an apparent destabilization of the e homotrimer, or to the different physical properties of membranes lacking cholesterol and sphingolipids. in flaviviruses, cholesterol facilitates fusion, but neither cholesterol nor sphingolipids are essential for fusion. many class ii viruses, especially the flaviviruses, represent important human pathogens such as dengue, hepatitis c, yellow fever, west nile, japanese encephalitis and tick-borne encephalitis viruses. for most of these viruses, there are no specific treatments for infection, their control by vaccination has proved elusive, and the number of infections is on the rise. recently determined three-dimensional structures of class ii fusion proteins suggest new strategies for inhibiting viral entry by blocking membrane fusion. one such strategy stems for the discovery in dengue virus e of a long, tapering channel lined with hydrophobic side chains. in the crystal structure, the channel is occupied by a molecule of the detergent n-octyl--d-glucoside. in the absence of detergent, a -hairpin covering the channel swings towards the protein, and closes up the channel. the location of this 'ligand-binding pocket' at the domain i-domain ii interface coincides with that of mutations affecting the ph threshold of fusion in various flaviviruses. [ ] [ ] [ ] [ ] [ ] [ ] most of these mutations involve side chains lining the ligand-binding pocket. the postfusion structure of dengue virus e shows that this region acts as a hinge between domains i and ii during the fusogenic conformational rearrangement (see above). the opening up of a ligand-binding pocket just at the locus of a hinge suggests that compounds tightly inserted at this position might hinder the conformational changes required for membrane fusion (fig. a) . the mechanism of action of such compounds might resemble that of some of the well-studied antipicornaviral compounds, which block a concerted structural transition in the icosahedral assembly. alternatively, small molecules that pry open the -hairpin on binding in the pocket may inhibit infection by facilitating the low-ph conformational change, causing premature triggering. knowledge of the structure of the binding pocket with a bound ligand will guide efforts to design derivative ligands with higher affinities for use as inhibitors of flaviviral membrane fusion. the postfusion structures of dengue and semliki forest viruses suggest a second possible strategy for fusion inhibition, related to an approach successful in developing an hiv antiviral compound. peptides corresponding to the stem region of the gp fusion protein inhibit hiv entry by binding to the trimeric, n-terminal 'inner core' of the protein and interfering with the folding back against it of the stem and c-terminal viral transmembrane anchor. the way in which the stem is likely to fold back in class ii viral fusion proteins (figs. , d-f) suggests that an analogous strategy may be successful with class ii viruses. peptides derived from stem sequences could block completion of the fusogenic conformational change, by competing with the stem for interaction with surfaces on domain ii, at the trimer interface (fig. b) . stem-like peptides or peptidomimetic compounds could thus inhibit viral membrane fusion in class ii enveloped viruses by preventing the final folding back of the fusion protein that is required to drive the viral and host-cell membranes together to fuse. all viral membrane fusion proteins use the same physical principles and topology to drive membrane fusion. class ii fusion proteins are structurally and evolutionarily distinct class of proteins found in flaviviridae, such as dengue, yellow fever, and west nile viruses, and on alphaviruses, such as semliki forest and sindbis viruses. unlike class i fusion proteins such as influenza ha, which undergoes extensive refolding during membrane fusion, the three domains of class ii fusion proteins retain most of their folded structures. instead, the domains undergo major rearrangements in their relative orientations, through flexion of the interdomain linkers. class ii fusion proteins rely on a hydrophobic fusion loop to anchor themselves in the target cellular membrane. like the class i fusion peptide, the class ii fusion loop penetrates only partway into the hydrocarbon layer of the target membrane. class ii fusion proteins drive membrane fusion in a foldback rearrangement of a trimeric protein assembly. crystal structures of class ii envelope proteins have suggested two specific strategies for fusion inhibition, with hydrophobic small molecules and "stem"-like peptides or peptidomimetics, respectively. a) the discovery of a ligand-binding pocket at the interface between domains i and ii in dengue virus e, just at the locus of a hinge motion required for fusion, suggests that compounds inserted in the pocket might hinder the hinge motion and hence inhibit the fusion transition. this approach would block the first step in the fusion mechanism ( fig. a-b) . b) peptides corresponding to the stem region of the fusion protein may inhibit viral entry by binding to the trimeric core of the protein in its postfusion conformation, , and interfering with the folding back against it of the fusion protein's own stem. an analogous strategy has been successful with hiv gp . , this approach would block the last step in the fusion mechanism ( fig. e-f) . flaviviridae: the viruses and their replication togaviridae: the viruses and their replication a quantitative model for membrane fusion based on low-energy intermediates a mechanism of protein-mediated fusion: coupling between refolding of the influenza hemagglutinin and lipid rearrangements structural basis for paramyxovirus-mediated membrane fusion evidence that the transition of hiv- gp into a six-helix bundle, not the bundle configuration, induces membrane fusion membrane fusion machines of paramyxoviruses: capture of intermediates of fusion receptor binding and membrane fusion in virus entry: the influenza hemagglutinin structure of the haemagglutinin membrane glycoprotein of influenza virus at a resolution the envelope glycoprotein from tick-borne encephalitis virus at a resolution structure of the haemagglutinin-esterase-fusion glycoprotein of influenza c virus the fusion glycoprotein shell of semliki forest virus: an icosahedral assembly primed for fusogenic activation at endosomal ph a ligand-binding pocket in the dengue virus envelope glycoprotein variable surface epitopes in the crystal structure of dengue virus type envelope glycoprotein structure of the parainfluenza virus f protein in its metastable, prefusion conformation structure of influenza haemagglutinin at the ph of membrane fusion retrovirus envelope domain at . angstrom resolution core structure of gp from the hiv envelope glycoprotein atomic structure of a thermostable subdomain of hiv- gp atomic structure of the ectodomain from hiv- gp crystal structure of the simian immunodeficiency virus (siv) gp core: conserved helical interactions underlie the broad inhibitory activity of gp peptides three-dimensional solution structure of the kda ectodomain of siv gp crystal structure of the ebola virus membrane fusion subunit, gp , from the envelope glycoprotein ectodomain crystal structure of human t cell leukemia virus type gp ectodomain crystallized as a maltose-binding protein chimera reveals structural evolution of retroviral transmembrane proteins n-and c-terminal residues combine in the fusion-ph influenza hemagglutinin ha( ) subunit to form an n cap that terminates the triple-stranded coiled coil structural characterization of the human respiratory syncytial virus fusion protein core crystal structure of severe acute respiratory syndrome coronavirus spike protein fusion core structural basis for coronavirus-mediated membrane fusion. crystal structure of mouse hepatitis virus spike protein fusion core structure of the dengue virus envelope protein after membrane fusion purification of the fusion protein of sendai virus: analysis of the nh -terminal sequence generated during precursor activation detection of a fusion peptide sequence in the transmembrane protein of human immunodeficiency virus structure of a proteolytically resistant core from the severe acute respiratory syndrome coronavirus s fusion protein structure and membrane interaction of the internal fusion peptide of avian sarcoma leukosis virus mutational evidence for an internal fusion peptide in flavivirus envelope protein e dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate dendritic-cell-specific icam -grabbing nonintegrin is essential for the productive infection of human dendritic cells by mosquito-cell-derived dengue viruses dc-sign (cd ) mediates dengue virus infection of human dendritic cells monoclonal antibodies that bind to domain iii of dengue virus e glycoprotein are the most efficient blockers of virus adsorption to vero cells an external loop region of domain iii of dengue virus type envelope protein is involved in serotype-specific binding to mosquito but not mammalian cells serotype-specific entry of dengue virus into liver cells: identification of the -kilodalton/ -kilodalton high-affinity laminin receptor as a dengue virus serotype receptor interaction of west nile virus with alpha v beta integrin mediates virus entry into cells mapping of functional elements in the stem-anchor region of tick-borne encephalitis virus envelope protein e proteolytic activation of tick-borne encephalitis virus by furin structures of immature flavivirus particles the first step: activation of the semliki forest virus spike protein precursor causes a localized conformational change in the trimeric spike structure of dengue virus: implications for flavivirus organization, maturation, and fusion visualization of membrane protein domains by cryo-electron microscopy of dengue virus structure of west nile virus cryo-electron microscopy reveals the functional organization of an enveloped virus, semliki forest virus placement of the structural proteins in sindbis virus cleavage of protein prm is necessary for infection of bhk- cells by tick-borne encephalitis virus fusion activity of flaviviruses: comparison of mature and immature (prm-containing) tick-borne encephalitis virions the murray valley encephalitis virus prm protein confers acid resistance to virus particles and alters the expression of epitopes within the r domain of e glycoprotein conformational change and protein-protein interactions of the fusion protein of semliki forest virus structure of a flavivirus envelope glycoprotein in its low-ph-induced membrane fusion conformation nucleotide changes responsible for loss of neuroinvasiveness in japanese encephalitis virus neutralization-resistant mutants mutations in the envelope protein of japanese encephalitis virus affect entry into cultured cells and virulence in mice changes in the dengue virus major envelope protein on passaging and their localization on the three-dimensional structure of the protein epitopes on the dengue virus envelope protein recognized by neutralizing igm monoclonal antibodies attenuation of murray valley encephalitis virus by site-directed mutagenesis of the hinge and putative receptor-binding regions of the envelope protein single mutation in the flavivirus envelope protein hinge region increases neurovirulence for mice and monkeys but decreases viscerotropism for monkeys: relevance to development and safety testing of live, attenuated vaccines structure of an analog of fusion peptide from hemagglutinin membrane structure and fusion-triggering conformational change of the fusion domain from influenza hemagglutinin structure and function of membrane fusion peptides studies on the structure of the influenza virus haemagglutinin at the ph of membrane fusion membrane interactions of the tick-borne encephalitis virus fusion protein e at low ph hiv entry and its inhibition membrane fusion mediated by the influenza virus hemagglutinin requires the concerted action of at least three hemagglutinin trimers dilation of the influenza hemagglutinin fusion pore revealed by the kinetics of individual cell-cell fusion events hemifusion between cells expressing hemagglutinin of influenza virus and planar membranes can precede the formation of fusion pores that subsequently fully enlarge lipid-anchored influenza hemagglutinin promotes hemifusion, not complete fusion gpi-anchored influenza hemagglutinin induces hemifusion to both red blood cell and planar bilayer membranes meta-stability of the hemifusion intermediate induced by glycosylphosphatidylinositol-anchored influenza hemagglutinin the transmembrane domain of influenza hemagglutinin exhibits a stringent length requirement to support the hemifusion to fusion transition the caenorhabditis elegans unc- locus encodes a syntaxin that interacts genetically with synaptobrevin close is not enough: snare-dependent membrane fusion requires an active mechanism that transduces force to membrane anchors mutations within the putative membrane-spanning domain of the simian immunodeficiency virus transmembrane glycoprotein define the minimal requirements for fusion, incorporation, and infectivity deletion of the cytoplasmic tail of the fusion protein of the paramyxovirus simian virus affects fusion pore enlargement role of the cytoplasmic tail of ecotropic moloney murine leukemia virus env protein in fusion pore formation the role of the cytoplasmic tail region of influenza virus hemagglutinin in formation and growth of fusion pores truncation of the cooh-terminal region of the paramyxovirus sv fusion protein leads to hemifusion but not complete fusion functional analysis of the cytoplasmic tail of moloney murine leukemia virus envelope protein membrane fusion of semliki forest virus requires sphingolipids in the target membrane a single point mutation controls the cholesterol dependence of semliki forest virus entry and exit a conserved histidine in the ij loop of the semliki forest virus e protein plays an important role in membrane fusion novel mutations that control the sphingolipid and cholesterol dependence of the semliki forest virus fusion protein involvement of lipids in different steps of the flavivirus fusion mechanism the site of attachment in human rhinovirus for antiviral agents that inhibit uncoating inhibiting hiv- entry with fusion inhibitors potent suppression of hiv- replication in humans by t- , a peptide inhibitor of gp -mediated virus entry crystal structure of west nile virus envelope glycoprotein reveals viral surface epitopes crystal structure of dengue virus type envelope protein in the postfusion conformation and its implications for membrane fusion glycoprotein organization of chikungunya virus particles revealed by x-ray crystallography structural changes of envelope proteins during alphavirus fusion structural insights into the neutralization mechanism of a higher primate antibody against dengue virus crystal structure of the japanese encephalitis virus envelope protein this chapter was originally written in and was updated in to add recent relevant advances. key: cord- -vlk cebh authors: kolter, thomas title: ganglioside biochemistry date: - - journal: isrn biochem doi: . / / sha: doc_id: cord_uid: vlk cebh gangliosides are sialic acid-containing glycosphingolipids. they occur especially on the cellular surfaces of neuronal cells, where they form a complex pattern, but are also found in many other cell types. the paper provides a general overview on their structures, occurrence, and metabolism. key functional, biochemical, and pathobiochemical aspects are summarized. together with glycoproteins and glycosaminoglycans, glycosphingolipids (gsls) contribute to the glycocalyx that covers eukaryotic cell surfaces. gangliosides are sialic acidcontaining glycosphingolipids and provide a significant part of cell surface glycans on neuronal cells. gsls are lipids that contain a sphingoid base and one or more sugar residues [ ] . sialic acids ( figure ) are nine-carbon sugars biosynthetically formed from n-acetylmannosamine and phosphoenolpyruvate [ , ] . with a mean pk a value of around . , they are more acidic than the majority of carboxylic acids and negatively charged at most physiological ph values. the name "ganglioside" was coined by the german biochemist klenk ( klenk ( - and assigned to a group of acidic gsls that he isolated from ganglion cells [ , ] and from the brains of patients who suffered from the so-called amaurotic idiocy [ , ] . sialic acid was first isolated from submaxillary mucin in [ ] . its structure was elucidated in the nineteen fifties by different groups and it was found to be identical to that of the n-acetylneuraminic acid isolated by klenk and faillard. the first structure of a ganglioside was elucidated in by kuhn and wiegandt [ ] . in , svennerholm suggested a nomenclature of brain gangliosides [ , ] . the biochemical defects underlying the diseases formerly known as amaurotic idiocy, gm gangliosidosis [ ] , tay-sachs- [ ] , and sandhoff disease [ ] were identified by sandhoff and others in the s. in their structures, gangliosides combine a glycan and a lipid portion and contribute to both, the cellular lipidome and the glycome/sialome [ ] . a great variety of carbohydrate sequences are found within the gsls [ ] , including the gangliosides [ ] . although carbohydrate residues of different structure, linkage, and anomeric configuration occur in gsls, only a limited number of the so-called series with characteristic carbohydrate sequences are found within evolutionary related organisms (table ) . within the gangliosides, sialic acids can be attached only to a few of the gsl series, in adult mammals especially to the ganglio series. among the sialic acids, n-acetylneuraminic acid is the most frequently found member in humans, but also nglycolylneuraminic acid is abundant in many other species (figure ) . a total of more than different sialic acids have been described [ , ] . they can be o-acetylated in positions , , or [ ] , but also n-deacetylated, omethylated, sulfated, or modified by lactonization [ ] (see figure ). the nomenclature of gsls specifies the glycan part of these lipids. two ganglioside nomenclature systems are currently in use to assign names to the corresponding structures. most researches prefer the short-hand nomenclature according to svennerholm, which was initially based on the migration order of ganglio-series gangliosides in chromatography [ ] . later on, it has been extended to other root structures. the more comprehensive iupac system [ ] is less frequently applied. according to svennerholm, a core structure of neutral sugars define the name of a respective series, in which the pyranose forms of dgalactose (gal), d-n-acetyl-glucosamine (glcnac), or d-n-acetylgalactosamine (galnac) are attached in defined order and linkage to lactosylceramide (galβ , glcβ cer) or β-galactosylceramide (galβ cer). the names contain information about the series ("g" = ganglio, "l" = lacto), the number of sialic acids ("a" = , "m" = , "d" = , "t" = , "q" = , "p" = , "h" = , "s" = ), and, indirectly, on the number of uncharged carbohydrates: initially it has been assumed that this number cannot exceed , so that the name "ganglioside gm " indicates that this ganglioside contains ( − " " = ) neutral sugars of the ganglio series. this series is defined by the sequence galβ - galnacβ - galβ - glccer. sialic acids can be attached once, twice, or severalfold to different positions within the core structures. most often, they are found in α , -linkage to the "inner" or "outer" galactosyl residue, and in α , -linkage to other sialic acids. ganglioside gm bears one sialic acid moiety connected to the -oh-group of the galactosyl residue in position ii of the gangliotetraose moiety (see also figure ). the corresponding iupac-iubmb short-hand name is ii neu acgg cer. structures of ganglio-series gangliosides can also be derived from the scheme of ganglioside biosynthesis (see below; figure ). in general, ganglio-series gsls of the -series bear no sialic acids on the galactose in position ii, of the a-series bear one, of the b-series bear two, and of the c-series bear three sialic acid residues. however, gm b and gd c have a "b" and "c" in their names, although both are -series gangliosides (see the scheme of ganglioside biosynthesis, figure ). gm is a gala-series ganglioside, although the "g" suggests ganglio series. figure shows the structure of ganglioside gq b, one of the most abundant gangliosides in adult human brain (g = ganglio series, q = sialic acids, − = neutral carbohydrate residues, and bseries = sialic acids attached to the "inner" galactose). ganglioside core structures can be additionally modified; they can be elongated, such as in gd agalnac [ ] ( figure ). this ganglioside occurs, for example, on spinal neurons [ , ] and can give rise to autoantibodies as a cause of variant forms of the guillain-barré syndrome [ , ] and other neuropathies [ ] . a modified gm derivative that contains taurine in amide linkage to the sialic acid carboxyl group has been identified in the brain of patients with tay-sachs disease [ ] . hybrid-type gsls and gangliosides with postglycosylation modifications add further complexity to this substance class [ ] . as an example, lacto-ganglio hybrid-type gangliosides have been identified in bovine brain [ ] . most gangliosides found in adult mammals belong to the ganglio, gala, lacto, and neolacto series. ganglioside gm (figure ), a member of the gala series, has the structure neuacα , galβ cer and is often found with an α-hydroxyfatty acid within the ceramide moiety. during development, also gangliosides with other core structures are transiently formed, such as the stage-specific embryonic antigen ssea- , a ganglioside of the globo series [ ] (figure ). in adults, globo-series gangliosides occur on human erythrocytes [ ] , in human kidney [ ] , and on various stem cells [ ] . for example, ssea- , but not ssea- or globo-h (figure ), is expressed in cord blood-derived mesenchymal stem cells [ ] . with the exception of echinoderms (marine organisms of typically pentaradial symmetry), gangliosides are usually absent from invertebrates. arthropods, for example, form acidic gsls with a manβ , glcβ , cer core, which contain glucuronic acid instead of sialic acids. for gangliosides of echinoderms [ ] [ ] [ ] [ ] [ ] , there is no systematic short-hand nomenclature. they show structural features uncommon to mammalian gangliosides, such as sialic acid residues within the oligosaccharide moieties (e.g., lg- , figure ), α , linked sialic acids (e.g., llg- , figure ), sialic acid methylation or sulfation, or a glycosyl inositolphosphoceramide core, for example, [ , ] . in cultured neurons, echinodermal gangliosides show neuritogenic and growth-inhibitory activities. in this regard, they are more potent than other figure : structure of gq b, one of the most abundant gangliosides in adult human brain, which is involved in long term potentiation, synaptic plasticity, and improvement of cognitive function [ ] . gangliosides [ , ] and potentiate the neuritogenic effect of nerve growth factor. heterogeneity is not only found within the glycan part, but also within the ceramide moiety. this can consist of different sphingoid bases [ ] , sphinganine, sphingosine, and phytosphingosine of different chain lengths ( figure ), which can be further modified by o-acetylation [ ] . in higher animals, c -and c -sphingosine are the most abundant sphingoid bases of gangliosides. the fatty acids found in the ceramide part of gangliosides are mostly saturated. α-hydroxylated fatty acids [ ] are not frequently found in brain gangliosides, but are, for example, abundant in gangliosides from intestine, liver, or kidney, and in gm . to specify the lipoform of a ganglioside, designations such as (d : / : )gm are used for a ii neu aclaccer with a sphingosine (d = dihydroxy, = one double bond; see also figure ) of carbons and a stearoyl residue ( : ) within the ceramide portion. the functional consequences of the heterogeneities in the lipid component are largely unknown, but the lipid part can mask the receptor function of ganglioside glycans via interaction with membrane cholesterol [ , ] . as another example, the ceramide portion of gm dictates retrograde transport of cholera toxin bound to gm , and only gm with unsaturated acyl chains is sorted from the plasma membrane to the trans-golgi network and the er [ ] . ganglioside profiling with respect to glycan and ceramide structures is more and more in the focus of ganglioside analysis. gangliosides are especially abundant in the brain, where their occurrence in the grey matter is about -fold higher than in white matter. in adult human brain regions, the β galt-v β galnact figure : formation of ssea- from globotriaosylceramide (gb cer). ssea, stage-specific embryonic antigen; t, transferase; fut, fucosyltransferase (modified from [ ] figure : examples for gangliosides from echinoderms: lg- from the starfish astropecten latespinosus [ ] and llg- from the starfish linckia laevigata [ ] [ ] [ ] . d-ribo-phytosphingosine values range from to μg lipid-bound sialic acid/mg protein [ ] . in the brain, ganglioside expression correlates with neurogenesis, synaptogenesis, synaptic transmission, and cell proliferation [ , ] . in cultured murine hippocampal neurons, axonogenesis, but not dendritogenesis, is accompanied by an increase in the formation of complex gangliosides and by a shift from the a-to b-series [ ] . in extraneural tissues, the ganglioside content is one-to twoorders of magnitude lower than in the brain; relatively high concentrations of ganglio-series gangliosides are found in bone marrow, erythrocytes, intestine, liver, spleen, and testis, gm in kidney, and ssea- in embryonic stem cells. cellular gangliosides form in part complex, cell-type-and tissue-specific glycan patterns [ ] . these are not stable with time, but change with physiological and pathophysiological processes such as cell growth, differentiation, viral transformation, ontogenesis, oncogenesis, embryogenesis [ , ] , lactation, or tumor progression [ ] . gangliosides of the ganglio-series are especially found in the nervous system, where they contribute to - % of the lipid content [ ] . during brain development, the ganglioside pattern changes from the prevalence of the simple gangliosides gm and gd to more complex ones such as gd a and gt b [ ] (for structures, see figures and ) . ganglioside content and composition of the brain change also during aging: for example, the amount of lipid-bound sialic acid decreased from μg/g wet weight in a -year-old healthy proband to μg/g wet weight in a -year-old individual. despite this, the concentrations of gq b, gt b, and gd b increase with age at the expense of gm and gd a [ ] (for structures, see figures and ) . changes in ganglioside composition with age also occur in liver [ ] . there are only indications on the functional consequences of such changes [ ] . gangliosides are also found in serum. there, especially gm , gd , gd a, gm , gt b, sialylneolactotetraosylceramide ( figure ), gd b, and gq b are present, where about % of them are transported by serum lipoproteins, predominantly by ldl ( %), followed by hdl ( %) and vldl ( %) [ ] . after the discovery of extracellular microvesicles (formerly called microparticles) [ ] , which were not distinguished from lipoproteins in earlier experiments, it might turn out that these assignments have to be revised. experiments in rats have shown that after injection of [ c]sialic acid-labeled gangliosides gm and [ h]sphingosine-containing labeled ganglioside gm , the gm and gm probes had serum half-lives of . and . h, respectively. after three hours, % of the gm and % of the gm probes were taken up by the liver, and a smaller extent in the central nervous system, kidneys, and lung [ ] . subcellularly, the majority of gangliosides resides in the plasma membrane [ ] . however, gangliosides also occur in organellar membranes such as in mitochondria, where gd regulates apoptosis [ ] , and in the nucleus, where they are involved in ca + balance [ , ] . the glycans found in gangliosides are sometimes modified by the acylation of sialic acid residues in different positions [ ] . o-acetylated sialic acids in gangliosides occur especially in growing cells and tissues and are regarded as oncofetal markers present on different tumors [ ] . they also serve as receptors for influenza c viruses or coronaviruses [ ] . another modified sialic acid is n-glycolylneuraminic acid (neu gc) [ ] . with the exception of certain tumors and in fetuses, it is found only in trace amounts in human tissues [ ] . as a component of glycoconjugates, neu gc is known as the hanganutziu-deicher antigen [ ] . it is abundant in many species of the deuterostome lineage, including simians, mice, rat, beef, pork, or lamb, but is nearly absent from birds and reptiles [ ] . neu gc on glycoconjugates contributes to xenoantigenicity in pig-human xenotransplantation [ ] , and in cats, neu gc distinguishes the blood groups a and b: [neu gc] gd is found in feline blood group a erythrocytes, [neu ac] gd on blood group b, and feline blood group ab erythrocyte membranes contain [neugc] gd , [neu ac,neu gc]gd , and [neuac] gd [ ] . humans cannot synthesize neu gc due to an irreversible inactivation of the cmah gene on chromosome p . encoding cytidine monophosphate-n-acetylneuraminic acid hydroxylase [ ] . this enzyme converts cmpneu ac to cmpneu gc and its function is thought to be lost during a "sialoquake" in human evolution [ , ] . determination of neu gc and neu ac-containing gangliosides is either achieved by classical chromatographic techniques combined with antibody staining [ ] , or, with higher sensitivity, by combination of chromatography with esi-ms [ ] . a potential application is the immunochemical detection of [neu gc]gm as biomarker of nonsmall-cell lung cancer [ ] . also ganglioside lactones ( figure ) have been detected in various tissues, for example, gd lactone in mouse brain [ ] and gd b lactone in human brain [ ] . ganglioside lactones are more immunogenic than gangliosides [ ] and occur on tumor cells such as melanoma as tumor-associated antigens. in vitro, lactonization of gangliosides can be followed by a strong negative cotton effect at nm in cd spectroscopy [ ] . temporal and spatial differences are also observed for the ganglioside lipid part. in undifferentiated neuronal cell cultures, gangliosides with c sphingosine are present only in trace amounts, but their content increases with the onset of cell differentiation [ ] . in rat brain, the fraction of gangliosides containing c sphingosine increases with age [ ] in cerebellum [ ] or forebrain [ ] . spatial differences regarding the sphingoid base chain length have also been detected in mice: while gangliosides containing c species were widely distributed throughout the frontal brain, c species are selectively localized along the entorhinalhippocampus projections [ ] . fatty acid and sphingoid base composition is also different between human motor and sensory nerves [ ] . nutrition. since gangliosides are components of most vertebrate cell types, they are ingested with the nutrition, for example, with egg yolk (gm , gm , and gd ), meat, or in milk [ ] . milk contains gangliosides, especially gd and gm , in the membrane fraction of the fat globule. dietary gangliosides modify the intestinal microflora and prevent infections during early infancy [ ] . in infants, more than % of dietary gangliosides survive the passage through the stomach, in part with acid-catalyzed lactonization, and are absorbed in the intestine [ ] . ingestion of dietary gangliosides leads to an increase of gangliosides in serum. in human nutrition, sialic acid derived from gangliosides and other glycoconjugates is an essential nutrient for the rapidly growing brain in infants [ ] . the pathophysiological consequences of nutritional neu gc uptake are unknown. in the past, ganglioside structures and levels were obtained by comprehensive chemical analysis, while nowadays this is attempted within lipidomics using mass spectrometry as the key technology [ ] . in general, gangliosides are isolated from tissues and body fluids by chloroform-methanol extraction [ , ] . extraction efficiency can increase when small amounts of water are present in the extraction solvent [ ] , for example, using the solvent system chloroform : methanol : water ( : : ) [ ] . when extraction is followed by a partition step such as that developed by folch et al. [ ] , gangliosides-in contrast to the majority of other lipid classes-partition into the upper, aqueous phase. from there, they can be isolated by a solid phase extraction and separated from neutral gsls by anion exchange chromatography [ ] , such as with deae (=diethylaminoethyl) sephadex [ ] . o-acetylation of sialic acids and also ganglioside lactonization [ ] are modifications that are lost under alkaline conditions. these are often applied to remove glycerophospholipids that contain fatty acids in ester linkage [ ] . if information on these modifications is desired, gangliosides from tissues can be determined without alkaline treatment, for example, after chloroform/methanol extraction in a ratio of : and a subsequent partition step [ ] . separation of gangliosides according to their glycan composition is achieved by thin layer chromatography (tlc) [ ] and by hplc and other techniques that can be coupled to mass spectrometry [ ] . this facilitates their identification by mass spectrometry and is required for their characterization by staining with suitable antibodies [ , ] , lectins, or other binding proteins [ ] . although not required for mass-spectrometric profiling, separated ganglioside classes can also be further separated according to their ceramide structure by reversed phase chromatography [ ] [ ] [ ] . quantification can be achieved by staining and densitometry, or-if suitable standard substances are availableby mass spectrometry. since the biosynthetic machinery generates heterogeneities within both, the lipid and the glycan part, comprehensive ganglioside analysis is a highly demanding task within lipidomics [ ] . in addition to glycoforms that are also well known from glycoprotein analysis, "lipoforms" [ ] become increasingly important to understand ganglioside metabolism and function. various protocols for ganglioside determination by mass spectrometry have been developed [ ] . they are largely based on electrospray mass spectrometry as ionization technique; but also maldi plays a role. the available methods range from preanalytics to bioinformatic data handling and include imaging methods using maldi and secondary ion mass spectrometry (sims) to determine the spatial distribution of the analytes [ ] . in addition to their constitution, little is known about the conformation of gangliosides in their native, membrane-bound surroundings. x-ray data are not available for gangliosides, although isolated glycans have been investigated by various means. for a simulation of gm conformations in a bilayer, compare [ ] , which, for example, shows that the glucose moiety of gm is buried within phosphatidylcholine head groups. the diversity of cell surface glycans, including that of gangliosides, is generated within the golgi apparatus [ ] , and the heterogeneities within the ceramide part result from the biosynthesis of ceramide at the endoplasmic reticulum (er). de novo synthesis of gangliosides can be distinguished from salvage processes [ , ] , in which sialic acids, sugars, fatty acids, and sphingoid bases are recycled. the latter process can predominate by far in differentiated cells. ganglioside biosynthesis starts with the formation of ceramide ( figure ) at the cytoplasmic leaflet of the er membrane [ ] [ ] [ ] . the first step, the condensation of l-serine and a coenzyme a-activated fatty acid is catalyzed by the pyridoxal phosphate-dependent serine palmitoyltransferase (spt) [ ] . the incorporation of l-serine into gsls can be used to monitor their de novo biosynthesis using l-serine radiolabelled in the position isrn biochemistry (the carbon in position is lost as carbon dioxide). in the brain, the external supply of l-serine by astrocytes is essential for neuronal lipid biosynthesis and brain development [ ] . in agreement with this observation, genetically engineered rodents with deficient phosphoglycerate dehydrogenase required for l-serine formation from d-glucose show drastically reduced ganglioside levels, defects in brain morphogenesis, and drastically reduced lifespan [ , ] . the next step in sphingolipid biosynthesis is the nadph-dependent reduction of -ketosphinganine to sphinganine by -ketosphinganine reductase, followed by acylation of sphinganine to dihydroceramides of different chain lengths [ ] . during salvage, also other sphingoid bases are acylated by n-acyltransferases of the lass family. lass encodes ceramide synthase , which is expressed in the brain and involved in the formation of the membrane anchor of gangliosides. in mice, spontaneous recessive mutations in the lass gene are associated with cerebellar ataxia and purkinje cell degeneration [ ] . although the ceramide part of brain gangliosides contains mostly nonhydroxylated fatty acids, apparently all members of the lass family are also able to transfer the corresponding -hydroxy-fatty acids [ ] . dihydroceramides are dehydrogenated to ceramide by the dihydroceramide desaturase des [ ] , or hydroxylated to phytoceramides by des . ceramide is the common precursor of gsls and sphingomyelin and is transported to the golgi apparatus at least in part in a protein-dependent manner by the transport protein cert [ ] [ ] [ ] . formation. gsl synthesis continues by the stepwise transfer of nucleotide-activated monosaccharide units first on ceramide and then on gsls with growing glycan chains. glycosidation is coupled to exocytosis through the golgi apparatus to the plasma membrane [ ] at the rate of bulk vesicle flow [ ] . the complex ganglioside and gsl glycoforms on eukaryotic cell surfaces are generated by only a few enzymes that act within a combinatorial biosynthetic pathway [ , ] . the first glycosyltransferases involved in ganglioside biosynthesis have been characterized in the laboratories of roseman and basu [ ] . according to the number of sialic acids connected to the "inner" galactosyl residue, ganglioseries gangliosides are classified into members of the -, a-, b-, and c-series ( figure ). b-series gangliosides contain the neu acα , neu ac sequence, which is commonly not found in glycoproteins. higher members of these different subseries can be formed by the action of the same glycosyltransferases, which show less specificity than those acting early in the pathway [ ] [ ] [ ] [ ] [ ] . the glycosyltransferases and sialyltransferases [ , ] of the ganglioside biosynthetic pathway are expressed in a cell-type-and developmental-dependent fashion. ganglioside pattern changes during the development of the brain [ ] , and after differentiation, differences in glycolipid composition have even been found between different neuronal cell types [ ] . in addition, ganglioside patterns vary between different cell types and change with the differentiation of the cell. as an example, β , -n-acetylglucosaminyl-transferase expression, which leads to the formation of glycolipids of the lacto and neolacto series ( figure ), is high during murine embryonic development and decreases after birth to undetectable levels in most cell types [ , ] . in adult animals, expression is high in spleen [ ] , and in cerebellum, it is restricted to purkinje cells [ ] . gsls including gangliosides are formed biosynthetically at intracellular membranes from which they are transported to the plasma membrane by exocytotic membrane flow [ ] . while many human diseases are known that are due to defects in gsl and sphingolipid degradation, the only known human disease caused by a defect glycosyltransferase of ganglioside biosynthesis is the human autosomal recessive infantile-onset symptomatic epilepsy syndrome, which is caused by a nonsense mutation in the gene encoding gm synthase [ ] . a principal difference between ganglioside biosynthesis in the golgi apparatus and degradation in the endolysosomal compartment is that during gsl formation, membranebound glycosyltransferases interact with their membranebound glycolipid substrates by diffusion within the twodimensional plane of the lipid bilayer. therefore, reaction rates can become independent of the reaction volume and obey two-dimensional enzyme kinetics. this means that kinetic constants can be normalized on lipid surface area instead of reaction volume, for example, in terms of the amount of membrane protein [ ] . as a consequence, glycosyltransferases that lack their transmembrane domain lose most of their activity towards membrane bound substrates [ ] . during degradation in endosomes and lysosomes, the glycosidases are soluble enzymes, and the substrates are membrane-bound. this explains in part the requirement for endosomal and lysosomal lipid-transfer proteins for the degradation of gsls with short glycan chains, which is not the case in biosynthesis. in addition to ganglioside biosynthesis in the golgi apparatus, there are also indications ganglioside formation by plasma membrane-associated glycosyltransferases [ ] . in the monoglycosylceramides glucosylceramide (glccer) and galactosylceramide (galcer), which are also called cerebrosides, the hexosyl residues are present in βanomeric configuration. galcers with α-configuration occur only in lower organisms [ ] and are highly immunogenic for mammals [ ] . most gangliosides are biosynthetically derived from glccer; only ganglioside gm is derived from galcer. ganglioside gm has been discovered as a minor component of human brain gangliosides [ ] , where it is localized within myelin [ ] . it also occurs, for example, on erythrocytes, kidney, and in the intestine and is abundant in some fish species. however, the most frequently found members of the gala series are galcer and sulfatide (galcer- -sulfate) in oligodendrocytes, schwann cells, kidney, testis, and intestine. they are present in high concentrations in the multilamellar layers of the myelin where they are required for glial adhesion [ ] , apparently via interaction between the carbohydrate head groups of sulfatide and galcer on different myelin layers [ ] . myelin lipids contain the highest fraction of -hydroxy-fatty acids, which are formed by fatty acid hydroxylase- [ ] . their presence in gala-series gsls contributes to carbohydrate-carbohydrate interactions between the gsls [ ] . in contrast to galcer synthase, glccer synthase appears to be dispensable for oligodendrocytes [ ] . while ceramide galactosylation catalyzed by udp-glucose:ceramide galactosyltransferase (galt ) [ ] occurs at the er membrane, the later steps of gala-series gsl biosynthesis, formation of sulfatide [ ] , digalactosylceramide [ ] , and ganglioside gm take place in the lumen of the golgi apparatus. in contrast to most glycosyltransferases in ganglioside biosynthesis, which are type ii transmembrane proteins, ceramide galactosyltransferase is a type i transmembrane protein with the catalytic domain on the luminal side of the er [ ] . according to data obtained in zebrafish and mice, gm can be formed by st gal v, which can also make gm . therefore, gm and gm formation appear to depend on the availability of their precursors, galcer and laccer [ ] . little is known about the function of gm . it can interact with the myelin basic protein, shows immunosuppressive properties, and can prevent experimental allergic encephalomyelitis in guinea pigs [ ] . glucosylceramide. the first step in the biosynthesis of most gangliosides is the transfer of a glucose residue from udp glucose to ceramide catalyzed by udp-glucose:ceramide glucosyltransferase [ , ] . although glccer and galcer synthases catalyze similar reactions, their cdnas share no sequence homology. ceramide glucosyltransferase is a type iii transmembrane protein. it forms noncovalent dimers or oligomers [ ] with their cterminal catalytic domains in the cytosol [ ] . since the formation of glccer occurs on the cytoplasmic face [ ] and that of laccer on the luminal site of the golgi membrane [ ] , glucosylceramide has to be translocated across a membrane. this is mediated by a flippase of unknown identity: the abc-transporters, abc-b and -c , translocate short chain glccer analogs through the golgi membrane [ , ] . transversal translocation can be carried out after transport of glccer by the cytoplasmic lipid-transfer protein fapp (four-phosphate adaptor protein ) either to the er [ ] , where it might be translocated by an uncharacterized flippase [ ] , or at the trans-golgi [ ] . a part of the glccer pool can reach the cytosolic leaflet of the plasma membrane where it can be degraded by the β-glucosidase gba [ ] . candidate cytosolic glccertransporters are the glycolipid transfer protein gltp and fapp . the biosynthesis of higher gangliosides occurs on the luminal face of the golgi apparatus [ ] , so that their glycan chains are orientated extracytoplasmic. laccer is formed by galactosyltransferase i, which transfers a galactose residue from udp galactose to glucosylceramide [ ] . further carbohydrate residues are transferred in a stepwise manner to the growing glycan chains. laccer and its sialylated derivatives, the hematosides gm , gd , and gt ( figure ) serve as precursors for complex gangliosides of the -, a-, b-, and c-series. these different series ( figure ) are characterized by the presence of no ( -series), one (a-series), two (b-series) or three sialic acids residues linked to the position of the "inner" galactosyl residue. in adult mammalian brain, gangliosides from the -and c-series are found only in trace amounts, and gm b and gd α are transiently expressed during chick brain biogenesis [ ] . -series gangliosides (gm b, gd c, and gd α) are found in genetically engineered mice deficient in st gal v (gm synthase), where they are present in amounts that correspond to the total ganglioside content of normal animals [ ] . these mice are not able to form gm and higher gangliosides of the a-c-series. they display altered glucose homeostasis with an accelerated insulin receptor signalling pathway, a key finding that demonstrates the inhibition of the insulin receptor by gm or a higher ganglioside derived from it in vivo [ ] . c-series gangliosides (figures and ) are formed during mammalian brain development where they are thought to be involved in growth, differentiation, and migration of neuronal cells. they are abundant in fish brain, and in adult rats; they occur in liver, kidney, and pancreas [ ] and in tumors such as glioma. the transferases that catalyze the first steps in ganglioside biosynthesis show high specificity towards their glycolipid substrates. the relative amounts of laccer, gm , gd , and gt seem to determine the amount of -, a-, b-, and c-series gangliosides. the glycosyltransferases that act late in this pathway represent a kind of assembly line and transfer the respective carbohydrates to glycosyl acceptors that differ only in the number of sialic acid residues bound to the "inner" galactose residue. the complex "α-"gangliosides with sialic acid moieties in α , -glycosidic linkage to n-acetylgalactosamine residues is specific for cholinergic neurons [ ] and has been added later to the biosynthetic scheme [ ] . in mice, the sialyltransferases that form gangliosides gd a and gt b have been identified as st gal ii and st gal iii [ ] . a significant advance towards understanding the function of the complex ganglioside pattern found in eukaryotic cells is the development of mice with defects in distinct biosynthetic steps [ ] . a mouse melanoma cell line deficient in glccer and glccer-derived gsls was viable and showed only minor changes in cellular morphology and growth rate. from these observations it was concluded that gsls including gangliosides are not essential for animal survival [ ] . later, it was reported that mice with targeted disruption of the ceramide glucosyltransferase gene displayed no cellular differentiation beyond the primitive germ layers and died around day . of embryonic development [ ] . mice deficient in b galnt i (gm synthase) are not able to form gm , gd , and higher gangliosides derived from them. although these animals show only subtle impairment of brain function [ ] , they exhibit multiple defects, such as axonal degeneration, defects in myelination [ ] and motor function [ ] , or an impaired response of t cells to interleukin [ ] , only to mention a few. later studies showed that cd -and cd -positive t cells require different ganglioside subsets for activation [ ] . the mutant male mice are sterile and also show morphological and functional defects in the testis [ ] . further examination of galnac-transferase deficient mice revealed that gm -deficiency is accompanied by parkinsonlike symptoms, which could be rescued by l-dopa or the membrane permeable gm -analog liga (see figure ) [ ] . this is in agreement with a series of reports that gm can alleviate symptoms in models of parkinson disease, for example, [ ] . in parkinson disease, anionic lipids and especially gm inhibit aggregation of α-synuclein to cytotoxic fibrils [ ] . mice deficient in st sia i (gd -synthase) do not form gd and b-series gangliosides. they have a normal life span and are without detectable developmental defects [ ] . when these mice were crossbred with mice carrying a disrupted b galnt i gene, the resulting double mutant mice express only ganglioside gm as their major ganglioside. these "gm -only-mice" are extremely susceptible to sound stimuli, develop lethal seizures, and display a sudden death phenotype [ ] . double knockout mouse deficient in b galnt i and st gal v (gm -synthase) are not able to form any ganglioside of the ganglio-series. these animals are severely diseased and show elevated levels of laccer, laccersulfate, and traces of other gangliosides that are present also in normal brain [ ] . sphingolipid biosynthesis is a highly regulated process and also coordinated with sterol and glycerolipid biosynthesis. sphingolipids are major regulators of lipid metabolism and activate sterol-regulatory element binding proteins (srebps) [ ] . the sphingomyelin synthaserelated synthase, the ceramide transporter cert, and proteins of the orosomucoid-(orm)-familie seem to play key roles in sphingolipid homeostasis [ ] . ganglioside pattern are characteristic for a cell type in a certain differentiation state, and, for example, mice deficient in gm -synthase that cannot form the typical brain gangliosides show a ganglioside content similar to that of normal animals [ ] . how exactly the relative amounts of gangliosides are controlled is not clear [ ] , but the transcriptional regulation of transferase genes seems to be a key point [ ] . the picture gets more complicated by the fact that different transferase isoforms with different properties can be present: three murine gm -synthase isoforms that arise from two transcripts have been characterized. one is resident in the er membrane, the two others in the golgi, but with different half-life [ ] . in addition, the kinetic parameters of the transferases, their topological organization within the golgi apparatus, or spatial neighborhood to other transferases will influence the resulting ganglioside pattern. an attempt has been made to calculate glycolipid pattern on the bases of the kinetic constants of the transferases, that were estimated from the steady state concentrations of the glycolipid substrates in intact cells [ ] . contradictory results have been reported on the subcellular localization of the glycosyltransferases involved in the biosynthesis of ganglioseries gangliosides [ ] . an additional feature of ganglioside biosynthesis and its regulation [ ] is the formation of functional complexes, as predicted by roseman [ ] . in these complexes [ ] , the glycosyltransferases do not only form functional platforms, but can also show altered activity and suborganellar localization [ ] . one of these complexes characterized in certain cho cells [ ] comprises b galnt i and b galt iv (figure ), so that it can accept gm and release gm . this might explain why the brain contains large amounts of gm and gd a, but little gm . also galt i, st gal v, and st sia i can form such a complex [ ] . . . general. the constitutive degradation of gangliosides takes place in endosomes and lysosomes. in addition, also the plasma membrane-associated sialidase neu [ , ] can degrade gangliosides and is, for example, highly expressed on melanoma cells [ ] . even the nuclear envelope contains sialidases, with neu in the inner and neu in the outer nuclear membrane [ ] . lysosomal ganglioside degradation takes place after the endocytosis of parts of the plasma membrane at intraendosomal and intralysosomal membranes and related lipid aggregates. this requires the presence of suitable glycosidases [ ] , of an appropriate ph, in some cases also of lipid-transfer proteins, and of an appropriate composition of the ganglioside-containing membranes [ ] . as proposed in , two different membrane pools are present in endosomes and lysosomes [ ] (figure ). they differ in lipid-and protein composition and function. while the luminal membrane pool that is derived from the plasma membrane or by autophagy is degraded, the perimeter membrane ( figure ) is protected from degradation by various means [ ] . this ensures the integrity of the compartment, which can be abolished during apoptosis [ ] . a marker lipid that is exclusively found in luminal membranes [ ] is bis(monoacylglycero)phosphate (bmp; figure ) , chemically incorrect also named as lysobisphosphatidic acid (lbpa). bmp plays a key role for membrane degradation [ ] and is formed from phosphatidylglycerol [ ] . due to its sn , sn configuration, it is only slowly degraded by lipases and persists on inner membranes, in which it can amount up to % of total phospholipids [ ] . with a predicted pk a value of about , bmp is negatively charged even at lysosomal ph. in vitro studies show that negatively charged lipids are required for binding of lysosomal proteins to membranes. although other negatively charged lipids such as dolichol phosphate or phosphatidylinositol can be present on luminal membranes, bmp appears to be the key factor that distinguishes this membrane pool from the perimeter membrane. on the other hand, the perimeter membrane of endosomes and lysosomes shows an entirely different lipid and protein composition. it is protected by a glycocalyx formed by highly n-glycosylated integral membrane proteins [ , ] , and ganglioside gm present in this membrane is resistant to degradation [ ] . ganglioside degradation starts with the action of glycosidases that cleave off monosaccharide units from the non-reducing end of the ganglioside glycan chains. this happens in a sequential manner, which explains the different human diseases that are associated with defects in this pathway. the glycosidases are soluble enzymes in the lumen of endosomes and lysosomes. it turned out that their activity is not sufficient towards gsl substrates with cleavage sites in proximity to the intralysosomal membrane surface. although also other factors play a role, this can be attributed to steric hindrance by adjacent membrane components that impede the access of the soluble enzyme. for example, in wild-type and gm -activator deficient fibroblasts, radiolabelled gd agalnac (figure ), which has the same terminal trisaccharide as gm , is degraded in the absence of the gm -activator protein, while the degradation of gm itself is strictly dependent on the presence of the activator [ ] . as glycosidase substrates, gsls with four carbohydrate residues or less require the additional presence of small lipid binding glycoproteins, either the gm activator protein or one of the four saposins a-d. these act in part as lipid-transfer proteins that extract the membrane-bound substrates and present them to the hydrolases. they have different specificities and mechanisms of action [ ] . in the case of gangliosides, at least the gm -activator protein and saposin-b participate in the degradation of gm , gm , and gm ( figure ). in vitro, in addition to enzymes and activator proteins, also an appropriate membrane-lipid composition of the ganglioside-containing membrane is required for degradation [ ] . saposin-a [ ] and saposin-b [ ] extract membrane lipids much better from membranes that are rich in bmp and poor in cholesterol. bmp also increases the ability of the gm activator to solubilize lipids [ ] and stimulates the hydrolysis of membrane-bound gm by gm β-galactosidase [ ] and of ganglioside gm by β-hexosaminidase a [ ] . bmp also stimulates hydrolysis of the kidney sulfatide with ganglio-series gsl-core sm (gangliotriaosylceramide-ii sulfate) by β-hexosaminidases a and s in the presence of the gm activator [ , ] . cholesterol, which is known to stabilize lipid bilayers, has to be transported from intraendosomal membranes to the npc protein resident in the endosomal perimeter membrane by the soluble lipid-transfer protein npc . in vitro, this transfer is greatly stimulated by bmp and strongly inhibited by sphingomyelin [ ] . saposins are small, water-soluble lysosomal lipid-binding and -transfer proteins of about - kda molecular weight. they are derived from a common precursor protein, prosaposin, by proteolytic processing. saposins belong to a family of proteins with conserved three-dimensional fold [ ] and occur as homo-and heterodimers and -oligomers. the first saposin has been characterized in as the so-called sulfatide activator since it enables the degradation of sulfatide by arylsulfatase a [ ] . today this protein is known as saposin-b or sap-b. saposin-b has many functions: it is a lipid-binding protein with broad specificity [ ] and forms water-soluble lipidprotein complexes [ ] . with respect to gangliosides, it is able to stimulate the degradation of ganglioside gm by gm -β-galactosidase [ ] . studies in cultured human skin fibroblast derived from saposin-b and prosaposin-deficient patients show that it is also required for the degradation of gm [ , ] . it is important to note that glycosylation of saposin-b is essential for some of its functions and that human patients without this postranslational modification die, although the unglycosylated variant protein is present in lysosomes [ ] . mechanistically, saposin-b dimers seem to act similar to the gm activator: x-ray data indicate that they can adopt two conformations, an open one and a closed one [ ] . according to a model view, the open conformation interacts directly with the membrane and extracts the lipid ligand. this is accompanied by a change to the closed conformation in which the ligand is exposed to the degrading enzyme in a water-soluble activator-lipid complex. human patients with an inherited deficiency of saposin-b develop an atypical form of metachromatic leukodystrophy with the accumulation of sulfatides, digalactosylceramide, and globotriaosylceramide [ ] (see figure for structures). saposin-b knockout mice show enhanced levels of sulfatides especially in brain and kidney [ ] . activator. the gm activator is a small glycoprotein of . kda in its deglycosylated form and is required for the degradation of ganglioside gm by βhexosaminidase a in vivo [ ] . inherited deficiency of the gm -activator protein leads to the ab variant of gm gangliosidoses [ ] . based on the x-ray structure [ , ] and data from photoaffinity labeling [ ] , in some respects the gm -activator acts in a way similar to saposin-b. a more detailed picture of the binding mode was derived from binding studies using a spin-labelled gm activator to phosphatidylcholine bilayers [ ] . the protein can extract a variety of lipids, which has been exploited for assay development [ ] . however, its major function is to form a water-soluble gm -protein complex that is the native michaelis-menten substrate of β-hexosaminidase a [ ] . negatively charged lipids such as bmp, dolichol phosphate, or phosphatidylinositol increase the extraction efficiency towards gm [ ] , gm [ ] , and other lipids [ ] from liposomal membranes. binding characteristics of the gm activator are altered by the presence of a his tag [ ] . in langmuir experiments, the gm -activator protein is able to penetrate into a phospholipid monolayer, but only when the lateral pressure is below a critical value, which depend on the lipid composition and is in the range from to mn/m [ ] . in addition to its function as a ganglioside-transfer protein, the gm activator binds also other lipids like phosphatidylcholine [ ] and platelet activating factor (paf) and inhibits its action [ , ] . it is not clear whether the gm activator displays inherent hydrolytic activity towards lipid substrates such as platelet activating factor [ ] or phosphatidylcholine [ ] . apparently unrelated to its gm transfer property is the function of the gm activator as adipokine [ ] . gm activator orthologs might serve different functions in other organisms, for example as a pheromone-binding protein in drosophila [ ] , or an inhibitor of paf-induced chemotaxis in nematodes [ ] . the saposins and the gm -activator play major roles in the transfer of lipid antigens to membrane-resident cd proteins [ , ] . degradation. gm -β-galactosidase is a protein of kda, which is derived from an -kda precursor [ , ] . an alternatively spliced, enzymatically inactive β-galactosidase form of kda is an elastin/laminin-binding protein [ ] . gm -β-galactosidase is part of a lysosomal multienzyme complex, together with the so-called protective protein (carboxypeptidase a), sialidase, and n-acetylaminogalactose- -sulfate sulfatase [ ] . gm -β-galactosidase catalyzes the hydrolytic cleavage of several β-galactosides. the hydrolysis of ganglioside gm to gm requires the presence of either the gm -activator protein, or saposin-b [ ] , or, in vitro, of an appropriate detergent. degradation. gm is degraded by the cleavage of the n-acetylgalactosaminyl residue by β-hexosaminidases. in mice, the substrate specificity of the murine lysosomal sialidase allows for a significant cleavage also of the sialic moiety in gm (to yield ga ) [ ] . cleavage of the galnac residue requires the presence of the gm -activator protein in vivo, or of an appropriate detergent in vitro. three gene products participate in gm hydrolysis, the β-hexosaminidase αand β-chains, and the gm -activator protein. β-hexosaminidases are dimers that result from the combination of their αand β-subunits and differ in properties such as stability and substrate specificity. β-hexosaminidase a with subunit composition α,β cleaves terminal β-glycosidically linked n-acetyl-glucosamine and n-acetylgalactosamine residues from negatively charged and uncharged glycoconjugates by a retaining doubledisplacement mechanism. the enzyme has two active sites, one on the α-chain and the other on the β-chain [ ] . β-hexosaminidase b (ββ) [ , ] predominantly cleaves uncharged substrates such as ga and oligosaccharides with terminal n-acetyl-hexosamine residues (see also figure ). β-hexosaminidase s (αα) is thermolabile and of secondary significance for gm degradation, but it contributes to the degradation of glycosaminoglycans and sulfated glycolipids [ ] . defects in enzymes and other proteins required for lysosomal degradation of complex lipids and of oligomeric or polymeric biomolecules lead to inherited diseases, the lysosomal storage diseases [ ] . they can be classified according to the stored substances, as sphingolipidoses [ ] , mucopolysaccharidoses, mucolipidoses, glycoprotein-, and glycogen-storage diseases [ , ] . ganglioside degradation is impaired in the gangliosidoses and secondarily also in other sphingolipid storage diseases [ ] . the principles [ ] governing pathogenesis [ , ] and therapy of sphingolipidoses [ ] are also valid for the ganglioside storage diseases. key factors are the residual activity of the degrading system, which determines the course of the disease [ , ] , and the cell-type-specific expression of storage material. due to the cell-type-specific expression of gangliosides, the central nervous system is especially affected in the gangliosidoses. in sphingolipidoses in general, the storage lipids coprecipitate other hydrophobic substances present in the endolysosomal compartment, lipids and proteins, as secondary storage products [ ] . in niemann-pick disease, type c, which is a primary defect of endosomal cholesterol transport, a secondary accumulation of sphingomyelin (therefore the name niemann-pick) and of gangliosides is observed that is also of therapeutic relevance [ , ] ; for a remarkable treatment of niemann pick c fibroblasts with a histone deacetylase inhibitor, compare [ ] . secondary storage of gangliosides gm and gm occurs also in hurler disease [ ] (mucopolysaccharidosis type i; α-liduronidase deficiency). lipid storage produces a kind of traffic jam [ , ] , which interferes with lipid transport and lysosomal function. primary and secondary storages substances can impair nutrient delivery via the endolysosomal system: as demonstrated in mouse models of gm gangliosidoses and in a variant form of the gm gangliosidoses, sandhoff disease, iron homeostasis is impaired in the animals, and supplementation of the animals with iron ions increased their life expectancy by nearly % [ ] . since also autophagy can be impaired in lysosomal storage diseases [ ] , both pathways may lead to a shortage of nutrients. ganglioside degradation is impaired in the gangliosidoses. in another disease, galactosialidosis, the primary defect of carboxypeptidase a (protective protein), leads to a secondary loss of β-galactosidase and sialidase neu accompanied by gm storage [ ] . gangliosidoses are caused by defects in the genes encoding glycosidases or lipidtransfer proteins that are required for lysosomal ganglioside degradation. the theoretical basis for the therapeutic approaches towards gangliosidoses is the "threshold theory" [ ] , which predicts that the ratio of substrate influx into the lysosomes and the degradation capacity determine the course of the diseases. both parameters can be addressed by different therapeutic approaches [ ] . . . gm gangliosidosis. gm gangliosidosis is caused by an inherited deficiency of gm -β-galactosidase (acid βgalactosidase; glb ; ec . . . ) [ ] . after the description of the first patients [ ] it became also known as landing diseases [ ] . it is a rare disease with an autosomal recessive mode of inheritance and characterized by the accumulation of gm and ga (figure ) in neuronal cells [ ] . according to the substrate specificity of the variant enzyme in the patients, an inherited defect of the β-galactosidase can also lead to another disease, morquio disease, type b. three clinical forms of gm gangliosidosis can be distinguished, infantile (type ) gm gangliosidosis with the developmental arrest and progressive deterioration of the nervous system in early infancy and a life expectancy of about years, late infantile/juvenile form (type ), and an adult/chronic form (type ). dysmorphic changes characteristic for morquio disease type b are less prominent or completely absent in these clinical forms. in addition to gm , other enzyme substrates accumulate, such as ga (figure ) [ ] , oligosaccharides from glycoproteins, and intermediates of keratin sulfate degradation [ ] . these substances are stored in different organs, according to their major site of biosynthesis. lysosomal gm accumulation in neurons leads to the degeneration of the nervous system. like in other storage diseases, an inflammatory response [ ] , neurorestorative properties of excess ganglioside gm [ ] in the plasma membrane, and an unfolded protein response [ ] contribute to pathogenesis. such as in other sphingolipidoses [ ] , severity and progression of the disease correlate with the residual enzymatic activity in cells and body fluids. morquio type b disease clinically resembles a mild phenotype of morquio a disease, where keratan sulfate accumulates due to n-acetyl-galactosamine- -sulfatase deficiency. like gm gangliosidosis, morquio type b is due to the inherited defect of gm -β-galactosidase. it is characterized by the predominant storage of keratan sulfate and oligosaccharides with terminal β-galactosyl residues. patients show generalized skeletal dysplasia without involvement of the nervous system and without hepatosplenomegaly; for a clinical description, compare [ ] . differences between gm gangliosidosis and morquio b disease can be attributed to a lower affinity and activity of β-galactosidase variants towards substrates with gal-β , -glcnac motifs in morquio patients compared to the gal-β , -galnac motive present in ganglioside gm [ ] . there is no causal therapy available for gm -gangliosidosis; however, progress is made towards the development of pharmacological chaperones also for this lysosomal disease [ ] [ ] [ ] . the gm -gangliosidoses are caused by defects in degradation of ganglioside gm [ ] . the three variant forms of the gm -gangliosidoses are named according to the hexosaminidase isoenzyme that remains intact. the b-variant, in its infantile course better known as tay-sachs disease, is caused by the deficiency of hexosaminidases a and s, but with normal hexosaminidase b. the variant, or sandhoff disease, is caused by the deficiency of the β-chain and the resulting deficient activity of β-hexosaminidases a and b (therefore, none of the major enzymes is intact), however with the remaining activity of β-hexosaminidase s. the ab-variant-β-hexosaminidases a and b (and s) intact-results from mutations in the gm -activator gene; so that tissue samples from the patients are able to degrade gm in detergent-containing enzyme assays. clinically, the b variant of gm gangliosidoses can be subclassified into infantile, juvenile, chronic, and adult onset forms. the infantile form, tay-sachs disease, has a higher prevalence among ashkenazi jews with a heterozygote frequency of : . affected children are normal at birth and show first symptoms, such as mild motor weakness, a cherry red spot in the central retina, and increased startle reaction between and months of life. progressive deterioration with weakness, hypotonia, or poor head control leads to a vegetative state and death often between the second and fourth year of life. juvenile and adult course is observed in patients with a higher residual activity of the variant hexosaminidase a [ ] . symptoms are very heterogeneous; for a clinical description, compare [ ] . the b variant of gm gangliosidoses [ , ] was very difficult to elucidate: synthetic uncharged substrates used for diagnosis such as mufglcnac (figure ; for kinetic parameters see [ ] ) were cleaved, suggesting the presence of β-hexosaminidase, and also the gm activator was present. as it turned out, the b variant differs enzymatically from the b variant by an altered substrate specificity of the variant β-hexosaminidase a. while uncharged substrates are cleaved, no activity is detected towards gm and towards sulfated, negatively charged [ ] synthetic fluorogenic substrates. in the b variant, the function of the α-chain active site is defective, but subunit association, enzyme processing, and the activity of the β-chain are not impaired. homozygous patients with the b mutation show the course of the juvenile disease; compound heterozygotes with a b and a null allele show a late infantile course. disease. the variant of gm gangliosidosis was the first gangliosidosis for which the underlying enzymatic defect was identified [ ] . due to the deficiency of two enzyme activities, β-hexosaminidases a and b, storage of negatively charged glycolipids characteristic for tay-sachs disease and, in addition, of uncharged substrates such as ga in the brain and globoside in visceral organs (figure ) is observed. in infantile sandhoff disease, patients show clinical and pathological manifestations of tay-sachs disease (infantile b variant) and in addition also organomegaly and slight bone deformations. for further symptoms and the description of juvenile and adult forms, compare [ ] . the ab variant is due to the deficiency of the gm -activator protein [ ] , with intact β-hexosaminidases a and b (and s), therefore the name. the disease is characterized by accumulation of gm and ga (for structures, see figure ). the clinical picture [ ] resembles that of tay-sachs disease with a delayed appearance of symptoms; an animal model is available [ ] . although lysosomal gm as the major storage compound in gm gangliosidoses is neither toxic nor immunogenic, its accumulation induces inflammatory responses as demonstrated for glycoconjugates in the murine model of sandhoff disease [ ] . huge axon hillock enlargements, the so-called meganeurites, have been observed in neurons of patients with different lysosomal storage diseases, which might be attributed to the storage substance gm and contribute to synaptic dysfunction [ ] . as in other sphingolipidoses [ ] , the corresponding (more toxic) lysolipid, in this case lysogm (figure ), is elevated [ , ] and contributes to the pathogenesis. lysogm has been suggested as a biomarker for tay-sachs and sandhoff disease [ ] ; for occurrence and role of lysogsls in acquired diseases, compare [ ] . despite naturally occurring animal models of gm gangliosidoses in dogs, cats, and pig, murine models are used for therapy studies. since the mouse model of tay-sachs disease is largely asymptomatic, the mouse model of sandhoff disease is used for most studies [ ] . despite some success in the experimental treatment of juvenile and adult patients as well as in the animal models, there is no causal therapy available for the severe forms of the gm gangliosidoses. the limitations of the substrate reduction approach, which reduces the gm influx into the lysosomal compartment, have been evaluated by a genetic experiment: sandhoff-disease mice were crossbred with mice defective in gm synthase. the lifespan of these animals was much longer than that of sandhoff-disease mice, but instead of gm storage they developed a oligosaccharide storage, neurological disease [ ] . therapeutic approaches such as bone-marrow transplantation [ ] , enzyme-replacement therapy with recombinant highly phosphomannosylated β-hexosaminidase a [ ] , or transplantation of neural stem cells [ ] have been investigated in the animal model of the variant, substrate-reduction therapy with n-butyl deoxynojirimycin [ , ] , and with pyrimethamine as pharmacological chaperone [ , ] in adult patients and gene therapy in endothelial cells [ ] . treatment of the accompanying inflammation is beneficial [ ] . aspects. in addition to inherited diseases, ganglioside levels can also be altered in several acquired diseases [ ] . for example, gangliosides play roles in neurological diseases such as alzheimer's [ ] , parkinson, or huntington's disease [ ] . in cancer, ganglioside expression can also be altered in tumor cells with an impact on signalling and tumor-host interactions [ ] . [neu gc]gm [ ] , gd , gd , gm , and fucosylgm are regarded as tumor-associated antigens [ ] and are targets for the immunotherapy of cancer [ ] . also several neuropathies including variant forms of guillain-barré and miller-fisher syndrome are caused by serum antibodies against gangliosides [ ] . there are only a few therapeutic roles for gangliosides, especially since they can induce neuropathies. in the past, gangliosides isolated from bovine brain have been investigated and also applied to human patients to improve neural repair and for the treatment of stroke [ , ] . also the direct application of ganglioside gm into the brain of patients with alzheimer disease has been evaluated [ ] . as indirect roles, the inhibition of ganglioside biosynthesis for the treatment of insulin resistance [ ] , the interference with microbial binding to gangliosides [ ] , or the reduction of neurotoxicity with liga [ ] has to be mentioned. a plethora of functions has been attributed to gangliosides [ ] , for example, for gm [ ] , the most abundant ganglioside in most mammalian cell types, but not in neurons, or for gm [ ] . in general, gangliosides mediate their function via interaction with soluble or membranebound binding molecules outside the cell ("trans" interaction), or by influencing properties of proteins within the same membrane ("cis" interaction) [ , [ ] [ ] [ ] [ ] . "trans" interactions occur between the glycan part of gangliosides on the one side with lectins on the other side. also gangliosides contribute to the chemical high-density sugar code of cell surfaces [ ] . for example, gm can be recognized by galectin- [ ] and sialic acids in α , figure : structure of liga , a semitruncated and halogenated gm analog that can pass the blood-brain barrier. are recognized by the sialic acid binding immunoglobulin lectins siglec- , α , -sialosides by siglec- , and α , sialosides by siglec- [ ] . also carbohydrate-carbohydrate interactions can play a role [ , ] . although interactions between individual carbohydrate residues [ ] are weak, clustering of gangliosides offer the possibility for multivalent interactions, if they are not buried under glycoprotein glycans. apparently, gm on mouse melanoma b cells can mediate cell adhesion to mouse lymphoma l cells by binding to ga (gangliotriaosylceramide, galnacβ , galβ , glcβ , cer; see figure or figure ) [ ] . within the nervous system, gangliosides act in a "trans" manner with the myelin-associated glycoprotein mag. mag recognizes neuacα - galβ - galnactermini on axonal gangliosides, an interaction that is essential for axon-myelin stability and axon regeneration [ ] . "cis" interactions can happen via a direct interaction, or, indirectly, via the properties of the membrane or of putative-membrane domains [ ] . this way gangliosides influence the activities of receptor-tyrosine kinases in the plasma membrane, such as the receptors of epidermalgrowth factor, nerve growth factor, and insulin and therefore cell signaling [ ] . for example, gm enhances trka neurotrophin receptor-activation in a "cis"-manner [ ] ; for a brief overview of proteins affected by certain gangliosides, compare, for example, [ ] . since the characterization of lipid microdomains in living cells is difficult, some conclusions can be drawn from in vitro experiments. for example, gm inhibits the autophosphorylation of purified egfr reconstituted into the proteoliposomes of defined lipid compositions, but not the egf binding [ ] . there are indications that gangliosides may not act only in an autonomous manner, but might also support the formation of distinct membrane phases, although it is a matter of debate to which extent this operates in vivo. it is believed that gangliosides are not homogeneously distributed on the cell surface, but segregate into membrane domains together with gpi-anchored proteins, sphingomyelin and cholesterol. such rafts have been supposed to be the physiological surroundings of many membrane proteins, although no rigid proof for their existence has been provided. ganglioside plays a largely unexplored role for membrane structure [ ] . due to their large hydrated head groups, they stabilize membrane areas with positive curvature [ ] . a multitude of reports propose a segregation of gangliosides and other gsls with cholesterol and gpi-anchored proteins into the lipid platforms known as "rafts" in the membranes of living cells [ ] [ ] [ ] [ ] . since most of the applied methods (detergent extraction, antibody, and toxin staining) constitute a bias towards the formation of such domains [ ] , the existence, size, and lifetime of rafts are a matter of debate. from thermodynamic considerations, it is clear that procedures such as detergent extraction can (or have to) produce artificial results and are not suitable for raft characterization [ ] [ ] [ ] [ ] . although it has been demonstrated that the treatment of cellular membranes with detergents causes the redistribution of gangliosides and gpi-anchored proteins [ , ] , these techniques are still applied and not always critically examined. experiments in living cells using sted microscopy [ ] and other visualization techniques [ ] point to an upper limit of lifetime and size of such domains in the range of ms and nm. in addition, sialic acids and oligosialic acids present on gangliosides can modulate membrane surface charge density, the ph at the membrane surface, and membrane potentials [ ] . in planar lipid bilayers, ganglioside gd a can increase the excitability of voltage-dependent sodium channels [ ] . . . infection. "cis" and "trans" interactions of gangliosides play multiple roles in the immune system [ ] and in infectious diseases [ , ] , where gangliosides act as cellular receptors and coreceptors for viruses, bacteria, and microbial toxins. the most prominent example is gm as the receptor for cholera toxin [ ] ; other examples are the toxin of clostridium botulinum and the saba adhesin of helicobacter pylori [ ] that bind to cell surface gangliosides of the host [ ] . binding of sialylated cell surface glycoconjugates to siglecs [ ] on white blood cells is used within innate and adaptive immune responses to distinguish between self and nonself and to dampen autoimmune responses [ ] . many pathogens use sialic acids on cell surface glycoconjugates for cellular entry, for example, periodontal pathogens [ ] . recent examples include ganglioside gt b, which seems to be the host cell receptor for the merkel cell polyomavirus [ ] . this virus has been identified as the cause of merkel cell carcinoma, an aggressive type of skin cancer. also sialidase-insensitive rotaviruses recognize sialic acid, for example, on ganglioside gm , which is not substrate of all sialidases due to its branched structure [ ] and the glycan present in ganglioside gd a serves as host receptor for the adenoviruses that cause epidemic keratoconjunctivitis [ ] . gangliosides are secondary gene products. their function can be analyzed by knockout experiments, where in cells, tissues, or organisms their formation or degradation is interrupted by genomic, posttranscriptional, or chemical strategies. especially valuable were genetically engineered mice with defects in ganglioside biosynthesis [ ] , which revealed, for example, a role of gangliosides in calcium homeostasis [ ] , neural repair [ ] , or neurological diseases [ ] . also investigations in human patients [ ] , genetically engineered mammals [ ] , and other organisms [ ] allowed insight into various aspects of ganglioside metabolism and transport. also mutant and silenced cells have been applied for functional studies. in vitro systems such as liposomes or planar monolayers allow investigations that are to difficult to be carried out in cells. for example, when gm is incorporated into liposomes, a phase separation into gm -rich and gm -poor phases occurs above a certain gm content [ ] . this would fit to reports on gm -enriched microdomains in living cells. in experimental approaches, ganglioside biosynthesis can be modulated by inhibitors [ ] . also the enhancement of ganglioside biosynthesis can be used, for example, chemically, or by the introduction of glycosyltransferase encoding cdna in cultured cells [ , ] . an enantiomer of the glucosylceramide synthase inhibitor d-threo-pdmp (pdmp = -phenyl- -decanoylamino- -morpholino- -propanol), lthreo-pdmp, acts as an enhancer of ganglioside biosynthesis by upregulating glycosyltransferases. this was accompanied by increased neurite outgrowth [ ] . additional possibilities are the generation of mutant cells, for example for gm synthase [ ] or by posttranscriptional silencing like rna interference. while even complex systems like cultured cells can survive without gsls, they are required for the development of multicellular organisms [ ] . gangliosides. structurally homogenous gangliosides and ganglioside probes that are modified by isotopes, fluorescence, chemical reporter groups, photoaffinity, or affinity ligands are valuable tools for the analysis of ganglioside function, metabolism, and transport. these tools are available by total or partial chemical synthesis, or by biosynthetic incorporation of suitable-for example, photolabile-n-acylmannosamine precursors into gangliosides [ ] using the methodology for biosynthetic sialic acid modification developed by kayser et al. [ ] . for enzymological, transport, and crosslinking studies, tritium and c are incorporated into different positions of gangliosides, but also radioiodination is possible in the presence of aryl residues [ , ] . ganglioside total synthesis is a time-consuming and demanding task and usually performed only in specialized laboratories for examples, see [ , ] . it relies predominantly on the sequential glycosidation of a -o-protected azidosphingosine [ ] with suitably protected and activated glycosyl donors. this includes the trichloroacetimidates [ ] as well as methods for α-selective sialylation reactions [ ] . also chemoenzymatic procedures have been developed, where different glycosidation steps are catalyzed by glycosyltransferases [ ] or glycosidases [ ] , or where oligosaccharyl fluorides are coupled to native or fluorescent ceramide anchors using an engineered endoglycoceramidase (glycosynthase) [ , ] . ganglioside oligosaccharides, such as those of gm , gm , gm , gd , and gt , can be produced by genetically engineered bacterial strains. for the biotechnological production of ganglioside head groups, lactose can be internalized in e. coli as a precursor to be used as acceptor for glycosyltransferases [ ] [ ] [ ] [ ] [ ] [ ] . an application of the ganglioside biosynthetic machinery is the preparative production of neoglycolipids with ganglioside head groups [ ] figure : example of a gm analog [ ] spin labelled with a , -dimethyl-oxazolidine- -oxyl-(doxyl-) residue and a fluorescent gm analog used for sted microscopy [ ] . using a lung squamous-cell carcinoma line (rerf-lc-a) and -azidododecyl β-lactoside as a suitable primer [ ] . gsls isolated from natural sources can be used for the preparation of chemically modified derivatives [ ] like labelled gsls [ , ] , or those of enhanced metabolic stability [ ] . the chemical release of the ganglioside glycan chain can be achieved by osmium tetroxide/periodate treatment of protected gangliosides [ , ] , or by ozonolysis of native gangliosides [ ] . initially, both methods give rise to ganglioside aldehydes, which are not isolated but subsequently fragmented by alkaline treatment, or, if desired, are isolated for further applications [ ] . the glycan part can also be released enzymatically by ceramide glycanase [ , ] . lysogangliosides that lack the acyl moiety at the sphingosine nitrogen can be prepared by chemical procedures [ , ] , or by enzymatic treatment of gangliosides with sphingolipid n-deacylase [ ] [ ] [ ] . lysogangliosides (see figure ) can be used for the introduction of fluorescence, spin, or radiolabels or other modifications into the lipid backbone of gangliosides. a semitruncated, dihalogenated gm analog that is able to pass the blood-brain barrier is liga [ ] (figure ). there are also several approaches to the synthesis of photoactivatable gsl derivatives [ ] . it has to be noted that when gangliosides are added to the medium of cultured cells, they are largely present as oligomers in the form of micelles or vesicles, as monomers bound to proteins, and as free monomers. in aqueous surroundings, gangliosides form aggregates of different size and shape [ , ] . in most cases these are micellar structures of - kda [ ] , in the case of the gangliosides with small head groups, gm and gm also vesicles [ , , ] . the critical micellar concentrations of gsls are in the range of − - − m [ ] and depend on temperature, ph, and, in part, on the method of determination. typical values are . × − m for gm to . × − m for gt b [ ] . uptake of exogenously added gangliosides by cells in culture [ ] can proceed in different ways [ , ] ( figure ) . with the aid of radiolabelled [ ] and spinlabelled gangliosides [ ] (figure ), three modes of adherence have been distinguished: - % of the exogenous ganglioside consist in loosely associated micelles and also monomers, which can be removed by delipidated serum proteins. a second fraction is attached to cellular proteins in a trypsin-labile fashion, and, finally, a trypsin-stable fraction is presumably inserted into the plasma membrane of the cell. only the last fraction is in the topologically correct, native orientation. when bound to proteins, the offrate of gangliosides with native alkyl chain lengths can be very low. this is not the case for synthetic, semitruncated derivatives of higher solubility, which are frequently used, but show different intracellular transport behaviour compared to native gangliosides [ ] . fluorescently labelled glycosphingolipids [ ] have been applied, but also their properties can differ significantly from the ones of native glycosphingolipids. gangliosides can also be transferred from cultured donor to acceptor cells that are separated by a membrane [ ] . by this process known as "shedding" [ ] , tumor cells can release up to . % of their gangliosides per hour [ ] . fluorescent ganglioside probes that bear the fluorophore in at the membrane-water interphase ( figure ) behave physicochemically more like native gangliosides. such compounds have been used as probes for sted microscopy [ ] or to quantify the transfer capacity of gm activator in a liposomal fret assay system [ ] . despite the fast development of analytical and biophysical tools, the analytical determination of ganglioside pattern, their spatial resolution, and their correlation with function is still a challenge. especially a convincing characterization of ganglioside-containing membrane domains in living cells and of their roles at the cellular level would constitute a considerable advance in the field. n-acetyl-neuraminic acid glc: glucose gal: galactose galnac: n-acetyl-galactosamine. the author has no direct financial relation with any commercial identity mentioned in the paper that might lead to a conflict of interests. a comprehensive classification system for lipids primer on genes encoding enzymes in sialic acid metabolism in mammals sialic acids Über die ganglioside, eine neue 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digestion metabolism, function and mass spectrometric analysis of bis(monoacylglycero)phosphate and cardiolipin separation and characterization of late endosomal membrane domains lysosome biogenesis and lysosomal membrane proteins: trafficking meets function lysosomal membrane proteins: life between acid and neutral conditions hoppe-seyler's zeitschrift für physiologische chemie the human g(m ) activator protein. a substrate specific cofactor of β-hexosaminidase a principles of lysosomal membrane digestion: stimulation of sphingolipid degradation by sphingolipid activator proteins and anionic lysosomal lipids saposin a mobilizes lipids from low cholesterol and high bis(monoacylglycerol)phosphate-containing membranes: patient variant saposin a lacks lipid extraction capacity saposin b mobilizes lipids from cholesterol-poor and bis(monoacylglycero) phosphate-rich membranes at acidic ph: unglycosylated patient variant saposin b lacks lipid-extraction capacity degradation of membrane-bound 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membraneorganizing principle lipids and cholesterol as regulators of traffic in the endomembrane system principles of microdomain formation in biological membranes-are there lipid liquid ordered domains in living cellular membranes? pip signaling in lipid domains: a critical reevaluation rafts defined: a report on the keystone symposium on lipid rafts and cell function detergentresistant membranes should not be identified with membrane rafts triton promotes domain formation in lipid raft mixtures membrane redistribution of gangliosides and glycosylphosphatidylinositol-anchored proteins in brain tissue sections under conditions of lipid raft isolation membrane redistribution of gangliosides and glycosylphosphatidylinositol-anchored proteins in brain tissue sections under conditions of lipid raft isolation imaging of mobile long-lived nanoplatforms in the live cell plasma membrane membrane oligo-and polysialic acids ganglioside gd a increases the excitability of voltage-dependent sodium channels multifarious roles of sialic acids in immunity sphingolipids in infectious diseases viruses and sialic acids: rules of engagement cholera toxin-a foe & a friend helicobacter pylori and complex gangliosides sialic acids in human health and disease siglecs and their roles in the immune system siglecs as sensors of self in innate and adaptive immune responses sialic acid, periodontal pathogens and tannerella forsythia: stick around and enjoy the feast! ganglioside gt b is a putative host cell receptor for the merkel cell polyomavirus sialic acid dependence in rotavirus host cell invasion the gd a glycan is a cellular receptor for adenoviruses causing epidemic keratoconjunctivitis knockout mice and glycolipids cerebellar neurons lacking complex gangliosides degenerate in the presence of depolarizing levels of potassium regulatory mechanisms of nervous systems with glycosphingolipids functions of sphingolipid metabolism in mammals -lessons from genetic defects sphingolipids and membrane biology as determined from genetic models phase behavior in multilamellar vesicles of dppc containing ganglioside gm with a c : sphingoid base and a : acyl chain (gm ( , )) observed by x-ray diffraction glycosphingolipids-nature, function, and pharmacological modulation enhancement of malignant properties of human osteosarcoma cells with disialyl gangliosides gd /gd gm /gd /ga synthase expression results in the reduced cancer phenotypes with modulation of composition and raft-localization of gangliosides in a melanoma cell line chapter neurotrophic and neuroprotective actions of an enhancer of ganglioside biosynthesis mutant ng - cells (ng-cr ) deficient in gm synthase respond aberrantly to axonogenic stimuli and are vulnerable to calcium-induced apoptosis: they are rescued with liga- biomolecule function: no reliable prediction from cell culture metabolism of diazirine-modified n-acetylmannosamine analogues to photocross-linking sialosides biosynthesis of a nonphysiological sialic acid in different rat organs, using n-propanoyl-d-hexosamines as precursors tubulin anchoring to glycolipid-enriched, detergent-resistant domains of the neuronal plasma membrane hplc-based procedure for the preparation of carbenegenerating photoreactive gm and gm ganglioside derivatives radioiodinated to high specific radioactivity with chloramine t as an oxidant azidosphingosine glycosylation in glycosphingolipid synthesis glycosyl trichloroacetimidates selective α-sialylation enzymatic approaches to o-glycoside introduction: glycosyltransferases enzymatic glycosylation by transferases glycosphingolipid synthesis employing a combination of recombinant glycosyltransferases and an endoglycoceramidase glycosynthase biosynthesis of conjugatable saccharidic moieties of gm and gm gangliosides by engineered e. coli the genetic bases for the variation in the lipo-oligosaccharide of the mucosal pathogen, campylobacter jejuni genetic engineering of escherichia coli for the economical production of sialylated oligosaccharides highly efficient biosynthesis of the oligosaccharide moiety of the gd ganglioside by using metabolically engineered escherichia coli a new fermentation process allows large-scale production of human milk oligosaccharides by metabolically engineered bacteria large-scale in vivo synthesis of the carbohydrate moieties of gangliosides gm and gm by metabolically engineered escherichia coli large scale biosynthesis of ganglioside analogues by rerf-lc-ai cells cultured in hyperflask use of sphingolipid analogs: benefits and risks a simple and novel method for tritium labeling of gangliosides and other sphingolipids preparation of radiolabeled gangliosides uptake and metabolism of exogenous glycosphingolipids by cultured cells structure and function of glycosphingolipids and sphingolipids: recollections and future trends release of carbohydrates from sphingoglycolipid by osmium-catalyzed periodate oxidation followed by treatment with mild alkali carbohydrate components of extraneuronal gangliosides from bovine and human spleen, and bovine kidney structural and functional glycosphingolipidomics by glycoblotting with an aminooxy-functionalized gold nanoparticle a unique glycosphingolipid-splitting enzyme (ceramide-glycanase from leech) cleaves the linkage between the oligosaccharide and the ceramide preparation of homogenous oligosaccharide chains from glycosphingolipids synthesis of lysogangliosides lysogangliosides: synthesis and use in preparing labeled gangliosides facile method for the preparation of lyso-gm and lyso-gm design of a covalently bonded glycosphingolipid microarray anti-ganglioside antibodies bind with enhanced affinity to gangliosides containing very long chain fatty acids sphingolipid photoaffinity labels thermodynamicgeometric correlations for the morphology of self-assembled structures of glycosphingolipids and their mixtures with dipalmitoylphosphatidylcholine aggregative properties of gangliosides in solution lipid membrane domains in glycobiology micellar properties of glycosphingolipids in aqueous media shedding and uptake of gangliosides and glycosylphosphatidylinositol-anchored proteins characterization of the cellular binding of exogenous gangliosides chemical synthesis of fluorescent glycero-and sphingolipids synthesis, shedding, and intercellular transfer of human medulloblastoma gangliosides: abrogation by a new inhibitor of glucosylceramide synthase shedding and immunoregulatory activity of yac- lymphoma cell gangliosides shedding of human neuroblastoma gangliosides synthesis of novel nbd-gm and nbd-gm for the transfer activity of gm -activator protein by a fret-based assay system support from professor dr. k. sandhoff, the dfg, and the european community ( th framework program "lipidomicnet", proposal no. ) is gratefully acknowledged. key: cord- -tid a authors: basso, luis g. m.; vicente, eduardo f.; crusca jr., edson; cilli, eduardo m.; costa-filho, antonio j. title: sars-cov fusion peptides induce membrane surface ordering and curvature date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: tid a viral membrane fusion is an orchestrated process triggered by membrane-anchored viral fusion glycoproteins. the s subunit of the spike glycoprotein from severe acute respiratory syndrome (sars) coronavirus (cov) contains internal domains called fusion peptides (fp) that play essential roles in virus entry. although membrane fusion has been broadly studied, there are still major gaps in the molecular details of lipid rearrangements in the bilayer during fusion peptide-membrane interactions. here we employed differential scanning calorimetry (dsc) and electron spin resonance (esr) to gather information on the membrane fusion mechanism promoted by two putative sars fps. dsc data showed the peptides strongly perturb the structural integrity of anionic vesicles and support the hypothesis that the peptides generate opposing curvature stresses on phosphatidylethanolamine membranes. esr showed that both fps increase lipid packing and head group ordering as well as reduce the intramembrane water content for anionic membranes. therefore, bending moment in the bilayer could be generated, promoting negative curvature. the significance of the ordering effect, membrane dehydration, changes in the curvature properties and the possible role of negatively charged phospholipids in helping to overcome the high kinetic barrier involved in the different stages of the sars-cov-mediated membrane fusion are discussed. corresponding to residues - (immediately positioned n-terminally to hr ) and to residues - (immediately positioned c-terminally to a second, internal cleavage site s ' at r ) , and another less conserved region corresponding to a hydrophobic stretch located between residues and (near the s /s boundary region at site r ) . these putative fps are thought to destabilize host cell membranes, driving the refolding of the s subunit into the post-fusion -hb configuration, one of the late steps in the viral membrane fusion process . although membrane fusion promoted by class i viral glycoproteins, such as sars-cov spike, human immunodeficiency virus (hiv) gp or influenza virus hemagglutinin (ha), has been broadly studied in recent years [ ] [ ] [ ] [ ] , many aspects of the molecular mechanism behind the virus-host cell membrane fusion remain unknown, including conformational changes of the lipid bilayers during peptide-membrane interactions. elucidating the nature of protein-lipid interactions as well as the conformational properties of both the membranotropic segments of the viral fusion proteins and the lipids in cell membranes can help to dissect the major steps of the orchestrated membrane fusion mechanism promoted by those biological machines. however, structural and dynamics information at the molecular level of peptide-induced membrane fusion in the context of the whole spike protein is difficult to obtain. thus, synthetic peptides corresponding to the putative fusion peptides might be very useful in providing detailed information on the interaction of those segments with lipid model membranes not only because the peptides themselves support membrane fusion, but also because there is a direct correlation between the effects of mutations in the intact protein and in the peptide analogues for membrane fusion [ ] [ ] [ ] . in the present study, we investigated the effects of two putative fusion peptides from sars-cov s glycoprotein, corresponding to residues - (sars fp ) and - (sars ifp ) , , , , on the structural dynamics, physicochemical properties, and thermotropic phase behavior of lipid model membranes by differential scanning calorimetry (dsc), continuous wave (cw) and pulsed electron spin resonance (esr) along with nonlinear least-squares (nlls) spectral fitting . we found that both peptides increase the lipid packing and decrease the water content inside the lipid bilayer only for membranes containing negatively charged lipids, as well as generating opposing curvature stresses on highly curved membranes containing non-bilayer-forming phospholipids. the significance of the ordering effect, membrane dehydration, changes in the curvature properties and the possible role of negatively charged phospholipids for the fusion mechanism mediated by the sars-cov s glycoprotein are discussed. we are interested in investigating the extent of perturbation of model membranes caused by a functional concentration of the peptides, i.e, peptide concentration that is already known to promote fusion of model membranes. at this concentration, what are the changes in the structural dynamics, curvature and hydration of the lipids and in the thermodynamic parameters of the membranes that lead to membrane fusion? it has been shown that sars fp and sars ifp are able to induce membrane fusion only at high peptide-to-lipid molar ratio , . thus, we chose a : lipid/peptide molar ratio for most of the experiments and compared the effects of the peptides on model membranes that present very different physicochemical properties. to examine the effects of the putative sars fusion peptides on the structural integrity of lipid model membranes, the thermotropic phase behavior of different multilamellar vesicles (mlv) in the absence and in the presence of mol% of peptides were determined (fig. ) . the thermodynamic parameters obtained from the analysis of the dsc curves are shown in table . it is worth mentioning that the membrane-associated peptides remain folded in the liquid crystalline phase of the phospholipids , . multilamellar vesicles of dppc and dppg exhibited two endothermic events in the temperature range studied. the low-enthalpic, broad pretransition arises from the conversion of the lamellar gel phase, l β' , to the ripple gel phase, p β' , and is observed at about . °c for dppc and at . °c for dppg (table ) . on the other hand, the more energetic and more cooperative (narrow) main phase transition arises from the conversion of p β' to the liquid-crystalline phase, l α , and is centered at . °c for dppc and at . °c for dppg. membranes composed of unsaturated lipids presented only a very asymmetric, low-enthalpic, and broad main phase transition at . °c for popa, at . °c for pope, and at ~ . °c for pops. these results reasonably agree with the literature [ ] [ ] [ ] [ ] . the slight discrepancies observed in comparison with literature data are likely due to differences in lipid preparations, buffers, and ionic strength [ ] [ ] [ ] . by contrast, the main phase transition of dpps vesicles is split into two endothermic events, one centered at . °c and the other centered at . °c (table ). this two-peak feature has been attributed to different protonation states of the head group polar moiety . binding of drugs, peptides, and proteins to membranes can promote structural and dynamic changes of lipid bilayers that significantly affect their thermotropic phase behavior , . shifts of the melting transition temperature of the lipids, for instance, can be related to alterations of the entropy change between the gel and fluid states, whereas a broadening of the dsc thermogram may be the result of a decreased transition cooperativity (~ /Δ t / ). as can be observed in fig. , incorporation of mol% of fusion peptides into the lipid bilayers did not strongly affect the main phase transition temperature of the liposomes. however, the enthalpy and entropy changes of the main phase transition were significantly altered, especially for membranes containing negatively charged lipids ( fig. and table ) . sars fp and sars ifp only slightly perturbed zwitterionic dppc liposomes. it was observed a small increase of both the melting temperature t m (less than %) and the calorimetric enthalpy change of the transition Δ h (less than . %), indicating a slight stabilization of the peptide-bound dppc gel phase. the increased pretransition temperature of the peptide-containing dppc vesicles also indicates structural changes in the dppc polar head group , . on the other hand, more prominent effects on the enthalpy change and on the cooperativity of the transition of zwitterionic pope vesicles were observed. sars fp decreased the transition Δ h by about %, whereas sars ifp decreased it by % (table ) . pope-sars fp interaction also markedly broadened the dsc endotherm (increase of Δ t / by ~ %). this result indicates that sars fp intercalates within pope bilayer and thus decreases the intermolecular cooperativity of the transition. overall, more significant effects on the thermodynamic (or dsc) parameters of the bilayer phase transitions were observed for membranes containing negatively charged lipids ( table ) : reduction of the calorimetric Δ h by about % ( %) for dppg and by about % ( %) for pops, for instance, after incorporation of sars fp (sars ifp ). unlike the previous cases, endotherms containing multiple peaks appeared when the peptides were mixed with popa vesicles (fig. ) . in this case, the lower-temperature endothermic peak certainly arises from peptide-bound membranes, whereas the higher-temperature peak can be due to peptide-free popa vesicles in the popa/sars fp samples and due to a mixture of peptide-free and peptide-bound popa vesicles in the popa/sars ifp samples. the two-component feature of dpps main phase transition remained in the peptide-containing dpps samples. although the calorimetric enthalpy change of the whole transition decreased in the presence of the peptides, the area under the higher-temperature endotherm increased. the peptides also promoted opposing effects on the cooperativity of the two endotherms: while sars fp narrowed both transitions (thus increasing lipid cooperativity), sars ifp broadened the endotherms. this may be related to different locations and topology of the peptides in the membranes. another interesting feature is the appearance of a third, low-enthalpic peak at around °c in dpps/peptide vesicles. this result indicates a different mechanism of ps-peptide interaction compared to the other negatively charged lipids. the low-enthalpy, higher-temperature peak ( °c) was also observed in dmso-treated peptide-containing dpps liposomes, but was not observed in the acetonitrile-treated or dmso-treated peptide-free dpps vesicles. this result indicates that the new peak is not due to the binding of acetonitrile or dmso from the peptide stock solutions to the dpps mlvs (see fig. s and table s in supplementary information). interestingly, a low-enthalpy peak also appeared in the peptide-bound pops vesicles at a temperature slightly higher than the t m of the peptide-free pops vesicles (fig. ) . thus, this more stable and much less energetic endothermic peak may be due to specific ps-peptide interaction. taken together, these results suggest different mechanisms for the interaction of the peptides with zwitterionic and negatively charged phospholipids. electrostatic interactions seem to play an important role for the sars-cov fusion peptide-membrane interactions, since both peptides are positively charged at ph . : sars fp , + e sars ifp , + . moreover, since both peptides also significantly changed the thermotropic phase behavior of dppg mlvs at high ionic strength ( mm nacl; reduction of Δ h by % for sars fp and by % for sars ifp - table ), hydrophobic interactions may also play an important role in peptide binding and penetration into membranes. interestingly, the peptides promoted different effects on the thermograms of dppg, dpps, pops, and popa, suggesting that not only the charge but also the lipid packing (dpps vs. pops) and the lipid polar head group (pg, ps, or pa) may contribute to the energetics of peptide-membrane interactions. membrane curvature-promoting properties. since changes in membrane curvature have been associated with the potential mechanistic role of fusion peptides in inducing membrane fusion , , we tested the ability of the sars-cov fusion peptides to promote curvature strain on dipope vesicles. below the lα -h ii phase transition temperature, t h , phosphatidylethanolamines, such as dipope, spontaneously form lipid bilayers in the liquid-crystalline state, whereas above t h , they usually pack together in a highly curved hexagonally inverted structure with negative membrane curvature , . stabilization of this concave curvature due to peptide binding, for instance, will favor the nonbilayer h ii phase, which will translate into a t h reduction . conversely, peptides that induce positive membrane curvature will increase t h of the dipope/peptide samples and thus will stabilize the liquid-crystalline bilayer phase . figure shows dsc traces of dipope without and with . or . mol% of sars fp and sars ifp at both low ( fig. a) and high (fig. b) effects of the peptides on the structural dynamics of lipid vesicles. the local ordering and rotational dynamics of the lipid head group and acyl chains were investigated by cw esr using the nitroxide-labeled lipids dpptc, -pcsl, and -pcsl (see structure of the spin probes in supplementary fig. s ), which monitor different regions of the lipid bilayers. dpptc reports on the lipid/water interface, whereas -pcsl and -pcsl monitor the hydrophobic core of the membranes at different depths of penetration , [ ] [ ] [ ] . despite the perturbation of the dppc thermotropic parameters caused by the peptides, esr spectra of the spin labels embedded into dppc and dppc/cholesterol / (mol/mol) mlvs at different temperatures did not show noticeable changes in the lipid structural dynamics of the peptide-containing vesicles as compared to the peptide-free lipid bilayers (see supplementary fig. s ). this means that the membrane-bound peptides do not significantly perturb the local structural dynamics of the head group or the hydrophobic core of the zwitterionic mlvs as investigated by cw esr. on the other hand, remarkable changes in the esr spectra of the spin labels in liposomes containing negatively charged lipids, such as dppg, dpps, and popa, were observed. the experimental and best-fit nlls simulations are presented in supplementary figs s and s and the best-fit magnetic tensor components, rotational diffusion rates, and order parameters are summarized in supplementary tables s -s . before analyzing the changes in the lipid ordering and mobility induced by the peptides, it is worth mentioning a general feature observed for the order parameter s of dpptc in the gel and the fluid phases of all lipid model membranes. we generally and consistently found a positive value for s in the gel phase of the lipids and a negative value in their fluid phase. for instance, we found s = − . for dpptc in dppg at °c (table s ) , s = − . in dpps at °c (table s ) , and s ~ − . in popa at °c and °c (table s ). the meaning of this negative value for s of dpptc was extensively discussed by ge and freed for this same spin label in the fluid phase of dppc dispersions (s = − . at °c) in terms of the analysis of the cartesian components of the restoring potential u(Ω) . in the gel phase, there is a molecular force field in the head group that tends to align the trimethyl ammonium (tma) group of the dpptc (supplementary fig. s ) perpendicular to the bilayer surface, i.e. parallel to the local director of the bilayer, giving rise to s > . however, in the fluid phase, that surface-orienting potential tends to align the tma group parallel to the surface, giving rise to a negative order parameter . by definition, s ≡ < ( cos θ − )/ > , hence s tends to − . when θ tends to ° and it is negative for ° < θ < ~ . °. thus, if we compare the s values of dpptc from our studies with that obtained by ge and freed, we might infer that the orientation of the tma group in dppc (s = − . ; ge and freed work) and in dpps (s = − . ; our work) lies in between the one in dppg (s = − . ) and the other in popa (s = − . ). those differences in the tma orientation are likely due to distinct orienting forces that arise from the particular hydrogen-bonding network provided by the pg, ps, and pa bilayers and the dipolar interactions between the zwitterionic phosphoryl-tempo-choline group of dpptc and the surrounding negatively charged head groups. additionally, irrespective of its sign, an increase of s (either by becoming more positive for s > or less negative for s < ) would lead to the same interpretation: a greater tendency of the preferential orienting axis of tma to orient along the local director of the membrane and an increased restriction of the amplitude of its rotational motion. figure shows the plots of the rotational diffusion rates r ⊥ and the changes in the order parameter s fig. ). thus, the dpptc esr spectra at °c may be regarded as a single averaged spectrum from which both gel-like and fluid-like populations are not resolved. therefore, the effect of the peptides on r ⊥ and s should be analyzed with caution in this case. the ordering effect of the peptides on the dppg head group region in the gel and fluid phases was also observed in the hydrophobic core of the bilayer at all temperatures, but with the most prominent changes in the ripple gel phase (fig. b ,c). the more fusogenic sars fp consistently increased the ordering of -pcsl and -pcsl more than sars ifp . particularly, since the -pcsl probe is more sensitive to molecular motions than dpptc and -pcsl , , the structural dynamics of the gel-like and fluid-like lipid states could be resolved and characterized (table s ) . although the peptides shifted the equilibrium between the two states towards the fluid-like one (the population of the fluid-like component increased from to ~ %), the packing (s ) of the center of the bilayer was also increased for both lipid states. as for the mobility, the peptides reduced the rotational diffusion of -and -pcsl mainly in the fluid phase. only sars fp was able to decrease r ⊥ below t m , with the most significant change observed for -pcsl at °c (r ⊥ decreased from . × s − to . × s − ; table s ). figure shows the r ⊥ and Δ s values for dpptc and -pcsl embedded in dpps at temperatures corresponding to the gel ( and °c) and fluid ( °c) phases of the membrane. the rotational mobility and order parameter of dpptc were both affected by the peptides at all temperatures, but the most striking changes were observed in the very ordered dpps gel phase (fig. a ). in the fluid phase, r ⊥ decreased by % and % for sars ifp and sars fp , respectively. at °c, r ⊥ decreased by - %: from . × s − to . × s − for sars ifp , and to . × s − for sars fp (table s ). the most prominent change in the dpps head group region induced by the peptides, based on our nlls simulations, took place on the molecular alignment of the dpptc tma group. firstly, the s at °c found in our simulations was . , much lower than that found in pure dppg at the same temperature (s = . ; table s ). ge and freed reported a value of . for dpptc in dppc dispersions in the gel phase ( °c) and a similar value was found by barroso et al. for a similar pc spin probe in an equimolar mixture of dppc/dpps (s = . ). this result indicates that the phosphoryl-tempo-choline group of dpptc is more loosely packed in the gel phase of the pure dpps bilayer than in the other model membranes reported. interestingly, peptide addition to pure dpps remarkably increased the ordering of the head group in the gel phase: s jumped from . to about . for both peptides at °c and similar changes were observed at °c. in the fluid phase, though, just a slight variation was obtained (Δ s = . - . ) (fig. a ). peptide binding to dpps head group caused a large ordering effect, thus restricting the mobility of the lipids. the spectra of -pcsl in the peptide-free dpps obtained at temperatures below t m presented very broad resonance lines (supplementary fig. s b ; °c). even in high ionic strength condition, a very broad lineshape in the ordered dpps gel phase persisted (not shown). this line broadening effect is due to strong dipolar interactions that arise from possible cluster formation of the pc spin labels in the dpps milieu. to test this hypothesis, we prepared dpps/ -pcsl samples with either . mol% or . mol% of the spin label. at the higher probe concentration, even broader lines were obtained as compared with the esr lineshape of . mol%, whereas narrower, but still broad, lines were found with . mol%. this result shows that -pcsl does not partition very well in the dpps gel phase but does so in the fluid phase, as illustrated in supplementary fig. s b (spectrum acquired at °c). after unsuccessful attempts to fit the -pcsl spectra with the addition of a heisenberg exchange coupling, we presented only the best-fit parameters from nlls simulations of the spectra acquired at °c (supplementary table s ). both r ⊥ and s were only slightly affected by the membrane-bound peptides at that temperature: r ⊥ slightly decreased from . × s − to . × s − for sars fp and s increased from ~ . to ~ . or . for sars ifp and sars fp , respectively (table s ) . interestingly, binding of the peptides to dpps promoted a better partition of -pcsl in the membrane. the structural organization of the dpps/peptide membranes altered in such a way that the pc spin probe became miscible in the binary system. as for the -pcsl in dpps, no change in the esr lineshape at both °c and °c upon peptide addition was observed (not shown), indicating no long range effect of the peptides on the center of the bilayer, despite the fact that they do bind and perturb the head group and the region around c of the lipid acyl chain. however, at °c, sars fp and sars ifp promote the appearance of a second, disordered component in the esr spectra table s ). in the fluid phase, the only parameter affected by peptide binding was the rotational mobility, which was diminished from . × s − to . or to . × s − for sars fp or sars fp , respectively. our results indicate the peptides bind to both gel and fluid phases of dpps, but peptide insertion into the membrane seems to be activated either by temperature or ultimately by loosening of the hydrophobic packing of the highly ordered dpps gel phase. as the temperature is increased, long range effects on r ⊥ of the acyl chain were observed in the bilayer center. we also studied the effect of the peptide binding on the structural dynamics of popa mlvs in the fluid phase ( °c and °c). as shown in table s , sars fp and sars ifp only changed the ordering and r ⊥ of the spin labels at °c (fig. ) . at °c, the esr spectra from peptide-free and peptide-bound vesicles are almost the same. differently from the other model membranes, peptide binding to popa head group causes an increase of the mobility: r ⊥ changed from . to . × s − for sars ifp and to . × s − for sars fp (fig. a ). this result is probably related to the lack of a bulky head group of popa (the surface area per lipid of pa head group is smaller than that of pg or ps), which may facilitate the rotational diffusion of the phosphoryl-tempo-choline group of dpptc, despite the small decrease in the amplitude of the tma molecular motion (s increased by about . to . ; fig. b ). on the other hand, the best-fit nlls simulations of -pcsl spectra showed a prominent effect on r ⊥ rather than s : while the rotational mobility diminished % for sars ifp , it decreased % for sars fp , whereas Δ s was only about . (supplementary table s ). as for the -pcsl, we found a very high value for s (− . ) in the peptide-free popa liposomes, indicating a large deviation from cylindrical symmetry of the molecular alignment of the end-chain label to the local director of the membrane. to gain further insights on the molecular orientation of the end-chain label we derived the orienting potentials along the cartesian directions from the potential coefficients obtained by the best fits of the nlls simulations (section si ): u x ≡ u( °, °)/kt = . , u y ≡ u( °, °)/kt = − . , and u z ≡ u( °, °)/kt = − . . this result indicates a strong preference for the rotational diffusion axes z r (parallel to the pz orbital of the nitrogen) and y r of the nitroxide moiety to align along the normal to the bilayer, while x r , which is parallel to the n-o bond, is prevented to align along the membrane local director. therefore, the unsaturation of the oleyl acyl chain of popa would allow for a dynamic bending of the end chain of the spin label that favors much more the alignment of both z r and y r axes to table s ). since the fluid-state lipids ( °c) present lower order (s = . ) and high degree of molecular misalignment (s = − . ) with r ⊥ ranging from . to . × s − (supplementary table s ), these results allow us to infer that this second component does not present the same properties of fluid-state lipids but rather peptide-bound lipids. that is, peptide binding to popa bilayers gives rise to peptide-enriched and peptide-free domains, where the latter is barely affected by the peptides. affected by the peptides. these results are in good agreement with our dsc experiments, in which, as discussed earlier, thermograms with multiple peaks arise from peptide-associated and peptide-free popa vesicles. additionally, it is important to investigate the effects of membrane fusion promoters and inhibitors on the bilayer properties in an attempt to identify the changes in the physicochemical parameters of the membrane that might be relevant for membrane fusion. generally speaking, insertion of inverted cone-shaped molecules such as the lysophosphatidylcholine -palmitoyl- -hydroxyl-pc (lpc) into the outer monolayer would prevent membrane fusion supposedly by promoting positive membrane curvature , . conversely, insertion of cone-shaped molecules such as phosphatidylethanolamine, arachidonic acid (aa), and linoleic acid (la) into the outer monolayer would facilitate membrane fusion presumably by inducing negative curvature . using esr, ge and freed have found opposing effects of lpc and aa on the order parameter of a headgroup spin label embedded in dmpc model membranes . in their work, an ordering (disordering) effect was observed for the fusion promoter (inhibitor) aa (lpc), which may help to induce (prevent) membrane fusion. since the putative fusion peptides from sars-cov s protein promote substantial membrane fusion only in the presence of negatively charged lipids, it is important to verify whether that correlation is also valid for negatively charged lipid-containing vesicles. to do so, we prepared equimolar mixtures of dppc/dppg and dppc/popa membranes and studied the effects of sars fp , sars ifp , la, and lpc on the order parameter s of dpptc at °c. the best-fit parameters of the esr spectra shown in fig. s are summarized in table s in supplementary information. figure shows that both fusion peptides and la caused an ordering effect on the head group of both model membranes. in contrast, lpc promoted a disordering effect of dpptc. these results are thus in accordance with those found by ge and freed for the zwitterionic dmpc and indicate that sars fp and sars ifp might facilitate membrane fusion similarly to how the fusion peptide from influenza hemagglutinin does. since the ordering effect of the lipid head group has been attributed to bilayer dehydration , we used eseem spectroscopy to find out whether the peptide-induced ordering of anionic vesicles is possibly related to membrane dehydration, as suggested as a general fusion mechanism of other class i fusion peptides , . eseem has been successfully applied to examine the penetration depth profile of deuterium-substituted molecules, such as water, glycerol, and sugar inside lipid bilayers [ ] [ ] [ ] [ ] as well as to investigate the water permeation at protein/peptide-lipid interface of membrane-interacting peptides and membrane proteins [ ] [ ] [ ] . here we used a d o-containing buffer to probe the deuterium environment surrounding the spin labels doptc, -pcsl, and -pcsl ( supplementary fig. s ) embedded in popc/popg / (mol/mol) mlvs. the different spin labels allowed investigating the changes promoted by the peptides in the water content from the lipid/water interface down to the hydrophobic core of the bilayers. changes in the modulation depth of the time-domain eseem spectra or in the deuterium spectral density yields information about d o molecules situated near the nitroxide within a distance range of up to . nm, which is the known spatial reach of the method . figure a shows the spectral density of the spin-labeled lipids in the peptide-free and peptide-containing lipid vesicles (the normalized time-domain stimulated echo signals are given in the supplementary fig. s a ). all spectra are dominated by two signals, one centered at . mhz, arising from the hyperfine interaction of the nitroxide with surrounding deuterium nuclei ( h-larmor frequency at mt), and the other centered at . mhz, which arises from matrix protons near the spin label. since lipids, peptides and possibly residual h o contribute to the spectral density at . mhz, the h signal was not further analyzed. two additional weak peaks were observed in the ft-spectra of doptc and -pcsl: one at ~ . mhz, which is assigned to n , and the other at ~ . mhz, which corresponds to the larmor frequency of p (fig. a) . those nuclei belong to the choline group of popc ( n) and to the phosphate group ( p) of the lipids. both peaks are absent in the -pcsl eseem spectra because the distance between the spin probe moiety in the bilayer midplane and the head group is beyond the limit of the technique. the deuterium peak is composed of two spectral components: a low-intensity broad signal that arises from a direct deuterium bond between d o and the nitroxide radical; and a narrow doublet of intensity i( h) and amplitude Δ (fig. a) , which arises from the quadrupole interaction between the electron spin and non- h-bonded water molecules inside the bilayer . both Δ and i( h) have been found to depend linearly and nonlinearly, respectively, on the concentration of free water molecules at a distance of . to . nm . the parameters i( h), measured at . mhz, and Δ for all lipid spin labels are shown in fig. b . as expected, the deuterium spectral density decreases from the bilayer/water interface toward the membrane center. particularly, i( h) is reduced from ( ± ) ns in the water/lipid interface to ( ± ) ns around th carbon position and to ( . ± . ) ns near the bilayer midplane in the peptide-free vesicles, indicating reduced water content in the hydrophobic core as compared to the head group region. those i( h) values are somewhat higher than previously reported spectral densities for popc/popg / (mol/mol) bilayers but are in the same order of magnitude of those for dppc , . the discrepancies are most likely due to different freezing protocols, sample preparation, and experimental conditions such as the choice of τ , the first interpulse delay, and the frequency chosen to measure the intensity of the spectral density . sars fp and sars ifp decreased both i( h) and Δ for the spin labels that monitor the hydrophobic region of the bilayer (fig. b) . this finding implies that both peptides interact with popc/popg membranes and displace free water molecules from their hydrophobic core. theoretical calculations from milov et al. indicated that nitroxide-deuterons interactions spread to around nm . therefore, the spin label at c position is able to monitor water density around c up to the level of the carbonyl-glycerol and phosphocholine regions (distant about . nm from c ) , . thus, the contribution from bulk water molecules to the spectral density of -pcsl is negligible , . the significant % decrease of Δ for -pcsl in the peptide-containing membranes, relative to the peptide-free vesicles (supplementary table s , fig. b ), allows us to infer that the intramembrane water in the outer membrane region is dramatically reduced upon peptide incorporation. the same reasoning holds true for the -pcsl. as for the head group spin label doptc, we found that the peptides surprisingly promoted opposing effects on i( h) and Δ : while they slightly raised the magnitude of the d o signal, Δ values were reduced (fig. b) . the source of this opposing effect remains unclear to us, but it may be of some interest to speculate on that. considering the electron density profile of pc lipids , , we may infer that the nitroxide radical of the doptc phosphoryl-tempo-choline group is in direct contact with both membrane surface and bulk water molecules. changes in the orientation of the phosphoryl-tempo-choline must therefore locally disturb the water density around the spin label. incorporation of cholesterol into pc membranes, for instance, decreases the ordering of the lipid head group, i.e., makes it less aligned along the bilayer director . this effect allows for water molecules to move from the bulk into the membrane (down to ~c ), thus increasing both i( h) and Δ (fig. s b and table s in supplementary information) . on the other hand, if the phosphoryl-tempo-choline group becomes more aligned along the bilayer normal, i.e. the s of doptc increases, membrane-surface dehydration takes place . in this situation, the concentration of membrane-surface water molecules is reduced in the 'membrane side' . in contrast, realignment of the head group dipole makes the nitroxide radical become more exposed to interact with bulk water molecules in the 'solvent side' . milov et al. have theoretically shown that the nonlinear dependence of the i( h) on water concentration is due to the formation of nitroxide-water complexes . therefore, if the water distribution around the nitroxide is locally perturbed in such a way that it allows for formation of nitroxide-water complexes (i.e., disturbances take place closer than . nm), it must affect mainly the i( h), but not Δ . that is, reorientation of phosphoryl-tempo-choline due to peptide-lipid interactions may be such that it allows formation of nitroxide-water complexes, affecting mainly i( h). this may correspond to the source of the increased i( h). the contribution to Δ stems mainly from water molecules belonging to the second hydration shell (> . nm) up to ~ nm. from the 'solvent side' , the second water coordination sphere for the water-exposed nitroxide radical should not change, thus the major contribution to Δ of doptc may arise from free water molecules located in the 'membrane side' . the reduced level of the head group hydration accounts therefore for the decrease of Δ values. however, further detailed studies are needed to investigate this hypothesis. viral membrane fusion is a concerted mechanism that involves remarkable protein and lipid conformational changes. the molecular mechanism by which viral fusion proteins catalyze the membrane fusion reaction and the molecular details of lipid rearrangements in the lipid bilayer are not fully understood yet, although the current model has been constantly revisited and refined [ ] [ ] [ ] [ ] [ ] [ ] . crystal structures of pre-and post-fusion states of class i viral fusion proteins along with nmr structures of fusion peptides in membrane mimetics have provided a mechanistic view of the membrane fusion process. in this process, different protein segments act in an orchestrated way to achieve the complex and kinetically unfavorable task of bringing together and fusing two lipid bilayers , . however, there are still major gaps in the molecular details involved in the fusion process. they include: the sequential order and kinetics of the events, changes in the structure, dynamics, and physicochemical properties of different protein domains and lipid bilayers during the whole membrane fusion mechanism, free energies of the corresponding pre-, post-(fusion pore) and intermediate (hemifusion) fusion states as well as the modulation of all those parameters by lipid composition. sars-cov s subunit possesses various membranotropic segments, i.e., relatively short hydrophobic domains that are able to bind to and perturb membranes. these segments can act independently from each other and may help to stabilize the contact between viral and cell membranes , [ ] [ ] [ ] [ ] . however, monitoring the structural rearrangements of different segments of the intact s subunit as well as the dynamics of their interaction with viral and cell membranes on a molecular level constitute a challenging task. the use of synthetic peptides and phospholipids has provided important thermodynamic, structural, biochemical, and functional details of peptide-membrane interactions from both peptide and lipid perspectives, which makes them a good platform to study membrane fusion , , . our results indicate that both sars fp and sars ifp significantly perturb the thermotropic phase behavior as well as the molecular ordering and phospholipid rotational mobility of model membranes containing negatively charged lipids, but only cause moderate effects on zwitterionic membranes. these findings are in agreement with previously reported studies that indicated greater disturbance and higher affinity of both peptides for model membranes containing anionic rather than zwitterionic phospholipids , . even though the peptides do not partition well into zwitterionic membranes, they are able to perturb the thermodynamic parameters of the zwitterionic dppc and pope phase transitions as well as to change the membrane curvature properties of dipope. on the other hand, our cw esr experiments showed that the peptides do not affect the structural dynamics of zwitterionic lipid bilayers, but they do promote an ordering effect on the lipid head group and on the acyl chains of negatively charged membranes. this latter effect seems to be correlated to membrane dehydration, as shown by our eseem experiments. ordering and dehydration effects have also been observed for other class i viral fusion peptides such as those from hiv-i gp and influenza ha glycoproteins , . the relevance of membrane-curvature induction and lipid dehydration for the viral membrane fusion of the sars-cov fusion peptides will be discussed below. peptides induce bending moment and membrane dehydration of anionic membranes. lipid molecules in bilayers experience a restoring torque from neighboring lipids that tends to reorient the lipid chain and/or the head group along the normal to the bilayer. the order parameter s , which is associated with the restoring torque through the orienting potential u(Ω), is a measure of the extent of that molecular alignment. generally, the higher the s the lower is the angular amplitude of the wobbling motion of the spin probe, i.e. the more aligned along the local director is the lipid segment to which the probe is attached. a better alignment can enhance the molecular interactions between lipid molecules in the bilayer. therefore, s directly reports on the lipid packing density. in particular, slight changes in s of the lipid head group would have a greater impact on the structural organization of the membrane than changes in the ordering of the acyl chain . this is primarily due to the strong hydrogen-bonding network in the head group region (~ . to . kcal/mol) . thus, although the molecular structure of dpptc head group is different from those of the pg, ps, and pa head groups, reorientation of dpptc due to conformational changes in the polar region might be associated with changes in the hydrogen-bonding network . that is, the more ordered the dpptc, the more condensed is the bilayer, which leads to changes in the ionic interactions between the head groups and between the head groups and surface-bound water molecules. by contrast, the van der waals interactions between the lipid acyl chains in the hydrophobic core of the membrane are much weaker, i.e. the strength of the interaction is about . kcal/mol, which is even smaller than the thermal energy at k (k b t ~ . kcal/mol) . nevertheless, enhancement of lipid-lipid interactions due to condensation of the hydrophobic core leads to an increased chain-packing energy. in our studies, the peptides were added into preformed vesicle solutions. therefore, the changes observed here are primarily due to the interaction of the peptides with the outer leaflet of the bilayers. sars fp and sars ifp increase the s of -pcsl and -pcsl in dppg and popa membranes, making the nonpolar core of the outer leaflet more solid-like. furthermore, both peptides increase the ordering of head group spin label dpptc in pure dppg, dpps, and popa as well as in dppg-and popa-containing membranes, but not in the zwitterionic dppc and dppc/chol. therefore, an increase of both the head group and acyl chain packing densities of the outer leaflet is the major effect of the peptides on the negatively charged phospholipid membranes investigated in this work, which is in agreement with previous studies , . interestingly, sars fp shows higher membrane fusion activity in pure pg or in ps-or phosphatidylinositol (pi)-containing membranes, but not in zwitterionic ones . thus, ordering of the lipid head group seems to be an important structural change in the bilayer necessary to induce membrane fusion , . indeed, we found the same ordering effect on the head group of membranes containing negatively charged lipids for the membrane fusion promoter la and an opposite effect for lpc, a known fusion inhibitor. this ordering or condensation effect on the outer monolayer leads to a shrinkage of its surface area that compresses the inner leaflet. because of the mechanical coupling between the two leaflets in a lipid bilayer vesicle, the inner layer counteracts the compressive force exerted by the outer monolayer and therefore creates a nonuniform tension in the two leaflets, which redistributes the stress profile across the bilayer. as a result, a uniform membrane bending moment toward the condensed leaflet is induced, which ultimately generates negative (positive) membrane curvature if the outer (inner) monolayer is condensed . due to the properties of the stress profile across the bilayer and to the nature of the molecular interactions in the head group and acyl chain regions, the largest contribution to the bending moment arises from the ordering of the head group region . thus, the increase of s of dpptc in the outer leaflet of negatively charged lipid membranes promoted by the sars-cov fusion peptides could potentially induce negative membrane curvature (please see section si of supplementary information for further details). this negative curvature effect induced by bending moment has also been proposed as the putative membrane fusion mechanism of other class i fusion peptides such as those from influenza ha or hiv- gp , . another important aspect to consider is the inverse correlation between head group ordering and hydration of lipid bilayers . if an increased head group packing density leads to membrane dehydration, this effect may help to overcome the high hydration repulsive energy that arises from membrane surface-bound water molecules . prior to fusion, two apposed lipid bilayers must approach. when the distance between the approaching bilayers is within to nm, a high hydration repulsion arises and dominates the interactions between them , thus preventing the membranes to make contact and to proceed to fusion. therefore, molecules that have the ability to overcome this hydration barrier could succeed in promoting membrane fusion. bilayer dehydration can be accomplished by different mechanisms depending on the molecular nature of the fusogenic molecule [ ] [ ] [ ] . particularly, hiv- gp and influenza ha fusion peptides promote membrane dehydration by increasing the ordering of the lipid bilayers , . that conclusion was inferred, though, from the aforementioned s /dehydration correlation and not by actually measuring the water content inside the bilayer. our eseem experiments, on the other hand, have undoubtedly shown that partial membrane dehydration actually takes place upon binding of the sars-cov fusion peptides to popc/popg membranes. this finding also provided a direct evidence of the correlation between head group ordering and membrane dehydration. however, in contrast to our eseem data, guillén et al have suggested that sars fp increases the water penetration depth into zwitterionic and anionic large unilamellar vesicles . this conclusion was based on the analysis of the fluorescence decay of diphenylhexatriene (dph) embedded in membranes, whose multi-component lifetimes were shortened in the presence of the peptide. although the increase of water penetration depth is a possible interpretation for their results , since the quantum yield of dph decreases in water, probe location and orientation might also influence the fluorescence lifetime of dph in lipid bilayers. conflicting results in the literature have indicated there is no consensus yet regarding the exact location of dph in pure model membranes [ ] [ ] [ ] [ ] . particularly, konopásek and coworkers , have shown that the short-lived component of dph embedded in lipid bilayers originates from a probe population located at the membrane-water interface. therefore, the subnanosecond short-lived component of dph in the very ordered gel phase of dimyristoyl phosphatidylglycerol (dmpg) found by guillen et al. in the absence of sars fp could thus potentially be due to a shallower interfacial location of dph in the dmpg bilayer. thus, the decrease of the dph lifetime in the presence of sars fp found by guillen and coworkers could potentially stem from the contact of the fluorophore with water molecules at the membrane-water interface due to a reorientational distribution of dph in the bilayer upon the condensing effect induced by the peptide. lastly, it is important to emphasize that the alterations of the bilayer structure observed for sars fp and sars ifp take place at high peptide concentrations and thus are most likely due to a cooperative behavior of membrane-bound self-associated β -sheet peptides. in fact, both peptides present membrane fusion activity only at high peptide-to-lipid molar ratio and regular extended β -sheet aggregates are the most populated membrane-bound peptide conformation , , . peptide oligomerization in membranes as extended β -like aggregates seems to play an important role in peptide-induced membrane fusion . peptide self-association in small areas of the membrane surface might actually be important in the very first stages of the membrane fusion process . indeed, since a high amount of work is required to merge large surface areas of two lipid bilayers, lipid merging most likely proceeds through local points of contact , , . thus, bending moments promoting negative curvature and membrane dehydration may not only help to decrease the hydration barrier between the proximal leaflets of the approaching bilayers but also minimize the work necessary for merging the monolayers. the latter is a consequence of the possible formation of point-like membrane protrusions, also referred to as local 'nipples' , a critical step that theoretically precedes the formation of the intermediate hemifusion stalk , , . peptides change membrane curvature of non-bilayer lipids in opposing ways. accumulating evidence suggests that membrane fusion involves the formation of strongly curved lipid bilayers in the pre-fusion, intermediate, and post-fusion states , . it is therefore tempting to investigate the potential ability of fusion peptides to generate membrane curvature, since that information can help to elucidate the role played by those molecules in the viral membrane fusion process. dsc experiments with non-bilayer-forming lipids such as phosphatidylethanolamines (pe) have been successfully used to indirectly probe changes in the intrinsic spontaneous curvature properties of pe vesicles by a wide variety of peptides , , . shift of the l α -to-h ii transition temperature is an excellent indicator of lipid phase changes and curvature alterations . we found that both sars fp and sars ifp peptides have the ability to bend dipope lipid bilayers. while sars fp induces positive curvature, sars ifp causes opposing stresses on the membrane depending on the ionic strength: it promotes positive (negative) curvature strain at low (high) ionic strength. the capacity of the sars fusion peptides to generate different curvature stresses on pe vesicles might actually be important in the context of the whole sars-cov s -mediated membrane fusion. the small stressed protrusions formed in the pre-fusion state are characterized by lipid domains possessing positive curvature flanked by negatively-curved lipid patches that stabilize the local 'nipple' and allow for the establishment of close intermembrane contact , , . therefore, both peptides can hypothetically act in this early stage by stabilizing the two curved domains depending on the ionic strength of the environment and on the exposure to pe or to negatively charged lipids . membrane fusion proceeds with the formation of the so-called hemifusion intermediate or lipidic stalk, which is characterized by lipid mixing between the outer leaflets of the two apposed bilayers with the distal monolayers remaining unfused. the resultant mixed outer leaflet presents a high degree of negative curvature , . stabilization of this intrinsic negative curvature has been traditionally interpreted as the common role played by various fusion peptides in the viral membrane fusion process , . the hemifusion intermediate would then subsequently progress to the formation of the fusion pore state , , which is characterized by lipid mixing of both the outer and inner leaflets of the merged bilayers, thus establishing the opening of a pore that allows content mixing between the two apposed membranes. in this pore state, both monolayers present strong and opposite curvature strains , . based on our findings, the outer, negatively-curved monolayer of both the lipidic stalk and the fusion pore states may be stabilized by the sars fusion peptides depending on the lipid composition: sars fp and sars ifp could act in membranes containing anionic lipids, and sars ifp could play a major role in pe-rich membranes at high ionic strength. on the other hand, the intrinsic positive curvature of the inner leaflet of the pore state can be stabilized by sars fp in the presence of pe lipids. that is, the more fusogenic sars fp peptide can also support membrane fusion by stabilization of porous structures. experimental and computational studies have also indicated stabilization of the pore state by fusion peptides from influenza ha , and parainfluenza virus (piv ) f protein . thus, our findings imply that both sars fusion peptides can act at different stages of the fusion process to facilitate membrane fusion. putative sars-cov membrane fusion model. taken together, our results reveal a functional plasticity of sars fp and sars ifp in helping to promote membrane fusion. both peptides can act in the early and late stages of the membrane fusion reaction by changing three properties of lipid bilayers, namely spontaneous curvature, hydration, and lipid packing density. incorporation of our findings into the currently proposed membrane fusion model induced by class i viral fusion proteins yields the following. upon receptor binding, s domain of sars-cov spike glycoprotein undergoes a large conformational change that releases and exposes sars fp and sars ifp to interact with target membranes . both peptides insert into but not significantly disturb (our data and data in references , and ) the highly-ordered, zwitterionic outer leaflet of the plasma membrane bilayer . this binding process bridges viral and cell membranes and thus facilitates trimerization of other s subunits. trimers are, in general, the fusion-active oligomeric state of class i fusion proteins . as a result, a trimeric extended prehairpin conformation is formed. at this point, it is important to emphasize that the actual conformational state of the sars-cov fusion peptides remains elusive. sars fp and sars ifp adopt, respectively, a v-shaped and a linear helical conformation in dodecylphosphatidylcholine micelles , but have a high tendency to aggregate and to form intramolecular β -sheets and extended β -strands stabilized by intermolecular interactions in models of lipid bilayers as well as to adopt, in small fractions, α -helical and unordered structures , . in the context of the intact protein, however, the structure and oligomerization state of those peptide segments still need to be addressed, although it has been proposed that membrane-bound self-associated peptides may provide the major driving force for trimerization of the whole protein , . peptide binding, conformational change and possibly aggregation into the membrane may trigger s refolding into a trimeric hairpin conformation. as a result, a six-helix bundle would form, bringing not only viral and target membranes into close proximity, but also the internal fusion peptide (sars ifp ) and the pretransmembrane (sars ptm ) domain of the s subunit . due to the high hydration repulsion of the closely apposed lipid bilayers and the requirement for bending membranes to minimize areas of strong interbilayer repulsion , , displacement of water molecules from the membrane surface and changes in membrane curvature seem to be the prerequisites for allowing close intermembrane contact and subsequent formation of the high-energy hemifusion intermediate state. interaction of sars fp and sars ifp with pe or with negatively charged lipids contained either in the plasma (via nonendocytic pathway) or in the endosome (via endocytic pathway) membranes may be important for the formation of point-like protrusions or for stabilization of the hemifusion stalk . since anionic phospholipids are mostly located in the inner leaflet of the membrane, it would be possible that the action of lipid flippases and scramblases could be endorsed by the peptide perturbation on the outer leaflet of the plasma membrane . the major effects of the peptides at the pre-fusion state could be the following: induction of positive curvature on pe-rich membranes, as indicated by our dsc data; and membrane dehydration and induction of bending moment on the outer leaflet of bilayers comprised of anionic lipids, as suggested by our esr data. the latter effects may also be responsible for triggering stalk formation, which is further stabilized by exposure of pe on the outer leaflet to sar ifp . hemifusion could be further facilitated by membrane interaction of a loop peptide segment located in between hr and hr domains and by a possible heteroligomerization of sars ifp with sars ptm . juxtaposition of sars ifp and sars ptm leads to a synergistic and cooperative action of both peptides that causes membrane destabilization and further peptide insertion . exposure of sars fp to the inner leaflet of the merged viral and cell membranes could have a great impact in the post-fusion state. indeed, sars fp could act by promoting positive curvature and stabilizing the high positively-curved inner leaflet that characterizes the porous state, thus facilitating pore formation (our dsc data). however, the molecular details of the above processes still need to be investigated. overall, the two putative fusion peptides from sars-cov s protein may help to regulate membrane fusion by acting in the early and late stages of the membrane fusion process. our main findings were: ( ) sars fusion peptides increase the ordering of the headgroup and acyl chain regions of mlvs containing negatively-charged, but not zwitterionic phospholipids; ( ) membrane fusion promoters induce similar effects on the head group ordering than do the fusion peptides, whereas membrane fusion inhibitors cause opposing effects; ( ) changes in the order parameters of the lipids are generally greater for the more fusogenic sars fp peptide than for sars ifp ; ( ) both peptides promote dehydration of pg-containing membranes and this effect is well correlated with the increased head group ordering; and ( ) dsc data support a hypothesis that sars fp induces positive curvature on dipope vesicles, whereas sars ifp promotes opposing stresses on the intrinsic negative curvature of dipope depending on the ionic strength. peptide-induced chain-packing energy and membrane surface ordering of the outer leaflet of negatively charged lipid bilayers promote partial membrane dehydration and could generate bending moment, as suggested by our esr studies. both effects may induce negative curvature and decrease the hydration repulsion of apposed bilayers. possible peptide involvement on the formation of the pre-fusion point-like protrusions and intermediate hemifusion stalk as well as on the stabilization of the fusion pore state suggest that the sars fusion peptides might play important roles in the whole membrane fusion process. taken together, our findings suggest that the sars fusion peptides have the ability to change the physicochemical properties of model membranes depending on the lipid composition and on the ionic strength. therefore, they can act in the early and late stages of the membrane fusion process, conferring them a functional plasticity that might be important to help overcome the high kinetic barrier involved in the sars-cov-induced membrane fusion. materials. n-terminally acetylated and c-terminally amidated sars fp ( mwktptlkyfggfnfsqil ) and sars ifp ( gaalqipfamqmayrf ) peptides were either purchased from genscript (piscataway township, nj) or manually synthesized according to the standard fmoc solid-phase peptide synthesis method on a rink-amide resin . the details of peptide synthesis are described in vicente et al. . purification was performed as described in supplementary section si . the phospholipids -palmitoyl- -hydroxy-sn-glycero- -phosphocholine (lpc), , -dipalmitoyl-snglycero-phosphatidylcholine (dppc), , -dipalmitoyl-sn-glycero- -phospho-( '-rac-glycerol) (dppg), , -dipalmitoyl-sn-glycero- -phospho-l-serine (dpps), , -dipalmitoleoyl-sn-glycero- -phosphoethanolamine (dipope), -palmitoyl- -oleoyl-sn-glycero- -phosphocholine (popc), -palmitoyl- -oleoyl-sn-glycero- -phospho-( '-rac-glycerol) (popg), -palmitoyl- -oleoyl-sn-glycero- -phospho-l-serine (pops), -palmitoyl- -oleoyl-sn-glycero- -phosphate (popa), and the spin labels -palmitoyl- -stearoyl(n-doxyl)-snglycero- -phosphocholine (n-pcsl, where n = and ), , -dioleoyl-sn-glycero- -phospho(tempo)choline (doptc), and , -dipalmitoyl-sn-glycero- -phospho(tempo)choline (dpptc) were purchased from avanti polar lipids, inc. (alabaster, al). cholesterol (chol) and linoleic acid (la) were obtained from sigma-aldrich (st. louis, mo). all reagents were used without further purification. sample preparation. phospholipids ( . mg for dsc and . mg for esr) and spin labels ( . mol% for cw esr and mol% for pulsed esr) either in chloroform or chloroform/methanol : (v/v) stock solutions were mixed in a glass tube. after dried under a n flow, the lipid film was ultracentrifuged under vacuum overnight to remove traces of solvent. for cw esr experiments, the sample was hydrated in mm potassium phosphate buffer, ph . , sonicated in a bath type sonicator for a few seconds and maintained at a temperature above the main phase transition of the lipid for at least two hours for complete hydration. samples were then subjected to at least six freeze-thaw cycles. a measured volume of sars fp or sars ifp stock solutions in dimethyl sulfoxide (dmso) was added to the preformed multilamellar lipid dispersions. for dsc experiments, peptides dissolved in either acetonitrile/water : (v/v) or in dmso solutions were diluted into buffer and added to the lipid film for hydration. samples were vortexed for few seconds, maintained at a temperature above the phase transition for each lipid during at least min, and subjected to six freeze-thaw cycles. the amount of phospholipid (final lipid concentration of mg/ml for esr and mg/ml for dsc) and peptides used provided a : lipid/peptide molar ratio for most of the experiments. it is worth mentioning that the same amount of dmso or acetonitrile/water : added in the peptide-containing samples was also used in the peptide-free samples as controls for the esr and dsc experiments. the control samples were prepared using the same protocol as those of the peptide-containing samples. for dipope/peptide samples, peptides and lipids dissolved in chloroform/methanol : (v/v) stock solutions were mixed in a glass tube, dried to a lipid film under n gas and lyophilized overnight. samples were hydrated in mm sodium phosphate buffer, ph . , with or without mm sodium chloride, and freeze-thaw cycled six times below the liquid crystalline-to-inverted hexagonal (lα -h ii ) phase transition temperature (t h ) of the lipid. dipope concentration was mg/ml and peptide concentration varied from . to . mol% ( : and : lipid/peptide molar ratio, respectively). for eseem experiments, popc/popg : mol/mol and peptides ( : lipid/peptide molar ratio) were prepared as above, but hydrated in mm sodium phosphate, mm nacl d o buffer, pd = . (actual ph measurement). peptide concentration was confirmed spectrophotometrically by using the theoretical molar extinction coefficients of , m − cm − for sars fp esr experiments. cw-esr experiments were carried out on a varian e- spectrometer operating at . ghz. temperature was controlled by a homemade temperature control unit coupled to the spectrometer, whose accuracy is about . °c. samples were transferred to glass capillaries ( . mm i.d.), which were set into a quartz tube containing a mineral oil bath to help stabilize the sample temperature. the following acquisition parameters were used: center field, , g; scan width, to g; modulation amplitude, . or . g; modulation frequency, khz; microwave power, or mw; time constant, ms, and acquisition time, s. nonlinear least-squares simulations (nlls) of the cw-esr spectra were performed using the multicomponent labview (national instruments) software developed by dr. christian altenbach (university of california, los angeles, california) , . the rotational diffusion rates (r ⊥ , r ∥ ) and order parameters (s , s ) were obtained as described in ref. with further details in the section si of supplementary information. seed values for the magnetic parameters of both -pcsl and -pcsl were obtained from earle et al. and those of dpptc were taken from ge and freed . the strategy of the nlls simulation was performed as described elsewhere . pulsed esr experiments were performed on a bruker elexsys x-band pulsed esr spectrometer equipped with the bruker flexline er x-ms split-ring resonator and the itc oxford cryogenic system for temperature control. samples were immersed into liquid nitrogen prior to the measurements at k. three pulse electron spin echo envelope modulation (eseem) experiments were carried out with the π / -τπ / -t -π / -τecho pulse sequence and using a four-step phase cycling to suppress unwanted echoes . the microwave power was adjusted to give ns π / pulses and an interpulse delay τ of ns, kept constant in all experiments, was chosen to maximize deuterium modulations at the magnetic field where the echo intensity is maximum. starting at time delay t = ns, points were recorded with Δ t = ns steps to obtain the three-pulse stimulated echo decays. the integration gate length was ns and the shot repetition time was , μ s. the number of accumulations varied from to depending on the signal-to-noise ratio and on the modulation depth. data analysis was performed as described in bartucci et al. . briefly, the contribution of the spin relaxation to the eseem signal was eliminated by dividing the time-dependent echo amplitudes, v(τ , t), by a bi-exponential decay, 〈 v(τ , t)〉 , followed by subtraction of unity, as v norm (τ , t) = v(τ , t)/〈 v(τ , t)〉 − . the remained oscillations about zero were apodized with a hamming window and zero-filled to increase the total number of points to about k. numerical fourier transformation was performed and the resultant magnitude spectrum was multiplied by the dwell time Δ t = ns to provide a spectral density in ns units. sars -beginning to understand a new virus characterization of a novel coronavirus associated with severe acute respiratory syndrome angiotensin-converting enzyme is a functional receptor for the sars coronavirus cd l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus the sars-cov s glycoprotein: expression and functional characterization sars coronavirus entry into host cells through a novel clathrin-and caveolae-independent endocytic pathway the life cycle of sars coronavirus in vero e cells the spike protein of severe acute respiratory syndrome (sars) is cleaved in virus infected vero-e cells furin cleavage of the sars coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry mechanisms of coronavirus cell entry mediated by the viral spike protein structure of a proteolytically resistant core from the severe acute respiratory syndrome coronavirus s 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the beginning of the melting process membrane fusion between liposomes composed of acidic phospholipids and neutral phospholipids induced by melittin: a differential scanning calorimetric study effects of the antimalarial drug primaquine on the dynamic structure of lipid model membranes interaction of a peptide model of a hydrophobic transmembrane alpha-helical segment of a membrane protein with phosphatidylethanolamine bilayers: differential scanning calorimetric and fourier transform infrared spectroscopic studies nature of the thermal pretransition of synthetic phospholipids: dimyristolyl-and dipalmitoyllecithin thermal analysis of lipids, proteins and biological membranes. a review and summary of some recent studies molecular view of the role of fusion peptides in promoting positive membrane curvature fusion peptides and the mechanism of viral fusion stability of lyotropic phases with curved interfaces the mechanism of lamellar-to-inverted hexagonal phase transitions in phosphatidylethanolamine: implications for membrane fusion mechanisms reciprocal effects of apolipoprotein and lytic peptide analogs on membranes. cross-sectional molecular shapes of amphipathic alpha helixes control membrane stability interactions of the antimalarial amodiaquine with lipid model membranes the two sides of a lipid-protein story -ghz electron spin resonance studies of polarity gradients along the aliphatic chains in phospholipid membranes polarity profiles in oriented and dispersed phosphatidylcholine bilayers are different: an electron spin resonance study fusion peptide from influenza hemagglutinin increases membrane surface order: an electron-spin resonance study hiv gp fusion peptide increases membrane ordering in a cholesterol-dependent fashion chain configuration and flexibility gradient in phospholipid membranes -comparison between spin-label electron spin resonance and deuteron nuclear magnetic resonance, and identification of new conformations an electron-spin-resonance study of interactions between phosphatidylcholine and phosphatidylserine in oriented membranes inhibition of membrane fusion by lysophosphatidylcholine lysophosphatidylcholine inhibits vesicles fusion induced by the nh -terminal extremity of siv/hiv fusogenic proteins control of baculovirus gp -induced syncytium formation by membrane lipid composition hydration, structure, and molecular interactions in the headgroup region of dioleoylphosphatidylcholine bilayers: an electron spin resonance study water concentration profiles in membranes measured by eseem of spin-labeled lipids glycerol penetration profile in phospholipid bilayers measured by eseem of spin-labelled lipids eseem measurements of local water concentration in d( ) o-containing spin-labeled systems membrane-sugar interactions probed by pulsed electron paramagnetic resonance of spin labels investigation of model membrane disruption mechanism by melittin using pulse electron paramagnetic resonance 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fusion: overcoming of the hydration barrier and local restructuring modulation of poly(ethylene glycol)-induced fusion by membrane hydration: importance of interbilayer separation thermodynamic and structural properties of phosphatidylserine bilayer membranes in the presence of lithium ions and protons a mechanism of divalent ion-induced phosphatidylserine membrane fusion fluorescence techniques for probing water penetration into lipid bilayers location of diphenylhexatriene (dph) and its derivatives within membranes: comparison of different fluorescence quenching analyses of membrane depth recent developments in molecular dynamics simulations of fluorescent membrane probes the origin of the diphenylhexatriene short lifetime component in membranes and solvents short-lived fluorescence component of dph reports on lipid-water interface of biological membranes mechanics of membrane fusion induction of negative curvature as a mechanism of cell toxicity by amyloidogenic peptides: the case of islet amyloid polypeptide induction of non-lamellar lipid phases by antimicrobial peptides: a potential link to mode of action membrane lipids: where they are and how they behave wild-type and mutant hemagglutinin fusion peptides alter bilayer structure as well as kinetics and activation thermodynamics of stalk and pore formation differently: mechanistic implications conformation and lipid interaction of the fusion peptide of the paramyxovirus piv in anionic and negative-curvature membranes from solid-state nmr nmr structures and localization of the potential fusion peptides and the pre-transmembrane region of sars-cov: implications in membrane fusion tryptophan-dependent membrane interaction and heteromerization with the internal fusion peptide by the membrane proximal external region of sars-cov spike protein protein-lipid interplay in fusion and fission of biological membranes biophysical properties of lipids and dynamic membranes synthesis of the antibacterial peptide cecropin-a( - ) n-terminal microdomain peptide from human dihydroorotate dehydrogenase: structure and model membrane interactions labview programs for the analysis of epr data polarity profiles in oriented and dispersed phosphatidylcholine bilayers are different -an electron spin resonance study elimination of unwanted echoes and reduction of dead time in three-pulse electron spin-echo spectroscopy key: cord- - isw wl authors: balgoma, david; gil-de-gómez, luis; montero, olimpio title: lipidomics issues on human positive ssrna virus infection: an update date: - - journal: metabolites doi: . /metabo sha: doc_id: cord_uid: isw wl the pathogenic mechanisms underlying the biology and biochemistry of viral infections are known to depend on the lipid metabolism of infected cells. from a lipidomics viewpoint, there are a variety of mechanisms involving virus infection that encompass virus entry, the disturbance of host cell lipid metabolism, and the role played by diverse lipids in regard to the infection effectiveness. all these aspects have currently been tackled separately as independent issues and focused on the function of proteins. here, we review the role of cholesterol and other lipids in ssrna+ infection. the ongoing covid- pandemic is developing (july ) worldwide with devastating global consequences, both for social organization and healthcare systems. covid- illness is brought about by infection with the severe acute respiratory syndrome coronavirus sars-cov- [ , ] , which is an enveloped positive single-stranded rna virus (ssrna+) [ ] . the most abundant studies related to human diseases induced by ssrna-positive viruses refer to picornaviridae, coronaviridae, and flaviviridae [ ] . this impact in a short time span has brought the biology and biochemistry of viral infection mechanisms to reach momentum. the infection mechanisms have been described for diverse unrelated viral families [ ] , with the majority of them being dna viruses. within picornaviridae, coronaviridae, and flaviviridae, rhino and poliovirus (picornaviridae), sars-cov, middle east respiratory syndrome coronavirus (mers-cov), hepatitis c virus (hcv), west nile virus (wnv) and dengue virus (denv) fall within the viruses whose life cycle biology is better known. nonetheless, knowledge regarding virus entry mechanisms and other related features of the virus life cycle has been gained from the research on the influenza virus from the orthomysoviridae family and the human immunodeficiency virus from the retroviridae family. consequently, these and other unrelated viruses will be also considered in this review from the point of view of the different aspects that affect the lipidomics of the viral infection. all ssrna+ viruses initially infect mammal cells through the interaction of virus proteins with any given host cell protein. further fusion of the virus and host cell membranes is required for the viral genetic material to get into the cell. once inside the cell, the genomic and subgenomic viral rnas are translated into the virus proteins; these then lead the virus replication, which is a process that involves modulation of the host cell lipid metabolism [ , , ] . consequently, along with other features, current lipid studies about the aforementioned virus infection focus their research on membrane fusion and modulation of the lipid metabolism of the host cell. these two processes are considered separated disciplines of the infection. the fight against the virus infection encompasses primarily the inhibition of the binding of the viral spike protein to the host cell's receptor protein. consequently, most of the current research focuses on the role played by viral proteins but the lipid environment, where the proteins carry out their function and regulation, is considered secondarily [ ] . nevertheless, improving the knowledge on how the lipids are involved in the mechanisms of infection may provide clues to develop treatments and better counteract the virus-induced pathology [ ] . to fill this gap, here, we review the main aspects regarding the lipidome regulation of the viral infection mechanism by ssrna+ viruses. the initial step in virus infection is the binding of any viral structural glycoprotein to a receptor of the host cell. the spike protein accounts for such function in coronaviruses (covs) and other enveloped viruses. after the virus is attached to the host cell protein, the process of membrane fusion starts to get the viral genome into the host cell. this process implies viral envelope and host cell membrane fusion, for which an energetically cost-effective barrier must be overcome. for example, in coronavirus, membrane fusion is driven by the fusion peptide (class i), which is localized within the spike protein (s protein) and becomes active after cleavage of the s protein at specific sites by host proteases or ph-dependent mechanisms [ , , ] . a different mechanism of attachment and endocytosis drives the virus entry in the case of hcv. this mechanism is more complex than that of coronaviruses and involves interaction of the virus envelope e and e proteins (class ii fusion loop) with several host cell proteins [ ] [ ] [ ] . however, a membrane fusion-driven pore is also required in hcv to deliver the viral genetic material into the host cell cytoplasm. two main mechanisms of membrane fusion have been described: viral endocytosis by host cell membrane (endocytic pathway), and both viral and host cell plasma membrane fusion (non-endocytic pathway). after docking of the virus to the attachment factor or the receptor on the host cell surface, the virus may internalize its genomic material or the entire particle [ ] [ ] [ ] . the non-endocytic pathway encompasses the direct delivery of the genetic material through a pore formed in the cell membrane by the induction of viral proteins at neutral ph. this pathway is typical of non-enveloped viruses. the endocytic pathway is more complex and harnesses the host cell endocytosis machinery for the virus internalization. three main ways have been described in the endocytic pathway, namely: the clathrin-mediated endocytosis (cme), the caveolae-mediated endocytosis (cavme), and the macropycnocytosis. the best-known endocytic mechanism is the clathrin-mediated endocytosis. the cme is used by small to intermediate-sized viruses. this mechanism uses vesicles coated by the protein clathrin, which forms a polyhedral lattice that surrounds the cell membrane-derived vesicle where the virus is internalized into the cell cytoplasm through the early endosomes. clathrin coating is coordinated by the adaptor protein (ap- ) and other adaptors; it is less commonly ap-independent. the protein dynamin is involved in regulating the clathrin-coated vesicle (ccv) formation as well as its scission from the membrane. some viruses proceed to membrane fusion at this stage for releasing their genome into the cytoplasm. the early endosomes have a ph of about . to . ; therefore, it is considered that membrane fusion is not strictly ph-dependent. other viruses need a lower ph for the membrane fusion to be effective; thus, it is considered ph-dependent. a further step leading to endosome maturation to become late endosomes with a ph of about has to proceed before the membrane fusion takes place and the genetic material is delivered to the cytoplasm. sequential acidification of the virus proteins from the early to late endosomes has also been suggested through the self-organized endosomal network. maturation of the early endosomes to late endosomes and trafficking between them is controled by the rab proteins, which are members of the ras superfamily of small g proteins. subsets of rab proteins differ between the early and late endosomes, and the rab subset change is accompanied for by formation of the phosphoinositide pi( , )p from the precursor pi( )p. regarding lipid composition, early endosome membrane lipids are primarily composed of unsaturated and short alkyl chains, whereas long and saturated alkyl chains, such as in gangliosides, are predominant in the membrane lipids of late endosmes. membrane fusion in some viruses requires a further step in which late endosomes are fused with lysosomes, this step giving rise to the late endosome/lysosome pathway. cholesterol depletion driven by its synthesis inhibition or extracting agents as methyl-β-cyclodextran (mβcd) is used to assess whether the virus entry takes place through the caveolae/raft endocytosis. this pathway in less known and encompasses the formation of initial endocytic vesicles enriched in cholesterol from lipid-rafts, with complex signaling routes that involve the activity of tyrosine kinases and phosphatases. thereafter, the cargo is transported to the endoplasmic reticulum (er) through early and late endosomes. most of the viruses using this endocytic pathway have different gangliosides as receptors, mainly gm , which has a high concentration in caveolae. polyomavirus, which are non-enveloped dna viruses that replicate in the nucleus, use preferently this endocytic pathway, but picornaviruses and the coronavirus hcov- e have also been reported to internalize through the caveolae-mediated endocytosis [ ] . macropinocytosis is a phagocytic-like mechanism of virus entry that is currently utilized by the cell to internalized fluids; it is dependent on actin and implies the actin cytoskeleton rearrangement to enable internalization of the virus particle [ ] . macropinocytic vacuoles (macropinosomes) are formed after membrane ruffles fold to reach at its end the membrane again, and the vacuole is closed through self-membrane fusion. these vacuoles containing the viral particle may traffick afterwards through the early and late endosome network. macropinocytosis is common to large-sized viruses. however, recent work [ ] has shown that ebola virus (ebov) may use a macropinocytosis-like process to entry the host cell in a clathrin, caveolae, and dynamin-independent manner, but dependent of actin and a lipid raft. conversely, this virus may use as well an endocytic pathway that is dependent on clathrin, caveolae, and dynamin. which endocytic route is used by this virus depends on the host cell type. description of the current methodologies used to study the entry route by viruses can be found in reference [ ] . some viruses may use different entry mechanisms, this feature being likely dependent upon the membrane lipid composition of the host cell they infect as well as the particular cell surface factor attachment used. cme is the entry route currently used by hcv, hiv- , ebov, rotaviruses, and some coronaviruses, even though other routes can also be used as for ebov (see above). a reaction between clathrin and actin seems to be necessary for the effective entry of these viruses. regulation by microtubules of the cme has been reported for flaviviruses. denv, wnv, and semliki forest virus (sfv, alphavirus family, togaviridae) have been found to depend on early endosomes (rab protein marker) for entry but not late endosomes (rab protein marker), which means that they do not have strict low ph requirements or depend on different acidification mechanisms for membrane fusion. conversely, influenza avian virus (iav) needs both early and late endosomes to entry, thus reflecting low ph dependence for membrane fusion. marburg virus (marv) may use for internalization a cme through the endo/lysosomal pathway. coronaviruses differ in their internalization mechanism among strains. thus, while hcov- e is known to use the cav-me route, sars-covs use an endocytic pathway that is clathrin-and caveolae-independent but receptor and ph-sensitive, with lipid rafts playing an essential role [ ] . this endocytic mechanism implies internalization of the receptor protein angiotensin-convering enzyme (ace ) along with the spike protein into the early endosomes, but the receptor is afterward recycled to the membrane via lysosomes. nonetheless, previous studies showed that sars-cov could enter through a ph-independent direct membrane fusion as it could infect cells that do not express ace , such as enterocytes and hepatocytes [ ] . recent research on the virus sars-cov- points to ph-independent direct cell and viral membrane fusion, which is a process that is driven by the subunit s of the spike protein after cleavage by the cellular serine protease tmprss [ ] . on the contrary, the infectious bronchitis virus (ibv), a gamma-coronavirus, was reported to use the cme pathway to entry, with vesicle scission being mediated by gtpase dynamin , and a dependence on low ph and lipid raft localization of the receptor. tracking of the virus trip inside the cell was followed by using diverse inhibitors, cholesterol sequestering agents, and virus particles labeled with fluorescent markers. membrane fusion takes place at the late endosome/lysosome step of the endocytic pathway, with deep rearrangement of the host cell cytoskeleton being induced by the endosomal viral cargo [ ] . accordingly, viruses may sequester on their own profit the diverse endocytic pathways that are currently used by the host cell, but variability of the proteins and even the general mechanisms may also exist as a consequence of virus specifity. membrane fusion has been described to proceed through the catalytic action of three different types of fusion peptides or fusion loops of class i, ii, or iii. these proteins afford the free energy necessary to overcome through conformational changes the kinetic barrier due to repulsive hydration strength. most of the knowledge on the viral and host membrane fusion has been gained from the influenza virus and its type i fusion peptide hemagglutinin. a detailed description of the three fusion peptide-guided mechanisms involved in membrane fusion has been previously reviewed in [ ] [ ] [ ] . bringing the viral and the host membranes closer enough (c.a. Å) for inducing the membrane fusion is a process that entails membrane curvature and changes in the lipid bilayer phase. they are driven by the insertion of a hydrophobic region of the fusion peptide, which requires dehydration of the inter-membrane space. nonetheless, from experiments with no-protein fusogens, such as polyethilen glycol, it seems that membrane curvature stabilization is not a key player in membrane pore opening. the calculated displacement of lipids in the outer leaflet of the host membrane accounts for no more than % of the membrane area (about Å ), which does not represent a substantial energetic demand [ ] . this energetic burden has been demonstrated to be afforded by the cooperation of three fusion peptides in influenza virus membrane fusion [ ] , whereas two adjacent trimers of the fusion protein are required in west nile virus [ ] . this result points to the fact that the viral membrane curvature may not actually impose a constraint for proceeding to the hemifusion step and the formation of a steep curvature stalk, where the outer leafleats are merged. by the mesurement of electron density profiles through x-ray reflectivity in stalks formed from bilayers in a lamellar state with different lipid compositions, aeffner et al. [ ] determined that the inter-bilayer separation should attain . ± . Å in order to facilitate dehydration and promote stalk formation. these authors also found that increasing the relative proportion of nonbilayer-forming, cone-shaped lipids, such as glycerophosphoethanolamine or cholesterol, favored the stalk formation by reducing the hydration energy barrier and, possibly, by contributing with their intrinsic negative curvature. as well, the energy required for dehydration was, in this study, found to decrease with the length of the acyl chains of the glycerophospholipids. however, the hemifusion stalk stage was not detected by gui et al. [ ] using fluorescence and electron microscopy. the results of this study show that such a stage might be an unstable intermediate that is quickly resolved toward the postfusion stage. contrarily, localized point-like contacts were abundantly visualized in this study, where the dimples formed in the target membrane, about nm wide, were drawn toward the virus surface. they were able to detect up to well-resolved four types of virus-target membrane contacts at ph . and . using liposomes of dioleylglycerophosphocholine, dopc, with % cholesterol. at the lowest ph, a tight contact of the two membranes through an extended length of about nm (catalogued by the authors as type iii) was the predominant interaction, whose abundance was increased by about -fold in cholesterol-containing liposomes in comparison to only dopc liposomes. using synthetic peptides that resemble the fusion peptide hemagglutinin and electron spin resonance (esr), ge and freed [ ] found that the most relevant effect of the synthetic fusion peptides was the induction of highly ordered membrane domains, which came motivated by virtue of electrostatic interactions between the peptide and negatively charged phospholipid headgroups. a similar effect was reported for two putative fusion peptides enclosed in the spike glycoprotein of sars-cov- . it was found in this study that the inner water content in the lipid bilayer was dropped by the insertion of the fusion peptide as a consequence of increased lipid packing, but only in membranes containing negatively charged lipids, whereas the water content was only slightly altered in zwitterionic dipalmitoylglycerophosphocholine (dppc) liposomes [ ] . additionally, the fusion peptides created opposing curvature stresses in the highly bended membranes containing nonbilayer-forming phospholipids. however, previous studies had pointed out that interaction with the lipid headgroups is not an essential factor in reaching the membrane hemifusion state [ , ] . in sars-cov, the possibility of existing two fusion peptides that act in coordination has been suggested [ ] ; one of the peptides would promote the dehydration process, while the other one would act in modifying/disturbing the lipid organization within the target membrane [ , , ] . hence, the catalytic role of the fusion peptide(s) is likely to tackle three properties of the target membrane in the virus entry machinery: (i) dehydration of the intermembrane space for the fusing membranes coming into the required proximity, (ii) to promote negative curvature to form the hemifusion stalk, and (iii) to alter the lipid packing density, which will be generated in the highly curved local dimples of the stalk [ , ] . the effectiveness of these three processes is likely to depend upon the membrane lipid composition. further research is devoted to this issue, and new clues are expected to come from electron and fluorescence microscopy [ ] . since the dominant phospholipid in the outer leaflet of most membranes is the bilayer-forming, positive charged diacylglycerophosphosphocholine (pc), the idea was raised that the viral docking to the receptor on the target cell and, consequently, the membrane fusion were likely to take place at specific microdomains with particular lipid composition, the so-called lipid rafts [ , [ ] [ ] [ ] [ ] [ ] . a special characteristic of the lipid rafts is the high content of cholesterol [ ] [ ] [ ] . even though a high content of sphingolipids and gangliosides is also a defining characteristic of lipid rafts (figure ), direct in vivo visualization still remains unresolved [ ] . metabolites , , x for peer review of altered in zwitterionic dipalmitoylglycerophosphocholine (dppc) liposomes [ ] . additionally, the fusion peptides created opposing curvature stresses in the highly bended membranes containing nonbilayer-forming phospholipids. however, previous studies had pointed out that interaction with the lipid headgroups is not an essential factor in reaching the membrane hemifusion state [ , ] . in sars-cov, the possibility of existing two fusion peptides that act in coordination has been suggested [ ] ; one of the peptides would promote the dehydration process, while the other one would act in modifying/disturbing the lipid organization within the target membrane [ , , ] . hence, the catalytic role of the fusion peptide(s) is likely to tackle three properties of the target membrane in the virus entry machinery: (i) dehydration of the intermembrane space for the fusing membranes coming into the required proximity, (ii) to promote negative curvature to form the hemifusion stalk, and (iii) to alter the lipid packing density, which will be generated in the highly curved local dimples of the stalk [ , ] . the effectiveness of these three processes is likely to depend upon the membrane lipid composition. further research is devoted to this issue, and new clues are expected to come from electron and fluorescence microscopy [ ] . since the dominant phospholipid in the outer leaflet of most membranes is the bilayer-forming, positive charged diacylglycerophosphosphocholine (pc), the idea was raised that the viral docking to the receptor on the target cell and, consequently, the membrane fusion were likely to take place at specific microdomains with particular lipid composition, the so-called lipid rafts [ , [ ] [ ] [ ] [ ] [ ] . a special characteristic of the lipid rafts is the high content of cholesterol [ ] [ ] [ ] . even though a high content of sphingolipids and gangliosides is also a defining characteristic of lipid rafts (figure ), direct in vivo visualization still remains unresolved [ ] . an unexplored possibility is that rafts do not have a permanent localized existence, but they arise under the induction of certain proteins such as the hydrophobic insert of the viral fusion peptide or the fusion loop. this fact might be also responsible for bringing negatively charged lipids from the inner leaflet of the bilayer to its outer leaflet by flip-flop mechanisms. this hypothesis would explain the promotion of virus entry by the interaction of the fusion peptide with the negatively charged phospholipid headgroups [ , ] as well as the kinetics of the membrane fusion [ ] . a number of studies have shown that the hemifusion step and pore widening are sped up after increasing the relative concentration of cholesterol in the bilayer composition, whereas either the depletion of cholesterol in the cell culture medium or the inhibition of cholesterol synthesis by an unexplored possibility is that rafts do not have a permanent localized existence, but they arise under the induction of certain proteins such as the hydrophobic insert of the viral fusion peptide or the fusion loop. this fact might be also responsible for bringing negatively charged lipids from the inner leaflet of the bilayer to its outer leaflet by flip-flop mechanisms. this hypothesis would explain the promotion of virus entry by the interaction of the fusion peptide with the negatively charged phospholipid headgroups [ , ] as well as the kinetics of the membrane fusion [ ] . a number of studies have shown that the hemifusion step and pore widening are sped up after increasing the relative concentration of cholesterol in the bilayer composition, whereas either the depletion of cholesterol in the cell culture medium or the inhibition of cholesterol synthesis by statins was able to halt the viral infection at the virus entry step [ ] [ ] [ ] , ] . the effect of cholesterol on promoting membrane merging has also been observed for bis-(monoacylglycero)-phosphate (bmp) [ ] . this particular phospholipid was shown to be strictly necessary for dengue virus (denv) entry even at low endosomal ph [ ] . as pointed out above, the exact role played by cholesterol is not known in detail, but its intrinsic negative curvature seems to be an essential characteristic in promoting the stalk formation during virus entry. however, a recent study shows that the cholesterol action is likely to involve a direct influence on the oligomeric state of the fusion peptide after insertion into the host cell membrane, as well as on the effects of the fusion peptide on the membrane reorganization and dynamics [ ] . in another recent study, a new lipid-label-free methodology was used to measure the kinetics of influenza virus infection [ ] . according to the results of this study, cholesterol is able to augment the efficiency of membrane fusion in a receptor binding-independent manner. nevertheless, the rate of membrane fusion was not altered. these results led the authors to conclude that the positive effect of cholesterol in membrane lipid mixing is related to its capability to induce negative curvature. since membrane mixing was achieved in this latter study without binding of the spike protein of the influenza virus to the host cell receptor, the catalytic effect of the fusion peptide might proceed in an independent way in this virus. cleavage of the spike protein in sars-cov- does not seem to be also necessary for the fusion peptide to become fusogenic, but rearrangement of disulfide bridges in the s peptide after receptor binding are likely involved in the conformational changes driving the fusion mechanism [ , ] . contrary to these latter results, which point to the fact that membrane fusion is independent of viral protein attachment to its receptor, guo et al. reported lipid raft-dependent viral protein binding with the suppression of viral infection if the lipid rafts were disrupted with cholesterol drug-induced depletion; lipid rafts, as recognized by the caveolin- marker, were the membrane domain where structural proteins of the infectious bronchitis virus (ibv) co-localized but the nonstructural proteins did not [ ] . the question regarding whether the lipid-raft domains may serve as platforms to concentrate the proteins required for viral entry and, even though some evidence exists, to activate signaling pathways inside the host cell still remains unsolved. sphingomyelins (sms) are also common lipids found in lipid rafts, which contribute to make these membrane microdomains detergent-resistant [ ] . the structure of a representative of this lipid class is illustrated in figure . the ganglioside gm , a sphingolipid, is used as a marker of lipid rafts [ ] . sphingolipids (sls) promote to an extent higher than chol the liquid-ordered phase in the outer leaflet of the membrane bilayer because of the long saturated acyl chains they currently contain (the r group in figure may extend to a length of up to c), in addition to their capability to form intermolecular hydrogen bonds [ ] . a relevant function of the lipid rafts has been suggested to be the connection between the events outside the cell with the pathways inside the cell, thus acting as 'signaling platforms'. with the aim of this function to be properly accomplished, the lipid rafts would act as concentrators of specific transmembrane proteins, mainly receptors, whose compatibility with the membrane phase would determine their selectivity. thus, sls would account for a role in connecting the outer leaflet with the inner leaflet through their long saturated acyl chains. regarding virus entry, research has been primarily focused toward the role played by cholesterol, but a number of studies have also enlightened the sm influence on this early step of viral infection. the displacement of cholesterol by sms and the other way round has been demonstrated, with the bilayer liquid-ordered phase being preferentially determined by the interaction between sm and cholesterol. this interaction would be controlled to a certain extent by the intracellular actin meshwork, which would also be responsible for the compartmentalization of the membrane into lipid-specific domains [ ] . furthermore, the actin role is possibly extended to the routing of the viral genomic material toward the replication place inside the host cell. the hydrolysis of sm by sphingomyelinases to render the corresponding ceramide in specific membrane domains is proposed to regulate the dynamics of cholesterol in the cell membrane, the effect of such regulation being the progressive disassembly of cholesterol from the liquid-ordered phase and its displacement. since the interaction of ceramides with cholesterol has been suggested to be an apoptotic regulator, it can be expected that viral proteins would act in recruiting cholesterol to displace the ceramide and to avoid the programmed cell death. this fact is added to the other characteristics conferred by cholesterol to the membrane mechanical properties discussed above. to study the influence of ceramide on membrane fusion during semliki forest virus (sfv, alphavirus family, togaviridae) infection, ceramide analogs have been used [ ] . according to this experiment, in which cholesterol-containing pc plus pe liposomes were used, the roles played by the -hydroxyl group and the , -trans carbon-carbon double bond of the sphingosine backbone ( figure ) were found to be essential in the fusion process. in additon, ceramide was the simplest sl to accomplish this significant contribution in mediating the fusion, independently of the length of the acyl chain. more recently, a ca + -dependent pathway of infection by the rubella virus (ruv, rubivirus family, togaviridae) was demonstrated to proceed through direct binding of the fusion loop in the viral e protein to sm/cholesterol-enriched membranes [ ] . however, the treatment of host cells with sphingomyelinase proved that sm is exclusively required for viral entry but is not required for the further steps of viral replication. sm in the host cell membrane and acid sphingomyelinase (asmase) activity have also been shown to be required by the ebola virus (ebov), a negative single-stranded rna virus belonging to the filoviridae family, to get into the host cell. the asmase activity renders ceramide that provoques raft enlargement and membrane invagination [ ] . this study also showed that the virus was able to recruit both sm and asmase to the raft where the viral attachment was happening. conversely, bovine herpesvirus (bohv- , herpesviridae family) seems to require sm in the virus envelope but does not in the host cell [ ] . the role played by ceramides is contradictory as they may enhance or inhibit virus replication, but this sl action seems to be related to the viral replication phase rather than to the internalization phase [ ] [ ] [ ] . in virus using the endocytic pathway, similar to the influenza virus or the ebola virus, it has been shown that activity of glucosylceramidase (gba) is required for viral entry and membrane fusion through the regulation of endocytosis, but in a virus-dependent manner. it was also shown that trafficking of the epidermal growth factor (egf) to late endosomes was impaired in gba-knockout cells, which negatively affects the virus entry through spoiling the endocytic pathway [ ] . indeed, co-clustering of the ha attachment factor and egf in submicrometer domains that overlap partially has been reported recently [ ] . accordingly, there is evidence that sls have a function in enveloped ssrna viruses at the early stage of infection that accounts for the viral entry modulation, but further research is still necessary to unveil the exact mechanisms of sl reactions. metabolites , , x for peer review of displace the ceramide and to avoid the programmed cell death. this fact is added to the other characteristics conferred by cholesterol to the membrane mechanical properties discussed above. to study the influence of ceramide on membrane fusion during semliki forest virus (sfv, alphavirus family, togaviridae) infection, ceramide analogs have been used [ ] . according to this experiment, in which cholesterol-containing pc plus pe liposomes were used, the roles played by the -hydroxyl group and the , -trans carbon-carbon double bond of the sphingosine backbone ( figure ) were found to be essential in the fusion process. in additon, ceramide was the simplest sl to accomplish this significant contribution in mediating the fusion, independently of the length of the acyl chain. more recently, a ca + -dependent pathway of infection by the rubella virus (ruv, rubivirus family, togaviridae) was demonstrated to proceed through direct binding of the fusion loop in the viral e protein to sm/cholesterol-enriched membranes [ ] . however, the treatment of host cells with sphingomyelinase proved that sm is exclusively required for viral entry but is not required for the further steps of viral replication. sm in the host cell membrane and acid sphingomyelinase (asmase) activity have also been shown to be required by the ebola virus (ebov), a negative single-stranded rna virus belonging to the filoviridae family, to get into the host cell. the asmase activity renders ceramide that provoques raft enlargement and membrane invagination [ ] . this study also showed that the virus was able to recruit both sm and asmase to the raft where the viral attachment was happening. conversely, bovine herpesvirus (bohv- , herpesviridae family) seems to require sm in the virus envelope but does not in the host cell [ ] . the role played by ceramides is contradictory as they may enhance or inhibit virus replication, but this sl action seems to be related to the viral replication phase rather than to the internalization phase [ ] [ ] [ ] . in virus using the endocytic pathway, similar to the influenza virus or the ebola virus, it has been shown that activity of glucosylceramidase (gba) is required for viral entry and membrane fusion through the regulation of endocytosis, but in a virus-dependent manner. it was also shown that trafficking of the epidermal growth factor (egf) to late endosomes was impaired in gba-knockout cells, which negatively affects the virus entry through spoiling the endocytic pathway [ ] . indeed, co-clustering of the ha attachment factor and egf in submicrometer domains that overlap partially has been reported recently [ ] . accordingly, there is evidence that sls have a function in enveloped ssrna viruses at the early stage of infection that accounts for the viral entry modulation, but further research is still necessary to unveil the exact mechanisms of sl reactions. some covs (hcov-oc and hcovhku ), as well as influenza a virus (whose fusion loop is hemagglutinin, ha) and other non-related viruses (i.e., non-enveloped simian virus sv- , of polyomavirus family), use the sialoglycan moiety ( -o-acetyl-sialic acid) of gangliosides or glycoproteins located in membrane lipid rafts as receptors for the spike protein. the amino acid trp in the domain a of the hcov-oc s protein was shown to be essential for receptor binding. however, despite the fact that binding to -o-acetyl-sialic acid is required for membrane fusion, further interaction of the virus protein with other host membrane sialoglycans or proteins is also necessary to induce the conformational changes leading to membrane fusion [ , ] . conversely, formation of the complex sv protein with the host cell ganglioside gm was found to be enough to induce the membrane curvature and invaginations required for membrane fusion [ ] . as already discussed above, some studies have depicted the possibility that interaction of the fusion peptide or fusion loop with negatively charged phospholipids on the host membrane might be required for an efficient membrane fusion [ ] . in this regard, phosphatidylserine (ps) contained in the virus envelope has been demonstrated to serve after externalization as a virus co-receptor through the t cell immunoglobulin mucin domain (tim- ) receptor in ebov and other viruses, even in an indispensable fashion [ ] [ ] [ ] [ ] . in the study of nanbo et al. [ ] , flipping of ps from the inner leaflet to the outer leaflet of the cell membrane for virion adquisition and incorporation to its envelope is proposed as a previous step to tim binding. in herpes simplex virus (hsv), phospholipid scramblase- (plscr ), after activation by hsv exposure, flips both ps and akt to the outside of the membrane in a ca + -dependent mechanism. ps is restored to the inner leaflet to h after infection to avoid apoptotic triggering [ ] , suggesting a different role for ps in relation to the tim- ps receptor. however, the function of tim- as an essential receptor for hav has been disputed [ ] due to the finding that quasi-enveloped ha virions (ehav) were able to infect tim -knockout vero cells to a similar extent to naked hav. hence, the authors proposed tim to be an accessory attachment factor by binding ps on the hav envelope rather than an essential virus protein receptor. in spite of these contradictory data, ps seems to act in any way in virus attachment and entry in certain virus families, at least contributing to the process efficiency, but the exact role may depend on every virus or it may be complementary to other factors. a phospholipid currently associated to the inner leaflet of the lipid rafts is phosphatidylinositol (pi), which is a negatively charged phospholipid with important and versatil signaling functions ( figure ) [ , ] . abundant data suggest that a derivative of pi, the phosphatidylinositol , -biphosphate (pip ), accumulates preferently in liquid-disordered phases (l d ) [ ] , where the cholesterol content is presumed to be low, and interplays with ps, which is rather localized in liquid-ordered phases (l o ). pis play an essential role also in endosome maturation, which is a requisite for efficient virus infection of those using the endosomal pathway [ , ] . during hiv infection, pip has been proposed to coordinate the actin cytoskeleton changes required for efficient virus entry in cd + t cells [ ] ; after virus attachment to the host cell receptor, pip is recruited to the binding membrane microdomain, and in this way, pip controls the protein reactions, leading to actin polymerization. as well in hiv- , the requisite of pip accumulation for the virus gag protein to be properly anchored and stabilized in the inner leaflet of the cell plasma membrane has been pointed out [ , ] . two isoforms, α and γ, of the phosphatidylinositol- -phosphate -kinase family type (pip k ) have recently been shown to participate in gag stabilization by pip through targeting the gag precursor pr gag to the cell plasma membrane [ ] . as commented above, interaction with the headgroup of negatively charged phospholipids such as ps or pi may also contribute to the dehydration process in the formation of the hemifusion stalk, with this contribution happening by promotion of the inverted hexagonal phase in the lipid bilayer and binding of ca + [ ] . in in vitro experiments with cos- cells and multilamellar vesicles (mlvs), unspecific binding of the marburg virus (marv) mvp protein to pip, pip , and even pip species present in the mlvs, both in the presence or absence of ps, has been reported. in this study, it was also found that with increasing ps concentration, the association of mvp to mlvs rose up to a threshold. furthermore, the addition of sphingosine with the aim to reduce the negative charge load in the inner leaftet of the cos- cells led to a decrease in the binding level. these facts suggest that the electronic density, rather than the specific lipid species, is a determinant factor for binding [ ] . activation of the pi k pathway for signaling is one of the most relevant features taking place for both entry and budding during infection by a number of viruses [ , [ ] [ ] [ ] . pi k converts pip into phosphatidylinositol , , -triphosphate (pip ). in addition to stabilizing proteins or serving as a binding factor, pip has been shown to collaborate with akt through the signaling pathway pi k/akt on avoiding apoptotic events, and in this way, keeping the host cell metabolically active for virus replication and budding [ ] [ ] [ ] . all these results clearly bring evidence that the lipid environment surrounding proteins involved in virus infection has a relevant function in the virus entry mechanism. different lipids are essential for virus docking to the cell receptor either serving directly as (co)-receptors or providing the appropriate environment (lipid rafts) for the necessary reactions (e.g., membrane curvature). in addition, the virus, through specific protein conformational changes, takes advantage of several cell signaling pathways controlled by diverse membrane lipids. this process allows the virus to govern the cell metabolism following endocytosis of the viral genetic molecules. after the virus or its genome gets inside the infected cell, ssrna+ viruses and other enveloped ones that replicate in the cytoplasm manage the cell metabolism to develop the replication scaffold, this membrane structure bolstering the so-called 'virus factory' [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . there is consensus on that the functions of these structures are (i) to compartmentalize the diverse processes involved in viral genome replication, its envelopment, and structural protein assembly; (ii) to increase virion concentration during budding before infecting naïve cells; and (iii) to create a protected environment to escape the innate immune recognition of the viral components. virus replication imposes an extra-energetic expenditure to the cell metabolism. hence, cell central metabolism is orchestated by viral proteins to redirect toward the generation of enough energy and metabolites that are required for virus replication. in particular, building the scaffold demands a high rate of new lipid synthesis. therefore, the lipid metabolism is hijacked by the virus proteins for the de novo synthesis of fatty acids in order to generate the scaffold membranes, the replication complexes (rcs), as well as for energy production in the β-oxidation pathway in the mitochondria. concurrently, the cell metabolism needs to be kept above a threshold level to avoid exhaustion of the host cell. full understanding of the mechanisms and related factors involved in virus-host interaction is a requisite for developing efficient antiviral infection therapies. the scaffold structure raised for building the viral factory varies between different virus in their morphology and possibly lipid composition. flaviviruses develop a so-called 'membranous network' (mn) in a spherule/invagination type, while coronavirus does through a quarter-like type delimited by 'double membrane vesicles' (dmvs). nonetheless, hcv (flaviviridae) uses dmvs instead [ ] ; hence, this morphological separation may have exceptions or be somewhat diffuse. an extended review of the different virus family-related morphologies of the mns as well as diverse factors influencing their formation can be found in [ , , ] . it should be remarked that the exact lipid composition of the rcs' membranes is not known in detail yet, although there is evidence that their lipid profile differs from that of the organelles from which they are generated. the enrichment of typical lipids such as cholesterol, sphingomyelins, and glycosphingolipids in the lipid rafts seems to be a common feature of these mns. the rcs' membranes may be originated from the endoplasmic reticulum (er) in the perinuclear area, as for example in sars-cov and faviviridae [ , , ] , from the golgi, giving rise to cytopathic vesicles (cpvs) as in togaviridae and picornaviridae [ ] , from mitochondria (nodaviridae) [ ] , or from the cell plasma membrane (cpvs in alphaviruses) [ ] . however, vesicle trafficking between the er and the golgi organelles may contribute to an undefinition in this regard. mns, and in particular dmvs, are connected to the cytosol through a pore, which is believed to serve as the gate to the replication scaffold for the requiered metabolites, in particular nucleotides. this pore-mediated gate has not been detected up to date in sars-cov's dmvs, which raises the concern of how the required metabolites get inside the rcs. there is evidence from a number of studies that dmvs are the site of replication, but it has also been shown that dmvs can be developed irrespective of whether rna replication takes place by the sole action of the viral proteins, at least for hcv [ , ] . viral nonstructural proteins nsp , nsp , and nsp are involved in dmv development in sars-cov- in a time-dependent manner and correlating with rna replication. timecourse events have been shown to run with the initial formation of single membrane vesicles (smvs) during the first - h after cell infection. these futher evolve to dmvs h after infection, and they ultimately turn into multimembraneous vesicles (mmvs) close to the cis-golgi at the budding stage - h after infection, this latter transformation being coincident with the formation of vesicle packets [ , , , ] . in hcv, ns a seems to be enough for dmv formation, but the collaboration of ns - b is required for completing efficient dmvs, whereas ns b is likely responsible for inducing the formation of smvs [ , , ] . even though particular hints can be likely associated to every particular virus, there are common features shared by all ssrna+ viruses regarding rcs' structure and buildup. enveloped viruses such as ssrna+ viruses have a membrane lipid whose profile is different to that of the original organelle membrane when the envelope is created. since the viral membrane is known to be enriched in cholesterol, sphingolipids, and phospholipids with saturated acyl chains, the dmv is believed to be also primarily composed of such classes of lipids. an unusual sphingolipid, dehydrosphingomyelin, along with ps and plasmalogens of pe were reported in the hiv envelope [ ] . a role for sphingomyelin-to-ceramide conversion has been proposed in wnv budding, as its envelope was found to be highly enriched in sphingomyelin [ ] . more recently, using multi-color super-resolution microscopy and mass spectrometry analysis, a substantial increase in pip (from % to %) and pip (from . % to . %) was reported in the hiv membrane as compared with the plasma membrane of the host cell [ ] ; this fact is related to the recruitment of gag protein for efficient membrane fusion as aforementioned ( figure ) . however, the most striking and known lipid-related factor associated to the mns' development is the pi kiii signaling pathway. the pi pα isoform, which is mainly expressed in the er, has been shown to be a key factor for hcv replication, whereas the pi kiiiβ is found in the golgi and is required by picornaviruses and some hcv strains [ ] . this enzyme interacts with the viral protein ns a, and disrupting this interaction prevents virus replication. the product of the pi k enzyme is pip ; enrichment in this pi has been shown to act in different processes regarding virus replication: membrane curvature, directly or indirectly through recluting cholesterol [ ] , glycosphingolipid transport to the rcs by the action of the fapp protein [ ] , and protein concentration. however, conversely to these studies, it has been shown that currently used inhibitors of pi kiiiα, enviroxime and bf , actually exert their inhibition against pi k [ ] . thus, this result points out a genomic dependence on the pi kinases in hcv; otherwise, the action on pi k is required only at the entry stage (see above). enviroxime-like inhibitors have been shown to halt enterovirus replication through the action against pi kβ [ ] . the de novo lipid synthesis has also been evidenced for wnv, from the flaviviridae family as hcv, to proceed in a pi p-independent fashion and, concurrently, it is not related to pi kiii signaling [ ] . there is no clear evidence on the fact that the pi k signaling pathway has a relevant function in mns' development. hence, while pi kiiiβ was shown to be important for sars-cov's dmv formation [ ] , another study did not find its metabolite, pi p, within the host factors involved in sars-cov replication, and the authors attibute to pi p a function rather in virus entry. however, the authors of this latter study acknowledge that sirna methodology may provide false negatives [ , ] . since dmvs are not common in healthy cells but they can be observed during authophagy, it has been suggested that sars-cov and other coronaviruses use the autophagy pathway for development of the dmvs; indeed, it has been shown that nsp in mhv or the equivalent nsp - in arteriviruses, which hits the er, can activate such a pathway [ , ] . nonetheless, dmvs are smaller than autophagosomes, and hence, they might be rather endoplasmic reticulum derived vesicles (edesomes) enriched in pi p and not follow exactly the same synthetic route [ ] . further work on coronaviruses and autopahgy found that only the lc -i protein, the microtubule-associated proteins a/ b light chain b, is localized on the replication membranes, but the active protein lipidated with phosphatidylethanolamine lc -ii inserted into the autophagosome membrane is absent. accordingly, present knowledge on coronaviruses in regard to autophagy suggests that they take benefit of the autophagocytic components but do not develop autophagosomes per se [ ] . the autophatocytic pathway has also been associated to the start of hcv infection, but it seems not to be necessary for the infection to go on [ ] . later on, it was shown that autophagy was key in rna replication at the onset of hcv infection [ ] , but the virus life cycle can go ahead afterward without the autophagy system intervention. further work has shown that hcv, and possibly denv, uses the autophagy system to evade the innate immune system [ ] . using immortalized human hepatocytes defective of the autophagy-related proteins either beclin (bcn ) or atg , it was shown in the latter study that disruption of the autophagy machinery elicites activation of the interferon signaling pathway and leads to apoptosis of the infected cells. triggering of the autophagy pathways takes place after binding of the virus to the cell surface via the downregulation of mtor and inactivation of akt signaling [ ] . conflicting results have been reported for the induction of autophagy by hcv in regard to the unfolded protein response (upr) [ ] . recent work [ ] has bound the induction of autophagy by hcv to golgi membrane fragmentation to render vesicles that colocalize with the hcv replicons. the immunity-related gtpase m protein (irgm) mediates the phosphorylation of the early autophagy initiator ulk as well as the golgi membrane fragmentation in response to hcv infection. the protein lc has also been detected in the replication membranes of the hiv- , and the association of lc -ii with gag-derived proteins seems to be a requisite for the efficient maturation of the gag subunit p [ , ] . members of the picornaviridae family, non-enveloped viruses, have been reported to subvert the autophagosome pathway as a means to exit the infected cell without membrane lysis; support for this spreading mechanism comes from the finding of numerous extracelular vesicles that are enriched in phosphatidylserine phospholipids [ ] . the best studied virus regarding autophagy is the dengue virus (denv). even though it was initially suggested that the denv replication complexes are developed from autophagosomes, further work pointed out that the replication of denv took place on invaginations arising from the endoplasmic reticulum (er), while autophagy was rather used by denv to modify the lipid metabolism in a way that is known as lipophagy [ , ] . lipophagy was first shown to be an active way to get energy under starvation [ ] through the association of autophagic components with lipid droplets (lds). recently, lipophagy has been demonstrated to regulate the fatty acid availability for the β-oxidation through contact sites between the mitochondria and the er [ ] . regarding virus-associated hijacking of the cell lipid metabolism, heaton and randall [ ] early showed that increased β-oxidation and the depletion of triglycerides was concurrent with and necessary for denv replication. then, these features were linked to the action of autophagy through the association with lipid droplets. a recent study by zhang et al. [ ] has found that aup , a type iii protein with signals for lds and er, plays a relevant role in lipophagy induced by denv and other flaviviruses such as wnv. unmodified aup is required for lipophagy triggering. a -fold increase in the content of diacylglycerophosphocholines (pcs) was measured in this study in infected cells containing unmodified aup , this increase being concomitant with a depletion of triacylglycerols and cholesterol esters, whereas the contents of free fatty acids and unesterified cholesterol rose. conversely, smaller lds, but not a reduction of their abundance, were observed in aup -knocked-out cells. thus, these data point to an augmented consumption of lds in the infected cells. this study unveils the mechanism that leads to the commented results; after the denv protein ns a associates with aup , the complex is relocalized from lds to autophagosomes, where the acyltransferase domain of aup is activated for the generation of phospholipids. this process was found to be dependent on the aup ubiquitylation status, with ns a inhibiting the ubiquitylation of aup . similar to viral entry, cholesterol has been found to be also relevant in the rcs' membranes [ , ] . up to a c.a. -fold enrichment of cholesterol was found in hcv-developed dmvs as compared to its content in the er membranes from which dmvs were originated [ ] . a key protein in cholesterol metabolism associated to non-vesicular transport is the oxysterol-binding protein (osbp). this protein has been described to transport cholesterol to pi p-enriched membranes, which would agree with its collaboration in delivering cholesterol to dmvs with an abundant content of this pi [ ] . the ceramide transfer protein (cert) and the four-phosphate adaptor protein (fapp ) are known to undergo a similar fate in hcv infection [ ] . an important protein involved in cellular lipid homeostasis is the sterol regulatory element binding protein (srebp), a bhlh-zip transcription factor with three isoforms; srebp c regulates the expression of fatty acid (fa) biosynthesis genes [ , ] , whereas srebp transactivates genes implied in cholesterol biosynthesis, intracellular lipid transport, and lipoprotein import [ ] . a recent study shows that the inhibition of srebp with the retinoid derivative and rar-α agonist am prevents mers-cov infection by avoiding the formation of functional dmvs [ ] . in this study, the lipid metabolism was the most affected pathway, with sterol biosynthesis being strengthened at expense of the glycerophospholipid metabolic pathways. fast activation of the lipid biosynthesis enzymes acetyl-coa carboxylase (acc), fatty acid synthase (fas), and hmg-coa synthase (hmgcs) was observed in such study, whose activity was partially blocked by am inhibition of srebp enzymes. promotion of lipid biosyntheis after infection had already been pointed out for hcv in an elegant proteomics and lipidomics study [ ] . hcv infection elicites changes in the proteome of host cells that resembled the warburg effect described in cancer cells toward lactate production and the support of continuous glycolysis; concurrently, the up-regulation of citrate synthase (cs) and other lipogenic enzymes h after infection was interpreted by the authors of the latter study as indicative of re-routing of the tricarboxylic acid (tca) cycle for cytosolic accumulation of citrate, which would be used in fa synthesis. the up-regulation of peroxisomal and mitochondrial fa oxidation pathways is concurrent with the other metabolic changes. an increase in pro-apoptotic ceramides was observed in the latter study as well; two possible interpretations were attributed to this finding, either a cytopathic effect after cell cycle arrest over time enough to complete virus offspring or a defense response of the host cell to avoid infection spread. blocking cholesterol suitability for the membraneous network or endosomes used for the virus replication and internalization has been demonstrated to inhibit the virus life cycle in a number of unrelated viruses. disruption of the srebp pathway restrains the andes virus (andv), an ssrnavirus, internalization, although it does not bind to the cell surface receptor [ ] . in addition to srebp , other components of this pathway were found to be necessary. the dependence of viral entry on the sterol regulatory element binding protein cleavage activating protein (scap) and the site protease (s p) was evidenced in cells null for these proteins. thus, in the study of petersen et al. [ ] , the virus was not internalized in cells lacking s p, this result pointing out that a complete cholesterol biosynthesis pathway is required. infectivity was also reduced -fold when the cells were treated with methyl-β-cyclodextrin (mβcd), a cholesterol sequestering agent, and comparable results were obtained after cell treatment with mevastatin or the s p inhibitor pf- . however, the s p dependence of virus infectivity does not seem to affect other viruses, thus this route being likely selective for hantaviruses [ ] . in this study, the genetic or pharmacological disruption of the srebp pathway at the site of the regulatory element membrane-bound transcription factor peptidase/site protesase (mbtps /s p) dramatically reduced viral infection, which is a feature that confirms the essential dependence of hantavirus on the high membrane cholesterol content for membrane fusion and effective infection. the down-regulation of sterol synthesis at the gene level after infection was found to be controlled by an interferon regulatory loop, in which a type i interferon-dependent mechanism down-regulates the expression of srebp [ ] , this result showing a link between the innate immune response and cholesterol biosynthesis after viral infection. this type i interferon response toward cholesterol synthesis down-regulation was dependent on the mevalonate-isoprenoid branch as a supply of mevalonate completely blocked the cholesterol synthesis, whereas a supply of cholesterol did not. additionally, in the presence of geranylgeraniol, the type i interferon inhibition of sterol biosynthesis was severely diminished. further research has shown that interferon may regulate the sterol synthesis pathway in multiple forms through micrornas [ ] . in particular, mir- - p was found to hit multiple srebp-independent targets of the mevalonate-sterol synthesis pathway after viral infection. the type i interferon response was also observed in regard to the impairment of the formation of double membrane structures induced by arteriviruses as replication sites [ ] . host cell fight against viral infection by a reduction of cholesterol availability has been also pointed out to come from the antiviral effector protein interferon-inducible transmembrane protein (ifitm ). this protein interacts with vesicle-membrane-protein-associated protein a (vapa), impeding its association with the oxysterol binding protein (osbp), and consequently, altering the normal function of osbp. as a result of the ifitm action, virus release into the cytosol is blocked by the accumulation of cholesterol in multivesicular bodies and endosomes. this effect restrains the membrane fusion of the intraluminal vesicles and that of the multivesicular bodies, which is a requisite for virus budding and release to the cytosol [ ] . the viral accesory protein of hiv nef competes with the cholesterol transporter abca to prime the transport of cholesterol to lipid rafts as a viral strategy to raise the replication membranes, thus overcoming the antiviral properties of abca [ ] . the replication of rabies virus (rabv), an ssrna virus, is halted by the action of viperin (virus inhibitory protein, endoplasmic reticulum-associated, ifn-inducible) in raw . cells. this protein is induced by the rabv, ifv, hiv, or hcv infection through promotion of the innate immune response bound to the tlr signaling pathway. the inhibitory activity of viperin on virus budding is related to its capability to substantially drop the contents of cholesterol and sphingomyelin in the replication membranes [ ] , thus pointing out the relevance of the membrane lipid composition for efficient virus replication. the induction of viperin has also been proven for hcv and ifav [ ] . however, viperin does not intervene in the inhibition of arterivirus-induced double membrane formation [ ] . remodeling of the lipid metabolism by virus infection may leave signals at the organism level even some years after healing. the metabolome profile of patients undergoing sars-cov- infection during the outbreak of - was assessed years after overcoming the pathology [ ] . an outstanding result of this study regarding disturbed lipid metabolism was the elevation of phosphatidylinositol (pi) and lysophosphatidylinositol (lpi) species concentrations in serum, which in turn correlated positively with the levels of very low-density lipoproteins (vldl); higher concentrations of products of the phospholipase a (pla ) such as lysophospholipids (lppls) and free arachidonic acid (aa) were also found in patients as compared to healthy voluntiers, with a correlation between the level of aa and the ratio of lpi( : ) to total : -pis being observed as well. these results show a potential high sensitivity of sars-cov patients to pla activity. in the general context, the metabolome of these patients pointed to hyperlipidemia, cardiovascular abnormalities, and glucose metabolism alteration as a delayed efffect of the viral infection. nonetheless, the authors acknowledge that some of the related metabolic disturbations are likely owed to the pharmacological treatment. high levels of pla group iid (pla g d) in lungs of middle-aged mice as compared to young mice had previously been associated to a fatal or worse outcome [ ] . the authors of this study conclude that the negative influence of this enzyme in sars-cov infection was to increase the concentration of anti-inflammatory lipid mediators, mainly protaglandin d (pgd ), which impaired the efficient function of the immune system [ ] . in the recent sars-cov- outbreak (covid- ), mortality has mostly affected aged people above years old, thus showing an age-related fatality as for sars-cov- and mers-cov [ ] . using a lipidomics approach, the effect of hcov- e and mers-cov infection on the host cell lipid profile was recently investigated in cell culture [ ] . the main conclusions of this study agree with the raised content of aa and lppls through plase activity, which indicates that the possible virus-induced activation of cpla favors virus replication as a factor required for dmvs' formation. in this study, linoleic acid (la) or aa supplementation to the culture cells suppressed replication, which is a result that may be interpreted as a demonstration of the perturbation of the la/aa axis of the lipid metabolism. in the covid- outbreak, it has been suggested that increasing the levels of vitamin d could help fighting against the sars-cov infection [ ] . this suggestion is based on the fact that -hydroxyvitamin d was found to protect huh cells against mers-cov [ ] . vitamin d is a lipid-related compound belonging to the group of fat-soluble secosteroids, with the most important form in humans being vitamin d (cholecalciferol) [ ] . in a recent study, high doses of vitamin d have shown protective effects against denv infection through regulation of the toll-like receptor expression as well as the modulation of pro-inflammatory cytokines release, which suggests that its action is focused toward the immune system modulation rather than to lipid metabolism [ ] . however, evidence on the beneficial effects of vitamin d uptake is still poor, and more studies are devoted to this issue. lipids, as components of membranes, are related to viroporins, which are specific viral proteins that are known to create ion channels for ion trafficking [ ] [ ] [ ] . the effect on cell metabolism of diverse viroporins differs among them, but there is evidence that they are closely related to viral pathogenity [ ] . viroporins may play a relevant role during virus infection, as they are involved in membrane permeability and calcium homeostasis. their participation in the development of vacuoles from the er during the dmvs' formation has been suggested, but data on this issue are still scarce. the regulation of ca + flux by viroporins might favor the membrane fusion through the interaction of this cation with the phospholipid headgroups and concurrently facilitate the required dehydratation reaction. viroporins are not required for virus replication with the exception of rotaviruses and picornaviruses; thus, whether this function is exerted through the ion channels or another property of viroporins remains an issue still unknown [ ] . the lipid composition of the membrane may influence the viroporin activity, leading to different versions of ion channels, which depends on the electric charge that the phospholipids confer to the membrane and curvature [ ] . a viroporin from rotavirus, nsp , was shown to co-localize with the autophagy marker protein lc in membranes accomodating virus replication; this viroporin is implicated in the sequestering of autophagy for the transport of proteins from the er to the replication sites [ ] . further research is necessary to understand the role played by viroporins in virus infection in order to consider them as potential therapeutic targets. remodeling of the virus-induced host cell lipid metabolism is a remarkable feature of the viral infection that affects viral entry, replication of the genomic material, and the releasing of progeny. a comperhensive view of the process is illustrated in figure . the main actors are well known to be cholesterol, sphingolipids, and pis, but other lipid species and their related pathways such as the la/aa axis are also relevant. how to target the lipid metabolism in a safe manner to avoid virus infection or reduce its pathogenity is a promising therapeutic tool, but it demands improving the knowledge on the actual pathways that are affected over the virus life cycle. the exact mechanism through which the enzyme inhibitors act on the key enzymes of the lipid metabolism is additionally required to develop more efficient and safe therapeutic drugs. since the lipid metabolism is essential for proper cell function, selective drugs targeting the virus or exclusively the infected cells have to be used to avoid 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autophagy in virus infection the autophagy machinery is required to initiate hepatitis c virus replication knockdown of autophagy enhances the innate immune response in hepatitis c virus-infected hepatocytes hepatitis c virus triggers golgi fragmentation and autophagy through the immunity-related gtpase m. proc. natl. acad. sci autophagy during viral infection-a double-edged sword dengue virus-induced autophagy regulates lipid metabolism dengue virus and autophagy autophagy regulates lipid metabolism autophagy regulates fatty acid availability for oxidative phosphorylation through mitochondria-endoplasmic reticulum contact sites flaviviruses exploit the lipid droplet protein aup to trigger lipophagy and drive virus production activators of the complete program of cholesterol and fatty acid synthesis in the liver common fatty markers in diseases with dysregulated lipogenesis coronavirus nsp proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate 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membrane channels: role and function in the virus life cycle autophagy hijacked through viroporin-activated calcium/calmodulin-dependent kinase kinase-signaling is required for rotavirus replication this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors thank m. s. crespo for helpful reading of the manuscript. the authors declare no conflict of interest. the authors thank m. s. crespo for helpful reading of the manuscript. the authors declare no conflict of interest. key: cord- -bcw f b authors: nan title: abstracts: th ebsa european biophysics congress, august rd– th , budapest, hungary date: - - journal: eur biophys j doi: . /s - - -z sha: doc_id: cord_uid: bcw f b nan o- structure determination of dynamic macromolecular complexes by single particle cryo-em holger stark max-planck-institute for biophysical chemistry, goettingen, germany macromolecular complexes are at the heart of central regulatory processes of the cell including translation, transcription, splicing, rna processing, silencing, cell cycle regulation and repair of genes. detailed understanding of such processes at a molecular level requires structural insights into large macromolecular assemblies consisting of many components such as proteins, rna and dna. single-particle electron cryomicroscopy is a powerful method for threedimensional structure determination of macromolecular assemblies involved in these essential cellular processes. it is very often the only available technique to determine the d structure because of the challenges in purification of complexes in the amounts and quality required for x-ray crystallographic studies. in recent years it was shown in a number of publications that it is possible to obtain near-atomic resolution structures of large and rigid macromolecules such as icosahedral viruses. due to a number of methodological advances there are now also great perspectives for high-resolution single particle cryo-em studies of large and dynamic macromolecules. successful high-resolution structure determination of dynamic complexes requires new biochemical purification strategies and protocols as well as state of the art electron microscopes and high-performance computing. in the future cryo-em will thus be able to provide structures at near-atomic resolution and information about the dynamic behavior of macromolecules simultaneously. detection and rapid manipulation of phosphoinositides with engineered molecular tools tamas balla section on molecular signal transduction, program for developmental neuroscience, nichd, nih, bethesda, md , usa polyphosphoinositides (ppis) are ubiquitous lipid regulators of a variety of cellular processes serving as docking sites and conformational switches for a large number of signaling proteins. the localization and dynamic changes in ppis in live cells have been followed with the use of protein domain gfp chimeras. in this presentation we will show experimental systems that allow rapid manipulation of the levels of ppis in specific membrane compartments. we are also actively pursuing strategies that will allow us to map the distribution and possible functional diversity of the phosphatidylinositol (ptdins) pools within intact cells since they are the precursors of ppis. we will show our most recent progress in addressing this question: the use of a ptdins specific plc enzyme isolated from listeria monocytogenes together with a highly sensitive diacylglycerol sensor to determine the distribution and also to alter the level of ptdins in living cells. these studies reveal that a significant metabolically highly active ptdins pool exists associated with tiny mobile structures within the cytoplasm in addition to the known er and pm ptdins pools. we will show our most recent data on the consequences of ptdins depletion within the various ptdins pools on ppi production and on the morphology and functions of various organelles. the functionality of proteins is known to be intimately related to the motion of their constituents on the atomic/molecular level. the study of microscopic motion in complex matter is often reduced to the observation of some average mean square atomic displacement, a first, very partial characterization of the dynamics. the marked crossover in the temperature dependence of such quantities in hydrated proteins around k, the so called ''dynamic transition'' has been originally observed a quarter of century ago. the origin, nature and the key characteristics of the atomic motions behind this remarkable evolution of the mean square displacement in proteins remained controversial over the past decades. recent analysis of mö ssbauer, dielectric relaxation and neutron scattering spectroscopic data provide unambiguous evidence that this phenomenon is caused by the temperature dependence of a relaxation process spread over several orders of magnitude in the time domain, similarly to the b relaxation process observed in glasses. the review and critical analysis of the available data highlights the inherent ambiguities of commonly used data fitting approaches. emerging evidence from model independent observations tend to exclude some of the proposed mechanisms. microbial rhodopsins: light-gated ion channels and pumps as optogenetic tools in neuro-and cell biology e. bamberg, c. bamann, r.e. dempski, k. feldbauer, s. kleinlogel, u. terpitz, p. wood department of biophysical chemistry, max-planck-institute of biophysics, frankfurt, germany microbial rhodopsins are widely used in these days as optogenetic tools in neuro and cell biology. we were able to show that rhodopsins from the unicellar alga chlamydomonas reinhardtii with the transmembrane helix motif act as light-gated ion channels, which we named channelrhodopsins(chr , ). together with the light driven clpump halorhodopsin chr is used for the non-invasive manipulation of excitable cells and living animals by light with high temporal resolution and more important with extremely high spatial resolution the functional and structural description of this new class of ion channels is given (electrophysiology, noise analysis,flash photolysis and d crystallography). new tools with increased spatial resolution and extremely enhanced light sensitivity in neurons are presented. a perspective for basic neurobiology and for medical applications is given. extracellular signals consists of the induction of specific gene expression patterns and the re-organization in space and time of stereo-specific macromolecular interactions that endow the cell with its specific morphology. we develop quantitative experimental and computational approaches to derive and conceptualize physical principles that underlie these dynamics of signal processing and cellular organization. we have an experimental emphasis on functional microscopic imaging approaches at multiple resolutions to study the localization and dynamics of protein reactions/ interactions, maintaining the inherent spatial organization of the cell. we have a strong recursion between computation of molecular dynamics in realistic cell geometries as sampled by microscopy, and experiments that reveal the dynamic properties of networks in living cells. we investigate the cellular topography of activities that transmit signals from receptors at the cell surface. here we ask, how spatial partitioning of intracellular signalling activities is achieved by the causality structure of the signalling network, and how this partitioning affects signal response. this entails the experimental elucidation of connections between reactions and the determination of enzyme kinetic parameters in living cells. o- molecular photovoltaics mimic photosynthesis michael grä tzel laboratory of photonics and interfaces, institute of chemical science and engineering, station , ecole polytechnique fé dé rale, ch- lausanne, switzerland e-mail: michael.graetzel@epfl.ch the field of photovoltaic cells has been dominated so far by solid state p-n junction devices made e.g. of crystalline or amorphous silicon, profiting from the experience and material availability of the semiconductor industry. however, there is an increasing awareness of the possible advantages of devices referred to as ''bulk'' junctions due to their interconnected three-dimensional structure. their embodiment departs completely from the conventional flat p-n junction solid-state cells, replacing them by interpenetrating networks. this lecture focuses on dye sensitized mesoscopic solar cells (dscs), which have been developed in our laboratory. imitating natural photosynthesis, this cell is the only photovoltaic device that uses a molecular chromophore to generate electric charges from sunlight and that accomplishes the separation of the optical absorption from the charge separation and carrier transport processes. it does so by associating the molecular dye with a film constituted of tiny particles of the white pigment titanium dioxide. the dsc has made phenomenal progress, present conversion efficiencies being over percent for single junction and percent for tandem cells, rendering the dsc a credible alternative to conventional p-n junction devices. molecularly single-molecule imaging and tracking techniques that are applicable to living cells are revolutionizing our understanding of the plasma membrane dynamics, structure, and signal transduction functions. the plasma membrane is considered the quasi- d non-ideal fluid that is associated with the actinbased membrane-skeleton meshwork, and its functions are likely made possible by the mechanisms based on such a unique dynamic structure, which i call membrane mechanisms. my group is largely responsible for advancing highspeed single molecule tracking, and based on the observations made by this approach, i propose a hierarchical architecture of three-tiered meso-scale ( - nm) domains as fundamental organizing principles of the plasma membrane. the three tiers i propose are the following. [tier ] - nm compartments made by partitioning the entire plasma membrane by the membrane-associated actinbased meshwork (membrane skeleton: fences) and its associated transmembrane proteins (pickets). since the entire plasma membrane is partitioned by these structures, and the membrane skeleton provides important platforms for the molecular interactions and pools, membrane compartments are the most basic tier for the plasma membrane organization. [tier ] meta-stable - nm raft domains that can be turned into stable * - -nm domains (receptor-cluster rafts), based on ligand-induced homo-dimers of glycosylphosphatidylinositol (gpi)-anchored receptors (coupling with [tier ]) and facilitated by raft-lipid interactions. [tier ] protein complexes of various sizes ( - nm) and lifetimes. i will also talk about how domains of tiers and are coupled to the membrane partitioning (tier ). the concept of the three-tiered domain architecture of the plasma membrane and the cooperative interactions of different tiers provides a good perspective for understanding the mechanisms for signal transduction and many other functions of the plasma membrane. introduction: in the present study we investigate the effects of electromagnetic fields (emf) on the binding of norfloxacin (nrf) to human serum albumin (hsa) by fluorescence, three-dimensional fluorescence and uv-visible spectroscopic approaches. hsa is the most abundant protein in human blood plasma which works as a carrier that transports different materials in the body. nrf is used to treat variety of bacterial infections. it works by stopping the bacterial growth. methods: hsa, nrf and potassium phosphate buffer were purchased from sigma. fluorescence spectrofluorometer, uv-vis spectrophotometer, three-dimensional fluorescence and a home-built emf generator apparatuses were used. results: results obtained from this study indicated that nrf has a strong ability to quench hsa in nm. in addition, there was a slight blue shift, which suggested that the microenvironment of protein became more hydrophobic after addition of nrf. moreover, synchronous fluorescence demonstrated that the microenvironment around tyrosine (tyr) had a trivial increase. these, and the results of hsa-nrf in the presence of emf with khz, illustrates the same results inferred from quenching and blue shift. however, there was a significant decrease in k sv of nrf with hsa in presence of emf exposure. moreover, the binding parameters including the number of binding sites and the binding constant were calculated form hill equation. conclusion: it was shown that nrf could induce conformational changes in hsa both in the absence and presence of emf with no significant difference. yet, the affinity is decrease significantly in the presence of emf. the clinical implications are discussed in detail. characterization of the biochemical properties and biological function of the formin homology domains of drosophila we characterised the properties of drosophila melanogaster daam-fh and daam-fh -fh fragments and their interactions with actin and profilin by using various biophysical methods and in vivo experiments. the results show that while the daam-fh fragment does not have any conspicuous effect on actin assembly in vivo, in cells expressing the daam-fh -fh fragment a profilindependent increase in the formation of actin structures is observed. the trachea specific expression of daam-fh -fh also induces phenotypic effects leading to the collapse of the tracheal tube and lethality in the larval stages. in vitro, both daam fragments catalyze actin nucleation but severely decrease both the elongation and depolymerisation rate of the filaments. profilin acts as a molecular switch in daam function. daam-fh -fh , remaining bound to barbed ends drives processive assembly of profilin-actin, while daam-fh forms an abortive complex with barbed ends that does not support profilinactin assembly. both daam fragments also bind to the sides of the actin filaments and induce actin bundling. these observations show that the drosophila melanogaster daam formin represents an extreme class of barbed end regulators gated by profilin. electron spin echo studies of free chain-labelled stearic acids interacting with b-lactoglobulin rita guzzi, luigi sportelli, rosa bartucci dipartimento di fisica, università della calabria, rende (cs), italy b-lactoglobulin (blg) binds non-covalently fatty acids within its central calyx, a cavity in the barrel formed by the strands ba-bh. we present results of pulsed electron paramagnetic resonance (epr) spectroscopy on the interaction of blg with stearic acids spin-labelled at selected positions, n, along the acyl chain (n-sasl, n = , , , , ). d o-electron spin echo envelope modulation (eseem) fourier transform spectra indicate that all segments of the bound chains in the protein binding site are accessible to the solvent. the extent of water penetration decreases progressively on moving from the first segments toward the terminal methyl end of the chain. about % of the nitroxides in the upper part of the chain (n = , ) are h-bonded by a single water molecule and this fraction reduces to % at the chain terminus (n = , ). a lower fraction of the nitroxides are h-bonded by two water molecules, and it decreases from about % to a vanishingly small value on going down the chain. echo-detected ed-epr spectra reveal subnanosecond librational motion of small amplitude for both -and -sasl in the protein cavity. the temperature dependence of the librations is more marked for -sasl and it arises mainly from an increase in librational amplitude with increasing temperature. fusion peptides (fp) pertaining to the spike glycoprotein from severe acute respiratory syndrome (sars) coronavirus are essential for the fusion between viral and host cellular membranes. here we report a biophysical characterization of the interaction of two putative fps with model membranes. fluorescence and dsc experiments showed that both peptides bind stronger to anionic than to zwitterionic lipid membranes. esr spectra showed that toac-sars ifp rotational dynamics is modulated by lipid composition and ph as compared to the spectrum of this peptide in solution. however, stearic acid spin labels reported no changes on the dynamic structure of zwitterionic micelles, whereas the whole chain of anionic surfactants was perturbed by the peptides. finally, cd data revealed a predominant b-strand structure for sars fp and an a-helix for sars ifp in the presence of micelles, in contrast to their disordered structures in buffer. overall the results point out that electrostatic and hydrophobic interactions are both important to the energetic behavior of peptide membrane interaction. these findings might provide a useful rationale for the elucidation of one of the steps involved in the fusion process, and thus help understanding the more general way of action of fps at a molecular level. interaction of filamentous actin and ezrin within surface modified cylindrical nanopores daniela behn , and claudia steinem institute for organic and biomolecular chemistry, university of gö ttingen, tammannstraße , gö ttingen, germany, ezrin is a member of the ezrin-radixin-moesin (erm) protein family that acts as a dynamic linker between the plasma membrane and the actin cytoskeleton and is hence involved in membrane organization, determination of shape and surface structures and other cellular processes. the protein is highly enriched in microvilli of polarized epithelial cells, where it binds filamentous actin (f-actin) with its c-terminal domain, while the n-terminal domain is connected to the plasma membrane via specific binding to l-a-phosphatidylinositol- , -bisphosphate (pip ). nanoporous anodic aluminum oxide (aao) films provide similar dimensions as microvilli and are thus a versatile template to investigate the interaction of ezrin with f-actin within spationally confined areas. owing to their optical transparency, functionalized aaos can be used to measure the binding process of ezrin to a pip containing solid supported membrane by means of time resolved optical waveguide spectroscopy (ows). confocal laser scanning microscopy (clsm) will elucidate, whether f-actin binding to ezrin takes place within or atop the nanopores. furthermore, elasticity mapping of f-actin filaments by means of atomic force microscopy will allow determining binding forces and the lateral tension of the actin cytoskeleton. in vitro application of porphyrin photosensitisers on mcf , hela and g tumour cell lines binder s., kolarova h., bajgar r., tomankova k., daskova a. deparment of medical biophysics, faculty of medicine of palacky university, olomouc, czech republic tumour treatment presents a challenge to all scientists and clinicians. contemporary methods like radiotherapy, chemotherapy or surgery have many undesirable side effects. photodynamic therapy (pdt) seems to be one of alternatives which can be helpful in malignant cell therapy. pdt is not only limited to cancer treatment but is also used as an alternative for cardiovascular, skin and eye disease treatment. pdt employs photosensitive agents which need to be activated by light which is not harmful to a patient. the activated photosensitive agent provokes a formation of reactive oxygen species leading to cell damage or death. the phototoxicity of the two porphyrin photosensitizer (tmpyp, zntpps . h o) on the malignant cell lines (g , hela, mcf ) irradiated with the jcm - doses was evaluated by ros production assay, mtt assay and comet assay. our results indicate higher efficiency of tmpyp over zntpps . h o. as for the photodynamic effectiveness of the used photosensitizers on chosen cell lines we found that hela cell line is the most sensitive to phototoxic damage induced by tmpyp. p- nmr analysis of the respiratory syncytial virus m - protein structure and of its interaction with some of its targets c. sizun the respiratory syncytial virus (rsv) is a major cause of acute respiratory tract infections (bronchiolitis, pneumonia) in human and a leading cause of viral death in infants and immunocompromised patients. rsv genome is formed of a single non-segmented negative strand rna which transcription and replication is ensured by a specific rna-dependent rna polymerase complex formed of the large (l) polymerase subunit and of several cofactors. this complex has no cellular counterpart and represents an ideal target for antiviral drugs. among the cofactors, m - acts as an antitermination factor and increases the polymerase processivity. its central domains has been shown, in vitro, to bind the phosphoprotein p and genomic rna in a competitive manner. here we report the nmr structure of this central domain and its interaction with p and rna fragments. m - shares structural similarity with vp , a transcription factor of ebola virus. the binding surfaces for rna and p are distinct but overlapping. rna binds to a basic cluster located next to residues found to be critical for transcription both in vitro and in vivo by mutational analysis. we speculate that m - might be recruited by p to the transcription complex, where interaction with rna takes place, stabilized by additional elements. force spectroscopy at the membrane-cytoskeleton interface: interactions between ezrin and filamentous actin julia a. braunger , , ingo mey and claudia steinem institute for organic and biomolecular chemistry, georg-august-university of gö ttingen, tammannstraße , gö ttingen, germany, ggnb doctoral program: imprs - ezrin, a member of the erm (ezrin/radixin/moesin) protein family, provides a regulated linkage between the plasma membrane and the actin cytoskeleton. it contributes to the organization of structurally and functionally distinct cortical domains participating in adhesion, motility and fundamental developmental processes. ezrin is negatively regulated by an intramolecular interaction of the terminal domains that masks the f-actin binding site. a known pathway for activation involves the interaction of ezrin with phosphatidylinositol , bisphosphate (pip ) in the membrane, followed by phosphorylation of the threonine residue in the c-terminal domain. to date, it is unclear to what extent both regulatory inputs contribute to the activation. we developed an in vitro system that facilitates the specific analysis of the interaction forces between ezrin and f-actin by means of atomic force spectroscopy (afm). applying ezrin wild type and the pseudophosphorylated mutant protein ezrin t d, respectively, permits to monitor the individual influence of phosphorylation on the f-actin-ezrin interaction. thus, a thorough characterization of the acting forces at the ezrin-actin interface will elucidate the activation mechanism of ezrin. delivery system even more efficient, we have constructed nano-carrier by coating of ldl by polyethylene glycol (peg) . the hydrophilicity of peg should reduce the interaction of ldl with other serum proteins and consequently decrease the redistribution of loaded drug from ldl to the (lipo)proteins. dynamic light scattering was used for determination of hydrodynamic radius of ldl-peg particles. cd spectroscopy measurements didn't reveal structural changes of apoliprotein b- (ligand for ldl receptors on cell surface), after conjugation of peg with ldl. interaction of ldl-peg complexes with hypericin (hyp) a natural photosensitizer was studied by fluorescence spectroscopy. we have demonstrated accumulation of higher number of hyp in ldl-peg than ldl particles. however, the kinetics of hyp redistribution from hyp/ldl-peg complex to free ldl have similar parameters as those for the kinetics of hyp transfer between non-modified ldl molecules. we suggest that hyp molecules are mostly localized in the vicinity of the surface of the ldl-peg particles and they are prone to redistribution to other serum proteins. grant support: lpp- - , vega- - . modification of the head-group of aminophospholipids by glycation and subsequent lipid oxidation affect membrane's structure causing cell death. these processes are involved in the pathogenesis of aging and diabetes. non-enzymatic glycation forms in the first step a schiff base (sb), which rearranges to a more stable ketoamine, amadori product, which leads to the formation of a heteregenous group of compounds (ages). although several studies have been focused on identification of aminophospholipid glycation products, less attention has been paid to kinetic mechanism of the reaction. for that reason, in the present work, we compare the kinetic reactivity of polar head-group of phosphatidylethanolamine (pe) and phosphatidylserine (ps), the two target phospholipids components of mammalian cell membranes. the reaction of pe and ps's head-group with glycating compounds (glucose and arabinose) was studied in physiological conditions by using nmr spectroscopy. the obtained formation rate constants for sb are lower than those determined for the sb of the peptide ac-phe-lys with the same carbohydrates. it suggests that the phosphate group may delay the glycation process. moreover, the ps's head-group has a carboxilic group in the structure, which affects the stability of the sb. we developed ultrasensitive, elisa-like nanoimmuno assays suitable for proteomics/interactomics studies in low sample volumes. we exploit the approach of dna microarray technologies applied to proteomics [ ] , in combination with atomic force microscopy (afm) to generate functional protein nanoarrays: semisynthetic dna-protein conjugates are immobilized by bioaffinity within a nanoarray of complementary ssdna oligomers produced by afm nanografting (ng). a nanoarray of different antibodies or synthetic molecular binders can be generated in a single operation, once the dna nanoarray is produced. moreover, ng allows adjusting the packing density of immobilized biomolecules to achieve optimum bio-recognition. afm-based immunoassays with these nanoarrays were shown to achieve detection limit of hundreds of femto molar, in few nanoliters volumes, with very high selectivity and specificity [ ] . to detect the hybridization efficiency of our devices, we run a combined experimental-computational study that provides quantitative relations for recovering the surface probe density from the mechanical response (afmcompressibility measurements) of the sample. nucleoside analogues used as anticancerous drugs can be rapidly degraded within treated cells, constituting a major obstacle of their therapeutic efficiency. among the enzymes responsible for this degradation, the cytosolic 'nucleotidase ii (cn-ii) catalyses the hydrolysis of some nucleoside monophosphates. in order to improve the efficacy of anticancerous drugs and to define the precise role of cn-ii, new original inhibitors have been developed against cn-ii. virtual screening of chemical libraries on the crystal structure has allowed us to identify very promising candidates that turned to be competitive inhibitors of cn-ii. one molecule was included in the anticancerous treatment of tumoral cell lines in order to evaluate the potential benefit and could induce in fine a sensitization of certain anticancerous drugs. we also explore other inhibitors targeting the allosteric sites of this enzyme using a strategy that takes into account the dynamics of cn-ii. the chemical structures of the newly identified allosteric inhibitors as well as the atomic interactions with enzyme residues will be presented. the final goal of this study is to find molecules that can freeze the enzyme in a conformation for which its dynamics is severely limited and therefore its function. native mass spectrometry to decipher interactions between biomolecules sarah cianferani laboratoire de spectromé trie de masse bio-organique, université de strasbourg, iphc, rue becquerel strasbourg, france. cnrs, umr , strasbourg, france mass spectrometry is generally understood as ''molecular mass spectrometry'' with multiple applications in biology (protein identification using proteomic approaches, recombinant protein and monoclonal antibody characterization). an original and unexpected application of mass spectrometry emerged some twenty years ago: the detection and the characterization of intact biological noncovalent complexes. with recent instrumental improvements, this approach, called native ms, is now fully integrated in structural biology programs as a complementary technique to more classical biophysical approaches (nmr, crystallography, calorimetry, spr, fluorescence, etc.). native ms provides high content information for multiprotein complexes characterization, including the determination of the binding stoichiometries or oligomerization states, sitespecificities and relative affinities. recent developments of ion mobility / mass spectrometry instruments (im-ms) provide a new additional level for ms-based structural characterization of biomolecular assemblies allowing size and shape information to be obtained through collisional cross section measurements. these different aspects of native ms for structural characterization of biomolecular assemblies will be illustrated through several examples, ranging from multiprotein-complexes to protein/nucleic acid assemblies. complex coacervation is a process which may result by electrostatic interaction between charged polysaccharides. it depends essential on ph, ionic strength and biopolymers properties like ratio, concentration and charge density. in this case, the main work was to study the structural properties of a colloidal system of opposite charge -chitosan and gum-arabic by atomic force microscopy (afm). according to some of complexes show tendency to agglomerate. this depends on the molar ration of the macromolecules and their relative molecular weights. afm micrographs show, too, that some formation of irregular aggregates by both polymers were due to presence of noncharged polar monomers in chitosan molecule. at higher gum-arabic/chitosan ratios biopolymer concentrations, coacervates appear like a core-shell miccelar structure composed of hydrophobic core (charge neutralized segments) stabilized by the excess component (positive zeta potential) and non-charged segments of gum arabic. interaction of human serum albumin with rutin theoretical and experimental approaches Ícaro p caruso human serum albumin (hsa) is the principal extracellular protein with a high concentration in blood plasma and carrier for many drugs to different molecular targets. flavonoids are a large class of naturally occurring polyphenols widely distributed in plants. rutin (quercetin- -rutinoside) is the glycoside between flavonoids quercetin and disaccharide rutinose. like other flavonoids, rutin displays anti-inflammatory and anti-oxidant properties. the interaction between hsa and rutin was investigated by fluorescence spectroscopy, ab initio and molecular modeling calculations. fluorescence titration was performed by keeping the hsa concentration ( lm) constant and stoichiometrically varying the rutin concentration ( - lm) . the emission spectra were obtained in the range of to nm, with the excitation wavelength at nm. the obtained fluorescence data were corrected for background fluorescence and for inner filter effects. the stern-volmer quenching constant values were . and . m - at and k, respectively. from the modified stern-volmer association constants . (at k) and . m - (at k) were calculated the thermodynamic parameters dh = . kj mol - , dg k = - . kj mol - and dg k = - . kj mol - , and ds = . kj mol - k - . fluorescence quenching method was used also to study the binding equilibria thus determining the number of binding sites . and . , and binding constant . m - and . m - at and k, respectively. the efficient quenching of the trp fluorescence by rutin indicates that the binding site for the flavonoid is situated within subdomain iia of hsa. the distance r = . nm between the donor (hsa) and the acceptor (rutin) was obtained according to fluorescence resonance energy transference (fret). wavelength shifts in synchronous fluorescence spectra showed the conformation of hsa molecules is changed in the presence of rutin. the structure of rutin utilized in molecular modeling calculation was obtained by gaussian program. the optimization geometry of rutin was performed in its ground states by using ab initio dft/b lyp functional with - g(d,p) basis set used in calculations. the molecular electrostatic potential (mep) was calculated to provide the molecular charge distribution of rutin. the gap energy value between the homo and lumo of the rutin molecule was about . ev which indicates that rutin is classified as a reactive molecule. from molecular modeling calculation the interaction between hsa and rutin was investigated using the autodock program package. the three-dimensional coordinates of human serum albumin were obtained from the protein data bank (entry pdb code ao ) and of rutin were obtained from output optimization geometry of dft. the best energy ranked result shows that rutin is localized in the proximity of single tryptophan residue (trp ) of hsa that is in agreement with the fluorescence quenching data analysis. the effect of toxofilin on the structure of monomeric actin lívia czimbalek, veronika kollá r, roland kardos, gá bor hild university of pé cs, faculty of medicine, department of biophysics, pé cs, hungary actin is one of the main components of the intracellular cytoskeleton. it plays an essential role in the cell motility, intracellular transport processes and cytokinesis as well. toxoplasma gondii is an intracellular parasite, which can utilise the actin cytoskeleton of the host cells for their own purposes. one of the expressed proteins of t. gondii is the kda-sized toxofilin. the long protein is a monomeric actinbinding protein involved in the host invasion. in our work we studied the effect of the actin-binding site of toxofilin - on the g-actin. we determined the affinity of toxofilin to the actin monomer. the flourescence of the actin bound e-atp was quenched with acrylamide in the presence or absence of toxofilin. in the presence of toxofilin the accessibility of the bound e-atp decreased, which indicates that the nucleotide binding cleft is shifted to a more closed conformational state. the results of the completed experiments can help us to understand in more details what kind of cytoskeletal changes can be caused in the host cell during the invasion of the host cells by intracellular parasites. t bacteriophage, as a surrogate on non-enveloped viruses was selected as a test system. both tmpcp and bmpcp and their peptide conjugates proved to be efficient photosensitizers of virus inactivation. the binding of porphyrin to phage dna was not a prerequisite of phage photosensitization, moreover, photoinactivation was more efficiently induced by free than by dna bound porphyrin. mechanism of photoreaction (type i. versus type ii) and the correlation between dna binding, singlet oxygen production and virus inactivation capacity was also analyzed. dna binding reduced the virus inactivation due to the reduced absorbance and singlet oxygen production of bound photosensitizer, and altered mechanism of photoinactivation. as optical melting studies of t nucleoprotein revealed, photoreactions of porphyrin derivatives affected the structural integrity of dna and also of viral proteins, even if the porphyrin did not bind to np or was selectively bound to dna. anesthesia is a medical milestone (friedman & friedland, medicine's greatest discoveries, ) and local anesthetics (la) are the most important compounds used to control pain in surgical procedures. however, systemic toxicity is still a limitation for la agents as well as low solubility, as for tetracaine (ttc). approaches to improve la effects include macrocyclic systems formation, such as in cyclodextrins (cd). we have studied complexes formed between ttc and b-cd or hydroxylpropyl (hp)-b-cd through nmr and other (uv-vis, fluorescence, dsc and x-ray diffraction) techniques. at ph . a : stoichiometry of complexation was detected for both complexes, with association constants of m - and m - for ttc:b-cd and ttc:hp-b-cd, respectively. the nuclear overhauser nmr data disclosed trough the space proximities between hydrogens h h and h iat the aromatic ring of ttc -and hydrogens from the inner cavity of the cyclodextrins, allowing us to propose the topology of ttc:cd interaction. complex formation did not curb ttc association with model (liposomes) and biological membranes since the total analgesic effect (infraorbital nerve blockade in rats) induced by mm ttc increased % upon complexation. supported by (fapesp # / - , - ) brazil. p- itc as a general thermodynamic and kinetic tool to study biomolecule interactions philippe dumas , dominique burnouf , eric ennifar , sondes guedich , guillaume bec , guillaume hoffmann isothermal titration calorimetry (itc) is a powerful technique for thermodynamic investigations that is little used to obtain kinetic information. we have shown that, in fact, the shape of the titration curves obtained after each ligand injection is strictly governed by the kinetics of interaction of the two partners. a simple analysis allowed us to explain several facts (e.g. the variation of time needed to return to equilibrium during a titration experiment). all simplifications were further released to obtain a very realistic simulation of an itc experiment. the method was first validated with the binding of the nevirapine inhibitor onto the hiv- reverse transcriptase by comparison with results obtained by biacore tm . importantly, for more complex systems, the new method yields results that cannot be obtained in another way. for example, with the e. coli transcription-regulator thiamine pyrophosphate riboswitch, we could resolve kinetically and thermodynamically the two important successive steps: ( ) the binding of the tpp ligand and ( ) the subsequent rna folding. our results show that initial tpp binding is controlled thermodynamically by tpp concentration, whereas the overall transcription regulation resulting from rna folding is kinetically controlled. gfps, due to their tendency to dimerize at high concentration. we have characterized for the first time the selfassociation properties of cfp (cyan fluorescent protein), the fluorescent protein mostly used as fret donor. we found that the fluorescence quenching observed at high expression level in the cell cytoplasm and the fluorescence depolarization measured at high concentration in vitro are insensitive to the a k mutation, shown to dissociate other gfp dimers. both phenomena are satisfactorily accounted for by a model of non-specific homo-fret between cfp monomers due to molecular proximity. modeling the expected contributions to fluorescence depolarization of rotational diffusion, homo-fret within a hypothetical dimer and proximity homo-fret shows that cfp has a homo-affinity at least times lower than gfp. this difference is due to an intrinsic mutation of cfp (n i), originally introduced to increase its brightness and that by chance also disrupts the dimers. biomolecular recognition typically proceeds in an aqueous environment, where hydration shells are a constitutive part of the interacting species. the coupling of hydration shell structure to conformation is particularly pronounced for dna with its large surface to volume ratio. conformational substates of the phosphodiester backbone in b-dna contribute to dna flexibility and are strongly dependent on hydration. we have studied by rapid scan ftir spectroscopy the isothermal b i -b ii transition on its intrinsic time scale of seconds. correlation analysis of ir absorption changes induced by an incremental growth of the dna hydration shell identifies water populations w (po --bound) and w (non-po --bound) exhibiting weaker and stronger h-bonds, respectively, than those in bulk water. the b ii substate is stabilized by w . the water h-bond imbalance of - kj mol - is equalized at little enthalpic cost upon formation of a contiguous water network (at - h o molecules per dna phosphate) of reduced !(oh) band width. in this state, hydration water cooperatively stabilizes the b i conformer via the entropically favored replacement of w -dna interactions by additional w -water contacts, rather than binding to b i -specific hydration sites. such water rearrangements contribute to the recognition of dna by indolicidin, an antimicrobial -mer peptide from bovine neutrophils which, despite little intrinsic structure, preferentially binds to the b i conformer in a water-mediated induced fit. in combination with cd-spectral titrations, the data indicate that in the absence of a bulk aqueous phase, as in molecular crowded environments, water relocation within the dna hydration shell allows for entropic contributions similar to those assigned to water upon dna ligand recognition in solution. segmental-labeling expression of sh domains of cd ap protein to study interaction with their ligand i.f. herranz-trillo , j.l. ortega-roldan , n.a.j. van transient and low affinity interactions within the cell can be enhanced by the combination of more than one domain. up to now most of the effort has been put on the study of the regulation in the affinity and specificity of the binding to isolated single domains but little is known about the effect of the presence of a second or third domain. multiple examples of proteins containing tandem domains exist in the genome like the cin /cms family of adaptor proteins. in this family all three n-terminal sh domains are involved in a wide variety of different interactions, they share higher similarity among themselves than to any other sh domains, suggesting an overlapping specificities in binding. cd associated protein (cd ap) is an adaptor protein and belongs to this family, its n-terminus consists of three sh domains and the interaction of each one of them with its target(-s) might be ultimately modulated by the presence of its next-door-neighbor. in this work we present the expression and purification of the tandem cd ap-sh a/ sh b produced by segmental labeling techniques that allow us to express the domains with different isotopic label, improving the nmr signal and facilitating to study the interaction of the natural ligand in the presence of nextdoor-neighbor domain. there are plenty of molecules that exert their effects at the cell membrane. the evaluation of these interactions, frequently quantified by the nernst lipid/water partition constant (kp), helps to elucidate the molecular basis of these processes. we present here a recently derived and tested method to determine kp for single solute partitions using fpotential measurements. the concept was then extended to the interaction of supramolecular complexes with model membranes. a simultaneous double partition with an aqueous equilibrium is considered in this partition model. the results were validated by dynamic light scattering -dls, f-potential, fluorescence spectroscopy and laser confocal microscopy experiments. we evaluated the interaction of supramolecular complexes (peptides derived from dengue virus proteins with oligonucleotides) with luv to study our biophysical models. dengue virus (dv) infects over - million people every year and may cause viral hemorrhagic fever. no effective treatment is available and several aspects of its cellular infection mechanism remain unclear. the extension of the interactions of these complexes with biomembranes helps to elucidate some steps of dv life cycle. the aggregation of amphotericin b in the lipid membranes induced by k + and na + ions: langmuir monolayers study marta arczewska, mariusz gagoś department of biophysics, university of life sciences in lublin, poland the polyene antibiotic amphotericin b (amb) is currently the drug of choice in the treatment of fungal infections despite its undesirable side effects. according to the general conviction, the biological action of the drug is based on the formation of transmembrane channels which affect physiological ion transport, especially k + ions. this work reports the results of langmuir monolayers study of the effect of k + and na + ions on the molecular organizations of amb in the model lipid membrane. the two-component monolayers containing amb and phospholipid (dppc) have been investigated by recording surface pressure-area isotherms spread on aqueous buffers containing physiological concentration of k + and na + ions. the strength of the amb-dppc interactions and the stability of the mixed monolayers were examined on the basis of surface pressure measurements, the compressional modulus and the excess free energy of mixing. the obtained results proved a high affinity of amb towards lipids in the presence of k + than na + ions. the most stable mixed monolayers were formed with the : and : stoichiometry in the presence of k + and na + ions, respectively. this research was financed by ministry of education and science of poland within the research project n n . microcalorimetric study of antibiotic amphotericin b complexes with na + , k + and cu + ions arkadiusz matwijczuk, grzegorz czernel, mariusz gagoś department of biophysics, university of life sciences in lublin, poland amphotericin b (amb) as a metabolite of streptomyces nodosus is one of the main polyene antibiotics applied in the treatment of deep-seated mycotic infections. we presented microcalorimetric (dsc) study of molecular organization of amphotericin b in lipid membranes induced by na + , k + and cu + ions. the analysis of dsc curves indicates the influence of na + and k + ions on the main phase transition of pure dppc lipid. for the molar fractions of , , , mol% amb in dppc we observed the thermal shift towards higher temperatures in respect to pure lipid, both in the presence of na + and k + ions. this result may be connected with the changes in dynamic properties of the model membrane system. in case of amb-cu + complexes in aqueous solution at two ph values, . and . , the dsc measurements reported endothermic heat effect. this phase transition was related to the dissociation process of amb-cu + complexes. the formation of amb-cu + complexes are accompanied by changes of the molecular organization of amb especially disaggregation. these all observed effects might be significant from a medical point of view. this research was financed by ministry of education and science of poland within the research project n n . membrane proteins and peptides are acting in an environment rich in other proteins or peptides. aim of our study was to understand how such molecular crowding and resulting intermolecular interactions can influence the behavior of membrane proteins, using various antimicrobial peptides and membrane proteins as examples. in the case of antimicrobial peptides we have previously described a change in their alignment in the membrane at a characteristic threshold concentration. to understand whether this change is due to unspecific crowding or specific peptidepeptide interactions, we tested if this re-alignment depends on the presence of additional peptides. in most cases we found a similar re-orientation behavior irrespective of the added peptide type, indicating unspecific crowding. when pairing pgla and magainin- , however, we observed a distinctly different sequence of pgla re-orientation in the membrane, indicating a specific interaction between these two peptides, which correlates well with their known synergistic activity. a rather different effect of crowding was observed for the larger channel protein mscl, which was found to form clusters of functionally active proteins in the membrane. we propose that this clustering is caused by lipid-mediated protein-protein interactions. water, hydrophobic interaction, and protein stability j. raul grigera and c. gaston ferrara instituto de física de líquidos y sistemas bioló gicos (iflysib), conicet-unlp, la plata, argentina although there are several forces maintaining protein structure, it is well know that hydrophobic interaction is the dominant force of protein folding. then, we can infer that any factor that alters hydrophobic interaction will affect the protein stability. we have study by computer simulation a model system consisting in solution of lenard-jones particles in water (spc/e model) at different pressures and temperatures and analyzed the solubility i.e. the aggregation properties, of such a system. from the obtained data we are able to build up the phase surface determining the critical point. the computing results where compared with experimental data of binary mix of non polar substance in water and of protein denaturation, finding high coincidence on the critical point. since the behavior of our model system can only be due to hydrophobic effects, the coincidences with the denaturation of proteins allow us to conclude that the dominant factor that determine temperature and pressure denaturation of proteins is the hydrophobic interaction. the temperature and pressures at which the denaturation, as well the disaggregation of simple non-polar particles, starts agree with what we could expect based on the cross over line of the low to high density structure water transition. the functional reconstitution of a mitochondrial membrane protein into a lipid bilayer was studied using a quartz crystal microbalance. the xhis-tagged protein was immobilised via specific binding to a cu + terminated sensor surface, with a change in frequency indicating approximately % coverage of the sensor surface by the protein. a lipid bilayer was reconstituted around the protein layer, with a final change in frequency that is consistent with the remaining area being filled by lipid. incubation with a specific ligand for the protein resulted in a significant change in frequency compared to the interaction with the surface or lipid alone. the change is greater than expected for the mass of the ligand, indicating a possible conformational change of the protein, such as the opening of a channel and increased water content of the layer. electrical impedance measurements on the same system have provided additional evidence of protein-lipid bilayer formation, and it is intended that this system will be studied with neutron reflectometry to characterise potential ligand induced channel formation. valuable functional and structural information about this membrane protein was obtained by using surface sensitive techniques to study the protein in a biomimetic lipid bilayer. visualizing and quantifying hiv-host interactions with fluorescence microscopy jelle hendrix , *, zeger debyser , johan hofkens and yves engelborghs laboratory for biomolecular dynamics, university of leuven, belgium, laboratory for molecular virology and gene therapy, university of leuven, belgium, laboratory for photochemistry and spectroscopy (*present address), university of leuven, belgium protein-chromatin interactions are classically studied with in vitro assays that only provide a static picture of chromatin binding. fluorescence correlation spectroscopy (fcs) is a non-invasive technique that can be used for the same purpose. being applicable inside living cells it provides dynamic real-time information on chromatin interactions. transcriptional co-activator ledgf/p has well characterized protein and chromatin interacting regions. we studied ledgf/p in vitro and inside living cells with fcs and other techniques (luminescent proximity assay, spot/half-nucleus fluorescence recovery after photobleaching, continuous photobleaching). protein-protein interactions in living cells can be monitored with fluorescence cross-correlation spectroscopy (fccs) using fluorescent proteins as genetic labels. advantages over using fö rster resonance energy transfer (fret) are the independence from intermolecular distance and knowledge of absolute protein concentrations. we characterized fccs with fluorescent proteins in vitro and then studied the intracellular complex of ledgf/p and the hiv- integrase (in) enzyme both with fret and fccs. nucleus and its compartment nucleolus are a seat of enormous biosynthetic activity in human cancer cells. nucleolar proteins, e.g. b or c , play an important role in regulation of cell division and proliferation. one of the strategies how to intermit malignant cell proliferation is affecting, e.g. by drug treatment, a net of intracellular protein interactions to bring the cell on a way of apoptosis. a cytostatic agent actinomycin d initiates apoptosis in human cancer cells, as well as in normal peripheral blood lymphocytes. at the same time, translocation of b and c into nucleoplasm is observed in the treated cells. therefore interaction between nucleolar and apoptotic proteins comes into a question. co-immunoprecipitation, fluorescence microscopy and yeast two hybrid analysis are used to answer it. in co-immunoprecipitation experiments, tumor suppressor p showed up to be a promising candidate for the interaction. fluorescence deposits mostly constituted by variants of transthyretin (ttr), a homotetrameric plasma protein implicated in the transport of thyroxine and retinol [ ] . nowadays, the only effective therapy for ttr amyloidosis is liver transplantation. new therapeutic strategies are being developed taking advantage of our current understanding of the molecular mechanisms of amyloid formation by ttr [ ] . a significant effort has been devoted to the search and rational design of compounds that might decrease ttr tetramer dissociation, for example, through ligand binding at the thyroxine binding sites of ttr [ , ] . here, we use isothermal titration calorimetry (itc) to characterize the thermodynamic binding signature of potential ttr tetramer stabilizers, previously predicted by computerassisted methods [ ] . itc allows the measurement of the magnitude of the binding affinity, but also affords the characterization of the thermodynamic binding profile of a protein-ligand interaction. high affinity/specificity ttr ligands, enthalpically and entropically optimized, may provide effective leads for the development of new and more effective drug candidates against ttr amyloidosis. we have established a set of vectors to promote easy cloning of ecfp and eyfp fusions with any protein of interest. we exploit these fluorescent fusion proteins to study protein-protein interactions by fluorescence lifetime of ecfp. the decrease of ecfp lifetime reveals fret between ecfp and eyfp and hence the interaction between proteins in question. groel-groes chaperonin complex is required for the proper folding of eschericia coli proteins. bacteriophage t and its distant relative coliphage rb encode co-chaperon proteins (respectively gp and coco) that can replace groes in the chaperonin complex. gp is also required in the folding of the major capsid protein of the phage. prd is a large membrane-containing bacteriophage infecting gram-negative bacteria such as e. coli and salmonella enterica. it has kb long linear dsdna genome and the capsid has an icosahedral symmetry. the groel-groes chaperonin complex is needed in the assembly of prd . we have found evidence that prd protein p can work similar way as other viral co-chaperones and substitute groes in chaperonin complex. fluorescence lifetime studies between proteins groel and p reveals an interaction that backs up the theory. structural modification of model membranes by fibrillar lysozyme as reaveled by fluorescence study a.p. kastorna v.n. karazin kharkiv national university, svobody sq., kharkiv, , ukraine recent experimental findings suggest that protein aggregation, leading to the formation and depositions of amyloids play a central role in the neurodegenerative diseases, type ii diabetes, systemic amyloidosis, etc. in the present study we focused our efforts on investigation of the influence of fibrillar lysozyme on the structural state of model lipid membranes composed of phosphatidylcholine and its mixtures with cardiolipin ( mol %) and cholesterol ( mol %). to achieve this purpose, two fluorescent probes with different bilayer location, , -diphenyl- , , -hexatriene (dph) distributing in membrane hydrocarbon core and -lauroyl- -dimethylaminonaphthalene (laurdan) locating at lipid-water interface, have been employed. the changes in membrane viscosity under the influence of amyloid lysozyme were characterized by fluorescence anisotropy of dph. this fluorescence parameter was not markedly affected by fibrillar protein in all types of model membranes. the changes in emission spectra of laurdan were analysed by the generalized polarization value (gp). it was found that adding of amyloid lysozyme resulted in the increment of gp value. our data suggest that lysozyme fibrils cause reduction of bilayer polarity and increase of lipid packing density. isothermal titration calorimetry (itc) is the gold standard for the quantitative characterisation of protein-ligand and protein-protein interactions. however, reliable determination of the dissociation constant (k d ) is typically limited to the range lm [ k d [ nm. nevertheless, interactions characterised by a higher or lower k d can be assessed indirectly, provided that a suitable competitive ligand is available whose k d falls within the directly accessible window. unfortunately, the established competitive itc assay requires that the high-affinity ligand be soluble at high concentrations in aqueous buffer containing only minimal amounts of organic solvent. this poses serious problems when studying protein binding of small-molecule ligands taken from compound libraries dissolved in organic solvents, as is usually the case during screening or drug development. here we introduce a new itc competition assay that overcomes this limitation, thus allowing for a precise thermodynamic description of highand low-affinity protein-ligand interactions involving poorly water-soluble compounds. we discuss the theoretical background of the approach and demonstrate some practical applications using examples of both high-and low-affinity protein-ligand interactions. interaction of myoglobin with oxidized polystyrene surfaces studied using rotating particles probe m. kemper , , d. spridon , l.j. van ijzendoorn , m.w.j. prins , eindhoven university of technology, department of applied physics, eindhoven, the netherlands, dutch polymer institute, eindhoven, the netherlands, philips research, eindhoven, the netherlands the interaction of proteins with polymer surfaces is of profound importance for the sensitivity of biosensors. polymer surfaces are often treated in order to tune their chemical and physical properties, for example by oxidation processes. to get a better understanding of the association of proteins to treated polymer surfaces, we use the rotating particles probe (x.j.a. janssen et al., colloids and surfaces a, vol. , p. , ). in this novel technique, protein coated magnetic particles are in contact with a substrate and the binding is recorded for all individual particles using a rotating magnetic field. we investigate the interaction of myoglobin coated magnetic particles to spincoated polystyrene surfaces that have been oxidized with a uv/ozone treatment. the surfaces have been characterized by xps, afm and water contact angle measurements. we will demonstrate a clear influence of polystyrene oxidation on the binding fractions of the myoglobin coated particles. we interpret the results in terms of dlvo-theory: electrostatic as well as electrodynamic properties of the surfaces will be influenced by the oxidation. interact with monomeric and/or filamentous actins. twinfilin is a - kda protein composed of two adf-homologue domains connected by a short linker. in our work we studied the effects of the mouse twinfilin- (twf ) on the monomeric actin. we determined the affinity of twf to the atp-actin monomer with fluorescence anisotropy measurement (k d = . lm). the fluorescence of the actin bound e-atp was quenched with acrylamide in the presence or absence of twf . in the presence of twinfilin the accessibility of the bound e-atp decreased, which indicates that the nucleotide binding cleft is shifted to a more closed conformational state. it was confirmed with stopped-flow experiments that the kinetics of nucleotide-exchange of actin decreased in the presence of twf . we determined the thermodynamic properties of twf and investigated the effect of twinfilin on the stability of actin monomer with differential scanning calorimetry. the twf stabilized the stucture of the g-actin. our results can help us to understand the regulation of actin cytoskeleton in more details. magnetic np have attracted attention due to their potential of contrast enhancement of magnetic resonance imaging and targeted drug delivery, e.g. tumor magnetic hyperthermia therapy. potential nephrotoxicity of single i.v. administration of fe o np was studied in female wistar rats i.v. administered either placebo ( % v/v rat serum in . % nacl), suspension of tio np (positive control, bimodal / nm distribution), or fe o np (bimodal / nm distribution) in doses of . , . or . mg/kg. rats were sacrificed h, -, -and -days after np injection (n= - /each group). administration of np did not alter kidney size significantly; renal function of np administered rats as monitored by plasma creatinine and urea concentrations, creatinine clearance and protein excretion rate did not differ significantly in either interval from rats administered placebo. one week after administration significant rise in plasma ca, its urinary and fractional excretion was observed in rats administered mg fe o /kg. plasma mg levels rose in this group and weeks after administration. no significant changes in the expression of tnf-a, tgf-b, and collagen iv genes in renal cortex were revealed. no obvious nephrotoxic effects were observed in rats after a single i.v. dose of fe o np. study was supported by fp ec eu: nanotest (development of methodology for alternative testing strategies for the assessment of the toxicological profile of nanoparticles used in medical diagnostics.), grant no.: . biomimetic supramolecular assemblies for studying membrane interactions in vitro and in vivo s. kolusheva, r. jelinek ben-gurion university, beer-sheva, israel we designed a novel biomimetic sensor, composed of conjugated polydiacetylene (pda) matrix embedded within lipid vesicles. the system is capable of detecting various compounds occurring within lipid membranes through rapid colorimetric as well as fluorescent transitions. the colorimetric response of the sensor is correlated to the extent of compound-membrane binding and permeation and quantified binding sensitivity to lipid composition. we describe a new disease diagnostic approach, denoted ''reactomics'', based upon reactions between blood sera and an array of vesicles comprising different lipids and polydiacetylene (pda), a chromatic polymer. we show that reactions between sera and such a lipid/pda vesicle array produce chromatic patterns which depend both upon the sera composition as well as the specific lipid constituents within the vesicles. through attachment of chromatic polydiacetylene (pda) nanopatches onto the plasma membrane, real-time visualization of surface processes in living cells is possible. the ras protein is mutated in % of human tumors. ras acts as a switch, transmitting a growth signal in an active gtp-bound form and turning the signal off in an inactive gdp-bound form. the switch off is accomplished by gtp hydrolysis, which is catalyzed by ras and can be further accelerated by gtpase activating proteins (gaps). mutations which prevent hydrolysis cause severe diseases including cancer. we investigate the reaction of the ras gap protein-protein complex by time-resolved ftir spectroscopy. detailed information on the mechanism and the thermodynamics of the reaction was revealed: first, the catalytic arginine-finger of gap has to move into the gtp binding pocket, then cleavage of gtp is fast and h po hydrogen-bonded in an eclipsed conformation to the b-phosphate of gdp is formed. further, we performed for the first time atr-ftir spectroscopy of ras in its native environment, a lipid membrane. in this setup we are able to do difference spectroscopy of the immobilized protein. interactions with other proteins can be determined in a similar way as in spr experiments but with the additional information from the infrared spectra. galectins are a family of animal lectins that specifically bind b-galactosides and have gained much attention due to their involvement in several biologic processes such as inflammation, cell adhesion and metastasis. in such processes, several issues are still not clear including the mechanisms of interaction with different carbohydrates. galectin- (gal- ) is a tandem-repeat type galectin that contains two carbohydrate recognition domains (crd-i and crd-ii) connected by a linking peptide. in this study, we performed spectroscopic studies of the carbohydrate-recognition domains from human gal- . our goals are two-fold: ( ) to monitor conformational changes in each domain upon its binding to specific ligands and then to correlate the observed changes with structural differences between the crds and ( ) to investigate the interaction between the crds and lipid model membranes. to achieve such objectives we used a combined approach of spectroscopic techniques involving circular dichroism and electron spin resonance. overall the results obtained so far show that crd-i and crd-ii have distinct behaviors in terms of carbohydrate recognition and membrane binding. this may be due to specific differences in their structures and certainly suggests a non-equivalent role in protein function. hemoglobin influence on lipid bilayer structure as revealed by fluorescence probe study o.k. kutsenko, g.p. gorbenko, v.m. trusova v.n. karazin kharkov national university, kharkov, ukraine hemoglobin (hb) is a red blood cell protein responsible for the oxygen transport. its affinity for lipid bilayers represents interest for gaining insight into protein biological function as well as for some applied aspects such as development of blood substitutes or biosensors. hb influence on lipid bilayer structure was investigated using fluorescent probes pyrene and prodan. model membranes were prepared of phosphatidylcholine (pc) and its mixtures with phosphatidylglycerol (pg) and cholesterol (chol). hb penetration into membrane interior is followed by the increase of relative intensity of pyrene vibronic bands and decrease of prodan general polarization value suggesting an enhancement of bilayer polarity. this implies that hb incorporation into membrane interior decreases packing density of lipid molecules, promoting water penetration into membrane core. chol condensing effect on lipid bilayer prevents protein embedment into bilayer, thus decreasing membrane hydration changes as compared to pc bilayers. in the presence of anionic lipid pg hb-induced increase of bilayer polarity was found to be most pronounced, pointing to the modulatory role of membrane composition in hb bilayer-modifying propensity. we present optimized sialic acid-based mimics binding in the low nanomolar range. molecular interactions were determined with surface plasmon resonance (spr), characterizing the affinity and the kinetics of binding. furthermore, isothermal titration calorimetry (itc) was applied to dissect the standard free energy of binding (dg°) into the standard enthalpy of binding (dh°) and the standard entropy of binding (ds°). in order to pass the cell membranes, most of these medicines has to be administrated to patients as nucleoside pro-drugs and not directly as triphosphorylated forms. because of the poor phosphorylation of the nucleoside analogues used in therapy, it is important to understand and to optimize their metabolism. our aim is to understand how compounds of chirality l turn away -phosphoglycerate kinase (pgk) from its normal glycolytic function to be converted into the triphosphate forms. in order to elucidate pgk mechanism and substrate specificity, we have measured the kinetics of the different steps of the enzymatic pathways by rapid mixing techniques and studied the influence of the nature of the nucleotide substrate thereon. we first performed an extensive study with d-and l-adp (see poster by p. lallemand). we are now extending the studies to other nucleotide diphosphates (some of them used in therapies). changes in the nature of the nucleobase or deletion of hydroxyl group of the sugar affect the efficiency of phosphorylation by pgk, either by decreasing dramatically their affinity or by altering the phospho-transfer step itself. structural explanations are given based on docking data. probing drug/lipid interactions at the molecular level represents an important challenge in pharmaceutical research, drug discovery and membrane biophysics. previous studies showed that enrofloxacin metalloantibiotic has potential as an antimicrobial agent candidate, since it exhibits antimicrobial effect comparable to that of free enrofloxacin but a different translocation route. these differences in uptake mechanism can be paramount in counteracting bacterial resistance. in view of lipids role in bacterial drug uptake, the interaction of these compounds with different e. coli model membranes were studied by fluorescence spectroscopy. partition coefficients determined showed that lipid/antibiotic interactions were sensitive to liposomes composition and that the metalloantibiotic had a higher partition than free enrofloxacin. these results corroborate the different mechanism of entry proposed and can be rationalized on the basis that an electrostatic interaction between the metalloantibiotic positively charged species, present at physiological ph, and the lipids negatively charged head groups clearly promotes the lipid/antimicrobial association. oligomerization and fibril assembly of amyloid b peptide amyloid b peptide (ab) forms a large amount of extracellular deposits in the brain of alzheimer's disease (ad) patients and it is believed that this peptide is related to the pathogenesis of that disease. the most abundant monomeric form of physiological ab (* %) is constituted by amino acids and is benign, but by an unknown mechanism this endogenous material becomes aggregated and neurotoxic. increasing evidence suggests that membrane interaction plays an important role in ab neurotoxicity. in this work it will be studied the interactions of ab( - ) with ctac (cationic), sds (anionic), pfoa (anionic with fluorine atoms) and og (nonionic) amphiphiles in monomeric and micellar forms. the results demonstrated that ab( - ) forms fibrils with different morphologies in the presence of micelles. in addition, the presence of micelles accelerates the formation of fibrils and decreases the lifetime of oligomers. we present here the exploitation of the powerful approach of surface plasmon resonance imaging and mass spectrometry coupling for protein fishing in biological fluids such as human plasma at the same sensitivity. on one hand, multiplex format spri analysis allows direct visualization and thermodynamic analysis of molecular avidity, and is advantageously used for ligand-fishing of captured bio-molecules on multiple immobilized receptors on a spri-biochip surface. on the other hand, maldi mass spectrometry is a powerful tool for identification and characterization of molecules captured on specific surface. therefore, the combination of spri and ms into one concerted procedure, using a unique dedicated surface, is of a great interest for functional and structural analysis at low femtomole level of bound molecules. to reach these goals, particular surface engineering has been engaged to maintain a high level of antibody grafting and reduce non-specific adsorption. thus, various chemistries have been tested and validated towards biological fluids such plasma, keeping in mind the capacity of the in situ investigation by ms. finally, signal to noise ratio was magnified leading to the characterization of protein lag , a potential marker of breast cancer, in human plasma. atenolol incorporation into pnipa nanoparticles investigated by isothermal titration calorimetry mihaela mic, ioan turcu, izabell craciunescu, rodica turcu, national institute for r&d of isotopic and molecular technologies, cluj-napoca, romania e-mail: mihaela.mic@itim-cj.ro poly(n-isopropylacrylamide) (pnipa) is a thermo-sensitive hydrogel undergoing a volume phase transition at about of °c close to the body temperature. this volume phase transition is envisaged as a key property for drug binding and release. the purpose of our research is the thermodynamic characterization of the binding of atenolol by pnipa polymeric nanoparticles. the thermodynamic parameters which characterize the binding process are obtained using the isothermal titration calorimetry (itc) as the main investigation technique. when polymeric nanoparticles bind drug molecules, heat is either generated or absorbed depending on the amount of bond molecules and also on the exothermic or endothermic character of the binding process. the heat measurement allows the determination of binding constants, reaction stoichiometry and the thermodynamic profile of the interaction. itc technique has been used to investigate the binding properties of nanoparticles which shrink from the swollen to the collapsed state. the capacity of such nanogels to bind atenolol molecules is directly related to relevant differences between the binding properties in the swollen and in the collapsed state respectively. aggregation study of x-(alkyldimethylamonium)alkylaldonamide bromides p. misiak , b. ró _ zycka-roszak , e. woźniak , r. skrzela , k.a. wilk department physics and biophysics, wrocław university of environmental and life sciences, wrocław, poland, department of chemistry, wrocław university of technology, wrocław, poland sugar-based surfactants are of considerable research interest because they have improved surface and performance properties, reduced environmental impact, and have potential pharmaceutical and biomedical applications. x-(alkyldimethylamonium)alkylaldonamide bromides (c n gab) with different chain lenghs (n = , , , ) belonging to cationic sugar-based surfactants were newly synthesised. the aggregation processes of c n gabs were studied by means of isothermal titration calorimetry (itc), electric conductance method and molecular modelling methods. the critical micelle concentrations (cmc), the degree of micelle ionization (b), the enthalpies (dh m ) and the entropies (ds m ) of micellization as well as the contributions of the headgroups to the gibbs free energies (dg m (hy)) were calculated. the obtained values were compared with those for dodecyldimethylethylammonium bromide and literature data for analogical glucocationic surfactants. the latest compounds differ from c n gab surfactants by substitution of sugar chain by gluco ring. molecular modelling methods were used to relate the molecular properties of the compounds with their experimentally studied properties in solution. this work was supported by grant n n . every year over million people are infected with dengue virus (denv), transmitted by a mosquito (aedes aegypti). this enveloped virus, member of the flaviviridae family, has four distinct serotypes. it has a single stranded positive rna molecule with a single open reading frame that encodes a single poliprotein, which, after appropriate processing by viral and host proteases, gives rise to three structural proteins (c, prm and e) and seven non-structural proteins (ns , ns a, ns b, ns , ns a, ns b and ns ) [ ] . the surface of the immature virion is composed of e and prm heterodimers that are arranged as trimers protruding from the membrane [ ] . the virus is thought to enter the host cell via a receptor-mediated endocytosis, although, if any, the specific dengue receptors have not been described. once inside the cell, the acidified environment inside the endocytic vesicle triggers an irreversible trimerization of the envelope (e) protein, inducing the release of the nucleocapsid (composed of viral rna and multiple copies of c protein) to the cytoplasm, thus starting the infection process, where the poliprotein is translated and processed, originating all viral proteins. considering the structural proteins c and e, these are essential for the viral infection, specifically, protein c is thought to be involved in the viral assembly and specific encapsidation of the genome and protein e (a class ii fusion protein) plays a major role in the fusion process. as recently described by some studies [ ] , protein c is composed of four a helices connected by four short loops and has a highly hydrophobic region forming a concave groove that could interact with lipid membranes and a region with an increased concentration of positive charges, possibly interacting with the viral rna. as for protein e, it is composed of three b stranded domains. it is proposed that the fusion loop is located in domain ii of this protein and the putative receptor binding sites, considered essential for the viral entry, are supposedly located in domain iii. in this work, we describe the identification of the membrane active regions of both these proteins, considering both theoretical studies, hydrophobic moments, hydrophobicity and interfaciality values as well as experimental ones, namely fluorescence spectroscopy, where a fluorescent probe is encapsulated in model membrane systems, and differential scanning calorimetry [ ] . we have found one region in protein c and four regions in protein e with membranotropic activity. this is the first work describing experimentally the putative membrane interacting zones of both these proteins. this work was funded by grant bfu - -bm from ministerio de ciencia y tecnologia, spain, granted to jose villalaín. investigation of membrane-membrane interaction mediated by coiled coil lipopeptides gesa pä hler, andreas janshoff georg-august-university, tammannstrasse , gö ttingen, germany e-mail: gpaehle@gwdg.de specific cellular membrane interaction and fusion are crucial points in vivo which are in eukaryotic cells mediated by snare proteins. the definite mechanism behind these processes is still poorly understood, but the coiled coil formation of a snare core complex consisting of four a-helices seems to generate a fusogenic driving force. this offers the possibility to design a straightforward experimental setup to mimic the complex protein-mediated membrane-membrane interaction by using mere protein fragments or peptides attached to artificial lipid bilayers which self-assemble to a coiled coil structure. in our approach, two artificial three heptad repeat coiled coil forming peptides were synthesized and attached to maleimide functionalized membranes via an in situ-coupling reaction. thus, secondary structure changes, kinetic characteristics and binding energetics were monitored during coiled coil formation with real time ellipsometry, ir and cd spectroscopy. the lipopeptide mediated membrane-membrane interaction itself is investigated by colloidal probe spectroscopy and tirfm. these techniques and the setup of our model system allow screening the energetic and structural properties of variable coiled coil forming peptides, i.e. linker-modified or biologically inspired sequences. enzymatic reactions in nanostructured surfaces: unzipping and cutting the double helix pietro parisse , matteo castronovo , bojana lucic , alessandro vindigni , giacinto scoles and loredana casalis sincrotrone trieste s.c.p.a., trieste, italy, temple university, philadelphia, usa, protein-dna interactions are vital for living organisms. from viruses to plants and humans, the interactions of these two different classes of biopolymers control processes as important and diverse as the expression of genes and the replication, rearrangement, and repair of dna itself. to understand these processes at the molecular level, and to follow changes in cellular pathways due to different kinds of perturbations and/or diseases it is necessary the identification and quantification of proteins and their complex network of interactions. we have exploited the high spatial resolution given by atomic force microscopy to generate dna arrays of variable density by means of nanografting. on such nanostructures, we investigate the mechanism of different enzymatic reactions (from restriction enzymes to helicases). registering with high precision the height variation due to the action of the enzyme onto the engineered dna sequences (in the case of restriction enzymes) or taking advantage of the different mechanical properties of single and double stranded dna (in the case of helicases, where for the first time kinetic data were obtained on recq human helicase), we were able to monitor either the activity and/or the action mechanisms of these two important classes of enzymes. in this study an attempt has been made to investigate the structure, dynamics and stability of cyclic peptide nanotubes (cpnts) formed by the self-assembly of cyclic peptides (cps) using classical molecular dynamics (md) simulation and semiempirical quantum chemistry calculation employing pm . the structure and energetics of monomer and various oligomeric cpnts have been investigated by considering the (cyclo-[(d-ala-l-ala) ]) peptide as the model for cp. various geometrical parameters extracted from the md simulation reveal that the terminal residues are loosely hydrogen bonded to the inner subunits regardless of degree of oligomerization. the hydrogen bonds present in the inner core regions are stronger than the terminal residues. as the degree of oligomerization increases, the stability of the tube increases due to the hydrogen bonding and stacking interactions between the subunits. the results show that the binding free energy increases with the extent of oligomerization and reaches saturation beyond cpnt . in addition, hydrophobic and electrostatic interactions play crucial roles in the formation of cpnts. analysis of both structural and energetics of formation of cpnts unveils that the selfassembly of dimer, trimer and tetramer cpnts are the essential steps in the growth of cnpts. monolayers on a langmuir trough constitute a great biomimetic model to characterize protein-protein or protein-lipid interaction, where the physical state of the interfacial layer is completely controlled. we present here three studies performed on monolayers, with a wide panel of experimental (optical, spectroscopical, rheological) techniques. i) surface properties and conformation of nephila clavipes spider recombinant silk proteins (maspi and masp ) was studied at the air-water interface: we show that the mechanism of assembly of both proteins is different, although both proteins share the same sequence pattern and a close hydrophobicity. they both exhibit a certain propensity to form b-sheets that may be important for the efficiency of the natural spinning process. ii) the dystrophin molecular organization and its anchoring in a lipidic environment depend on the rod fragment used and on the lipid nature. moreover the interaction is guided by the lateral surface pressure. this lipid packing variation is essential to understand the role of the dystrophin during compression-extension cycle of the muscle membrane. iii) we evidence that non additive behavior of mixtures of food globular proteins leads to enhanced foaming properties or to self assembled objects. nucleolar-targeting peptides (nrtps) were designed by structural dissection of crotamine, a toxin from the venom of a south-american rattlesnake. at lm concentration, nrtps penetrate different cell types and exhibit exquisite nucleolar localization. the aim of this work was to pursue with the study of nrtps molecular mechanism for translocation, as well as to determine the ability of nrtp to delivery large molecules into cells. for the translocation experiments, rhodamine b-labeled nrtps were used and tested with giant multilamellar vesicles. confocal microscopy results show that there is an efficient translocation across model membranes. high levels of intracellular peptide were also seen in different cell lines and pbmc, soon after incubation with nrtp. furthermore, a conjugate of nrtp (nrtp c) bound to b-galactosidase was prepared by chemical synthesis and tested in hela cells. this conjugate maintains enzymatic activity and is stable at °c for several days. the work done so far with this new family of cell-penetrating peptides revealed strong interaction and translocation with lipid model systems. moreover, successfully cellular delivery of bgalactosidase was observed and quantified. interaction of zinc phthalocyanine with ionic and non ionic surfactants: uv-vis absorption and fluorescence spectroscopy for application in photodynamic therapy m. p. romero, s. r. w. louro physic department, pontifícia universidade cató lica de rio de janeiro puc-rio, brazil among the second-generation photosensitizer (ps) developed for the treatment of neoplastic diseases by photodynamic therapy (pdt), metallo-phthalocyanines (mpc) have been proposed as an alternative to the currently used ps in clinical application. unsubstituted mpc are not soluble in physiological solvents and their in vivo administration relies upon their incorporation into carriers or their chemical conversion into water-soluble dyes by the attachment of selected substituents. in this work, uv-vis absorption and fluorescence spectroscopy were used to study the ability of different micelles for dispersing zinc phthalocyanine (znpc). the following surfactants were tested: sds, ctab, hps, tween , tween , and pluronic f . znpc has low solubility in virtually all solvents, but dmf and dmso are observed to dissolve znpc in concentrations of the order of . and . mmol/l, respectively. stock solutions of znpc in dmf and dmso were prepared. micelles of the different surfactants containing znpc were prepared by dissolving in aqueous medium (milli-q water or phosphate buffer ph . ) small amounts of the stock solutions previously mixed with each surfactant. the amounts of each surfactant were calculated to give an average ratio of one znpc molecule per micelle in the final solution. the absorption and fluorescence spectra of znpc in the micellar systems were obtained, and were observed to change in time. immediately after dissolution the spectra are characteristic of monomeric znpc, suggesting formation znpccontaining nanoemulsions with the mixture of znpc-organic solvent in the hydrophobic region of the micelle. since dmso and dmf are miscible with water, the solvent diffuses out of the micelle and znpc stays inside the micelle in a monomeric or aggregated form. the different surfactants lead to different time evolution of znpc aggregation. aggregation lifetimes vary from one hour, in the case of pluronic f , to more than twelve hours, in the case of ctab and hps. it was observed that the ionic surfactants were more efficient than non ionic ones for monomeric delivery of znpc . work partially supported by cnpq, inami and faperj. nucleobase-containing peptides are an important class of molecules comprising both artificial (synthetic nucleopeptides) and natural (peptidyl nucleosides and willardiine-containing peptides) compounds characterized in many cases by interesting biological properties. , in this work, we report a spectroscopic study on the properties of a chiral nucleobase-bearing peptide obtained by chemical synthesis starting from commercial sources. the findings of this research strongly encourage further efforts in the field of the use of nucleopeptides as supramolecular assembling systems and open the way to novel drug delivery approaches based on nucleobase recognition. conformational plasticity. their structure depends tremendously on their local environment and confinment, and may accommodate several unrelated conformations, that are a strong challenge for the traditional characterizations of structure, supramolecular assembly and biorecognition phenomena. atomic force microscopy (afm) has been successfully exploited for both highly controllable nanolithography of biomolecules and for biorecognition studies, such as oriented prion protein -antibody interaction (sanavio et al., acs nano ( ) ( ): , bano et al. nano lett ( ) ( ): - ). here, we report different strategies for selective, oriented confinement of alphasynuclein at the nanoscale for sensitive and accurate direct detection, via precise topographic measurements on ultraflat surfaces, of biomolecular interactions in confined assemblies. a new class of cell penetrating peptides (cpps) was generated by splicing the ( - ) and ( - ) segments of crotamine, a toxin from crotalus durissus terrificus venom [ ] . as they localize preferably on the nucleolus, these novel cpps were named nucleolar-targeting peptides (nrtps). the extent of nrtp partition to zwitterionic (popc; popc:cholesterol : ) and anionic (popg; popc:popg : ) lipid vesicles was studied following the intrinsic tyr or trp fluorescence of the peptides. the partition curves into popc zwitterionic vesicles were characterized by downward slopes and higher partition coefficients (k p * - ). for pure popg, an upward curve and smaller partition coefficient point out for a different type of membrane-peptide interaction. popc:popg membranes present characteristics of both types of interaction. from red edge excitation shift and quenching experiments similar conclusions were attained. leakage assays ruled out lipid vesicle disruption by crotamine or nrtps. further studies on nrtp cellular translocation mechanism and large molecule delivery are currently in progress. dystrophin is essential to skeletal muscle function and confers resistance to the sarcolemma by interacting with cytoskeleton and membrane. we characterized the behaviour of dys r - , a five spectrin-like repeats from the central domain of human dystrophin, in the presence of liposomes and monolayers as membrane models. interaction of dys r - depends on the lipid nature, anionic or zwitterionic, with suvs, and on the lipid packing when comparing luvs to suvs. lateral pressure of lipid monolayers modifies the protein organization and leads dys r - to form a regular network as revealed by afm. trypsin proteolysis assays show that the protein conformation is modified following its binding to monolayer and suvs. label free quantification by nano-lc/ms-ms allowed identifying the helical amino acid sequences in repeats and that are involved in the interaction with anionic suvs. results indicate that dys r - constitutes a part of dystrophin that interacts with anionic as well as zwitterionic lipids and adapts its interaction and organization depending on lipid-packing and lipid nature. we provide here strong experimental evidence for a physiological role of the central domain of dystrophin on sarcolemma scaffolding through modulation of lipid-protein interactions. extracellular matrix proteins. overexpression of the mmps has been associated with a variety of diseases ranging from periodontal disease and arthritis to tumor invasion and metastasis. the majority of the more powerful synthetic inhibitors of mmps incorporate a hydroxamate group, but exhibit low selectivity and are toxic. in a recent modeling study, astaxanthin (ast), a carotenoid with potent antioxidant property, has been shown to be a potential inhibitor of mmp- function by occupying a binding site near the active center of the enzyme (bika di et al. ). in our ongoing project, we investigate the binding of ast to the catalytic domain of mmps using biochemical and ultimately crystallization to validate the proposed action of ast. along these lines, the catalytic domain of mmp- (cdmmp- ) was expressed in e.coli bl (de ) codon-plus and refolded using a novel effective refolding method. our results reveal that ast has a potent inhibitory effect on cdmmp- activity, however, determination of ic % or k i is difficult due to fast oxidation and structural instability of ast. ongoing work aims at optimizing the inhibition conditions and improving the refolding yield to allow analyzing structure and function of the ast-bound mmp- in more detail. hyaluronic acid (hyaluronan, ha) is a linear polysaccharide with a molar mass in the range of to da and is built from alternating units of glucuronic acid and n-acetylglucosamine. synthesized in the cellular plasma membrane, it is a network-forming and space-filling component in the extracellular matrix of animal tissues. here, we create hyaluronic acid films atop a porous alumina substrate, where they act as a barrier for macromolecular transport depending on their length and geometry. the geometry of the hyaluronic acid switches between a fully stretched and a mushroomlike state and is dependent on the concentration of hyaluronic acid. to bind hyaluronic acid selectively atop the nanoporous anodic aluminum oxide (aao), the aao is orthogonally functionalized by silane chemistry. by means of time resolved optical waveguide spectroscopy (ows) the transport of macromolecules, e.g. avidin, across the hyaluronic acid barriers can be recorded as a function of the pore diameter and hyaluronic acid concentration in a time resolved and label free manner. confocal laser scanning microscopy (clsm) provides an alternative method to investigate the orthogonal functionalization of the pores and to elucidate whether a molecule can cross the barrier at the pore entrance. we functionalized gold surfaces with a hydroxy-terminated self-assembled thiol monolayer exposing an adjustable fraction of biotin moieties. [ ] by in-situ acetylation or fluorination, surface properties could be fine-tuned to different protein immobilization scenarios. using streptavidin as a linker protein, immobilization of human abcc [ ] in liposomal and planar bilayer systems was possible. abcc -containing proteoliposomes doped with a biotinylated anchor lipid were successfully tethered to our streptavidin-coated surfaces. biotinylation of the extracellular glycosylation of abcc allowed direct immobilization with inside-up orientation and subsequent assembly of a lipid bilayer. outside-up orientation was achieved by exploiting the c-terminal histidin tag of recombinant abcc for immobilization via ni + and biotinnitrilotriacetate. all systems were thoroughly characterized by quartz crystal microbalance, atomic force microscopy and surface plasmon resonance techniques with respect to monitoring abcc mediated substrate transport in real time. because of its role in the apoptotic pathway, conformational transitions of cytochrome c (cyt c) have gain on interest. in native cyt c, met and his residues serve as heme axial ligands. cyt c interaction with the membrane causes the disruption of the iron-met bond. this allows the binding of others endogenous ligands forming alternative low spin species , or induces peroxidase activity through the formation of a five coordinated high spin iron specie. acquisition of this peroxydase activity by cyt c has been shown to be a key stage before its release from the mitochondria . in order to study these non native low spin species by checking the possible amino acids able to bind the human cyt c heme, different mutants have been designed and produced: h q, h n, and the double one h q/h n. sds in countries where seafood is an integrate part of the diet, fish are among the most common food allergen sources. the major fish allergen parvalbumins are abundant in the white muscle of many fish species. parvalbumin belongs to the family of ef-hand proteins and has a globular shape containing six helical parts. high pressure is known to unfold proteins. we performed high pressure ftir experiments, to explore the p-t phase diagram of cod parvalbumin, gad m , to test the possibility of its inactivation by high pressure treatment. the infrared spectrum of parvalbumin is characteristic for the helical conformation, in agreement with the crystal structure. a marked transition in the structure of the parvalbumin was observed with the central point of . gpa (at room temperature). the amide i position shifts to a wavenumber which is between the helical and the unfolded position. we assign this change to a native-molten globule transition. it was reversible as seen from the infrared spectra. it has been proven in the past that reflectometric interference spectroscopy (rifs) is a powerful measurement system for the detection of protein-protein interactions . we present here the development of a reflectometric sensor which allows for the detection of membrane-protein interactions in the micromolar regime. in this study we employ two different instrumental assemblies. the first installation enables direct detection and quantification of the interaction of membrane proteins with solid supported lipid bilayers. in the second assembly the original instrument is combined with an upright fluorescence microscope. the advantages of this installation are the direct optical control of the experiment as well as a smaller sensing area. the set-up allows for the detection of interactions on lipid-patches of just several micrometers in diameter. the aim of this work is an experimental system that enables the measurement of transport processes through lipid membranes. we attempt to achieve this by covering a closed porous substrate with a lipid membrane. the first steps to reach this goal were done by spanning membranes over anodized aluminum oxide substrates. initiation of actin polimerization in cells requires nucleation factors. a pointed-end-binding protein of f-actin -the lei-omodin -acts as a strong filament nucleator in muscle cells. the dynamical, structural and kinetic properties of a protein can provide important information to understand the intramolecular events underlying its function. we are interested in how does the leiomodin regulate the actin polimerization. our aim is to determine the dissociation constant of the actin-leiomodin complex, and study a possible side-binding effect of the leiomodin . the cardiac leiomodin of rattus norvegicus is a kda molecular weight protein, which contains a kda n-terminal, a kda leucin reach repeat (lrr) and a kda c-terminal region. the n-term and the lrr regions are together tropomodulin homologues. we expressed the wild type the n-term+lrr the lrr+c-term and the c-term protein fragments by using a ptyb vector that contains an amplicillin resistance gene. the expression of the proteins was carried out with the twin-cn (ne bio-labs) kit, which is a chitin-intein self-cleavage and purification system. the nucleation activity of leiomodin and the polimerization speed of actin in the presence of tropomyosin and leiomodin were studied by using pyrene-actin polimerization assay. we measured the stoichiometric, conformational and kinetic properties of the leiomodin-actin complexes with co-sedimentation assay, fluorescence spectroscopic and rapid-kinetic methods. the results showed that the rate of actin polimerization depended on the leiomodin concentration. the nucleator activity of leiomo-din was ionic strength dependent. the data also confirmed that leiomodin is a side-binding and pointed-end binding protein of f-actin. the binding of leiomodin to the sides of the actin filaments was slower than to the pointed-end of the f-actin. the structure of f-actin was changed by the sidebound leiomodin . these observations will contribute to the better understanding of the development and function of thin filaments in cardiac and other muscle tissues. leukemias are one of the most common malignancy worldwide. there is a substantial need for new chemotherapeutic drugs effective against these diseases. doxorubicin (dox), used for treatment of leukemias and solid tumors is poorly efficacious when administered systemically at conventional doses. therefore, in our study, to overcome these limitations, we used transferrin (trf) as a drug carrier. we compared the effect of dox and doxorubicin-transferrin conjugate (dox-trf) on human leukemic lymphoblasts (ccrf-cem). the in vitro growth-inhibition test, xtt assay, indicated that dox-trf was more cytotoxic for leukemia cells than dox alone. in our researches we also evaluated the alternations of mitochondrial transmembrane potential (dw m) , and production of reactive oxygen species (ros). we monitored the dw m using dye jc- ( , ', , '-tetrachloro- , ', , '-tetraethylbenzimidazolcarbocyanine) . the level of ros was studied using the fluorescent probe dcfh -da ( ', 'dichlorodihydrofluorescein diacetate). the results demonstrate that dox-trf induced, decrease of mitochondrial membrane potential and significantly higher production of ros compared with dox treated cells. moreover, all these results seem to be correlated with dna fragmentation analyzed by dna ladder. the tested processes were partially inhibited by the antioxidant, n -acetylocysteine (nac). the changes induced by dox-trf conjugate and free drug were suggest the different mechanism of action of dox alone and conjugated with transferrin. time-resolved detection of protein-protein interaction masahide terazima kyoto university, kyoto, - , japan revealing molecular mechanism of a protein reaction has been a central issue in biophysics. for that purpose, a variety of time-resolved spectroscopic methods have been developed. however, most of them can monitor only dynamics associated with an optical transition and it has been very difficult to trace processes without optical transition. we used the pulsed laser induced transient grating (tg) method to study spectrally silent reactions of various proteins in time-domain. here we will show studies on pixd. pixd is a kda short protein which consists of the bluf domain and additional short helices, and is involved in phototactic movement. the photochemical reaction studied by absorption spectroscopy revealed that this protein exhibits typical photochemistry of the bluf proteins. the red-shifted intermediate is generated within a ps. the spectrum does not change after this initial reaction, and returns back to the dark state with a time constant of s at room temperature. we studied the reaction of this protein by our method and found that the proteinprotein interaction is drastically changed during the reaction. the details and the biological meaning will be presented. human ileal bile acid-binding protein (i-babp) plays a key role in the enterohepatic circulation of bile salts. previously we have shown that the protein has two binding sites and triand dihydroxy bile salts bind with strong and moderate positive cooperativity, respectively. positive cooperativity is thought to be related to a slow conformational change in the protein. our current study is directed at the structural and dynamic aspects of molecular recognition in human i-babp using nmr spectroscopy and other biophysical techniques. as a first step in the investigation, n relaxation nmr experiments have been employed to characterize the backbone motion in the apo and holo protein on a wide range of timescales. our results show a moderately decreased ps-ns flexibility in the ligated protein, with most significant ordering near the portal region. in addition, the measurements indicate a slow ls-ms fluctuation at four distinct segments in the apo protein, a motion not observed in the doubly-ligated form at room temperature. our studies support the hypothesis of an allosteric mechanism of binding cooperativity in human i-babp. to shed more light on the molecular details of the binding mechanism, a site-directed mutagenesis study is in progress. cationic porphyrin-peptide conjugates were recently shown to enhance the delivery of peptide moiety to the close vicinity of nucleic acids but their interaction with dna is not yet studied. we synthesized two cationic porphyrin-peptide conjugates: tetra-peptides were linked to the tri-cationic meso-tri( -n-methylpyridyl)-mono-( -carboxyphenyl)porphyrin and bi-cationic meso- , -bis( -n-methylpyridyl)- , di-( -carboxyphenyl)porphyrin. dna binding of porphyrins, and their peptide conjugates was investigated with comprehensive spectroscopic methods. evidences provided by the decomposition of absorption spectra, fluorescence decay components, fluorescence energy transfer and cd signals reveal that peptide conjugates of di-and tricationic porphyrins bind to dna by two distinct binding modes which can be identified as intercalation and external binding. the peptide moiety does not oppose the interaction between the dna and cationic porphyrins. we compared the effect of complexation on structural stability of dna and nucleoprotein complex : hela nucleosomes and t phage. uv and cd melting studies revealed that the porphyrin binding increases the melting temperature of dna and destabilizes the dna protein interaction in the nucleosomes but not in the t phage. the wide nucleotide specificity of -phosphoglycerate kinase (pgk) allows its contribution to the effective phosphorylation (activation) of nucleotide-based pro-drugs against hiv. here the structural basis of the nucleotide-pgk interaction is characterised in comparison to other kinases, namely pyruvate kinase (pk) and creatine kinase (ck) by enzyme kinetic and structural modelling studies. the results evidenced favouring the purine vs. pyrimidine base containing nucleotides for pgk rather than for pk or ck. this is due to the exceptional ability of pgk in forming the hydrophobic contacts of the nucleotide rings that assures the appropriate positioning of the connected phosphate chain for catalysis. the unnatural l-configurations of the nucleotides (both purine and pyrimidine) are better accepted by pgk than either by pk or ck. further, for the l-forms the absence of the ribose oh-groups with pgk is better tolerated for the nucleotides with purine rather than pyrimidine base. on the other hand, positioning the phosphate chain of both purines and pyrimidines with l-configuration is even more important for pgk, as deduced from the kinetic studies with various nucleotide-site mutants. these characteristics of the kinase-nucleotide interactions can provide a guideline in drug-design. the role of the enzyme types atp-ases in the muscle contraction g. vincze-tiszay , j. vincze , e. balogh hheif, budapest, hungary, nové zá mky, slovakia the myofibrilla assuring muscle contraction gains energy to the slipping in mechanisms and the degree of efficiency of this process will decisively be determined by the velocity of recombination of the atp molecule. in this there play a particular part the na + -k + -atp-ase and mg ++ -atp-ase enzymes. chemical reactions taking place in the living organism are catalyzed by enzymes, so the recombination from adp to atp, too. this transport process can be modelled from the energetic point of view on the basis of the general transport theorem through the following formula: grad a x dx dt: from the point of view of muscle contraction it is of interest that, dependent from the type of the motions whether the length of time is very short, some seconds, or we can speak about a long lasting process. in the first case one can compare the decomposition of the atp with the avalanche effect while in the spot. its degree of efficiency is determined by the migration and linkage velocity of the ions. conclusion: the degree of efficiency of the muscle contraction is determined by the quantities of the two enzymes (na + -k + -atp-ase and mg ++ -atp-ase) as related to each other. [ ] . experiments with deletion mutants have shown that the aminoterminal domain contains a beta sheet with an ordered array of acidic residues, which mediates the attachment to basic calcium phosphates [ , ] . the inhibition is based on the formation of nanometer-sized, spherical mineral-fetuin-a colloids, denoted as calciprotein particles (cpps) [ , ] . the initially formed cpps show hydrodynamic radii in the range of nm and are only transiently stable. after a distinct lag time, they are subject to a morphological change towards larger prolate ellipsoids with hydrodynamic radii of - nm [ ] . in this context, we studied the role of fetuin-a in the formation and ripening of cpps. on the one hand, dynamic light scattering (dls) was used to study the influence of temperature, fetuin-a concentration and mineral ion supersaturation on the kinetics of cpp ripening [ ] . on the other hand, the protein fetuin-a was investigated by means of small angle x-ray scattering (saxs) and fluorescence correlation spectroscopy (fcs degradation of the mrna cap (mrna ' end) by dcps (decapping scavenger) enzyme is an important process of the gene expression regulation, but little is known about its mechanism. the biological role of dcps and its potential therapeutic applications, e.g. as a novel therapeutic target for spinal muscular atrophy, make it an interesting object for biophysical investigations. the ability of dcps to act on various short capped mrnas will be presented. we have examined the substrate specificity and binding affinity of the enzyme in a quantitative manner, employing experimental physics' resources, such as atomic force microscopy (afm) and fluorescence spectroscopy for enzyme kinetics and timesynchronized-titration method (tst ) . in this study we extended the application of mqd-ihc to investigate potential biomarkers associated with prostate cancer (pca) invasiveness and lethal progression. objectives: to establish a mqd-ihc protocol using qd light-emitting nanoparticles ) to detect the expression/activation of critical cell signaling proteins at the single cell level; ) to image the plasticity and lethal progression of human pca with specific emphasis on emt and c-met signaling; and ) to examine the utility of mqd-ihc in clinical pca specimens to determine its invasion ability and predict its metastatic capability. results: we analyzed the co-expression and activation of osteomimicry associated biomarkers: b -microlgobulin (b -m), phosphorylated cyclic amp responsive element binding protein (pcreb) and androgen receptor (ar) in , cells from localized pca tissue areas (gleason and ) of patients with known metastatic status. the overall median % triple positive for b -m + /pcreb + /ar + cells was . %. the median triple positive for the samples with metastatic potential was % compared with those without metastatic potential (median = %); p = . by a wilcoxon rank sum test. the results were confirmed in pca bone metastatic specimens. we also investigated the c-met signaling in castration-resistant human pca model or crpc xenografts and the clinical pca specimens and found that the downstream signal components including pakt and mcl- were activated. conclusion: to validate our findings, additional clinical specimens with confirmed survival data will be analyzed and the cell-signaling-network-based mqd-ihc will be automated by vectra image analysis system in a high throughput manner with the hope to predict the lethal progression of pca prior to clinical manifestation of distant metastases. protein ligand binding is an important field of biopharmaceutical research. there are many techniques for quantitative determination of the ligand binding. the combination of isothermal titration calorimetry (itc) and thermal shift assay provides a robust estimate of the binding constant. many binding reactions are coupled to the absorption or release of protons by the protein or the ligand, conformational changes of the protein and other processes. to correlate the structural features of binding with the energetics of binding one needs to carry out a detailed thermodynamic study of the binding reaction and to determine dependencies such as ph, buffer and temperature. here we present a detailed thermodynamic description of radicicol binding to human heat shock protein hsp and determined proton linkage contributions to observed binding thermodynamics. we calculated the pk a of the group responsible for proton linkage, the protonation enthalpy of this group and intrinsic thermodynamic parameters for radicicol binding. the intrinsic enthalpy of radicicol binding to hsp is one of the largest enthalpies observed for any protein -ligand binding. the structural features responsible for such large binding enthalpy and very favorable intrinsic binding gibbs free energy are discussed. neuronal systems and modelling o- optogenetic electrophysiology walther akemann, amelie perron, hiroki mutoh, and thomas knö pfel laboratory for neuronal circuit dynamics, riken brain science institute, japan the combination of optical imaging methods with targeted expression of protein-based fluorescent probes enables the functional analysis of selected cell populations within intact neuronal circuitries. we previously demonstrated optogenetic monitoring of electrical activity in isolated cells, brain slices and living animals using voltage-sensitive fluorescent proteins (vsfps) generated by fusing fluorescent proteins with a membrane-integrated voltage sensor domain. however, several properties of these voltage reporters remained suboptimal, limiting the spatiotemporal resolution of vsfpbased voltage imaging. a major limitation of vsfps had been a reduced signal-to-noise ratio arising from intracellular aggregation and poor membrane targeting upon long-term expression in vivo. to address this limitation, we generated a series of enhanced genetically-encoded sensors for membrane voltage (named vsfp-butterflies) based on a novel molecular design that combines the advantageous features of vsfp s and vsfp s with molecular trafficking strategies. the new sensors exhibit faster response kinetics at subthreshold membrane potentials and enhanced localization to neuronal plasma membranes after long-term expression in vivo, enabling the optical recording of action potentials from individual neurons in single sweeps. vsfp-butterflies provide optical readouts of population activity such as sensoryevoked responses and neocortical slow-wave oscillations with signal amplitudes exceeding % dr/r in anesthetized mice. vsfp-butterflies will empower optogenetic electrophysiology by enabling new type of experiments bridging cellular and systems neuroscience and illuminating the function of neural circuits across multiple scales. opsin molecules are a burgeoning new tool for temporally precise neuronal stimulation or inhibition. opsin properties are commonly characterized in cell culture or acute brain slice preparations using whole cell patch clamp techniques, where neuronal membrane voltage is fixed at the resting potential. however, in vivo, where neurons are firing action potentials, opsins are exposed to large fluctuations in membrane voltage and transmembrane ionic concentrations which can influence opsin function. in the case of implanted light delivery devices, stimulation light power varies as a function of brain tissue volume. we therefore investigated the stability of opsin properties across a variety of in vivo-like stimulation conditions. we find that off-kinetics of excitatory opsins vary significantly with holding membrane potential; channelrhodopsin (chr ) slowing with depolarisation and chief (chr /chr hybrid) in contrast, accelerating. new chr point mutation variants demonstrate stability across all membrane potentials. we additionally explore responses to initial and subsequent light pulses and find that chief has the unique property of accelerating kinetics after the first light stimulation. inhibitory opsins vary in sensitivity to light in a manner which correlates with their off-kinetics. slower opsins, such as mac (leptosphaeria maculans), have higher sensitivity at low light power densities, saturating early relative to fast inhibitory opsins such as arch (archaeorhodopsin) and nphr (halorhodopsin). we discuss the relative merits of stability versus versatility of opsins under variable stimulation conditions. it has been previously shown that overexpression of ndm ncrna in a sknbe -derived neuroblastoma (nb) cell line leads to cell differentiation, with a decrease of malignant potential. here we use the patch-recording technique to characterize the ionic channel apparatus of nb cells expressing ndm at its basal level (mock cells) or at . fold higher levels (s cells). the two cell lines shared very similar pools of functional k channels, but s cells displayed larger ttx-sensitive na currents and were able to generate action potentials, while mock cells were not. in addition, while mock cells barely express functional gaba receptors, in the majority of s rapid application of gaba elicited a current with a ec = . lm; this current was antagonized by bicuculline ( lm) and potentiated by zaleplon (ec = nm). in mock cells, real time pcr evidenced a high level of gaba a a subunit, while in s cells a significant expression of a and a was detected, whereas a mrna was downregulated by %, confirming the development of functional gaba a receptors. in the same cell lines, the presence of specific markers and the secretion of specific cytokines confirmed that ndm expression leads to a differentiation process toward a neuron-like, rather than glial-like, phenotype. was planned therefore to reconstitute a model of brain tumors in rats by orthotopic implantation of xenogenic transformed human cells. iron is important element used for chemical reaction catalysis and physiological cell functions. the reason of iron deposition is still unknown. under conditions prevailing in human brain it is expected the formation of an amorphous or minute crystalline phase. we used light, scanning (sem) and transmission electron microscopy (tem), energy-dispersive microanalysis, electron diffraction and electron paramagnetic resonance (epr) for investigation of iron deposits in globus pallidus of human brain. sem revealed iron rich particles with na, si, p, s, cl, ca and cu around glial cells. tem revealed bumpy, solid particles of platy and sometimes rounded shape with the size of lm to lm. these ones were identified as hematite. epr measurements showed the presence of fe(iii) and cu(ii), but little amount of fe(ii) can not be excluded. we consider low-temperature process of hematite formation in human globus pallidus in aqueous environment influenced by organic and inorganic factors. chemical processes leading to nanoparticles formation can be associated with neurodegenerations such as alzheimer or parkinson disease. over the past years our understanding of the basic biophysical mechanisms governing the spatio-temporal dynamics of neuronal membrane potentials and synaptic efficacies has significantly expanded and improved. much research has focussed on how ionic currents contribute to the generation and propagation of action potentials, how subthreshold signals propagate along dendritic trees, how the active properties of dendrites shape the integration of incoming signals in a neuron, and how pre-and postsynaptic activities â and potential heterosynaptic effects a determine the way synaptic efficacies change on the short-and long-term. yet, despite these advances, there have been no systematic efforts to relate the basic dynamical repertoire of neurons to the computational challenges neural circuits face, and in particular to explain systematically how the biophysical properties of neurons are adapted to process information efficiently under the constraints of noise and uncertainty in the nervous system. as an initial step in this direction, i will show how various biophysical properties of neurons, in particular short-term synaptic plasticity and dendritic non-linearities, can be seen as adaptations to resolve an important bottleneck in neuronal information processing: the loss of information entailed by the conversion of analogue membrane potentials to digital spike trains. the optogenetic toolbox has greatly expanded since the first demonstration of genetically-targeted optical manipulation of neural activity. in addition to the cation channel channelrhodopsin- (chr ), the panel of excitatory opsins now includes an array of chr variants with mutations in critical residues, in addition to other, related cation channels, and channel hybrids. the inhibitory opsin panel has similarly expanded beyond the first-described halorhodopsin (nphr), a chloride pump, to include trafficking-enhanced versions of nphr as well as the proton pumps mac and arch. while the expansion of available opsins offers researchers an increasingly powerful and diverse selection of tools, it has also made it increasingly difficult to select the optimal tool for a given experiment. one cannot extract a comparison of opsins from the current literature, since studies differ across multiple variables known to contribute to opsin performance (e.g. expression method, light power density, stimulation protocols, etc.). here, we provide the first empirical comparison of both excitatory and inhibitory opsins under standardized conditions. furthermore, we identify the set of parameters that describe the properties of an opsin in a way that is maximally relevant for biological application. o- subcellular compartment-specific distribution of voltage-gated ion channels zoltan nusser institute of experimental medicine, hungarian academy of sciences, budapest, hungary voltage-gated na + (nav) channels are essential for generating the output signal of nerve cells, the action potential (ap). in most nerve cells, aps are initiated in the axon initial segment (ais). in vitro electrophysiological and imaging studies have demonstrated that dendritic nav channels support active backpropagation of aps into the dendrites, but the subunit composition of these channels remained elusive. here, i will present evidence for the somato-dendritic location of nav channels in hippocampal pyramidal cells (pcs). using a highly sensitive electron microscopic immunogold localization technique, we revealed the presence of the nav . subunit in pc proximal and distal dendrites, where their density is -fold lower than that found in aiss. a gradual decrease in nav . density along the proximo-distal axis of the dendritic tree was also detected. we have also investigated the subcellular distribution of kv . voltage-gated k + channel subunit and found a somato-dendritic localization. in contrast to that of nav . channels, the density of kv . first increases then decreases as a function of distance from the somata of pcs. such subcellular compartment-specific distribution of voltage-gated ion channels increases the computational power of nerve cells. keywords: memory, extra cellular matrix, random walk we expose first a biological model of memory based on one hand of the mechanical oscillations of axons during action potential and on the other hand on the changes in the extra cellular matrix composition when a mechanical strain is applied on it. due to these changes, the stiffness of the extra cellular matrix along the most excited neurons will increase close to these neurons due to the growth of astrocytes around them and to the elastoplastic behavior of collagen. this will create preferential paths linked to a memory effect. in a second part, we expose a physical model based on random walk of the action potential on the array composed of dendrites and axons. this last model shows that repetition of the same event leads to long time memory of this event and that paradoxical sleep leads to the linking of different events put into memory. myelinated nerve fibres were studied with fluorescent microscopy and laser interference microscopy. ca + redistribution during prolonged stimulation, changes in morphology and rearrangement of cytoplasmic structures were compared in normal conditions and after membrane modification by lysolecithin and methyl-b-cyclodextrin. lysolecithin is a detergent known to provoke demyelination, and methyl-b-cyclodextrin extracts cholesterol from membranes. cholesterol extraction could lead to disruption of membrane caveolae-like microdomains or ''rafts'' and solubilisation of different proteins connected to them. our data suggest that methyl-b-cyclodextrin and lysolecithin lead to different changes in morphology and distribution of cytoplasmic structures. the effect was different for different regions of the nerve (node of ranvier, paranodal and internodal regions). the agents also altered the kinetics of ca + response to stimulation in myelinated fibres. extracellular carbonic anhydrase contributes to the regulation of ca + homeostasis and salivation in submandibular salivary gland nataliya fedirko, olga kopach, nana, voitenko lviv national university, human and animal physiology, lviv, ukraine the maintenance of ph in the oral cavity is important for the oral heath since even a minor drop in ph can result in dental caries and damage to the teeth. submandibular salivary gland (smg) is main source of fluid and electrolytes enriched saliva therefore its core for oral ph homeostasis. smg secretion is activated by acetylcholine (ach) in [ca + ] idependend manner and accompanied with oral ph acidic shifts. ph shifts could be due to changes in buffering capacity that is regulated by carbonic anhydrase (ca). despite the expression of different subtypes of ca in smg the role of ca in the regulation of smg function is unclear yet. we found that ca inhibition by benzolamide (bz) decreased of fluid secretion in vivo extracellular na + concentration in situ. the latter confirm the ability of ca to modify both primarily and final saliva secretion. we also found correlation between the secretion and ca + -homeostasis since bz-induced decrease of: in striated muscle ca + release from the sarcoplasmic reticulum (sr) occurs when ryanodine receptors (ryr-s) open either spontaneously or upon the stimulation from dihydropyridine receptors that are located in the adjacent transverse-tubular membrane and change their conformation when the cell is depolarized. recent observations demonstrated that muscles from animal models of ptdinsp phosphatase deficiency suffer from altered ca + homeostasis and excitation-contraction coupling, raising the possibility that ptdinsp-s could modulate voltage-activated sr ca + release in mammalian muscle. the openings of a single or a cluster of ryr-s can be detected as ca + release events on images recorded from fibres loaded with fluorescent ca + indicators. to elucidate the effects of ptdinsp-s on ca + release events, images were recorded from skeletal muscle fibers enzymatically isolated from the m. flexor digitorum breavis of mice utilizing a super-fast scanning technique. a wavelet-based detection method was used to automatically identify the events on the images. three different ptdinsp-s (ptdins p, ptdins p, and ptdins( , )p) were tested. all these ptdinsp compounds decreased the frequency of spontaneous ca + release events. supported by the hungarian national science fund (otka ), té t. calcium sparks elicited by mmol/l caffeine and by a depolarization to - mv were recorded at high time resolution on both x-y ( frames/s) and line-scan images ( lines/ ms) on intact skeletal muscle fibers of the frog. while a typical spark appeared in one frame only, . and . % of spark positions overlapped on consecutive frames following caffeine treatment or depolarization, respectively. while both caffeine and depolarization increased the frequency of sparks, as estimated from x-y images, the morphology of sparks was different under the two conditions. both the amplitude (in df/f ; . ± . vs. . ± . ; n = vs. ; mean ± sem, p \ . ) and the full width at half maximum (in lm; parallel with fiber axis: . ± . vs. . ± . ; perpendicular to fiber axis: . ± . vs. . ± . ) of sparks was significantly greater after caffeine treatment than on depolarized cells. these observations were confirmed on sparks identified in line-scan images. in addition, x-t images were used to analyze the time course of these events. calcium sparks had significantly slower rising phase under both conditions as compared to the control. on the other hand, while the rate of rise of signal mass was decreased after depolarization, it increased in the presence of caffeine. prolonged depolarisation of skeletal muscle cells induces entry of extracellular calcium into muscle cells, an event referred to as excitation-coupled calcium entry. skeletal muscle excitation-coupled calcium entry relies on the interaction between the , dihydropyridine receptor on the sarcolemma and the ryanodine receptor on the sarcoplasmic reticulum membrane. in this study we exploited tirf microscopy to monitor with high spatial resolution excitationcoupled calcium entry (ecce) in primary coltures of human skeletal muscle cells harbouring mutation in the ryr gene linked to malignant hyperthermia and central core disease. we found that excitation-coupled calcium entry is strongly enhanced in cells from patients with central core disease compared to individuals with malignant hyperthermia and controls. in addition, excitation-coupled calcium entry induces generation of reactive nitrogen species and causes the nuclear translocation of nfatc . the activation of nfatc dependent genes is consistent with an increase of the il secretion from primary coltures human myotubes from ccd patients and with fibre type predominance of skeletal muscle of ccd patients. membrane lipids, microdomains & signalling p- ftir and calorimetric investigation of the effects of trehalose and multivalent cations on lipid structure sawsan abu sharkh, jana oertel, and karim fahmy division of biophysics, institute of radiochemistry, helmholtz-zentrum dresden-rossendorf, germany e-mail: s.sharkh@hzdr.de the structure of membrane lipids is of fundamental importance for the integrity of cell and organelle membranes in living organisms. membrane lipids are typically hydrated and their headgroup charges counter-balanced by solvated ions. consequently, water loss can induce severe structural changes in lipid packing (lyotropic transitions) and can lead to the damage of lipid membranes even after rehydration. this can be one out of several factors that affect the viability of organisms undergoing desiccation. many organisms, however, are resistant to even extreme water loss. some of them synthesize trehalose which has been shown to be associated with survival of desiccation in phylogenetically diverse organisms (yeast, nematodes, brine shrimp, insect larvae, resurrection plants, and others). here we have studied hydration sensitive transitions in model lipids to determine the effect of trehalose and electrostatics on lipid order. hydration pulse-induced time-resolved fourier-transform infrared (ftir) difference spectroscopy was used to address hydration-dependent lipid structure as a function of trehalose. in combination with differential scanning calorimetry and studies of langmuir-blodget films we arrive at a structural and energetically consistent picture of how trehalose can affects lipidic phase behaviour and support a native lipid structure under water loss. experiments were performed on model lipids with different headgroups and native lipids from desiccation-tolerant organisms. controlled self-assembly and membrane organization of lipophilic nucleosides and nucleic acids: perspectives for applications martin loew , paula pescador , matthias schade , julian appelfeller , jü rgen liebscher , oliver seitz , daniel huster , andreas herrmann , and anna arbuzova humboldt universitä t zu berlin, institute of biology/ biophysics, berlin, germany, humboldt universitä t zu berlin, institute of chemistry, berlin, germany, universitä t leipzig, department of medical physics and biophysics, leipzig, germany lipophilic conjugates of nucleosides and nucleic acids such as dna, rna, and peptide nucleic acid (pna) -combining assembly properties of amphiphiles and specific molecular recognition properties of nucleic acids -allow numerous applications in medicine and biotechnology. we recently observed self-assembly of microtubes, stable cylindrical structures with outer diameters of nm and - lm and a length of - lm, from a cholesterol-modified nucleoside and phospholipids. morphology and properties of these microtubes and functionalization with lipophilic dna will be characterized. we also observed that lipophilic nucleic acids, pna and dna differing in their lipophilic moieties, partition into different lipid domains in model and biological membranes as visualized by hybridization with respective complementary fluorescently-labeled dna strands. upon heating, domains vanished and both lipophilic nucleic acid structures intermixed with each other. reformation of the lipid domains by cooling led again to separation of membrane-anchored nucleic acids. by linking specific functions to complementary strands, this approach offers a reversible tool for triggering interactions among the structures and for the arrangement of reactions and signaling cascades on biomimetic surfaces. conformational dependent trafficking of p-glycoprotein with rafts zsuzsanna gutayné tó th, orsolya bá rsony, katalin goda, gá bor szabó and zsolt bacsó university of debrecen, mhsc, department of biophysics and cell biology, debrecen, hungary p-glycoprotein (pgp), an abc-transporter playing a prominent role in multidrug resistance, demonstrate conformationdependent endocytosis on the surface of t -mdr cells. these cell surface transporters have a uic conformationsensitive-antibody-recognizable subpopulation, which is about one-third of the rest persisting long on the cell surface and perform fast internalization via rafts. we have identified that the rapid internalization is followed by quick exocytosis, in which the other subpopulation is not or only slightly involved. the exocytosis presents a cholesterol depletion dependent intensification, in contrast to the internalization, which is inhibited by cyclodextrin treatment. this continuous recycling examined by total internal reflection (tirf) microscopy increases the amount of the raft associated subpopulation of pgps in the plasma membrane, and it might have a role in restoring the cholesterol content of the membrane after cholesterol depletion. our presentation will summarize related endocytotic, exocytotic and recycling processes and that how does our data fit into our current notions regarding to the cholesterol and sphingomyelin trafficking. membrane nanodomains based on phase-segregating lipid mixtures have been emerged as a key organizing principle of the plasma membrane. they have been shown to play important roles in signal transduction and membrane trafficking. we have developed lipid-like probes carrying multivalent nitrilotriacetic acid (tris-nta) head groups for selective targeting of histagged proteins into liquid ordered or liquid disordered phases. in giant unilamellar vesicles strong partitioning of tris-nta lipids into different lipid phases was observed. for a saturated tris-nta lipid, at least -fold preference for the liquid ordered phase was found. in contrast, an unsaturated nta lipid shows a comparable preference for the liquid disordered phase. partitioning into submicroscopic membrane domains formed in solid supported membranes was confirmed by superresolution imaging. single molecule tracking of his-tagged proteins tethered to solid supported membranes revealed clear differences in the diffusion behavior of the different nta-lipids. by using bsa as a carrier, multivalent nta lipids were efficiently incorporated into the plasma membrane of live cells. based on this approach, we established versatile methods for probing and manipulating the spatiotemporal dynamics of membrane nano domains in live cells. il- is a multifunctional cytokine with pleiotropic effects on t cells. the il- r consists of the cytokine-specific a-subunit and the c c -chain shared with other cytokines, including il- and - , important regulators of t cells. we have previously shown the preassembly of the heterotrimeric il- and il- r, as well as their participation in common superclusters with mhc glycoproteins in lipid rafts of human t lymphoma cells. integrity of lipid rafts was shown to be important in il- signaling. we could hypothesize that other members of the c c cytokine receptor family, such as the il- r complex, may also fulfill their tasks in a similar environment, maybe in the same superclusters. co-localization of il- r with lipid rafts as well as with the il- r/mhc superclusters was determined by clsm. molecular scale interactions of il- ra with il- r and mhc molecules were determined by microscopic and flow cytometric fret experiments. the role of lipid rafts in il- r signaling was assessed by following the effect of cholesterol depletion on il- induced stat phosphorylation. our results suggest the possibility that preassembly of the receptor complexes in common membrane microdomains with mhc glycoproteins may be a general property of c c cytokines in t cells. to unravel the molecular processes leading to fas clustering in lipid rafts, a -mer peptide corresponding to the single transmembrane domain of the death receptor was reconstituted into model membranes that display liquid-disordered/ liquid-ordered phase coexistence, i.e. mimicking cells plasma membranes. using the intrinsic fluorescence of the peptide two tryptophans residues (trp and trp ), fas membrane lateral organization, conformation and dynamics was studied by steady-state and time-resolved fluorescence techniques. our results show that the fas has preferential localization to liquid disordered membrane regions, and that it undergoes a conformational change from a bilayer inserted state in liquid-disordered membranes to an interfacial state in liquidordered membranes. this is a result of the strong hydrophobic mismatch between the (hydrophobic) peptide length and the hydrophobic thickness of liquid-ordered membranes. in addition, we show that ceramide, a sphingolipid intimately involved in fas oligomerization and apoptosis triggering, does not affect fas membrane organization. overall, our results highlight ceramide role as an enhancer of fas oligomerization, and unravel the protective function of fas transmembrane domain against non-ligand induced fas apoptosis. organization and dynamics of membrane-bound bovine a-lactalbumin: a fluorescence approach arunima chaudhuri and amitabha chattopadhyay centre for cellular and molecular biology, hyderabad , india e-mail: amit@ccmb.res.in many soluble proteins are known to interact with membranes in partially disordered states, and the mechanism and relevance of such interactions in cellular processes are beginning to be understood. interestingly, apo-bovine alactalbumin (bla), a soluble protein, specifically interacts with negatively charged membranes and the membranebound protein exhibits a molten globule conformation. we have used the wavelength-selective fluorescence approach to monitor the molten globule conformation of bla upon binding to negatively charged membranes as compared to zwitterionic membranes. tryptophans in bla exhibit differential red edge excitation shift (rees) upon binding to negatively charged and zwitterionic membranes, implying differential rates of solvent relaxation around the tryptophan residues. our results utilizing fluorescence anisotropy, lifetime and depth analysis by the parallax approach of the tryptophans further support the differential organization and dynamics of the membrane-bound bla forms. in addition, dipole potential measurements and dye leakage assays are being used in our ongoing experiments to explore the mechanism of bla binding to membranes. these results assume significance in the light of antimicrobial and tumoricidal functions of a-lactalbumin. role of long-range effective protein-protein forces in the formation and stability of membrane protein nano-domains nicolas destainvill laboratoire de physique thé orique, université paul sabatier toulouse -cnrs, toulouse, france we discuss a realistic scenario, accounting for the existence of sub-micro-metric protein domains in plasma membranes. we propose that proteins embedded in bio-membranes can spontaneously self-organize into stable small clusters, due to the competition between short-range attractive and intermediate-range repulsive forces between proteins, specific to these systems. in addition, membrane domains are supposedly specialized, in order to perform a determined biological task, in the sense that they gather one or a few protein species out of the hundreds of different ones that a cell membrane may contain. by analyzing the balance between mixing entropy and protein affinities, we propose that protein sorting in distinct domains, leading to domain specialization, can be explained without appealing to preexisting lipidic micro-phase separations, as in the lipid raft scenario. we show that the proposed scenario is compatible with known physical interactions between membrane proteins, even if thousands of different species coexist. lipid rafts are cholesterol and sphingolipid-enriched functional microdomains present in biomembranes. rafts have been operationally defined as membrane fractions that are detergent insoluble at low temperature. here we have characterized drms from erythrocytes treated with the nonionic detergents brij and brij , at °c and °c, and compared them to drms obtained with triton x- (tx ). we have also investigated the effect of cholesterol depletion in drms formation. brij drms were enriched in cholesterol as well as tx drms. hptlc analysis showed a very similar distribution of phosphatidylcholine-pc, phosphatidylethanolamine-pe and sphingomyelin-sm in brij drms to that found in ghost membranes. sm-enriched drms were obtained only with tx while pe content was decreased in tx drms, in comparison to brij drms. immunoblot essays revealed that rafts markers (flotillin- and stomatin) were present in all drms. contrary to tx drms, analysis of electron paramagnetic resonance spectra (with -doxyl stearate spin label) revealed that brij drms are not in the liquid-ordered state, evincing the differential extraction of membrane lipids promoted by these detergents. supported by fapesp/cnpq (brazil). several biological membrane mimics have been built to investigate the topology of molecules in membranes. among them ''bicelles'', i.e., mixtures of long-chain and short-chain saturated phospholipids hydrated up to %, became very popular because they orient spontaneously in magnetic fields. disk-shaped systems of - nm diameter and - nm thickness have been measured by electron microscopy and solid state nmr and can be oriented by magnetic fields with the disc-plane normal perpendicular to the field. we have been developing recently lipids that contain in one of their chains a biphenyl group (tbbpc) affording an orientation parallel to the magnetic field, in the absence of lanthanides. a large number of hydrophobic molecules including membrane proteins have been successfully embedded and static nmr afforded finding the orientation of protein helices in membranes; mas nmr provided the d structure of peptides in bicelles. biphenyl bicelles keep their macroscopic orientation for days outside the field, thus leading to combined nmr and x-rays experiments. tbbpc allows also construction of lm vesicles showing a remarkable oblate deformation in magnetic fields (anisotropy of - ) and opens the way to applications for structural biology or drug delivery under mri. imaging membrane heterogeneities and domains by super-resolution sted nanoscopy christian eggeling, veronika mueller, alf honigmann, stefan w. hell department of nanobiophotonics, max planck institute for biophysical chemistry, am fassberg , gö ttingen, germany cholesterol-assisted lipid and protein interactions such as the integration into lipid nanodomains are considered to play a functional part in a whole range of membrane-associated processes, but their direct and non-invasive observation in living cells is impeded by the resolution limit of [ nm of a conventional far-field optical microscope. we report the detection of the membrane heterogeneities in nanosized areas in the plasma membrane of living cells using the superior spatial resolution of stimulated emission depletion (sted) far-field nanoscopy. by combining a (tunable) resolution of down to nm with tools such as fluorescence correlation spectroscopy (fcs), we obtain new details of molecular membrane dynamics. sphingolipids or other proteins are transiently (* ms) trapped on the nanoscale in cholesterol-mediated molecular complexes, while others diffuse freely or show a kind of hopping diffusion. the results are compared to sted experiments on model membranes, which highlight potential influences of the fluorescent tag. the novel observations shed new light on the role of lipid-protein interactions and nanodomains for membrane bioactivity. ca + controlled all-or-none like recruitment of synaptotagmin- c ab to membranes sune m. christensen, nicky ehrlich, dimitrios stamou bio-nanotechnology laboratory, department of neuroscience and pharmacology, nano-science center, lundbeck foundation center biomembranes in nanomedicine, university of copenhagen, copenhagen, denmark e-mail: ehrlich@nano.ku.dk & stamou@nano.ku.dk synaptotagmin- (syt) is the major ca + sensor that triggers the fast, synchronous fusion of synaptic vesicle with the presynaptic membrane upon ca +-mediated membrane recruitment of the cytosolic c ab domain. the ca +-dependent recruitment of syts c ab domain to membranes has so far been investigated by ensemble assays. here we revisited binding of wild type c ab and different c ab mutants of syt to lipid membranes using a recently developed single vesicle assay. the hallmark of the single vesicle approach is that it provides unique information on heterogeneous properties that would otherwise be hidden due to ensemble averaging. we found that c ab does not bind to all vesicles in a homogenous manner, but in an all-or-none like fashion to a fraction of the vesicles. the fraction of vesicles with bound c ab is regulated by the amount of negatively charged lipids in the membrane as well as by [ca +] . this ca + controlled all-ornone like recruitment of syt to membranes provides a possible explanation for the strongly heterogeneous behavior of the in vitro model system for neuronal membrane fusion. furthermore, heterogeneity in release probability among synaptic vesicles is a critical property in determining the output of a neuronal signaling event. new insights in the transport mechanism of ciprofloxacin revealed by fluorescence spectroscopy mariana ferreira, sílvia c. lopes, paula gameiro requimte, faculdade de ciê ncias da universidade do porto, porto, portugal keywords: fluoroquinolones; liposomes; proteoliposomes; ompf; ciprofloxacin; fluorescence; anisotropy. fluoroquinolones are antibiotics that have a large spectrum of action against gram negative and some gram positive bacteria. the interaction between these species and liposomes has been cited as a reference in the understanding of their diffusion through phospholipid bilayer and can be quantified by the determination of partition coefficients between a hydrophobic phase (liposomes) and an aqueous solution. it is also known that some porins of the bacterial membranes are involved in transport mechanism of many fluoroquinolones. ompf, a well characterized membrane protein characteristic of the outer membrane of gram negative bacteria assumes the conformation of homo-trimer, whose monomers have two tryptophan residues (one located at the interface of monomers and the other at the interface lipid/protein). thus, we proceeded to study the interaction of ciprofloxacin, a second generation fluoroquinolone, with unilamellar liposomal vesicles and ompf proteoliposomes of pope/popg, pope/popg/cardiolipin and e. coli total. partition coefficients (kp's) and the association with ompf proteoliposomes were determined by steady state fluorescence spectroscopy under physiological conditions (t= °c; ph . ). the membrane mimetic systems used were characterized by dls and fluorescence anisotropy. motivation is whether there exist differences in the patternforming capabilities of two adhesion molecules of different roles: cd , mediating ,,dynamic'' adhesion in cell rolling and icam- , mediating ,,static'' adhesion during the formation of immune-synapse. homo-and hetero-associations of cd , icam- and the mhci is investigated on the nm-and lmdistance levels on ls t colon carcinoma cells in two different conditions of lymphocyte homing: ( ) with ifnc and tnfa, both lymphokines up-regulating the expression level of mhci and icam- and down-regulating that of cd . ( ) crosslinking of cd and icam- representing receptor engagement. the observations are explained by assuming the existence of a kinase cascade-level crosstalk between the cd and icam- molecules which manifests in characteristic complementary changes in the properties of cell surface receptor patterns. for the characterisation of cluster morphology new colocalization approaches were developed: (i) ,,number of first neighbours'' distribution curves, (ii) ,,acceptor photobleaching fret-fluorescence intensity fluctuation product'' correlation diagrams, and (iii) ,,random gradient-kernel smoothing assisted decay'' of pearson-correlations, and (iv) k-function formalism. analyzing janus kinases in living cells with single-color fcs using confocal and tir-illumination thomas weidemann , hetvi gandhi , remigiusz worch , , robert weinmeister , christian bö kel , and petra schwille biophysics research group, technische universitä t dresden, germany, crtd, center for regenerative therapies dresden, technische universitä t dresden, germany, institute of physics, polish academy of science, warsaw, poland cytokine receptors of the hematopoietic superfamily transduce their signal through non-covalently bound janus kinases. there are only such kinases in humans (jak , , and tyk ), which associate to different cytokine receptor chains. here we study the dynamics of gfp-tagged jak and jak in epithelial cells with fluorescence correlation spectroscopy (fcs). jak and jak behave differently in various aspects: in the absence of receptors, jak still binds the membrane, whereas jak diffuses homogeneously in the cytoplasm. we used fcs under total internal reflection illumination (tir-fcs) and determined the membrane binding affinity of jak to be ± nm. the association of jak with the common gamma chain (c c ) is very tight as shown by fluorescence recovery after photobleaching (frap). molecular brightness analysis of single-point fcs shows that jak diffuses as a monomer in the rather small cytoplasmic pool, whereas jak diffuses as dimers, which undergo a defined oligomerization. the degree of oligomerization decays at higher concentrations, indicating that some unknown, saturable scaffold is involved. characterizing the binding and mobility schemes of the janus kinases may be important to further elucidate their specific and redundant effects in signal transduction. plasma membrane (pm)-enriched fraction obtained through subcellular fractioning protocols are commonly used in studies investigating the ability of a compound to bind to a receptor. however, the presence of mitochondria membranes (mi) in the pm-enriched fraction may compromise several experimental results because mi may also contain the interest binding proteins. aiming to analyze the subcellular fractioning quality of a standard sucrose density based protocol, we investigated (a) the na + k + -atpase (pm marker) and succinate dehydrogenase -sd (mi marker) activities; (b) the immunocontent of the adenine nucleotide translocator (ant -mi membrane marker) in both pm-and mi-enriched fractions. since several binding protocols may require long incubation period, we verified the quality of both fractions after hours of incubation in adequate buffer. our results show that pmand mi-enriched fractions exhibit contamination with mi or pm, respectively. we did not observe any effect of incubation on na + k + -atpase activity and ant content in both fractions. surprisingly, sd activity was preserved in the pm-but not in mi-enriched fraction after incubation. these data suggest the need of more careful use of pm-enriched fraction preparation in studies involving pm proteins characterization. human neutrophil peptide (hnp ) is a human cationic defensin that present microbial activity against various bacteria (both gram-positive and negative), fungi and viruses. hnp is stored in the cytoplasmic azurophilic granules of neutrophils and epithelial cells. in order to elucidate the mode of action of this antimicrobial peptide (amp), studies based on its lipid selectivity were carried out. large unilamellar vesicles (luv) with different lipid compositions were used as biomembranes model systems (mammal, fungal and bacterial models). changes on the intrinsic fluorescence of the tryptophan residues present in hnp upon membrane binding/insertion were followed, showing that hnp have quite distinct preferences for mammalian and fungal membrane model systems. hnp showed low interaction with glucosylceramide rich membranes, but high sterol selectivity: it has a high partition for ergosterol-containing membranes (as fungal membranes) and low interaction with cholesterolcontaining membranes (as in mammalian cells). these results reveal that lipid selectivity is the first step after interaction with the membrane. further insights on the hnp membrane interaction process were given by fluorescence quenching measurements using acrylamide, -doxylstearic acid ( ns) or ( ns). nanoparticles (np) are currently used in many industrial or research applications (paints, cosmetics, drug delivery materials…). recent papers demonstrate clearly their activity with biological membranes (nanoscale holes, membrane thinning, disruption). different studies of the np-membrane interaction suggest that parameters are particularly important, such as the np size, their surface properties or their aggregation state. composition of biological membranes being particularly complex, supported lipid bilayers (slb) composed of a restricted number of lipids are usually used as simplified membrane model. moreover, these two-dimensional systems are convenient for surface analysis techniques, such as atomic force microscopy (afm), giving information on the morphology of the slb and its mechanical properties. in this work, we study the behaviour of slbs made of lipids representative of the membrane fluid phase (popc) or of the raft phase (sphingomyelin). these slbs are deposited on planar surfaces (mica or glass) previously recovered with silica beads ( or nm in diameter) in order to mimick the np-membrane interaction. we will present in this work our first results obtained by afm and fluorescence microscopy. it is well known that the eukaryotic nuclei are the sphere of lipids active metabolism. the investigations demonstrated the existence of numerous enzymes in nuclei which modulate the changes of nuclear lipids during different cellular processes. although the nuclear membrane is accepted as the main place of the lipids localization, nearly % of nuclear lipids are discovered in chromatin fraction. the ability of chromatin phospolipids to regulate dna replication and transcription was already demonstrated. the chromatin phospolipids seems to play an important role in cell proliferation and differentiation as well as in apoptosis. it seems also possible that chromatin phospholipids may be participated in realization of cisplatin antitumor effects. the -hour in vivo effect of cisplatin on rat liver chromatin phospholipids was investigated. the phospholipids of rat liver chromatin were fractionated by microtlc technique. the quantitative estimation of fractionated phospholipids was carried out by computer program fugifilm science lab. image gauge v . . the alteration of total phospholipids content as well as the quantitative changes among the individual phospholipids fractions in rat liver chromatin after in vivo action of cisplatin was established. the total content of chromatin phospholipids was significantly decreased after the cisplatin action. four from five individual phospholipids fractions were markedly changed after the drug action. two cholin-content phospholipids, particularly phosphatidylcholine and sphingomyelin exhibit diversity in sensitivity to this drug: the increase of sphingomyelin content accompanied by quantitative decrease of phosphatidylcholine. the quantity of cardiolipin was markedly increased while the amount of phosphatidylinositol was decreased after the cisplatin treatment. the phosphatidylethanolamin content remained unchanged after the drug action. it seems that high sensitivity of chromatin phospholipids exhibited to cisplatin action may play an important role in antitumor effects of this drug. membrane lipids and drug resistance in staphylococci r. d. harvey institute of pharmaceutical science, king's college london, stanford street, london se nh, uk staphylococci express numerous resistance mechanisms against common antimicrobials, including peptide components of the innate immune system which have been trumpeted as being likely candidates to replace our increasingly ineffective antibiotics. the membrane phospholipid lysylphosphatidylglycerol (l-pg) appears to play a key role in staphylococcal drug resistance, since its absence in mutant bacteria renders them susceptible to a range of cationic antimicrobials. the current assumption about the role l-pg plays in drug resistance is that of facilitating charge neutralisation of the plasma membrane, leading to loss of affinity towards cationic moieties. we have investigated this phenomenon using a range of model membrane systems composed of both synthetic lipids and reconstituted natural lipid extracts, using such techniques as stopped-flow fluorescence, circular dichroism and neutron scattering. our conclusions indicate that the initial assumptions about the role of l-pg in drug resistance are over-simplistic and certainly do not tell the whole story of the physical and biological properties of this fascinating moderator or membrane behaviour. our findings show that l-pg does not inhibit antimicrobial drug action by charge dampening, hinting at a different protective mechanism. modulation of a-toxin binding by membrane composition m. schwiering, a. brack, c. beck, h. decker, n. hellmann institute for molecular biophysics, university of mainz, mainz, germany although the alpha-toxin from s. aureus was the first poreforming toxin identified, its mode of interaction with membranes is still not fully understood. the toxin forms heptameric pores on cellular and artificial membranes. the present hypothesis is that the initial binding to the membrane occurs with low affinity, and that an efficient oligomerisation, relying on clusters of binding sites, is the reason for the overall high affinity of the binding process. in order to separate the effects of increasing concentration of binding sites from this topological effect, we investigated the oligomer formation based on pyrene-fluorescence for a series of lipid compositions, where the fraction of toxin binding lipids (egg phospatidylcholine (epc) or egg sphingomyelin (esm)) was varied while their concentration remained constant. the results indicate that an increased local density of toxin binding sites occurring due to phase separation facilitates oligomer formation. furthermore, the change in local environment (number of neighboring cholesterol molecules) upon domain formation also enhances oligomer formation.we thank the dfg (sfb ) for financial support, s. bhakdi and a. valeva for production of the toxin and helpful discussions. we explored quercetin effects on lipid bilayers containing cholesterol using a spectrofluorimetric approach. we used the fluorescent probe laurdan which is able to detect changes in membrane phase properties. when incorporated in lipid bilayers, laurdan emits from two different excited states, a non-relaxed one when the bilayer packing is tight and a relaxed state when the bilayer packing is loose. this behavior is seen in recorded spectra as a shift of maximum emission fluorescence from nm at temperatures below lipids phase transition to nm at temperatures above lipids phase transition values. emission spectra of laurdan were analyzed as a sum of two gaussian bands, centered on the two emission wavelengths allowing a good evaluation of the relative presence of each population. our results show that both laurdan emission states are present with different shares in a wide temperature range for dmpc liposomes with cholesterol. quercetin leads to a decrease in the phase transition temperature of liposomes, acting in the same time as a quencher on laurdan fluorescence. this paper is supported by the sectorial operational programme human resources development, financed from the caveolins are essential membrane proteins found in caveolae. the caveolin scaffolding domain of caveolin- includes a short sequence containing a crac motif (v tkywfyr ) at its c-terminal end. to investigate the role of this motif in the caveolin-membrane interaction at the atomic level, we performed a detailed structural and dynamics characterization of a cav- (v -l ) nonapeptide encompassing this motif and including the first residue of cav- hydrophobic domain (l ), in dodecylphosphocholine (dpc) micelles and in dmpc/dhpc bicelles, as membrane mimics. nmr data revealed that this peptide folded as an amphipathic helix located in the polar head group region. the two tyrosine sidechains, flanked by arginine and lysine residues, are situated on one face of this helix, whereas the phenylalanine and tryptophan side-chains are located on the opposite face (le lan c. et al., , eur. biophys. j., , - ). to investigate the interactions between the crac motif and the lipids, we performed molecular dynamics simulations in two different environment: a dpc micelle and a popc bilayer. the results obtained are in good agreement with nmr data and the comparison between both systems provided insight into the orientation of the crac motif at the membrane interface and into its interactions with lipids. this work was partially supported by the strategic grant posdru/ / . our study suggests that conformation and positioning of hydroxyl groups significantly affects thermotropic properties of sphingolipids and sterol interaction. the polymorphism of a new series of bolaamphiphile molecules based on n-( -betainylamino-dodecane)-octyl b-d-glucofuranosiduronamide chloride is investigated. the length of the main bridging chain is varied in order to modify the hydrophilic/lipophilic balance. the other chemical modification was to introduce a diacetylenic unit in the middle of the bridging chain to study the influence of the pp stacking on the supramolecular organisation of these molecules. dry bolaamphiphiles self-organize in supramolecular structures such as lamellar crystalline structure, lamellar fluid structure and lamellar gel structure. the thermal dependence of these structures, as well as the phase transition is followed by smallangle and wide-angle x-ray scattering. once the thermal cycle is accomplished, the system remains in the kinetically stabilized undercooled high-temperature phase at temperature of °c. subsequently, the time dependence of the relaxation to the thermodynamically stable phase is followed and very slow relaxation on the order of hours or days is observed. the study of polymorphism and the stability of various phases of this new series of bolaamphiphiles is interesting for potential application in health, cosmetics or food industry, is undertaken in this work. alkylphospholipids have shown promising results in several clinical studies and among them perifosine (opp) is promising for breast cancer therapy. antitumor effect was much better in estrogen receptor negative (er-) than in estrogen receptor positive (er+) tumors in vivo. it is believed that apl do not target dna, but they insert in the plasma membrane and ultimately lead to cell death. liposomes made of opp and different amount of cholesterol (ch) showed diminished hemolytic activity as compared to micellar opp, but in most cases cytotoxic activity was lower. in order to find optimal liposomal composition and to understand better the difference in the response of er+ and er-cells the interaction opp liposomes with er+ and er-cells was studied. for liposomes with high amount of ch both cell types showed slow release of the liposome entrapped spin probe into the cytoplasm. liposmes with low amount of ch interact better with cells but the release is faster for er-as for er+ cells at °c. experiments with nitroxide-labeled opp (sl-opp) liposomes suggest that the exchange of sl-opp between liposomes and cellular membranes is fast. however, translocation of sl-opp across the plasma membrane is slow, but seems to be faster for opp resistent, er+ cells as for er-cells at °c. estimation of a membrane pore size based on the law of conservation of mass krystian kubica , artur wrona and olga hrydziuszko institute of biomedical engineering and instrumentation, wroclaw university of technology, wroclaw, poland, centre for systems biology, university of birmingham, birmingham, uk the size of biomembrane pores determines which solutes or active compounds may enter the cell. here, using a mathematical model of a lipid bilayer and the law of conservation of mass, we calculate the radius of a membrane pore created by rearranging the lipid molecules (the pore wall was formed out of the lipid heads taken from the membrane regions situated directly above and below the pore, prior its formation). assuming a constant number of lipid molecules per bilayer (with or without the pore) and based on the literature data ( % decrease in the area per chain for a fluid-to-gel transition and a matching change of one chain volume not exceeding %) we have shown that the pore radius can measure up to . nm (for a nm thick lipid bilayer) without the lipid molecules undergoing a phase transition. a further assumption of area per chain being modified as a consequence of the lipids conformational changes has resulted in an increase of the calculated radius up to . nm. finally, a comparison of the pore volume with the corresponding volume of the lipid bilayer has led to a conclusion that for the system under consideration the membrane pore can only be created with the lipids undergoing fluidto-gel conformational changes. the key signaling pathway involves tyrosine phosphorylation of signal transducers and activators of transcription (stat and stat ) by receptor-associated janus kinases. we aim to unveil of the very early events of signal activation including ligand-induced receptor assembly and the recruitment of the cytoplasmic effector proteins stat and stat in living cells. to this end, we have explored the spatiotemporal dynamics of stat recruitment at the membrane on a single molecule level. highly transient interaction of stats to membrane-proximal sites was detected by tirf-microscopy, allowing for localizing and tracking individual molecules beyond the diffraction limit. thus, we obtained a pattern of the spatio-temporal recruitment of stat molecules to the plasma membrane revealing distinct submicroscopic structures and hotspots of stat interaction with overlapping recruitment sites for stat and stat . strikingly, these stat binding sites were independent on receptor localization and expression level. simultaneous superresolution imaging of the cytoskeleton revealed the organization of stat recruitment sites within the cortical actin skeleton. characterization of molecular dynamics on living cell membranes at the nanoscale level is fundamental to unravel the mechanisms of membrane organization andcompartmentalization. we have recently demonstr ated the feasibility of fluorescence correlation spectroscopy (fcs) based on the nanometric illumination of near-field scanning optical microscopy (nsom) probes on intact living cells [ ] . nsom-fcs was applied to study the diffusion of fluorescent lipid analogs on living cho cells. the experiments allowed to reveal details of the diffusion hidden by larger illumination areas and associated with nanoscale membrane compartmentalization. the technique also offers the unique advantages of straightforward implementation of multiple color excitation, opening the possibility to study nanoscale molecular cross-correlation. furthermore, the nsom probe evanescent axial illumination allows to extend diffusion study to the membrane-proximal cytosolic region. as such, nsom-fcs represents a novel powerful tool to characterize the details of many biological processes in which molecular diffusion plays a relevant role. the growing interest in supported lipid bilayers (slbs) on conductive substrates, such as gold, is due to the possibility of designing lipid-based biosensor interfaces with electrochemical transduction. due to the hydrophobicity of gold it is still a challenge to deposit planar and continuous bilayers without previous surface modification. most studies on gold concern single-phase slbs without cholesterol or gangliosides, two vital components of biomembranes. in this work the experimental conditions suitable for the formation of complex slbs with phase-separation directly on gold are exploited. the mixtures dopc/dppc/cholesterol ( : : ) with or mol % of ganglioside gm , which should yield lipid raft-like domains according to reported phase diagrams, were studied. slb with lipid rafts were successfully formed onto bare au ( ), although surface modification with -mercapto-undecanoic acid sam stabilized the slbs due to its charge and hydrophilicity. the different experimental conditions tested had an impact on nano/microdomains organization observed by atomic force microscopy in buffer solution. surface characterization through the combined use of ellipsometry, cyclic voltammetry and afm allowed to optimize the conditions for the formation of more planar and compact slbs. it is widely accepted that the conversion of the soluble, nontoxic amyloid b-protein (ab) monomer to aggregated toxic ab rich in b-sheet structures is central to the development of alzheimer's disease. however, the mechanism of the abnormal aggregation of ab in vivo is not well understood. we have proposed that ganglioside clusters in lipid rafts mediate the formation of amyloid fibrils by ab, the toxicity and physicochemical properties of which are different from those of ab amyloids formed in solution [ , ] . in this presentation, we report a detailed mechanism by which ab-( - ) fibrillizes in raft-like lipid bilayers composed of gm /cholesterol/sphingomyelin. at lower concentrations, ab formed an a helix-rich structure, which was cooperatively converted to a b sheet-rich structure above a threshold concentration. the structure was further changed to a seed-prone b structure at higher concentrations. the seed recruited ab in solution to form amyloid fibrils. [ hepatitis c virus (hcv) has a great impact on public health, affecting more than million people worldwide since it is the cause of liver-related diseases such as chronic hepatitis, cirrhosis and hepatocarcinoma. hcv entry into the host cell is achieved by the fusion of viral and cellular membranes, replicates its genome in a membrane associated replication complex, and morphogenesis has been suggested to take place in the endoplasmic reticulum (er) or modified er membranes. the variability of the hcv proteins gives the virus the ability to escape the host immune surveillance system and notably hampers the development of an efficient vaccine. hcv has a single-stranded genome which encode a polyprotein, cleaved by a combination of cellular and viral proteases to produce the mature structural proteins (core, e , e , and p ) and non-structural ones (ns , ns , ns a, ns b, ns a and ns b), the latter associated with the membrane originated from the er in the emerging virus. the ns b protein, a fundamental player in the hcv replicative process and the least characterized hcv protein, is a highly hydrophobic protein associated with er membranes. it has recently been shown that the c-terminal is palmitoylated and the n-terminal region has potent polymerization activity. the expression of ns b induces the formation of the so called membranous web, which has been postulated to be the hcv rna replication complex. thus, a function of ns b might be to induce a specific membrane alteration that serves as a scaffold for the formation of the hcv replication complex and therefore has critical role in the hcv cycle. due to the high hydrophobic nature of ns b, a detailed structure determination of this protein is very difficult. the ns b protein is an integral membrane protein with four or more transmembrane domains. the c-terminal region of ns b is constituted by two a helices, h (approximately from amino acid to ) and h (approximately from amino acid to ), which have been studied as potential targets for inhibiting hcv replication. previous studies from our group, based on the study of the effect of ns b peptide libraries on model membrane integrity, have allowed us to propose the location of different segments in this protein that would be implicated in lipid-protein interaction. additionally, the h region could be an essential constituent in the interaction between protein and membrane. in this study we show that peptides derived from the c-terminal domain of ns b protein of hcv are able to interact with high affinity to biomembranes, significantly destabilizing them and affecting their biophysical properties. there were also differences in the interaction of the peptide depending on the lipid composition of the membranes studied. we have also applied fluorescence spectroscopy, infrared spectroscopy and differential scanning calorimetry which have given as a detailed biophysical study of the interaction of the peptide with model biomembranes. this work was supported by grant bfu - -bmc (ministerio de ciencia y tecnología, spain) to j.v. a semi-quantitative theory describing the adhesion kinetics between soft objects as living cells or vesicles was developed. the nucleation-like mechanism has been described in the framework of a non-equilibrium fokker-planck approach accounting for the adhesion patch growth and dissolution (a. raudino, m. pannuzzo, j. chem. phys. , ( )). a well known puzzling effect is the dramatic enhancement of the adhesion/fusion rate of lipid membranes by water-soluble polymers that do not specifically interact with the membrane surface. we extend the previous approach by molecular dynamics simulations in the framework of a coarse-grained picture of the system (lipid+polymer+ions embedded in an explicit water medium) in order to test and support our previous analytical results. simulations show that the osmotic pressure due to the polymer exclusion from the inter-membrane spacing is partially balanced by an electrostatic pressure. however, we also evidenced an interesting coupling between osmotic forces and electrostatic effects. indeed, when charged membranes are considered, polymers of low dielectric permittivity are partially excluded from the inter-membrane space because of the increased local salt concentration. the increased salt concentration means also a larger density of divalent ions which form a bridge at the contact region (stronger adhesion). the overall effect is a smaller membrane repulsion. this effect disappears when neutral membranes are considered. the model could explain the fusion kinetics between lipid vesicles, provided the short-range adhesion transition is the rate-limiting step to the whole fusion process. the role of ceramide acyl chain length and unsaturation on membrane structure sandra n. ceramide fatty acid composition selectively regulates distinct cell processes by a yet unknown mechanism. however, evidence suggests that biophysical processes are important in the activation of signalling pathways. indeed, ceramide strongly affects membrane order, induces gel/fluid phase separation and forms highly-ordered gel domains. the impact of ceramide n-acyl chain in the biophysical properties of a fluid membrane was studied in popc membranes mixed with distinct ceramides. our results show that: i) saturated ceramide has a stronger impact on the fluid membrane, increasing its order and promoting gel/fluid phase separation, while their unsaturated counterparts have a lower (c : ceramide) or no (c : ceramide) ability to form gel domains at physiological temperature, ii) differences between distinct saturated species are smaller and are related mainly to domain morphology, and iii) very long chain ceramide induces the formation of tubular structures probably associated with interdigitation. these results suggest that generation of different ceramide species in cell membranes has a distinct biophysical impact with acyl chain saturation dictating membrane lateral organization, and chain asymmetry governing interdigitation and membrane morphology. extra high content of cholesterol (chol) in fiber-cell membranes in the eye lens leads to chol saturation and formation of cholesterol bilayer domains (cbds). it is hypothesized that high enrichment in cholesterol helps to maintain lens transparency and protect against cataractogenesis. in model studies, the cbd is formed in a phospholipid bilayer when cholesterol content exceeding the cholesterol solubility threshold, thus, the cbd is surrounded by phospholipid bilayer saturated with cholesterol. in the present study, we carried out molecular dynamics (md) simulation of two bilayers: a palmitoyloleoylphosphatidylcholine (popc) bilayer (reference) and a : popc-chol bilayer, to investigate the smoothing effect of the saturating amount of cholesterol on the bilayer. to our knowledge, this effect has not been studied so far so this study is certainly providing new results. our results indicate that saturation with cholesterol significantly narrows the distribution of vertical positions of the center-of-mass of the popc molecules and the popc atoms in the bilayer and smoothes the bilayer surface. we hypothesize that this smoothing effect decreases light-scattering and helps to maintain lens transparency. the phospholipid content of staphylococcus aureus membranes displays a high degree of variability ( - ). the major phospholipids found in s. aureus are phosphatidylglycerol (pg), cardiolipin (cl) and lysylphosphatidylglycerol (lpg), ( ) the concentrations of which are environment dependent and see fluctuations on exposure to high concentrations of positively charged moieties ( ) . up regulation of lpg has a suspected role in neutralisation of the plasma membrane in response to cationic threats. studies have been conducted to probe biomimetic models of this theory however our focus is to look at atomic details of membrane extracts in the presence of magaininf w. s. aureus lipid extracts from cells grown at ph . and . , studies by neutron diffraction with and without peptide at two contrasts. d-spacings were assessed by vogt area fitting and bragg's law. bilayer separation at low ph was * - Å less than ph . . with peptide, bilayer separations of ph . and . extracts were reduced by * Å and * Å , respectively. reduced pg content of low ph extracts is suggested to reduce d-spacing, however presence of peptide further reduces this, possibly by an anion neutralisation effect. abnormal d-spacing on increased humidity may be due to the breakdown of lpg. activation of neutrophils releasing hocl and apoptosis of vein endothelial cells are the events documented to occur in the course of atherosclerosis. as lipid chlorohydrins, which are the key products of the reaction between hocl and unsaturated fatty acid residues, were found in atherosclerotic plaques, we decided to check their biological activity in the context of their ability to act as the mediators of hoclinduced oxidative stress and apoptosis in the culture of immortalized human umbilical vein endothelial cells (hu-vec-st). the concentration of reactive oxygen species was found to be elevated after h cell incubation with phospahtidylcholine chlorohydrins. this effect was at least partially caused by the leakage of superoxide anion from mitochondria and followed by depletion of gsh and total thiols. the significant decrease of antioxidant capacity of cell extracts was also observed. the intracellular red-ox imbalance was accompanied by the increase of the ratio between phosphorylated and dephosphorylated forms of p map kinase. after longer incubation a significant number of apoptotic cells appeared. summing up, phosphatidylcholine chlorohydrins may be regarded as signaling molecules, able to initiate signalling pathways by induction of oxidative stress. giant unilamellar vesicles (guvs) are a valuable tool in the study of lateral distribution of biological membrane components. guv dimensions are comparable to typical cell plasma membranes and lipid phase separation can be observed through fluorescence microscopy. guv studies frequently require immobilization of the vesicles, and several methods are available for that effect. one of the most common methodologies for vesicle immobilization is the use of avidin/streptavidin coated surfaces and biotin labeled lipids at very low concentration in the vesicles. here, we analyze the effect of using this methodology on lipid domain distribution for different lipid compositions. we show that as a result of non-homogeneous distribution of biotin labeled lipids between liquid disordered, liquid ordered and gel phases, distribution of lipid domains inside guvs can be dramatically affected. monitoring membrane permeability: development of a sicm approach christoph saßen , and claudia steinem institute for organic and biomolecular chemistry, university of gö ttingen, tammannstraße , gö ttingen, germany, ggnb doctoral program: imprs, physics of biological and complex systems scanning ion conductance microscopy (sicm) utilises a nanopipette containing an electrode as a probe for surface investigations with resolutions of / of the inner pipette diameter. experiments are conducted under physiological conditions, in situ and without mechanical contact of probe and sample. hence, sicm serves as a well-suited technique for the investigation of soft objects such as cells or artificial lipid membranes. using pore-suspending membranes (psm) as a model system, interactions of melittin as an example for cell penetrating peptides (cpps) and lipid membranes are investigated by means of sicm. formation of a range of solvent free psm from lipid vesicles has been achieved as confirmed by means of fluorescence microscopy and sicm. application of melittin results in rupturing of the lipid bilayer. putative insights gained from this assay are critical concentrations of membrane permeabilising ccps and answers to mechanistic questions, e.g. whether ccps translocate only or form pores within the lipid bilayer. positioning of the z-ring in escherichia coli prior to cell division is regulated by intracellular pole-to-pole oscillation and membrane binding of min proteins, allowing assembly of ftsz filaments only at the center plane of the cell. in order to investigate the influence of membrane geometry on the dynamic behavior of membrane binding of min proteins, we combined concepts of synthetic biology and microfabrication technology. glass slides were patterned by a gold coating with microscopic windows of different geometries, and supported lipid bilayers (slb) were formed on these microstructures. on slbs, min-proteins organize into parallel waves. confinement of the artificial membranes determined the direction of propagation.min-waves could be guided along curved membrane stripes, in circles and even along slalom-geometries. in elongated membrane structures, the protein waves always propagate along the longest axis. coupling of protein waves across spatially separated membrane patches was observed, dependent on gap size and viscosity of the aqueous media above the bilayer. this indicates the existence of an inhomogeneous and dynamic protein gradient above the membrane. minimal systems for membrane associated cellular processes petra schwille bio technology center biotec, technical university of dresden, germany the strive for identifying minimal biological systems, particularly of subcellular structures or modules, has in the past years been very successful, and crucial in vitro experiments with reduced complexity can nowadays be performed, e.g., on reconstituted cytoskeleton and membrane systems. in this overview talk, i will first discuss the virtues of minimal membrane systems, such as guvs and supported membranes, in quantitatively understand protein-lipid interactions, in particular lipid domain formation and its relevance on protein function. membrane transformations, such as vesicle fusion and fission, but also vesicle splitting, can be reconstituted in these simple subsystems, due to the inherent physical properties of selfassembled lipids, and it is compelling question how simple a protein machinery may be that is still able to regulate these transformations. as an exciting example of the power of minimal systems, i show how the interplay between a membrane and only two antagonistic proteins from the bacterial cell division machinery can result in emergence of protein self-organization and pattern formation, and discuss the possibility of reconstituting a minimal divisome. quantitative microscopic analysis reveals cell confluence regulated divergence of pdgfr-initiated signaling pathways with logically streamlined cellular outputs Á rpá d szö } or, lá szló ujalky-nagy, já nos szö ll} osi, gyö rgy vereb university of debrecen, department of biophysics and cell biology, debrecen, hungary platelet derived growth factor receptors (pdgfr) play an important role in proliferation and survival of tumor cells. pdgf-bb stimulation caused a redistribution of pdgf receptors towards gm rich domains, which was more prominent in confluent monolayers. pdgf-bb stimulation significantly increased relative receptor phosphorylation of the ras / mapk pathway specific tyr residues and the pi -kinase / akt pathway specific tyr residues in nonconfluent cultures. tyr residues that serve as adaptors for ras-gap which inactivates the mapk pathway and tyr residues feeding into the plc-gamma / camk-pkc pathway were the docking sites significantly hyperphosphorylation following ligand stimulation in confluent cells. we found that p-akt facilitated cell survival and pmapk dependent proliferation is more activated in dispersed cells, while phospholipase c-gamma mediated calcium release and pkc-dependent rhoa activation are the prominent output features pdgf stimulus achieves in confluent cultures. these observations suggest that the same stimulus is able to promote distinctly relevant signaling outputs, namely, cell division and survival in sparse cultures and inhibition of proliferation joined with promotion of migration in confluent monolayers that appear contact inhibited. a thermodynamic approach to phase coexistence in ternary cholesterol-phospholipid mixtures jean wolff, carlos m. marques and fabrice thalmann institut charles sadron, université de strasbourg, cnrs upr, , rue du loess, strasbourg cedex, f- , france e-mail: thalmann@ics-cnrs.unistra.fr we present a simple and predictive model for describing the phase stability of ternary cholesterol-phospholipid mixtures. assuming that competition between the liquid and gel organizations of the phospholipids is the main driving force behind lipid segregation, we derive a phenomenological gibbs free-energy of mixing, based on the calorimetric properties of the lipids main transition. gibbs phase diagrams are numerically obtained that reproduces the most important experimental features of dppc-dopc-chol membranes, such as regions of triple coexistence and liquid orderedliquid disordered segregation. based on this approach, we present a scenario for the evolution of the phase diagram with temperature. results for other phospholipid species, such as popc or psm will also be presented. interleukin- and - receptors play a central role in the activation, survival and death of t lymphocytes. they form supramolecular clusters with mhc i and ii glycoproteins in t cells. in damaged or inflamed tissues the extracellular k+ concentration increases, which can depolarize the membrane. the common signaling beta and gamma chains of il- / r are phosphorylated upon cytokine binding and get a permanent dipole moment, thus their conformation, interactions, mobility and activity may be sensitive to the membrane potential. we induced depolarization on ft . t lymphoma cells by increasing the ec. k+ level or by blocking kv . voltage gated k+ channels with margatoxin. fcs measuremens showed that the lateral mobility of fab-labeled il- / r and mhc i and ii decreased upon depolarization, while that of gpi-linked cd did not change. fret efficiency measured between some elements of the il-receptor/mhc cluster increased, which may reflect an increase of cluster size. il- -induced receptor activity, as monitored by measuring stat -phosphorylation, increased upon depolarization, whereas il- induced phosphorylation did not change. our results may reveal a novel regulatory mechanism of receptor function by the membrane potential. cytokines play an important role in t cell activation and immunological memory, whereas mhcs are known for the role in antigen presentation. we applied rnai to silence the expression of mhc i in order to study its possible role in receptor assembly and function. fret data indicated that the association of il- r and il- r with mhc i as well as between il- r and il- r weakened. fcs indicated an increase of receptor mobility also suggesting the partial disassembly of the clusters. mhc i gene silencing lead to a remarkable increase of il- /il- induced phosphorylation of stat transcription factors. in search for the molecular background of this inhibition of signaling by mhc i we checked il- binding and the formation of the receptor complex (il- r alpha -il- r beta association), but we did not find a difference as compared to the control. our results suggest that mhc i plays an organizing role in maintaining supramolecular receptor clusters and inhibits il- r signaling, revealing a nonclassic new function of mhc i beyond its classical role in antigen presentation. interleukin- (il- ) is an important cytokine involved in adaptive immunity. il- binds with high affinity the singlepass transmembrane receptor il- ra. the occupied complex, il- /il- ra then engages either il- rc or il- ra , to form an activated type i or ii receptor, respectively. this formation of heterodimers is believed to trigger cross-activation of intracellular janus kinases. here we follow a fluorescently labeled ligand through various stages of receptor activation in hek t: using fluorescence correlation spectroscopy (fcs), we see that the receptor chains diffuse as monomers within the plasma membrane. using dual-color fccs provides direct evidence for ligand induced co-diffusion of occupied il- ra and il- ra . in contrast, type i complexes containing il- rc could not be observed. however, ectopic expression of gfp-tagged il- rc/jak induced stable fluorescent speckles in or close to the plasma membrane. we identified these structures as early sorting endosomes by colocalization of surface markers like eea and rab gtpases. the il- ra chain is continuously trafficking into these compartments. these observations suggest that the formation of a type i il- r heterodimer may require internalization and that early endosomes serve as a platform for il- signaling. among the membrane associated proteins, the ras family, which is lipid-anchored g protein, plays a key role in a large range of physiological processes and, more importantly, is deregulated in a large variety of cancer. in this context, plasma membrane heterogeneity appears as a central concept since it ultimately tunes the specification and regulation of ras-dependent signaling processes. therefore, to investigate the dynamic and complex membrane lateral organization in living cells, we have developed an original approach based on molecule diffusion measurements performed by fluorescence correlation spectroscopy at different spatial scales (spot variable fcs, svfcs) ( ). we have shown in a variety of cell types that lipidbased nanodomains are instrumental for cell membrane compartmentalization. we have also observed that thesenanodomains are critically involved in the activation of signaling pathways and are essential for physiological responses ( - ). more recently, weextend the application of svfcs to characterizethe dynamics of k-rasproteinat the plasma membrane. as major result, we demonstrated that the rate of k-ras association/dissociation from the membrane is fast but vary as a functional of the activation state of the molecule as well as of specific intracellular protein interactions. we have so demonstrated that an helical lid sub-domain in the sbd is essential for monomeric as binding, but not for the anti-aggregation activity of the chaperone, suggesting that hsp is able to interact with pre-fibrillar oligomeric species formed during as aggregation and that, then, the mechanism of binding for these species is different from that of the monomeric protein. aggregation of the acylphosphatase from sulfolobus solfataricus (sso acp) into amyloid-like protofibrils is induced by the establishment of an intermolecular interaction between a -residue unfolded segment at the n-terminus and the globular unit of another molecule. we have used data from hydrogen/deuterium exchange experiments, intermolecular paramagnetic relaxation enhancements and isothermal titration calorimetry measurements on an aggregation-resistant sso acp variant lacking the -residue n-terminus to characterize the initial steps of the aggregation reaction. under solution conditions that favour aggregation of the wild-type protein, the truncated protein was found to interact with a peptide corresponding to the n-terminal residues of the full length protein. this interaction involves the fourth strand of the main b-sheet structure of the protein and the loop following this region and induces a slight decrease in protein flexibility. we suggest that the amyloidogenic state populated by sso acp prior to aggregation does not present local unfolding but is characterized by increased dynamics throughout the sequence that allow the protein to establish new interactions, leading to the aggregation reaction. amyloid-like aggregates alter the membrane mobility of gm gangliosides martino calamai and francesco pavone university of florence, lens -european laboratory for non-linear spectroscopy, sesto fiorentino, florence, italy neuronal dysfunction in neurodegenerative pathologies such as alzheimer's disease is currently attributed to the interaction of amyloid aggregates with the plasma membrane. amongst the variety of toxic mechanisms proposed, one involves the binding of amyloid species to gm gangliosides. gm takes part into the formation of membrane rafts, and exerts antineurotoxic, neuroprotective, and neurorestorative effects on various central neurotransmitter systems. in this study, we investigated the effects of amyloid-like aggregates formed by the highly amyloidogenic structural motif of the yeast prion sup (sup nm) on the mobility of gm on the plasmamembrane of living cells. preformed sup nm aggregates were incubated with cells and gm molecules were subsequently labeled with biotinylated ctx-b and streptavidin quantum dots (qds). single qds bound to gm were then tracked. the mobility of gm was found to decrease dramatically in the presence of sup nm aggregates, switching from brownian to mainly confined motion. the considerable interference of amyloid-like aggregates with the lateral diffusion of gm might imply a consequent loss of function of gm , thus contributing to explain the toxic mechanism ascribed to this particular interaction. insights into the early stages of fibrillogenesis of insulin using mass spectrometry harriet l. insulin is a vital hormone in metabolic processes as it regulates the glucose levels in the body. insulin is stored in the b cells of the pancreas as a hexamer, however its biologically active form is the monomer. the formation of fibrillar aggregates of insulin rarely occurs in the body; however localised amyloidosis at the site of injection for diabetes patients and aggregation of pharmaceutical insulin stocks present problems. in the current study oligomers formed early in the process of fibril assembly in vitro are observed by mass spectrometry (ms). ms is the only technique which allows early species to be characterised as it can identify different oligomeric orders by mass to charge ratio and show protein abundance and aggregation propensity. on mobility ms is used to examine rotationally averaged collision cross sections of oligomers in the aggregating solution. a wide array of oligomers is observed and the stability of specific species is remarked. the presence of multiple conformations for the highly charged oligomers is particularly noted and their assignment confirmed using fourier transform ion cyclotron resonance ms and collision induced dissociation. molecular modelling has been used to further explore the conformational space the oligomers inhabit. amyloid fibrils consisting of different proteins have been recognized as an accompanying feature of several neurodegenerative diseases. many proteins without known connection to any diseases have been found to form amyloid fibrils in vitro, leading to suggestion that the ability to form fibrils is the inherent property of polypeptide chain. the observed common character of protein amyloid formation enables to seek further clues of fibrillation mechanism by studying generalized sequenceless polypeptide models, e.g. polylysine. we have studied conformational transitions of polylysine, with different chain length at various ph, ionic strength and temperature by means of novel approachviscometric method. this polypeptide undergoes alfa-helix to beta-sheet transition and forms amyloid fibrils in special conditions. temperature induced a-helix to b-sheet transitions occurs at ph interval form to . and with increasing chain length is slightly shifted to the lower ph. we have found narrow ph interval, in which the thermal transition is fully reversible, suggesting the high sensitivity of polypeptide conformation on subtle changes in charge on its side chains. this work was supported within the projects vega , and , cex sas nanofluid and by esf project no. . understanding the mechanisms of the conversion from the native state of a protein to the amyloidal state represents a fundamental step in improving the purification, storage and delivery of protein-based drugs and it is also of great relevance for developing strategies to prevent in vivo protein aggregation. amyloid fibrils have a structural arrangement of cross b-sheet but they can also experience different packing into three dimensional superstructures, i.e. polymorphism. it is well known that, among others, both the geometric confinement of the molecules and shear forces can affect the final morphology of the aggregates. importantly, due to the complexity and crowding of the cellular region, such parameters also play a crucial role in in vivo processes. we present an experimental approach to study in vitro amyloid aggregation in a controlled and uniform shear force field and within microscale environments. in particular we focus on the effect of these two parameters on the formation of spherical aggregates, known as spherulites. using micro channels of different cross-sections from to lm x lm and flow rates in the range of hundreds of ll/min, the number and diameter of spherulites within the channels have been characterized using crossed polarizers optical microscopy. inhibition of insulin amyloid fibrillization by albumin magnetic fluid k. insulin amyloid aggregation causes serious problems for patient with insulin dependent diabetes undergoing long-term treatment by injection, in production and storage of this drug and in application of insulin pumps. recent studies indicate that protein amyloid aggregation causes the cell impairment and death; however, the prevention of amyloid aggregation is beneficial. we have investigated ability of albumin magnetic fluid (amf) to inhibit insulin amyloid aggregation by spectroscopic and microscopic techniques. albumin magnetic fluid consists of magnetic fe o nanoparticles sterically stabilized by sodium oleate and functionalized with bovine serum albumin (bsa) at various weight ratios bsa/fe o . we have found the positive correlation between inhibiting activity of afm and nanoparticle diameter and zeta potential. the ability of amf to inhibit formation of amyloid fibrils exhibits concentration dependence with ic values comparable to insulin concentration. the observed features make amf of potential interest as agents effective in the solving of problems associated with insulin amyloid aggregation. (this work was supported within the projects vega , and , cex sas nanofluid, apvv- - , sk-ro- - and esf project ). amyloid formation of peptides causes diseases like alzheimer's and parkinson's disease. however, the conditions for the onset of the neurotoxic beta-sheet formation are poorly understood. we focus on aggregation triggers and their interplay: interactions with hydrophobic-hydrophilic interfaces, orientation of peptides in d, metal ion complexation and lipid layers. the tailor-made model peptides exhibit defined secondary structure propensities and metal ion binding sites. the interactions of the peptide with the air-water interface and with metal ions are studied using surface sensitive methods connected to film balance measurements. x-ray diffraction, x-ray reflection, infrared reflection-absorption spectroscopy and total reflection x-ray fluorescence were applied to reveal the layer structure, peptide conformations and metal ion binding at the interface. we found that amyloid formation in d is dominated by the hydrophobic-hydrophilic interface and not comparable to the bulk behaviour. the interface can enhance or inhibit betasheet formation. the effect of metal ion complexation depends on the arrangement of the binding sites in the peptide and the preferred metal complexation geometry. the two triggers interface and metal ion complexation, can oppose each other. effect of apoe isoform and lipidation status on proteolytic clearance of the amyloid-beta peptide hubin, e. alzheimer's disease (ad) is the most common type of dementia in the elderly. the most important genetic risk factor identified for ad is the isoform, e , e or e , of apolipoprotein e (apoe), a lipid-carrying protein. one hallmark of ad is the accumulation of amyloid-beta peptide (ab) in the brain which is thought to result from an imbalance between the production of ab and its clearance. previous studies report an important role for apoe in ab degradation. we sought to determine the effect of apoe isoform and lipidation status on the degradation of soluble ab by proteinases such as insulin-degrading enzyme and neprilysin. in this study an in vitro ab clearance assay based on the competition between ab and a fluorogenic peptide substrate is developed to quantify ab degradation. to elucidate the proteolytic clearance mechanism, the fragments resulting from cleavage are identified by mass spectrometry and further analyzed to identify the interacting stretch of the ab sequence with the different apoe isoforms. the results suggest that apoe influences the rate of ab degradation. the aggregation of proteins into fibrillar nanostructures is a general form of behaviour encountered for a range of different polypeptide chains. the formation of these structures is associated with pathological processes in the context of alzheimer's and parkinson's diseases but is also involved in biologically beneficial roles which include functional coatings and catalytical scaffolds. this talk focuses on recent work directed at understanding the kinetics of this process through the development and application of experimental biophysical methods and their combination with kinetic theories for linear growth phenomena. lbs contains not only as, but also other proteins including - - proteins. - - proteins exist mainly as a dimer and its exact functions are remain unclear. however, recent work has shown that the association of - - (eta) with as in lbs. herein we show how - - (eta) can modulate as in vitro aggregation behavior, by rerouting it toward the formation of stable non-fibrillar aggregates. we also show that the resulting populations of fibrillar and pre-fibrillar aggregates exhibit a modified toxicity in vivo with respect to the unperturbed aggregates. interestingly, - - (eta) does not show any binding affinity for monomeric as, nor for the mature fibrillar aggregates. we provide evidence that it acts on the oligomeric species which form during the amyloidogenesis process of aggregation. since - - (eta) can influence the toxicity of amyloidogenesis without perturbing the functional as monomers, we are convinced that once fully understood, its mode of action could represent a promising model to mimic with synthetic drugs and peptides. what makes an amyloid toxic: morphology, structure or interaction with membranes? more than human diseases are related to amyloids. in order to understand why some amyloids may become toxic to their host and some others are not, we first developed a genetical approach in the yeast saccharomyces cerevisiae. we have chosen the amyloid/prion protein het-s prion forming domain ( - ) from the fungi podospora anserina, which is not toxic in yeast. some toxic amyloids mutants were generated by random mutagenesis. in vitro the most toxic mutant called ''m '' displays very peculiar nanofibers, which polymerized mainly in amyloid antiparallel b-sheets whereas the non-toxic wt exhibits a parallel polymerization. we further established the dynamic of assembling of the m toxic amyloid, in comparison to the wt non-toxic amyloid, and showed the presence of specific oligomeric intermediates also organized in antiparallel b-sheet structures. a more global structure/toxicity study on more than mutants clearly identified an antiparallel b-sheet signature for all the toxic mutants. therefore size, intermediates and antiparallel structures may account for amyloid toxicity in yeast but we still wonder what their cellular targets are. recently, we established the first evidences that toxic mutants may specifically bind in vitro to lipids, particularly negatively charged. interconnected mechanisms in abeta ( - ) we present an experimental study on the fibril formation of ab( - ) peptide at ph . . the kinetics of this process is characterized by the occurrence of multiple transient species that give rise to final aggregates whose morphology and molecular structure are strongly affected by the growth conditions. to observe in details the aggregation pathway as a function of solution conditions, we have used different experimental techniques as light scattering, thioflavin t fluorescence, circular dichroism and two-photon fluorescence microscopy. this approach gives information on the time evolution of conformational changes at molecular level, on the aggregates/fibrils growth and on their morphologies. the selected experimental conditions allowed us to highlight the existence of at least three different aggregation mechanisms acting in competition. a first assembly stage, which implies conformational conversion of native peptides, leads to the formation of small ordered oligomers representing an activated conformation to proceed towards fibril growth. this process constitutes the rate limiting step for two distinct fibril nucleation mechanisms that probably implicate spatially heterogeneous mechanisms. the formation of amyloid fibrils of amylin - was studied by means of molecular dynamics (md) and energy partition on three peptide ß-sheet stack systems with the same amino acid composition: wild type amylin - (amyl - ), reverse amylin - (rev-amyl - ) and scrambled amylin version scr-amyl - . the results show that for amylin - peptides, amino acid composition determines the propensity of a peptide to form amyloid fibrils independent of their sequence. the sequence of amino acids defines the shape and the strength of amyloid protofibril, which conforms with the atom force microscope (afm) data [ ] . md show that the x revamyl- - stack has looser selfassembly than the x amyl- - stack, which conforms with the results of fourier transform infrared spectroscopy (ftir) measurements for the peptides studied [ ] . the results of md show that x amyl- - could have a turn, which consists with ftir data [ ] . data on ab aggregation kinetics have been characterized by a large spread between experiments on identical samples that contain so many molecules that stochastic behaviour is difficult to explain unless caused by uncontrolled amounts of impurities or interfaces. we have therefore spent considerable effort to eliminate sources of inhomogeneity and reached a level of reproducibility between identical samples and between experiments on separate occasions that we can now collect data that can lead to mechanistic insights into the aggregation process per se, and into the mechanism of action of inhibitors. data on ab aggregation will be shown that give insight into the influence of physical parameters like peptide concentration, shear and ionic strength, as well as the effect of inhibitory proteins, model membranes and the effects of sequence variations. monte carlo simulations of amyloid formation from model peptides corroborate the finding from experiments and underscore that the very high level of predictability and reproducibility comes from multiple parallel processes. negatively-charged membranes were reported to catalyze ''amyloid-like'' fiber formation by non-amyloidogenic proteins [ ] . our study aims to elucidate the factors that govern the formation of these amyloid-like fibers. lysozyme was selected as a model of non-amyloidogenic protein and was fluorescently-labeled with alexa fluor (a -lz). first, a -lz partition towards phosphatidylserine-containing liposomes was characterized quantitatively using fluorescence correlation spectroscopy (fcs), in order to calculate the protein coverage of liposomes. secondly, the interaction between a -lz and negatively-charged lipid membranes was studied using both steady-state and time-resolved fluorescence techniques. this interaction was found to switch from a peripheral binding to the anionic headgroups, at high lipid/ protein molar ratio (l/p), to a partial insertion of protein into the hydrophobic core of the membrane, at low l/p. finally, the lipidprotein supramolecular complexes formed at low l/p were characterized by fluorescence lifetime imaging microscopy (flim). the mean lifetime of a -lz in these supramolecular structures is much lower compared to the values obtained for the free and bound a -lz at high l/p. the fiber characterization will be complemented by fcs studies. [ the conversion of normal prp c to its pathological isoform prp sc is a key event in prion diseases and is proposed to occur at the cell surface or more probably in acidic late endosomes. a convergence of evidence strongly suggests that the early events leading to the structural conversion of the prp seem to be in relation with more or less stable soluble oligomers, which could mediate neurotoxicity. as commonly shared by other amyloidogenic proteins, membrane-bound monomers undergo a series of lipid-dependent conformational changes, leading to the formation of oligomers of varying toxicity rich in b-sheet structures (annular pores, amyloid fibrils). here, we have used a combination of biophysical techniques (dynamic and static light scattering, fluorescence techniques, and quartz crystral microbalance) to elucidate the interaction of native monomeric prp and that of purified b-rich oligomeric prp on model lipid membranes. under well established conditions, three b-sheet-rich oligomers were generated from the partial unfolding of the monomer in solution, which were found to form in parallel. from single mutation and/or truncation of the full length prp, the polymerization pathway is strongly affected, revealing the high conformational diversity of prp. in our previous work, we identify the minimal region of the prp protein leading to the same polymerization pattern of the full length prp. soluble -subunits and -subunits oligomers were obtained depending on the single mutation or truncation and purified. we compare their structural properties (ftir, cd) when associated with anionic lipid bilayers and study their propensities to permeabilize the membrane. fluorescence kinetics suggest different mechanisms of membrane perturbation for the monomer and the prp oligomers. deciphering this complex network of lipid interactions and conformational diversity of the prp protein will help for understanding of how amyloidogenic proteins induce neurotoxicity. the traditional view of the lipid bilayer described as a ''sea'' of lipids where proteins may float freely, is going to be inadequate to describe the increasingly large number of complex phenomena which are known to take place in biological membranes. membrane-assisted protein-protein interactions, formation of lipid clusters, protein-induced variation of the membrane shape, abnormal membrane permeabilities and conformational transitions of membrane-embedded proteins are only a few examples of the variegated ensemble of events whose tightly regulated cross-talk is essential for cell structure and function. experimental work on the above mentionated problems is very difficult and some time not accessible, especially when the studied systems have a fast dynamics. due to the large size of the systems usually involved in this multifaceted framework, a detailed molecular description of these phenomena is beyond the possibilities of conventional amyloid aggregation, a generic behavior of proteins, is related to incurable human pathologies -amyloid-related diseases, associated with formation of amyloid deposits in the body. all types of amyloid aggregates possess rich bsheet structural motif. the recent data confirm the toxic effect of aggregates on the cells, however, it was found that reduction of amyloid aggregates plays important role in prevention as well as therapy of amyloidosis. we have investigated effect of phytoalexin derivatives on amyloid fibrillization of two proteins, human insulin and chicken egg lysozyme, by tht and ans fluorescence assays. we have found that amyloid aggregation of both studied proteins was significantly inhibited by phytoalexin derivates cyclobrassinin and benzocamalexin. for most effective phytoalexins the estimated ic values were at low micromolar concentration. the observed inhibiting activity was confirmed by transmission electron microscopy. our data suggest the potential therapeutic use of the most effective phytoalexins in the reduction of amyloid aggregation. (this work was supported within the projects vega , , cex sas nanofluid, apvv- - , sk-ro- - and esf project ). the amyloid pore hypothesis suggests that interactions of oligomeric alpha-synuclein (as) with membranes play an important role in parkinson's disease. oligomers are thought to permeabilize membranes and interfere with ca + pathways. permeabilization by as requires the presence of negatively charged phospholipids. whether as can bind and permeabilize membranes with physiologically relevant lipid compositions has not been extensively explored. here we report on the binding of as to giant unilamellar vesicles (guvs) with physiologically relevant lipid compositions. comparing different protocols of oligomer preparation, leakage assays on both large unilamellar vesicles (calcein release) and guvs (hpts efflux assay) show that as is not able to permeabilize these membranes. the presence of cholesterol has a stabilizing effect on these membrane systems. in agreement with these findings, we do not observe concentration dependent as toxicity using in vivo mts assays. however, in the calcein release assay, different as preparations show differences in kinetics and as concentrations that cause % leakage. these results motivate us to critically reassess the amyloid pore hypothesis, and suggest that membrane permeabilization may be attributable only to a very specific as species. alpha-synuclein oligomers impair membrane integrity-a mechanistic view martin t. stö ckl, mireille m. a. e. claessens, vinod subramaniam nanobiophysics, mesa+ institute for nanotechnology, university of twente, enschede, the netherlands one of the most prevalent neurodegenerative diseases is parkinson's disease (pd), which is accompanied with the loss of dopaminergic neurons. although the mechanisms leading to the death of these cells are still unclear, the protein alpha-synuclein (as) is one of the pivotal factors. previous studies indicate that especially oligomeric forms of as show a detrimental effect on membrane integrity. as an intact membrane is crucial to many cellular processes, the impairment of the membrane integrity is a likely pathway for neuronal death. we use different phospholipid bilayer model systems to investigate the mechanisms underlying this process. atomic force microscopy in combination with suspended asymmetric phospholipid bilayers, which closely mimic the plasma membrane, allows the identification of the binding sites, the measurement of penetration depths of the as oligomers into the phospholipid bilayer, and the detection of membrane thinning or creation of membrane defects. using an approach based on phospholipid vesicles we were able to observe for the first time that as oligomers cause an enhanced lipid-flip flop. suggesting that the loss of lipid asymmetry is a novel mechanism which may contribute to or trigger neuronal death in pd. amyloid protein-membrane interactions and structuretoxicity relationship study h.p. ta (toxic) has a much higher and more specific effect on negatively charged phospholipids (dops, dopi and dopg) than in the case of wt (non-toxic). therefore the insertion of protein into phospholipid monolayers, which occurred similarly for both wt and m , is not a key factor for these effects (h.p. ta et al. langmuir, in press). we are now using unilamellar vesicles as a membrane model to investigate the amyloid protein (toxic and nontoxic) -phospholipid interactions. results confirmed the high specific and strong effects of m on negatively-charged membrane. in this project, we study the chemical, physical and biological properties of fibrillar networks. the formation and the network mechanics are investigated by combining droplet-based microfluidics with optical microscopy and small angle x-ray scattering (saxs). the chosen system, fibrin network formation, plays an important role in blood coagulation processes. crosslinking of fibrinogen induced by an enzymatic reaction with thrombin leads to d fibrin network formation. the fibrillar networks are formed within picoliter droplets of aqueous solutions in an continuous oil phase. droplets containing fibrinogen and thrombin can be produced of different sizes and stored for fibrin network formation. the formation of the fibrillar networks is imaged by fluorescence microscopy. to analyze the elastic properties of the networks, the droplets flow through a microchannel device of alternating width in order to squeeze and stretch the networks. additionally, saxs experiments will give structural information about the molecular dimensions of the networks. the amyloid beta peptide (ab), implicated in alzheimer's disease (ad), is released from the amyloid precursor protein (app) by secretase-induced cleavage. this process results in the release of a range of ab peptides varying in length. the brains of ad patients often contain longer ab peptides while the total concentration of ab is unaffected. longer peptides are more hydrophobic having far-reaching consequences for their toxicity and aggregation. as ab is necessary for normal neuronal function, research activities into ad therapeutic development currently explore the possibilities of modulating c-secretase activity to produce short ab peptides. whether such an approach effectively ameliorates the toxic effect of ab has not been explored yet. to answer this question, we studied the impact of heterogeneity in ab pools in an in vitro biophysical and in cellulo context using microelectrode array to assay the synaptic activity of primary neurons. we show that various lengths of the ab peptide and mixtures thereof aggregate with distinct kinetics and notoriously affect synaptotoxic and cytotoxic response. we also show that small amounts of less abundant peptides ab and ab induce aggregation and toxicity of ab while the behavior of ab is unaffected. one of the most important irreversible oxidative modifications of proteins is carbonylation, a process of introducing the carbonyl group in reaction with reactive oxygen species. importantly, carbonylation increases with the age of cells and is associated with the formation of intracellular protein aggregates and the pathogenesis of age-related disorders such as neurodegenerative diseases and cancer. however, it is still largely unclear how carbonylation affects protein structure, dynamics and aggregability on the atomic level. here, we use classical molecular dynamics simulations to study structure and dynamics of the carbonylated headpiece domain of villin, a key actin-organizing protein. we perform an exhaustive set of molecular dynamics simulations of native villin headpiece together with every single combination containing carbonylated versions of its seven lysine, arginine and proline residues, the quantitatively most important carbonylable amino acids. surprisingly, our results suggest that high levels of carbonylation, far above those associated with cell death in vivo, may be required to destabilize and unfold protein structure through the disruption of specific stabilizing elements, such as salt bridges or proline kinks, or tampering with the hydrophobic effect. on the other hand, by using thermodynamic integration and molecular hydrophobicity potential approaches, we quantitatively show that carbonylation of hydrophilic lysine and arginine residues is equivalent to introducing hydrophobic, chargeneutral mutations in their place, and, by comparison with experimental results, demonstrate that this by itself significantly increases intrinsic aggregation propensity of both structured, native proteins and their unfolded states. finally, our results provide a foundation for a novel experimental strategy to study the effects of carbonylation on protein structure, dynamics and aggregability using site-directed mutagenesis. septins are an evolutionarily conserved family of gtp-binding proteins involved in important cellular processes, such as cytokinesis and exocytosis, and have been implicated in neurological diseases, such as alzheimer's and parkinson's diseases. the focus of this study was two septins of schistosoma mansoni, (the causative agent of schistosomiasis in south america) named smsept and smsept , which were produced in a recombinant system. our objective was to verify if these septins from a simpler organism display similar characteristics to human septins. analysis of protein structure by circular dichroism showed that both recombinant smseptins produced were folded. the gtpase activity assay showed that smsept was able to hydrolyze gtp, whereas smsept was not. aggregation studies for amyloid fibril detection by right angle light scattering and thioflavin t fluorescence assay were performed. both proteins showed a temperature dependent increase in light scattering and fluorescence emission of tht probe. this indicated that s. mansoni septins tend to aggregate into amyloid-like fibers in high temperatures, with thresholds of °c for smsept and °c for smsept . these results are in accordance to that previously reported for human septins. in our work we investigated the response to standard chemotherapy of blood lymphocytes of patients suffering with melanoma. dna single and double strand breaks were determined using comet assay; intracellular levels of marker proteins were detected using immunocytochemistry. ultimately this set of parameters allows to characterize two mechanisms of dna repair (base excision repair, ber and mismatch repair, mmr) which together with apoptosis proneness underlie response of tumor cells to chemotherapy. cell death caused by o mg adducts is promoted by mmr system by inducing unrepaired double strand breaks in dna. there was a linear correlation between the level of dsdna breaks in lymphocytes after -st cycle of chemotherapy and mmr efficiency in them. the level of double strand breaks in dna after -st cycle of chemotherapy is predictive of clinical outcome. otherwise damage at the level of ssdna (ap-sites and single strand breaks) and ber mechanism associated with it couldn't be a good prognostic factor of chemotherapy. high level of double strand breaks in dna in blood lymphocytes of melanoma patients hours after -st cycle of chemotherapy appears to be a marker of a good prognosis. self-assembly and stability of g-quadruplex: counterions, pressure and temperature effects e. baldassarri jr., p. mariani, f. spinozzi, m. g. ortore saifet dept. & cnism, marche polytechnic university, ancona, italy the important role of g-quadruplex in biological systems is based on two main features: composition and stability of telomeres, and activity of telomerase. the g-quadruplex structures are formed by supramolecular organization of basic units called g-quartets that are planar rings constituted by four guanosines linked by hoogsten hydrogen bonds. gquadruplex requires the presence of monovalent cations playing a key role in stabilizing these structures, since they give rise to coordination bonds needed for the stacking of more tetrads. we performed x-ray diffraction experiments at different pressures (ranging from to bar), and small angle x-ray scattering (saxs) changing the temperature (between - °c retinoic acid receptor (rar) is a member of the nuclear receptor superfamily. this ligand-inducible transcription factor binds to dna as a heterodimer with the retinoid x receptor (rxr) in the nucleus. the nucleus is a dynamic compartment and live-cell imaging techniques make it possible to investigate transcription factor action in real-time. we studied the diffusion of egfp-rar by fluorescence correlation spectroscopy (fcs) in order to uncover the molecular interactions determining receptor mobility. in the absence of ligand we identified two distinct species with different mobilities. the fast component has a diffusion coefficient of d = . - lm /s corresponding to small oligomeric forms, whereas the slow component with d = . - . lm /s corresponds to interactions of rar with the chromatin or other large structures. the rar ligand binding domain fragment also has a slow component probably as a result of indirect dna-binding via rxr, with lower affinity than the intact rar:rxr complex. importantly, rar-agonist treatment shifts the equilibrium towards the slow population of the wild type receptor, but without significantly changing the mobility of either the fast or the slow population. by using a series of mutant forms of the receptor with altered dna-or coregulator-binding capacity we found that the slow component is probably related to chromatin binding, and that coregulator exchange, specifically the binding of the coactivator complex, is the main determinant contributing to the redistribution of rar during ligand activation. formation of inactive nuclear with high level of dna compaction in sperm cells is accompanied by a substitution of linker histones h by a number of other proteins. among them sperm-specific histones (ssh), which are characterized by elongated arginine-rich polypeptide chain compared to the somatic h . the secondary and tertiary structure of the ssh and their interactions with dna were studied using spectroscopic and thermodynamic approaches. the histones were isolated from sperm of marine invertebrates and rat thymus. all studied ssh demonstrate no considerable compaction of dna in solutions of low ionic strength. however, at physiological conditions, ssh h from s.intermedius and a.japonica compact dna more intensively than other ssh. the somatic h from rat thymus revealed a minimal ability to compact the dna. we suggest that the ssh h are able to interact with dna not only in the major groove but also in the minor groove of the double helix inducing considerable structural changes in dna and facilitating the formation of the supercompact sperm chromatin. the authors are grateful for the financial support from the russian foundation for basic research (grants § - - and - - ) and from administration of saint-petersburg. ionizing radiation causes modification and destruction of nitrogenous bases in dna molecule. there are also local breakages of hydrogen bonds both in the lesion sites mentioned above and in other sites of the macromolecule. to reveal the amount of some of these damages we applied cd and uv absorption spectroscopy. radiation-induced changes in dna structure influence its uv absorption spectrum in different ways: partial denaturation causes hyperchromic effect, while destruction of the bases results in hypochromic shift. at the same time both of them result in the same changes in dna cd spectra: the decrease in intensity. we attempted to segregate the described damages in dna structure and studied the influence of dna ionic surroundings on the radiation effect. it is shown that the radiation efficiency of base destruction and partial denaturation increases with decreasing concentration of nacl in irradiated solution. udu (ugly duckling) has been first identified from a zebrafish mutant and shown to play an essential role during erythroid development; however, its roles in other cellular processes remain largely unexplored. facs analysis showed that the loss of udu function resulted in defective cell cycle progression and comet assay indicated the presence of increased dna damage in udu mutants. we further showed that the extensive p -dependent apoptosis found in udu mutants is a consequence of activation in the atm-chk pathway. udu appears not to be required for dna repair, because both wild-type and udu embryos similarly respond to and recover from uv treatment. yeast two-hybrid and coimmunoprecipitation data demonstrated that pah-l repeats and sant-l domain of udu interacts with mcm and mcm . furthermore, udu was colocalized with brdu and heterochromatin during dna replication, suggesting a role in maintaining genome integrity. recently, we started to work on the second zebrafish homolog, udu , and its mammalian counterpart, gon l. preliminary data showed that udu and gon l mrna injection can rescue zebrafish udu mutant phenotypes. furthermore, pah-l and sant-l domains of udu and gon l can bind to mcm and mcm and they are localized in the nucleus. these data suggest that udu and gon l are functionally equivalent to zebrafish udu. their molecular mechanism leading to udu phenotypes is currently under investigation. chromatin condensation: general polyelectrolyte association and histone-tail specific folding nikolay korolev, nikolay berezhnoy, abdollah allahverdi, renliang yang, chuan-fa liu, james p. tam, lars nordenskiö ld school of biological sciences, nanyang technological university, nanyang drive, , singapore the major component of chromatin, dna, is a densely charged polyanion. electrostatic interactions between dna and dnapackaging proteins contribute decisively to formation of its elementary unit, the nucleosome, and are also important in chromatin folding into higher-order structures. we investigate condensation of dna and chromatin and find that electrostatics and polyelectrolyte character of dna play dominant role in both dna and chromatin condensation. by comprehensive experimental studies and using novel oligocationic ligands, we suggest simple universal equation describing dna condensation as a function of oligocation, dna and monovalent salt concentrations and including the ligand-dna binding constant. we found that similar dependence was also observed in condensation of the nucleosome arrays. next, we studied how general electrostatic and specific structural alterations caused by lysine acetylations in the histone tails influence formation of -nm chromatin fibre and intermolecular nucleosome array association. for the first time, a structural model is suggested which explains critical dependence of chromatin fibre folding on acetylation of the single lysine at position of the histone h . exceptional importance of the h lys acetylation in general and gene specific transcriptional activation has been known for many years but no structural basis for this effect has yet been proposed. detection of specific dna sequences is central to modern molecular diagnostic. ultrasensitive raman measurements of nucleic acids are possible through exploiting the effect of surface-enhanced raman scattering (sers). in this work, the sers spectra of genomic dnas from leaves of different apple trees grown in the field, have been analyzed [ ] . a detailed comparative analysis of sers signatures of genomic dnas is given. sers wavenumbers (cm - ) are reported here for all types of vibrations of plant genomic dnas, including bands assigned to localized vibrations of the purine and pyrimidine residues, localized vibrations of the deoxyribosephosphate moiety, etc. proposed band assignments are given. a strong dependence of the sers spectra on dna concentration and on time have been observed. in biochemical fields, nucleic acids might be used to explore the interaction between dna and small molecules, which is important in connection with probing the accurate local structure of dna and with understanding the natural dnamediated biological mechanisms [ ] . the ph-dependent structure of dna studied by fourier transform infrared spectroscopy the region of the infrared spectrum studied covered the wave number range from cm - to cm - . ir spectra show that in ph . - . interval carbonyl (c=o) band at - cm (assigned to guanine) is reduced in intensity and slightly shifted to lower frequencies. at the ph . significantly decreases band intensity at cm - due to unbounded c =o of thymine and shifts to lower frequencies, indicating at the transition of this group in bounded form, supposedly by means of excess polarized hydroxyl ions. together this, in basic region a new intense absorption band has been observed in - cm - frequency interval, corresponding to o-h group in-plane bending vibration ( - cm - ). as for acidic conditions, it was observed that under the extreme ph (* . ) value carbonyl absorption region shifts to higher frequencies and absorption intensity significantly increased, indicated at releasing of c=o groups from h-bonding between base pairs. moreover, bands intensity at cm - and cm - corresponding to out-of-plan deformation of nh groups increased due to rupture of connections between the dna strands. during the last decade it was found that in many cases specific structural organization of multi-molecular protein and dna-protein complexes determines their functioning in living cells. although these functioning structures are usually unique, it is often possible to identify their common structural elements. one of the interesting examples of such universal elements are hmgb domains: structurally conservative functional domains of non-histone proteins hmgb / also identified in many nuclear proteins. using afm, thermodynamic approaches, circular dichroism and molecular absorption spectroscopy in far-uv and mid-ir regions we have studied structural organization of the complexes between dna and different proteins, including hmgb , hmgb-domain recombinant proteins and linker histone h . we have demonstrated, that interaction with dna leads to increasing both a-helicity of the proteins and thermal stability of dna. also, this interaction may result in formation of highly ordered supramolecular complexes facilitated by hmgb-domains. the c-terminal sequence of hmgb / regulates affinity of the proteins to dna and can be ''inactivated'' by interaction with histone h . based on the data obtained a model of the interaction of multy-domain hmgb-proteins with dna is suggested. darmstadt, germany, lmu biozentrum; munich, germany *these authors contributed equally to this work chromatin in living cells displays considerable mobility on a local scale. this movement is consistent with a constrained diffusion model, in that individual loci execute multiple, random jumps. to investigate the connection between local chromatin diffusion (lcd) and the changes in nuclear organization, we established a stable hela cell line expressing gfp-pcna. this protein, a core component of the replication machinery, serves as a cell-cycle marker and allows us to visualize sites of ongoing dna synthesis within the nucleus. to monitor lcd, we labeled discrete genomic loci through incorporation of cy -dutp. this experimental system, in conjunction with particle tracking analysis, has enabled us to quantitatively measure chromatin mobility throughout the cell cycle. our results demonstrate that lcd is significantly decreased in s-phase. to explore the connection between dna replication and reduced chromatin movement, we undertook a more detailed examination of lcd in s-phase nuclei, correlating chromatin mobility with sites of replication. our results demonstrate that labeled chromatin in close proximity to gfp-pcna foci exhibit significantly decreased mobility. we therefore conclude that presence of active replication forks constrains the movement of adjacent chromatin. single-molecule studies of dna replication antoine m. van oijen zernike institute for advanced materials, groningen university, nijenborgh . ag groningen, the netherlands e-mail: a.m.van.oijen@rug.nl advances in optical imaging and molecular manipulation techniques have made it possible to observe individual enzymes and record molecular movies that provide new insight into their dynamics and reaction mechanisms. in a biological context, most of these enzymes function in concert with other enzymes in multi-protein complexes, so an important future direction will be the utilization of single-molecule techniques to unravel the orchestration of large macromolecular assemblies. we are applying a single-molecule approach to study dna replication. i will present recent results of single-molecule studies of replication in bacterial and eukaryotic systems. by combining the stretching of individual dna molecules with the fluorescence observation of individual proteins, we visualize the dynamic interaction of replication factors with the fork. in the bacteriophage t replication system, we show that dna polymerases dynamically associate with and dissociate from the fork during replication. further, i will present new data from single-molecule replication studies in x. laevis oocyte extracts. we have developed a novel imaging scheme that permits single-molecule fluorescence experiments at concentrations of labeled protein that were hitherto inaccessible. using this method, we visualize, in real time, origin firing and fork movement. in force-extension diagrams of reference models of naked dna (freely jointed chain, wormlike chain) as well as extensionrotation diagrams of naked dna have been successfully recovered. of note, plectonemic structures are most efficiently simulated thanks to ode's collision detection code. new insights into nucleosome and chromatin fiber structure and dynamics will be presented. the study of the pkm. plasmid effect on the repair of dna j. vincze , i, francia , g. vincze-tiszay hheif, budapest, hungary, univ. debrecen, debrecen, hungary in our experiments was studied the effect of pkm. plasmid on repair of single strand breaks in dna induced by cogamma irradiation in e.coli k ab (wild type) and its different rec mutant cells. the pkm. resistant-factor in case of the control decreases the sensitivity of radiation, which as we suppose, is reached by the help of dna conformation change. it can be supposed from the well known effect of radiation biology that by the effect of pkm. , the ratio of dna radiation sensitive volumes by appearing its new conformation decreases. the pkm. r-factor in rec mutants in case of gamma irradiation shows effects in two ways. one is the ''chemical'' connection between the r-factor and dna, though the other relate to positive and negative ''induced'' radiation resistance from the local type effect of the connection of an r-factor and a rec mutant, and the two radiation resistant effects are added algebraically. as a result from the view of biology we have to categorize the radiation resistance and the connected repair processes as two different classes according to the change either in the chemical or in the induced radiation resistant effect. recent studies have indicated that two trimethylated peptides (k , k ), derived from the parental hybrid peptide ca( - )m( - ), strongly interact with a bacterial membrane model (mixture of zwitterionic and negatively charged lipids), but not with a membrane model of mammalian erythrocytes (zwitterionic lipids) [ ] . a reduction of the cytotoxicity effect and an improvement of the therapeutic index have also been reported for the derivatives when compared with the parental ca( - )m( - ) [ ] . in this work, with the aim of providing insight on the interaction phenomena of the indicated peptides with zwitterionic and negatively charged membrane models, a systematic molecular dynamics study was carried out. full hydrated bilayers of dmpc:dmpg ( : ) and pope:popg( : ) were studied in the presence of each peptide, and results analyzed in terms of peptide structure and membrane composition. lipid-water and lipid-lipid interactions at the membrane/water interface play important role in maintaining the bilayer structure, however, this region is not easily available for experimental studies. we performed molecular dynamics simulations of two bilayers composed of two different types of lipids: ( ) dioleoylphosphatidylcholine (dopc); ( ) galactolipid monogalactosyldiacylglycerol (mgdg). to investigate the properties of the membrane/water interface region, we performed analysis of lipid-lipid interactions: direct, via charge pairs (dopc) and hydrogen bonds (mgdg) as well as indirect, via water bridges. we also examined water-lipid interactions. existence of well-defined entities (lipids) linked by different types of interactions (hydrogen bonds, charge pairs, water bridges) makes the analysis of the membrane/ water interface region a suitable for a graph theoretical description. we applied a network analysis approach for comparative analysis of simulated systems. we note a marked difference between the organization as well as the dynamics of the interfacial region of the two bilayers. l-nucleoside analogues form an important class of antiviral and anticancer drug candidates. to be pharmacologically active, they need to be phosphorylated in multiple steps by cellular kinases. human phosphoglycerate kinase (hpgk) was shown to exhibit low specificity for nucleotide diphosphate analogues and its catalytic efficiency in phosphorylation was also affected. to elucidate the effect of ligand chirality on dynamics and catalytic efficiency, molecular dynamics simulations were performed on four different nucleotides (d-/l-adp and d-/l-cdp) in complex with hpgk and , -bisphospho-d-glycerate (bpg). the simulation results confirm high affinity for the natural substrate (d-adp), while l-adp shows only moderate affinity for hpgk. the observed short residence time of both cdp enantiomers at the active site suggests very weak binding affinity which may result in poor catalytic efficiency shown for hpgk with d-/l-cdp. analysis of the simulations unravels important dynamic conditions for efficient phosphorylation replacing the single requirement of a tight binding. using the van der waals density functional based on the semilocal exchange functional pw together with a longrange component of the correlation energy [ ] implemented in the siesta program code, we have calculated the band structure of the double stranded dna. the unit cell was built by taking together gc (or at) homogenous base pairs and we have considered the translational symmetry as the periodic boundary condition. the results obtained are compared with the oligomer calculations taking up to seven base pairs. the band structure obtained with this van der waals density functional is also compared with results obtained with other exchange-correlation functionals as well as with band structure obtained by the hartree-fock crystal-orbital method taking into account the helical symmetry of the double stranded dna. the role of different parts of dna (base pairs, sugar-phosphate backbone, na ions) is also presented. transmembrane (tm) proteins comprise some % to % of the proteome but owing to technical difficulties, relatively few of these structures have been determined experimentally. computational modeling techniques can be used to provide the essential structural data needed to shed light on structurefunction relationships in tm proteins. low-resolution electrondensity maps, obtained from cryo-electron microscopy (cryo-em) or preliminary x-ray diffraction studies, can be used to restrict the search in conformational space. at the right resolution, the locations of tm helices can be roughly determined even when the amino acids are not visible. when these data are combined with physicochemical characteristics of amino acids (such as their hydrophobicity) and with evolutionary conservation analysis of the protein family, the location of the amino acids can be modeled. the modelstructure may provide molecular interpretations of the effects of mutations. moreover, it can be used to suggest molecular mechanisms and to design new mutations to examine them. the overall approach will be demonstrated using two human proteins: copper transporter (ctr ), which is the main copper transporter in the human cell, and the kda translocator protein (tspo) of the outer mitochondria. modelstructures of these proteins and their functional implications in health and disease will be discussed. calcium channels play a crucial role in many physiological functions and their selectivity mechanism is still an unresolved question and a subject of debate. a physical model of selective ''ion binding'' in the l-type calcium channel is constructed, and consequences of the model are compared with experimental data. this reduced model treats only ions and the carboxylate oxygens of the eeee locus explicitly and restricts interactions to hard-core repulsion and ion-ion and ion-dielectric electrostatic forces. according to the charge/space competition mechanism, the charge of structural ions attracts cations into the filter, while excluded volume effects are trying to keep them out. this is a competition between energy and entropy, where the balance of these terms minimizes free energy and determines selectivity. experimental conditions involving binary mixtures of alkali and/or alkaline earth metal ions are computed. the model pore rejects alkali metal ions in the presence of biological concentrations of ca + and predicts the blockade of alkali metal ion currents by micromolar ca +. conductance patterns observed in varied mixtures containing na+ and li+, or ba + and ca +, are predicted. ca + is substantially more potent in blocking na+ current than ba +. in apparent contrast to experiments sing buffered ca + solutions, the predicted potency of ca + in blocking alkali metal ion currents depends on the species and concentration of the alkali metal ion, as is expected if these ions compete with ca + for the pore. these experiments depend on the problematic estimation of ca + activity in solutions buffered for ca + and ph in a varying background of ulk salt. equilibrium binding affinity (expressed as the occupancy of the selectivity filter by various ions) is computed by equilibrium grand canonical monte carlo (gcmc) simulations. the conductivity of the channel is estimated from the equilibrium concentration profiles using the integrated nernst-planck equation. our simulations show that the selectivity of l-type calcium channels can arise from an interplay of electrostatic and hard-core repulsion forces among ions and a few crucial channel atoms. the reduced system selects for the cation that delivers the largest charge in the smallest ion volume. we have also performed dynamic monte carlo (dmc) simulations for a model ca channel to simulate current directly and present our results for the dynamical selectivity (expressed as the flux carried by various ions). we show that the binding affinity of ca + versus na+ is always larger than the dynamical selectivity because ca + ions are tightly bound to the binding site of the selectivity filter of the channel and, at the same time, their mobility and drift velocity is smaller in this region. carotenoids are used in light-harvesting complexes with the twofold aim to extend the spectral range of the antenna and to avoid radiation damages. the effect of the polarity and conformation of the environment is supposed to be responsible for the tuning of the electronic, optical and vibrational properties of peridinin carotenoid both in solution and in protein matrix. we investigate the effect of vibrational properties of peridinin in different solvents by means of vibrational spectroscopies and qm/mm molecular dynamics simulations . the shift of vibrational fingerprints in the - cm - frequency region, due to the solvent polarity and proticity, is studied in three cases: cyclohexane (apolar/aprotic), deuterated acetonitrile (polar/aprotic) and methanol (polar/ protic). the frequencies and vibrational modes of the carbonyl, the allene, and the polyene chain were identified using effective normal mode analysis and compared with the present and previous experimental data . on the basis of our calculations and experiments in different solvents, we propose a classification of the four peridinins of the high-salt pcp form. the controlled self-assembly of functional molecular species on well defined surfaces is a promising approach toward the design of nanoscale architectures. by using this methodology, regular low-dimensional systems such as supramolecular clusters, chains, or nanoporous arrays can be fabricated. small biological molecules such as amino acids represent an important class of building blocks that are of interest for molecular architectonic on surfaces because they inherently qualify for molecular recognition and selfassembly [ ] . the interaction between amino acids and solid surfaces is decisive for the development of bioanalytical devices or biocompatible materials as well as for a fundamental understanding of protein-surface bonding. we investigate the adsorbtion mechanism of the cysteine on au( ) surfaces by means of the dft [ ] . our main concern is to describe the molecule-metal bonding mechanism. therefore we present a complex study, including the full determination of the density of states for the free and adsorbed molecule, the determination of molecule-surface bonding energy. the method of crystal orbital overlap populations is used in order to determine the contribution of specific atomic orbitals to the molecule-metal bond. it is now widely accepted that myoglobin (mb) is not simply an o storage/delivery system but, depending on oxygen concentration, it exerts other fundamental physiological roles. recent studies revealed a widespread expression and, in particular, an over-expression in response to hypoxia, in various non-muscle tissues, including tumor cells. in human five different mb isoforms are present. the two most expressed ([ %) differ only at the th position, k (mb-i) and e (mb-ii) respectively. since high-altitude natives from tibet are characterized by a higher mb concentration and locomotion efficiency, together with the observation that the mb overexpression is totally attributable to mb-ii, the idea that the latter might be one of the responses to high-altitude evolutionary adaptation, i.e. hypoxic environment, started to emerge. however, this is not yet supported by any structure/function investigation. we performed hundred nanoseconds md simulations on human mbs to investigate the structure and dynamics of both protein and surface water. important differences have been protein kinases play key roles in cell signaling and constitute crucial therapeutic targets. in normal cell, upon substrate binding, tyrosine kinase receptor kit undergoes extensive structural rearrangement leading to receptor dimerization and activation. this process is initiated by the departure of the juxta membrane region (jmr) from the active site, allowing the activation loop (aloop) deployment. the deregulation of kit activity is associated with various forms of cancer provoked by abnormalities in signal transduction pathways. mutations v g (jmr) and d h/v (a-loop) have been reported as oncogenic and/or drug-resistant. to contribute further in the understanding of kit activation/ deactivation mechanisms, we applied a multi-approach protocol combining molecular dynamics (md), normal modes analysis (nma) and pocket detection. disturbing structural effects, both local (a-loop) and long-range (jmr), were evidenced for kit d h/v in the inactive state. nma showed that jmr is able to depart its position more easily in the mutants than in the wild type. pockets analysis revealed that this detachment is sufficient to open an access to the atp binding site. our results provided a plausible conception of mutant dimerization and a way to explore putative allosteric binding sites. transmembrane association of leukocyte integrin heterodimer might be mediated by a polar interaction choon-peng chng and suet-mien tan biophysics group, a*star institute of high performance computing, and, school of biological sciences, nanyang technological university, republic of singapore the lateral association of transmembrane (tm) helices is important to the folding of membrane proteins as well as a means for signaling across the cell membrane. for integrin, a hetero-dimeric protein important for cell adhesion and migration, the association of its a-and b-subunits' tm helices plays a key role in mediating bi-directional mechanical signaling across the membrane. we found evidence from experiment and simulation for a polar interaction (hydrogenbond) across leukocyte integrin alb tm that is absent in the better-studied platelet integrin aiibb [ ] . our coarse-grained molecular dynamics simulations of tm helix-helix selfassembly showed more native-like packing achieved by alb within the simulation timescale as compared to its 'lossof-function' b t g mutant or aiibb [ ] . association free energy profiles also showed a deeper minimum at a smaller helix-helix separation for alb , suggestive of tighter packing. the likely conservation of this polar interaction across the b integrin family further reinforces its importance to the proper functioning of leukocyte integrins. active extrusion of drugs through efflux pumps constitutes one of the main mechanisms of multidrug resistance in cells. in recent years, large efforts have been devoted to the biochemical and structural characterization of rnd efflux pumps in gram-negative bacteria, in particular the acrb/a-tolc system of e.coli. specific attention has been addressed to the active part of the efflux system, constituted by the acrb unit. despite the presence of several data, crucial questions concerning its functioning are still open. the understanding of the structure-dynamics-function relationship of mexb, the analogous transporter in p. aeruginosa, encounters even more difficulties, because of the lack of structural data of the transporter in complex with substrates. to shade some light on the activity of mexb, we performed computational studies on mexb interacting with two compounds, meropenem and imipenem, the first known to be a good substrate, and the second a modest one. several techniques were used in the present work, ranging from flexible docking [ ] to standard and targeted molecular dynamics (md) simulations. starting from the published crystal structure [ ] we identified the most probable poses of the two compounds in both the original experimental and in the md-equilibrated structures. we used information from acrb binding pocket in order to find relevant binding sites of the two compounds in the analogous binding pocket of mexb. meropenem frequently lies with appropriate orientation in a pocket similar to the one identified for doxorubicin in acrb [ ] , while the occurrence of imipenem poses in the same pocket is very scarce. additionally, when present in the pocket, imipenem is located in a position that renders very unlikely its extrusion toward the oprm docking domain during the simulation of the functional peristalsis. the analysis of the trajectories has provided a complete inventory of the transporter and antibiotic hot spots, which is key information in terms of screening and design of antibiotics and inhibitors. clathrins are three-legged proteins with the intriguing ability to self-assemble into a wide variety of polyhedral cages. the nucleation and growth of a clathrin lattice against the cytosolic face of a cell membrane enables the endocytosis of membrane proteins and various external molecules, by wrapping the membrane around the cargo to produce a coated transport vesicle within the cell. clathrins can also self-assemble, in slightly acidic solutions devoid of auxiliary proteins, into empty cages. our simulations of this process, using a highly coarsegrained model, indicate that the key to self-assembly is neither calthrin's characteristic puckered triskelion shape, nor the alignment of four legs along all cage edges, but an asymmetric distribution of interaction sites around the leg's circumference. based on the critical assembly concentration, the binding strength in these cages is estimated at to k b t per clathrin. the simulations also answer the long-standing conundrum of how flat patches of purely hexagonal clathrin lattices, which in cryo electron microscopy are frequently seen to decorate cell membranes, can produce highly curved cages containing twelve pentagonal faces interdispersed between hexagonal faces. we present experimental evidence supporting this pathway. in eukaryotic cells, the exchange of macromolecules between the cytoplasm and the nucleus is mediated by specialized transport factors. by binding to these transporters, cargo molecules, which are otherwise excluded from entering the nucleus, can traverse the nuclear pore complex efficiently. most of the proteins mediating nuclear import and export exhibit a characteristic _-solenoid fold, which provides them with an unusual intrinsic flexibility. crm is an essential nuclear export receptor, which recognizes a very broad range of export cargoes. crm -dependent nuclear export is ran-gtpase-driven, and recognition of rangtp and cargo is highly cooperative. however, recent crystal structures show that the binding sites for export cargos and rangtp are located at distant parts of crm [ ] [ ] [ ] . we have used a combined approach of all-atom molecular dynamics simulations and small-angle x-ray scattering to study rangtp and cargo binding to crm . we have found that the allosteric effect in crm -dependent nuclear export arises from a combination of subtle structural rearrangements and changes in the dynamic properties of crm . light-induced phototactic responds in green algae chlamydomonas reinhardtii are mediated via microbial-type rhodopsins, termed channelrhodopsin- (chr ) and channelrhodopsin- (chr ) , which carry the chromophore retinal covalently linked to lysin via a schiff base and were shown to be directly light-gated ion-channels . the n-terminal putative seven-transmembrane region of chr was shown to be responsible for the generation of photocurrents and exhibits sequence similarity to the well understood proton pump bacteriorhodopsin (br) and the sensory rhodopsin anabaena sensory rhodopsin (asr) . as for the majority of membrane proteins, there is no d-structural data for chr available yet. here we present homology models of chr using two high-resolution x-ray template structures of br ( qhj ) respectively asr ( xio ) in order to get structural and functional insights into chr . with both homology models we performed molecular dynamics (md) simulations in a native membrane/solvent environment using gromacs . . . comparison of energetic and structural results revealed obvious advantages of the br-based homology model of chr . here we show that the br-based homology model is a reliable model of chr exhibiting structural features already found experimen-tally . our br-based homology model of chr allows predictions of putative crucial residues within chr . so we proposed several mutations within the chr sequence which are already accomplished. electrophysiologic and spectroscopic studies of these mutations are underway in order to confirm the functional relevance of these residues and to contribute to an optimized usage of chr as a powerful tool in optogenetics. ( neuroglobin is a recently discovered globin protein predominantly expressed in brain. its biological function is still elusive. despite the fact that neuroglobin shares very little sequence homology to the well-known globins as mioglobin and hemoglobin, they all have a characteristic globin fold with heme molecule bound to the distal pocket. the structural investigations and co binding kinetics revealed existence of cavities and tunnels within the protein matrix, where small ligands can be stored even for hundreds of microseconds [ ] . in human neuroglobin there is one internal disulfide bond possible which existence is proved to have significant effect on ligand affinity [ ] . in this study effects of temperature, ph, distal histidine mutation and presence of disulfide bond on co rebinding to neuroglobin are investigated by flash photolysis experiments. in parallel, the molecular dynamics simulations are performed in corresponding conditions in order to investigate structural change of neuroglobin and especially changes in distribution of internal tunnels and cavities able to bind diatomic ligands in response to different physical conditions listed above. the thrombospondin family, being extracelluar proteins, is known to be implicated in various physiological processes such as wound healing, inflammation, angiogenesis and neoplasia. the signature domain of thrombospondins shows high sequence identities and thus allows us to transfer results obtained, studying this complex calcium reach part of the proteins, from one member of the family to the other. the domain is known to play a key role in hereditary diseases such as psach or med. in this part of thrombospondins lies a binding site to integrins, important for cell attachment. it is further known that the lectin like globe binds to cd- , a feature known to be important in cancer research. as the theoretical unit we are trying to resolve these problems by numerical means and are constantly challenged by the size, where thrombospondin can be a huge trimeric protein as one strand can measure kda, and the large variety of subdomains found in this proteins. we are thus facing a multiscale problem which can range from solving, by means of quantum mechanics a specific ion binding site, to large scale abstraction by continuum mechanics. in our talk we will show you our newest results that we obtained by simulating calcium rich c-terminal domain which is known to be conserved across the entire family, and give you an outlook into the future of our research. the process of swift heavy ions energy deposition while penetrating a solid or scattering on its surface can result in a strong and nonequilibrium excitation of matter. an extremely localized character of this excitation, meanwhile, can make possible both selective changes in chemistry of matter and its surface nanomodifications . since possible applications have been found in bio-and it-technologies (cancer curing and nanostructuring respectively) in the last decade, the heavy ion bombardment technique has attracted a lot of scientific interest , . the processes of fast energy deposition into the solid and its further dissipation, however, are essentially perturbed with highly excited and nonequilibrium state of both lattice and electron systems. at such conditions therefore, the precision in treatment of processes of electron thermalization, fast electron heat conduction, and phase transformation of the overheated solid becomes crucial. having several physical models to handle the mentioned processes, it is nevertheless difficult to describe all of them within a scale of a single computational approach. our work is aimed on elaboration of the atomistic-continuum model of heavy ion bombardment of solids. in particular, the model will be applied to study the formation of nanohillocks in the experiments on swift heavy ion xe + scattering on srtio surface . [ ] g. aquaporins are protein channels located across the cell membrane with the role of conducting water or other small sugar alcohol molecules (aquaglyceroporins). the presence of the human aquaporin (hsaqp ) in cells proximal to airinteracting surfaces (eyes, lacrimal glands, salivary glands, lungs, stomach etc.) suggest its potentially important role in ''wetting'' these surfaces. the high-resolution x-ray structure of the hsaqp tetramer (pdb code d s) exhibits two important features: (i) lack of the four fold symmetry, common in most of the aquaporins, and (ii) occlusion of the central pore by a phosphatidylserine lipid tail. in this study we investigate the importance of these two features on the transport properties of the human aqp by means of molecular dynamics simulations. we found that the asymmetry in the tetramer leads to a distribution of monomeric channel structures characterized by different free energy landscapes felt by the water molecules passing through the channel. furthermore, the structures' distribution is influenced both by the presence/absence of the lipid tail in the central pore, and by the lipid composition of the bilayer that solvates the hsaqp tetramer. elucidating the modular structure of the protein g c fragment and human igg fc domain binding site using computer simulations hiqmet kamberaj faculty of technical sciences, international balkan university, skopje, r. of macedonia protein-protein recognition plays an important role in most biological processes. although the structures of many protein-protein complexes have been solved in molecular detail, general rules describing affinity and selectivity of proteinprotein interactions break down when applied to a larger set of protein-protein complexes with extremely diverse nature of the interfaces. in this work, we will analyze the non-linear clustering of the residues at the interface between proteins. the boundaries between clusters are defined by clustering the mutual information of the protein-protein interface. we will show that the mutations in one module do not affect residues located in a neighboring module by studying the structural and energetic consequences of the mutation. to the contrary, within their module, we will show that the mutations cause complex energetic and structural consequences. in this study, this is shown on the interaction between protein g c fragment and human igg fc domain by combining molecular dynamics simulations and mutual information theory, and computational alanine scanning technique. the modular architecture of binding sites, which resembles human engineering design, greatly simplifies the design of new protein interactions and provides a feasible view of how these interactions evolved. the results test our understanding of the dominant contributions to the free energy of protein-protein interactions, can guide experiments aimed at the design of protein interaction inhibitors, and provide a stepping-stone to important applications such as interface redesign. membrane proteins can form large multimeric assemblies in native membranes that are implicated in a wide range of biological processes, from signal transduction to organelle structure. hydrophobic mismatch and membrane curvature are involved in long range forces largely contributing to such segregation. however, the existing assembly specificity is thought to be coded in the atomic details of protein surface and topology. these are best described in high resolution structures and atomistic molecular dynamics simulations. in order to explore more systematically such forces and energetics arising at intermediate time scales and resolution, we use coarse grained molecular dynamics simulations applied to membrane systems spanning over to us. as a first glimpse we study how proteins induce lipid perturbations using a previously developed conformational entropy estimator. we show that in the model membrane where hydrophobic mismatch is present, lipid perturbations extend up to * a from the protein surface. however, significant variations in perturbation profiles are seen. parameters such as protein shape, surface topology, and amino acid physicochemical properties are studied to discover the parameters governing such perturbations. crossing energy barriers with self-guided langevin dynamics gerhard kö nig, xiongwu wu, bernard brooks national institutes of health, national heart, lung and blood institute, laboratory of computational biology, rockville, md, usa even with modern computer power, the applicability of molecular dynamics simulations is restricted by their ability to sample the conformational space of biomolecules. often high energy barriers cause normal molecular dynamics simulations to stay trapped in local energy minima, leading to biased results. to address this problem, self-guided langevin dynamics (sgld) were developed. it enhances conformational transitions by accelerating the slow systematic motions in the system. this is achieved by calculating the the local average of velocities and adding a guiding force along the direction of systematic motions. thus, the efficiency of sgld is governed by three factors: a.) the friction constant involved in the langevin dynamics b.) the local averaging time and c.) the guiding factor that determines the guiding force. however, the guiding force also causes deviations from the original ensemble that have to be corrected by reweighting the data, thus decreasing the efficiency. here, we explore the three-dimensional parameter space of sgld for several benchmark systems with particularly rough energy surfaces. based on our data, we supply guidelines for the optimal selection of sgld parameters, to allow the extension of our method to other biological problems of interest. propagation of d v/h mutation effects across kit receptor e. laine, i. c. de beauchê ne, c. auclair and l. tchertanov lbpa, cnrs -ens de cachan, france receptor tyrosine kinases (rtks) regulate critical biological processes. constitutive activation of rtks provokes cancers, inflammatory diseases and neuronal disorders. biological data evidenced that oncogenic mutations of the rtk kit, located either in the juxtamembrane region (jmr) or in the activation loop (a-loop) -as is the case of d v/h, displace the equilibrium of conformational states toward the active form. we present a multi-approach study that combines molecular dynamics (md), normal modes (nm) and pocket detection to characterize and compare the impact of d v/h on the structure, dynamics and thermodynamics of kit. we have evidenced a local structural destabilization of a-loop induced by the mutation and a long-range effect on the structure and position of jmr. we have further correlated these observations with experimental data and decipher some details about the activation mechanisms of the mutants, involving leverage of the jmr negative regulation and release of an access to the catalytic site. through the identification of ''local dynamic domains'' and the recording of interactions within the protein, we propose a model of the mutational effects propagation, which highlights the importance of both structural distortion and local conformational fluctuations. investigation of biological active azolidinones and related heterocycles refer to one of the most successful scientific projects of dh lnmu. it is based on three strategic vectors: organic synthesis, pharmacological research, rational design of ''drug-like'' molecules (including in silico approaches). while applying the research strategy we succeeded in gaining a number of interesting results that make possible to extend the field, especially in the scope of ''drug-like'' molecules design, notably it has focused on the search of new anticancer agents. anticancer activity screening was carried out for more than compounds (us nci protocol (dtp) based on obtained directed library over new compounds, among them compounds showed high activity level. for the purpose of optimization and rational design directions of highly active molecules with optimal ''drug-like'' characteristics and discovering of possible mechanism of action sar, compare analysis, molecular docking and qsa(p)r were carried out. obtained results allowed to form main directions of possible anticancer activity mechanisms, which probable are apoptosis-related. nowadays the investigation of cellular and molecular aspects of anticancer effects is in progress. regulation of (bio)chemical reactions by mechanical force has been proposed to be fundamental to cellular functions [ , , ] . atomic force microscopy experiments have identified the effect of mechanical force on the reactivity of thiol/disulfide exchange, a biologically important reaction [ ] . in order to understand the influence of the force at an atomistic level, we have performed hybrid quantum mechanicsmolecular mechanics (qm/mm) transition path sampling simulation of the reaction under external forces. the results of the simulations supported the experimental findings and demonstrated that the location of the transition state on the free energy surface was shifted due to force [ ] . in contrast to our findings, however, a recent experimental study suggests only a weak coupling between the mechanical force and the reaction rate [ ] . in this study, the reactants were covalently linked to a stilbene molecule. in this system a force can be applied by photo-isomerization from the relaxed trans to the strained cis configuration. a drawback of this system is that one cannot easily determine the forces that acting on the reaction coordinate. therefore, we have developed a force distribution analysis method for quantum mechanical molecular dynamics simulations. the results of the analysis show how isomerization of stilbene alters the forces acting on the reacting atoms. the force distribution is essential for understanding how chemistry is controlled by external forces. [ conformational space modelling (csm) is a promising method for membrane protein structure determination. it is based on the concept of the side-chain conformational space (sccs), which is formed by the allowed side-chain conformations of a given residue. each sccs can be calculated from a d structure or measured via epr-sdsl experiments. for structure determination a set of singly spinlabelled mutants is needed. the final structure is obtained by altering an initial (possibly random) d structure until the best fit between the calculated and measured sccs for the whole set is found. such optimization is computationally intensive; therefore csm includes several empirical approximations. one of them describes the effect of the lipid tails on the sccs. the implementation is not trivial as lipids diffuse in the plane of the membrane and the lipid tails behave differently at different membrane depths. to unravel this relationship adaptive biasing force md simulations were used. an alanine peptide helix was made in silico, spin-labelled at the middle and inserted perpendicularly into a dmpc membrane. the free energy of the spin-label orientation at various membrane depths was calculated. a d free energy surface describing the membrane ''depth'' effect was obtained. it is known that b-cyclodextrins (bcds) are able to modify the cholesterol content of cell or model membranes. however the molecular mechanism of this process is still not resolved. using molecular dynamics simulations, we have been able to study the bcd-mediated cholesterol extraction from cholesterol monolayers and lipid-cholesterol monolayer mixtures. we have investigated many conditions that would affect this process (e.g. lipid-cholesterol ratio, lipid chain unsaturation level) our results can be summarized as follow: i) dimerization of bcds, ii) binding of the dimers to the membrane surface assuming either a tilted (parallel to the membrane normal) or untilted ( °respect to the normal of the membrane) configuration, iii) the latter configuration is suitable to extract cholesterol at a reasonable computational time scale ( - ns), however, this process may be affected by unfavorable energy barriers (from to kj mol - ), iv) desorption of the complex brings cholesterol in solution, v) the bcd-cholesterol complex is able to transfer cholesterol to other membranes. with a clearer understanding of the basic molecular mechanism of the bcd mediated process of cholesterol extraction, we can begin to rationalize the design of more efficient bcds in numerous applications. the mechanism of the complex formation of biopolymers with ligands including the solvent molecules is an actual problem of modern biophysical and biological science. polypeptides form a secondary structure and mimic the motifs of the protein architecture. the study of complexation between polypeptides and solvent molecules leads to deeper understanding of the basic interaction of proteins with environment at atomic level. besides polypeptides are promising for the development of applications which encompass some of the following desirable features: anti-fouling, biocompatibility, biodegradability, specific biomolecular sensitivity. on this account polypeptides have a great significance for a variety modern applications ranging from the nanoscale medicine devises up to food technology and others. we compare the results of calculations of complexes between helical polypeptides (polyglutamic acid in neutral form and poly-c-benzyl-l-glutamate) and water molecules at dft pbe level and the results of ftir-spectroscopic study of the film of wetted polypeptides. vibrational spectroscopy is one of the most useful experimental tools to study non-covalent bonded complexes, and calculated spectra in comparison with experimental data are reliable test for reality of simulated complexes. platelet aggregation at the site of vascular injury is vital to prevent bleeding. excessive platelet function, however, may lead to thrombus formation after surgery. therefore, an accurate measure and control of platelet aggregation is important. in vitro platelet aggregometry monitors aggregate formation in platelet rich plasma triggered by agonists such as adp, epinephrine or collagen. the fraction of aggregated platelets is plotted versus time, and platelet function is assessed by analyzing the plot's morphology. we propose new measures of platelet function based on a compartmental kinetic model of platelet aggregation induced by adp. our model includes three compartments: single, aggregated and deaggregated platelets. it is simpler than earlier models and agrees with experimental data. the model parameters were determined by non-linear least squares fitting of published data. we associated the kinetic parameters with the activity of the adp receptors p y and p y . to this end, we studied published data obtained in the presence and in the absence of specific antagonists of these receptors. comparison of kinetic parameters of healthy subjects with those of patients with myeloproliferative disorder (mpd) shows that the function of p y is significantly reduced in mpd. coarse-grained modeling of drug-membrane interactions manuel n. melo, durba sengupta, siewert-jan marrink groningen biomolecular sciences and biotechnology institute, university of groningen, groningen, the netherlands the martini coarse-grained (cg) forcefield was used to simulate the actions of the antimicrobial peptide alamethicin and of the anti-tumoral drug doxorubicin. both drugs were shown to interact strongly with a fluid phospholipid bilayer, and aggregate there, in agreement with experimental results. because doxorubicin may establish intermolecular h-bonding, and thus lower its dipole moment, the cg representation of a doxorubicin dimer was adjusted. this less polar dimer was then tested for translocation and/or pore formation. contrary to results of atomistic simulations, alamethicin aggregates did not spontaneously open pores. they did so, however, when the size of the water beads was decreased. several small independent pores could then be observed. the magnitude of the permeability of these pores is analyzed and compared to experimental values. the occurrence of multiple small pores could indicate that the different conductance levels experimentally observed for alamethicin might simply result from the association of different numbers of these small pores. polarization refers to the asymmetric changes in cellular organization in response to external or internal signals. neuronal polarization begins with the growth of a single neurite shortly after cell division, followed by the growth of a second neurite at the opposite pole. this early bipolar shape is critical for brain function, as it defines axis of migration and consequently proper three dimensional organization and nerve circuitry. however, it is not known if a direct relationship exists between the formation of the second, opposite, neurite and the mechanisms involved in the formation of the first. we tackled this issue through mathematical modeling, based on membrane traffic (exocytosis-endocytosis), and lateral diffusion. with this approach, we demonstrated that a single pole of molecular asymmetry is sufficient to induce a second one at the opposite side, upon induction of growth from the first pole. our work gives mathematical proof that the occurrence of a single asymmetry in a round cell is sufficient to warrant morphological bipolarism. trypsin is one of the best characterized serine proteases. enzyme acylation process is required for substrate degradation. there is a lot of information about how this process undergoes. however, in order to obtain a more detailed description of the catalytic triad mechanism, a qm/mm approach was used. we used the hybrid qm/mm potential implemented in amber package. in the qm calculations a dft hamiltonian was used. we develop an approach based on the adaptively biased md in order to obtain the free energy surface of the conformational space defined by the reaction coordinates. this approach presents some characteristics of steered md and umbrella sampling procedures. our results offer information about the lowest energy trajectory, the barrier profile of the reaction, and the geometry of the transition state. this method also provides a further insight into the role of specific residues in the reaction. substituting asp , member of the catalytic triad, for ala we were able to detect an increase of the barrier profile. this was due to the loss of the interaction of carbonyl group of asp with nd of his , which make ne of this residue a worst proton acceptor. this results show our approach as a valuable method in the study of enzymatic mechanisms. the intracellular media comprise a great variety of macromolecular species that are not individually concentrated, but being preset in the same compartment they exclude each other's volume and produce crowding. crowding has a profound impact on protein structure and determines conformational transitions and macromolecular association. we investigated macromolecular association on a % w/w bovine serum albumin (bsa) solution by time-domain terahertz (thz) spectroscopy and molecular modeling. molecular crowding was simulated by including two bsa molecules in the same water box. we generated * such dimeric models, computed their thz spectra by normal modes analysis and compared them with the experimental data. the best bsa dimer model was selected based on the agreement with the experiment in the lowest frequencies domain of up to thz. symmetry constraints improve accuracy of ion channels models. application to two-pore-domain channels adina l. ion channels are important drug targets. structural information required for structure-based drug design is often filled by homology models (hm). making accurate hm is challenging because few templates are available and these often have substantial structural differences. besides, in molecular dynamics (md) simulations channels deviate from ideal symmetry and accumulate thermal defects, which complicate hm refinement using md. we evaluate the ability of symmetry-constrained md simulations to improve hm accuracy, using an approach conceptually similar to casp competition: build hm of channels with known structure and evaluate the efficiency of various symmetry-constrained md methods in improving hms accuracy (measured as deviation from x-ray structure). results indicate that unrestrained md does not improve accuracy, instantaneous symmetrization improves accuracy but not stability during subsequent md, while gradually imposing symmetry constraints improves both accuracy (by - %) and stability. moreover, accuracy and stability are strongly correlated, making stability a reliable criterion in predicting hm accuracy. we further used this method to refine hm of trek channels. we also propose a gating mechanism for mechanosensitive channels that was further experimentally confirmed. nucleotide modifications and trna anticodon-mrna codon interactions on the ribosome olof allné r and lennart nilsson karolinska institutet, stockholm, sweden molecular dynamics simulations of the trna anticodon and mrna codon have been used to study the effect of the common trna modifications cmo u and m a . in trna val these modifications allow all four nucleotides to be successfully read at the wobble position in a codon. previous data suggest entropic effects are mainly responsible for the extended reading capabilities but detailed mechanisms have remained unknown. the aim of this paper is to elucidate the details of these mechanisms on an atomic level and quantify their effects. we have applied: extensive free energy perturbation coupled with umbrella sampling, entropic calculations of trna (free and bound to the ribosome) and thorough structural analysis of the ribosomal decoding center. human neuroserpin (hns) is a serine protease inhibitor (serpin) of a tissue-type plasminogen activator (tpa). the conformational flexibility and the metastable state of this proteins underlies to misfolding and to dysfunctional mutations causing a class of rare genetic diseases which share the same molecular basis. the conformational transition of the native form, triggered upon the cleavage at reactive center loop (rlc), releases a complex of the cleaved form bound to the inactivated target protease. without rlc cleavage a stable inactive latent form can be obtained by intra/inter molecular loop insertion leading to polymerization. this work concerns the study of the three above mentioned forms of hns by md simulations to investigate the relation between their conformational stability and. the starting native and cleaved configurations are based on the x-ray structure, while the latent form is here modelled. the results of the simulation reveal a striking conformational stability along with the intrinsic flexibility of selected regions of the fold.the analysis of the essential collective modes of the native hns shows that the initial opening of the b-sheet a coincides with several changes in the local pattern of salt bridges and of hydrogen bonds. regulation of ubiquitin-conjugating enzymes: a common mechanism based on a pattern of hydrophobic and acidic residues enzyme temperature adaptation generally involves a modulation of intramolecular interactions, affecting proteins dynamics, stability and activity [ ] [ ] . in this contribution, we discuss studies of different classes of extremophilic enzymes, focusing on cold-adapted variants, as well as their mesophilic-like mutants, performed by all-atom molecular dynamics simulations with particular attention to structural communication among residues within the threedimensional architecture [ ] [ ] . common adaptation strategies turned out to be based on improved local flexibility in the proximity of the functional sites, decrease in interconnected electrostatic interactions, and modulation of correlated motions and networks of communicating residues. specific differences related to the diverse protein folds can also be detected. bneurexins and neuroligins are cell adhesion molecules and play important role in synapse junction formation, maturation and signal transduction between neurons. mutations in genes coding these proteins occurs in patients with cognitive diseases like autism disorders, asperger syndrome and mental retardation [ ] . it has been found that the complex bneurexin-neuroligin has also an important role in angiogenesis [ ] . herein we will present molecular foundations of bneurexin-neuroligin interactions obtained from all-atom molecular dynamics simulations of bneurexin, neuroligin and their complex ( b q) [ ] . ns md trajectories (charmm force field) were analyzed and roles of ca + and n-actetyl-d-glucosamine posttranslational modifications in intermolecular interactions were scrutinized. advances in hardware and software have enabled increasingly long atomistic molecular dynamics simulations of biomolecules, allowing the exploration of processes occurring on timescales of hundreds of microseconds to a few milliseconds. increasing the length of simulations beyond the microsecond time scale has exposed a number of limitations in the accuracy of commonly employed force fields. such limitations become more severe as the size of the systems investigated and the length of the simulations increase. here i will describe the force field problems that we have encountered in our studies, how we identified and addressed them, and what we have learned in the process about the biophysics of the systems we are investigating. while the quest for a ''perfect'' force field is not over (and may never be), our work has greatly improved the accuracy and range of applicability of simple physics-based force fields, to the point that reliable predictions can now be obtained from millisecond-timescale simulations of biomolecules. local anesthetics (la) are pain-relief drugs, widely used in medicine and dentistry. the relatively short duration of analgesia still restricts their clinical use for the treatment of chronic pain. nowadays, intensive research is focused on anesthetics entrapped into liposomes to enhance their activity and pharmacokinetic properties [ ] . in this work, we investigated the encapsulation of prilocaine (plc), an aminoamide local anesthetic, into a small unilamellar liposome. on the line of our previous work [ ] , we have carried out molecular dynamics (md) simulations using a coarse grain model up the microsecond time scale. in this way, we compare the effects of the concentration of la at fisiological ph. we were able to capture important features of the plc-vesicle interactions. the behavior of plcs at fisiological ph is essentially a combination of high and low ph: we found that all neutral plc molecules rapidly diffuse into the hydrophobic region of the vesicle adopting an asymmetric bimodal density distribution. protonated plc molecules (pplc) initially placed in water were instead only found on the external monolayer, with a high rate of exchange with the water phase and no access to the inner part of the liposome in a concentration dependent way. we focus on applications of molecular and mesoscale simulation methodologies to the cellular transport process of endocytosis, i.e., active transport mechanisms characterized by vesicle nucleation and budding of the cell membrane orchestrated by protein-interaction networks, and functionalized carrier adhesion to cell surfaces. we discuss theoretical and computational methodologies for quantitatively describing how cell-membrane topologies are actively mediated and manipulated by intracellular protein assemblies. we also discuss methods for computing absolute binding free energies for carrier adhesion. we present rigorous validation of our models by comparing to diverse range of experiments. the importance of delta-opioid receptors as target of a large number of drugs is well recognized, but the molecular details of interaction and action of the compounds are largely unknown. in an effort to shade some light on this important issue we performed an extensive computational study on the interaction of two compounds, clozapine and desmetilclozapine, with a delta-opioid receptor. according to experiments, the lacking of a single methyl group in desmetilclozapine with respect of clozapine makes the former more active than the latter, providing a system well suited for a comparative study. we investigated stable configurations of the two drugs inside the receptor by simulating their escape routes by metadynamics, an algorithm that allows the simulation of events that are otherwise out of range for standard molecular dynamics simulations. our results point out that the action of the compound is related to the spatial distribution of the affinity sites it visits during its permanency. desmetilclozapine has a larger distribution of residues, which is interacting with, than clozapine. however, large conformational changes of the receptor were not observed in the presence of both compounds. thus, a more dynamical picture of ligand-receptor affinity is proposed on the basis of the results obtained, involving the competition among different stable states as well as the interaction with the solvents. such information might be useful to provide hints and insights that can be exploited in more structure-and-dynamics-oriented drug design. the coupling between the mechanical properties of enzymes and their biological activity is a well-established feature that has been the object of numerous experimental and theoretical works. in particular, recent experiments show that enzymatic function can be modulated anisotropically by mechanical stress. we study such phenomena using a method or investigating local flexibility on the residue scale, which combines a reduced protein representation with brownian dynamics simulations. we performed calculations on the enzyme guanylate kinase in order to study its mechanical response when submitted to anisotropic deformations. the resulting modifications of the protein's rigidity profile can be related to the changes in substrate binding affinities that were observed experimentally. further analysis of the principal components of motion of the trajectories shows how the application of a mechanical constraint on the protein can disrupt its dynamics, thus leading to a decrease of the enzyme's catalytic rate. eventually, a systematic probe of the protein surface led to the prediction of potential hotspots where the application of an external constraint would produce a large functional response both from the mechan- hiv- protease autocatalyses its own release from gag and gagpol precursor polyproteins into mature functional proteins. as it is functional in the dimeric form, whilst initially, only a single monomer is embedded within each gagpol chain, the question arises as to what cut's the cutter. two individual monomers in different gagpol chains are known to come together to form an embedded-dimer precursor protease. mature-like protease activity is concomitant with n-terminal intramolecular cleavage of this transient embedded-dimer precursor, but how this crucial maturation-initiating step is physically achieved, has remained unknown. here, we show via all-atom explicit solvent molecular dynamics simulation runs of ns each that the n-terminal of an immature-like protease, with the n-terminal initially unbound as in the gagpol polyprotein, can self-associate to the active site and therefore be cleaved under conditions of thermodynamic equilibrium, identifying possible binding pathways at atomic resolution, in agreement with previous indirect experimental evidence [ ] . the binding pathway predominantly makes use of the open conformation of the beta-hairpin flaps characterised by us previously [ ] , and the n-terminal binds across the entire active site in good agreement with crystal structures of a cleavage-site peptidebound protease. the n-terminus serves two roles, firstly in the maturation of the protease itself by self-associating to the protein and then as a stabilizing component of the dimer interface in a mature protease. targeting the prior mechanism could be the focus of a novel therapeutic strategy involving immature protease inhibition. [ knotted proteins are the object of an increasing number of experimental and theoretical studies, because of their ability to fold reversibly in the same topologically entangled conformation. the topological constraint significantly affects their folding landscape, thus providing new insight and challenges into the funnel folding theory [ ] . recently developed experimental methods to trap and detect knots have suggested that denaturated ensembles of the knotted proteins may be knotted [ ] . we present numerical simulations of the early stage of folding of the knotted proteins belonging to protein families mtase (methyltransferase) and sotcase (succinyl-ornithine transcarbamylase), and of their unknotted homologues [ ] . our results show that native interactions are not sufficient to generate the knot in the denaturated configurations. however, when non-native interactions are included we observe formation of knots only in the chains whose native state is knotted. in addition, we find that the knots are formed through a universal mechanism. such a knot formation mechanism correctly predicts the fraction of the knotted proteins found in nature and can be used to make qualitative prediction on their folding times. shape and motility and also for numerous signaling processes. adhesion is based on non-covalent interactions between transmembrane proteins and the extracellular matrix. cells are able to create two-dimensional assemblies of integrins, so called focal adhesions, which they use to stick to the substrate and collect information about the environmental properties. the goal of this work is a deeper understanding of the formation and the stability of these adhesion clusters. bond cluster formation and disintegration is dynamically modeled with the aid of monte carlo simulations. in the model, a membrane is attached to a flat surface via a variable number of adhesion bonds. the spacial configuration of these adhesion points subjected to an inhomogeneous stress field maps into a distribution of local membrane/ surface distances. we introduce a model which explicitly accounts for the membrane elasticity and demonstrate that such models are able to explain the spontaneous formation of adhesion bond clusters. structure based models are successful at conjugating the essence of the energy landscape theory of protein folding [ ] with an easy and efficient implementation. recently their realm expanded beyond single protein structure, been used profitably to widely study large conformational transitions [ ] [ ] . still, when dealing with conformational transition between two well-defined structures an unbiased and realistic description of the local backbone and sidechain interactions is necessary. the proposed model merges a precise description of these interactions with a structure-based long range potential that takes into account different conformers. we present the results of the activation of the catalytic domain of human csrc tyrosine kinase for which we reconstructed the transition free energy and the description of the activation loop flexibility. the excellent model performances in terms of speed and the satisfactory accuracy of the description of the system and its flexibility are promising for a more systematic study of the tyrosine kinase family activation mechanisms. [ we introduce a previously undescribed technique for modelling the kinetics of stochastic chemical systems. we apply richardson extrapolation, a sequence acceleration method for ordinary differential equations, to a fixed-step tau-leaping algorithm, to produce an extrapolated tau-leaping method which has weak order of accuracy two. we prove this mathematically for the case of linear propensity functions. we use four numerical examples, two linear and two nonlinear, to show the higher accuracy of our technique in practice. we illustrate this by using plots of absolute error for a fixed-step tau-leap and the extrapolated tau-leap. in all cases, the errors for our method are lower than for a fixedstep tau-leap; in most cases they are second order of accuracy. the major tripartite efflux pump acrab-tolc is responsible for the intrinsic and acquired multidrug resistance in escherichia coli. at heart of the extrusion machinery there is the homotrimeric transporter acrb, which is in charge of the selective binding of structurally and chemically different substrates and energy transduction. the effects of conformational changes, which have been proposed as the key features of the extrusion of drugs, are investigated at molecular level using different computational methods like targeted molecular dynamics. simulations, including almost half a million atoms, have been used to assess several hypotheses concerning the structure-dynamics-function relationship of the acrb protein. the results indicate that, upon induction of conformational changes, the substrate detaches from the binding pocket and approaches the gate to the central funnel. in addition, we provide evidence for the proposed peristaltic transport involving a zipper-like closure of the binding pocket, responsible for the displacement of the drug. using these atomistic simulations the role of specific amino acids during the transitions can be identified, providing an interpretation of sitedirected mutagenesis experiments. additionally, we discuss a possible role of water molecules in the extrusion process. virus inhibitory peptide (virip), a amino acid peptide, binds to the fusion peptide (fp) of human immunodeficiency virus type (hiv- ) gp and blocks viral entry. molecular dynamics (md) and molecular mechanics/poisson-boltzmann surface area (mm/pbsa) free energy calculations were executed to explore the binding interaction between several virip derivatives and gp fp. a promising correlation between antiviral activity and simulated binding free energy was established thanks to restriction of the flexibility of the peptides, inclusion of configurational entropy calculations and the use of multiple internal dielectric constants for the mm/pbsa calculations depending on the amino acids sequence. bases on these results, a virtual screening experiment was carried out to design enhanced virip analogues. a selection of peptides was tested for inhibitory activity and several improved virip derivatives were identified. these results demonstrate that computational modelling strategies using an improved mm/pbsa methodology can be used for the simulation of peptide complexes. as such, we were able to obtain enhanced hiv- entry inhibitor peptides via an mm/pbsa based virtual screening approach. an essential step during the hiv life cycle is the integration of the viral cdna into the human genome. hiv- integrase mediates integration in a tight complex with the cellular cofactor: ledgf/p [ ] . disruption of the interaction interferes with hiv replication and therefore provides an interesting new drug target for antiretroviral therapy [ , ] . here we present the structure based discovery and optimization of a series of small molecule inhibitors that bind to hiv- integrase and block the interaction with ledgf/p . the work flow was set up according to a funnel principle in which a series of virtual screening tools were applied in such way to discard at each step molecules unlikely to be active against the desired target (including d filtering, pharmacophore modelling and molecular docking) the activity and selectivity of the selected molecules were confirmed in an alpha screen based assay, that measure protein-protein interaction in vitro, and furthermore by in vivo experiments. active compounds proceeded towards crystallographic soaking into the receptor protein crystals. these crystal structures not only validated the binding mode and activity of the hit compounds, but furthermore were used as a platform for structure based drug design which resulted in the rational discovery of new hit compounds and optimized lead compounds. in vitro and in vivo experiments validated the mechanism of action of these compounds and show that they are a novel class of antiretroviral compounds with in vivo inhibitory activity by targeting the interaction between ledgf/p and hiv- integrase. cross resistance profiling indicate that these compounds are active against current resistant viral strains. [ ] currently the most potent inhibitors show an in vivo ic of nm. these compounds are promising for future pharmaceutical optimizations to be used in the clinic as new antiretroviral agents. crystallography was used to validate the binding mode of the discovered inhibitors. insights in the interaction of the ligand-protein complex allowed for rational design of optimized inhibitors. ligand-induced structural changes are small, thermal fluctuations can play a dominant role in determining allosteric signalling. in thermodynamic terms, the entropy change for subsequent binding is influenced by global vibrational modes being either damped or activated by an initial binding event. one advantage of such a mechanism is the possibility for long range allosteric signalling. here, changes to slow internal motion can be harnessed to provide signalling across long distances. this paper considers homotropic allostery in homodimeric proteins, and presents results from a theoretical approach designed to understand the mechanisms responsible for both cooperativity and anticooperativity. theoretical results are presented for the binding of camp to the catabolite activator protein (cap) [ ] , where it is shown that coupling strength within a dimer is of key importance in determining the nature of the allosteric response. results from theory are presented along side both atomistic simulations and simple coarse-grained models, designed to show how fluctuations can play a key role in allosteric signalling in homodimeric proteins. [ reversibly switchable fluorescent proteins (rsfps) can be switched between a fluorescent (on) and a nonfluorescent (off) state which is accompanied by a cis-trans isomerization of the chromophore on the molecular level , . this unique property has already provided new aspects to various microscopy techniques, such as high resolution microscopy, fcs or monochromatic multicolor microscopy - . despite of their established potential, rsfps still have a major drawback: the wavelength for fluorescence excitation is always one of the two switching wavelengths. the imaging process thus inevitably results in the switching of a small fraction of the rsfps, which might hinder or complicate some experiments. we developed a new reversibly switchable fluorescent protein which eliminates the problem of the coupling between switching and fluorescence excitation. this fluorescent protein follows an unusual and currently unknown mechanism of switching between a fluorescent and a nonfluorescent state. it is brightly fluorescent and exhibits an excellent signal to noise ratio. in parallel studies [ ] , qd-based ligands (egf, mabs) were targeted to egfr in gliomas. cell-cultures, animal models and ex vivo biopsies of human high-grade as well as low-grade gliomas showed high probe specificity.. the aim is to define more precisely the tumor boundaries at the time of resection. we used the programmable array microscope designed for sensitive, high-speed optical sectioning, particularly of living cells. the pam is based on structured illumination and conjugate detection using a digital micromirror device (dmd) [ ] located in a primary image plane. the unique feature is the rapid, (re)programmable adjustment of local excitation intensity, dynamic, on-the-fly optimization is thus achieved, e.g. multipoint frap [ ] , light exposure minimization and object tracking [ ] , or super-resolution strategies. the features and operation of the rd generation pam will be presented. contraction of muscle cells, motility of microorganisms, neuronal activity, and other fast cellular processes require microscopic imaging of a three-dimensional ( d) volume with a video-rate scanning. we present d video-rate investigations of structural dynamics in biological samples with the multicontrast third-and second-harmonic generation as well as fluorescence microscope. the multidepth scanning is achieved by two combined laser beams with staggered femtosecond pulses. each of the beams is equipped with a pair of deformable mirrors for dynamic wavefront manipulation enabling multidepth refocusing with simultaneous corrections for optical aberrations. combined, more than frames per second lateral scanning with fast refocusing enables the d video-rate imaging of dynamically moving structures. in addition, combination of two laser beams is accomplished at two perpendicular polarizations enabling live imaging of sample anisotropy, which is important for structural studies particularly with the second harmonic generation microscopy. investigations of beating chick embryo hearts with the d video-rate scanning microscope revealed multidirectional cardiomyocyte contraction dynamics in myocardial tissue. intricate synchronization of contractions between different layers of myocytes in the tissue will be presented. the video-rate d microscopy opens new possibilities of imaging fast biological processes in living organisms. confocal fluorescence microscopy is an invaluable tool to study biological systems at cellular level also thanks to the synthesis of always new specific fluorescent probes, e.g. multiprobe labelling enables complex system characterization. however, only the recent employment of narrowband tunable filters overcomes the problems due to the use of the broadband ones. the possibility of acquiring the emission spectra in a spatially resolved way extends simple image intensity studies into characterization of complex probeenvironment relationship through the sensitivity of fluorescence spectra to the local molecular environment differences. consequently, fluorescence microspectroscopy (fms) is able to provide the spectral information in a well defined spatial region allowing the researcher to simultaneously obtain spatial and spectroscopic information. our instrument has been specially built to study live cells and their interaction with nanomaterials, drug carriers and modified cell environment. other main characteristics are reducing the bleaching effect and employing a white light source that does not limit the use to specific probes. graphical tools, such as colour coded images, have also been introduced to provide explicit and straightforward visual information. high speed fpga based multi-tau correlation for singlephoton avalanche diode arrays jan buchholz , , jan krieger we demonstrate the use of fret-imaging (forster resonance energy transfer) as an assay to directly monitor the dynamics of cross-bridge conformational changes in single fibres of skeletal muscle. we measured nm-distances of several fret pairs located at strategic positions to sense myosin head conformational changes: we focused our attention on the essential light chain, elc (specifically labelling a modified elc and exchanging it with the natural elc of the fibre) and we investigated its interaction with the sh helix, with the nucleotide binding pocket and with actin. we characterized fret in single rigor muscle fibres, determining distances in agreement with those from the crystallographic data. the results demonstrate the viability of the approach in sensing different fret efficiencies over a few nanometres, an essential requirement to follow the expected small fret variations in contracting muscle fibres. we are now performing dynamic experiments on rigor and active fibres by applying small stretch/release cycles to alter the interaction distances (estimated time resolution of nearly ms/frame). in this configuration, it will be possible to measure functional changes, shedding light on the myosin head dynamics during contraction. focal stimulation of cultured neurons is crucial since it mimics physiological molecules release. indeed, the nervous system finely tunes the activity of each synapse by regulating the secretion of molecules spatially and temporally. currently used techniques have some drawbacks such as a poor spatial resolution or a low flexibility. we propose a novel approach based on optical tweezers (ot) [ ] to overcome these limitations. ot allow an ease manipulation with sub-micrometric precision of silica beads, which can be functionalized with any protein. for a proof-of-principle study we coated , lm large beads with brain-derived neurotrophic factor (bdnf) or bovine serum album (bsa) as control. we showed that a single bead was able to activate the bdnf receptor trkb, inducing its phosphorylation. moreover bdnf beads but not control beads were able to induce c-fos translocation into the nucleus [ ] , indicating that the whole pathway was activated. finally, we positioned vectors in proximity to the growth cones of cultured hippocampal neurons [ ] . control beads didn't affect the normal development of these structures while bdnf beads significantly did. these findings support the use of the ot technology for long-term, localized stimulation of specific subcellular neuronal compartments. a key role of its photoactivity, due to the singlet oxygen production, which has a very short lifetime (ns-ls, depending of hyp environment). hyp sub-cellular localization depends on its concentration in the medium, incubation time and used delivery system. variations in activity of protein kinase c, (anti-apoptotic pkca and pro-apoptotic pkcd) in correlation with the activity of bcl- protein, cytochrome c release from mitochondria and decreasing of mitochondrial membrane potential after photodynamic action were monitored. the study was performed for two different delivery modes of hyp to u- mg glioma cells-hyp alone (membrane diffusion) vs. hyp loaded in low density lipoprotein (ldl) (endocytosis). confocal fluorescence microscopy, flow-cytometry and specific fluorescence labeling were used as main experimental techniques. our results show that hyp photoaction strongly affects apoptotic response of the cells and that the dynamics of this action significantly depends on used delivery system. correlation analysis between the monitored parameters (see above) determined for both delivery system is presented and critically discussed. surface contamination by bacteria is a natural and spontaneous process occurring in any natural environment on biotic (mucosa, tissues…) and abiotic surfaces (medical equipments, food surfaces…). whatever the bacterial nature (gram-positive or -negative), the environmental fluid (air, water, blood…) and the receptor surface (implants, medical equipments, food surfaces…), the surface contamination initiated by the first adherent bacteria can evolve to a three dimensional structure named biofilm (cohesive bacteria assembly ensured by an autoproduced extracellular organic matrix). the mechanisms by which these biofilms offer protective environment to viral particles or hypertolerance to antimicrobial action are not yet elucidated. to reach a better understanding of biofilm reactivity, we reported for the first time successful applications of correlative time-resolved optical microscopy approach by time-lapse (tl), frap, fcs, flim, for real-time analysis of molecular mobility and reaction inside biofilms. by means of non-biological or biological (virus, biocides and antibiotics) reactive compounds, significant advances to understand the roles of the extracellular matrix and bacteria physiological properties were obtained, an important step to improve pathogenic biofilm inactivation. here we present a feasibility study to develop two-photon microscopy ( pm) into a standard diagnostic tool for noninvasive, skin cancer diagnosis. the goal is defining experimental parameters to maximize image quality of optical biopsies in human skin, while avoiding tissue damage. possible diagnostic indicators will be compared for healthy tissue, benign, and malignant melanocytic lesions. we report on preliminary results of a study on pm imaging of ''ex-vivo'' biopsy samples, were autofluorescence intensity and contrast between lesion and surrounding tissues were optimised varying excitation wavelength, detection band, and dispersion pre-compensation. moreover, we determined modulation functions for laser power and detector gain to compensate losses in deep tissue imaging. as the main process of photo-damage, thermo-mechanical modifications were quantified and damage threshold powers were determined. in order to image structural changes in ordered tissue like collagen fibres, second-harmonic generation signals were recorded and optimised. in-vivo two-photon imaging of the honeybee antennal we adapted a two-photon microscope for in-vivo imaging of the honeybee olfactory system, focusing on its primary centres, the antennal lobes. the setup allowed obtaining both d-tomographic measurements of the antennal lobe morphology and time-resolved in-vivo calcium imaging of its neuronal activity. the morphological data were used to precisely measure the glomerular volume in both sides of the brain, investigating the question of volumetric lateralization. functional calcium imaging allowed to record the characteristic glomerular response maps to external odour stimuli applied to the bees' antennae. compared to previous neural imaging experiments in the honeybee, this work enhanced spatial and temporal resolution, penetration depth, and it minimized photo-damage. final goal of this study is the extension of the existing functional atlases of the antennal lobe to d and into the temporal dimension by investigating the time-resolved activity pattern. the use of voltage sensitive fluorescent dyes (vsd) for noninvasive measurement of the action potential (ap) in blood perfused heart have been hindered by low interrogation depth, high absorption and auto-fluorescence of cardiac tissue. these limitations are diminished using new near infrared (nir) vsd (di- -anbdqbs). here we validated toxicity and photo-toxicity of these dyes in guinea pig and human cardiac muscle slabs. application of nir vsd showed no effect on cardiac muscle contraction force or relaxation. optical action potentials closely tracked kinetics of microelectrode recorded aps in both field and electrode stimulated preparations. for phototoxicity assessment dye ( lm) preloaded cardiac slabs were exposed to prolonged laser radiation of various power. microelectrode ap recordings show that exposure to prolonged laser radiation ( min.; mw/mm ) of dye loaded tissue had no statistically significant effect on apd or conduction velocity, thus indicating no or weak photo-toxicity on the nir vsd. in contrast, exposure to min. laser radiation of phototoxic dyes (mitotracker deep-red) preloaded tissue caused significant reduction in apd (by %) and conduction velocity ( %). thus, due to the low photo-toxicity, nir vsd are well suited for in vivo cardiac imaging. streptomycesetes are filamentous gram-positive soil bacteria well known for their complex morphological development and secondary metabolite production. during their life cycle spores germinate to form a network of hyphae, which later develops into aerial mycelium when cross-walls are generated and spores are formed. we have examined and compared the last stage of the differentiation process in a wild-type s. coelicolor (m ) and its dcabb mutant lacking a calmodulin-like calcium binding protein. the strains were grown on four kinds of media: smms, smms with % saccharose, r and r with reduced calcium in order to study the effect of environment and osmotic stress on the sporulation of the two strains to assess the function of cabb protein. from the cultures pictures were taken at hours and after days using phase contrast, atomic force and confocal laser scanning microscope and the sizes of spores were measured. our results showed that the dcabb mutant made smaller spores, its differentiation and stress response were slower. we could conclude from it that the aberrant protein slows the metabolism, the signal transduction and has effects on sporulation, septation and air-mycelium formation. based on it we can tell that the cabb has a significant role in normal development. the mobility and reaction parameters of molecules inside living cells can be conveniently measured using fluorescent probes. typically fluorescence correlation spectroscopy (fcs) based on confocal microscopy is used for such measurements. this implies high time-resolution but only for a single spot at a time. in order to achieve a high time-resolution at multiple spots, we built a single plane illumination microscope (spim) equipped with high-speed image acquisition devices and a high-na detection optics. this allows us to do parallel fcs measurements in a thin plane (width * - lm) of the sample. our setup is equipped with a fast emccd camera (full frame time resolution ls) and a pixel array of spads. the spad array has a full frame time resolution of - ls, which is even fast enough to resolve the typical motion time-scale of small molecules (like egfp) inside living cells. the performance of the system is characterized by diffusion measurements of water-soluble fluorescent beads, as well as fcs measurements in living cells. our data acquisition system uses programmable hardware for some tasks and is fast enough to allow real-time correlation of pixels, as well as saving the complete dataset for later evaluation. electron cryo-microscopy (cryo-em) covers a larger size range than any other technique in structural biology, from atomic resolution structures of membrane proteins, to large noncrystalline single molecules, entire organelles or even cells. electron crystallography of two-dimensional ( d) crystals makes it possible to examine membrane proteins in the quasi-native environment of a lipid bilayer at high to moderately high resolution ( . - Å ). recently, we have used electron crystallography to investigate functionally important conformational changes in membrane transport proteins such as the sodium/proton antiporters nhaa and nhap, or the structure of channelrhodopsin. ''single particle'' cryo-em is well suited to study the structure of large macromolecular assemblies in the . to Å resolution range. a recent example is our Å map of a mitochondrial respiratory chain supercomplex consisting of one copy of complex , two copies of complex iii, and one of complex iv. the fit of the x-ray structures to our map indicates short pathways for efficient electron shuttling between complex i and iii by ubiquinol, and between complex iii and iv by cytochrome c. electron cryo-tomography can visualize large protein complexes in their cellular context at - Å resolution, and thus bridges the gap between protein crystallography and light microscopy. cryo-et is particularly suitable for studying biological membranes and large membrane protein complexes in situ. we found that long rows of atp synthase dimers along ridges of inner membrane cristae are an ubiquitous feature of mitochondria from all species we investigated ( mammals, fungi, plant). the proton pumps of the respiratory chain appear to be confined to the flat membrane regions on either side of the ridges. this highly conserved pattern suggests a fundamental role of the mitochondrial cristae as proton traps for efficient atp synthesis. single-particle analysis: advanced fluorescence imaging, including subdiffraction microscopy, relies on fluorophores with controllable emission properties. chief among these fluorophores are the on/off photoswitchable and green-to-red photoconvertible fluorescent proteins. irisfp was recently reported as the first fluorescent protein to combine irreversible photoconversion from a green-emitting to a red-emitting state with reversible on/off photoswitching in both the green and red states. the introduction of this protein resulted in new applications such as super-resolution pulse-chase imaging, but the properties of irisfp are far from being optimal from a spectroscopic point of view and its tetrameric organization complicates its use as a fusion tag. we have demonstrated how four-state optical highlighting can be rationally introduced into photoconvertible fluorescent proteins by developing and characterizing a new set of such enhanced optical highlighters derived from meo-sfp and dendra . one of these, which we called nijifp, was identified as a promising new multi-photoactivatable fluorescent protein with optical properties that make it ideal for advanced fluorescence-based imaging applications. introducing to medicine and biology concept of optical markers in tremendous way has changed the recent status of these two important disciplines. this was mainly due to strong development in imaging techniques which recently allow us to investigate both static as well dynamic properties of living cells, their components and their interactions with external factors. recently used molecular markers including organic dyes, fluorescent proteins or chelates containing lanthanide ions have several significant limitations. one of the alternatives for molecular markers are inorganic quantum dots (ie. cdse, cds) which are recently commonly used in many academic works. however, even if they are much better from physicochemical point of view, from the application point of view at this moment they are rather useless mainly because of their high risk of toxicity. one of the solution combining advantages of both concepts is to make nontoxic inorganic nanocrystals doped by lanthanide ions. in this work, we will present optical results obtained for nayf nanocrystals doped by different lanthanide ions. the aim of this work was to design and to synthesize these markers and to understand physical processes responsible for their emission and to optimize these processes to the physical limits. intravital microscopy has fostered a full blown of publication regarding the behavior of cells in different tissues and physiological conditions. however, a few papers describe how the motility parameters can be used to understand whether an interaction is occurring and, on balance, the distinction between interacting and non interacting cells is performed visually on the image time stack. here we describe a multi-parameter approach that allows to discern among different cell behaviors on an objective ground and we demonstrate its effectiveness valuating the mutual fate of natural killer (nk) and dendritic (dc) cells at the draining lymph-nodes in inflammatory and stationary conditions. the method is time saving and allows a wide scale characterization of the lymphocyte tracks and to build up statistics of the cell-cell interaction duration. this has allowed the development of a numerical model of the nk-dc interaction, based on a molecular-stochastic dynamic approach, whose output can be directly compared to the data. hemozoin is formed during malaria infection of red blood cells, the malaria parasite cleaves hemoglobin, leaving free heme which is then toxic to the parasite. the free heme is then bio-crystalized to form hemozoin which allows the parasite to remain viable. the hemozoin is released during the breakdown of the red blood cells, is small and can be difficult to resolve spatially. since it contains an abundance of heme protein, which has a strong absorbance at nm, it can be readily detected and tracked by using resonant raman scattering spectroscopy. here we use slit-scanning confocal raman imaging to detect the hemozoin and resolve it against the background molecules. inside a red blood cell, hemoglobin is the strongest background signal since it also contains large amounts of heme. nevertheless, the discrimination is possible, and the time-resolved observation of hemozoin is an important tool to understand effects of malaria since the hemozoin can trigger the immune response and cause inflammation in tissue. muscle performance at the molecular level is determined by the elementary displacement (working stroke) produced by the motor protein myosin ii, and its dependence on load. we developed a laser trap assay (the optical leash) capable of applying controlled loads to a single myosin head before the working stroke is initiated and probing actin-myosin interaction on the microsecond time scale. we found that the working stroke size depends both on load and on the detachment pathway followed by myosin. in a first pathway, myosin detaches very rapidly from actin (\ ms) without producing any movement. in a second pathway, myosin steps and remains bound to actin for a time inversely proportional to atp concentration; the working stroke remains constant (* nm) as the load is increased, until it suddenly vanishes as the isometric force is reached ( . ± . pn). a third dissociation pathway becomes more populated as the force is increased, due to premature unbinding of myosin from actin, resulting in a working stroke that decreases with load. taken together, these results give a new insight into the molecular mechanism of load dependence of the myosin working stroke, which is a primary determinant of skeletal muscle performance and efficiency. previously we have deleted either or both of these terminal helices genetically. surprisingly, all mutants rotated in the correct direction, showing that the shaft portion is dispensable. here we inquire if the rest of the c rotor, the globular protrusion that accounts for * % of the c residues, is also dispensable. keeping the n-and c-terminal helices that constitute the shaft, we have replaced the middle * residues with a short helix-turn-helix motif borrowed from a different protein. the protrusion-less mutant thus made retained a high atpase activity and rotated fast in the correct direction. this may not be unexpected because, in crystal structures, most of the removed residues do not contact with the a b ring. combined with the previous results, however, the present results indicate that none of the c residues are needed for rotation. the rotary mechanism of a molecular engine, the vacuolar proton-atpase, working in a biomembrane csilla ferencz , pá l petrovszki , zoltá n kó ta the rotary mechanism of vacuolar proton-atpase (v-atpase) couples atp hydrolysis and trans-membrane proton translocation. we tested the effect of oscillating electric (ac) field on v-atpase activity in yeast vacuoles. the ac technique has several advantages over direct observations: it can be applied on native membranes, there are no labels and attachments involved, and the target protein is in its natural environment. this was/is the first of its kind of experiment on v-atpase, and we got strikingly different results from previous studies on other proteins: both low and high frequency ac field reduces atpase activity in a wide frequency range. a sharp resonance is seen at . hz, where the atpase activity reaches or exceeds the control (no ac) level. we think that the resonance happens at that of the degrees rotor steps, meaning that the rotation rate of the rotor is around hz, under the given conditions. synchronisation of individual atpases by slow or matching, but not fast ac is likely via a hold-and-release mechanism. we can explain the above observations by assuming that the ac field interacts with the proton movements, and if we consider the estimated geometry of the hydrophilic proton channels and the proton binding cites on the rotor. [ ] . the ttss constitutes a continuous protein transport channel of constant length through the bacterial envelope [ ] . the needle of the type three secretion system is made of a single small protein (protomer). we analyzed the assembly and the structure of the ttss needle using different biophysical methods including fourier transform-infrared spectroscopy, nmr spectroscopy and x-ray crystallography. we show that the ttss needle protomer refolds spontaneously to extend the needle from the distal end. the protomer refolds from a-helix into b-strand conformation to form the ttss needle [ ] . regulated secretion of virulence factors requires the presence of additional protein at the ttss needle tip. x-ray crystal structure analysis of the tip complex revealed major conformational changes in both the needle and the tip proteins during assembly of the s. typhimurium ttss. our structural analysis provides the first detailed insight into both the open state of the ttss needle tip and the conformational changes occurring at the pathogen-host interface [ ] . [ the membrane-bound component f o of the atp synthase works as a rotary motor and plays the central role of driving the f component to transform chemi-osmotic energy into atp synthesis. we have conducted molecular dynamics simulations of the membrane-bound f o sector with explicit lipid bilayer, in which the particular interest was to observe the onset of helix motion in the c ring upon the change of the protonation state of asp of the c subunit, which is the essential element of the boyer's binding-change mechanism. to investigate the influence of transmembrane potential and ph gradient, i.e., the proton motive force, on the structure and dynamics of the a-c complex, different electric fields have been applied along the membrane normal. correlation map analysis indicated that the correlated motions of residues in the interface of the a-c complex were significantly reduced by external electric fields. the deuterium order parameter (s cd ) profile calculated by averaging all the lipids in the f o -bound bilayer was not very different from that of the pure bilayer system, which agrees with recent h solid-state nmr experiments. however, by delineating the lipid properties according to their vicinity to f o , we found that the s cd profiles of different lipid shells are prominently different. lipids close to f o formed a more ordered structure. similarly, the lateral diffusion of lipids on the membrane surface also followed a shell-dependent behavior. the lipids in the proximity of f o exhibited very significantly reduced diffusional motion. the numerical value of s cd was anti-correlated with that of the diffusion coefficient, i.e., the more ordered lipid structures led to slower lipid diffusion. our findings will not only help for elucidating the dynamics of f o depending on the protonation states and electric fields, but may also shed some light to the interactions between the motor f o and its surrounding lipids under physiological condition, which could help to rationalize its extraordinary energy conversion efficiency. this work has been published in march , and it was selected as one of the two featured articles of that issue. the research project is directed toward the construction of a synthetic bio-inorganic machine that consists in a single actin filament that interacts with a linear array of myosin ii motors regularly disposed on a nano-structured device. the motor array is intended to simulate the unique properties of the ensemble of motor proteins in the half-sarcomere of the muscle by providing the condition for developing steady force and shortening by cyclic interactions with the actin filament. the mechanical outputs in the range . - pn force and - , nm shortening will be measured and controlled the bacterial flagellar motor is a membrane-embedded molecular machine that rotates filaments, providing a propulsive force for bacteria to swim. the molecular mechanism of torque (turning force) generation is being investigated through the study of the properties and three-dimensional structure of the motor's stator unit. we are taking both topdown and bottom-up approaches, combining data from the molecular genetics studies, cross-linking, x-ray protein crystallography and molecular dynamics simulations. we have recently determined the first crystal structure of the protein domain that anchors the proton-motive-force-generating mechanism of the flagellar motor to the cell wall, and formulated a model of how the stator attaches to peptidoglycan. the work presented at the meeting will inform the audience on our latest work that establishes the relationship between the structure, dynamics and function of a key component of the bacterial flagellar motor, the motility protein b (motb). this work will be put in the perspective of the mechanism of rotation, stator assembly, anchoring to peptidoglycan and interaction with the rotor, and discussed in the light of the elementary events composing the cycle of electrochemical-to-mechanical energy conversion that drives flagellar rotation. members of the conserved kinesin- family fulfill essential roles in mitotic spindle morphogenesis and dynamics and were thought to be slow, processive microtubule (mt)-plusend directed motors. the mechanisms that regulate kinesin- function are still not well understood. we have examined in vitro and in vivo functions of the saccharomyces cerevisiae kinesin- cin using single-molecule motility assays and single-molecule fluorescence microscopy and found that cin motility is exceptional in the kinesin- family. in vitro, individual cin motors could be switched by ionic conditions from rapid (up to lm/min) and processive minus-end, to bidirectional, to slow plus-end motion. deletion of the uniquely large insert of amino acids in loop of cin induced bias towards minus-end motility and strongly affected the directional switching of cin both in vivo and in vitro. we further found that deletion of the functionally overlapping kinesin- kip and of the spindle-organizing protein ase affected cin velocity and processivity, but directionality was not affected. the entirely unexpected finding of switching of cin directionality in vivo and in vitro demonstrates that the ''gear box'' of kinesins is much more complex and versatile than thought. many biological motor molecules move within cells using stepsizes predictable from their structures. myosin-vi, however, has much larger and more broadly-distributed stepsizes than those predicted from its short lever arms. we explain the discrepancy by monitoring qdots and gold nano-particles attached to the myosin vi motor domains using high-sensitivity nano-imaging. the large stepsizes were attributed to an extended and relatively rigid lever arm; their variability to two stepsizes, one large ( nm) and one small ( nm). these results suggest there exist two tilt-angles during myosin-vi stepping, which correspond to the pre-and post-powerstrokes states and regulate the leading head. the large steps are consistent with the previously reported hand-over-hand mechanism, while the small steps follow an inchworm-like mechanism and increase in frequency with adp. switching between these two mechanisms in a strain sensitive, adpdependent manner allows myosin-vi to fulfill its multiple cellular tasks including vesicle transport and membrane anchoring. http://www.fbs.osaka-u.ac.jp/labs/yanagida/, http:// www.qbic.riken.jp/. ferritin deposits iron in oxyhydroxide iron core surrounded by protein shell. the iron core structure may vary in different ferritins in both normal and pathological cases. to study iron core variations the mö ssbauer spectroscopy with a high velocity resolution was applied for comparative analysis of normal and leukemia chicken liver and spleen tissues, human liver ferritin and commercial pharmaceutical products imferon, maltoferÒ and ferrum lek as ferritin models. mö ssbauer spectra of these samples measured with a high velocity resolution at room temperature were fitted using two models: homogenous iron core (one quadrupole doublet) and heterogeneous iron core (several quadrupole doublets). the results of both fits demonstrated small variations of mö ssbauer hyperfine parameters related to structural variations of the iron cores. these small structural variations may be a result of different degree of crystallinity, the way of iron package, nonequivalent iron position, etc. obtained small differences for normal and leukemia tissues may be useful for distinguishing ferritins from normal and pathological cases. this work was supported in part by the russian foundation for basic research (grant # - - -a). invasion of epithelial cells by salmonella enterica is mediated by bacterial ''effector'' proteins that are delivered into the host cell by a type iii secretion system (ttss). the collaborative action of these translocated effectors modulates a variety of cellular processes leading to bacterial uptake into mammalian cells. type iii effectors require the presence in the bacterial cytosol of specific tts chaperones. effectors are known to interact with their chaperone via a chaperone binding domain (cbd) situated at their n-terminus. this work focus on sopb, an effector with phosphoinositide phosphatase activity and particularly its interaction with the specific chaperone sige by biochemical, biophysical and structural approaches. we have co-expressed sopb with its specific chaperone sige and purified the complex, determined the limits of the cbd and purified the sopb cbd /sige complex. the structure of sige has been solved previously but no crystals could be obtained for structure determination of both complexes. we used saxs experiment combined with biophysical approach to analyse the interaction between sopb and its chaperone as well as the quaternary structure on the complex that will be described in this presentation. guanylate monophosphate kinase (gmpk) is a cytosolic enzyme involved in nucleotide metabolic pathways. one of the physiological roles of gmpks is the reversible phosphoryl group transfer from atp to gmp (its specific ligand), yielding adp and gdp. the gmpk from haemophilus influenzae is a small protein, with a number of amino acids in the primary structure. in order to determine the secondary structure changes of this enzyme, as well as some physical characteristics of its complexes with gmp and atp ligands, circular dichroism (cd) and atr -ftir studies were performed. the enzyme and its ligands were dissolved in tris -hcl buffer, at ph . and °c. from the cd spectra the content of the secondary structure elements of gmpk and gmpk/gmp, gmpk/atp (with and without mg + ) were determined. the major secondary structure elements of gmpk from haemophilus influenzae were a-helix (* %) and b-sheet (* %). atr -ftir experiments show that the amide i and amide ii bands of the gmpk are typical for a protein with great a-helix content. from the second derivative spectra, the content of the secondary structure elements were estimated. these data were in agreement with those obtained by cd. assembly of the mature human immunodeficiency virus type capsid involves the oligomerization of the capsid protein, ca. the c-terminal domain of ca, ctd, participates both in the formation of ca hexamers, and in the joining of hexamers through homodimerization. intact ca and the isolated ctd are able to homodimerize in solution with similar affinity (dissociation constant in the order of lm); ctd homodimerization involves mainly an a-helical region. in this work, we show that peptides derived from the dimerization helix (which keep residues energetically important for dimerization and with higher helical propensities than the wild-type sequence) are able to self-associate with affinities similar to that of the whole ctd. moreover, the peptides have a higher helicity than that of the wild-type sequence, although is not as high as the theoretically predicted. interesting enough, the peptides bind to ctd, but binding in all peptides, but one, does not occur at the dimerization interface of ctd (helix ). rather, binding occurs at the last helical region of ctd, even for the wild-type peptide, as shown by hsqc-nmr. as a consequence, all peptides, but one, are unable to inhibit capsid assembly of the whole ca in vitro. the peptide whose binding occurs at the ctd dimerization helix has an val?arg mutation in position , which has been involved in dimer-dimer contacts. these findings suggest that event keeping the energetically important residues to attain ctd dimerization within a more largely populated helical structure is not enough to hamper dimerization of ctd. putp is an integral membrane protein located in the cytoplasmic membrane of escherichia coli, being responsible for the coupled transport of na + and proline. it belongs to the family of sodium solute symporters (sss). structural data for putp is not available, but secondary structure predictions together with biochemical and biophysical analyses suggest a transmembrane motif. from a recent homology model based on the x-ray structure of the related na + /galactose symporter vsglt, previously published electron paramagnetic resonance (epr) studies, and recent crystallographic and epr studies on the cognate bacterial homolog of a neurotransmitter:na + symporter, leut, it has been proposed that helices viii and ix as well as the interconnecting ''loop '' region determine the accessibility of the periplasmic cavities which bind sodium and proline. we performed site-directed spin labeling of ''loop '' in combination with epr spectroscopy to investigate the structural features of this region and possible conformational changes induced by sodium and proline. analyses of spin label mobility and polarity as well as accessibility to paramagnetic quenchers allow us to refine this region in the present homology model. furthermore, our data suggest conformational changes in this region upon substrate binding including an overall motion of a helical segment. fatty acid-binding proteins (fabp) are a family of low molecular weight proteins that share structural homology and the ability to bind fatty acids. the common structural feature is a b-barrel of antiparallel b-strands forming a large inner cavity that accommodates nonpolar ligands, capped by a portal region, comprising two short a-helices. b-fabp exhibits high affinity for the docosahexaenoic (dha) and oleic acid (oa). it is also postulated that b-fabp may interact with nuclear receptors from ppar family. in the present work, we used molecular biology and spectroscopic techniques to correlate structure, dynamics and function. site-directed mutagenesis was used to produce mutants of b-fabp with a nitroxide spin probe (mtsl) selectively attached to residues located at the portal region. esr spectra of the labeled b-fabp mutants were sensitive to the location of the mutation and were able to monitor interactions in three cases: ( ) shsp are ubiquitous proteins involved in cellular resistance to various stress (oxidative, heat, osmotic…). they are able to prevent aggregation of non-native proteins through the ability of forming large soluble complexes and preventing their nonspecific and insoluble aggregation. in consequence to this molecular chaperone function, they can regulate many processes (resistance to chemotherapy, modulation of the cellular adhesion and invasion, inflammatory response in skin), and the modulation of their expression has been found to be a molecular marker in cancers, spermatogenesis, or cartilage degeneration. furthermore, they are involved in several pathologies: myopathies, neuropathies, cancers, cataracts. among the human members (hspb - ), this study focused on hspb (hsp involved in some cancers), hspb (lens specific), hspb (lens, muscle, heart, lung) and hspb -r g (responsible for a desmin-related myopathy and a cataract). as shsp form large, soluble (but polydisperse in mammals) hetero-oligomers, molecular biology, biochemistry, biophysics and bioinformatics were successfully combined to compare the functional/dysfunctional assemblies in order to understand the critical parameters between shsp members depending upon their tissue and cellular localization. ionizing radiation is a type of radiation that contains enough energy to displace electrons and break chemical bonds. this can promote the removal of at least one electron from an atom or molecule, creating radical species, namely, reactive oxygen species (ros) [ , ] . these are often associated with damages at cellular level, such as, dna mutations, cell cycle modifications and, in animal cells, cancer. to overcome this problem, organisms developed different protection/repair mechanisms that enable them to survive to these threats. dna glycosylases are enzymes that are part of base excision repair (ber) system, mainly responsible for dna repair. they can recognize a dna lesion and, in some cases, are able to remove the mutated base. here we propose to study one of those enzymes, endonuclease iii, which contains a [ fe- s] cluster [ , ] . samples were exposed to different doses of uv-c radiation and the effects were studied by electrophoretic and spectroscopic methods. [ na,k-atpase is an integral protein present in the plasma membrane of animal cells, and consists of two main subunits: the a and b. cholesterol is an essential constituent of the animal membrane cells. in order to study the interaction between na,k-atpase and cholesterol, we have used the dsc technique, and a proteoliposome system composed by the enzyme and dppc:dppe, with different percentage in mol of cholesterol. the heat capacity of purified na,k-atpase profile exhibits three transitions with , and kcal/mol at , and °c. multiple components in the unfolding transition could be attributed either to different steps in the pathway or to independent unfolding of different domains. this denaturation of na,k-atpase is an irreversible process. for the proteoliposome, we also observed three peaks, with , and kcal/mol and , and °c. this increase in dh indicates that the lipids stabilize the protein. when cholesterol was used (from to mol %), the first transition was shifted to a lower temperature value around °c. these results confirm that cholesterol has an influence on the packing and fluidity of lipid bilayer and changes in lipid microenvironment alter the thermostability as well as the activity of na,k-atpase. financial support: fapesp. we have undertaken to study the structure and function of peroxisomal multifunctional enzyme type (mfe- ) from different organisms. mfe- is a key enzyme in long-and branched-chain fatty acids breakdown in peroxisomes. it contains two enzymes in the same polypeptide and also consists of differing amount of domains depending on the species. crystal structure and enzyme kinetics of drosophila melanogaster mfe- has revealed the domain assembly and raised a question about existence of a substrate channeling mechanism. small-angle x-ray scattering studies have further resolved the assembly of domains in the human mfe- . mutations in the mfe- -coding gene in humans may cause d-bifunctional protein deficiency -a metabolic disease characterized by accumulation of fatty acyl-coa intermediates due to inactive or residually active mfe- protein. we have also studied the structure, stability and dynamics of such mutant proteins both experimentally and in silico. the latest results on all these studies will be presented. ftsz is a protein that plays a key role in bacterial division, forming a protein ring directly related to the constriction of the membrane. this process has been observed to occur without the help of molecular motors. nonetheless, the details of the self-assembly and subsequent force generation of the septal ring are still obscure. afm observations allows the study of the behaviour of ftsz solutions on a substrate with unprecedented resolution, permitting the identification of individual protein filaments. the different resulting structures can be compared to monte carlo models for a d lattice accounting for the essential interactions between monomers. these include a strong longitudinal bond that allows a limited flexibility (i.e., curvature of the filaments) and a weaker lateral interaction. the work we present follows this approach, focussing on the latest experiments with ftsz mutants. by using these mutants, it is possible to choose the specific region of the monomer that will anchor to the substrate, thus generating new structures that provide an insight into monomer-monomer interactions. in this way, we explore the anisotropy of the lateral bond in ftsz, a factor that has not been taken into account before but may prove to be of importance in fstz behaviour in vivo. modeling protein structures and their complexes with limited experimental data dominik gront university of warsaw, pasteura , - warsaw, poland conventional methods for protein structure determination require collecting huge amounts of high-quality experimental data. in many cases the data (possibly fragmentary and/or ambiguous) on itself cannot discriminate between alternative conformations and a unique structure cannot be determined. small angle xray scattering is an example of such a ''weak'' experiment. the spectrum encodes only several independent degrees of freedom that provide a global description of a molecular geometry in a very synthetic way. in this contribution we utilized both local information obtained from nmr measurements and global description of a macromolecule as given by saxs profile combined with a knowledge-based bimolecular force field to determine tertiary and quaternary structure of model protein systems. saxs curve as well as various kinds of local nmr data such as isotropic chemical shifts and their tensors, j-couplings, rdc, backbone noe and redor from nmr in solid phase are parsed with the ''experimental'' module of bioshell toolkit and utilized by rosetta modeling suite to generate plausible conformations. obtained results show the new protocol is capable to deliver very accurate models. noenet-use of noe networks for nmr resonance assignment of proteins with known d structure dirk stratmann, carine van heijenoort and eric guittet structural genomics programs today yield an increasing number of protein structures, obtained by x-ray diffraction, whose functions remain to be elucidated. nmr plays here a crucial role through its ability to readily identify binding sites in their complexes or to map dynamic features on the structure. an important nmr limiting step is the often fastidious assignment of the nmr spectra. for proteins whose d structures are already known, the matching of experimental and back-calculated data allows a straightforward assignment of the nmr spectra. we developed noenet, a structure-based assignment approach. it is based on a complete search algorithm, robust against assignment errors, even for sparse input data. it allows functional studies, like modeling of protein-complexes or protein dynamics studies for proteins as large as kd. almost any type of additional restraints can be used as filters to speed up the procedure or restrict the assignment ensemble. noenet being mainly based on nmr data (noes) orthogonal to those used in triple resonance experiments (jcouplings), its combination even with a low number of ambiguous j-coupling based sequential connectivities yields a high precision assignment ensemble. we observed, that t. thermophilus isopropylmalate dehydrogenase (ipmdh) has higher rigidity and lower enzyme activity at room temperature than its mesophilic counterpart (e. coli), while the enzymes have nearly identical flexibilities under their respective physiological working conditions, suggesting that evolutionary adaptation tends to maintain optimum activity by adjusting a ''corresponding state'' regarding conformational flexibility. in order to reveal the nature of the conformational flexibility change related to enzymatic activity, we designed a series of mutations involving non conserved prolines specific to thermophilic ipmdh. proline to glycine mutations substantially increased conformational flexibility and decreased conformational stability. the mutant enzyme variants did not show enhanced catalytic activity, but the non arrhenius temperature dependence of enzyme activity of the wild type was abolished. this phenomenon underlines the fact that the delicate balance between flexibility, stability and activity which is required for the environmental adaptation of enzymes can be easily disrupted with mutations even distant from the active site, providing further evidence that optimization of proper functional motions are also a selective force in the evolution of enzymes. the kinetoplastids trypanosoma brucei, t. cruzi and leishmania major are responsible for causing great morbidity and mortality in developing countries. the all a-helical dimeric dutpases from these organisms represent promising drug targets due to their essential nature and markedly different structural and biochemical properties compared to the trimeric human enzyme. to aid in the development of dutpase inhibitors we have been structurally characterizing the enzymes from these species. here we present the structure of the t. brucei enzyme in open and closed conformations, completing the view of the enzymes from the kinetoplastids. furthermore, we sought to probe the reaction mechanism for this family of enzymes as a mechanism has been proposed based on previous structural work but has not received any further verification. the proposed scheme is similar to that of the trimeric enzyme but differs in detail. using tryptophan fluorescence quenching in the presence of the transition state mimic alf we have been able to identify which species is the likely transition state in the reaction. the crystal structure of t. brucei in complex with this transition state analogue confirms the nature of the nucleophilic attack clearly showing how it differs from trimeric enzymes. the structure of factor h-c d complex explains regulation of immune complement alternative pathway circular dichroism (cd) spectroscopy is a widely used technique for studying the secondary structure (ss) of proteins. numerous algorithms have been developed for the estimation of the ss composition from the cd spectra. although, these methods give more or less accurate estimation for proteins rich in a-helical structure, they often fail to provide acceptable results on mixed or b-rich proteins. the problem arises from the diversity of b-structures, which is thought to be an intrinsic limitation of the technique. the worst predictions are provided for proteins of unusual b-structures and for amyloid fibrils. our aim was to develop a new algorithm for the more accurate estimation of ss contents for a broader range of protein folds with special interest to amyloid fibrils. using synchrotron radiation cd (srcd), we were able to collect high quality spectra of amyloid fibrils with good s/n ratios down to nm. the novel reference dataset with spectra that significantly differ from present reference sets, extends the information content for ss determination. our algorithm takes into account the diverse twist of the b-sheets that has a great influence on the spectral features. for the first time, we can reliably distinguish parallel and antiparallel b-structure using cd spectroscopy. monitoring the assembly of the membrane protein insertase alexej kedrov , marko sustarsic , arnold j.m. driessen groningen biomolecular sciences and bioengineering institute, university of groningen, the netherlands, university of oxford, uk molecular forces that govern membrane protein integration and folding remain a major question in current molecular biology and biophysics. each nascent polypeptide chain should acquire its unique three-dimensional folded state within a complex environment formed by the anisotropic lipid membrane and the membrane-water interface. secyeg translocase and members of a recently described yidc/oxa /alb chaperone family are recognized as primary players in the membrane protein genesis. these proteins, so called insertases, serve as membraneembedded molecular pores where the newly synthesized protein is loaded prior its release into the bilayer. here we apply fluorescence correlation spectroscopy to monitor the assembly of the insertase:ribosome:nascent polypeptide chain complexes in solution and reconstituted into nanodiscs and model membranes. results provide insights on molecular mechanisms and dynamics of the insertase functioning. conformational changes during gtpase activity induced self-assembly of human guanylate binding protein revealed by epr spectroscopy correct assembly and regulation of multi-component molecular machines is essential for many biological activities. the type iii secretion system (t ss) is a complex molecular machine that is a key virulence determinant for important gram-negative pathogens including shigella, yersinia and salmonella species [ ] [ ] . the t ss consists of multiple copies of * different proteins (totalling * mda), spans both bacterial membranes and drives insertion of a contiguous pore into the host-cell membrane. virulence factors are secreted via this apparatus directly into the host cell. in all t ss various levels of regulation occur with switching between secretion off and on states overlaid on control of which substrates are secreted. genes involved in a variety of these switches have been identified but the molecular mechanisms underlying these functions is poorly understood. we are studying the t ss of shigella flexneri, the causative agent of dysentery and will present the structure of the so-called ''gatekeeper protein'' mxia. the diacylglycerolacyltransferase (dgat ) is an integral protein from the reticulum endoplasmic membrane that plays an essential role in triacylglyceride synthesis. in cattle, this enzyme is associated to the fat content regulation on milk and meat. in this study, synthetic peptides corresponding to both dgat binding sites (sit and sit ) were designed, purified and employed to investigate the enzyme interaction with substrates and membrane models. different binding specificities in the interaction with phospholipid vesicles and micelles were noted. sit showed to bind more strongly in nonpolar membrane models, while sit was electrostatically attracted to negative phospholipid surfaces. the binding of both peptides was followed by significant conformational changes (like unordered to helix transition) in circular dichroism spectra and a nm blue shift in fluorescence emission. the binding of sit and sit peptides to negative liposome gave dissociation constants (k d ) of and . lm, respectively, and a leakage action -fold higher to sit . the difference in specificity is related to the features of the putative substrates (acyl-coas and diacylglycerol) and can be attributed to the distinct role of each dgat binding site during lipid synthesis. supported by fapesp. ftsz, the bacterial homologue of tubulin, assembles into polymers in the bacterial division ring. the interfaces between monomers include a gtp molecule, although the relationship between polymerization and gtpase activity is still controversial. a set of short ftsz polymers were modelled and the formation of active gtpase structures was monitored using molecular dynamics. only the interfaces nearest the polymer ends exhibited geometry adequate for gtp hydrolysis. conversion of a mid-polymer interface to a close-to-end interface resulted in its spontaneous rearrangement from an inactive to an active conformation. fluorescent proteins (fps) have become extremely valuable tools in the life sciences. due to the latest advances in the light microscopy, there is a steady need for fps with improved spectral properties. mirisfp is a monomeric fp that can be switched reversibly between a bright green fluorescent and a dark state by illumination with light of specific wavelengths [ ] . structurally, this photo-switching is based on a cis-trans isomerization of the chromophore. upon illumination with violet light, mirisfp can be irreversibly photoconverted from the green-emitting to a red-emitting form. the red form can again be switched reversibly between a fluorescent and a dark state. to elucidate the mechanistic details of the photoinduced reactions, we have generated mirisgfp . this variant can still undergo reversible photoswitching, but lacks the ability to photoconvert to the red state so that the photoinduced transitions of the green form can be studied without 'artifacts' due to green-to-red photoconversion. using uv/visible spectroscopy, we have characterized the on-and off-switching processes in great detail. several light-activated reaction pathways have been identified. they are highly intertwined so that the net effect achieved with light of a particular wavelength depends on the relative probabilities to photoinduce the various processes. phosducin (pd) is a g t bc-binding protein that is highly expressed in photoreceptors. pd is phosphorylated in dark-adapted retina and is dephosphorylated in response to light. dephosphorylated pd binds g t protein bc-heterodimer with high affinity and inhibits its interaction with g t a or other effectors, whereas phosphorylated pd does not. therefore pd down-regulates the light response in photoreceptors. phosphorylation of pd at s and s leads to the binding of the - - protein. the - - proteins function as scaffolds modulating the activity of their binding partners and their role in pd regulation is still unclear. the - - protein binding may serve to sequester phosphorylated pd from g t bc or to decrease the rate of pd dephosphorylation and degradation. we performed several biophysical studies of the - - :pd complex. analytical ultracentrifugation was used to determine the complex stoichiometry and dissociation constant. conformational changes of pd induced both by the phosphorylation itself and by - - binding were studied using the time-resolved fluorescence spectroscopy techniques. mö ssbauer spectroscopy with a high velocity resolution was used for comparative study of various oxyhemoglobins for analysis of the heme iron electronic structure and protein structure-function relationship. samples of pig, rabbit and normal human oxyhemoglobins and oxyhemoglobins from patients with chronic myeloleukemia and multiple myeloma were measured using mö ssbauer spectrometric system with a high velocity resolution at k. mö ssbauer spectra were fitted using two models: one quadrupole doublet (model of equivalent iron electronic structure in a-and b-subunits of hemoglobins) and superposition of two quadrupole doublets (model of non-equivalent iron electronic structure in a-and b-subunits of hemoglobins). in both models small variations of mö ssbauer hyperfine parameters (quadrupole splitting and isomer shift) were observed for normal human, rabbit and pig oxyhemoglobins and related to different heme iron stereochemistry and oxygen affinity. small variations of mö ssbauer hyperfine parameters for oxyhemoglobins from patients were related to possible variations in the heme iron stereochemistry and function. the different types of silk produced by orb-weaving spiders display various mechanical properties to fulfill diverse functions. for example, the dragline silk produced by the major ampulate glands exhibits high toughness that comes from a good trade-off between stiffness and extensibility. on the other hand, flagelliform silk of the capture spirals of the web is highly elastic due to the presence of proline and glycine residues. these properties are completely dictated by the structural organization of the fiber (crystallinity, degree of molecular orientation, secondary structure, microstructure), which in turn results from the protein primary structure and the mechanism of spinning. although the spinning process of dragline silk begins to be understood, the molecular events occurring in the secretory glands and leading to the formation of other silk fibers are unknown, mainly due to a lack of information regarding their initial and final structures. taking advantage of the efficiency of raman spectromicroscopy to investigate micrometer-sized biological samples, we have determined the conformation of proteins in the complete set of glands of the orb-weaving spider nephila clavipes as well as in the fibers that are spun from these glands. domain of rgs at . Å resolution was solved. the stoichiometry of - - f /rgs protein complex was elucidated using the analytical ultracentrifugation. to map the interaction between - - f and rgs protein we performed a wide range of biophysical measurements: h/d exchange and cross link experiments coupled to mass spectrometry, fret (fö rster resonance energy transfer) time-resolved fluorescence experiments, time-resolved tryptophan fluorescence spectroscopy and saxs (small angle x-ray scattering) measurements. based on all these results we build d model of - - f /rgs protein complex. our model revealed new details on architecture of complex formed by - - proteins. to date all known structure of - - proteins complexes suggests that the ligand is docked in the central channel of - - protein. our results indicate that the rgs domain of rgs protein is located outside the central channel of - - f protein interacting with less-conserved residues of - - f. the receptor for advanced glycation end-products (rage) is a multiligand cell surface receptor involved in various human diseases. the major alternative splice product of rage comprises its extracellular region that occurs as a soluble protein (srage). although the structures of srage domains were available, their assembly into the functional full length protein remained unknown. here we employed synchrotron small-angle x-ray scattering to characterize the solution structure of human srage. the protein revealed concentration-dependent oligomerization behaviour, which was also mediated by the presence of ca + ions. rigid body models of monomeric and dimeric srage were obtained from the scattering data recorded at different solvent conditions. the monomer displays a j-like shape while the dimer is formed through the association of the two n-terminal domains and has an elongated structure. the results provided insight into the assembly of i) the heterodimer srage:rage, which is responsible for blockage of the receptor signalling, and ii) rage homodimer, which is necessary for signal transduction, paving the way for the design of therapeutical strategies for a large number of different pathologies. clpb is a hexameric aaa? atpase that extracts unfolded polypeptides from aggregates by threading them through its central pore. the contribution of coiled-coil m domains is fundamental for the functional mechanism of this chaperone, and its location within the protein structure in previous structural models is contradictory. we present cryo-electron microscopy structural analysis of clpb from e. coli in several nucleotide states. the study reveals a novel architecture for clpb and shows that m domains form an internal scaffold located in the central chamber of clpb hexamers. this inner structure transmits local signals due to atp binding and hydrolysis by aaa? domains. surprisingly, coiled-coil m domains are seen to bend significantly around a hinge region that separates two structural motifs. our results present a new framework to understand clpb-mediated protein disaggregation. streptomyces clavuligerus isoenzymes involved in clavulanic acid biosynthesis: a structural approach clavulanic acid (ca) is a potent b-lactamase inhibitor produced by streptomyces clavuligerus. n -( -carboxyethyl) arginine synthase (ceas) and proclavaminate amidino hydrolase (pah) catalyze the initial steps in the biosynthesis of ca. recently ceas and pah genes (paralogous of ceas and pah) were related to the ca biosynthesis but their products have not been studied yet. here we present the initial structural analysis of ceas and pah using biophysical techniques. pah and ceas genes were isolated from the genomic dna of s. clavuligerus and overexpressed in e. coli. the recombinant proteins were purified by affinity chromatography and analyzed by size exclusion chromatography, non-denaturing page, dynamic light scattering, far-uv circular dichroism (cd) and fluorescence spectroscopy. our results showed that pah and ceas were obtained as hexamer and dimer respectively. both proteins showed an a/b folding, being stable up to °c. above this temperature protein unfolding was observed but the complete unfolding was not observed, even at °c. moreover ceas and pah showed to be stable over a wide ph range (ph . - . ). we are currently working on improving ceas crystals which are a promising step towards the elucidation of the ceas structure. supported by fapesp. • synchrotron radiation circular dichroism • mass spectrometry following vuv photoionisation • fluorescence imaging with lifetime and spectral measurements here we will present the srcd experiment. high photon flux of photons / sec, improved detector performances as well as user-orientated software developments have proven to be the garants for successful data collections,which considerably increased the information content obtained. the exploration into the charge transfer region of the peptide bonds is adding specifically new insights. low sample volumes of as little as ll per spectra as well as convenient sample chamber handling allow for economic and efficient data collections. typical spectra acquisition from to nm, last for min for three scans with a nm step size. prior to high resolution based techniques, srcd spectrawill answer questions about folding states of macromolecules including dna, rna and sugar macromolecules as well as their complexes with proteins and specially membrane proteins. sporulation in bacillus subtilis begins with an asymmetric cell division producing a smaller cell called the forespore, which initially lies side-by-side with the larger mother cell. in a phagocytosis-like event, the mother cell engulfs the forespore so that the latter is internalised as a cell-within-a-cell. engulfment involves the migration of the mother cell membrane around the forespore until the leading edges of this engulfing membrane meet and fuse. this releases the forespore, now surrounded by a double membrane, into the mother cell cytoplasm. membrane migration during engulfment is facilitated by the interacting proteins spoiiq and spoiiiah that are membrane-associated and expressed in the forespore and the mother cell respectively . they interact in the intercellular space and function initially as a molecular zipper and later they participate in a more elaborate complex in which spoiiq and spoiiiah are integral components of an intercellular channel. this channel is a topic of much current interest, having initially been proposed as a conduit for the passage from the mother cell to the forespore of a specific, but putative, regulator of the rna polymerase sigma factor, r g and later as a gap junction-like feeding tube through which the mother cell supplies molecules for the biosynthetic needs of the forespore. here we present data on the structure and interactions of spoiiq and spoiiiah gleaned from biophysical methods and protein crystallography. these data lead to a plausible model for the intercellular channel. the glycine receptor (glyr) is a chloride permeable ligand gated ion channel and that can mediate synaptic inhibition. due to a possible involvement in the pathophysiology of temporal lobe epilepsy, the different properties of glyrs containing alpha l and k subunit isoforms are currently investigated. previous characterizations of homomeric receptors consisting of these isoforms have shown a difference in their electrophysiological properties and their membrane distribution as observed by diffraction-limited fluorescence microscopy. we studied these isoforms, when separately expressed in transfected hek t cells, by using single molecule tracking (smt) in living cells and direct stochastic optical reconstruction microscopy (dstorm) on fixated cells. for both techniques the glyrs are stained using a primary antibody directly labeled with alexa fluor dyes. the dstorm experiments support the observation that alpha l glyr are clustered, while the alpha k glyrs are more uniformly spread. the analysis of the short range diffusion coefficients obtained by smt reveals the presence of heterogeneous motion for both isoforms. the k-isoform has a higher fraction of fast diffusion. in contrast, the l-isoform is more associated with slow diffusion and appears to undergo hindered diffusion. since nanoparticles are suitable for tumor therapy due to their passive targeting to cancer cells by enhanced permeability and retention effect [ ] , it is important to understand mechanisms of their delivery into the living cancer cells. in this respect we have developed a modular spectral imaging system based on a white light spinning disk confocal fluorescence microscope and a narrow tunable emission filter. firstly, interaction of polymer nanoparticles and cells labeled with spectrally overlapping probes was examined. the use of fluorescence microspectroscopy (fms) allowed co-localization, which showed that the size of polymer nanoparticles strongly influences their transfer across the cell plasma membrane. next, the delivery of liposomes (composed of cancerostatic alkylphospholipid (opp) and cholesterol) labeled with environment-sensitive fluorescent probe was monitored. we were able to detect a very small shift in emission spectra of cholesterol-poor opp liposomes inside and outside the cells, which would not be possible without the use of fms. this shift implies that the delivery of these liposomes into cancer cells is based on fusion with the cell membrane [ ] . [ high-resolution optical imaging techniques make now accessible the detection of nanofeatures in bio-and soft-matter by non-ionizing visible radiation. however, high-resolution imaging is critically dependent by the fluorescent probes used for reporting on the nano-environment. on account of our long-standing interest in the development of fluorescent probes, we set out to design and engineer new fluorescent systems for nanoscale imaging and sensing of biological specimens and soft-matter. these fluorophores report on fast subtle changes of their nanoscale environment at excited state and are meant to fulfill these requirements: a) optical responses (intensity, wavelength-shift, lifetime, anisotropy) predictably related to the environmental polarity, viscosity, macromolecular structure, b) high brightness allowing for single-molecule detection, c) easily conjugable to biomolecules or macromolecules of interest. notably, we aim at conjugating these properties with the capability of nanoscopy imaging based on stimulated emission depletion or stochastic reconstruction optical microscopy. in this lecture the main features and applications of the engineered probes will be reviewed and future developments in this exciting field will be discussed. foamy virus (fv) is an atypical retrovirus which shares similarities with hiv and hepatitis b viruses. despite numerous biochemical studies, its entry pathway remains unclear, namely membrane fusion or endocytosis. to tackle this issue, dual color fluorescent viruses were engineered with a gfp labeled capsid and a mcherry labeled envelope. using high resolution d imaging and d single virus tracing, we followed the entry of the fluorescent viruses in living cells with a precision of nm in the plane and nm along the optical axis. to distinguish between the two possible pathways, we developed a novel colocalization analysis method for determining the moment along every single trace where the colors separate, i.e. the fusion event. the combination of this dynamical colocalization information with the instantaneous velocity of the particle and its position within the reconstructed d cell shape allows us to determine whether the separation of capsid and envelope happens at the cell membrane or from endosomes. we then compared two types of fv and demonstrated, consistently with previous ph-dependency studies, that the prototype fv can enter the cell by endocytosis and membrane fusion, whereas the simian fv was only observed to fuse after endocytosis. phosphatidylinositol , -bisphosphate (pi( , )p ) is a minor component of the plasma membrane known to a critical agent in the regulation of synaptic transmission. clustering of pi( , )p in synaptic active zones is important for synaptic transmission. however, pi( , )p does not spontaneously segregate in fluid lipid membranes and another mechanism must be responsible for the lateral segregation of this lipid in active zones. clustering of pi( , )p is expected to be associated with lipid-protein interactions and possibly partition towards lipid rafts in the plasma membrane. here we analyze the influence of protein palmitoylation on the formation of pi( , )p clusters and on synaptic protein-pi( , )p interaction by means of fö rster resonance energy transfer measurements by fluorescence lifetime imaging (fret-flim) and fret confocal microscopy. . during sporulation an entire chromosome is transferred into the forespore. this process starts by the formation of an asymmetrically located division septum that leads to the formation of two unequally sized compartments: a large mothercell and a smaller forespore. the septum traps about % of the chromosome to be transferred into the forespore. the remaining (* mbp) are then translocated from the mother cell into the forespore by an active mechanism involving the spo-iiie dna translocase. the mechanisms of translocation, particularly the control of the directionality, still remains unknown and various models have been proposed so far. since each model predicts very different distribution of spoiiie proteins at the sporulation septum, we used palm microscopy (photoactivated localization microscopy) to investigate proteins localization in live-sporulating bacteria. using this technique, we showed that spoiiie proteins are forming a single tight focus at the septum with a characteristic size of around nm. more surprising, the focus is usually localized in the mother cell compartment and the mean distance between the spoiiie focus and the septum is nm. our data suggest that during the translocation process, spoiiie proteins are only forming stable complexes on the mother cell side, allowing then for a control of the chromosome translocation from the mother cell to the forespore. morphogenetic gradients determine cell identity in a concentration-dependent manner and do so in a way that is both incredibly precise and remarkably robust. in order to understand how they achieve this feat, one needs to establish the sequence of molecular mechanisms starting with morphogen gradient formation and leading to the expression of downstream target genes. in fruit flies, the transcription factor bicoid (bcd) is a crucial morphogen that forms an exponential concentration gradient along the embryo ap axis and turns on cascades of target genes in distinct anterior domains. we measured bcd-egfp mobility in live d. melanogaster embryos using fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. we found that bcd-egfp molecules had a diffusion coefficient on the order of * lm /s during nuclear cycles - , both in the cytoplasm and in nuclei. this value is large enough to explain the stable establishment of the bcd gradient simply by diffusion. on the other hand, in the context of the extremely precise orchestration of the transcription of the hunchback bcd target gene, it is too slow to explain how a precise reading of bicoid concentration could be achieved at each interphase without the existence of a memorization process. single molecule studies of key processes during the initiation of innate and adaptive immune response the two pillars of the vertebrate immune system are the innate and adaptive immune response, which confer resistance to pathogens and play a role in numerous diseases. here we exploit single molecule fluorescence imaging on live cells to study the key molecular processes that underpin these responses. the first project looks at the changes in the organisation of toll-like receptor (tlr ) on the cell surface of macrophages upon activation via lipopolysaccharide (lps), as it is currently not known whether a higher level of tlr organisation is required for the signalling process. macrophages natively express tlr at a low level which allows for oligomerisation to be analysed in live cells by dynamic single molecule colocalisation (dysco) using data obtained by totalinternal reflection fluorescence (tirf) microscopy. the experiments of the second project aim at determining the critical initial events in t-cell triggering by labelling key proteins like the tcr receptor and cd on the surface of live t-cells and following how their spatial distribution changes following the binding of the t-cell to a surface. this enables us to distinguish between the different models of t-cell triggering which are based on aggregation, segregation or a conformational change of the tcr. the study of cells using scanning force microscopy the motility of unicellular parasites in mammalians seems very interesting, yet very complex. in a world, were inertia cannot be used for propulsion, in a world at low reynolds numbers, most of our everyday strategies of self-propulsion do not work. one class of parasites that know their way around, the flagellate trypansome, manage not only to survive in the blood stream, which is a lot faster than its own propulsion velocity and where the trypanosome is constantly attacked by its host's immune response, but also to penetrate the bloodbrain-barrier, which actually should be to tight to enter. even though trypanosomes are known for more than years, their motility behaviour is not completely elucidated yet. now, using high-speed darkfield-microscopy in combination with optical tweezers in microfluidic devices and analyzing the recorded data, new light has been shed on the motility of these parasites. astonishing results show that trypanosomes are very well adapted to their hosts environment, they even can abuse red blood cells for their self-propulsion and use the bloodstream itself to drag antibodies bound to their surface to their cell mouth, where the antibodies are endocytosed and digested. the first part of the presentation will discuss nanoparticle (quantum dot, qd) biosensors and nanoactuators that exploit novel and unusual fret phenomena in the induction/detection of protein aggregation [ ] , reversible on-off qd photoswitching [ ] , and ph sensing [ ] . the second part of the presentation will feature the application of an integrated chemical biological fret approach for the in situ (in/on living cells) detection of conformational changes in the ectodomain of a receptor tyrosine kinase (the receptor for the growth factor egf) induced by ligand binding [ ] . the measurements were conducted with a two-photon scanning microscope equipped with tcspc detection; novel methods for lifetime analysis ad interpretation were employed to confirm the concerted domain rearrangements predicted from x-ray crystallography. the study of protein-protein interactions in vivo is often hindered by the limited acquisition speed of typical instrumentation used, for instance, for lifetime imaging microscopy. anisotropy polarization is altered by the occurrence of foerster resonance energy transfer (fret) and anisotropy imaging was shown to be comparatively fast and simple to implement. here, we present the adaptation of a spinning disc confocal microscope for fluorescence anisotropy imaging that allowed to achieve in vivo imaging at high spatial and temporal resolution. we demonstrate the capabilities of this system and in-house developed analysis software by imaging living caenorhabditis elegans expressing constitutive dimeric and monomeric proteins that are tagged with gfp. measuring intracellular viscosity: from molecular rotors to photodynamic therapy of cancer marina k. kuimova department of chemistry, imperial college london, exhibition road, sw az, uk viscosity is one of the main factors which influence diffusion in condensed media and is crucial for cell function. however, mapping viscosity on a single-cell scale is a challenge. we have imaged viscosity inside live cells using fluorescent probes, called molecular rotors, in which the speed of rotation about a sterically hindered bond is viscosity-dependent [ ] [ ] [ ] . this approach enabled us to demonstrate that viscosity distribution in a cell is highly heterogeneous and that the local microviscosity in hydrophobic cell domains can be up to higher than that of water. we demonstrated that the intracellular viscosity increases dramatically during light activated cancer treatment, called photodynamic therapy (pdt) [ ] . we also demonstrated that the ability of a fluorophore to induce apoptosis in cells during pdt [ ] , or to act as a benign molecular rotor, measuring viscosity, can be controlled by carefully selecting the excitation wavelength in viscous medium [ ] . in the field of biophysics and nanomedicine, the cellular reaction and the kinetics of gene expression after transfection of live cells with plasmid dna or gene-silencing sirna is of great interest. in a previous study on the transfection kinetics of non-viral gene-transfer [ ] we realised that the development of single-cell arrays would be a great step towards easy-to-analyse, high-throughput transfection studies. the regular arrangement of single cells would overcome the limitations in image-analysis that arise from whole populations of cells. in addition to that, the analysis of expression kinetics at the single-cell can help to identify the cell-to-cell variability within a cell population. in order to develop suitable single-cell arrays, we are currently adjusting the different parameters of such a microenvironment (e.g. size, shape, surface-functionalisation) in order to end up with a defined surrounding for single-cell transfection studies. in addition to that, we try to find the optimal uptake pathway for each of the different applications. the neurodegenerative disorder alzheimer's disease (ad) causes cognitive impairment such as loss of episodic memory with ultimately fatal consequences. accumulation and aggregation of two proteins in the brain -amyloid beta and tau -is a characteristic feature. these soluble proteins aggregate during the course of the disease and assemble into amyloid-like filaments. recently it was found that the toxicity of soluble amyloid beta oligomers must also be taken into account for the pathogenesis of cognitive failure in ad. if oligomers are the predominant toxic species it would be pertinent to determine how they disrupt and impair neuronal function. the prion protein (prp) receptor has been proposed to mediate amyloid beta binding to neuronal cells. we have characterised the interaction of the amyloid beta and the prp receptor expressed on hippocampal and neuroblastoma cells at the single-molecule level. we do not detect any colocalisation of either the or amino acids variants with the prp receptor. bacterial biofilms are of the utmost importance in the study of environmental bioremediation and the design of materials for medical applications. the understanding of the mechanisms that govern cell adhesion must be analysed from the physics point of view in order to obtain quantitative descriptors. the genus rhodococcus is widely spread in natural environments. the species are metabolically diverse and thus they can degrade a wide range of pollutants. due to their high hydrophobicity, these cells are very resistant to harsh conditions, are able to degrade hydrophobic substances (e.g. oil) and attach to high-contact angle surfaces. the hydrophobicity of several strains of rhodococcus is measured and mapped using chemical force microscopy (cfm) in the present study. cfm relies on the functionalisation of scanning force microscopy (sfm) tips using hydrophobic or hydrophilic groups. in cfm, the microscope is operated in the forcevolume mode, which combines adhesion data with topographic images. the careful control of the tip chemistry permits the study of interactions between the functional groups on the tip and the bacterial surface, thus allowing the assessment of hydrophobicity. in order to perform a cfm study, the cells need to be firmly anchored to a substrate under physiological conditions (i.e. under a nutrient media or a saline buffer). to this end, several adhesive surfaces have been tested in order to find the one that gives the best results. optical microscopy is arguably the most important technique for the study of living systems because it allows d imaging of cells and tissues under physiological conditions under minimally invasive conditions. conventional far-field microscopy is diffraction-limited; only structures larger than * nm can be resolved, which is insufficient for many applications. recently, techniques featuring image resolutions down to * nm have been introduced such as localization microscopy (palm, storm) and reversible saturable optical fluorescent transition microscopy (resolft, sted). these methods are well suited for live-cell imaging and narrow the resolution gap between light and electron microscopy significantly. we have used palm imaging to study the formation and disassembly of focal adhesions of live hela cells in a high resolution pulse-chase experiment using monomeric irisfp [ ] . mirisfp is a photoactivatable fluorescent protein that combines irreversible photoconversion from a green-to a red-emitting form with reversible photoswitching between a fluorescent and a nonfluorescent state in both forms. in our experiments a subpopulation of mirisfp molecules is photoconverted to the red form by irradiating a specified region of the cell with a pulse of violet light. migration of tagged proteins out of the conversion region can be studied by subsequently localizing the proteins in other regions of the cell by palm imaging, now using the photoswitching capability of the red species. real-time image reconstruction developed in our lab [ ] allowed instant control imaging parameters. live cell imaging of cancer cells is often used for in-vitro studies in connection with photodynamic diagnostic and therapy (pdd and pdt). especially in presence of a photosensitizer, this live cell imaging can only be performed over relatively short duration (at most hour). this restriction comes from the light-induced cell damages (photodamages) that result from rapid fluorescence photobleaching of photosensitizer. while these studies reveal exciting results, it takes several hours to discover the detailed effects of the photosensitizer on cell damage. up to our knowledge, however, there is no general guideline for modification of excitation light dose to achieve that. in this paper, the relation between excitation light doses, photobleaching of photosensitizer (pvp-hyperycin) and cell vitality are investigated using human lung epithelial carcinoma cells (a ). the strategy of this paper is to reduce the excitation light dose by using a low-power pulsed blue led such that the structures are visible in time-lapse images. fluorescence signals and image quality are improved by labelling the cells with an additional non-toxic marker called carboxyfluorescein-diacetate-succinimidyl-ester (cfse). in total we collected time-lapse images (time intervals min) of dual-marked a cells under three different light intensities ( . , . and . mw/cm ) and a variety of pulse lengths ( . , . , , . and ms) over five hours. we have found that there is a nonlinear relationship between the amount of excitation light dose and cell vitality. cells are healthy, i.e. they commence and complete mitosis, when exposed to low light intensities and brief pulses of light. light intensities higher than . mw/cm together with pulse durations longer than ms often cause cell vesiculation, blebbing and apoptosis. in all other cases, however, we found no cell death. in the future, this striking nonlinearity will be studied in more detail. progressive advances in scanning ion conductance microscopy (sicm) [ ] enabled us to convert ordinary scanning probe microscope (spm) in to versatile multifunctional technique. as an imaging tool, ion conductance microscopy is capable to deliver highest possible topographical resolution on living cell membranes among any other microscopy techniques [ ] . also, it can visualize surfaces complexity of those makes them impossible to image by other spms [ ] . ion conductance microscopy combined with a battery of powerful methods such as fluorescence resonance energy transfer (fret) [ ] , patch-clamp, force mapping, localized drug delivery, nano-deposition and nano-sensing is unique among current imaging techniques. the rich combination of ion conductance imaging with other imaging techniques such as laser confocal and electrochemical [ ] will facilitate the study of living cells and tissues at nanoscale. ) and coleoptiles of wheat (triticum aestivum l.) seedlings, which were growing in light and dark conditions, were used to determine fluorescence of whole cells. fluorescence emission spectrum was monitored by fluorescent microscopy using the spectrometer usb . fluorescence intensity f , f , f and f was determined and data was statistically analyzed in annova. we observed that bgf, rf and frf intensity increased in the first leaves with the age of the seedlings. in the coleoptiles was observed great bgf intensity increase with the age of the seedlings. in the coleoptiles decreased rf intensity of the and hours old seedlings, and bgf intensity decreased of hours old seedlings. it was found that emission spectrum and fluorescence intensity changes are induced by the lack of light and salt (nacl) stress. analysis of fluorescence spectrum can quickly and accurately indicate the outset of light and salt stress in plants. there are analogical changes in fluorescence emission spectrum of plant cells in senescence and stress conditions. it was assumed that environmental stress and senescence have common mechanisms in plants. this changes can be monitored by fluorescent microscopy. triple-colour super-resolution imaging in living cells markus staufenbiel, stephan wilmes, domenik lisse, friedrich roder, oliver beutel, christian richter and jacob piehler universitä t osnabrü ck, fachbereich biologie, barbarastraße , , germany, markus.staufenbiel@biologie.uniosnabrü ck.de super-resolution fluorescence imaging techniques based on single molecule localisation has opened tremendous insight into the sub-micrometre organisation of the cell. live cell imaging techniques such as fluorescence photoactivation localization microscopy (fpalm) are currently limited to dual-colour detection due to the restricted availability of red-fluorescent photoswitchable proteins. we employed photoswitching of the oxazine dye atto under reducing conditions for super-resolution imaging in the cytoplasm of living cells. for efficient and specific covalent labelling of target proteins, we have made use of the halotag system. atto was coupled to the halotag ligand (htl) and fast reaction of htl-atto with the halotag enyzme was confirmed in vitro by solid phase binding assays. efficient labelling of the membrane cytoskeleton using lifeact fused to the halotag was observed and super-resolution imaging was readily achieved. based on this approach, we managed to follow the nanoscale dynamics of the actin cytoskeleton as well as clathrincoated pits using clathrin light chain fused to the halotag. we combined this technique with fpalm for triplecolour super-resolution imaging of the spatial distribution of membrane receptors in context of the membrane skeleton. the erbb family of receptor tyrosine kinases consists of four transmembrane proteins that transduce signals across the membrane to control cell fate. growth factor binding results in homo-and hetero-interactions between these receptors at the membrane. erbb receptors are implicated in many cancers, making them a target for therapeutic drugs. to date, studies of erbb interactions have been limited to individual family members or specific pairs, giving an incomplete picture of the highly complex behaviour controlling positive and negative feedback loops and signalling outcomes. to investigate erbb receptor interactions, we have developed tirf-based single molecule fluorescence microscopes capable of simultaneously imaging three, and soon five, fluorescence probes in live cells. we have also developed a catalogue of extrinsic fluorescent probes for : labelling of both endogenous and transfected erbb family members in mammalian cells, plus a bayesian approach to the analysis of single molecule data. this allows us to track active and inactive erbb family members at the basal surface of a model breast cancer cell line that expresses physiological levels of all four receptors. we present here initial characterisation of the entire erbb family together in the cell membrane. the human genome contains more than g proteincoupled receptors (gpcrs); overall, - % of the mammalian genome encodes these molecules. processes controlled by gpcrs include neurotransmission, cellular metabolism, secretion, and immune responses. however it is the stoichiometry of these receptors that is the most controversial. the starting point for understanding gpcr function was the idea that these receptors are monomeric. on the other hand a lot of recent studies favour the concept that gpcr form dimers and are not capable of signalling as independent monomers. recent single molecule studies try to solve this dilemma by suggesting that gpcrs form transient dimers with a lifetime of * ms. however questions remain about the physiological relevance of the preparations necessary for these studies, since they have not been performed on endogenous receptors. here, we directly image individual endogenous receptors using an equimolar mixture of two colour fluorescent fab fragments. we can then determine the receptors stoichiometry by quantifying its dynamic single molecule colocalisation (dy-sco) recorded by total-internal reflection fluorescence (tirf) microscopy. we have recently investigated the domain dynamics of pgk ( ) . structural analysis by small angle neutron scattering revealed that the structure of the holoprotein in solution is more compact as compared to the crystal structure, but would not allow the functionally important phosphoryl transfer between the substrates, if the protein would be static. brownian large scale domain fluctuations on a timescale of ns was revealed by neutron spin echo spectroscopy. the observed dynamics shows that the protein has the flexibility to allow fluctuations and displacements that seem to enable function. [ many physiological and pathological processes involve insertion and translocation of soluble proteins into and across biological membranes. however, the molecular mechanisms of protein membrane insertion and translocation remain poorly understood. here, we describe the ph-dependent membrane insertion of the diphtheria toxin t domain in lipid bilayers by specular neutron reflectometry and solid-state nmr spectroscopy. we gained unprecedented structural resolution using contrast-variation techniques that allow us to propose a sequential model of the membrane-insertion process at angstrom resolution along the perpendicular axis of the membrane. at ph , the native tertiary structure of the t domain unfolds, allowing its binding to the membrane. the membrane-bound state is characterized by a localization of the c-terminal hydrophobic helices within the outer third of the cis fatty acyl-chain region, and these helices are oriented predominantly parallel to the plane of the membrane. in contrast, the amphiphilic n-terminal helices remain in the buffer, above the polar headgroups due to repulsive electrostatic interactions. at ph , repulsive interactions vanish; the n-terminal helices penetrate the headgroup region and are oriented parallel to the plane of the membrane. the c-terminal helices penetrate deeper into the bilayer and occupy about two thirds of the acyl-chain region. these helices do not adopt a transmembrane orientation. interestingly, the t domain induces disorder in the surrounding phospholipids and creates a continuum of water molecules spanning the membrane. we propose that this local destabilization permeabilizes the lipid bilayer and facilitates the translocation of the catalytic domain across the membrane. the limited stability in vitro of mps motivates the search of new surfactants ( ) ( ) ( ) ( ) . fss with a polymeric hydrophilic head proved to be mild towards mps ( ) .new fss were designed with chemically defined polar heads for structural applications. lac-derivative was efficient in keeping several mps water soluble and active but formed elongated rods ( ) . the glu-family was synthesized, characterized in by sans and auc and for its biochemical interest. the formation of rods is related to the low volumetric ratio between the polar head and hydrophobic tail. the surfactant bearing two glucose moieties is the most promising one, leading to both homogeneous and stable complexes for both br and the b f. it was also shown be of particular interest for the structural investigation of membrane proteins using sans ( ). by combining elastic and quasi-elastic neutron scattering data, and by applying theory originally developed to investigate dynamics in glassy polymers, we have shown that in lyophilised apoferritin above t * k the dynamic response observed in the pico-to nano-second time regime is driven by ch dynamics alone, where the methyl species exhibit a distribution of activation energies. our results suggest that over the temporal and spatial range studied the main apoferritin peptide chain remains rigid. interestingly, similar results are reported for other smaller, more flexible lyophilised bio-materials. we believe this work elucidates fundamental aspects of the dynamic landscape in apoferritin which will aid development of complex molecular dynamic model simulations of super-molecules. a detailed appreciation of the relationships between dynamics and biological function will require analysis based on such models that realize the full complexity of macromolecular material. biological systems must often be stored for extended periods of time. this is done by lyophilisation in the presence of lyoprotectants, such as sugars, which results in stable products at ambient conditions. [ ] in an effort to understand the mechanism of preservation and stabilization, the interactions between sugars and liposome vesicles, which serve as a simple membrane model, have been studied extensively. amongst the common sugars, trehalose has superior preservative effects [ ] and accumulates to high concentrations in many anhydrobiotic organisms. despite many experimental and numerical studies three mechanisms are proposed: vitrification [ ] , preferential exclusion [ ] and water replacement [ ] . to gain more insight into the stabilization mechanism we have recently investigated the effect of trehalose on the bending elasticity of fully hydrated unilamellar vesicles of , -dipalmitoyl-phosphatidylcholine (dppc) in d o at temperatures below and above the lipid melting transition (tm) using neutron spin-echo. the data was analyzed using the zilman-granek theory. at all temperatures measured, trehalose stiffens the bilayer suggesting strong interactions between trehalose and the lipid. trehalose appears to broaden the melting transition but does not change the tm. this agrees with observations using differential scanning calorimetry. influence of macromolecular crowding on protein stability sté phane longeville, clé mence le coeur laboratoire lé on brillouin, gif-sur-yvette, france cell interior is a complex environment filled with a variety of different objects with respect to shape and size. macromolecules are present at a total concentration up to several hundred grams per litre and the overall occupied volume fraction can reach ö & . - . . under crowding environment protein-protein interaction play a fundamental role. the crowding environment can affect some physical, chemical, and biological properties of biological macromolecules [ , ] . traditionally, protein folding is studied in vitro at very low concentration of proteins. under such conditions, small globular single chain proteins can unfold and refold quite rapidly depending mainly to the nature of the solvent. such processes have been very intensively studied, since folding of proteins into their native structure is the mechanism, which transforms polypeptide into its biologically active structure. protein misfolding is involved in a very high number of diseases [ ] (e.g. alzheimer, parkinson, and kreuzfeld-jacob diseases, type ii diabetes, …). theoretically, the problem was studied by the introduction of the concept of excluded volume [ ] . in recent papers [ , ] , minton uses statistical thermodynamic models to address the question. he predicted that inert cosolutes stabilize the native state of proteins against unfolding state mainly by destabilizing the unfolded state and that the dimension of the unfolded state decreases with increasing the concentration of cosolute in a measurable way. small angle neutron scattering (sans) is a technique of choice for such study because, by using appropriate mixtures of light and heavy water, it is possible to match the scattering length density of the solvent to the one of the cosolute and thus to measure the conformation of a molecule at low concentration in a presence of a high concentration of another one. we will present a complete experimental study of the mechanism that leads to protein stabilization by macromolecular crowding [ , ] . coupled dynamics of protein and hydration water studied by inelastic neutron scattering and molecular dynamics simulation hiroshi nakagawa , , mikio kataoka , japan atomic energy agency, tokai, japan, juelich centre for neutron science, forschungszentrum juelich gmbh, nara institute of science and technology, ikoma, japan proteins work in an aqueous environment at ambient temperature. it is widely accepted that the proteins are flexible and mobile. the flexibility and mobility, that is, protein dynamics are essential for protein functions. neutron incoherent scattering is one of the most powerful techniques to observe protein dynamics quantitatively. here i will talk about dynamics of protein and its hydration water. the structure of a soluble protein thermally fluctuates in the solvated environment of a living cell. understanding the effects of hydration water on protein dynamics is essential to determine the molecular basis of life. however, the precise relationship between hydration water and protein dynamics is still unknown because hydration water is ubiquitously configured on the protein surface. we found that hydration level dependence of the onset of the protein dynamical transition is correlated with the hydration water network. hydration water dynamics change above the threshold hydration level, and water dynamics control protein dynamics. these findings lead to the conclusion that the hydration water network formation is an essential property that activates the anharmonic motions of a protein, which are responsible for protein function. thermal motions and stability of hemoglobin of three endotherms (platypus -ornithorhynchus anatinus, domestic chicken -gallus gallus domesticus and human -homo sapiens) and an ectotherm (salt water crocodile -crocodylus porosus) were investigated using neutron scattering and circular dichroism. the results revealed a direct correlation between the dynamic parameters, melting -, and body temperatures. on one hand, a certain flexibility of the protein is mandatory for biological function and activity. on the other hand, intramolecular forces must be strong enough to stabilize the structure of the protein and to prevent unfolding. our study presents experimental evidence which support the hypothesis that the specific amino acid composition of hb has a significant influence on thermal fluctuations of the protein. the amino acid sequence of hb seems to have evolved to permit an optimal flexibility of the protein at body temperature. macromolecular resilience was found to increase with body and melting temperatures, thus regulating hb dynamics. where k is the trap spring constant, a is the subdiffusion exponent and e a is the mittag-leffler function. the parameters obtained by fitting this equation to the experimental msds are summarized in table . at short lag times we have not found any difference between the two cell types, contrarily to the previous results obtained by afm . for both cell lines the subdiffusion exponent, a was found close to , the value predicted by the theory of semiflexible polymers. but the crossover frequency x , was found smaller for the cancerous cells for all datasets. it corresponds to passage to the confined regime at longer times. we attribute it to the bigger impact of molecular motors. we study the spatiotemporal evolution of fibrous protein networks present in the intracellular and extracellular matrices. here, we focus on the in vitro actin network dynamics and evolution. in order to study the hierarchical self-assembly of the network formation in a confined environment and provide external stimuli affecting the system minimally, we introduce a new microfluidic design. the microfluidic setup consists of a controlling channel to which microchambers of different shapes and sizes are connected through narrow channels. this design results in having mainly convective transport in the controlling channel and diffusive transport into the microchamber. rhodamine labeled actin monomers diffuse into the chamber. after polymerization is induced, they form a confined entangled network. cross-linking proteins can then be added to increase the network complexity. moreover, we can generate gradients of reactants across the microchambers and vasodilator-stimulated phosphoprotein (vasp) is a crucial regulator of actin dynamics. it is important in cellular processes such as axon guidance and migration, promoting assembly of filopodia and lamellipodia. vasp's multiple domain structure increases the range of interactions it has with actin monomers, filaments, and other proteins and it displays multiple binding modes both in vitro and in vivo, including barbed end elongation and filament bundling. however, it is not fully understood how vasp affects the structural and mechanical properties of actin networks. we characterize vasp-mediated bundling of actin networks in a simplified in vitro system using confocal microscopy and quantify mechanical properties with rheology measurements. we show that the network properties differ from other actin bundling proteins and reflect vasp's multiple domain structure, displaying a complex bundling phase space that depends upon solution conditions. we observe the formation of large bundle aggregates accompanied by a reduction in network elasticity at high protein ratios. in addition, we change vasp's actin binding mode and eliminate bundling by introducing free actin monomers. finally, we show preliminary results from a biomimetic system that extends the range of actin-vasp interaction. cell migration or proliferation? the go or grow hypothesis in cancer cell cultures tamá s garay, É va juhá sz, jó zsef tímá r, balá zs heged} us nd department of pathology, semmelweis university, budapest, hungary background: cancer related death is constantly growing in the past decades. the mortality of solid tumors is mostly due to the metastatic potential of tumor cells which requires a fine adjustment between cell migration and cell proliferation. as the metabolic processes in the cell provide a limited amount of available energy (i.e. atp) the various biological processes like cell motility or dna synthesis compete for the atp available. the go or grow hypothesis postulates that tumor cells show either high migration or proliferation potential. in our study we investigate on a large series of tumor cell lines whether this assumption stands for malignant cells. materials and methods: twenty tumor cell lines derived from malignant mesothelioma (mesodermal origin) and malignant melanoma (neuroectodermal origin) were subjected to three-days-long time-lapse videomicroscopic recordings. cell motility and proliferation were characterized by the probability of cell division within hours and the -hour migration distance of the cells. results: we found a wide range in both the cell migratory activity and the proliferation capacity in our series. the -hour migration distance ranged from to micron and from to micron in mesothelioma and melanoma cells, respectively. the lowest -hour cell division probability was found to be . in both the melanoma and mesothelioma series while the highest proliferation activity reached . and . in melanoma and mesothelioma, respectively. interestingly, in the melanoma cell lines we found a significant positive correlation (r= , ; p= , ) between cell proliferation and cell migration. in contrast our mesothelioma cell lines displayed no correlation between these two cellular processes. conclusions: in summary our findings demonstrate that the investigated tumor cells do not defer cell proliferation for cell migration. important to note the tumor cells derived from various organ systems may differ in terms of regulation of cell migration and cell proliferation. furthermore our observation is in line with the general observation of pathologists that the highly proliferative tumors often display significant invasion of the surrounding normal tissue. many cell types are sensitive to mechanical signals. one striking example is the modulation of cell proliferation, morphology, motility, and protein expression in response to substrate stiffness. changing the elastic moduli of substrates alters the formation of focal adhesions, the formation of actin filament bundles, and the stability of intermediate filaments. the range of stiffness over which different primary cell types respond can vary over a wide range and generally reflects the elastic modulus of the tissue from which these cells were isolated. mechanosensing also depends on the type of adhesion receptor by which the cell binds, and therefore on the molecular composition of the specific extracellular matrix. the viscoelastic properties of different extracellular matrices and cytoskeletal elements also influence the response of cells to mechanical signals, and the unusual non-linear elasticity of many biopolymer gels, characterized by strain-stiffening leads to novel mechanisms by which cells alter their stiffness by engagement of molecular motors that produce internal stresses. the molecular mechanisms by which cells detect substrate stiffness are largely uncharacterized, but simultaneous control of substrate stiffness and adhesive patterns suggests that stiffness sensing occurs on a length scale much larger than single molecular linkages and that the time needed for mechanosensing is on the order of a few seconds. to explore potential role of cytoskeletal component in cardiomyocyte for adaptation to extreme conditions was carried out the comparative study of expression of cytoskeletal sarcomeric protein titin in myocardium of ground squirrels during hibernation and gerbils after spaceflight. we have revealed a two-fold increase in content of long n ba titin isoform as compared to short n b titin isoform in different heart chambers of hibernating ground squirrels. the prevalence of the long titin isoform is known to determine the larger extensibility of heart muscle that promotes, according to frank-starling law, the increase in force of heart contractility for pumping higher viscous blood during torpor and adapting the myocardium to greater mechanical loads during awakening. moreover, titin mrna level showed seasonal downregulation in which all hibernating stages differed significantly from summer active level. it is possible that the decline of mrna and protein synthesis during hibernation may be regarded as the accommodation for minimization of energetic expenditures. we have not revealed differences in titin mrna levels between control gerbils and gerbils after spaceflight. but we have also observed the two-fold growth in the amount of n ba titin isoform in left ventricle of gerbils after spaceflight that is likely to be directed to the restoration of the reduced heart contractility at zero-gravity. these results suggest that the increase of the content of the long n ba titin isoform may serve as universal adaptive mechanism for regulating of heart function in response to the extreme conditions. nuclear migration is a general term for a non-random movement of the nucleus toward specific sites in the cell. this phenomenon has been described throughout the eukaryotes from yeast to mammals. the process is however still poorly understood in mammalian cells. by using microcontact printing we are able to regulate the geometry and spreading of cultured cells. adhesive micropatterns of fibronectin provide an attachment surface for the cells whereas the passivation of the surface by pll-peg prevents protein, thus cell adhesion. live cell imaging by time-lapse microscopy has shown that under these conditions cells gain a bipolar shape, and more interestingly, the nuclei of the cells showed auto-reversed motion. our research tries to understand the molecular cues and mechanisms behind the observed cellular and nuclear movement. we have already shown that the cytoskeleton plays an important role in this phenomena but the exact players and the detailed mechanism remain to be clarified. in order to identify the most important components and their relationship have drug treatments and sirna experiments have been applied. although our research focuses mainly on the motility of the nucleus, it may also help to get a better understanding of the general theme of cell migration. cell motility involves a number of strategies that cells use to move in their environments in order to seek nutrients, escape danger and fulfil morphogenetic roles. when these processes become uncontrolled, pathological behaviours, like cancer or metastasis of cancerous cells, can occur. here we present a new method for the contextual quantification of cellular motility, membrane fluidity and intracellular redox state, by using the ratiometric, redox-sensitive protein rxyfp and the ratiometric fluidity-sensitive probe laurdan. we provide evidence that dynamic redox and fluidity changes are correlated with signaling processes involved in cellular motility. these findings may pave the way to novel approaches for the pharmacological control of cell invasiveness and metastasis. manipulation of cellular mechanics anna pietuch, andreas janshoff georg-august university, tammanstraße , gö ttingen, germany, e-mail: anna.pietuch@chemie.unigoettingen.de rheological properties of cells determined by the underlying cytoskeleton (cortex) are key features in cellular processes like cell migration, cell division, and cell morphology. today it is possible to investigate local cellular elastic properties under almost physiological conditions using the afm. by performing force indentation curves on local areas on a cell surface the use of contact mechanic models provides the young's modulus, comprising information about the elastic properties of cells. the administration of cytoskeleton modifying substances into the cell is achieved by microinjection. we are also investigating morphological changes and rearrangements of the cytoskeleton in time resolved impedance measurements. electric cell-substrate impedance sensing is a label-free and minimal invasive technique which allows monitoring morphological changes of cells in real time. readout of the impedance is sensitive to changes in cell-substrate contacts as well as density of cell-cell contacts yielding important information about the integrity of the cell layer and changes in the properties of the cell membrane. we are studying the cellular response to modification of the cytoskeleton e.g. by introducing proteins which affect directly the organization of the actin structure like ezrin. mechanical characterization of actin gels by a magnetic colloids technique thomas pujol, olivia du roure, julien heuvingh pmmh, espci-cnrs-umpc-p . paris, france the actin polymer is central in cell biology: it is a major component of cytoskeleton and it plays a fundamental role in motility, division, mechanotransduction…. its polymerization just beneath the cell membrane generates forces responsible for cell movement. actin filaments form a network whose architecture is defined by the nature of the binding proteins and depends on the location in the cell. for example, in the structure which leads cell migration, the lamellipodium, the gel is branched due to its interaction with arp / protein complex. determining the mechanical properties of such actin network is a crucial interest to understand how forces are generated and transmitted in living cells. we grow a branched actin network from the surface of colloids using the arp / machinery. the particles are super paramagnetic and they attract each other via dipole-dipole interaction to form chain. by increasing the magnetic field we apply an increasing force to the gel and we optically measure the resulting deformation. from those measurements we deduce a young modulus for a large amount of data. we are characterizing different networks by varying the concentration of the capping and branching proteins and we show how mechanics can be regulated by the different proteins. microtubules (mts) are central to the organization of the eukaryotic intracellular space and are involved in the control of cell morphology. in fission yeasts cells mts transport polarity factors to poles where growth is located, thus ensuring the establishment and maintenance of the characteristic spherocylindrical shape. for this purpose, mt polymerization dynamics is tightly regulated. using automated image analysis software, we investigated the spatial dependence of mt dynamics in interphase fission yeast cells. we evidenced that compressive forces generated by mts growing against the cell pole locally reduce mt growth velocities and enhance catastrophe frequencies. in addition, our systematic and quantitative analysis (in combination with genetic modifications) provides a tool to study the role of ?tips (plus-end tracking proteins) such as mal and tip in the spatial regulation of mt dynamics. we further use this system to decipher how the linear transport by mt interferes with the feedback circuitry that assures the correct spatial distribution of tea , the main polarity factor in fission yeast cells. the dynamics of the cytoskeleton are largely driven by cytoskeletal motor proteins. complex cellular functions, such as mitotsis, need a high degree of control of these motors. the versatility and sophistication of biological nanomachines still challenges our understanding. kinesin- motors fulfill essential roles in mitotic spindle morphogenesis and dynamics and were thought to be slow, processive microtubule (mt)-plus-end directed motors. here we have examined in vitro and in vivo functions of the saccharomyces cerevisiae kinesin- cin using single-molecule motility assays and single-molecule fluorescence microscopy. in vivo, the majority of cin motors moved slowly towards mt plus-ends, but we also observed occasional minus-end directed motility episodes. in vitro, individual cin motors could be switched by ionic conditions from rapid (up to lm/min) and processive minus-end, to bidirectional, to slow plus-end motion. deletion of the uniquely large insert in loop of cin induced bias towards minus-end motility and strongly affected the directional switching of cin both in vivo and in vitro. the entirely unexpected in vivo and in vitro switching of cin directionality and speed demonstrate that kinesins are much more complex than thought. these results will force us to rethink molecular models of motor function and will move the regulation of motors into the limelight as pivotal for understanding cytoskeleton-based machineries. morphological and dynamical changes during tgf-b induced epithelial-to-mesenchymal transition david schneider , marco tarantola , and andreas janshoff institute of physical chemistry, georg-august-university, gö ttingen, germany, max planck institute for dynamics and self-organization, laboratory for fluid dynamics, pattern formation and nanobiocomplexity (lfpn), goettingen, germany the epithelial-to-mesenchymal transition (emt) is a program of cellular development associated with loss of cell-cell contacts, a decreased cell adhesion and substantial morphological changes. besides its importance for numerous developmental processes like embryogenesis, emt has also been held responsible for the development and progression of tumors and formation of metastases. the influence of the cytokine transforming growth factor (tgf-b ) induced emt on structure, migration, cytoskeletal dynamics and long-term correlations of the mammalian epithelial cell lines nmumg, a and mda-mb was investigated by time-resolved impedance analysis and atomic force microscopy (afm) performing force-indentation measurements. the three cell lines display important differences in cellular morphology mirrored in changes of their elastic response (young modulus), as well as their dynamics upon tgf-b treatment. impedance based measurements of micromotility reveal a complex dynamic response to tgf-b exposure which leads to a transient increase in fluctuation amplitude and long-term correlation. additionally, the investigation of cellular elasticity via afm depicts the different cytoskeletal alterations depending on the metastatic potential of the used cell type. physics of cellular mechanosensitivity studied with biomimetic actin-filled liposomes bjö rn stuhrmann, feng-ching tsai, guido mul, gijsje koenderink fom institute amolf, amsterdam, the netherlands biological cells actively probe the mechanical properties of their tissue environment by exerting contractile forces on it, and use this information to decide whether to grow, migrate, or proliferate. the physical basis for cell contractility is the actin cytoskeleton, which transmits motor generated stresses to mechanosensitive adhesions sites that anchor the cell to the tissue. the origins of mechanosensing are far from understood due to the complex interplay of mechanical effects and biochemical signaling that occurs far from equilibrium. we use a quantitative biophysical approach based on biomimetic constructs to elucidate physical principles that underlie active mechanosensing in biological cells. we have built realistic in vitro models of contractile cells by encapsulating cross-linked actin networks together with myosin motors in cell-sized membranous containers (liposomes). our method has several advantages over prior methods, including high liposome yield, compatibility with physiological buffers, and chemical control over protein/lipid coupling. i will show contour fluctuation spectra of constructs and first data on mechanical response obtained by laser tweezers microrheology. our work will yield novel insights into stress generation and stiffness sensing of cells. setting up a system to reconstitute cytoskeleton-based protein delivery and patterning in vitro nú ria taberner, liedewij laan, marileen dogterom fom institute amolf, amsterdam, the netherlands keywords: microtubules, fission yeast, cell polarity, protein patterning, plus end binding proteins. many different cell types, from mobile fibroblasts [ ] to fission yeast cells [ ] , display non-homogenous protein patterns on their cell cortex. in fission yeast the cell-end marker protein tea that among others is responsible for recruiting the actin dependent cell-growth machinery, is specifically located at the growing cell ends. tea travels at the tips of growing microtubules and is delivered to the cell ends [ ] . we aim to in vitro reconstitute a minimal microtubule plus-end tracking system that leads to cortical protein patterning in functionalized microfabricated chambers. our model will allow us to perturb microtubule-based transport and diffusion independently and evaluate the resulting protein patterns. [ the tropomyosins (tm) are dimeric actin-binding proteins that form longitudinal polymers along the actin filament groove. there is a great variety of isoforms, but the division of labour between the individual tms and their significance is poorly understood. as in most cell types, also in the neurons several isoforms are present, whose spatio-temporal localisation is differentially regulated. the neuron-specific brain isoform (tmbr- ) can be found in the axon of the mature cells. we aimed to clone, express and charaterise this protein in terms of its effects on the kinetic parameters of the actin filament. using a pet a construct we purified native, tag-free protein, and examined if it influences the rate of actin polymerisation or the stability of the filaments in the presence of either gelsolin or latrunculin-a, two depolymerising agents. in cosedimentation experiments the affinity of tmbr- to actin was* lm, about six times that of skeletal muscle tropomyosin. the net rate of actin polymerisation was reduced by % in the presence of tmbr- . the depolymerisation induced by gelsolin or latrunculin-a was inhibited in a concentration-dependent manner. tmbr- seems to stabilise actin filaments against disassembly without significant effect on the net polymerisation. cell mobility and metastatic spreading: a study on human neoplastic cells using optical tweezers f. tavano the primary causes of death in cancer patients are local invasion and metastasis but their mechanisms are not yet completely understood. metastatization is accompanied by alterations of the cytoskeleton and membrane structure leading to changes in their biomechanical properties [ ] . in this study we analyzed by means of optical tweezers the mechanical properties of two different breast adenocarcinoma cell lines corresponding to different metastatic potential. ot were used to grab the plasma membrane by a , um silica bead and form a plasma membrane tether. we measured the force exerted by the cell membrane on the bead and drew the force-elongation curves. fitting data in the kelvin body model [ ] we found out the values for the viscoelastic parameters influencing the pulling of the membrane tethers. the first cell line analyzed, mcf- , associated to a low metastatic potential showed tether stiffness of pn/um in average. the second cell line, mda-mb , poorly differentiated with a high metastatic potential had a tether stiffness of pn/um in average, that is a four times lower value. these results seems to confirm the hypotesis that metastasis prone cells are softer than less aggressive cancer cells, and support the use of ot for these measurements for its sub-pn force resolution and because cells are manipulated without damage. [ tubulin polymerization promoting protein (tppp/p ) is a brain-specific protein that primary targets the microtubule network modulating its dynamics and stability. tppp/p is a disordered protein with extended unstructured segments localized at the n-and c-terminals straddling a flexible region. tppp/p is primarily expressed in oligodendrocytes where its multifunctional features such as tubulin polymerization promoting and microtubule bundling activities are crucial for the development of the projections in the course of oligodendrocyte differentiation enabling the ensheathment of axons with a myelin sheath that is indispensable for the normal function of the central nervous system. microtubule network, a major constituent of the cytoskeleton, displays multiple physiological and pathological functions in eukaryotic cells. the distinct functions of the microtubular structures are attained by static and dynamic associations of macromolecules and ligands as well as by post-translational modifications. tppp/p is actively involved in the regulation of microtubule dynamics not exclusively by its bundling activity, but also by its tubulin acetylation-promoting activity. atypical histone deacetylases, such as nad-dependent sirt and histone deacetylase- , function outside of the nucleus and control the acetylation level of cytosolic proteins, such as tubulin. acetylation-driven regulation of the microtubule network during cellular differentiation is an ambiguous issue. tppp/p has been recently identified as an interacting partner and inhibitor of these deacetylases and their interaction decreased the growth velocity of the microtubule plus ends and the motility of the cells. we have established cell models for the quantification of the acetylation degree of microtubule network in correlation with its dynamics and stability as well as in relation to aggresome formation, that mimics the pathological inclusion formation. the intracellular level of tppp/p is controlled at posttranscription level by microrna and at protein level by the proteosome machinery. under pathological circumstances this disordered protein displays additional moonlighting function that is independent of its association with microtubule system or deacetylases; it enters aberrant proteinprotein interaction with a-synuclein forming toxic aggregates within the neuronal and glial cells leading to the formation of inclusions characteristic for parkinson's disease and multiple system atrophy, respectively. the cell membrane separates the intracellular from the extracellular environment while intimately interacting with the cytoskeleton in numerous cellular functions, including cell division and motility. cell shape changes are for a large part mediated by the contractile actomyosin network forming the cortex underneath the cell membrane. to uncover molecular mechanisms of cell shape control based on actin-membrane interactions, we built a novel biomimetic model system: a cell-sized liposome encapsulating an actively contracting actin-myosin network. our fabrication method is inspired by a recent report of liposome preparation by swelling of lipid layers in agarose hydrogel films. we extensively characterize important liposomal properties, finding diameters between and lm, unilamellarity, and excellent and uniform encapsulation efficiency. we further demonstrate chemical control of actin network anchoring to the membrane. the resulting liposomes allow quantitative tests of physical models of cell shape generation and mechanics. in the cohesive structure of the cytoskeleton functionally distinct actin arrays orchestrate fundamental cell functions in a spatiotemporally controlled manner. emerging evidences emphasize that protein isoforms are essential for the functional polymorphism of the actin cytoskeleton. the generation of diverse actin networks is catalyzed by different nucleation factors, like formins and arp / complex. these actin arrays also exhibit qualitative and quantitative differences in the associated tropomyosin (tm) isoforms. how the molecular composition and the function of actin networks are coupled is not completely understood. we investigated the effects of different tm isoforms (skeletal muscle, cytoskeletal nm and br ) on the activity of mdia formin and arp / complex using fluorescence spectroscopic approaches. the results show that the studied tm isoforms have different effects on the mdia -, and arp / complexmediated actin assembly. the activity of the arp / complex is inhibited by sktm and tm nm , while tmbr does not have any effect. all three tm isoforms inhibited the activity of mdia . these results contribute to the understanding of the mechanisms by which tropomyosin isoforms regulate the functional diversity of the actin cytoskeleton. chronic thromboembolic pulmonary hypertension (cteph) is a dual pulmonary vascular disorder, which combines major vascular remodelling with small-vessel arteriopathy. the presence of fibroblasts in the clot, occluding the pulmonary arteries, and its composition create a microenvironment with increased collagen level, which might affect the local endothelial function. in this study, human pulmonary artery endothelial cells (hpaec) were exposed to collagen type i to address the effect of the thrombotic microenvironment on the vessel wall forming cells. the hpaecs, cultured under standard conditions were treated with , and lg/ml of collagen type i for h and h. the changes in the endothelial cell barrier function were investigated by performing permeability and migration test as well as ve-cadherin staining. collagen type i treatment led to a decrease in ve-cadherin signal in hpaec. the loosening of cell-cell contacts could be proven with a significant increase in permeability after h of collagen treatment with different concentrations. besides the loosening of the cell-cell contacts, the hpaec migration was also dose dependently retarded by collagen application over time. our data show that collagen-rich microenvironment leads to a disruption of the junctional proteins in hpaecs, indicating an environmental induced possible alteration in the function of endothelial cells in the clots of cteph patients. the implementation of miniaturisation and high throughput screening has quickened the pace of protein structure determination. however, for most proteins the process still requires milligram quantities of protein with purity [ %. these amounts are required as a result of unavoidable losses during purification and for the extensive screening of crystallisation space. for integral membrane proteins (imps), one of the initial steps in the structure determination procedure is still a major bottleneck -the over-expression of the target protein in the milligram quantity range. with a view of developing guidelines for over-expression of human imps, a systematic approach using the three most common laboratory expression systems (e. coli, s. cerevisiae, sf insect cells) was implemented. initial expression levels were determined by either partial purification using ni ? -nta (e. coli), green fluorescence protein (gfp) fluorescence using a c-terminal gfp tagged protein (s. cerevisiae) or flag tagged partial purification (sf cells). the results show that e. coli is suitable for the over-expression of human imps in the required quantity range however protein size and complexity is an important factor. the yeast system is fast and affordable but, for the group of human imps tested, the expression levels were borderline. finally for the insect cell system, the timelines are slower and it is in comparison costly to run, however, it can produce relatively large quantities of human imps. the cu?-atpase copb of enterococcus hirae is a bacterial p-type atpase involved in resistance to high levels of environmental copper by expelling excess copper. the membrane protein copb was purified from an over-expressing strain and solubilized in dodecyl-maltoside. by uv circular dichroism the secondary structure is predicted to contain - % a-helices and - % b-sheets in agreement with estimates based on homology with the ca atpase serca . we present cd-spectroscopic data on thermal unfolding of the protein to address the influence of the binding of the atp analogs atpcs and the fluorescent analog mant-atp on the protein stability. such analogs are used to mimic functional states of the atpase but undergo different interactions with the binding site that are not well characterized. we propose a competition-based assay for nucleotide binding using cdspectroscopy to deduce the occupancy of the nucleotidebinding site by non-fluorescent nucleotides. alternatively, the change of intrinsic fluorescence of mant-atp upon binding to the atpase is exploited in these assays. finally, we show how the simultaneous measurement of protein cd and nucleotide fluorescence in thermal denaturation experiments may help to determine the stability of several functional conformational states of copb. showing the steady-state distribution of electric potential, ionic concentrations are obtained efficiently. channel current, a summation of drift and diffusive currents, can be further computed from the flux of ionic concentrations. the influence of finite size effect will be also addressed. effect of cholesterol and cytoskeleton on k v . membrane distribution jimé nez-garduño am , , pardo la , ortega a , stü hmer w unam, mexico-city, mexico, mpi-em, gö ttingen, germany the potassium channel k v . is expressed nearly exclusively in the central nervous system. besides its function as an ion channel, k v . has also been associated with non-canonical signaling functions. various membrane proteins associated with cholesterol-sphingolipids enriched microdomains are involved in signaling pathways. in this work we studied the membrane distribution of k v . in highly purified brain-tissue plasma membranes as a function of cholesterol content versus cytoskeletal proteins. the results show that one fraction of kv . associates to cholesterol-rich domains or detergent resistant membranes (drm) and another fraction to non-drm domains. the kv . fraction inserted in drm is dependent on cholesterol as well as on cytoskeleton proteins. depletion of cholesterol leads to a doubling of k v . current density. we suggest that k v . coexists in two different populations: one where the transmembrane domain fits cholesterol enriched membranes and another able to fit into a less packed lipid bilayer. the importance of this distribution on signaling processes needs to be further investigated. we use the reduced model of an axis-symmetric water-filled channel whose protein wall has a single charged site. the channel length, radius and fixed charge are selected to match experimental data for gramicidin a. the ion current, occupancy and escape rate are simulated by the d self-consistent bd technique with account taken of the electrostatic ion-ion interaction. the bath with non-zero ion concentration on one side of the channel is modelled via the smoluchowski arrival rate. it is shown that: a) the occupancy saturates with michaelis-menten kinetics. b) the escape rate starts from the kramers value at small concentrations and then increases with concentration due to the electrostatic amplification of charge fluctuations. the resulting dynamics of the current can be described by modified reaction rate theory accounting for ionic escape over the fluctuating barrier [ ] . many membrane-protein functions are amenable to biophysical and biochemical investigation only after the protein of interest has been reconstituted from a detergent-solubilised state into artificial lipid bilayers. unfortunately, functional reconstitution has remained one of the main bottlenecks in the handling of numerous membrane proteins. in particular, gauging the success of reconstitution experiments has thus far been limited to trial-and-error approaches. to address this problem, we have established high-sensitivity isothermal titration calorimetry (itc) as a powerful method for monitoring the reconstitution of membrane proteins into liposomes. itc has previously been employed for characterising liposome solubilisation and reconstitution in the absence of protein. here we show that itc is also excellently suited for tracking the complex process of membrane-protein reconstitution in a non-invasive and fully automated manner. the approach is exemplified for the prokaryotic potassium channel kcsa, which we first purified in detergent micelles and then reconstituted into stable proteoliposomes at very high protein densities. electrophysiological experiments performed in planar lipid membranes confirmed that kcsa regained its functional activity upon itc-guided reconstitution. gating currents of low-voltage-activated t-type calcium channels family má ria karmažínová , Ľ ubica lacinová institute of molecular physiology and genetics, sav, bratislava, slovak republic t-type calcium channels are distinguished by relatively low voltage threshold for an activation and steep voltage dependence of activation and inactivation kinetics just above the activation threshold. kinetics and voltage dependence of macroscopic inward calcium current through ca v channels was described in a detail. in contrast, very little information is available on gating current of these channels. therefore we compared gating currents measured from all three ca v . , ca v . and ca v . channels. voltage dependencies of charge movement differ dramatically from those for macroscopic current. first, their slope factors are several-fold bigger that slope factors of macroscopic current activation. second, activation mid-point for ca v . channels on-gating is shifted to more positive membrane potentials by about mv compare to ca v . and ca v . channels, whose activation mid-points are similar. the same is truth for off-gating voltage dependences. kinetics of both onand off-gating is remarkably faster for ca v . and ca v . channels compare to ca v . channels. further, more charge is moved per unit of macroscopic current amplitude in ca v . channels compare to ca v . and ca v . channels. supported by apvv- - and vega / / . the local anaesthetic lidocaine (lid) is generally believed to reach its binding site in the intracellular vestibule of the voltage-gated sodium channel via the cell membrane. qx (qx) is a permanently charged, quaternary amine analogue of lid, that can access this binding site via a hydrophilic route across the channel protein. the mutation i e of the adult rat muscle-type sodium channel (rna v . ) opens such a hydrophilic pathway. when bound to the internal vestibule, lid stabilizes both fast and slow inactivated states. we wondered whether qx, once bound to the internal vestibule, exerts a similar modulatory action on inactivated states as lid. the construct i e was expressed in tsa cells and studied by means of the patch-clamp technique. when applied from the extracellular side lm qx stabilized the slow but not the fast inactivated state in i e. when applied internally, qx entered the channel, but stabilization of inactivated states could not be observed. these results suggest that binding site for use-dependent block is in the inner vestibule of the channel, fast inactivation is modulated only by the hydrophobic form of lid, and the binding site for modulation of slow inactivation by qx is only accessible from the extracellular side of the channel. we observed that lipophilicity (quantified by the logarithm of the calculated water-octanol partition coefficient, logp) is important in determining both kr and ki, but had a greater effect on ki. distribution coefficients (logd) discriminated better between kr and ki than partition coefficients (logp). the ratio of positively charged/neutral forms (quantified by the acidic dissociation constant, pka) was a significant determinant of resting affinity: predominantly charged compounds tended to be more potent against resting channels, while neutral compounds tended to be more state-dependent. aromaticity was more important for inactivated state affinity. the acidification of intracellular compartments is critical for a wide range of cellular processes. a recent candidate for ph regulation within early endosomes and tgn is the highly conserved intracellular na ? /h ? exchanger isoform (nhe ), whose mutation leads to neurological syndromes in human patients. however, due to its intracellular localization, nhe biochemical features are still poorly characterized and its biological function remains elusive. we have developed somatic cell genetic techniques that enable the selection of variant cells able to resist h ? killing through plasma membrane expression of h ? extruders. this enabled us to obtain stable cell lines with forced plasma membrane expression of nhe . we used them to measure its functional and pharmacological parameters with high accuracy, using fast transport kinetics. to summarize, this exchanger displays unique features within the nhe family, especially with respect to its affinity for its substrates, lithium, sodium and protons and for its guanidine-derived inhibitors. taken together with our results on the subcellular localization of the native nhe , these unique biochemical features provide new insights on the biological function and pathological implications of this intracellular na ? /h ? exchanger. analysis of the collective behaviour of ion channels j. miśkiewicz, z. trela, s. przestalski wrocław university of environmental and life sciences, physics and biophysics department, ul. norwida , - wrocław, poland a novel approach to the analysis of the ion current recordings is proposed. the main goal of the standard patch clamp technique is to measure single channel activity (however the whole cell configuration is also used in various researches). in the presented study the ion channels time series recordings were several (up to four) ion channels were present are analysed and the collective behaviour of ion channels is investigated. the time ion current time series are converted into dwelltime series and the channel activity is analysed. the hypothesis of collective ion channels behaviour is verified and the influence of organolead compounds (met pbcl) on collective ion channel activity is measured. the analysis is performed on the sv cation channels of vacuolar membrane of beta vulgaris. the aim of our computed study was to examine the possible binding site of primaquine (pq) using a combined homology protein modeling, automated docking and experimental approach. the target models of wild-type and mutant-types of the voltage-dependent sodium channel in rat skeletal muscle (rna v . ) were based on previous work by tikhonov and zhorov. docking was carried out on the p-loop into the structure model of rna v . channel, in the open state configuration, to identify those amino acidic residues important for primaquine binding. the threedimensional models of the p-loop segment of wild types and mutant types (w . w c, w c and w a at the outer tryptophan-rich lip, as well as d c, e c, k c and a c of the deka motif) helped us to identify residues playing a key role in aminoquinoline binding. in good agreement with experimental results, a -fold inhibition loss was observed, tryptophan is crucial for the reversible blocking effects of pq. as a result, w c abolished the blocking effect of primaquine in voltage-clamp assays. hydrogen bond formation accelerates channel opening of the bacterial mechanosensitive channel mscl yasuyuki sawada and masahiro sokabe department of physiology, nagoya university graduate school of medicine, nagoya, japan the bacterial mechanosensitive channel mscl is constituted of homopentamer of a subunit with two transmembrane inner and outer (tm , tm ) a-helices, and its d structure of the closed state has been resolved. the major issue of mscl is to understand the gating mechanism driven by tension in the membrane. to address this question, we performed molecular dynamics (md) simulations for the opening of mscl embedded in the lipid bilayer. in the closed state of mscl, neighboring tm inner helices are crossed each other near the cytoplasmic side and leu and val in the constricted part form a stable hydrophobic environment called gate. upon membrane stretch, phe in tm outer helices was dragged by lipids, leading to an opening of mscl. thus phe was concluded to be the major tension sensor. during opening, tm inner helices were also dragged and tilted, accompanied by the outward sliding of the crossings. this led to a slight expansion of the gate associated with an exposure of oxygen atoms of the backbone to the inner surface of the gate. this allows water penetration in the gate and formation of hydrogen bonds between water and the exposed oxygen, which in turn weakened the hydrophobic interaction at the crossings, causing a further opening of the gate and water permeation. mitochondrial bk ca , mitobk ca has been proposed to be cardioprotective and formed by proteins of * to * kda. thus, we investigated the molecular characteristics of this channel in isolated mitochondria from murine heart. labeling of adult mouse cardiomyocytes with plasmalemma bk ca antibodies, mitotracker, and wheat germ agglutinin yielded remarkable mitochondrial but not plasma membrane localization. nanoscale fluorescence microscopy (stimulated emission depletion) revealed to of * - nm bk ca clusters per mitochondria. further, western blot analysis of purified mitochondria showed the presence of a full length * kda protein. systematic rt-pcr exon scanning of isolated cardiomyocyte mrnas were consistent with a full length * kda alpha-subunit protein and revealed the expression of three splice inserts. insertless-bk ca robustly localized to the plasma membrane of cho cells but when a c-terminal splice insert was present bk ca was readily targeted to the mitochondria (protein proximity index was * . indicating % colocalization). hence, cardiac mitobk ca is composed by full-length bk ca protein but with splice inserts which may facilitate its targeting to mitochondria. supported by nih and aha. patch-clamp technique was used to examine effect of trimethyl-lead and -tin on the sv channel activity in the red beet (beta vulgaris l.) taproot vacuoles. it was found that the addition of both investigated compounds to the bath solution inhibit, in a concentration-dependent manner, sv currents. when single channel properties were analyzed, only little channel activity can be recorded in the presence of lm of organometal. compounds investigated decreased significantly (by about one order of magnitude) the open probability of single channels. the recordings of single channel activity obtained in the presence and in the absence of organometal showed that compounds only slightly (by ca. %) decreased the unitary conductance of single channels. it was also found that organometal diminished significantly the number of sv channel openings, whereas it did not change the opening times of the channels. taken together, these results suggest that organometal binding site is located outside the channel's selectivity filter and that the inhibitory effect of both compounds investigated on sv channel activity probably results from organometal-induced disorder in compatibility between membrane lipids and membrane proteins. the research was financed by polish ministry of science and higher education by grant no. nn . electrophysiological investigation of the hvdac ion channel in pore-spanning membranes conrad weichbrodt, claudia steinem georg-august-universitä t gö ttingen, iobc, tammannstr. , d- gö ttingen, germany, e-mail: cweichb@gwdg.de the human voltage-dependent anion channel (hvdac ) plays an important role in cell life and apoptosis since it is the main porin of the outer mitochondrial membrane. as hvdac is believed to play a pivotal role in apoptosis-related diseases such as stroke, alzheimer, parkinson and cancer, the alterations of its electrophysiological properties under different conditions are of great value. to perform different investigations, refolded hvdac is reconstituted in artificial membranes which typically consist of dphpc with a cholesterol fraction of - %. they are prepared via the mü ller-rudin-technique on a functionalized porous alumina substrate containing pores with a diameter of nm. the quality of these so-called nano-black-lipid-membranes (nano-blms) is verified via electrochemical impedance spectroscopy (eis), hvdac is reconstituted and single channel recordings are made. membranes are also prepared by spreading proteoliposomes on hydrophobized porous silicon nitride with pores of lm diameter. information about altered gating-characteristics and related conductivities is gained by application of holding potentials up to ± mv and evaluation of the resulting currents. the hvdac was a kind gift of prof. c. griesinger, mpibpc, gö ttingen. pharmacological inhibition of cardiac herg k ? channels is associated with increased risk of arrhythmias. many drugs bind directly to the channel, thereby blocking ion conduction. ala-scanning mutagenesis identified residues important for drug block. two aromatic residues y and f were found to be crucial for block of most compounds. surprisingly, some cavity blocking drugs are only weakly affected by mutation y a. in this study we provide a structural interpretation for this observation. md simulations on the y a mutant suggest side chain rearrangements of f located one helical turn below y . loss of p-p stacking induces reorientation of f from a cavity facing to a cavity lining conformation, thereby substantially altering the shape of the binding site. docking studies reveal that due to their rigid shape and compactness y insensitive drugs can still favorably interact with the reoriented f aryl groups, while molecules with more extended geometries cannot. the ankyrin transient receptor potential channel trpa is a transmembrane protein that plays a key role in the transduction of noxious chemical and thermal stimuli in nociceptors. in addition to chemical activation, trpa can be activated by highly depolarizing voltages but the molecular basis of this regulation is unclear. the transmembrane part of the tetrameric trpa is structurally related to the voltagegated k ? channels in which the conserved charged residues within the fourth transmembrane region (s ) constitute part of a voltage sensor. compared to these channels, the voltage-dependence of trpa is very weak (apparent number of gating charges * . versus in k ? channels) and its putative voltage-sensing domain most likely lies outside the s because trpa completely lacks positively charged residues in this region. in the present study we used homology modelling and molecular dynamics to create models of the transmembrane part and the proximal cytoplasmic c terminus of trpa . in combination with electrophysiological data obtained from whole cell patchclamp measurements we were able to point out several positively charged residues which mutation strongly alter the voltage sensitivity of trpa channel. these may be candidates for as yet unrecognized voltage sensor. photosynthesis p- action of double stress on photosystem aliyeva samira a., gasanov ralphreed a. institute of botany, azerbaijan national academy of sciences, baku, azerbaijan the simultaneous effect of photoinhibitory illumination and toxic action of heavy metals ions (cd ? and co ? ) on activity of ps in vitro measuring by millisecond delayed fluorescence (ms-df) of chlorophyll a was studied. during action on chloroplasts only of cd ? ( - m) the fast component of ms-df, which originates via radiative recombination of reaction center with the camn -cluster or y z on donor side of ps , is inhibited stronger than at action of only co ? . the steadystate level at cd ? treatment is remain stable, while at co ? action it is increased. simultaneous action of cd ? and photoinhibitory illumination ( lmol photons m - s - ) have shown that fast component of ms-df was inhibited faster with time than in case of action of co ? and excess light. result indicates that damage sites of action cd ? and co ? are donor and acceptor side of ps , accordingly. we assume that binding site of cd ? is y z or camn -cluster, one of the recombination partners with p ? on the donor side of ps . thereby, action of cd ions on donor side of ps leads mainly to development of mechanism of donor-side photoinhibition. field instrument for determination of the photosynthetic capacity of intact photosynthetic bacteria e. asztalos , z. gingl and p. maró ti department of medical physics and informatics, university of szeged, hungary, department of experimental physics, university of szeged, hungary a combined pump and probe fluorometer and spectrophotometer with high power laser diodes has been constructed to measure fast induction and relaxation of the bacteriochlorophyll fluorescence yield and light-induced absorption changes in intact cells of photosynthetic bacteria. the construction is the upgraded version of our previous set up with better time resolution ( ls). the compact design of the mechanics, optics, electronics and data processing makes the device easy to use as outdoor instrument or to integrate into larger measuring systems. the versatility and excellent performance of the apparatus will be demonstrated on different fields: ) organization and redox state of the photosynthetic apparatus of the whole cells under different growth conditions deduced from fluorescence characteristics including the lag phase, the amplitude and the rise time of the variable fluorescence, ) electron transfer in the reaction center, cytochrome bc complex and in between obtained from relaxation of the fluorescence and ) re-reduction kinetics of the oxidized primary donor of the reaction center and energetization and relaxation of the intracytoplasmic membrane tracked by absorption changes at and nm, respectively. previous work has established that the iron stress induced protein a (isia) synthesized by cyanobacteria under stress conditions, has at least two functions: light harvesting [ ] and photoprotection [ ] . under prolonged iron starvation isia becomes the main chlorophyll-binding protein in the cell and occurs without a photosystem association. these isia aggregates have a strong ability to dissipate light energy and there is evidence of carotenoid participation in the quenching mechanism via downhill energy transfer from chlorophyll to the s state of a carotenoid [ ] . in the present work we have measured the temperature dependence of the fluorescence of carotenoid depleted mutants (echinenone and/or zeaxanthin) and isia monomers in order to investigate the role of carotenoid and aggregation in the quenching process. pigment analysis confirms the absence of the carotenoid mutated in its biosynthesis but shows that it is mainly replaced. the monomers are lacking two carotenoids, echinenone and one of the two ß-carotenes found previously in isia aggregates. temperature dependent fluorescence shows that quenching properties are affected in the monomers and the mutants lacking zeaxanthin. soon exhausted oil resources and global climate change have stimulated research aiming at production of alternative fuels, ideally driven by solar energy. production of solar fuels needs to involve the splitting of water into protons, energized electrons and dioxygen. in photosynthetic organisms, solar-energy conversion and catalysis of water splitting (or water oxidation) proceed in an impressive cofactor-protein complex denoted as photosystem ii (psii). the heart of biological water-oxidation is a protein-bound manganese-calcium complex working at technically unmatched efficiency. in an attempt to learn from nature, the natural paragon is intensely studied using advanced biophysical methods. structural studies by x-ray spectroscopy with synchrotron radiation play a prominent role in this endeavor. time-resolved methods provide insights in the formation of intermediate states of the reaction cycle. an overview is presented focusing on (i) the efficiency of solar energy usage in psii, (ii) the interrelation between electron transfer and proton relocations, and (iii) the mechanism of water oxidation. as an outlook, new results on water oxidation by biomimetic manganese and cobalt oxides, which may become a key element in future solar-fuel systems, are presented. the peridinin-chlorophyll-protein (pcp) is a light-harvesting complex (lhc) that works as antenna in the photosynthetic process of dinoflagellates. the protein contains both chlorophylls and carotenoids molecules, the latter being responsible to extend the spectral range of captured light to regions where chlorophylls are transparent. pcp crystal structures [ ] reveal that each chlorophyll is surrounded by or molecules of the carotenoid peridinin, located in non-equivalent positions. the different protein environment of the sites might be responsible of a spectral shift of the pigments with the functional role to extend the absorption spectra of the complex and enhance its light harvesting capabilities. high resolution x-ray diffraction data on a reconstructed pcp, the refolded peridinin-chlorophyll a-protein (rfpcp) [ ] , and on the less common high salt-pcp (hspcp) opened the way to the mechanistic understanding of peridinin spectral tuning, peridinin chlorophyll energy transfer and photoprotective mechanism [ ] . the two pcp forms differ in various features: spectral properties, molar mass, amino acid sequence and, above all, pigment stoichiometry, the peridinin:chlorophyll ratio being : for the rfpcp and : for the hspcp [ ] . in the present work we perform classical molecular dynamics simulations of the rfpcp and the hspcp in explicit water solution. we analyse the structure and dynamics of the proteins and of their pigments to characterize the different peridinin sites in both pcp forms in terms of quantities that can affect the chromophore spectra, such as distorsion, fluctuations and nature of the protein environment. the comparison between the data suggests correspondences between the pigments of the two forms. quantum and mixed quantum/ classical molecular dynamics simulations are also under progress to investigate the effect of the protein environment on the electronic and optical properties of the pcp pigments. peculiar applications, like in optoelectronics, biosensors, photovoltaics. among the existing carrier matrices conductive metal oxides (e.g. indium tin oxide, ito), carbon nanotubes, graphenes, silicon (si) are the most frequently used materials because of their unique characteristics such as good conductivity, good optical properties and excellent adhesion to substrates. in our work we combined purified photosynthetic reaction center protein (rc) and porous silicon (psi) investigating the morphology and optoelectronic properties of the bio-nanocomposite material. ftir spectroscopy, scanning electron microscopy and energydispersive x-ray spectroscopy indicated the binding of the protein to the psi. specular reflectance spectra showed a red shift in the characteristic interference peak of the psi microcavity which was saturated at higher concentration of the protein. the binding was more effective if the functionalization was done by the si-specific oligopeptide compared to the classical covalent binding via aminopropyl-triethoxysilane (aptes). excitation by single saturation flashes indicated that the rc still exhibited photochemical turnover after the binding. the role of reactive oxygen species (ros) in plant stress, both as damaging agent and as potential signal molecule is often assessed in experiments using photosensitized elicitor dyes. for these studies, it is essential to know how efficiently these chemicals generate ros, whether they are specific ros sources, as well as their cellular localization and additional side effects. the present study addresses these issues using a variety of dyes known and traditionally applied as singlet oxygen ( o ) sources. rose bengal (rb), methylene violet (mvi), methylene blue (mb), neutral red (nr) and indigo carmine (ic) were studied as putative ros sources in tobacco leaves. ros products of photosensitized dyes were measured in vitro, using spin trapping epr spectroscopy. dye concentrations and irradiation concentration leading to equal absorbed excitation quanta were determined spectrophotometrically. in vivo studies were carried out using tobacco leaves infiltrated with water solutions of the putative o sources. cellular localizations were identified on the basis of the dyes' fluorescence. rb, nr and mvi reached into mesophyll cells and were used to study the effects of these dyes on photosynthesis. photochemical yields and quenching processes were compared before and after photosensitization of the elicitor dyes inside the leaf samples. chlorophyll-chlorophyll charge transfer quenching is the main mechanism of non-photochemical quenching in higher plants alfred r. holzwarth max-planck-institut fü r bioanorganischechemie, stiftstraße - , d- mü lheim a.d.ruhr, germany non-photochemical quenching (npq) in plants protects against photochemical destruction of the photosynthetic apparatus under excess light conditions.while one location of the npq process has been shown to be centered on the major light harvesting complex ii (lhcii) (q type or qequenching), an additional quenching center responsible for qi type (identical to q center) quenchinghas been suggested to be located on the minor light-harvesting complexes upon accumulation ofzeaxanthin (zx), in particularon cp and cp we have performed femtosecond transient absorption and time-resolved fluorescence measurements of npq quenching in intact leaves of higher plants, on isolated light harvesting complexes in the minor (non-aggregated)light harvesting complex cp reconstituted with violaxanthin (vx) or zx, and in the isolated major lhc ii complex in the aggregated state. in all of these situations we find the formation of chl-chl charge transfer (ct) states to be the dominant quenching mechanism. the yield of formation of carotenoid cation states and/or carotenoid s state is either extremely low or absent, thus excluding their involvement in npq quenching as a major quenching mechanism. single-molecule spectroscopy (sms) is a powerful technique that allows investigation of fluorescence properties from single fluorescing systems. this technique enabled us to investigate the dynamics of the fluorescence intensity and spectral profiles of single, isolated light harvesting complex (lhc) on timescales of milliseconds to seconds, during continuous laser illumination. we were able to observe how each complex can rapidly switch between different emission states [ , ] and to characterise the intensity and the spectral dynamics of major and minor antenna complexes from plants, in two different environments, mimicking the light harvesting and the light dissipating state, respectively. the results will be discussed with respect to the current models for nonphotochemical quenching (npq) mechanisms [ , , ] , a vital photoprotection mechanism during which the lhcs of plants switch between a state of very efficient light utilisation and one in which excess absorbed excitation energy is harmlessly dissipated as heat. phaeodactylum tricornutum is one of the most utilized model organisms in diatom photosynthesis research mainly due to availability of its genome sequence ( ). it's photosynthetic antennae are the fucoxanthin chlorophyll a/c binding proteins (fcps) which share a high degree of homology with lhcs of higher plants and green algae ( ) . for detailed investigation of the antenna system of p.tricornutum, a transgenic strain expressing recombinant his-tagged fcpa protein was created which simplified the purification of a specific stable trimeric fcp complex consisting of fcpa and fcpe proteins. excitation energy coupling between fucoxanthin and chlorophyll a was intact and the existence of a chlorophyll a/fucoxanthin excitonic dimer was demonstrated ( ). we investigated in detail the existence of specific antenna system for psi and psii in p.tricornutum as in case of higher plants. our studies indicated that at least the main light harvesting proteins fcpa and fcpe are most probably shared as a common antenna by both psi and psii. harvesting complex ii (lhciib) from spinach or in native thylakoid membranes by picosecond time-resolved fluorescence. the domain size was estimated by monitoring the efficiency of added exogeneous singlet excitation quenchers -phenyl-p-benzoquinone (ppq) and dinitrobenzene (dnb). the fluorescence decay kinetics of the systems under study were registered without quenchers and with quenchers added in a range of concentrations. stern-volmer constants, k sv and k sv -for aggregates (membranes) and detergentsolubilized complexes, respectively, were determined from the concentration dependence of the ratio of the mean fluorescence lifetimes without/with quencher (s , s). the ratio k k sv • s /s was suggested as a measure of the functional domain size. values in the range of - were found for lhcii macroaggregates and - for native thylakoid membranes, corresponding to domain sizes of - chlorophylls. although substantial, the determined functional domain size is still orders of magnitude smaller than the number of physically connected pigment-protein complexes; thus our results imply that the physical size of an antenna system beyond these numbers has little or no effect on improving the light-harvesting efficiency. the interaction between photosynthetic reaction center proteins (rcs) purified from purple bacterium rhodobacter sphaeroides r- and functionalized and non-functionalized (single (swnt) and multiple (mwnt) walled) carbon nanotubes (cnt-s) has been investigated. both structural (afm, tem and sem microscopy) and functional (flash photolysis and conductivity) techniques showed that rcs can be bound effectively to different cnts. both physical sorption and binding through -nh or -cooh groups gave similar results. however, it appeared that by physical sorption some sections of the cnts were covered by multiple layers of rcs. after the binding the rcs kept their photochemical activity for a long time (at least for three months, even in dried form) and there is a redox interaction between the cnt and rcs. the attachment of rc to cnts results in an accumulation of positive and negative charges followed by slow reorganization of the protein structure after excitation. in the absence of cnt the secondary quinone activity decays quickly as a function of time after drying the rc onto a glass surface. the special electronic properties of the swnt/protein complexes open the possibility for several applications, e.g. in microelectronics, analytics or energy conversion and storage. the decay of the high fluorescence state generated by actinic illumination of different durations was measured in whole cells of various strains and mutants of photosynthetic purple bacteria. although similar method is used in higher plants, its application in photosynthetic bacteria is novel and highly challanging. the available data are restricted and only the re-oxidation of the reduced primary quinone (q a -? q a ) is usually blamed for the decay kinetics. here, we will analyse the complexity of the kinetics over a very broad time range (from ls to s) and show that the dark relaxation of the bacteriochlorophyll fluorescence reflects the overlap of several processes attributed to the intra-and interproteinous electron transfer processes of the reaction center (rc) and cytochrome bc complex of the bacterium. in the shorter (\ ms) time scale, the dominating effect is the re-reduction of the oxidized primary donor (p ? ? p) that is followed by the re-oxidation of the acceptor complex of the rc by the cytochrome bc complex. as the life times and amplitudes of the components depend on the physiological state of the photosynthetic apparatus, the relaxation of the fluorescence can be used to monitor the photosynthetic capacity of the photosynthetic bacteria in vivo. circular dichroism (cd) spectroscopy is an indispensable tool to probe molecular architecture. at the molecular level chirality results in intrinsic cd, pigment-pigment interactions in protein complexes give rise to excitonic cd, whereas ''psitype'' cd originates from large densely packed chiral aggregates. it has been well established that the anisotropic cd (acd), measured on samples with defined orientation, carries specific information on the architecture of molecules. however, acd can easily be distorted by linear dichroism of the sample or instrumental imperfections -which might be the reason why it is rarely studied in photosynthesis research. here we present acd spectra of isolated intact and washed, unstacked thylakoid membranes, photosystem ii membranes (bby), and tightly-stacked lamellar macroaggregates of the main light-harvesting complex ii (lhcii). we show that the acd spectra of face-and edge-aligned stacked thylakoid membranes and lhcii lamellae exhibit profound differences in their psi-type cd bands. marked differences are also seen in the excitonic cd of bby and washed thylakoid membranes. thus acd provides an additional dimension to the light induced conformation changes of quinone depleted photosynthetic reaction centers (rcs) purified from the carotenoid-less rhodobacter sphaeroides r- were investigated by transient absorption (ta) and grating (tg) methods. surprisingly, the decay of the ta signal measured at nm was divided into a ns and a ls components. the latter coincides with the life time of the tg signal, which was assigned earlier [nagy et al. ( ) febs lett. , - ] to spectrally silent conformational changes. the nature of the ls phase was investigated further. although, the probability of chlorophyll triplet formation under our measuring conditions was small, possible contribution of the triplet states was also studied. the presence of carotenoid in the wild type rcs eliminated the ls component indicating the role of carotenoid in the energy transfer within the rcs. there was no significant effect of the molecular oxygen on the ta. this fact may be explained if the chlorophyll triplets inside the protein have reduced accessibility to molecular oxygen. a differential effect of osmotic potential and viscosity on conformation changes accompanying the primary charge separation was measured by the effect of ficoll, glucose and glycerol as compared the ta to the tg signals. variable chlorophyll fluorescence: in part a yield change due to light-induced conformational change gert schansker , szilvia z. tó th , lá szló ková cs , alfred r. holzwarth and gy} oz} o garab institute of plant biology, biological research center, hungarian academy of sciences, szeged, hungary, max-planck-institut fü r bioanorganische chemie, mü lheim an der ruhr, germany on a dark-to-light transition the chlorophyll fluorescence rises from a minimum intensity (f ) to a maximum intensity (f m ). conventionally, this rise is interpreted to arise from the reduction of the primary quinone acceptor, q a , of photosystem ii -although this cannot explain all presently available observations. in untreated leaves, at room temperature, the fluorescence rise follows the reduction of the electron transport chain (etc). once induced, * - % of the variable fluorescence intensity relaxes within ms in darkness and can be re-induced within ms as long as the etc remains reduced. analyzing the fluorescence relaxation kinetics, ?/-dcmu, * % of the amplitude cannot be explained by q a -re-oxidation. special properties of this phase determined on dcmu-inhibited samples: at cryogenic temperatures (below - °c), where the q a -/s recombination is blocked, it still relaxes and it exhibits a strong temperature dependence with an apparent e a & kj/mol, whereas the reduction of q a is nearly temperature insensitive. a fluorescence yield change, driven by light-induced conformational change in the reaction center complex, can explain all these observations. tuning function in bacterial light-harvesting complexes katia duquesne , edward o'reilly , cecile blanchard , alexandra olaya-castro and james n. sturgis lism, cnrs and aix-marseille university, marseille, france, department of physics, university college, london, uk purple photosynthetic bacteria are able to synthesize a variety of different light-harvesting complexes, sometimes referred to as lh , lh and lh . here we have investigated the structural origins of these different forms and the manner in which the sequence tunes the absorption spectrum of the light-harvesting system. we then consider the functional consequences of this tuning for the organization of the light harvesting system and on the ecology of the organisms. specifically by spectroscopic techniques, in particular circular dichroism and resonance raman spectroscopy, we have been able to obtain information on the organization of the bacteriochlorophyll binding sites in the unusual lh of roseobacter denitrificans. this provides a picture of how different peripheral light-harvesting complexes are able to modulate the absorption spectrum. the structure and organization of this complex is the put in the context of the the recently published variability of the light-harvesting complexes. in particular the observation of their ability to form mixed complexes containing different polypeptides. we examine quantitatively the possible reasons for maintaining such variability by considering the transport properties of membranes containing either pure or mixed complexes and show that mixed complexes can permit light-harvesting to continue during adaptation. we then consider the different constraints that may be behind this type of adaptation in different bacteria and the conditions under which different types of antenna system might be optimal finally we integrate this into the evolutionary context of adaptation to variable light intensity and the ecological niches where such organisms are found. interaction between photosynthetic reaction centers and ito t. szabo , g. bencsik , g. kozak , cs. visy , z. gingl , k. hernadi , k. nagy , gy. varo and l. nagy departments of medical physics and informatics, physical chemistry and materials science, technical informatics and of, applied and environmental chemistry, university of szeged, hungary, institute of biophysics, has biological research center, szeged, hungary photosynthetic reaction center proteins (rc) purified from rhodobacter sphaeroides purple bacterium were deposited on the surface of indium-tin-oxide (ito), a transparent conductive oxide, and the photochemical/-physical properties of the composite was investigated. the kinetics of the light induced absorption change indicated that the rc was still active in the composite and there was an interaction between the protein cofactors and the ito. the electrochromic response of the bacteriopheophytine absorption at nm showed an increased electric field perturbation around this chromophore on the surface of ito compared to the one measured in solution. this absorption change is associated with the chargecompensating relaxation events inside the protein. similar life time, but smaller magnitude of this absorption change was measured on the surface of borosilicate glass. the light induced change in the conductivity of the composite as a function of the concentration showed the typical sigmoid saturation characteristics unlike if the chlorophyll was layered on the ito which compound is photochemically inactive. in this later case the light induced change in the conductivity was oppositely proportional to the chlorophyll concentration due to the thermal dissipation of the excitation energy. the supramolecular organization of photosystem ii in vivo studied by circular dichroism spectroscopy tü nde tó th , , herbert van amerongen , , gy} oz} o garab , lá szló ková cs institute of plant biology, biological research center, hungarian academy of sciences, hungary, wageningen university, laboratory of biophysics, wageningen, the netherlands, microspectroscopy centre, wageningen university, wageningen, the netherlands the light reactions of photosynthesis in higher plants take place in granal chloroplast thylakoid membranes, which contain chirally organized macrodomains composed of photosystem ii (psii) supercomplexes associated with light harvesting antenna complexes (lhciis). the physiological relevance of this hierarchic organization, which often manifest itself in semicrystalline assemblies, has not been elucidated but the diversity of the supramolecular structures and their reorganizations under different conditions indicates its regulatory role. the present work focuses on the structural and functional roles of different components of lhcii-psii supercomplexes. we used various growth conditions, influencing the protein composition, and different arabidopsis mutants (kocp , kocp , kopsbw, kopsbx, dgd ), with altered organization of the membranes, and measured their circular dichroism (cd) spectra as well as their chlorophyll fluorescence kinetics to characterize the chiral macro-organization of the chromophores and the functional parameters of the membranes, respectively. we show that the formation of chiral macrodomains require the presence of supercomplexes. our data also reveal specific functions of some of the protein or lipid compounds in the light adaptation processes of plants. excitation energy transfer and non-photochemical quenching in photosynthesis rienk van grondelle department of physics, vu university, de boelelaan , hv, amsterdam, the netherlands the success of photosynthesis relies on two ultrafast processes: excitation energy transfer in the light-harvesting antenna followed by charge separation in the reaction center. lhcii, the peripheral light-harvesting complex of photosystem ii, plays a major role. at the same time, the same light-harvesting system can be 'switched' into a quenching state, which effectively protects the reaction center of photosystem ii from over-excitation and photodamage. in this talk i will demonstrate how lhcii collects and transfers excitation energy. using single molecule spectroscopy we have discovered how lhcii can switch between this light-harvesting state, a quenched state and a red-shifted state. we show that the switching properties between the light-harvesting state and the quenched state depend strongly on the environmental conditions, where the quenched state is favoured under 'npq-like' conditions. it is argued that this is the mechanism of non-photochemical quenching in plants. photobiology in the soil: arrested chlorophyll biosynthesis in pea epicotyl sections beá ta vitá nyi, katalin solymosi, annamá ria kó sa, bé la bö ddi eö tvö s university, institute of biology, department of plant anatomy, pá zmá ny p. s. /c, h- budapest, hungary the key regulatory step of chlorophyll (chl) biosynthesis is the nadph:protochlorophyllide oxidoreductase (por) catalyzed reduction of protochlorophyllide (pchlide)which is light activated in angiosperms. this process is usually described on artificially dark-grown plants. in this work, we studied epicotyl segments developed under the soil surface, which were dissected from pea plants grown under natural light conditions. using k fluorescence spectroscopy, pigment analyses, electron microscopy and fluorescence microscopy, we found that upper segments showed transitional developmental stages, i.e. chl appeared besides pchl(ide) and etio-chloroplasts were typical. in regions under cm depth, however, the characteristics of the segments were similar to those of plants germinated artificially in complete darkness, i.e. only pchl(ide) and etioplasts were present. the results of this work prove that these latter symptoms may occur in shaded tissues of fully developed, photosynthetically active plants grown under natural conditions. in this overview talk it will be shown how atomistic computations can complement experimental measurements in our quest to understand biological electron and proton transfer reactions. at first the molecular simulation methods for calculation of important electron transfer parameter such as reorganization free energy, electronic coupling matrix elements and reduction potentials will be explained. then three applications will be discussed where such computations help interpret experimental data on a molecular level. the first example concerns electron tunneling between heme a and heme a in the proton pump cytochrome c oxidase. this reaction is very fast, occurring on the nano-second time scale, and it is unclear if this is due to an unusually low reorganization free energy or due to high electronic coupling. carrying out large-scale all-atom molecular dynamics simulation of oxidase embedded in a membrane, we do not find evidence for unusually small values of reorganization energy as proposed previously, implying that the nanosecond tunneling rate between heme a and a is supported by very efficient electronic coupling. the second example is on electron transport in a deca-heme 'wire'-like protein, used by certain anaerobic bacteria to transport electrons from the inside of the cell to extracellular substrates. the crystal structure of such a protein has been solved recently for the first time. however, it is unclear if and in which direction the wire structure supports electron transport. here we present results of heme reduction potential calculations that help us reveal the possible electron flow in this protein. in a third example we explain how quantum mechanical/molecular mechanical methods (qm/mm) recently helped us understand why the catalase from h. pylori is prone to undergo an undesired protein radical migration reaction during catalysis. proton pumping activity of purple and brown membranes regenerated with retinal analogues k. bryl , k.yoshihara university of warmia and mazury, department of physics and biophysics, olsztyn, poland, suntory institute for bioorganic research, wakayamadai, osaka , japan the retinal protein bacteriorhodopsin (br) acts as a light-driven proton pump in the purple membrane (pm) of halobacterium salinarium (h.s.). the aim of these studies was to clarify whether the specific crystalline structure of protein and protein-substrate interactions are significant for h ? transfer into the aqueous bulk phase. two membrane systems were prepared: purple membranes (br arranged in a two-dimensional hexagonal lattice) and brown membranes (br not arranged in a crystalline lattice) were regenerated with -fuororetinals. light-induced proton release and reuptake as well as surface potential changes inherent in the regenerated systems reaction cycles were measured. signals of optical ph indicators residing in the aqueous bulk phase were compared with signals of ph indicator covalently linked to the extracellular surface of proteins and with surface potential changes detected by the potentiometric probe. the energies of activation of proton transfer have been calculated. experimental results and thermodynamic parameters (energies of activation) suggest the different mechanism of proton transfer into the aqueous bulk phase in these two systems. the implications for models of localized-delocalized energy coupling by proton gradients will be discussed. iron regulation is a vital process in organisms and in most of them it is accomplished through the metal solubilisation and storage by ferroxidase enzymes of the ferritin family, which have the ability of sequester, oxidize and mineralize ferrous ions using oxygen or hydrogen peroxide as substrate. dnabinding proteins from starved cells (dps) belong to this ferritin's family. dps belongs to the sub-type designated as miniferritins and, besides iron storage and release capability, is responsible for hydrogen peroxide resistance showing the ability to form stable complexes with dna. the preferable cosubstrate of this enzyme is h o although the reaction can occur in the presence of oxygen with lower rate [ , ] . in this work, the electrochemical behaviour of the recombinant dps from pseudomonas nautica, was assessed as a function of metal content in anaerobic environment with h o as co-substrate. the obtained electrochemical results together with spectroscopic studies allowed inferring some new hypothesis on the dps iron uptake mechanism. for myoglobin electrostatically immobilized on au-deposited mixed self-assembled monolayers (sams) of the composition: -s-(ch ) -cooh/-s-(ch ) -oh. our approach allows for a soft switching of the haem group charge state and accurate probing of the accompanying reorganizational dynamics of conformational (quasi-diffusional) and quantum (e.g. protonrelated) modes. the electron transfer rate constants were determined with h o or d o as solvent, under the variable temperature ( - k) or pressure ( - mpa) conditions, revealing the overall reorganization free energy of . ± . ev, activation volume of - . ± . cm mol - and inverse solvent kinetic isotope effect of . ± . ( °c). on the grounds of an extended charge-transfer theory, we propose specific protoncoupled et mechanism additionally coupled to the slow conformational dynamics of the protein matrix accompanied by translocation(s) of haem-adjacent water molecule(s). proton gradients across pore spanning membranes: towards on-chip energy conversion daniel frese, claudia steinem institute for organic and biomolecular chemistry, georg-august-university goettingen, germany in cell organelles, chemiosmotic potentials resulting from proton gradients across membranes are widely used to fix chemical energy in forms of atp. the high efficiency of this protein-mediated energy conversion raises interest for artificial proton gradient setups. to investigate proton transport across artificial membranes, we prepared pore spanning membranes (psms) on porous silicon substrates via painting technique. this allowed us to trap aqueous content of well-defined composition and volume inside the substrates microcavities. nigericin, a peptide that acts as an h ? -k ? -antiporter and bacteriorhodopsin, a transmembrane protein which is well known to be a light driven proton pump, were reconstituted into the pre-formed psms to achieve proton transport from one aqueous compartment to the other. changes of proton concentrations inside the pores were monitored by means of confocal laser scanning microscopy (clsm). therefore, pores were filled with pyranine, a ph-sensitive fluorescence dye and variations in intensity were measured to analyze proton translocation. we were able to show that both, nigericin and bacteriorhodopsin are capable of building up a proton gradient across psms and plan to co-reconstitute atp synthases for on-chip energy conversion by formation of atp. application of the gibbs free energy profiles to sequential biochemical reactions pé ter farkas, tamá s kiss and eugene hamori department of biological physics, eö tvö s ló rá nd tudomá ny egyetem, budapest, hungary, and department of biochemistry, tulane university, new orleans, la., usa the full understanding of the energetic details of complex metabolic reaction sequences requires a step-by-step analysis of the gibbs free energy (g) changes of the ''parasystem'' (i.e., a collection of atoms comprising all the molecules participating in a given reaction) as it gradually changes from its initialreactants state to its final-products state along the reaction pathway. knowing the respective equilibrium constants of each of the participating reaction steps and also the actual in vivo concentrations of the metabolites involved, a free-energy profile can be constructed that will reveal important information about the progress of the reaction as driven by thermodynamic forces. this approach will be illustrated on some biochemical reactions including the glycolytic/gluconeogenetic pathways. furthermore, the often misleading text-book representation of enzymatic catalysis will be reexamined and explained in thermodynamic terms using the free-energy profiles of both the non-catalyzed and the enzyme-catalyzed reactions. redox-active proteins can be diversely functionalized at metaldeposited self-assembled monolayers (sams) of a widely variable composition and thickness. the voltammetric methodology in combination with the advanced data processing procedures allow for comprehensive kinetic data within the congruent series of nano-devices and the subsequent calculation of the key physical parameters, such as the medium reorganization energy of et, the donor-acceptor electronic coupling, effective relaxation time (related to the protein's and environment's fluctuational dynamics), etc. in our studies the ''model'' redox protein, cytochrome c (cytc), was either freely diffusing to sam terminal groups (mode ), or attached to sams through the electrostatic interaction (mode ), or specific ''wiring'' (mode ). another redox-active protein, azurin, was confined at terminal sam groups through the hydrophobic interaction (mode ). diverse experimental strategies including the variation of sam thickness, solution viscosity temperature and hydrostatic pressure, allowed for a severe demonstration of the full adiabatic and nonadiabatic control (thinner and thicker sams, respectively), and the intermediary regime, in a nice agreement with the major theoretical predictions. proton transfers in a light-driven proton pump j.k. lanyi dept. physiology & biophysics, university of california, irvine, usa illumination of bacteriorhodopsin causes isomerization of alltrans retinal to -cis, -anti and a cyclic reaction ensues, in which the protein and the chromophore undergo conformational changes with an overall ten millisecond turn-over time and a proton is transported from one membrane side to the other. with crystal structures of six trapped intermediate states and plausible structural models for the remaining two intermediates, structures are now available for the initial bacteriorhodopsin state and all intermediates. they reveal the molecular events that underlie the light-induced transport: protonation of the retinal schiff base by asp , proton release to the extracellular membrane surface, a switch event that allows reprotonation of the schiff base from the cytoplasmic side, side-chain and main-chain motions initiated in the cytoplasmic region, formation of a single-file chain of hydrogen-bonded water molecules that conducts the proton of asp to the schiff base, and reprotonation of asp from the cytoplasmic surface. the observed changes can be summarized as a detailed atomic-level movie in which gradual relaxation of the distorted retinal causes a cascade of displacements of water and protein atoms results in vectorial proton transfers to and from the schiff base. electron transfer (et) processes are fundamental in photosynthesis, respiration and enzyme catalysis. the relative importance of superexchange and sequential mechanisms in biological et is still a matter of debate. the identification of any ''stepping stones'' necessary for electron hopping is a key point in the understanding of long range et. hence, the study of a single event in the sequence of reactions occurring in these phenomena is a fundamental but formidable task. muon spin relaxation (lsr) has been shown to be sensitive to charge transport on a molecular lengthscale. the muon is a very sensitive probe of electron transport, as any changes to the electronic density sampled by the muon can change its spin polarization, which can easily be measured. in this context, a very useful tool is the detection of the so-called avoided level crossing (alc) resonances [ ] . the enhancement in the loss of polarization of the muon's spin on these resonances dramatically increases sensitivity. we show that a laser pump -lsr probe technique can measure et processes at particular, and most importantly known, sites within the amino acids chain, and therefore track the time evolution of the electron over the molecule. keywords: photosynthesis, reaction center, electron transfer, proton transfer, fourier transform infrared, l dn, isotopic labeling, band assignment, histidine, mechanism in photosynthesis, the central step in transforming light energy into chemical energy is the coupling of light-induced electron transfer to proton uptake. in the photosynthetic reaction center (rc) of rhodobacter sphaeroides, fast formation of the charge separated state p ? q a is followed by a slower electron transfer from the primary quinone q a to the secondary quinone q b and the uptake of a proton from the cytoplasm to q b . previous fourier transform infrared (ftir) measurements on rc suggested an intermediate x in the q a -q b to q a q b transition. mutation of the amino acid aspl to asn (l dn mutant) slows down proton uptake and oxidation of q a -. using time-resolved ftir spectroscopy we characterized this rc mutant and proposed specific ir bands that belong to the intermediate x. to study the role of the iron-histidine complex located between q a and q b , we performed fast-scan ftir experiments on the l dn mutant marked with isotopically labelled histidine. we assigned ir bands of the intermediate x between cm - and cm - to histidine vibrations. these bands show the protonation of a histidine, most likely hisl , during the q a -q b to q a q b transition. based on these results we propose a new mechanism of the coupling of electron and proton transfer in photosynthesis. complex i of respiratory chains is an energy transducing enzyme present in most bacteria and in all mitochondria. it is the least understood complex of the aerobic respiratory chain, even though the crystallographic a-helical structures of bacterial and mitochondrial complexes have been recently determined [ ] [ ] . this complex catalyses the oxidation of nadh and the reduction of quinone, coupled to cation translocation across the membrane. rhodothermus marinus complex i, our main model system, is a nadh:menaquinone oxidoreductase and has been extensively characterized. we have made an exhaustive study in order to identify all the subunits present in the complex [ ] . the nature of the coupling charge of r. marinus complex i was investigated using inside-out membrane vesicles, which were active with respect to nadh oxidation and capable of creating and maintain an nadh-driven membrane potential (dw) positive inside. it was observed that this bacterial complex i is able of h ? and na ? transport, although to opposite directions. the coupling ion of the system was shown to be the h ? being transported to the periplasm, contributing in this way to the establishment of the electrochemical potential difference, while na ? is translocated to the cytoplasm [ ] . the sodium ion extrusion from the membrane vesicles was due to the activity of complex i, since it was sensitive to its inhibitor rotenone, and it was still observed when the complex i segment of the respiratory chain was isolated by the simultaneous presence of cyanide and external quinones. additional studies have shown that although neither the catalytic reaction nor the establishment of the dph requires the presence of na ? , the presence of this ion increased the proton transport. combining all these results, a model for the coupling mechanism of complex i was proposed, suggesting the presence of two different energy coupling sites, one that works only as a proton pump (na ? independent), and the other functioning as a na ? /h ? antiporter (na ? dependent) [ ] . this model was reinforced by further studies performed in the presence of the na ? /h ? antiporter inhibitor, -(n-ethyl-n-isopropyl)-amiloride (eipa) [ ] . a deeper inside into the coupling mechanism of this enzyme was provided by studying the influence of sodium ions on energy transduction by complexes i from escherichia coli and paracoccus denitrificans. it was observed that the na ? / h ? antiporter activity is not exclusive of r. marinus complex i, since the e. coli enzyme is also capable of such a transport, but is not a general property given that the p. denitrificans enzyme does not performed sodium translocation [ ] . due to the fact that r. marinus and e. coli enzymes reduce menaquinone while p. denitrificans complex i reduce ubiquinone, it is suggested that the na ? /h ? antiporter activity may be correlated with the type of quinone used as substrate. under anaerobic conditions some bacteria can use nitrate instead of oxygen in a process called denitrification. during denitrification, the reduction of no to n o is catalyzed by a membrane-bound enzyme nitric oxide reductase (nor). this enzyme is an important step in the evolution of a respiratory system: nor belongs to the superfamily of o -reducing heme-copper oxidases and is assumed to be the evolutionary ancestor of cytochrome c oxidase. the understanding of nor functioning was limited by the lack of structural information, but recently the first structures (cnor type from ps. aerug. and qnor type from g. stearoth.) were solved [ ] [ ] . we will present results of the first computational studies of nor (both cnor and qnor types) [ ] [ ] . the studies include: (i) large-scale all-atom md simulations of proteins in their natural environment (i.e. embedded in membrane and solvent), which were performed to describe water dynamics inside the protein and to identify potential proton transfer pathways, and (ii) free-energy calculations by the empirical valence bond (evb) method [ ] for the explicit proton translocations along the pathways established by md. among important findings are new proton pathways, which were not predicted from the x-ray structure and could be identified only by means of computer simulations. simulations also reveal that, despite a high structural similarity between cnor and qnor, these enzymes utilize strikingly different proton uptake mechanisms. our results provide insights into the functional conversion between no and o reductases, and into evolution proton transfer mechanisms and respiratory enzymes in general. the genome of the bacterium geobacter sulfurreducens (gs) encodes for c-type cytochromes ( ). genetic studies using cytochrome deficient gs strains and proteomic studies identified cytochromes that were produced under specific growth conditions ( ) ( ) ( ) . a putative outer-membrane cytochrome, omcf, is crucial for fe ? and u ? reduction and also for microbial electricity production ( ). omcf is a monoheme c-type cytochrome with sequence similarity to soluble cytochromes c of photosynthetic algae and cyanobacteria ( ). the structure of oxidized omcf was determined ( ) being the first example of a cytochrome c -like structure from a nonphotosynthetic organism. the structural features of omcf hinted a different function compared to cytochromes c from photosynthetic organisms, being an excellent example of how structurally related proteins are specifically design by nature to perform different physiological functions. in order to elucidate omcf structural-functional mechanism, isotopic labeled protein ( n and c) was produced and its structure in the reduced form determined by nmr. single point-mutations at key residues were produced by site-directed mutagenesis and their impact on the structural and functional properties of omcf will be presented. in the early s, the search for the source of nitrogen monoxide (no) production in mammals led to the discovery of three major isoforms of no-synthases (nos): the neuronal nos (nnos), the inducible nos (inos), and the endothelial nos (enos) ( ( )). years later, based on genomic analysis, numerous nos-like proteins have been identified in the genome other organism and in particular of several bacteria (bacillus anthrax, staphylococcus aureus… ( )). in spite of superimposable d structures and the ability to catalyse no production, all these enzymes are carrying different (if not opposite) physiological activities including cgmp signalling, cytotoxic activities, anti-oxidant defence, metabolism… moreover, noss become increasingly associated to oxidative-stress related pathologies ranging from neurodegenerative disorders, cardiovascular and inflammatory diseases, diabetes, cancers ( )…. this apparent paradox seems related to the belief that the strong similarity of sequence and structure of no-synthases must lead to a unique and identical functioning (no production) for all isoforms. this is blatant for bacterial nos-like proteins that are lacking the essential components required for no biosynthesis but remain considered as genuine no synthases. this approach might remain an obstacle to the understanding of nos actual biological role and could prevent the design of efficient nos-targeted therapeutic strategies. to elucidate this ''nos paradox'' our group has initiated a multidisciplinary approach that aims to relate the wide diversity of nos biological activities to variations in the catalytic mechanism of noss, to modifications of their regulation patterns and to adaptations to their physiological environment. in this context we have been investigating the mechanism of bacillus subtilis nos-like proteins with a special focus on the features that are specific to nos mechanism: i) electron and proton transfers and the role of the substrate and the pterin cofactor ii) oxygen activation and the role of the proximal ligand iii) the very molecular mechanism and the variations in the nature of reaction intermediates. for that matter we have been using a combination of radiolytical techniques (cryoreduction with co y-irradiation, pulse-radiolysis with the elyse electron accelerator), stateof-the-art spectroscopies (epr, atr-ftir and resonance raman, and picoseconds uv-visible absorption spectrosocopies), organic synthesis (synthesized substrates and cofactors analogues) biochemistry and molecular biology (site-directed mutagenesis). we will present our results on the coupling of electron and proton transfers, on the tuning of the proximal ''push effect'' and we will discuss the conditions that favour for each nos isoform no production versus other reactive nitrogen and oxygen species. photosynthetic iron oxidation (pio) is an ancient form of photosynthesis with relevant consequences in the shaping of the planet. this form of metabolism may have been involved in the deposition of geological structures known as banded iron formations, which hold key information regarding the co-evolution of photosynthesis and earth. rhodopseudomonas palustris tie- and rhodobacter ferroxidans sw both use ferrous iron as an electron donor to support photosynthetic growth (i.e. photoferrotrophy). the sw foxeyz operon can stimulate light-dependent iron oxidation by other bacteria. it codes a two-heme cytochrome, a pyrroloquinoline quinone protein and an inner membrane transporter, respectively. in tie- the pioabc operon is required for photoferrotrophy. it codes for a ten-heme cytochrome, an outer membrane beta-barrel and a high potential iron-sulfur protein (hipip) respectively. here we present functional and structural characterization of proteins involved in pio. this molecular characterization is essential for understanding this mode of bioenergetic metabolism, and may one day aid the development of biotechnological applications like microbial fuel cells and bioremediation. alongside classical, cytochrome respiratory pathway, phycomyces blakesleeanus possess alternative, cyanideresistant respiration (crr) facilitated by alternative oxidase (aox). in order to study role of oxygen in regulation of crr, the effects of cyanide on respiration of h old mycelia in aerated (control), hypoxic and anoxic conditions were measured. mycelium was incubated in these conditions for . h, h and h. after . h, aox activity was increased only in specimens incubated in anoxic conditions ( . %). after h, increase in aox activity was significant in both hypoxic and anoxic specimens ( . % and . %, respectively), with even greater increase after h, . % for hypoxic and . % for specimens in anoxic conditions. mycelia treated for h was then oxygenated for minutes. this induced decrease in aox activity of % in anoxic and even . % in hypoxic mycelia. aox is recognized as one of the mechanisms for maintaining low levels of reduced ubiquione which can function in conditions in which cytochrome chain is disabled, such as anoxia. this is in concordance with results obtained on p. blakesleeanus, where aox levels rise in hypoxic and anoxic conditions and decrease close to control level shortly after introduction of oxygen into the system. influence of escherichia coli f f -atpase on hydrogenase activity during glycerol fermentation k. trchounian , , g. sawers , a. trchounian department of biophysics, yerevan state university, yerevan, armenia, institute for microbiology, martin-luther university halle-wittenberg, haale, germany e. coli encodes four hydrogenases (hyd); only three of these, hyd- , hyd- and hyd- have been well characterized. hyd- has been shown recently to reversibly evolve hydrogen during glycerol fermentation at ph . [ ] . proton reduction was inhibited by n,n'dicyclohexylcarbodiimide suggesting a link with the proton-translocating f f -atpase. indeed, at ph . in an e. coli mutant (dk ) lacking f f overall hyd-activity was reduced to approximately % of the wild type activity; hyd- , but not hyd- , was detected in an in-gel activity assay. f f is therefore suggested to be required for hyd- activity. at ph . in glycerol medium hyd-activity in dk was * % of wild type activity and hyd- and hyd- exhibited only weak activity. this indicated a significant f f contribution towards hyd-activity as ph decreased. furthermore, at ph . hydactivity was negligible and only a very weak activity band corresponding to hyd- could be observed. these results suggest that f f -atpase is essential for hydrogenase activity during glycerol fermentation at ph . . taken together, the results suggest an interdependence between hyd- , hyd- and f f -atpase activity. [ ion channels and transporters control many facets of cancer cell biology and blocking the activity of these impairs tumor cell growth in vitro and in vivo. this new paradigm has opened new opportunities for pharmaceutical research in oncology , . we have contributed to this field showing that k v . (herg ) channels are aberrantly expressed in several human cancers where they control different aspects of the neoplastic cell biology such as proliferation and apoptosis, invasiveness and angiogenesis, the latter through the regulation of vegf secretion (reviewed in ). the herg dependent effects were shown in vitro and, more recently, in vivo. in preclinical models of both leukemia and colorectal cancer , herg overexpression confers a higher malignancy to neoplastic cells. moreover, herg blockers have therapeutic potential, since preclinical tests showed that treatment with specific herg blockers overcame chemoresistance in acute leukemias as well as reduced gi cancer growth, angiogenesis and metastatic spread . the overall message emerging from our data is that the herg protein represents a novel biomarker and drug target in oncology. up to now, herg was considered an ''antitarget'' due to the cardiac side effects that many (but not all) herg blockers produce and that result in lengthening the electrocardiographic qt interval. we report here recent studies on known and newly developed herg blockers that exhibit no cardiotoxicity and are more specific for the herg channels expressed in cancer cells. we reported previously that the increase of the cholesterol content of the cell membrane (in vitro) modified the biophysical parameters of the gating of kvl. k ? ion channels in human t-lymphocytes. in our present study we aimed to determine the effect of hypercholesterolemia on the biophysical parameters of kv . gating and the proliferation of t cells. t-lymphocytes were isolated from the peripheral blood of patients with cholesterol level considered normal (\ . mmol/l,control group) and patients with hypercholesterinaemia (hc). whole-cell k? currents were measured in patchclamped t cells and the kinetic (activation and inactivation kinetics) and equilibrium parameters (voltage-dependence of steady-state activation) of kv . gating were determined. lymphocyte proliferation was measured using cfse staining with and without anti-cd and anti-cd stimulation. our results indicate that the biophysical parameters of kv . gating are similar in the control group and in the 'hc' samples. the cfse-based assay showed that hypercholesterolemic t cells had higher spontenaous activation rate compared to control group. however, t cells from high cholesterol level patients challenged by anti-cd and anti-cd exhibited lower proliferation rate than control cells. generalized epilepsy with febrile seizures plus (gefs?, omim ) is a childhood genetic epilepsy syndrome correlated to mutations in the ancillary b-subunit of neuronal voltage-gated sodium channels (nachs). b -subunit is non-covalently associated with nach a-subunits, serving as modulator of channel activity, regulator of channel cell surface expression and as cell adhesion molecule. the first and best characterized gefs? mutation is the c w. this mutation changed a conserved cysteine residue into a tryptophan, disrupting a putative disulphide bridge that should normally maintain an extracellular immunoglobulinlike fold. in this study, we investigated the presence of this putative disulphide bond using -d-diagonal-sds-page, where the proteins were separated in the first dimension in absence of a reduction agent and in presence of it in the second dimension. this method allows to visualize the protein above the diagonal experimentally confirming that the disulphide bond is intramolecular. duchenne muscular dystrophy (dmd) is associated with severe cardiac complications. recent research suggests that impaired voltage-gated ion channels in dystrophic cardiomyocytes accompany cardiac pathology. it is, however, unknown if ion channel defects are primary effects of dystrophic gene mutations, or secondary effects of the developing cardiomyopathy. here, we studied na and ca channel impairments in dystrophic neonatal cardiomyocytes, derived from dmd mouse models, prior to cardiomyopathy development. dystrophin-deficiency reduced na current density. in addition, extra utrophin-deficiency altered na channel gating. moreover, also ca channel inactivation was reduced, suggesting that ion channel abnormalities are universal primary effects of dystrophic gene mutations. to assess developmental changes, we also studied na channel impairments in dystrophic adult cardiomyocytes, and found a stronger na current reduction than in neonatal ones. the described na channel impairments slowed the action potential upstroke in adult cardiomyocytes, and only in dystrophic adult mice, the qrs interval of the ecg was prolonged. ion channel impairments precede pathology development in the dystrophic heart, and may be considered cardiomyopathy triggers. supported by austrian fwf (p ). it has been over years since sensory neuron-specific sodium channel na v . was identified. since then na v . has been shown to play a crucial role in pain pathways, and it became a prominent drug target for novel pain killers. in contrast to myelinated neurons, mechanisms that target voltagegated sodium channels to the unmyelinated c-fibre axons are largely unknown. we investigated the localisation of na v . in unmyelinated primary sensory neurons. na v . was found to be clustered in lipid rafts on unmyelinated axons. when the lipid rafts are disrupted, remarkable reduction in both the conduction velocity and the number of cells responsive to mechanical stimuli to the unmyelinated axons were seen. using a compartment culture system, we also found that disruption of rafts in the middle region of the sensory axons caused a significant reduction in the responsiveness of the neurons to chemical stimuli to nerve endings. this is due to the failure of action potential propagation through the axons. these data suggest that clustering of na v . in lipid rafts of unmyelinated fibres is a key factor for the functional properties of the channel, which may due to a change in the voltage threshold. disruption of the na v . cluster and modifying lipid rafts in primary sensory neurons may be a useful new approach to control the excitability of nociceptive neurons. ion currents in are crucially important for activation of t-lymphocytes. our aim was to investigate how the blockage of various ion channels in isolation or in combination affects mitogen-dependent activation and proliferation of t-cells. we activated human peripheral blood lymphocytes using monoclonal antibodies against the tcr-cd complex and cd . we applied specific channel blockers inhibiting the major ion channels of the t-cell: either the kv . (tea or anuroctoxin), ikca (tram- ), or the crac channel ( -apb) alone or in combination. five days after the stimulus we measured the change in cell size and cellular granulation with flow cytometry along with the proportion of dividing cells, using cfse (carboxyfluorescein succinimidyl ester) dilution assay. our measurements indicated that the ion channel blockers suppressed the proportion of dividing cells in a dosedependent manner. increasing the strength of the stimulation reduced the potency of the blockers to inhibit cell proliferation and eventually the blockers were ineffective in decreasing lymphocyte proliferation. we experienced the greatest amount of inhibition using the combination of the blockers, which indicates synergy in the regulation pathway of the various ion channels. recently, sodium dependent phosphate transporter napi b was revealed as potential marker for breast, thyroid and ovarian cancer. in vivo, napi b is involved in maintenance of phosphate homeostasis and mutations or aberrant expressions of its gene (slc a ) are associated with several diseases including cancer. however, data about napi b mrna expression in different types of cancer and correspondent normal tissues are controversial and restricted. we investigated slc a gene expression level in normal ovarian tissues and different histomorphological types of ovarian tumors using real-time pcr analysis. it was found that slc a gene was highly expressed in well-differentiated endometrioid and papillary serous tumors, but was not expressed in low-differentiated tumors, benign tumors and in most normal tissues. mrna expression of slc a in serouse and endometrioid ovarian tumors accurately correlated with protein expression that was detected in these tumor samples by western blot analysis and immunohistochemistry in our previous investigation. upregulation of slc a gene expression in well-differentiated tumors may reflect cell differentiation processes during ovarian cancerogenesis and could serve as potential marker for ovarian cancer diagnosis and prognosis. in the present contribution a procedure for molecular motion characterization based on the evaluation of the mean square displacement (msd), through the self-distribution function (sdf), is presented. it is shown how msd, which represents an important observable for the characterization of dynamical properties, can be decomposed into different partial contributions associated to system dynamical processes within a specific spatial scale. it is shown how the sdf procedure allows us to evaluate both total msd and partial msds through total and partial sdfs. as a result, total msd is the weighed sum of partial msds in which the weights are obtained by the fitting procedure of measured elastic incoherent neutron scattering (eins) intensity. we apply sdf procedure to data collected, by in , in and in spectrometers (institute laue langevin), on aqueous mixtures of two homologous disaccharides (sucrose and trehalose) and on dry and hydrated (h o and d o) lysozyme with and without disaccharides. the nature of the dynamical transition is highlighted and it is shown that it occurs when the system relaxation time becomes shorter than the instrumental energy time. finally, the bioprotectants effect on protein dynamics and the amplitude of vibrations in lysozyme are presented. we evaluated q for genotoxicity in mcf- breast cancer cells in the presence or absence of doxorubicin (dox), docetaxel (dtx) and paclitaxel (ptx), anticancer drugs commonly used in chemotherapy of different solid tumors. damage to dna was determinated by comet assay. the cells, after treatment with investigated compounds, were washed up and cultured in fresh medium for , , , and hours. we have found that q by itself caused significant dna damage. moreover flavonol enhanced genotoxic effect of anticancer drugs. the highest amount of dna in the comet tail was observed h after treatment with combination of q with dox. similar changes were found in cells incubated with combination of q with taxanes -ptx or dtx. however, damage to dna in this case was considerably lower than damage caused by combination of q with dtx. our results confirmed anticancer and genotoxic activity of quercetin, which makes it a promising candidate for a potential use as a modulator of cytotoxicity and anticancer activity of anthracycline and taxane chemotherapeutics. although the health effects of low-frequency and intensity electromagnetic fields (lfi-emfs) are controversial, increasing evidence suggests that lfi-emfs are capable for initiating various healing processes. many (bio)physical ideas were suggested to explain the influence of lfi-emfs in living systems but the main effect of lfi-emfs on cell functions remains vague. however, some effects of lfi-emfs may be explained by redox and membrane processes. during diseases, cells not only demonstrate altered biochemical processes but also produce altered non-linear bioelectromagnetic complex patterns. thus, it is reasonable to use non-linear bioelectric and bioelectromagnetic signals from cells of the body for potential therapeutic applications that may be more effective than the artificial lfi-emfs signals. our novel emost (electromagnetic-own-signal-treatment) method is based on the utilization of the non-linear, bioelectric and bioelectromagnetic signals of the patients without any electromagnetic wave modulation and inversion of recorded output signals of subjects. here, we report our some restorative results after emost application. we also suggest that the possible effects of the emost may be achieved via redox-related processes. background or leak potassium conductances are a major determinant of resting potential and input resistance, two key components of cell excitability. these currents are not passive but finely tuned and adapted to cell specific functions. k p channels producing these currents are tightly regulated by a variety of chemical and physical stimuli including temperature, membrane stretch, free fatty acids, ph, ca ? , neurotransmitters and hormones as well as protein partners. these different stimuli converge on gating mechanisms that show remarkable conservation between intracellular k p channels (twik channels) and k p channels located at the plasma membrane (trek / channels). living at the edge: volume the conduction system of interfacial forces into the alveolar type ii cell in previous investigations, using new microscopic approaches, we found that the presence of an a li leads to a paradoxical situation: it is a potential threat that may cause cell injury, but also a important stimulus: at ii cells respond promptly, and show sustained ca ?signals that activate exocytosis. exocytosed surfactant, in turn, clearly prolonged the time to irreversible cell damage, and may be an adaptive and evolutionary defense mechanism against the harmful nature of surface forces. recently we published that at ii cells are sensing the a li but how this stimulus is conducted and converted into the cell, is still obscure. currently we are searching for potential calcium sources and it seems that the cells signal ca ? by extracellular ca ? entry probably through mechanosensitive channels. specialdesigned gene chips allowed a whole genome profiling of a li exposed single cells. these cells react with rapid changes on the transcriptional level: cellular pathways that are involved include e.g. defense response and lipid metabolism, and we identified genes associated with several lung diseases and injuries. we summarize, interface forces are strong, they are acting on the cells and triggering cellular events that are closely related with classical concepts in mechanotransduction, it is very plausible that those forces play a crucial role in the lung surfactant homeostasis. calorimetric method and instrumentation were worked out and applied for investigation aqueous solutions of proteins. thermal effects were analyzed by own construction heat-fluxtype dsc cell designed for temperature range from the boiling point of water down to k. the achieved sensitivity of heat flow rate (hf) of the instrument is better than %w tested using ll of mm aqueous solution of nacl. from the integral value of hf -the total enthalpy change dh total , the enthalpies of transitions were separated from the heat capacities. using the method, several types of proteins (bsa, erd , ubq, a-, b-casein, and wt, a t, a-synuclein mutants) were investigated in that temperature range. the results are shown in detail as an illustrative example. potential applications are outlined, which include (i) the distinction between the solvent accessible surfaces of globular and intrinsically disordered proteins, (ii) the distinction between protein mutants, and (iii) the identification of monomer and polymer protein states. this method provides a possibility to study the polymerization process (amyloid formation) and to investigate in-situ the reason and circumstances of that. morphometry due to self gravity in living organism is an integral part in understanding self gravitation bio. computational studies of biological system, especially in ab-initio embryo as self gravitating mass, floating over amniotic fluid, as if maintaining anti -gravitation (extrinsic) mechanism, would be an interesting one to explore biomechanics of intrinsic gravity. since the work would be of exploratory nature, both from the point of view of identifying appropriate stage of development of embryological morulla and available computational logics, it was contemplated to initiate functional study with a few reported ultrasound evidential works on small animals like mice, grasshopper including compact human embryo as mathematical structure that may allow the formal definition of concepts such as convergence, mapping and continuity. as many of the finite-dimensional function analysis in topological vector spaces are available, we initiated our simulation and studies on concentrating locally compact banach spaces. institute for x-ray physics, university of gö ttingen, germany we report on hard x-ray phase contrast imaging of black lipid membranes (blms), which are freely suspended over a micro machined aperture in an aqueous solution. this new way of membrane structure analysis allows investigating bio molecular and organic substances in aqueous environments by parallel and divergent beam propagation imaging, using partially coherent multi-kev x-ray radiation. the width of the thinning film is significantly smaller than the detector pixel size, but can be resolved from quantitative analysis of the intensity fringes in the fresnel diffraction regime down to its native thickness of about nm. to our knowledge this is the first time that such small features of a very weak phase object have been visualized by direct x-ray imaging techniques. we have put forward a simplified but extendable model, which enables the theoretical description of image formation and characterization of membrane thickness and its decrease during the thinning process from a bulk to a bimolecular film. on the basis of recent experiments, future investigations will be performed to study the interactions of membranes, as they are for example known from synaptic fusion, with high spatial resolution. shown that the acid incorporates mainly in the exterior part of the erythrocyte membrane, inducing creation of echinocytes. this suggests that it interacts predominantly with the outer part of the lipid layer of erythrocytes and liposomes. it was also shown that the cga decreases the packing order of the hydrophobic part of the membranes, without changing the anisotropic fluorescence of the hydrophobic part. one of the unique features of single molecule absorption/ emission is their anisotropy due to the well-defined transition dipoles for both processes allowing the determination of the molecule's d orientation. therefore, several techniques have been proposed in order to determine the full d orientation of dipole emitters on a single molecule level. we recently demonstrated a technique that combines emission distribution and polarization detection [ , , ] . as the method is an intensity distribution technique and based on single photon detection in principle, one can extend the d orientation determination to fluorescence correlation spectroscopy (fcs) as well as dynamical anisotropy measurements. this allows for the determination of the dynamics in d orientation of single molecules down to a nanosecond timescales. the d orientation is particularly interesting in non-isotropic environments. a lipid membrane is such a non-isotropic environment of enormous importance in biological systems. we therefore use giant unilamellar vesicle (guv) labeled with dyes like dio as a model system. due to the defined curvature of such vesicles all possible dipole orientations can be achieved. this allows us to show the capabilities of our method on different timescales and to quantify the error in determination of d orientation dynamics in lipid membranes. [ the aim of the studies was to determine the changes that occur in a biological membrane and the model lipid membrane as a result of interaction with strawberry leaf extract. numerous studies conducted all over the world have documented a beneficial effect of polyphenolic compounds on the human organism. however, the mechanism of the interaction on the molecular and cell level is not yet known. in the work presented, the effect of strawberry leaf extract on the erythrocyte and black lipid membranes has been investigated. the applied methods -spectroscopic, fluorimetric and electric -allowed to determine the hemolytic and antioxidant activity, and the packing order in the erythrocyte membrane as well as the electric capacity of blms. the results obtained indicate that the extract is efficient in protecting membrane lipids against oxidation, does not induce hemolysis, increases osmotic resistance and decreases packing order in the hydrophilic region of the erythrocyte membrane. moreover, it increases stability and life-time of flat lipid membranes, without altering their specific capacity. supported lipid bilayers are an abundant research platform for understanding the behavior of cell membranes as they allow for additional mechanical stability and enable characterization techniques not reachable otherwise. however, in computer simulations these systems have been studied only rarely up to now. we present systematic studies on different length scales of the changes that a support inflicts on a phospholipid bilayer using molecular modeling. we characterize the density and pressure profiles as well as the density imbalance induced by the support. it turns out that the changes in pressure profile are strong enough that protein function should be impacted leading to a previously neglected mechanism of transmembrane protein malfunction in supported bilayers. we determine the diffusion coefficients and characterize the influence of corrugation of the support. we also measure the free energy of transfer of phospholipids between leaflets using the coarsegrained martini model. it turns out that there is at equilibrium about a - % higher density in the proximal leaflet. these results are in agreement with data obtained by very large scale modeling using a water free model where flip-flop can be observed directly. we are additionally characterizing the intermediate states which determine the barrier height and therefore the rate of translocation. we also study the influence of surface roughness and curvature on the behavior. simulations in atomistic detail are performed for selected systems in order to confirm the findings. [ the inverse bar (i-bar) domain is part of the superfamily of the membrane-deforming protein is bin-amphiphysin-rvs (bar) proteins which induce either positive or negative membrane curvature both in vitro and in cells. generation of membrane curvature by these membrane deforming proteins often works together with actin dynamics. i-bar shares its function between actin bundling and membrane binding but it is still obscured what molecular mechanisms are responsible for these functions. the aim of our project is to investigate the detailed membrane binding properties of the i-bar of irsp and its relations to the actin cytoskeleton. in vitro fret experiments and fluorescence quenching studies were carried out between the i-bar and liposomes made up from different lipid constructs. we have found that the i-bar has preference to bind to the negatively charged lipids however it can also bind to the uncharged lipids. the fluorescence quenching studies reflected that the accessibility of the i-bar surface was higher toward the negatively charged lipids than for the uncharged ones. tns fluorescence assay reflected that the i-bar domain binds to the surface of the micells rather than penetrating into its core. lipid bilayers present a well-known order-disorder chain transition at ambient temperatures. this transition may become anomalous if the lipid head-group presents ionic dissociation at low ionic strength, as detected by several experimental techniques: between the gel and the liquid phases an intermediate phase appears as a shoulder in the specific heat, a deep in turbulence or a maximum in conductivity. we propose a statistical model which allows ionic dissociation of the polar group on the membrane surface and thus introduces competition between the hydrophobic interaction of hydrocarbonic chains, which favours the gel phase, at low temperatures, and the electrostatic interaction of charged head-groups, which favours the fluid phase, at higher temperatures. the model presents an intermediate fluid phase with higher dissociation and charge ordering on the membrane surface, beyond a sharp gel-fluid transition. the model presents increasing temperature of the main transition by addition of salt, as well as the shrinking of the anomalous region as chain length increases. model thermodynamic behavior is compared to results for pgs, phospholipids with a glycerol head-group. the well-programmed membrane fusion systems, operating in a weakly acidic environment, have attracted attention in the fields of biochemistry, biophysics, and pharmacy because these acidic conditions are generally observed in endosomal membranes or tumor tissues. we have reported a selective liposomal membrane fusion system toward a sugar-like cyclic cis-diol structure on the target liposome. this system consists of a lapidated phenyl boronic acid derivative as membrane -bound fusogen and phosphatidylinositol as target. here we report the preparation of a boronic acid / ph-responsive polypeptide conjugate as a novel membrane fusion device and the development of a target selective liposomal membrane fusion system with endosomal ph-responsiveness. during the course of lipid-mixing, inner-leaflet lipid-mixing, and contents-mixing assays to characterize membrane fusion behavior, we clearly observed a liposomal membrane fusion phenomenon when the ph of the experimental system was changed from . (physiological) to . (endosomal). our highly effective methods, which include a target selective liposomal membrane fusion, can be useful in the area of nanomedicine such as hybridoma technology and liposomebased drug or gene delivery. complete and reversible chemical denaturation of an a-helical membrane protein jana broecker, sebastian fiedler, sandro keller molecular biophysics, university of kaiserslautern, erwin-schrö dinger-str. , kaiserslautern, germany the question of how an unordered polypeptide chain assumes its native, biologically active conformation is one of the greatest challenges in molecular biophysics and cell biology. this is particularly true for membrane proteins. chemical denaturants such as urea have been used successfully for in vitro un-and refolding studies of soluble proteins and b-barrel membrane proteins. in stark contrast with these two protein classes, in vitro unfolding of a-helical membrane proteins by urea is often irreversible, and alternative denaturation assays using the harsh detergent sodium dodecyl sulphate suffer from a lack of a common reference state. here we present the complete and reversible chemical denaturation of the bacterial a-helical membrane protein mistic out of different micellar environments by urea. we applied multidimensional spectroscopy and techniques typically used in b-barrel membrane protein unfolding. mistic unfolds reversibly following a two-state equilibrium that exhibits the same unfolded reference state. this allows for a direct comparison of the folding energetics in different membrane-mimetic systems and contributes to our understanding of how a-helical membrane proteins fold as compared with both b-barrel membrane proteins and water-soluble proteins. in recent years, buckwheat has been of great interest in the world markets of healthy food, due to its high energy value, the content of unsaturated fatty acids, mineral constituents and vitamins. its seeds contain flavonoids which are natural, efficient antioxidants. the aim of the present studies was to investigate the effect of buckwheat extracts on the properties of biological membrane, which is the main site of the interaction between the substances buckwheat contains and the organism. the research was conducted on red blood cells and their isolated membranes, using the spectrophotometric, microscopic and fluorimetric methods. from the results obtained it follows that the compounds contained in buckwheat extracts increase the osmotic resistance of erythrocytes, making them less sensitive to the medium's osmotic pressure, induce changes in cell shape, producing increased number of echinocytes, and decrease the packing order of the polar heads of membrane lipids. it can thus be inferred that the compounds contained in the extracts penetrate the hydrophobic region of the erythrocyte membrane and alter its properties. .due to its small size, symmetric structure, amphipathicity, proteolytic stability and testable mode of activity, the gs backbone is a convenient model system to examine the structure-activity relationship of individual amino acid substitutions. we have previously reported the structure analysis of two gs analogues in which either the val or leu residues on the hydrophobic face of the molecule were substituted by the aliphatic f-labeled amino acid f-phg. using f-ssnmr in oriented lipid bilayers, we observed a re-alignment of the peptide that is compatible with the formation of a putative pore [top.curr.chem. : ]. here, we present novel analogs of gs with different f-prolines in the b-turn region, and with cf -bpg in place of leu. based on these f-ssnmr results and supported by cd, dsc and activity tests, we could demonstrate that all analogues are structurally intact and antimicrobially active. we observe, however, differences in the re-alignment propensity when comparing these gs analogues in dlpc and dmpc bilayers. these differences can be rationalized in terms of molecular shape being changed upon incorporation of unnatural amino acids at various sites of the molecule. the beta-propiolactone (bpl) is an inactivating reagent commonly used to produce viral vaccine preparations (whole virions or split-virions). although bpl has been reported to inactivate nucleic acids, its mechanism of action on proteins and the outcome on viral infection remains ill-defined. in this work, h n /victoria/ / influenza virus strain has been submitted to various bpl inactivation conditions (from lm to mm). cell infection ability was progressively reduced and entirely abolished at mm bpl. to clarify the bpl effect, we focused on membrane fusion infection steps using kinetic fluorescence molecule leakage from liposome and lipid fret assays combined with cryo electron microscopy. membrane fusion measured at ph on gm liposomes was reduced in a dose-dependent manner. interestingly the fusion activity was partially restored using the proton-ionophore monensin as confirmed by cryoem images. in addition, a decrease of molecule leakage irrespective to bpl concentration was measured suggesting that the hemagglutinin affinity for gm was slightly modified even at low bpl concentration. altogether these results strongly suggest that bpl treatment impairs m protein activity likely by preventing proton transport and bring new light on the mechanism of action of bpl. cellular membranes have a heterogeneous lipid composition, potentially forming nano-domains or membrane rafts, believed to be platforms of altered fluidity involved in protein sorting and trafficking . an alternative mechanism, potentially leading to protein sorting, has recently been proposed, suggesting that the curvature of membranes can also actively regulate protein localization . recently we showed that a variety of protein anchoring motifs are membrane-curvature sensors and thus up concentrate in regions of high membrane curvature . furthermore the curvature sensing ability of the anchoring motifs persisted independently of their structural characteristics. this leads us to speculate that curvature sensing might be an inherent property of any curved membrane and as a consequence, the lipid composition of the bilayer could potentially regulate this recruitment by membrane curvature. thus there might be an intimate, yet unrecognized, link between the way raft-like membrane domains and membrane-curvature promotes the localization of membrane-anchored proteins. we examined how changing the lipid composition of liposomes influenced the recruitment by membrane curvature of a model amphiphilic protein-anchoring motif. employing our single liposome curvature assay, we tested lipid mixtures with different ratios of dopc, sphingomyelin and cholesterol, giving rise to liposome populations of different phase-states. we found an amplified recruitment by membrane curvature for all raft-like l o phase-state mixtures when compared to the l d phase-state counterparts. based on these findings we suggest a synergetic effect when combining a raft-like lipid phase-state and high membrane curvature, resulting in a highly potent mechanism for selective localization of membrane-anchored proteins. keywords: non-lamellar lipid structure, phase transition, minerval, -hydroxylated fatty acid. minerval ( -hydroxyoleic acid), a potent antitumoral drug, is known to modulate the lipid membrane structure by decreasing the lamellar-to-non-lamellar phase transition temperature (t h ). a series of -hydroxy fatty acid derivatives, varying in acyl chain length and degree of unsaturation, have been analyzed in terms of their ability to stabilize the inverted hexagonal (h ii ) phase in palmitoyl-oleoyl-phosphatidylethanolamine membranes. differential scanning calorimetry and p-nuclear magnetic resonance showed that mono-and polyunsaturated, but not saturated, -hydroxylated fatty acid molecules were able to decrease the t h . lipid vesicles mimicking the lipid composition of a cell membrane were solubilized at °c in the presence of triton x- . the results demonstrated that the amount of detergent-resistant membranes, which are related to liquid ordered (lo) structures, decreased in the presence of -hydroxylated fatty acids. the so-called lipid membrane therapy focuses on the reversion of cell disfunction through the modulation of the membrane structure, thus altering the activity of membrane-associated proteins. the ability to modify the biophysical properties of a lipid membrane makes the studied -hydroxylated fatty acid molecules be prospective candidates for the use in the lipid membrane therapy. ]. however, a significant downward divergence occurs above mol % of probe content, which might indicate deviations to ideal mixing in fluid phase. results for b-py-c -hpc, in mixtures of popc with and mol % pops were indistinguishable from those obtained with pure popc vesicles; however excimer formation in pure pops bilayers appears to be appreciably higher. we also compared the excimer formation findings with quenching of the same probes by low concentrations of doxyl quencher groups labeled acyl phospholipid chain at the same depth of the pyrenyl group. the results are also scrutinized by the same two-dimensional kinetic formalism and good correlation was also found. derek marsh max-planck-institut fü r biophysikalische chemie, gö ttingen, germany the amassing of comprehensive data on the lipid composition of biological membranes by lipidomics initiatives provides a potent challenge to the membrane biophysicist interested in lipid structure. this resolves itself essentially into two aspects. the first systematises the dependence of membrane biophysical parameters on lipid molecular structure. lipid volumes, membrane dimensions, chain-melting temperatures and enthalpies, nonlamellar phase formation and structure, critical micelle concentrations and thermodynamics of membrane formation, membrane-membrane interactions and lipid transfer are amongst the properties of central biophysical interest. the relevant structural parameters are lipid chain length, degree of unsaturation, chain branching and headgroup configuration. the second, more complex and less well developed, aspect concerns the lipidlipid interactions that determine the membrane properties of lipid mixtures. in part, these can be obtained from binary phase diagrams, and the more limited number of ternary phase diagrams -notably with cholesterol -that are available. extrapolation to higher order mixtures lies in the future. i shall attempt to summarise some of the progress in these directions. the immediate aim is a second edition of my handbook of lipid bilayers, which, in addition to a vastly expanded database, will include interpretative features and will be available in the early part of next year. lateral diffusion dynamics in phosphatidylcholine/cholesterol bilayers has been mostly accessed by means of epr, nmr and fcs spectroscopic techniques. reliable stady-state florescence quenching analysis of diffusion-controlled processes has been ampered by the lack of a self-consistent kinetic formalism for the two-dimensional ( d) counterpart of the classical stern-volmer analysis for three-dimensional ( d) solvents. we studied the excimer formation of phospholipid-labeled pyrenyl probes (proportion of mol %) in mixed popc/cholestrol mlv liposomes by combined steady-state and timeresolved fluorescence the findings are in very good agreement with the theoretical predictions of the kinetic formalism specific for fluorescence quenching processes occurring in the small hydrophobic head group, the closely packed acyl chains, and the capability of interfacial hydrogen bonding have been suggested to govern the characteristic membrane behavior of long chain saturated ceramides: the self-segregation, and the formation of hexagonal phases and highly ordered gel phases. while it has been shown that structural alterations of the ceramide acyl chains induce position dependent effects on their behavior, we wanted to study the effect of interfacial properties, including hydrogen bonding, on ceramide membrane properties. the h-bond donor functions of nh and oh in the sphingosine backbone of palmitoylceramide were disrupted either separately or simultaneously by replacing the hydrogen with a methyl-group. when the lateral phase behavior of mixed bilayers containing cholesterol/sphingomyelin-rich domains was studied in the presence of the ceramide analogs, the o-methylated ceramide appeared to form a thermally stable, sterol-excluding gel phase with sphingomyelin, whereas the o-methylated ceramide failed in both thermal stabilization and sterol displacement. the doubly methylated analog was the poorest ceramide mimic. together with the possible steric effects induced by the methylations, the lack of nh h-bond donor function impaired ceramide membrane behavior to a greater degree than the lack of oh h-bond donor function. sugar-based surfactants are made from renewable resources using the ''green chemistry'' methods, are easily biodegradable and used in washing agents, cosmetics, and drug carriers. besides, there are attempts to use them as nonviral vectors in gene therapy. we studied the influence of new x-(alkyldimethylamonium)alkylaldonamide bromides (c n gab) with different chain lengths (n = , , , ) on the thermotropic phase behavior of dppc, and dppc/chol bilayers by means of differential scanning calorimetry. the surfactants were added either to the water phase or directly to the lipid phase (a mixed film was formed). we analyzed the changes in the temperatures, enthalpies and shapes of the main phase transitions as a function of concentration. molecular modeling methods were also used. cytotoxicicity of the c n gabs was determined in the cell line l and a . for cytotoxicity test, the cells were seeded in -well plates ml of Á cells/ml in the culture medium eagle'a or dulbecco with % calf serum, penicillin and streptomycin was deposited into each plates. the cells were treated with various doses of surfactants and incubated. the minimal concentration which was toxic to approximately % of cells was taken as tccd . this work was supported by grant n n . membrane fusion is ubiquitous in life requiring remodeling of two phospholipid bilayers. as supported by many experimental results and theoretical analyses, merging of membranes seems to proceed via similar sequential intermediates. contacting membranes form a stalk between the proximal leaflets which expand radially into a hemifusion diaphragm (hd) and subsequently open to a fusion pore. direct experimental verification of the hd is difficult due to its transient nature. using confocal fluorescence microscopy we have investigated the fusion of giant unilamellar vesicles (guvs) containing fluorescent membrane protein anchors and fluorescent lipid analogues in the presence of divalent cations. time resolved imaging revealed that fusion was preceded by displacement of peptides and lipid analogues from the guv-guv contact region being of several lm in size. a detailed analysis showed that this structure is consistent with the formation of an hd. a quantitative model of the hemifusion equilibrium and kinetics of the growing hd was developed. bilayer tension could be shown to drive hd expansion and interleaflet tension was found to act as a counterforce, because the outer leaflets are compressed upon hd growth. the model and its predictions fit nicely with observations above. concentration effects of trehalose in the equivalent polarity of fluid popc bilayers c. nobre , d. arrais , j. martins , ibb -cbme, faro, portugal, dcbb -fct, universidade do algarve, faro, portugal trehalose is an important disaccharide, formed by two units of glucose linked by a a- , glicosidic bond. it is capable of replacing water molecules in the hydration shell of the phospholipid headgroups, in cases of extreme dehydration, by establishing hydrogen bonds with their -co and -po groups, preserving this way the membrane structure. the polarity gradient is a significant feature of lipid bilayers and is influenced by the amounts of water within this medium. it is therefore important to understand the effects of different concentrations of trehalose in simple model membranes. using the pyrene empirical polarity scale, we monitorized changes in the polarity values when varying trehalose concentration in the bounding aqueous phase. for lower concentrations (until . m), we observed a decrease in polarity, comparing with popc bilayers in pure water. for higher trehalose concentrations (above . m), the polarity values are indistinguishable from those popc in water. using the freeze and thaw technique we obtained the same results, except for the lower trehalose concentrations. general anesthetics are indispensible tools of daily surgery. yet, their molecular mode of action remains elusive. while one school favors specific (direct) interactions with proteins of the central nervous system, another school adheres to a nonspecific modulation of biophysical membrane properties. one of the strongest arguments against lipid theories is the absence of stereo-specific effects in model membranes, as opposed to their detection by electrophysiological measurements on ionchannels. we have combined x-ray scattering and molecular dynamics simulations on palmitoyl-oleoyl-phosphatidylcholine bilayers with fluorescence microscopy on live cells to study the effects of the stereoisomers of ketamine on membrane properties. we find significant effects of both enantiomers on the distribution of lateral pressures at clinically relevant concentrations, being more pronounced for s-(?)-ketamine. we further calculated the effect of the lateral pressure profile changes on the opening probability of an ion-channel using crystallographic information. the observed channel inhibition compares remarkably well with clinically observed effects of the enantiomers. we thus provide first evidence for a stereo-specific, but indirect effect of general anesthetics on ion-channels. dependence of gramicidin a channel lifetime on membrane structure obtained from x-ray scattering measurements horia i. petrache department of physics, indiana university purdue university indianapolis, in , usa the activity of ion channels, in particular the lifetime of their conducting (open) state depends on the physical properties of lipid bilayers [ , ] which in turn depend on lipid headgroup and acyl chain composition. in order to investigate this dependence, we have performed measurements of gramicidin a (ga) channel lifetimes in three different lipid series. in each series, the lipid headgroups were phosphatidylcholine (pc), phosphatidylethanolamine (pe), and phosphatidylserine (ps), while the acyl chains consisted of symmetric monounsaturated di( : ), mixed ( : )( : ), and methylated di( : - me). in order to minimize the effect of headgroup electrostatics, measurements where performed in m kcl salts. we show how ga lifetimes depend on headgroup and acyl chain composition and on structural parameters determined by x-ray scattering. for the lipids considered, ga lifetimes cover a range from . seconds in the dope lipid to seconds in dphps. in this range, we find a gaussian dependence of ga lifetime on bilayer thickness, consistent with hydrophobic matching models. we discuss different aspects of channel-lipid interactions and to what extent measurements of ga lifetime in binary mixtures are consistent with measurements in pure lipid systems. [ the aim of the studies was to determine the effect of chlorogenic acid (cga), which is the main constituent of plant extracts, on properties of the model membranes. its effect was studied on temperature of the main phase transition of various lipids with and without presence of cholesterol, using the differential scanning calorimetry (dsc) method and the fluorimetric method. in particular, the degree of packing order of the hydrophilic phase of liposomes was determined using the laurdan and prodan probes, and fluorescence anisotropy of the hydrophobic phase with the probes dph and tma-dph. it had also been studied the effect of chlorogenic acid on the structure and capacity of black lipid membranes (blms), formed of egg lecithin and lipids extracted from erythrocytes. the results obtained indicate that cga lowers the main phase transition temperature slightly, without changing the fluorescence anisotropy in the hydrophobic part of the bilyer, and causes a decease in the packing order of the hydrophilic phase. by monitoring the capacity during blm formation we have found that the presence of chlorogenic acid accelerates the process of lipid self-organization into a bilayer, and increases stability and life of the blms. however, the was no effect of cga on specific capacity of the membranes, and thus on thickness of the liposome membrane hydrophobic layer. this work was sponsored by the ministry of science and education, scientific project no. n n and n n . many examples have recently been found where biological processes in the lipid bilayer are affected by the changes in the physicochemical properties of the membrane, e.g. the local curvature, the membrane tension and certainly the membrane structure. it has been shown that the activity of polyene antibiotics is strongly correlated to the phase diagram in a membrane composed of a mixture of popc and ergosterol or cholesterol (j membrane biol, : - , ( )). it is known that polyene action is quite sensitive to the type of sterol in the membrane, which enables its medical use, mainly as antifungals. it has been proposed that this selectivity of the drug to fungi is related to structure modulation by the sterols (see for example, biophys. j., , , ( )) and therefore the correlation found could be due to structural differences between popc/ergosterol and popc/ cholesterol along the corresponding phase diagrams. to investigate this, molecular dynamics simulations of the above mixtures along their phase diagrams were performed. it was found that there are indeed marked differences in structure along the phase diagrams, but for the sterol-sterol distribution function. an analysis of the behavior of this observable and the implications on polyene action is discussed. acyl transfer from lipids without enzyme catalysis: a new paradigm for membrane protein ageing? john sanderson, catherine pridmore, jackie mosely, paul yeo durham university, department of chemistry, durham, uk membrane proteins are recycled in cellulo with half-lives ranging from minutes to days. in other systems, such as enveloped viruses, proteins may equally remain membranebound for periods of days. it is therefore of interest to examine the behaviour of proteins in model membranes over extended periods in order to determine the long-term stability of the mixed systems, both in kinetic terms (attainment of equilibrium states) and chemical terms (reactivity). the reactivity of proteins towards membranes has been examined using the peptide melittin as a model for membrane proteins. acyl transfer from phospholipids to the peptide was found to occur over a period of several days, in the absence of any enzyme catalysis. transfer was detectable after days and reached % conversion in days. using tandem mass spectrometry approaches, the sites of melittin modification were localised. these sites included the side chain of lysine, opening the possibility that this residue may be modified in any membrane protein where this residue has an appropriate disposition. these observations challenge preconceptions concerning the membrane as an inert medium and highlight potential new mechanisms for membrane protein ageing. interaction of poly(l-arginine) with negatively charged bilayers studied by ft-ir spectroscopy christian schwieger, alfred blume martin-luther-university, halle-wittenberg, institute of chemistry, van-dankelman-platz , halle / saale, germany e-mail: christian.schwieger@chemie.uni-halle.de oligoarginine residues attached to macromolecules are known to facilitate the transport through lipid membranes. since the mechanism of this transport is still unclear, the effect is often called ''arginine magic''. we studied the interaction of poly(larginine) (pla) of different molecular weight with negatively charged lipid bilayers. we have shown by calorimetric and monolayer techniques that the interaction is due to a combination of electrostatic and hydrophobic forces. now we present an ft-ir spectroscopic study to reveal the effect of pla binding on membrane organisation and peptide conformation. we will show that pla binding reduces the lipid miscibility of negatively charged (pg or pa) and zwitterionic (pc) lipids within the bilayer. from the shift of the c=o stretching vibration we deduce that arginine side chains penetrate into the hydrophobic/ hydrophilic interface and replace hydration water molecules. the binding reduces the rotational freedom of the lipid molecules, as could be shown by an analysis of the ch -streching vibrations. pla binds in a b-sheet conformation to pg or pa gel phase membranes whereas its structure in bulk is random coil. the shift of the guanidyl vibration frequencies shows that also hydrogen bonds contribute to the pla -lipid interactions. neutron scattering studies of model membrane as a function of hydration and temperature federica sebastiani , , alessandra filabozzi and giovanna fragneto dipartimento di fisica, università degli studi di roma ''tor vergata'', roma, italy, institut laue-langevin, grenoble, france cell membranes carry out highly specialised functions in living materials. the composition of bacterial membranes is essential to understand the mechanism of action of antimicrobial peptides. in order to understand the role of the various components contributing to the overall behaviour, we have reproduced the membrane of bacillus subtilis and carried out neutron diffraction studies on d (small momentum-transfer diffractometer) and d (reflectometer used as a diffractometer), at ill. an ordered and homogeneous sample has been obtained by using the widely studied dmpc. the measured d-spacing of dmpc as a function of the relative humidity (rh) is related to the physical and chemical conditions affecting the sample. consequently the reliability of the humidity chamber, which has been previously upgraded, has been stated. moreover, the most suitable preparation technique has been set up. in order to investigate the component roles within bacillus subtilis membrane, three samples of phospholipids were prepared (with pope, popg and cardiolipin). neutron diffraction measurements, performed at controlled rh and temperature, suggested the presence of interesting phase transitions or coexistence of phases. the rupture of membrane vesicles near solid surfaces annamá ria taká ts-nyeste, imre deré nyi department of biological physics, eö tvö s university, h- budapest pazmany p. stny. /a, hungary the behavior of lipid membranes near solid surfaces has a great significance both in medicine and in technology. in spite of the widespread use and study of such membrane phenomena, their theoretical analysis is rather scarce. our main goal here is to understand the process during which membrane vesicles first adhere to solid surfaces, then rupture (or go through a series of transient ruptures) due to the mechanical tension induced by the adhesion, and finally spread along the surface forming a supported lipid bilayer. in our theoretical description we simultaneously consider the dynamics of spontaneous pore opening and closing; volume loss via leakage through the pores; and the advancement of the adhesion front. all these processes are supposed to follow an overdamped dynamics and coupled to each other through membrane tension. our numerical simulations reveal that the rupture process consists of three well distinguishable phases: a fast initial volume loss; followed by a slow volume loss; ending with a final burst and surface spreading. the second phase can be skipped if either the first phase advances far enough or the third phase sets in early enough. the smaller the vesicle, the further the first phase can advance. the third phase can start earlier if either the surface is smooth enough, or the adhesion energy is large enough, or the line tension is small enough. when the second phase is not skipped the time needed for the rupture process can take very long with a large variance. in the realistic range of the material properties (line tension, bending rigidity) the process is qualitatively always the same, so the most decisive parameter remains the size of the vesicle: the smaller the vesicle the faster and easier it ruptures. ( )). we chose four plant-derived polyphenols (flavonoids and stilbenes) of documented biological activity to study their influence on lipid domain number, area, shape, and borderlength. we found that resveratrol elevated the number of domains per vesicle, decreased their area and markedly increased the total length of domain border without affecting domains' circular shape. surprisingly, no such effect was observed for piceatannol differing from resveratrol by one hydroxyl group only. neither genistein nor -prenylnaringenin changed the morphology of lipid domains significantly. the possible mechanism of resveratrol-induced effect on lipid domains' morphology could be its selective accumulation in the interfacial regions between liquid ordered and liquid disordered domains. putative cholesterol recognition amino acid consensus (crac) motif in hiv coreceptors cxcr and ccr mikhail a. zhukovsky, albrecht ott biological experimental physics department, saarland university, saarbruecken, germany we identified a cholesterol recognition amino acid consensus (crac) motif in transmembrane domain (tmd ) of two g protein-coupled receptors (gpcrs), human chemokine receptors cxcr and ccr , coreceptors of human immunodeficiency virus (hiv). we suggest that residues belonging to this crac motif are involved in cholesterol binding to cxcr and ccr that is responsible for cholesterol requirement for cxcr and ccr conformation and function and for the role that cell cholesterol plays in the cell entry of cxcr -using and ccr -using hiv strains. putative crac sequences involve residues v /l -y -k in cxcr and l /v /v -y -k in ccr . in cxcr , crac motif is highly conserved across chordata species, whereas in ccr , crac motif is less conserved. t curve describe quantitatively the interfacial landscape around the protein molecules and can be used for the distinction between the globular and idp states. the behavior of the t and t data showed that there are two reorientation types present for every protein solutions below °c, irrespective for the nature of the protein or the solvent composition. local field fluctuation and the bpp models were applied, which failed for the buffered protein solutions and for the idps dissolved in water. a main cause of the failure is the changing h in the analyzed temperature range. this case is valid for the solutions of idps and for buffered solutions of both protein types. another cause can be the active relaxation channels other than dipolar when ions of quadrupolar nuclei are present. ligand-induced disorder-to-order transition plays a key role in the biological functions of many proteins that contain intrinsically disordered regions. this trait is exhibited by rtx (repeat in toxin) motifs found in more than virulence factors secreted by gram-negative pathogenic bacteria. we investigated several cyaa rtx polypeptides of different lengths ranging from to residues. we showed that the rtx proteins exhibit the hallmarks of intrinsically disordered proteins in the absence of calcium: they adopt premolten globule conformations and exhibit a strong timeaveraged apparent hydration, due in part to the internal electrostatic repulsions between negatively charged residues, as revealed by the high mean net charge. calcium binding triggers a strong reduction of the mean net charge, dehydration and compaction, folding and stabilization of secondary and tertiary structures of the rtx proteins. we propose that the intrinsically disordered character of the rtx proteins may facilitate the uptake and secretion of virulence factors through the bacterial secretion machinery. these results support the hypothesis that the folding reaction is achieved upon protein secretion and, in the case of proteins containing rtx motifs, could be finely regulated by the calcium gradient across bacterial cell wall. occupational exposure to heavy metals has been recognized to be a risk factor for parkinson's disease via metaltriggered deposition of alpha-synuclein (as) , . in the present work, al ? induced conformational change and instant oligomerization of as have been studied using fret and fcs as main techniques. donor and acceptor were labeled in the c-terminal at positions a c and a c. the average lifetime of donor in the presence of acceptor increases with the increase of al ? concentration, indicating as adopts a more extended conformation upon al ? binding. the intrinsic tyr fluorescence rises sharply within the mixing dead time, reflecting an enhanced hydrophobicity of the tyr environment and a fast conformational change of as. al ? also induces an immediate oligomerization of as as monitored by fcs. the diffusion coefficient of as changes from ± lm /s as monomer state to ± lm /s as oligomer state. the oligomerization is supposed to be induced by the ligand bridging of trivalent al ions. nearly % of human genes encode protein-kinases (pk), enzymes involved in cellular signaling and several other vital biochemical functions, which transfer phosphate groups from atp to specific target molecules, modifying their activity. [ ] deregulated pk have been linked to numerous diseases including cancer and diabetes, making them attractive targets for drug design. [ ] conformational transitions play a central role in regulating the phosphorylation activity. pk adopt an on state that is maximally active and one or more inactive states that shows minimal activity. [ ] the similarity of the relatively rigid and largely conserved atp binding site makes the design of selective inhibitors binding to the active state very difficult. indeed some of the best cancer therapies available are based on inhibitors, as imatinib, that bind to inactive states peculiar to a small subset of pk (abl, c-kit and pdgfr in the case on imatinib). thus, understanding the atomic details of the active to inactive transitions in kinases has a great importance. here we study a particular active-toinactive transition of c-src, a fundamental proto-oncogene involved in cancer and metastasis, by using multi-microsecond long fully solvated molecular dynamics simulations, metadynamics and ptmetad calculations [ , ] . the results, validated by mutagenesis, x-ray crystallography and binding kinetics, are suggestive of a functional role for the conformational transition. moreover, we were able to single out the most important residues affecting the conformational transition and to show that even a very conservative amino-acid substitution can have a dramatic effect on the conformational free energy landscape. the time-scales of protein folding events range over many orders of magnitude. in order to understand the complex folding mechanisms, peptides with well-defined secondary structure are often used as model systems as they may be regarded as smallest folding units of proteins. the formation of secondary structure elements occur on the nanosecond to low microsecond time scale. thus, stopped-flow techniques are too slow whereas pulsed laser techniques are capable to trigger folding processes in nanoseconds and to analyze faster folding events. we study ns-to-ls peptide dynamics by temperature-jump infrared spectroscopy. after initiation of a nanosecond temperature jump, the spectral response is monitored at single wavelengths in the amide i region reflecting the dynamics of the peptide backbone. relaxation rates are obtained. the helix-to-coil relaxation of polyglutamic acid is a multi-step process and requires more complex models than two-state kinetics. however, there are kinetic steps that are well described by single-exponential behavior and a two-state model. we demonstrate how equilibrium and time-resolved infrared spectroscopic data can be combined to deduce folding rates. unfolding and refolding studies using chemical denaturants have contributed tremendously to our understanding of the thermodynamics and kinetics of protein folding and stability. however, a major limitation of this approach lies in the large uncertainty inherent in the extrapolation of the free energy of unfolding in the absence of denaturant from free energy values measured at finite denaturant concentrations. here we show that this limitation can be overcome by combining multiple spectroscopic signals-including fluorescence, circular dichroism, and absorbance-recorded in a quasi-simultaneous and fully automated way at different wavelengths. we have optimised the number of wavelength values used, the integration time per data point, the increment in the denaturant concentration, and the weighting scheme applied for global data fitting. compared with the traditional approach based on the use of a single or a few wavelengths, we could thus improve the precision of the free energy value by an order of magnitude. we exemplify and validate this novel approach using representative, well-studied globular proteins and explain how it can be exploited to quantify subtle changes in membrane-protein stability which have thus far remained elusive. the rates of protein conformational changes are usually not only limited by external but also internal friction, however, the origin and significance of this latter phenomenon is poorly understood. it is often found experimentally that a linear fit to the reciprocal of the reaction rate as a function of the viscosity of the external medium has a non-zero , the physical basis of pressure unfolding is still largely unknown. we report here a specific study of cavities contributions to the volume difference between unfolded and folded states (dv u ), using four single point mutants of staphylococcus nuclease (snase). each mutation is localised in a strategic position on the protein structure and was designed to change a large buried hydrophobic side chain into alanine, thus opening tunable cavities in the snase d structure. measuring hsqcs peaks intensities up to bar monitored the equilibrium high pressure unfolding and leads us to precise estimations of dv u for more than two-thirds of the residues of each mutant. so-fast hmqc experiments were also performed to measure folding and unfolding rates from bar pressure jumps. high-pressure fluorescence experiments were done on six additional alanine mutants to complement the nmr study, allowing a more complete exploration of the local pressure sensitivity along the protein d structure. all these highly reliable measurements shed light on the real signification of the thermodynamic parameter dvu, and bring an unprecedented complex and heterogeneous picture at a residue level of the apparent two-state folding process of snase. determination of contributing factors to the volume change magnitude between unfolded and folded states (dv u ) is a longstanding question in the high-pressure field. we provide here new experimental and computational data using two wellcharacterized model proteins: notch ankyrin repeat domain (nank) and staphylococcal nuclease (snase). the repetitive nature of the nank protein was used to study influence of the protein size on dv u in a systematic way with a set of deletion mutants. high-pressure fluorescence data provided new evidences that neither peptide bonds hydration nor side chains differential hydration could be considered as major contributor to the measured dv u value. additional molecular dynamics (md) simulations rather suggested that the heterogeneous distribution of void volume in the folded states structures could explain the dv u variations among the nank deletion mutants. the specific issue of the void volume contribution to dv u values was studied using cavity mutants in snase, allowing a large structural mapping of the alanine mutations on this globular protein. combination of x-ray crystallography, highpressure fluorescence, high-pressure nmr and md simulations provided a first clear determination of the void volume contribution to the dv u values. these results also bring an unprecedented complex and heterogeneous picture at a residue level of the apparent two-state folding process of snase. we expressed an ig domain (i ) and a -residue-long fragment of the pevk domain in order to investigate the effect of temperature and pressure on their conformation. ftir spectroscopy is a useful method for investigating the secondary structure of proteins. we analyzed the amide i band to obtain information on protein structure. fluorescence labeling was also used in some experiments. to generate high pressures, a diamond anvil cell was employed. the ftir and fluorescence spectra of the protein fragments were recorded across the pressure and temperature ranges of - gpa and - °c, respectively. moderate changes were observed in the conformation of the pevk fragments in the explored range of the t-p plane, suggesting that the domain is a highly flexible, random-coil across the entire studied t-p range. by contrast, the i domain showed quite stable secondary structure. intrinsically disordered proteins participate in important regulatory functions in the cell, including regulation of transcription, translation, the cell cycle, and numerous signal transduction events. disordered proteins often undergo coupled folding and binding transitions upon interaction with their cellular targets. the lack of stable globular structure can confer numerous functional advantages, including, paradoxically, both binding promiscuity and high specificity in target interactions. nmr is unique in being able to provide detailed insights into the intrinsic conformational preferences and dynamics of unfolded and partly folded proteins, and into the mechanism of coupled folding and binding. the function of intrinsically disordered protein domains in transcriptional regulation and signaling will be described, with particular reference to the general transcriptional coactivators cbp and p , the tumor suppressor p , and the adenovirus e a oncoprotein. the globular domains of cbp/p are targets for coupled folding and binding of disordered transactivation motifs of numerous transcription factors and viral oncogenes, which compete for binding to limiting amounts of cbp/p . many intrinsically disordered proteins contain multipartite interaction motifs that perform an essential function in the integration of complex signaling networks. the role of multipartite binding motifs and post translational modifications in regulation of p -mediated signaling pathways will be discussed. the early vascular network is one of the simplest functioning organs in the embryo. its formation involves only one cell type and it can be readily observed and manipulated in avian embryos or in vitro explants. the early vascular network of warm-blooded vertebrates self-organizes by the collective motility of cell streams, or multicellular ''sprouts''. the elongation of these future vascular network segments depends on a continuous supply of cells, moving along the sprout towards its tip. to understand the observed self-organization process, we investigate computational models containing interactions between adherent, polarized and self-propelled cells. by comparing the simulations with data from in vivo or simplistic in vitro experiments, we explore the role of active migration, leader cells, invasion of the ecm, and cell guidance by micromechanical properties of adjacent cell surfaces. boron neutron capture therapy (bnct) is a promising method for treating the highly fatal brain tumor; glioblastoma multiform. it is a binary modality; in which use is made of two components simultaneously; viz. thermal neutrons and boron- . the biophysics of bnct is very complicated; primarily due to the complexity of element composition of the brain. moreover; numerous components contributes to the over all radiation dose both to normal brain and to tumor. simple algebraic summation cannot be applied to these dose components, since each component should at first be weighed by its relative biological effectiveness (rbe) value. unfortunately, there is no worldwide agreement on these rbe values. thermal neutrons were formerly employed for bnct, but they failed to prove therapeutic efficacy. later on; epithermal neutrons were suggested proposing that they would be enough thermalized while transporting in the brain tissues. however; debate aroused regarding the optimum source neutrons energy for treating brain tumors located at different depths in brain. insufficient knowledge regarding the rbe values of different bnct dose components was a major obstacle. a new concept was adopted for estimating the optimum source neutrons energy appropriate for different circumstances of bnct. four postulations on the optimum source neutrons energy were worked out, almost entirely independent of the rbe values of the different dose components. four corresponding condition on the optimum source neutrons energy were deduced. an energy escalation study was carried out investigating different source neutron energies, between . ev and . mev. mcnp b monte_carlo neutron transport code was utilized to study the behavior of these neutrons in the brain. the deduced four conditions were applied to the results. a source neutron energy range of few electron volts (ev) to about kev was estimated to be optimum for bnct of brain tumors located at different depths in brain. simulation of mutation induction by inhaled radon progenies in the bronchial epithelium balá zs g. madas, Á rpá d farkas and imre balá shá zy hungarian academy of sciences kfki atomic energy research institute, konkoly-thege mikló s ú t - ., budapest, h- , hungary radon is considered as the second most important cause of lung cancer after smoking. to understand the mechanisms leading from radon exposure to cancer formation is of crucial importance. this study focuses on the description of mutation induction by radon progenies in the bronchial epithelium. computational fluid and particle dynamics approach was applied to determine the radio-aerosol deposition distribution in the central airways. a numerical replica of a small fragment of the bronchial epithelium was prepared based on experimental data. microdosimetric computations were performed to quantify the cellular radiation burdens at the very site of deposition accumulation. a mutagenesis model was applied supposing that radiation induces dna damages and enhances the cell turnover rate. the results show that both considered mutagenic effects of densely ionising radiation contribute significantly to mutation induction and mutation rate depends non-linearly on exposure rate. furthermore, simulations suggest that the local maintenance capacity of the bronchial epithelium can be exhausted by chronic exposure to radon progenies with activity concentration characteristic of some uranium mines. the present work demonstrates possible applications of numerical modelling in radon related carcinogenesis studies. the neural crest is a group of cells found in all vertebrate embryos. it forms in the neural folds at the border of the neural plate and gives rise to a huge variety of cells, tissues and organs. one of the astonishing characteristic of neural crest cells is that they are able to migrate very long distances in the embryo. the neural crest has been called the ''explorer of the embryo'' as it is one of the embryonic cell types that migrate most during development, eventually colonizing almost every tissue. in this talk i will discuss our recent finding about neural crest migration. we have shown that neural crest cells, classically described as mesenchymal cells, migrate in large clusters cytokinesis relies on tight regulation of the mechanical properties of the cell cortex, a thin acto-myosin network lying under the plasma membrane. although most studies of cytokinetic mechanics focus on force generation at the equatorial acto-myosin ring, a contractile cortex remains at the poles of dividing cells throughout cytokinesis. whether polar forces influence cytokinetic cell shape is poorly understood. combining cell biology and biophysics, we demonstrate that the polar cortex makes cytokinesis inherently unstable and that any imbalance in contractile forces between the poles compromises furrow positioning. we show that limited asymmetric polar contractions occur during normal cytokinesis, and that perturbing the polar cortex leads to cell shape oscillations and division failure. a theoretical model based on a competition between cortex turnover and contraction dynamics accurately accounts for the oscillations. we further propose that blebs, membrane protrusions that commonly form at the poles of dividing cells, stabilise the position of the cleavage furrow by acting as valves releasing cortical contractility. taken together, our findings show that the physical properties of the entire cell are integrated into a finetuned mechanical system ensuring successful cytokinesis. collective motion of individual cells marks the onset of the transition to multicellularity in many microorganisms. this transition is often mediated by intercellular communication signals between cells. here, we show, in contrast, that the transition from single cell to collective motion in an ensemble of gliding bacterial cells can be understood as a dynamical selfassembly process of self-propelled rods. experiments were carried out with a mutant of the bacterium myxococcus xanthus moving by means of the a-motility system only and without undergoing reversals. the collective motion phase is confined to a monolayer and is characterized by the organization of cells into larger moving clusters. a transition to collective motion is detected in experiments by image analysis, that reveals a qualitative change of the cluster-size distribution at a critical cell packing fraction around %. this transition is characterized by a scale-free power-law cluster size distribution with an exponent . . we provide a theoretical model for cluster formation of self-propelled rods that reproduces the experimental findings for the cluster size distribution. our findings suggest that the interplay of selfpropulsion of bacteria and volume exclusion effects of the rodshaped cell bodies is sufficient to explain the onset of collective motion and the related changes in the cluster statistics. despite much speculation on the existence of structurally distinct oligomeric species associated with the conversion of certain monomeric proteins into amyloid fibrils, it has not previously been possible to observe them directly or to relate them to any key mechanistic steps involved in the interconversion process. we have developed a novel application of singlemolecule intermolecular fret to investigate in unprecedented detail the aggregation and disaggregation of alpha-synuclein, the protein whose pathogenic deposition as intracellular lewy bodies is a characteristic feature of parkinson's disease. our study reveals that a range of oligomers of different size and structure are formed, even at physiologically relevant concentrations. interestingly, the resistance to degradation of the aggregated state of alpha-synuclein, which is a well- we focused on the structure-dynamics interplay and showed how the fractal-like properties of proteins lead to such anomalous dynamics. we used diffusion, a method sensitive to the structural features of the protein fold and them alone, in order to probe protein structure. conducting a large scale study of diffusion on over pdb structures we found it to be anomalous, an indication of a fractal-like structure. taking advantage of known and newly derived relations between vibrational dynamics and diffusion, we demonstrated the equivalence of our findings to the existence of structurally originated anomalies in the vibrational dynamics of proteins. more specifically, the time dependent vibrational mean square displacement (msd) of an amino acid is predicted to be subdiffusive. the thermal variance in the instantaneous distance between amino acids is shown to grow as a power law of the equilibrium distance. the autocorrelation function in time of the instantaneous distance between amino acids is shown to decay anomalously. our analysis offers a practical tool that may aid in the identification of amino acid pairs involved in large conformational changes. more recently, we studied the effect of the hydrodynamic interaction between amino acids using a zimm-type model. we computed the time-dependent msd of an amino acid and the time-dependent autocorrelation function of the distance between two amino acids, and showed that these dynamic quantities evolve anomalously, similar to the rouse-type behavior, yet with modified dynamic exponents. we also studied the dynamic structure factor s(k,t) of proteins at large wavenumbers k, kr g [ [ , with r g the gyration radius, that are sensitive to the protein internal dynamics. we showed that the decay of s(k,t) is dominated by the spatially averaged msd of an amino acid. as a result, s(k,t) effectively decays as a stretched exponential. we compared our theory with recent neutron spin-echo studies of myoglobin and hemoglobin for the rouse and zimm models of hydrodynamic friction. in addition, i will mention two other projects currently underway: (i) a new elastic network model that accounts for the tensorial aspects of protein elasticity and is a combination of stretch-compress springs and bond-bending energies. (ii) the unfolding of a protein under the exertion of a large pulling force. allosteric regulation of enzymatic activity is crucial for controlling a multitude of fundamental cellular processes. yet the molecular level details underlying regulation often remain poorly understood. here we employed single molecule activity studies to dissect the mechanistic origin of enzymatic activity regulation. as a model system we employed a lipase and measured its activity as a function of accessibility to surface tethered liposomes ( ), which are known regulators of its activity. our results surprisingly revealed that the lipase oscillates between states of different activity. we accurately quantified for the first time both the interconversion rates between activity states and the inherent activity of these states. based on these we calculated the energetic landscape of the entire reaction pathway and identified that regulatory interactions redistributed the probability to reside on preexisting enzymatic activity states but did not alter the activity of these states. our findings provide the missing link between conformational and activity substates supporting and represent the first direct validation of the textbook hypothesis of conformational selection for regulation of enzymatic activity to identify the potential targets of cgmp in arabidopsis plants we adopted a proteomic approach to isolate possible cgmp-binding proteins. purification of soluble cgmp-binding proteins was performed using cgmp-agarose-based affinity chromatography procedure. next eluted proteins were analyzed by sds-page which revealed ten bands. we focused the subsequent analysis on low-molecular peptides of , and kda which were bound cgmp more intensively. after d-ief-page of the proteins isolated by cgmp-agaroseaffinity chromatography eight most abundant protein spots in the low-molecular area were visualized. these spots of interest were excised from the gel and in gel digested by trypsin. then tryptic peptides were analyzed by maldi-tof mass spectrometry and identified as isoforms of nucleoside diphosphate kinase (ndpk) from arabidopsis. thus, our data suggest that ndpk is a potential target of cgmp signaling in arabidopsis. dual-color fluorescence-burst analysis (dcfba) was applied to measure the quaternary structure and high affinity binding of the bacterial motor protein seca to the protein-conducting channel secyeg reconstituted into lipid vesicles. dcfba is an equilibrium technique that enables the direct observation and quantification of protein-protein interactions at the single molecule level. seca binds to secyeg as a dimer with a nucleotideand preprotein-dependent dissociation constant. one of the seca protomers binds secyeg in a salt-resistant manner, while binding of the second protomer is salt-sensitive. since protein translocation is salt-sensitive we conclude that the dimeric state of seca is required for protein translocation. a structural model for the dimeric assembly of seca while bound to secyeg is proposed based on the crystal structures of the thermatoga maritima seca-secyeg and the escherichia coli seca dimer. • dcfba is a flurorescence based single molecule technique that allows assessment of the stoichiometry of ligands bound to membrane receptors • dimeric seca binds asymmetrically to the protein-conducting membrane channel secyeg • monomeric seca binds secyeg but dimeric seca is required for protein translocation • protein translocation depends on receptor cycling of the dimeric seca if the dna charge is sufficiently neutralized by counter-ions, electrostatic interactions between helical charge patterns can cause attraction [ ] . helix specific interactions also cause tilt, in one direction, between two dna fragments [ ] . in braids and supercoils, this impetus to tilt breaks positive-negative supercoil symmetry. we show that these effects may cause spontaneous braiding of two molecules, lowering the dna pairing energy [ ] . the pairing is more energetically favourable for homologues (same base pair text) than for nonhomologous pairs. this might explain pairing between only homologues observed in nacl solution [ ] . also, we construct a simple model for a closed loop supercoil, including chiral electrostatic interactions. there are very interesting effects, for sufficient charge neutralization and groove localization of counter-ions. i.) positive super-coils are more energetically favourable than negative ones. ii.) a transition between loosely and tightly wound supercoils as one moves from negative to positive values of the supercoiling density. iii.) in positive super-coils the chiral interaction underwinds dna. [ von willebrand factor (vwf) is a large multimeric protein that is crucial for the force sensing cascade triggering primary hemostasis. it mediates binding of activated thrombocytes to injured epithelial tissue and serves as a transporter for coagulation factor viii. while it was shown that the hemostatic activity of vwf is affected by shear stress [ ] , the exact impact that shear forces have on the inflammatory cascade remains unclear. it is assumed that hydrodynamic forces lead to partial unfolding of vwf, which in consequence exposes more binding sites. in order to observe shear-induced changes of the protein's functionality, we measure conformational changes of vwf under flow with fluorescence correlation spectroscopy (fcs). we aim to measure the degree of uncoiling of vwf multimers under various buffer conditions, e.g. in the presence of colloids, vesicles or platelets. as only large multimers show significant hemostatic activity we intend to monitor the molecular weight distribution of vwf. shifts in this distribution indicate various pathological conditions making our multimer analysis a fast diagnostical tool for vwf-related diseases. this will serve as a basis for studies of vwf binding to collagen, fviii, gpib, vesicles and membranecoated nanoparticles under shear flow. [ data on mechanical properties of medically important proteins located in neural junctions are very limited. contactins (cntn) and paranodin proteins, located in extracellular part of ranvier nodes, are important for proper brain wiring. here we study a new series of fniii modules from human cntn- and - using a single molecule afm force spectroscopy and advanced, all-atom steered molecular dynamics (smd) computer simulations. mutations in cntns are responsible for numerous brain disorders including autism or pathological development of odor maps. perhaps mechanical properties of individual fniii mutated protein modules are compromised, thus we address this problem. a comparison of our afm force spectra with those of reference proteins will be presented [ ] [ ] , and the molecular level interpretation fniii nanomechanics, based on our smd data will be given. we believe that these data should help to understand a role of cntn in regulation of sodium ion channels in both normal and autistic subjects. supported in part by polish ministry of education and science, grant no. n , the computational center task in gdansk and license for accelrys software. recent achievements in rational dna-motors engineering demonstrate the possibility to design nano-motors and nanorobots capable of performing externally controlled or programmed tasks. a major obstacle in developing such a complex molecular machine is the difficulty in characterizing the intermediates, the final products and their activity. typically, non in-situ gel and afm and in-situ bulk fluorescence methods are used. i will present two dna-motors recently developed and studied using in-situ single-molecule fluorescence resonance energy transfer (smfret), alternating laser excitation (alex) and total internal reflection fluorescence spectroscopy (tirf), and will demonstrate that these methods can improve the way we design, construct, measure and understand highly complex dna-based machines. a motor made of bipedal dna-walker, which walks on a dna track embedded on a dna-origami, capable of long walking distance and maintaining structural stability, will be presented. the motor is non-autonomous; it receives ss-dna fuel/anti-fuel commands from outside (as in shin & pierce, jacs, ). the motors assembly stages and singlemotor's walking steps are monitored using smfret. the second motor is based on published bipedal autonomous dna-motor (seeman, science ). it is characterized by coordinated activity between the different motor domains leading to processive, linear and synchronized movement along a directionally polar track. to prove that the motor indeed walks, the authors chemically froze the motor at each step and use a complicated radioactive gel assay. i will demonstrate that using single-molecule approach, we are able to directly and in-situ measure single-motor's movements in few simple experimental steps, and measure its structural dynamics and kinetics. translation by a single eukaryotic ribosome using single molecule total internal reflection fluorescence microscopy, we observed translation of a short messenger rna (mrna) strand by single eukaryotic ribosomes. the ribosome-mrnas complexes are fixed to a microscope coverslip through the mrna, and mrnas are located through fluorescently labelled oligonucleotides hybridized to it downstream start codon. because of the ribosome helicase activity, the double strand formed by the oligonucleotide and the mrna is opened while the ribosome translates this region of the mrna. thus, the loss of the fluorescence signal allows us to measure the distribution of translation speed of single ribosomes. careful attention was given to photobleaching for the data analysis. this experiment opens the door to the study of eukaryotic translation at the single molecule level. erythrocyte hyperaggregation, a cardiovascular risk factor, has been associated to high plasma concentrations of fibrinogen. using atomic force microscopy (afm)-based force spectroscopy measurements, we have recently identified the erythrocyte membrane receptor for fibrinogen, an integrin with a a or a -like subunit [ ] . after this, we extended the study to the influence of erythrocyte aging on fibrinogen binding [ ] . force spectroscopy measurements showed that upon erythrocyte aging, there is a decrease of the binding to fibrinogen by decreasing the frequency of its occurrence (from . % to . %) but not its force. this observation is reinforced by zeta-potential and fluorescence spectroscopy measurements. knowing that younger erythrocytes bind more to fibrinogen, we could presume that this population is the main contributor to the cardiovascular diseases associated with increased fibrinogen blood content, which disturbs the blood flow. our data also show that sialic acid residues on the erythrocyte membrane contribute for the interaction with fibrinogen, possibly by facilitating the binding to its receptor. antimicrobial peptides are usually polycationic and amphiphilic with high affinity for bacterial membranes. in order to characterize their therapeutic potential it is crucial to disclose which properties of the peptide/lipids are important for target selectivity, and to examine the peptide structure and its association with lipid bilayers. in this work, first experiments have been carried out on a promising peptide called sb , which might represent the basis for developing a novel class of antibiotics. with the goal of enhancing the activity of a new semi-synthetic sequence, two identical peptides (wkkirvrlsa) were assembled via a lysine-linker, carrying also an octanoyl-lipid anchor. a highly active compound was obtained, but its structure and mode-of-action remain unexplored. this dendrimeric peptide and its linear deca-peptide counterpart are being studied in parallel to highlight the relevant properties and differences between dendrimeric structure and the sequence. monolayer intercalation is investigated with microtensiometry, fluorescence spectroscopy is applied to study thermodynamics and kinetics of the binding process. circular dichroism, nmr and md simulations are employed with the aim of elucidating the d structure in the membrane-bound state. the capability of proteins to build structures via self-organization is fascinating biophysicists since decades. with the advent of single-molecule methods, namely fluorescence correlation spectroscopy (fcs) and fluorescence resonance energy transfer (fret), the process of complex formation is becoming accessible to direct observation. coronaviruses (cov) are enveloped positive-stranded rna viruses. for sars-cov, it was shown that coronaviruses encode a rna-dependent rna-polymerase (rdrp) build from non-structural protein (nsp ) and non-structural protein (nsp ). this hexadecameric nsp -nsp complex is a hollow, cylinder-like structure assembled from eight copies of nsp and held together by eight nsp molecules [ , ] . we are aiming at understanding the assembly process and conformational changes of the complex for the related feline coronavirus. first results implicate that nsp alone forms a dimer, where interchain fret is more efficient than intrachain fret. for the complex the results indicate that nsp -nsp form a heterodimer which is different from sars-cov. our experiments highlight the potential of single-molecule fret for the study of protein complex formation. diffracted x-ray tracking (dxt) has been considered as a powerful technique for detecting subtle dynamic motion of the target protein at single molecular level. in dxt, the dynamics of a single protein can be monitored through trajectory of the laue spot from the nanocrystal which was labeled on the objective protein. in this study, dxt was applied to the group ii chaperonin, a protein machinery that captures an unfolded protein and refolds it to the correct conformation in an atp dependent manner. a mutant group ii chaperonin from thermococcus strain ks- with a cys residue at the tip of the helical protrusion was immobilized on the gold substrate surface and was labeled with a gold nanocrystal. we monitored diffracted spots from the nanocrystal as dynamic motion of the chaperonin, and found that the torsional motion of the chaperonin in the presence of atp condition was times larger than that in the absence of atp condition. and uv-light triggered dxt study using caged atp revealed that the chaperonin twisted counterclockwisely (from the top view of chaperonin) when the chaperonin closed its chamber, and the angular velocity from open to closed state was % faster than that from closed to open state. peptides or proteins may convert (under some conditions) from their soluble forms into highly ordered fibrillar aggregates. in vivo such transitions can lead to neurodegenerative disorders such as alzheimer's disease. alzheimer's disease is characterised by the extracellular deposition of abeta peptide in amyloid plaques, and the intracellular formation of neurofibrillary tangle (nft) deposits within neurons, the latter correlating well with disease severity. the major constituent of nft deposits are paired helical filaments (phf) composed of a microtubule-associated protein known as tau. studying the process by which tau forms these large aggregates may be an essential step in understanding the molecular basis of alzheimer's disease and other tauopathies. we have applied a two-colour single molecule fluorescence technique, and single molecule intermolecular fret measurements to study the soluble oligomers of tau which are formed during the aggregation and disaggregation of phf's. the neuronal protein alpha-synuclein is considered to play a critical role in the onset and progression of parkinson's disease. fibrillar aggregates of alpha-synuclein are the main constituents of the lewy bodies that are found in the brains of parkinson patients. however, there is growing evidence suggesting that oligomeric aggregates are significantly more toxic to cells than fibrillar aggregates. very little is known about the structure and composition of these oligomeric aggregates. we present results using single-molecule photobleaching approaches to determine the number of monomeric subunits constituting the oligomers. our results show that the oligomers have a narrow size distribution, consisting of * - monomers per oligomer. fluorescence correlation spectroscopy data confirm the narrow size distribution and additionally indicate a very loose packing of the oligomers. in combination with bulk fluorescence spectroscopy results of tryptophan containing mutants of alpha-synuclein, we present a structural model for the alpha-synuclein oligomer. gold colloids are widely used for in vitro and in vivo imaging. compared to the traditional optical tags sers-coded nanoparticles show a narrow emission bandwidth with structured spectra typical of the molecule used, a wider excitation bandwidth, higher emission intensity, a better photo-stability, and a lower toxicity. this is why in cancer therapy, besides being considered good tools for the delivery of anti-tumor drugs, aunp can be also good optical tags for the analyses of both np localization by laser scanning microscopy and the process of drug release inside the cells by raman. in our work we used nm diameter aunp loaded with rhodamine g, a molecule with a high raman and fluorescence efficiency, and with a chemical structure similar to doxorubicin, the antitumoral drug used in our system. the data showed that aunp are internalized by cells and sers can be performed. nm and nm diameter aunp loaded with doxorubicin were incubated at different time points with a cell line (human adenocarcinomic alveolar basal epithelial cells). only nm aunp showed intense raman emission typical of the doxorubicin phonon transitions. in recent years biomedical applications of diamond nanoparticles have become of significant interest, which rises questions of their biocompatibility and mechanisms of interactions with cells. the aim of this study was to compare the effect of nonmodified diamond nanoparticles (dnps) and dnps modified by the fenton reaction on human endothelial cells. dnps (\ nm particle size, sigma) were modified by the fenton reaction introducing surface -oh groups. immortalized human endothelial cells (huvec-st) were incubated with - lg/ml dnps in the optimem medium. diamond nanoparticles modified by the fenton reaction had smaller hydrodynamic diameter estimated by dynamic light scattering and the surface potential (zeta potential) measured using laser-doppler electrophoresis. they were more cytotoxic as evaluated by the mtt reduction assay. dnps augmented generation of reactive oxygen species in the cells, estimated by oxidation of ', '-dichlorofluorescin, the effect being higher for the fenton-modified dnps after -h incubation. cellular production of nitric oxide, estimated with daf-fm, was also affected by dnps; after h, fentonmodified oh, in contrast to non-modified diamond, decreased no production. diamond nanoparticles affected also the cellular level of glutathione and activities of main antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione s-transferase). we aim at investigating how photoreactions of proteins can be controlled by means of intense thz radiation tuned in resonance to specific vibrational modes, much in analogy to coherent control experiments conducted by fs nir laser pulses [ ] . for this we will combine a time-resolved ir difference spectroscopic setup with uniquely intense, tunable narrow bandwidth thz radiation ( - lm) at the ps beamline of the thz free electron laser felbe. these experiments will be performed on bacteriorhodopsin (br) which is the sole protein of the purple membrane of the archaebacterium halobacterium salinarum [ ] . upon illumination, the chromophore retinal isomerizes around the c -c double bond [ ] and br pumps a proton from the cytoplasmic to the extracellular side. this proton gradient is used by the bacterium to drive photosynthetic atp production under low oxygen tension [ ] . in our experiment, the photoreaction is initiated by a visible laser pulse as in standard experiments, but then the sample will be irradiated by a thz pulse from the free electron laser tuned into resonance with low-energy vibrational modes which is supposed to influence the photoreaction [ ] . such vibrational control will be monitored by time-resolved ftir spectroscopy using the step-scan technique [ ] . liposomes are increasingly studied as nanoscale drug delivery systems and biomembrane models. however the exact structure dynamics and mechanical behavior of liposomes is little known. atomic force microscopy (afm) is a powerful tool to characterize nanoscale morphology and enables the mechanical manipulation of submicron-sized vesicles. a drawback of afm, however, is that liposomes may flatten and rupture on substrates to form patches or supported planar bilayers (spb). our aim was to obtain better understanding of factors affecting liposomes on substrates and find experimental conditions at which liposomes preserve their structural integrity. in the presence of divalent cations dppc liposomes formed spb on mica. vesicles sedimented subsequently preserved their integrity and showed stronger attachment to spb. in addition to cross-bridging lipid head groups, divalent cations influence the surface charge of liposomes, thereby modulating liposome-substrate and liposome-liposome interfacial interactions. preserved vesicles stabilized by divalent cations may provide a unique experimental system for studying membrane-protein interactions. the influence of e.g. ph and ionic strength on various chromatographic bead -biomass combinations. to analyze the force curves, possible elastic contributions, e.g. from deforming cellular membranes, have to be decoupled from the interaction forces. then, bead-biomass interactions will be modeled using (extended) dlvo theory and resulting data can also be compared to real-life eba processes. the project aims for a better understanding of the interaction forces in chromatography and might help to improve the process quality of eba. long-term non-invasive in vivo monitoring of the survival, migration, homing and fate of transplanted cells is of key importance for the success of cell therapy and regenerative medicine. tools for in vivo magnetic resonance (mr) imaging of labeled cells are therefore being developed. we have prepared superparamagnetic iron oxide nanoparticles by the coprecipitation of fe(ii) and fe(iii) salts and oxidation. to stabilize the particles and to facilitate their internalization by the cells, the nanoparticles were coated with several novel low-and highmolecular weight compounds including d-mannose, poly(llysin), poly(n,n-dimethylacrylamide) and dopamine-hyaluronate conjugate. the surface-modified magnetic nanoparticles were thoroughly characterized by a range of physico-chemical methods, which proved the presence of the coating on the particles. the particles were then investigated in stem cell experiments in terms of real time cell proliferation analysis, viability, labeling efficiency and differentiation. the iron oxide concentration of the labeled cells was assessed using mr relaxometry. the advantages/disadvantages of particular iron oxide coatings will be discussed and the optimal coating suggested. excellent contrast was achieved by labeling the cells with dopamine-hyaluronate-coated nanoparticles. support of the as cr (no. kan ) is acknowledged. in the last decades the interest towards the fabrication of innovative bio-sensors with improved sensitivity and reliability for medical-diagnostics applications has been constantly risen. among the different techniques, microfluidic systems are playing a major role. in order to detect extremely low concentrations of biomolecules (pm and fm), attention should be placed on the controlled, selective functionalization of micro-and nano-channels. in this work we propose a new approach to functionalize gold patches inside fluidic channels. we start from self-assembled monolayers (sams) of thiolated molecules on a gold electrode deposited inside the channel. then, by using an electrochemical approach [ , ] we remove molecules from the sam at selected locations, by applying a negative voltage to the electrode. the newly exposed gold surface can be re-functionalized by using a thiolated biomolecule (i.e an antibody) capable to bind specific proteins flowing inside the channel. the cycle can be applied to other electrodes in the microfluidic system, creating a multiplexing device which, as we will show, can differentially measure ionic current flows in different channels. optically actuated micromanipulation and micro-probing of biological samples are increasingly important methods in the today's laboratory. microbeads as probes are the most commonly used tools in this field, although the only manipulative motion they allow is translation. we present polymerized d microstructures which can also be used for optical micromanipulation with more degree of freedom than microbeads. the two-photon polymerization (tpp), based on focused fs laser beam into appropriate photopolymers is a powerful method to build structures of arbitrary complexity with submicrometer resolution. the presented tools have the advantage of being capable of twisting and rotational manipulative motion, and also that the position of biological manipulation and optical trapping is spatially separated. different manipulative interfaces, the positioning stability and surface activation of the manipulators will be discussed. the potential application of multiplexed quantum dot labeling, mqdl, in clinical detection, prognosis and monitoring therapeutic response has attracted high interests from bioengineers, pathologists and cancer biologists. mqdl is superior comparing with conventional organic dye staining in its narrow emission bandwidths, wide signal dynamic ranges, high detection sensitivity, and low noise to signal ratios. however, the majority of the mqdl application has been limited to identification of specific cell type or cancer subtype and the improvement in labeling methodology. in this study, we focused on simultaneous detection and analysis of proteins in the c-met activation pathway, i.e. rankl, vegf, nrpln- , p-c-met and mcl- , which are known to be associated with human prostate cancer progression and metastasis. two experimental systems were analyzed: ) fixed xenograft tissues from an established ltl castration resistance human prostate cancer or crpc model; and ) clinical prostate tissue specimens from localized cancer and bone metastasis. in the presentation we will report our experience in ) the mqdl protocol optimization for the sequential reactions of individual primary antibody, the biotinylated secondary antibody and streptavidin-coated qd conjugate with nuclear dapi staining; and ) the multiplexed image catching, image unmixing, and subsequent per cell base quantification. for future multi-specimen analyses and validation, we will introduce a high throughput vectra image analysis system. carbon nanotubes (cnts) are already quite popular among many scientific and technological disciplines . in recent years they have been targeted for biotechnological and medical applications. in this work we have investigated the nanostructural self assemblies of biological lipid molecules in presence of cnts. advantage of using highly aligned cnts for this purpose being the possibility of studying the interactions of lipid molecules on the macromolecular surface as well as in the confinement of aligned cnts. we have observed various lyotropic nanostructures that are found for corresponding lipids in the bulk under dry and hydrated conditions. nanostructural studies were mainly performed using small and wide angle x-ray scattering techniques. this work is crucial for designing the nano-micro-fluidic architectures and supported model membranes where both -functionalization of cnts and nanostructural assembling of lipids, could be employed simultaneously. alternative of the commonly used measuring tools in protein research and medical diagnostics. thazolidone derivatives are novel synthetic compounds possessing various biological activities. we selected three such compounds les- , , which passed national cancer institute in vitro tests. annexin v/pi and dapi staining, dna electrophoresis in agarose gel, and western-blot analysis using specific antibodies against cellular proteins involved in apoptosis were applied to study molecular mechanisms of tumor cell death induced by these compounds. it was found that molecular targets of thiazolidones in target cells strongly depend on structure of their side groups: les- containing isatine fragment, activated caspase- involved in receptor-mediated apoptosis, while les- possessing benzthiazol residue, induced mitochondrial apoptosis mediated by caspase- , and les- which has a unique chlorine atom in side chain, also led to mitochondrial apoptosis mediated by aif (apoptosis-inducing factor). to increase anticancer potential of these molecules, in silico study was performed and most active groups of les- and les- were combined into one molecule. in vitro studies showed that such hybrid molecule called les- possessed times higher anticancer potential (ic = lm) comparing with initial compounds. we report on the synthesis and characterization of vaterite microcontainers for controlled drug release. moreover, we present experiments on possible release strategies of encapsulated substances via recrystallization, ph controlled, or by desorption methods. vaterite spherical particles were fabricated with controllable average sizes from ± nm till ± hm. we considered two ways of functionalization of the containers: encapsulation of the substances during the vaterite synthesis or their adsorption onto the prepared particles. as model experiments, vaterite containers, encapsulating rhodamine g, were imaged by two-photon microscopy, showing dye release into the aqueous medium due to recrystallization to calcite within days. differently, in ethanol only small amounts of the encapsulated markers were diffusion released after one week. the release mechanisms can be further controlled by covering the microcontainers with additional polymer layers to increase diffusion and recrystallization time. a change of the ph from neutral to acid conditions leads to the destruction of the vaterite matrix followed by a quick release of the encapsulated materials. these flexible control mechanisms make this system an interesting candidate for pharmaceutical applications. magnetic nanoparticles (np) in combination with therapeutic molecules represent one of most promising methods for targeted drug delivery. one of major current limitations of magnetic drug targeting is to achieve efficient concentration of magnetic carrier-drug complexes at the targeted sites due to poor mobility of nanoparticles in tissue structures. interstitial delivery is hindered by microscopic extracellular matrix, which represents a major barrier for nanoparticles motilities. in order to achieve efficient magnetic drug targeting it is crucial to know particle mobility in a given in vivo environment as well as to apply magnetic field having appropriate field gradient which drags magnetic nps. we used gel magnetophoresis in order to measure motilities of different magnetic nps (co-ferrite, c-fe o ) in agarose gel. numerical modeling using fem method was used to determine appropriate settings of magnets, which generate sufficient magnetic field gradient. further, we used the numerical modeling to evaluate the magnetic force on the nps for different geometries. we obtained that one of crucial factors which determines final mobility in tissue is formation of larger aggregates of nanoparticles under physiological conditions and interaction of nanoparticles with surrounding matrix. defining the forces required to gate mechanosensitive channels in mammalian sensory neurons kate poole and gary lewin department of neuroscience, max delbrueck center for molecular medicine, robert-roessle str , , berlin-buch, germany our sense of touch and mechanical pain is based on mechano-electrical transduction (met) at the terminal endings of subsets of dorsal root ganglion (drg) neurons innervating the skin. to quantify the stimulus strengths required to gate mechanosensitive channels in these subsets of neurons, we developed an approach using microstructured surfaces. the drg neurons are grown on laminin-coated pdms pillar arrays, mechanical stimuli are applied by deflecting individual pili and the deflection is monitored using light microscopy. as the pili behave as light-guides, the center of each pilus can be determined from a fit of the intensity values, allowing detection of movements of a few nanometers. the response to such stimuli is monitored using whole-cell patch-clamp. pili deflections of nm can gate the rapidly adapting-current in mechanoreceptor cells, while deflections above nm are required for gating of slowly adapting-currents in nociceptors. smaller stimuli are required to generate currents via pili deflection ( nm) vs neurite indentation ( - nm), suggesting that gating occurs at the cell-substrate interface. we have also characterized the met currents present in n a cells which we show are modulated by the substrate to which the cells are attached. enhanced stimulation of toll-like receptor via immunostimulatory nanoparticles jan rother, anna pietuch, andreas janshoff georg-august university gö ttingen, institute of physical chemistry, tammanstr. , d- gö ttingen, germany e-mail: jrother@gwdg.de among the toll-like receptor family (tlrs), the tlr has been subject of intensive research because of its predominant localization in the lysosomes of immune cells and its ligand rendering it a potential candidate for immunotherapy of autoimmune diseases and cancer. additionally, a use as an adjuvant in vaccination is aimed using synthetic cpg-oligodeoxynucleotides (cpg-odn's). albeit, immunostimulatory cpg-odns already showed promising results in animal experiments and clinical trials, several groups found that tlr is also expressed by tumor cells. first experiments show that activation of tlr displayed by cancerous cells leads to a decreased apoptosis rate and proliferation posing unpredictable threat to tumor patients exposed to cpg-odns. therefore, detailed knowledge about the impact of cpg-odns on cancer cells is inevitable for a save use in pharmaceutics. herein, we describe a sophisticated way to address tlr in cancer cells using cpg-odn functionalized ''superparamagnetic'' mno-and c-fe o -nanoparticles (nps) to stimulate tlr in a cells. analysis of impedimetric measurements revealed a cytotoxic effect of the mno-nps. cells treated with immunostimulatory fe o -nps showed an increased micromotility as well as a higher long-term correleation of the impedance signal. biomedical diagnostics like high-sensitivity, single-molecule study, easy sample preparation. furthermore, sers allows to conduct non-invasive studies of conformations of the molecules without destruction of living cells, i.e. in vivo [ ] . this work presents a sers study of cytosolic hemoglobin (hb c ) using silver nanoparticles (agnps). the hb c was isolated from cytoplasm of red blood cells taken from rat erythrocytes and diluted. agnps were prepared by developing leopold and lendl method [ ] . three types of colloids were prepared at various temperatures ( , and °c). the resulted agnps were characterized by uv-vis-, ftirspectroscopy, dls and tem. reduction of ag ions leads to the formation of predominantly spherical agnps but also silver nanorods, faceted and aggregated agnps in small quantities with a surface plasmon resonance band in the range of - nm. for agnps synthesized at °c, for example, a bimodal size distribution was observed (about and nm medium sizes, respectively). sers measurements were optimized for each type of agnps. it was demonstrated that agnps gave strong raman enhancement from hb c and types of sers spectra differ from each other. nano-zno is characterized by unique properties, low toxicity and high biocompatibility instead of a lot of others nanomaterials. for this fact nanoparticles of zno have great potential for applications in biosystems, for example biolabeling, biosensoring, delivery systems and others, which can be used in genetics, pathology, criminology, safety of food and many other industries. for these bioapplications are necessary surface modifications, which can made to the nanostructures to better suit their integration with biological systems, leading to such interesting properties as enhanced aqueous solubility, bio-recognition or applicability for biological systems. for synthesis of zno nanoparticles in aqueous solution we used -mercapto-undecanoic acid (mua) as stabilizing agent. the coating of nanoparticles with mua could allow their solubility in the water and the binding through carboxyl groups present in its structure. we defined the optimal ph for mua modificated nano-zno solubility and their ability interaction with positive charges. we studied the optical properties of pure and surface modificated nanoparticles and their conjugates with cytochrome c and also the effect of ph on the interaction between nano-mua and horse cytochrome c. the permeation of water soluble molecules across cell membranes is controlled by channel forming proteins and particularly the channel surface determines the selectivity. an adequate method to study properties of these channels is electrophysiology and in particular analyzing the ion current fluctuation in the presence of permeating solutes provides information on possible interactions with the channel surface. as the binding of antibiotic molecules in the channels of interest is significantly weaker than that of preferentially diffusing nutrients in substrate-specific pores, the resolution of conductance measurements has to be significantly increased to be able to resolve the events in all cases. due to the limited time resolution, fast permeation events are not visible. here we demonstrate that miniaturization of the lipid bilayer; varying the temperature or changing the solvent may enhance the resolution. although electrophysiology is considered as a single molecule technique, it does not provide atomic resolution. molecular details of solute permeation can be revealed by combining electrophysiology and all atom computer modeling. [ novel functionalized nanocomposites (nc) were designed and synthesized on the basis of polymeric surface-active oligoelectrolytes. the developed technology permits controlling: ) quality and quantity of structural blocks of nc, and size unimodality; ) branching at specific sites in polymer chain of nc; ) providing nc with reactive chemical groups; ) covalent conjugation of specific bio-targeting molecules. provided bioactive elements were: a) specific anticancer drugs, antibiotics, alkaloids; b) dna and sirna; c) immunoglobulins and lectins; d) lipids and amino acids; e) polyethylene glycol. fluorescent, luminescent, super-paramagnetic, or x-ray detectable compounds were also incorporated in nc to make them detectable and measurable. biocompatible nc possessing low toxicity towards mammalian cells in vitro and in vivo (mice) were created. they were effective in delivery of: ) drugs (doxorubicine and antibiotics) for chemotherapy in vitro and in vivo; ) dna for transfection of mammalian, yeast and bacterial cells; ) protein antigens for animal immunization and specific lectins for targeting apoptotic cells. these and other approaches in application of developed nc and nanobiotechnologies are considered. this work was supported by stcu grants # , # , # . in the anti-cancer drug delivery domain, nanotechnologies are a promising tool, providing a good tissue distribution and a low toxicity. drug delivery vehicles relying on solid nanoparticles have been proposed, among which diamond nanoparticle (size\ nm) is a very promising candidate [ ] . we have investigated the delivery of sirna by nanodiamonds (nd) into cells in culture, in the context of the treatment of a rare child bone cancer (ewing sarcoma), by such a gene therapy. sirna was bound to nds after nds coating by cationic polymers, so that the interaction is strong enough to pass the cell membrane without loss of the drug and does not prevent its subsequent release. the cellular studies showed a specific inhibition of the gene expression at the mrna and protein level by the nd vectorized sirna. we also uses the fluorescence of color center created in the nanodiamonds [ ] to monitor the release of fluorescently-labeled sirna in the intracellular medium. this technique brings a quantitative insight in the efficiency of sirna to stop cell proliferation. considering the success of the cell model we recently started the drug delivery in tumor xenografted on nude mice. silica nanoparticles are stable aqueous suspension of condensed siloxane nanocomposites, having an average diameter between and nm. particles containing organic functional groups on their surface are called organically modified silica nanoparticles (ormosil). due to the various chemical and physical properties of the surface groups, ormosil nanoparticles may have an enormous variety of biological applications, such as in vivo bioimaging, non-viral gene delivery or targeted drug delivery. our aim was to synthesize both void and fluorescent dye doped amino functionalized ormosil nanoparticles through the microemulsion method and use them for gene delivery. the obtained nanoparticles have been characterized by transmission electron microscopy and dynamic light scattering. furthermore, the nanoparticles have been investigated to exploit their transfection efficiency and the possible toxicity caused by surfactants used in the synthesis. the transfection efficiency was tested on various cell cultures. our further aim is the in vivo transfection of salivary glands using ormosil nanoparticles. our work has shown that the nanomedicine approach, with nanoparticles acting as a dna-delivery tool is a promising direction for targeted gene therapy. in vivo amperoetric cells for detection of fast diffusing, physiologically important small molecules lívia nagy, bernadett bareith, tü nde angyal, erika pinté r, gé za nagy university of pé cs, pé cs, hungary h s is a naturally occurring gas that is toxic in high concentration. it exists also in different tissues of living animals sometimes in concentrations as high as lm. it is generally accepted, that h s has important roles in modulating different, physiologically important biochemical processes similarly to other, fast diffusing molecules like no, co and h o . for investigation of the physiological effects of these species their local concentration in the studied biological media is important to know. this means methods needed for measuring the instantaneous concentration with high spatial resolution in living tissues without major invasion. electrometric micro, and ultramicro sensors are often gain application in experimental life sciences for measurement of local ion concentration or following neurotransmitter species in vivo measurements. in our work efforts are being carried out to improve the applicability of selective electrometric sensors in life science experiments. as a result of these work an improved h s measuring cell and improved electrode and method was developed for measurement of electroactive small molecules like no or h o . in the poster to be presented the structure, the working principles and the performances of the different sensors mentioned will be described. bacteriorhodopsin (br) is the only protein in the purple membrane of the halophilic organism halobacterium salinarium. it is a light-driven proton pump converting light into a transmembrane proton gradient through isomerization of the covently bound retinal chromophore. its stability, as well as its photoactivity in dried films, has made br an attractive material for biomolecular devices. such studies, however, have used br within the membrane, on relatively large surfaces. here, conducting-probe atomic force microscopy (c-afm) analysis was performed after isolating the protein from its native membrane environment while keeping its basic trimeric structure, and demonstrated that the molecular conductance of br can be reversibly photoswitched with predictable wavelength sensitivity. intimate and robust coupling to gold electrodes was achieved by using a strategically engineered cysteine mutant located on the intracellular side of the protein which, combined with a % delipidation, generated protein trimers homogenously orientated on the surface. c-afm proximal probe analysis showed a reproducible fold drop of br mean resistance over * cycles of interspersed illuminations at the same gold-br-gold junction when k[ nm, while no shift was observed with other wavelengths. capture of circulating tumor cells with a highly efficient nanostructured silicon substrates with integrated chaotic micromixers shutao wang ). this core technology shows significantly improved sensitivity in detecting rare ctcs from whole blood, thus provides an alternative for monitoring cancer progression. by assembling a capture-agent-coated nanostructured substrate with a microfluidic chaotic mixer, this integrated microchip can be applied to isolate ctcs from whole blood with superb efficiency. ultimately, the application of this approach will open up opportunities for early detection of cancer metastasis and for isolation of rare populations of cells that cannot feasibly be done using existing technologies. this technology helped to find a needle in a haystack and will open up the opportunity for single cell genomic and epigenetic sequencing and gene expression profiling. results from further development of this technology will assist the physicians in follow-up patients and testing vigorously the concept of personalized oncology with individualized therapy. this novel technology has recently been reviewed and highlighted by nature medicine the growing crisis in organ transplantation and the aging population have driven a search for new and alternative therapies by using advanced bioengineering methods. the formation of organized and functional tissues is a very complex task: the cellular environment requires suitable physiological conditions that, presently, can be achieved and maintained by using properly-designed bioreactors reproducing all specific functions and bioactive factors that assure viability/regeneration of cells cultured in an appropriate scaffold. the creation of biomimetic environment requires the use of biomaterials such as membranes with specific physico-chemical, morphological and transport properties on the basis of the targeted tissue or organ. tailor-made membranes (organic, functionalized with specific biomolecules, in hollow-fiber configuration), designed and operated according to well-defined engineering criteria are able to sustain specific biotransformations, to provide adequate transport of oxygen, nutrients and catabolites throughout the cellular compartment, and to supply appropriate biomechanical stimuli of the developing tissue. in this talk the author will show the development of membrane engineered constructs focusing on liver and neuronal systems. the role of membrane surface and transport properties in providing instructive signals to the cells for the guiding of proliferation and differentiation will be discussed. membrane bioreactors, which through the fluid dynamics modulation may simulate the in vivo complex physiological environment ensuring an adequate mass transfer of nutrients and metabolites and the molecular and mechanical regulatory signals, will be presented. here we present a novel but simple system for cell-based assays enabling simultaneous testing of multiple samples on a same tissue without cross-contamination between neighbouring assays, as well as sequenced or repeated assays at the same tissue location. the principle of this method lies in the spatially-controlled diffusion of test compounds through a porous matrix to the target cells. a simple microfabrication technology was used to define areas where diffusion processes are allowed or inhibited. we performed proof-of-principle experiments on madin-darby canine kidney (mdck) epithelial cells using hoechst nuclear staining and calcein-am cell viability assay. fluorescent staining superimposed properly on membrane pattern with a dose-dependent response, indicating that both compounds specifically and selectively diffused to the target cells. mdck cells similarly treated with cytochalasin b showed their actin network rapidly altered, thus demonstrating the suitability of this system for drug screening applications. such a well-less cell-based screening system enabling multiple compounds testing on a same tissue and requiring very small volumes of test samples appears interesting for studying potential combined effects of different biochemicals applied separately or sequentially. it is generally believed that all-optical data processing is the most promising direction to achieve serious improvements both in capacity and speed of internet data traffic. one of the bottlenecks of the state-of-the-art photonic integration technology is to find the proper nonlinear optical (nlo) materials that are supposed to serve as cladding media in waveguidebased integrated optical circuits performing light-controlled active functions. recently, the unique chromoprotein bacteriorhodopsin (br) has been proposed to be used as an active, programmable nlo material in all-optical integrated circuits. in integrated optical applications of br, its light-induced refractive index change is utilized. in this paper we exploit the refractive index changes of a dried br film accompanying the ultrafast transitions to intermediates i and k, which allows even sub-ps switching, leading beyond tbit/s communication rate. in the experiments direct pulses of a femtosecond laser system at nm were used along with synchronized ultrafast laser pulses at nm. we believe that the results may be the basis for the future realization of a protein-based integrated optical device, and represent the first steps to a conceptual paradigm change in optical communication technologies. last years, such autoantibodies attract an increasing attention of researchers as potential cancer biomarkers. since the sera of cancer patients typically contain a unique set of antibodies that reflect the tumor-associated antigens expressed in a particular malignant tissue, diagnosing and predicting the outcome of disease such as breast cancer based on serum autoantibody profiling is an attractive concept. to create a representative panel of antigens for detecting of breast cancer autoantibody profile we selected breast cancer associated antigens. these antigens were identified by screening of tumor cdna libraries with autologous sera using serex (serological investigation of recombinantly expressed clones) approach. all antigens were cloned, expressed, purified in bacteria and tested with sera of breast cancer patients and healthy donors in large-scale allogenic screening using elisa. the utility of selected tumor associated antigens for detecting of autoantibody profile in different types of breast cancer was evaluated. (controlled by architectural software) is carried out according to a design template, consistent with the geometry and composition of the desired organ module. structure formation occurs by the post-printing fusion of the discrete bio-ink units. when the bio-ink units contain more than one cell type, fusion is accompanied by sorting of the cells into the physiologically relevant pattern. thus structure formation takes place through self-assembly processes akin to those utilized in early embryonic morphogenesis. we demonstrate the technology by detailing the construction of vascular and nerve grafts. spherical and cylindrical bio-ink units have been employed to build fully biological linear and branching vascular tubular conduits and multiluminal nerve grafts. upon perfusion in a bioreactor the constructs achieved desirable biomechanical and biochemical properties that allowed implantation into animal models. our results show that the printing of conveniently prepared cellular units is feasible and may represent a promising tissue and organ engineering technology. femtosecond lasers have become important tools for noncontact microprocessing of biological specimens. due to the short pulse length and intensity-dependent nature of the multiphoton ionization process, fs-laser pulses affect only a small volume of a treated cell, providing a high degree of spatial localization. we employed fs-laser to address topical bioengineering and biomedical problems such as cell fusion and embryo biopsy respectively. a tightly focused laser beam (cr:f seed oscillator and a regenerative amplifier, nm, fs, hz) was used for a fusion of blastomeres of two-cell mouse embryos and for a polar body (pb) biopsy. in order to fuse blastomeres the contact border of cells was perforated by a single laser pulse. the fusion process usually completed within * min. in order to perform a noncontact laser based pb biopsy we initially drilled an opening in the zona pellucida with a set of laser pulses, and then extracted the pb out of zygote by means of optical tweezer (cw laser, nm). the energy of laser pulses was thoroughly optimized to prevent cell damage and increase the fusion and biopsy rates. the proposed techniques demonstrate high efficiency and selectivity and show a great potential for using fs lasers as a microsurgical tool. new insights into mechanisms of electric field mediated gene delivery maš a kanduš er and mojca pavlin university of ljubljana, faculty of electrical engineering, si- ljubljana, slovenia gene electrotransfer is widely used for transfer of genetic material in biological cells by local application of electric pulses and is currently the most promising non-viral delivery method for gene therapy for a series of diseases as well as for dna vaccination. current description of the process defines several steps: electropermeabilization, dna-membrane interaction, translocation, trafficking to nucleus and into nucleus. but the mechanisms of electrotransfer are still not fully understood. we present results of the systematic in vitro analysis using pegfp of all steps involved in electrotransfection from electropermeabilization, analysis of different pulsing protocols, theoretical analysis of plasmid mobility to visualization of the processes of dna-membrane interaction. we demonstrate that in order to translate in vitro results to tissue level sub-optimal plasmid concentrations have to be used. furthermore, sofar the method of dna entry into cytoplasm was only speculated. our results suggest that it is crucial that first, membrane is electropermeabilized, then sufficient electrophoretic force is crucial for insertion of dna into destabilized lipid bilayer followed by dna translocation into cytoplasm via a slow process. efficiency of electrotransfer depends also on the stage of of cell culture -cells in dividing phase are easer to electrotransfect. gentamicin interaction with b f cell membrane studied by dielectrophoresis dielectrophoresis (dep) is the translational motion of polarizable particles due to an electric field gradient. positive-dep and negative-dep correspond to particle movement forward or backward the region of high field intensity, respectively. our study reveals some of the cell membrane modifications induced by gentamicin (gt), as they are reflected in the crossover frequency f co of b f murine cells incubated with gt for different concentrations and durations. f co is the ac frequency when cells turn from positive-dep to negative-dep. gentamicin is a positively charged aminoglycosidic antibiotic, with concentration-dependent killing action; it is widely used because of its low cost and reliable bactericidal activity. gt drawbacks consist in high toxicity for renal and hearing cells; the molecular mechanisms of this toxicity are still unclear. for low external medium conductivities (& . s/m), f co of control and gt-cells was found to range from to khz. f co shifts to higher frequencies with the increase of gt concentration and incubation time. cells dielectrophoretic behavior is discussed using the cell singleshell based model. extracellular matrix (ecm) is a major obstacle for succesful delivery of genes. chitosan is a versatile and biocompatible polysaccharide derived from chitin and is a promising gene carrier. chitosan-dna interactions, and hence dna polyplexation and release can be controlled through chitosan de-acetylation degree, molecular weight and functionalization of chitosan cationic groups. grafting of poly(ethylene glycol) peg to gene delivery vectors increases circulation time of gene delivery systems in blood vessels and reduces polyplexes charge. diffusion and unpacking of pegylated and non-pegylated chitosan-dna polyplexes through articial ecms based on collagen and collagen-hyaluronic acid (ha) gels were compared using fluorescence correlation microscopy, confocal microscopy and colocalization analysis. non-pegylated polyplexes were immobilized in the gels whereas pegylated polyplexes were diffusing. the smaller charge of pegylated polyplexes seems to decrease interactions between polyplexes and ecm components. furthermore, ha might also screen collagen fibers-pegylated polyplexes interactions. pegylated polyplexes also showed a higher degree of unpacking in gels, probably due to a looser compaction of dna by pegylated chitosan compared to non-pegylated chitosan. fabrication of vesicles, a close membrane made of an amphiphile bilayer, has great potentiality for encapsulation and controlled release in chemical, food or biomedical industries but also from a more fundamental point of view for the design of biomimetic objects. methods based on lipid film hydratation , inverse emulsion techniques and more recently microfluidic techniques such as double emulsion or jetting method are limited either by a low yield, a low reproducibility, a poor control on the size, or by the presence of remaining solvent or defects. we propose a fast and robust method easy to implement: continuous droplet interface crossing encapsulation (cdice), that allows the production of defect-free vesicles at high-yield with a control in size and content. the vesicles have controlled bilayer composition with a polydispersity in size lower than %. we have shown that solutions as diverse as actin, cells, micrometric colloids, protein and high ionic strength solutions can easily be encapsulated using this process. by adjusting the parameters of our set-up, we are able to produce vesicles in the range - lm in diameter, stable for weeks. we believe this method open new perspectives for the design of biomimetic systems and even artificial tissues. under appropriate conditions. the extremely variable d domain of flagellin subunits, comprising residues - , protrudes at the outer surface of flagellar filaments. the d domain has no significant role in the construction of the filament structure. thus, replacement of d may offer a promising approach for insertion of heterologous proteins or domains without disturbing the self-assembly of flagellin subunits. our work aims at the construction of flagellinbased fusion proteins which preserve the polymerization ability of flagellin and maintain the functional properties of the fusion partner as well. in this work a fusion construct of flagellin and the superfolder mutant of green fluorescent protein (gfp) was created. the obtained gfp variant was highly fluorescent and capable of forming filamentous assemblies. our results imply that other proteins (enzymes, binding domains etc.) can also be endowed by polymerization ability in a similar way. this approach opens up the way for construction of multifunctional filamentous nanostructures. generation polyamidoamine (pamam) dendrimer has been shown to be highly efficient nonviral carriers in gene delivery. however, their toxicity limits their applications. in this study, to improve their characteristics as gene delivery carriers, g pamam dendrimer was modified with anti-tag nanobody through hetrobifunctional peg, then complexed with t-bid coding pdna, yielding pamam-peg-anti-tag nanobody/pdna nanoparticles (nps). nuclear magnetic resonance (nmr) spectroscopy, zeta sizing and gel retardation assay results provided evidence that the nanovector was successfully constructed. the transfection efficiency of vector/pdna complexes were evaluated in vitro. real time pcr results also demonstrated that anti-tag nanobody modified nps are more efficient in t-bid killer gene expressing in colon cancer cell line than the unmodified nps. in conclusion, pamam-peg-anti-tag nanobody showed great potential to be applied in designing tumour-targeting gene delivery system. dept of chemistry, faculty of science, national university of singapore, singapore, dept of biochemistry, yong loo lin school of medicine, national university of singapore, singapore, division of bioengineering, faculty of engineering, national university of singapore, singapore macromolecular crowding (mmc) is a biophysical tool which has been used extensively to enhance chemical reactions and biological processes by means of the excluded volume effect (eve). the in vivo stem cell microenvironment contains macromolecules which are crucial for stem cell selfrenewal and cell fate determination. in order to mimic this physiological microenvironment, crowders are included in cell culture medium. we have observed that the ex vivo differentiation of human mesenchymal stem cells (hmscs) into the adipogenic lineage is significantly amplified when a crowder mixture comprising ficoll and ficoll is added to the culture medium. stem cell differentiation is modulated by soluble chemical substances as well as interactions between cells and the extracellular matrix (ecm), and both these external influences may be affected by mmc. measurements we have performed by fluorescence correlation spectroscopy (fcs) show that ficoll additives cause anomalous subdiffusion within a crowder concentration range of to mg/ml. the diffusion of fluorophorelabelled molecules in artificial lipid bilayers and membranes of living cells is not changed by crowders, suggesting that these crowders do not directly alter membrane properties and cell surface signalling. however, we have data to suggest that crowders increase actin polymerization reaction rates in vitro. we have also observed that crowders are taken up by stem cells and that they localize to specific compartments. based upon our observations, we hypothesize that crowders can influence stem cell differentiation by influencing molecular kinetics. lignocellulose-based composites are becoming extremely important and perspective sustainable and renewable natural materials. fibre modification enhancing their existing properties can be obtained to broaden the application areas. in response to shortcomings of traditional chemical and physical methods, enzymes and chemo-enzymatic methods have emerged as eco-friendly catalysts working under mild conditions and enable tailoring of the material surface properties by substrate specificity and regional selectivity. recently, binding of different functional molecules to lignin-rich fibres by using an oxidative enzyme (e.g. laccase) has been reported leading to their functionalisation through free radical reactions. by the application of electron paramagnetic resonance spectroscopy (epr) laccase action was inspected. consumption of substrates was investigated and their polymerization traced. stable radical intermediates were detected with epr when substrate molecules were in contact with active enzymes. secondly, oxidation of mediators like nitroxides was determined via epr spectroscopy of stable water-soluble nitroxide radicals. finally, the generation of short-lived radicals as well as their reduction was measured via epr spin trapping using dmpo as sensitive water soluble spin trap. mammalian ovary hormone stimulation (ohs) is known to be an inalienable stage of reproductive biotechnology as well as human infertility treatment. the basic aim of the ohs is to receive a stock of valuable oocytes and early embryos for subsequent utilization in the reproductive technology, experimental work et al. however, it is known that ohs itself affects the character of ovulation and oocyte quality, which in its turn affects the development of embryos and even has distant consequences. the wideness of cell parameters and appropriate methods for investigation of gamete/embryo quality are very important. the aim of this study is determination of specific electric conductivity of mouse oocytes and early embryos which have been received after ohs in comparison with the ones that have been received in natural animal sex cycle. using techniques of electroporation the dependence of specific electric conductivity of mouse oocytes, zygotes, -cell and -cell embryos on the external electric field intensity has been studied. it is shown that the whole pool of oocytes that were obtained in the result of ohs consists of two groups of oocytes that don't differ from each other morphologically, but differ by their electric parameters and resistance to electric breakdown. at the zygote stage, dividing of embryos into two groups is preserved, but is less expressed. at the stage of -cell and -cell dividing of embryos into two groups on their electric conductivity disappeared but certain scattering of the parameters due to individual embryo peculiarities is observed. the obtained data show that ohs may lead to latent changes of oocyte state that in their turn affect embryo quality. many microbes synthesize and accumulate granules of polyhydroxyalkanoates (pha, biodegradable storage materials alternative to traditional plastics), which help them survive under stresses. in particular, the plant-growth-promoting rhizobacterium azospirillum brasilense, that is under investigation worldwide owing to its agricultural and biotechnological significance, can produce poly- hydroxybutyrate (phb) [ ] . in our work, phb synthesis in a. brasilense cells was studied under various stresses using diffuse reflectance ftir spectroscopy. phb in cells was determined from the band intensity ratio of the polyester m(c=o) at * cm - to that of cell proteins (amide ii band at * cm - ), showing a. brasilense to be able to produce phb up to over % of cells' dry weight. stresses induced phb accumulation, enhancing ir absorption in phb specific regions. analysis of a few structure-sensitive phb vibration bands revealed changes in the degree of intracellular phb crystallinity (related to its enzymatic digestion rate) at different stages of bacterial growth, reflecting a novel trait of the bacterial adaptability to an enhancing stress, which is of great importance to agricultural biotechnology. the aim of this work is to furnish enzymes with polymerization ability by creating fusion constructs with the polymerizable protein, flagellin, the main component of bacterial flagellar filaments. the d domain of flagellin, exposed on the surface of flagellar filaments, is formed by the hypervariable central portion of the polypeptide chain. d is not essential for filament formation. the concept in this project is to replace the d domain with suitable monomeric enzymes without adversely affecting polymerization ability, and to assemble these chimeric flagellins into tubular nanostructures. to test the feasibility of this approach, xylanase a (xyna) from b. subtilis was chosen as a model enzyme for insertion. with the help of genetic engineering, a fusion construct was created in which the d domain was replaced by xyna. the flic(xyna) chimera exhibited catalytic activity as well as polymerization ability. these results demonstrate that polymerization ability can be introduced into various proteins, and building blocks for rationally designed assembly of filamentous nanostructures can be created ( table ) . the support of the hungarian national office for research and technology and the hungarian scientific research fund (otka) (grants ck , nk , nanoflag) is acknowledged. cluster phases of membrane proteins an alternative scenario for the formation of specialized protein nano-domains (cluster phases) in biomembranes dna fragmentation induced in human fibroblasts by accelerated fe ions of differing energies swift heavy ion irradiation of srtio under grazing incidence'' proc. natl. acad. sci. usa forespore engulfment mediated by a ratchet-like mechanism a channel connecting the mother cell and forespore during bacterial endospore formation a feeding tube model for activation of a cell-specific transcription factor during sporulation in bacillus subtilis the scanning ion-conductance microscope imaging proteins in membranes of living cells by high-resolution scanning ion conductance microscopy nanoscale live-cell imaging using hopping probe ion conductance microscopy beta -adrenergic receptor redistribution in heart failure changes camp compartmentation simultaneous noncontact topography and electrochemical imaging by secm/sicm featuring ion current feedback regulation timasheff in protein-solvent interactions the roles of water in foods on motor and electrical oscillations in urinary tract: computer evaluation daniele martin , , viktor foltin , , erich gornik , rumen stainov , , tanya zlateva nü rnberg. icsd e.v. postfach (pob) , d- mü nchen method: parameters: motor patterns (guinea-pig) -frequency/f, amplitudes/a (% init. length, isot. & intracell. rec.) of spontaneous phasic/spc & tonic/stc contractions, also electrical spikes/s, bursts/b, burst plateaus/bp (neu et al. biophys.j. / a/jan stretch ( - mn), k-/ca-influence induced specific changes in motor/electrical parameters. special computer programme reflects exactly biophysical parameters. conclusion: acc. to earlier/recent results mechano-sensitive ca ?? -activated k ? -channels participate in electrical oscillations of detrusor/ureteral myocytes. further experiments/evaluations incl effect of hydrophobic mismatch on the light-induced structural changes in bacterial reaction centers s. s. deshmukh, h. akhavein, and lá szló ká lmá n department of physics mechanism of proton transfer in nitric oxide reductase: computational study andrei pisliakov riken advanced science institute, wako-shi proteins , . acknowledgments this work was supported by project grant ptdc/qui/ / (cas) and doctoral grants sfrh th anniversary conference aicr this work was supported by the italian association for cancer research (airc), the istituto toscano tumori and the associazione noi per voi differential hydration, void volume: which factor provides the main contribution to dv u ? inserm umr fr; roumestand@cbs.cnrs.fr introduction: globalization needs new organizational models also for biophysics. reports on necessity of int. institutes for biophysics (iib) c/o int. universities (proposed by british nobel laureate b.russell) are given conception: proposals for ebsa-discussion: . enlargement of executive committee by a. honorary & presidents (permanent - : moral support & - fixed term), b. interdisciplinary commission: scientists from biology, medicine, physics, etc. (feps/iups, iuphar, iupab, etc). . implication of interdisciplinary topics to esba/iupab congressprogrammes, . also for biophysical journals. . organization of common interdisciplinary sessions not only to biophysical, but also to other congresses. . co-operation between esba/iupab with int. interdisciplinary organisations (waas, icsd/ias, eur. academies) for creation of iib by network of national ones: successive common personnel, possibility for whole life work, etc. conclusion: realization of proposals .- . could increase scientific/political authority of ebsa/iupab, leading to model for renewal of scientific organizations collective migration of neural crest cells: a balance between repulsion and attraction roberto mayor university college london goodilin , , olga v single-molecule cut-and-paste surface assembly (smcp) has been also used to build up a biotin scaffold that streptavidin utilizing specific molecular interactions, for example between dna-binding proteins and dna or antibodies and antigens, this technique is capable of providing a scaffold for the controlled self-assembly of functional complexes. furthermore, this allows for the introduction of smcp into protein science. we aim to employ dna-binding zinc-finger variants and gfp-binding nanobodies as shuttle-tags fused to the proteins of interest. thus a fully expressible system that can be used for the step-wise assembly of individual building blocks to form single-molecule cut-and-paste surface assembly optically monitoring the mechanical assembly of single molecules nanoparticle self-assembly on a dna-scaffold written by single-molecule cut-and-paste torsional motion analysis of group ii chaperonin using diffracted x-ray tracking nanomechanical manipulation of mason-pfizer monkey retroviral rna fragment with optical tweezers melinda simon , zsolt má rtonfalvi , pasquale bianco , beá ta vé rtessy , mikló s kellermayer micro-viscosimeter generated and manipulated by light andrá s buzá s , lá szló oroszi , ló rá nd kelemen , pá l ormos temesvá ri krt proc. natl. acad. sci. usa neural signal recordings with a novel multisite silicon probe gergely má rton , anita pongrá cz , lá szló grand , , É va vá zsonyi pé ter pá zmá ny catholic university, faculty of information technology, h- , /a prá ter st multiscale pattern fabrication for life-science applications francesco valle , beatrice chelli , michele bianchi , eva bystrenova , marianna barbalinardo , arian shehu , tobias cramer , mauro murgia , giulia foschi miroslava kuricova , jana tulinska , aurelia liskova , eva neubauerova , maria dusinska , , ladislava wsolova acceleration neuronal precursors differentiation induced by substrate nanotopography gianluca grenci , jelena ban , elisabetta ruaro , massimo tormen , marco lazzarino , and vincent torre light-induced structural changes are reported near the primary electron donor of bacterial reaction centers (brc) dispersed in detergent micelles and in liposomes from lipids with different fatty acid chain lengths. in this study we present evidence for the correlation between the light-induced increase of the local dielectric constant, determined by the analysis of the electrochromic absorption changes, and the lifetime of the charge-separated state at physiologically relevant temperatures. the increase of the local dielectric constant induced a significant decrease of the oxidation potential of the primary electron donor and a slow proton release, which appears to be the rate limiting step in the overall process. systematic selection of the head group charges of detergents and lipids, as well as the thickness of the fatty acid chains of the liposome forming lipids can increase the lifetime of the charge-separated state by up to orders of magnitude. such extensions of the lifetime of the charge-separated state were reported earlier only at cryogenic temperatures and can provide new opportunities to utilize the brc in energy storage. ontogenesis of photosynthetic bacteria tracked by absorption and fluorescence kinetics m. kis, e. asztalos, p. maró ti department of medical physics and informatics, university of szeged, hungarythe development of photosynthetic membrane of rhodobacter sphaeroides was studied by absorption spectroscopy and fast induction of bacteriochlorophyll fluorescence in different phases of the growth, under various growing conditions (oxygen content, light intensity etc.) and in synchronous cell population. the results are: ) the newly synthesized components of the membranes were imbedded immediately into the proteinous scaffold independently on the age of the cell (no ,,transient'' membranes were observed). ) under aerobic conditions, the pigments were bleached and under anaerobic conditions the pigment systems showed greening. the relative variable fluorescence (f v /f max ) had small age-dependent (but not cellcycle-related) changes. the fluorescence induction kinetics was sensitive marker of the aerobiosis: the f v /f max ratio dropped from . to . and the photochemical rate constant from Á s - to Á s - with an apparent halftime of about - hours after change from anaerobic to aerobic atmosphere. ) the electrogenic signal (absorption change at nm) reflected the energetization of the membrane which showed cell-cycle dependent changes. that included periodic production and arrangement of protein-lipid components of the membrane synchronized to the cell division. interfacial water in b-casein molecular surfaces: wide-line nmr, relaxation and dsc characterization t. verebé lyi , m. bokor , p. kamasa , p. tompa , k. tompa research institute for solid state physics and optics, hungarian academy of sciences, pob. , budapest,hungary, institute of enzymology, biological research center, hungarian academy of sciences, pob. , budapest, hungarywide-line proton nmr fid, echoes, spin-lattice and spin-spin relaxation times were measured at . mhz frequency in the - °c to ? °c temperature range, in lyophilized bcasein and aqueous and buffered solutions, and dsc method were also applied. the motivation for the selection of b-casein is the uncertainty of structural order/disorder. naturally, the nmr and thermal characteristics were also evaluated. the melting of hydration water could be detected well below °c and the quantity of mobile water molecules (hydration) was measured. the hydration vs. melting temperature curve has informed us on the bonding character between the protein surfaces and water molecules. the generally used local field fluctuation model and the bpp theory were applied in the interpretation, and the limits of the models were concluded. the torsional properties of dna play an important role in cellular processes such as transcription, replication, and repair. to access these properties, a number of single-molecule techniques such as magnetic tweezers have been developed to apply torque to dna and coil it. i will briefly refer to investigations of dna-protein interactions using these techniques, and describe what has been learnt. i will then focus on the development of novel magnetic techniques that go beyond standard magnetic tweezers, such as the magnetic torque tweezers and the freely-orbiting magnetic tweezers . these approaches allow one to quantify conjugate variables such as twist and torque. for example, the magnetic torque tweezers rely on high-resolution tracking of the position and rotation angle of magnetic particles in a low stiffness angular clamp. we demonstrate the experimental implementation of this technique and the resolution of the angular tracking. subsequently, we employ this technique to measure the torsional stiffness c of both dsdna molecules and reca heteroduplex filaments. lastly, i will describe novel applications of the optical torque wrench , . the optical torque wrench is a laser trapping technique developed at cornell capable of applying and directly measuring torque on microscopic birefringent particles via spin momentum transfer. we have focused on the angular dynamics of the trapped birefringent particle , demonstrating its excitability in the vicinity of a critical point. this links the optical torque wrench to non-linear dynamical systems such as neuronal and cardiovascular tissues, non-linear optics and chemical reactions, which all display an excitable binary ('all-or-none') response to input perturbations. based on this dynamical feature, we devise a conceptually novel sensing technique capable of detecting single perturbation events with high signal-to-noise ratio and continuously adjustable sensitivity.for the first time we report a comparative approach based on surface enhanced raman spectroscopy (sers) and raman spectroscopy to study different types of haemoglobin molecules in living erythrocytes. in erythrocytes there are two fractions of haemoglobin: cytosolic (hb c ) and membranebound (hb m ). the concentration of hb m is less then , % and therefore it is impossible to study hb m with traditional optical techniques. modifications of cellular membrane can affect conformation of hb m . therefore, it can be used as a sensitive marker of pathologies. firstly, we investigated enhancement of sers signal of hb m depending on ag nanoparticles' size. we found that the intensity of sers spectra of hb m and enhancement factor increase with the decrease in ag nanoparticles' size. secondly, we investigated the dependence of haemoporphyrin conformation in both hb c and hb m ion ph values. we observed different sensitivity of hb c and hb m to the ph and found that conformational movements of haemoporphyrin (vibrations of pyrrol rings and side radicals) in hb m are sterically hampered comparably with hb c . our observation is an evidence of a benefit of application of surface enhanced raman spectroscopy to investigate properties of the hb m in erythrocytes and provide new information about conformational changes and functional properties of hb m . rna nanotechnology is an emerging field with high potential for nanomedicine applications. however, the prediction of rna three-dimensional nanostructure assembly is still a challenging task that requires a thorough understanding the rules that govern molecular folding on a rough energy landscape. in this work, we present a comprehensive analysis of the free energy landscape of the human mitochondrial trna lys , which possesses two different folded states in addition to the unfolded one. we have quantitatively analyzed the degree of rna tertiary structure stabilization, firstly, for different types of cations, and, secondly, for several naturally-occurring nucleotide modifications in the structural core of the trna lys . , thus, notable variations in the rna binding specificity was observed for the divalent ions of mg ? , ca ? and mn ? , that can be attributed to their sizes and coordination properties to specific ligands. furthermore, we observed that the presence of m g modification together with the principal stabilizing m a modification facilitates the rna folding into the biologically functional cloverleaf shape to a larger extent than the sum of individual contributions of these modifications. in order to elucidate the mechanism of the recognition, we used diffracted x-ray tracking method (dxt) that monitors real-time movements of individual proteins in solution at the single-molecule level. we found that peptides move distinctly from i-a k , and the rotational motions of peptides correlate with the type b t cell activation. in the case of diabetogenic i-a g , immediately after peptide exchange, all the peptides moves magnificently but the motion ceased in a week, then new ordered motion appears; the rotational motion of peptides correlate to t cell activation, which is analogous to the peptide in i-a k . the rotational motion of peptides may create transient conformation of peptide/mhc that recognized by a population of t cells. dxt measurement of peptide/mhc complex well correlated to other biological phenomenon too. our finding is the first observation that fluctuations at the level of brownian motion affect to the functions of proteins.mason-pfizer monkey virus (mpmv) is an excellent model for the analysis of retrovirus assembly and maturation. however, neither the structure of the viral rna, nor its modulation by capsid-protein binding are exactly known. to explore the structure of the mpmv genome, here we manipulated individual molecules of its packaging signal sequence with optical tweezers. the -base-long segment of mpmv rna corresponding to the packaging signal, extended on each side with -base-long indifferent gene segments for use as molecular handles, was cloned into a pet b vector. rna was synthesized in an in vitro transcription system. rna/ dna handles were obtained by hybridization in a pcr with complementary dna initiated with primers labeled with either digoxigenin or biotin. the complex was manipulated in repetitive stretch and relaxation cycles across a force range of - pn. during stretch, transition occurred which increased the rna chain length and likely corresponds to unfolding. the length gain associated with the unfolding steps distributed across three main peaks at * , , nm, corresponding to * , , bases, respectively. often reverse transitions were observed during mechanical relaxation, indicating that refolding against force proceeds in a quasi-equilibrium process. structural investigation of gpcr transmembrane signaling by use of nanobodies jan steyaert , structural biology brussels, vrije universiteit brussel, pleinlaan , brussel, belgium, department of structural biology, vib, pleinlaan , brussel, belgiumin , scientists at the vrije universiteit brussel discovered the occurence of bona fide antibodies devoid of light chains in camelidae. the small and rigid recombinant antigen binding fragments ( kd) of these heavy chain only antibodies -known as vhhs or nanobodies -proved to be unique research tools in structural biology. by rigidifying flexible regions and obscuring aggregative surfaces, nanobody complexes warrant conformationally uniform samples that are key to protein structure determination by x-ray crystallography:• nanobodies bind cryptic epitopes and lock proteins in unique native conformations • nbs increase the stability of soluble proteins and solubilized membrane proteins • nbs reduce the conformational complexity of soluble proteins and solubilized membrane proteins • nbs increase the polar surface enabling the growth of diffracting crystals • nbs allow to affinity-trap active protein i will focus my talk on the use of nbs for the structural investigation of gpcr transmembrane signaling to illustrate the power of the nanobody platform for generating diffracting quality crystals of the most challenging targets including gpcrs and their complexes with downstream signaling partners. dynamics of the type i interferon receptor assembly in the plasma membrane stephan wilmes, sara lö chte, oliver beutel, changjiang you, christian paolo richter and jacob piehler university of osnabrü ck, division of biophysics, barbarastrasse , osnabrü ck, germanytype i interferons (ifn) are key cytokines in the innate immune response and play a critical role in host defense. all ifns bind to a shared cell surface receptor comprised of two subunits, ifnar and ifnar . detailed structure-function analysis of ifns has established that the ifn-receptor interaction dynamics plays a critical role for signalling specificities.here we have explored the dynamics of receptor diffusion and ifn assembly in living cells. by using highly specific orthogonal posttranslational labelling approaches combined with tirf-microscopy we probed the spatio-temporal dynamics of receptor diffusion and interaction in the plasma membrane of live cells on the single molecule level. for this purpose, we employed posttranslational labelling with photostable organic fluorophores. this allowed us to map diffusion and lateral distribution of ifnar and ifnar with very high spatial and temporal resolution by using single particle tracking (spt) and single molecule localization imaging. observed events of ''transient confinement'' and co-localization with the membrane-proximal actin-meshwork suggest partitioning of ifnar / in specialized microcompartments. this will be investigated in terms influence on receptor assembly and recruitment of cytoplasmic effector proteins. cytoplasmic dynein moves through uncoordinated action of the aaa ring domains ahmet yildiz department of physics, and department of molecular and cell biology, university of california, berkeley, ca usa cytoplasmic dynein is a homodimeric aaa? motor that moves processively toward the microtubule minus end. the mechanism by which the two catalytic head domains interact and move relative to each other remains unresolved. by tracking the positions of both heads at nanometer resolution, we found that the heads remain widely separated and move independently along the microtubule, a mechanism different from that of kinesin and myosin. the direction and size of steps vary as a function of interhead separation. dynein processivity is maintained with only one active head, which drags its inactive partner head forward. these results challenge established views of motor processivity and show that dynein is a unique motor that moves without strictly coordinating the mechanochemical cycles of its two heads.o- self-controlled monofunctionalization of quantum dots and their applicaitons in studying protein-protein interaction in live cells changjiang you, stephan wilmes, sara loechte, oliver beutel, domenik lisse, christian paolo richter, and jacob piehler universitä t osnabrü ck, fachbereich biologie, barbarastrasse , osnabrü ck, germanyindividual proteins labeled with semiconductor nanocrystals (quantum dots, qd) greatly facilitate studying protein-protein interactions with ultrahigh spatial and temporal resolution. multiplex single molecule tracking and imaging require monovalent quantum dots (mvqd) capable of orthogonally labeling proteins with high yield. for this purpose, we prepared monovalance qd-trisnta by a chemical conjugation method. our results indicated that monovalent qd-trisnta was obtained in high yield by restricting the coupling by means of electrostatic repulsion. monovalent functionalization of the qd-trisnta was confirmed by assays in vitro and in vivo. two-color qd tracking of interferon receptors ifnar and ifnar based on mvqd-trisnta were realized on live cell [ ] .to broaden the multiplex toolbox of mvqds, we extended the electrostatic-repulsion induced self-control concept for mono-functionalizing quantum dots with different affinity moieties. as a first instance, we used negatively-charged biotin peptide to produce qd with biotin mono-functionalization. we confirmed our approach was a general method to rend qd monovalent by single molecule assays based on stepwise photobleaching. these mvqds facilitate obtaining spatiotemporal information of ifnars' organization in live cells. by orthogonal labeling u a cells stably expressing ifnar at low level with biotin mvqd and mvqd-trisnta-ifn, we verified colocalization and colocomotion of individual ifn and ifnar at minute scale. combined with super-resolution imaging of ifnars' cytosolic effecter stat , we observed the dynamic coming-and-going contact between the microcompartments of ifnar and stat .a micron sized viscometer was fabricated using the couette type geometry that is capable of measuring the complex viscosity of fluids. the viscometer was produced by two photon polymerization of su photopolymer using a femtosecond laser system, a high na objective and a piezo translator stage. the viscometer was manipulated by holographic optical tweezers and operated in the . - hz frequency range. video analysis algorithm was used to evaluate our measurements. we tested the viscometer with water-glycerol solutions. one of the main reasons for lack of reliability in protein analysis for disease diagnostics or monitoring is a lack of test sensitivity. this is because, for many tests, to be reliable, they need to be performed on a homogeneous, and therefore very small, sample. current in-vitro techniques fail in accurately identifying small differences in protein content, function and interactions starting from samples constituted of few or even single cells. a nanotechnology approach may overcome the current limits in low abundance protein detection. we aim at designing a microwell device for the trapping (in native environment) and the parallel characterization of rare cells (e.g. adult stem cells). such versatile device, based on soft and nanolithography, will promote cell adhesion and viability on differently functionalized bio-compatible materials, allowing for the morphological characterization of the cells, at a single cell level. in parallel, by facing our microwell device with a protein nanoarray, produced via atomic force microscopy nanolithography, we can run proteomic studies at a single/few cells level. moreover, we could foresee the possibility to deliver different stimuli to each cell, correlating the changes in chemistry/ morphology with the protein profile at a single cell level. using an electrophysiological assay the activity of nhaa was tested in a wide ph range from ph . to . . forward and reverse transport directions were investigated at zero membrane potential using preparations with inside out and right side out oriented transporters with na ? or h ? gradients as the driving force. under symmetrical ph conditions with a na ? gradient for activation, both the wt and the ph-shifted g s variant exhibit highly symmetrical transport activity with bell shaped ph dependencies, but the optimal ph was shifted . ph units to the acidic range in the variant. in both strains the ph dependence was associated with a systematic increase of the k m for na ? at acidic ph. under symmetrical na ? concentration with a ph gradient for nhaa activation an unexpected novel characteristic of the antiporter was revealed; rather than being down regulated it remained active even at ph as low as . these data allowed to advance a transport mechanism based on competing na ? and h ? binding to a common transport site and to develop a kinetic model quantitatively explaining the experimental results. in support of these results both alkaline ph and na ? induce the conformational change of nhaa associated with nhaa cation translocation as demonstrated here by trypsin digestion. furthermore, na ? translocation was found to be associated with the displacement of a negative charge. in conclusion, the electrophysiological assay allowed to reveal the mechanism of nhaa antiport and sheds new light on the concept of nhaa ph regulation. swimming motility is widespread among bacteria. however, in confined or structured habitats bacteria often come in contact with solid surfaces which has an effect on the swimming characteristics. we used microfabrication technology to quantitatively study the interaction of swimming cells with solid boundaries. we tracked bacteria near surfaces with various engineered topologies, including flat and curved shapes. we were able to study several surface related phenomena such as hydrodynamic trapping and correlated motion. we think that our results may help to understand how physical effects play a role in surface related biological processes involving bacteria such as biofilm formation. cell labeling efficiency of oppositely charged magnetic iron oxide nanoparticles-a comparative study raimo hartmann , christoph schweiger , feng zhang , wolfgang. j. parak , thomas kissel ,# , pilar rivera_gil ,# biophotonics, institute of physics, philipps university of marburg, pharmaceutical technology, institute of pharmacy, philipps university of marburg e-mail: kissel@staff.uni-marburg.de; pilar.riveragil@physik.uni-marburg.dethe interaction of nanomaterials with cells is a key factor when considering their translocation into clinical applications. especially an effective accumulation of nanoparticles inside certain tissues is beneficial for a great number of applications. predominantly size, shape and surface charge of nanoparticles influence their cellular internalization and distribution. to investigate this, two series of maghemite (c-fe o ) nanoparticles were synthesized either via aqueous coprecipitation or via thermal decomposition of organometallic precursor molecules. size and the spherical shape of both nanoparticle types were kept constant whereas the charge was changed by modifying the surface of the nanoparticles with polymers of opposite charge, in detail poly(ethylene imine) (pei) and a polymaleic anhydride derivative (pma). the positively and negatively charged c-fe o nanoparticles were characterized with respect to size, zeta potential, colloidal stability and magnetic properties. furthermore, the uptake rate and localization of both formulations into a carcinoma cells after fluorescent labeling of the carriers as well as the resulting alteration in mr-relaxation times were evaluated. membrane proteins are the target of more than % of all drugs and are encoded by about % of the human genome. electrophysiological techniques, like patch-clamp, unravelled many functional aspects of membrane proteins but usually suffer from poor structural sensitivity. we have developed surface enhanced infrared difference absorption spectroscopy (seidas) , to probe potential-induced structural changes of a protein on the level of a monolayer. a novel concept is introduced to incorporate membrane proteins into solid supported lipid bilayers in an orientated manner via the affinity of the his-tag to the ni-nta terminated gold surface . full functionality of surface-tethered cytochrome c oxidase is demonstrated by cyclic voltammetry after binding of the natural electron donor cytochrome c. general applicability of the methodological approach is shown by tethering photosystem ii to the gold surface . in conjunction with hydrogenase, the basis is set towards a biomimetic system for h -production. recently, we succeeded to record ir difference spectra of a monolayer of sensory rhodopsin ii under voltage-clamp conditions . this approach opens an avenue towards mechanistic studies of voltage-gated ion channels with unprecedented structural and temporal sensitivity. initial vibrational studies on the novel light-gated channelrhodopsin- will be presented . probing biomass-chromatographic bead interactions by afm force spectroscopy gesa helms, marcelo ferná ndez-lahore, rami reddy vennapusa, and jü rgen fritz school of engineering and science, jacobs university bremen, bremen, germany e-mail: g.helms@jacobs-university.dein expanded bed adsorption (eba), bioproducts are purified from an unclarified fermentation broth by their adsorption on chromatographic beads in a fluidized bed. the unspecific deposition of biomass onto the adsorbent matrix can severely affect the process performance, leading to a poor system hydrodynamics which then decreases the success of this unit operation. to quantify the bead-biomass interactions different chromatographic beads are attached to afm cantilevers, and force spectroscopy experiments are performed with these colloidal probes on model surfaces and cells in solution. the experiments are conducted under varying conditions to study uncovering physiological processes at the cellular level is essential in order to study complex brain mechanisms. using multisite signal recording techniques in the extracellular space, functional connectivity between different brain areas can be revealed. a novel microfabrication process flow, based on the combination of wet chemical etching methods was developed, which yields highly reproducible and mechanically robust silicon-based multielectrode devices. the fabricated shaft of the probe is lm wide, lm thick, has rounded edges and ends in a yacht-bow like, sharp tip. its unique shape provides decreased invasivity. the sensor contains platinum recording sites at precisely defined locations. murine in vivo experiments showed that the probes could easily penetrate the meninges. high quality signals, providing local field potential, multi-and single unit activities, were recorded. the interfaces between the tissue and the platinum contacts were further improved by electrochemical etching and carbon nanotube coating of the metal sites. the integrated optical mach-zehnder interferometer is a highly sensitive device, considered a powerful lab-on-a-chip tool for specific detection of various chemical and biochemical reactions. despite its advantages, there is no commercially available biosensor based on this technique. the main reason is the inherent instability of the device due to slight changes of environmental parameters. in this paper we offer a solution to this problem that enables the optimal adjustment of the working point of the sensor prior to the measurement. the key feature is a control unit made of a thin film of the lightsensitive chromoprotein bacteriorhodopsin deposited on the reference arm of the interferometer. after showing the transfer characteristics of such a device, we demonstrate its applicability to sensing of specific protein-protein interactions. we expect our method to become a rapid and cost-efficientthe combination of unconventional fabrication technology and biomaterials allows both to realize state-of-the-art devices with highly controlled lateral features and performances and to study the main properties of the biomolecules themselves by operating at a scale level comparable with the one crucial for their activity. soft lithography and microfluidic devices offer a tool-box both to study biomolecules under highly confined environments [ ] and to fabricate in an easy way topographic features with locally controlled mechanical and chemical surface properties, thus leading to a finer control of the interplay of mechanics and chemistry. i will present an application of this technology to the control of cell fate that is becoming a key issue in regenerative medicine in the perspective of generating novel artificial tissues. patterns of extracellular matrix (ecm) proteins have been fabricated, by a modified lithographically controlled wetting (lcw), on the highly antifouling surface of teflon-af to guide the adhesion, growth and differentiation of neural cells (shsy y, n , ne- c) achieving an extremely accurate guidance [ ] . local surface topography is also known to influence the cell fate [ ] , thus, integrating this parameter in the substrate fabrication could increase the complexity of the signals supplied to the cells. in this perspective we have developed a novel fabrication technique, named lithographically controlled etching (lce), allowing, in one step, to engrave and to functionalize the substrate surface over different lengthscales and with different functionalities. i will conclude showing how we have been developing ultrathin film organic field effect transistors (ofets) as label-free biological transducers and sensors of biological systems. ofets are low dimensional devices where ordered conjugated molecules act as charge transport material. unconventional patterning techniques and microfluidics have been adapted to proteins and nucleic acids to dose the molecules on the ofet channel with a high control of the concentration. in another set of experiments, we have also been addressing the signalling from neural cells and networks grown on pentacene ultra-thin film transistosr [ , ] .advances in nanotechnology are beginning to exert a significant impact in medicine. increasing use of nanomaterials in treatment of diseases has raised concerns about their potential risks to human health. in our study, the effect of poly(lactic-co-glycolic acid) (plga) and titanium dioxide (tio ) nanoparticles (nps) on function of b-and t-lymphocytes was investigated in vitro. human blood cultures were treated with plga and tio nps in concentrations: . ; and lg/cm for h. lymphocyte transformation assay was used to assess the effect of nps on lymphocyte function. lymphocytes were stimulated with mitogens: concanavalin a, phytohaemmagglutinin (t-cell response) and pokeweed mitogen (b-cell response). our findings indicate immunomodulatory effect of plga nps. proliferative response of t-and b-lymphocytes exposed in vitro to the highest dose of plga for h was suppressed significantly (p. , p. ). on the other hand, we observed stimulative effect of exposure to middle dose of plga nps on b-lymphocyte proliferation (p. ). no alteration was found in lymphocyte proliferation treated in vitro with tio nps for h. in conclusion, proliferation of lymphocytes in vitro might be one of the relevant tests for evaluation of nps immunotoxicity.embryonic stem (es) cell differentiation in specific cell lineage is still a major challenge in regenerative medicine. differentiation is usually achieved by using biochemical factors (bf) which concentration and sides effects are not completely understood. therefore, we produced patterns in polydimethylsiloxane (pdms) consisting of groove and pillar arrays of sub-micrometric lateral resolution as substrates for cell cultures. we analyzed the effect of different nanostructures on differentiation of es-derived neuronal precursors into neuronal lineage without adding biochemical factors. neuronal precursors adhere on pdms more effectively than on glass coverslips but the elastomeric material itself doesn't enhance neuronal differentiation. nano-pillars increase both precursors differentiation and survival with respect to grooves. we demonstrated that neuronal yield was enhanced by increasing pillars height from to nm. on higher pillar neuronal differentiation reaches * % hours after plating and the largest differentiation enhancement of pillars over flat pdms was observed during the first hours of culture. we conclude that pdms nanopillars accelerate and increase neuronal differentiation. key: cord- -dw txm authors: wolf, michael w; reichl, udo title: downstream processing of cell culture-derived virus particles date: - - journal: expert rev vaccines doi: . /erv. . sha: doc_id: cord_uid: dw txm manufacturing of cell culture-derived virus particles for vaccination and gene therapy is a rapidly growing field in the biopharmaceutical industry. the process involves a number of complex tasks and unit operations ranging from selection of host cells and virus strains for the cultivation in bioreactors to the purification and formulation of the final product. for the majority of cell culture-derived products, efforts focused on maximization of bioreactor yields, whereas design and optimization of downstream processes were often neglected. owing to this biased focus, downstream procedures today often constitute a bottleneck in various manufacturing processes and account for the majority of the overall production costs. for efficient production methods, particularly in sight of constantly increasing economic pressure within human healthcare systems, highly productive downstream schemes have to be developed. here, we discuss unit operations and downstream trains to purify virus particles for use as vaccines and vectors for gene therapy. production procedures for viral vaccines and gene therapy vectors. one striking example is the development of cell culture-derived influenza vaccines. while conventional production processes rely on egg-based systems, optimized cell culture systems are currently being established to cope with sudden demands for pandemic vaccines and increasing supply of seasonal vaccines. with advances in upstream procedures to increase yields and harvest volumes for influenza vaccines, as well as for other vaccines and viral vectors, downstream processing (dsp) is becoming an important factor in the race for higher overall productivity and decreased cost of goods. the general aim of dsp is the recovery and purification of biological products from process-and product-related impurities. process-related impurities might originate from cell culture reagents and additives (e.g., antibiotics, bovine serum albumin and benzonase ® [merck kgaa, darmstadt, germany]), from the purification process (e.g., extractables and leachables in chromatography), or from the cell substrate (e.g., host cell protein, nucleic acids, proteoglycans and glycosaminoglycans). examples for virus particle-related impurities include free envelope proteins, virus aggregates or empty capsids, and virus particles that contain nucleic acid sequences other than the intended virus particles are currently used for medical [ ] [ ] [ ] [ ] [ ] , analytical and scientific applications [ ] [ ] [ ] [ ] , and as bioinsecticides [ , ] . recently, medical applications are gaining an increasing interest owing to the growing markets for viral vaccines (table ) and the potential broad usage of viral gene therapy vectors. vaccines, administered to prevent or treat viral diseases, are mainly based on attenuated or killed viruses, membrane fractions derived from purified virus particles, or recombinant viral proteins expressed in various hosts. examples of successful attenuated or killed virus vaccines are influenza, measles, mumps, rubella, rotavirus, yellow fever and varicella [ , , , ] . gene therapy involves the transfer of genetic information to cells or tissues of individuals to achieve a therapeutic effect [ ] . therefore, required genes are largely delivered by viral vector systems based, for example, on the herpes simplex virus, adenovirus, adeno-associated virus (aav), retrovirus (e.g., lentivirus) and vaccinia virus [ , ] . currently, numerous clinical gene therapy trials are conducted to investigate the treatment of diseases such as cancer, cystic fibrosis, alzheimer's, parkinson's, hemophilia and hiv/aids [ , , ] . the broad spectrum of these applications and the current expansions of medical markets underline the ongoing efforts to improve review genome [ ] [ ] [ ] . naturally, the requirements on product purity and product safety depend on the particular application. vaccines and viral vectors need to meet the stringent guidelines of regulatory authorities such as the us fda and ema. viruses are complex bioparticles with varying size, shape, composition and surface structure. the virus surface defines their individual physicochemical characteristics including number and distribution of charges, hydrophobic residues and post-translational modifications (i.e., glycans) of surface proteins. purification of virus particles based on these unique characteristics and removal of contaminants according to the regulatory guidelines can only be achieved by a combination of different unit operations. this article provides an overview of individual unit operations currently used for dsp of viral vaccines and gene therapy vectors. this includes methods such as precipitation, flocculation, extraction, centrifugation, microfiltration, ultrafiltration, bead-based and membrane-based chromatography, and the use of monoliths. in addition, it addresses issues concerning the use of continuous chromatography methods, that is, simulated moving bed chromatography, and the utilization of kits for small-and medium-scale purifications and concentrations of virus particles and vectors for gene therapy. finally, examples for complete purification trains are presented for dsp of virus particles for therapeutic applications and vaccine manufacturing. owing to the complexity of large bioparticles and the necessity to maintain the specific immunogenicity and infectivity of virus particles and viral vectors, precipitation and flocculation are rarely used in dsp. even for purification of recombinant proteins, use of these methods is considered problematic owing to potential losses in biological activity. however, the latest advances in upstream processes for recombinant proteins in terms of high cell density cultures and drastically improved product levels triggered the re-evaluation of these methods, particularly for purification of monoclonal antibodies [ , ] turkey coronavirus was precipitated with ammonium sulfate but with low recoveries compared with ultracentrifugation ( % sucrose cushion) [ ] . other virus particles were precipitated with varying combinations of polyethylene glycol (peg) and sodium chloride. in particular, peg precipitation of bovine rotavirus particles resulted in an approximately tenfold better yield than pelleting by high-speed centrifugation based on results determined by the tissue culture infective dose (tcid ) assay [ ] . this could be owing to increased damage of virus particles during high-speed centrifugation [ ] or an irreversible aggregation of virus particles leading to overall reduced tcid measurements. furthermore, precipitation of aav vectors via peg can lead to an enhanced purity compared with the classically conducted cesium chloride (cscl) gradient ultracentrifugation [ ] . an alternative to the precipitation of the target virus is the removal of contaminating host cell dna or protein from crude culture harvests. a recent study focusing on the precipitation of host cell dna from a clarified cultivation broth of influenza virus particles has been conducted by kröber et al. [ ] . they investigated cationic reagents with respect to their ability to selectively precipitate host cell dna and observed successful dna reduction applying the cationic polymers protamine sulfate and polyethyleneimine [ ] . however, as discussed by the authors, the acidic isoelectric point of influenza virus particles (table ) may have resulted in co-precipitation of virus particles with cationic polymers [ ] , and therefore product losses. in the past effective purification of encephalomyocarditis virus from cellular components [ ] , and the removal of cellular dna from poliovirus produced in continuous cell lines [ ] as well as from preparations of inactivated vaccines against tick-borne encephalitis [ ] by protamine sulfate precipitations was demonstrated. considering the continuous efforts to increase harvest volumes and cell concentration in vaccine and gene therapy vector production, precipitation represents an interesting approach for reduction of the high loads of host cell dna and proteins. however, process robustness (i.e., the specificity of the applied precipitants) needs to be improved, and nontoxic compounds for precipitation processes need to be identified for economic applications in pharmaceutical production. [ , ] hepatitis c virus flaviviridae - na spherical yes (+)ssrna [ ] herpes simplex virus yes dsdna [ , ] influenza virus orthomyxoviridae - (h n ) a /singapore/ : . pleomorphic, spherical yes (-)ssrna [ ] [ ] [ ] [ ] [ ] [ ] [ ] (h n ) a/pol/l/ : . - . [ , ] retroviridae - na spherical yes (+)ssrna [ ] papillomavirus papillomaviridae - papillomavirus: . icosahedral no dsdna [ , , ] poliovirus picornaviridae - pv- : . and . icosahedral no (+)ssrna as for precipitation, the system's complexity, which is mainly dependent on the structural and physicochemical diversity of virus particles, constrains this technique to become widely used. further drawbacks of applying extraction methods for the purification of virus particles are the possible losses in viral infectivity, immunogenicity or transduction efficiency, high levels of co-extracted contaminants and the high cost for recycling of the utilized organic solvents [ , ] . nevertheless, extractions via aqueous two-phase systems with peg, dextran, salt or polyvenyl alcohol are used for the separation of virus particles from cultivation medium. examples for these applications are the purification or concentration of adenovirus [ ] , bovine leukemia virus [ ] , feline leukemia virus [ ] , and human and simian immunodeficiency virus [ ] . the number of publications [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and patents [ ] [ ] [ ] describing the purification or concentration of virus particles by centrifugation methods demonstrates that these procedures are extensively used at industrial-and small-scale levels for viral vectors and vaccine production processes. for industrial scales only continuous flow centrifugation methods can be considered as they allow handling of larger volumes. however, these methods require high investment costs and suffer in some cases from losses of infectivity [ , , ] , leading to an increased usage of ultrafiltration techniques particularly outside of laboratory scales. a clear advantage of centrifigation methods is their potential to separate some assembled viral vectors from their empty capsids, which is commonly challenging with other separation techniques [ ] . another advantage is in the dsp of viruses with frequently changing strains (e.g., influenza) as these centrifugation methods are robust in relation to viral strain differences, which resulted in a rediscovery of this technology for a number of processes. the most common centrifugation method for the purification of macromolecules and virus particles is density gradient centrifugation using cscl, iodixanol, or sucrose gradients. these can be classified into two categories: rate-zonal separation and isopycnic separation. the latter is based on the buoyant density of the particle and the rate zonal separation on the particle size and mass. examples of virus particles that were purified by gradient centrifugation techniques are feline leukemia virus [ ] , gibbon ape lymphoma virus [ ] , hepatitis b virus [ ] , influenza virus [ ] , japanese encephalitis virus [ ] , mammalian type c virus [ ] , mouse mammary tumor virus [ ] , mouse oncorna virus [ ] , mumps virus [ ] , murine leukemia virus [ ] , tick-borne encephalitis virus [ ] , turkey coronavirus [ ], rabies virus [ ] and vaccinia virus [ ] . an alternative centrifugation method is the differential centrifugation where pelleting (epstein-barr virus [ ] and hepatitis a virus [ ] ) or simple sedimentation onto a sucrose cushion (moloney murine sarcoma virus [ ] ) is done once or repeatedly at different centrifugation speeds. current examples for the concentration or purification of virus particles via centrifugation are: adenovirus and aav [ , ] , bromovirus [ ], hepatitis c virus [ ] , herpes-like virus [ ] , retrovirus [ ] [ ] [ ] , rotavirus [ ] and vaccinia virus [ ] . microfiltration is commonly used for clarification in biotechnological production processes as an alternative to centrifugation to remove microcarriers, producer cells, cell debris and organelles. no dsrna [ , ] rubella virus togaviridae (rubivirus) - na quasispherical yes (+)ssrna [ , ] semliki forest virus [ , [ ] [ ] [ ] [ ] [ ] [ ] connaught: . lister: . lister: . lister (egg): . lister (rabbit): . wr: . yes dsdna [ ] † data on isoelectric points of different viruses taken from michen and graule [ ] . ‡ this is a brick-shaped viral particle therefore the dimension are shown in terms of axis x-y-z. d: diameter; l: length; na: not available. [ ] kda kda tangential flow filtration (millipore; pellicon ii filter module) polyethersulfone; flat sheet kda not provided [ ] tangential flow filtration (millipore; ultra- centrifugal filter unit) regenerated cellulose; flat sheet kda [ ] tangential flow filtration (millipore; pellicon ii filter module) polyethersulfone; flat sheet kda not provided [ ] kda not provided tangential flow filtration (individual setup) polysulfone (ge-healthcare) kda [ ] polysulfone frequently, membranes with pore sizes in the range of . - µm are applied, although this technique involves the risk of viral losses [ , , ] . recoveries depend on the initial membrane pore size, type of membrane (material), quality of the crude stock, virus (i.e., size), virus subtype and its aggregation behavior, applied buffer conditions, filtration rate, nature and level of protein concentration in the filtrate, and extent of pore obstruction as demonstrated for retroviral vectors [ ] . virus losses are mainly attributed to mechanical disruption, particularly for active virus particles (i.e., gene therapy vectors), exclusion of virus particles [ ] and membrane entrapment as well as unspecific virus adsorption. losses due to pore obstruction can be reduced by applying membrane cascades with decreasing pore sizes or filter capsules with dual membranes to minimize membrane fouling [ , , ] and applying larger membrane areas (i.e., frequent changes of the filtration units or usage of larger units) to limit the volume of supernatant per membrane surface. ultrafiltration is one of the preferred methods for virus removal in biological production methods [ ] [ ] [ ] and for virus concentration and buffer exchanges in vaccine and viral gene transfer vector manufacturing processes (table ). here, virus particles are usually enriched or maintained in the retentate while water and small-molecular-weight molecules are removed with the permeate. although ultrafiltration has a low resolution, the size difference of virus particles (table ) compared with soluble biopolymers (i.e., nucleic acids and proteins) allows a relatively efficient and economic separation of the virus. the key parameters for an ultrafiltration process are the transmembrane pressure (tmp), feed (retentate) and permeate flux as well as the nominal molecular weight limit, also sometimes referred to as molecular weight cutoff (mwco). pore sizes defining the mwco are normally distributed about the mean pore size, which varies depending on the production method for the membrane, and therefore also between the manufacturers. in order to minimize product losses via the permeate, the mwco has to be significantly smaller than the virus particle. however, if the mwco selected is too small, permeate fluxes are reduced,, which leads to longer filtration times, increased tmp and decreases the possibility of reducing the amount of contaminating proteins and nucleic acids. furthermore, membrane fouling, which decreases the permeate flux, can often be greater for larger pore size membranes than for smaller ones. here, small virus particles but also cell and virus review debris are able to enter the pores where they are eventually trapped in constricted channels. asymmetric ultrafiltration membranes with the finer filtering surface facing the feed suspension should be applied where possible to reduce internal fouling. important for concentration of active virus particles is a gentle processing for which tmp and retentate flux have to be optimized for low wall shear rates during filtration. the filtration performance and in particular the permeate flux is heavily affected by the type or components of the cultivation medium, thereby underlining the importance of a tight interaction between the up-and down-stream development. nowadays, serum containing growth media are rarely used for production processes. serum-free media often contain defined proteins such as bovine albumin and insulin, but also protein hydrolysates. protein-free media only include defined polypeptides and amino acids. the higher the concentration of proteins, target virus, and nucleic acids and the larger the size of contaminating dna fragments in the retentate, the higher the sample viscosity, leading to a continuously decreasing permeate flux at constant tmp. additionally, high retentate protein concentrations result in an increased degree of concentration polarization, which also reduces the permeate flux [ ] . finally, ionic strength, type of ions and the ph in the suspending buffer influence the sieving coefficient of proteins [ ] [ ] [ ] [ ] and probably also macromolecular biomolecules and virus particles. ultrafiltration can be carried out by a wide variety of filtration devices (table ). for small scales (up to ml) centrifugal filtration devices are well suited, and for small to medium volumes ( - ml) stirred cell tanks are ideal. larger volumes are usually concentrated by tangential flow filtration. there are three types of membranes available: tubular membranes, flat sheets (cassettes) and hollow-fiber membranes from which the latter two are commonly used in the biopharmaceutical industry. generally, flat-sheet and tubular tangential-flow membrane modules have higher mass transfer coefficients at low cross-flow rates compared with hollowfiber modules. the latter have wide feed flow paths, providing laminar flow with low shear rates [ ] , which are crucial processing life vaccines and gene therapy vectors. high shear rates can significantly reduce their efficacy and overall yield. a general advantage of hollow-fiber membrane modules compared with flat sheets is the high membrane surface-to-module volume ratio. however, individual modules of flat-sheet tangential-flow membrane modules are easier to clean and replacement of defective membrane elements is uncomplicated. overall, the type of membranes eventually used for a specific process depends largely on the viscosity, solid content and volume of the feed as well as the product stability (shear-sensitivity). thus, for the concentration or diafiltration of active virus particles, hollow-fiber membranes are superior to flat-sheet membranes. a potential improvement of the classical tangential-flow filtration is the high-performance tangential-flow filtration, which achieves a superior separation factor [ , ] . in high-performance tangential-flow filtration, part of the permeate is circulated cocurrent to the feed on the permeate side of the module, resulting in a more constant tmp throughout the module due to an axial pressure drop along the permeate flow channel [ ] . this allows, by careful optimization of the operating conditions, a more stable transmembrane flux over time and in some cases leads to an improved selectivity for the purification of virus particles (table ). in summary, the main advantages of ultrafiltration compared with other methods are their high-throughput and (for the concentration of active virus particles) the gentle processing at optimal operating conditions [ , ] that results in improved efficacies for purification of viral vectors for gene therapy. another advantage of utilizing size differences of virus particles to other soluble components in the culture medium for their purification and concentration is its broad applicability among different types of viruses, virus subtypes or different recombinant forms of a virus. this opens the path for the establishment of generic platform technologies. considering a complete purification train for the production of vaccines or gene therapy vectors (figure ) , current improvements of the dynamic binding capacities in chromatography media might facilitate the removal of the initial concentration step within the downstream process. however, later concentration steps and buffer exchanges for final formulations will continue to be done via ultrafiltration. chromatography is the most popular methodology for largescale purification of vaccines and viral gene therapy vectors. chromatographic separation is based on differences in the interaction of target virus and other components to the applied stationary phase. the specific characteristics of the virus particles exploited are described by the physicochemical properties of the outer particle surface, electrostatic properties of the whole particle, or their large hydrodynamic volume compared with other soluble process components. there are four main points of chromatography that have to be considered for a successful purification [ ] : • physical structure of the stationary phase including the morphology (i.e., beads, membranes and monoliths), pore size, porosity, mechanical and chemical properties (e.g., resistance to pressure and surface charge of the mobile phase), and specificity for resin particle size and particle distribution; • surface chemistry of the stationary phase and the composition of the mobile phase; • mode of operation such as adsorption or partitioning, type of elution; • column/housing and system hardware. for each application a balance between these individual aspects must be found to obtain an efficient chromatographic procedure. in the following sections, however, we will focus on the first two points. chromatographic purification of virus particles is mainly achieved by use of three different types of stationary phases: packed beds, membrane adsorbers and monoliths. in the case of packed beds the matrix consists of resins (beads), which are filled into a column. for bioseparations polymer and inorganic resin materials are commonly used. polymer-based beads either consist of natural polymers (agarose, agarose-dextran composites, cellulose and dextran) or synthetic polymers (polyacrylamide, polystyrene divinylbenzene, derivatives of polyacrylamide derivatives and polymethacrylate) [ ] . examples for inorganic matrices used for the purification of virus particles are hydroxyapatite [ ] [ ] [ ] , silica [ ] and controlled-pore glass [ , ] . beads can be spherical and nonspherical shaped and are available as porous, nonporous and solidcore resins. pore sizes of conventional porous beads range from to nm [ ] , although specific particles with pore diameters of up to nm are available [ ] . virus particles vary generally from nm to greater than nm (table ) . hence, the material transport of virus particles is limited by pore-diffusion or pore-exclusion effects. even small virus particles, which are able to enter large pores, diffuse through the intra-particular channels two-to -fold slower than proteins [ ] . in fact, the majority of chromatography resins currently available are optimized for purification of proteins rather than virus particles, which primarily adsorb to the outer bead surface [ ] . furthermore, the inter-particle porosity for packed beds can reach values of maximally %, whereas for stationary phases such as monoliths this value can be up to % or more [ ] . monoliths are continuous stationary phases of homologous columns, where interconnecting channels allow for convective flow of the mobile phase through the entire matrix [ , ] . commercially available monoliths are made of polymethacrylate copolymers (bia separations, ljubljana, slovenia; dionex sunnyvale, ca, usa), polyacrylamide (bio-rad, hercules, ca, usa; protista biotechnology ab, lund, sweden), polystyrene (dionex, sunnyvale, ca, usa), polystyrene-divinylbenzene (dionex, sunnyvale, ca, usa), modified cellulose (sepragen, hayward, ca, usa) and silica (merck, darmstadt, germany; phenomenex, torrence, ca, usa) [ , ] . depending on the manufacturer they are sold as disks, tubes and rods with pore sizes ranging from nm (silica, merck) [ ] to µm (polymethacrylate, bia separations). membrane materials for chromatographic bioseparations include cellulose, polyamine, polysulfone, hydrazide and composite membranes such as polyethylene oxide and polyethersulfone coated with hydroxyethyl-cellulose [ , ] . there are three types of chromatographic membrane adsorbers used for the purification of bioproducts: flat sheets (e.g., pall, dreieich, germany; sartorius, göttingen, germany), hollow fibers (e.g., kinetic systems inc., boston, ma, usa) and radial-flow devices (e.g., m, neuss, germany) [ , ] . the advantages of hollow-fiber membrane adsorbers are the high surface area and the reduction in particle accumulation near the pore entrance owing to the cross-flow principle used during separation. however, the breakthrough of target compounds is generally broadened, leading to poor adsorber utilization compared with flat sheets [ ] . furthermore, eluted products are heavily diluted and internal mixing within the fiber housing results in low resolutions, and the application of gradient elution is very limited. radial-flow adsorbers have flat-sheet membranes spirally wound over a cylindrical core [ ] whereby the available surface area can be significantly increased. thus, the volume-to-surface area for spiral-wound membrane adsorbers is better than for the majority of hollow-fiber adsorbers. still, products are diluted and gradient elutions are accordingly challenging. flat sheets are mainly used as stacks of numerous individual sheets providing a larger adsorbent volume. in flat-sheet membrane adsorbers the liquid is commonly introduced vertical to the membrane adsorber surface, allowing a minimum dead volume in an optimally designed membrane module. thus, these membrane adsorbers do not suffer from diluted product fractions and can be used for gradient elution procedures. the main advantages of membrane adsorbers and monoliths for the separation of virus particles are the predominance of convective material transport with nearly no diffusion limitations, [ , , [ ] [ ] [ ] [ ] owing to large pores, and well-interconnected channels with small constrictions and high dynamic binding capacities [ , , ] , whereby the latter is sometimes lower for membrane adsorbers than for comparable monoliths [ ] . the pore structure of membrane adsorbers and monoliths facilitates the accessibility of large virus particles to bound ligands in contrast to typical resins where they are excluded from the internal bead pores [ ] . except for monoliths, eddies are formed in the majority of chromato graphic matrices including membrane adsorbers. as a result, labile virus particles can be deteriorated and separation impaired by peak broadening. stacks of membrane adsorbers can ac (sulfated cellulose) sulfated cellulose ma - [ ] ac sartobind zn-ida ma [ ] ac different ligands immobilized to monoliths ¶ not provided [ ] parvoviridae (aedes aegypti densonucleosis virus) sartobind q, d sartobind s, c not provided [ , ] parvoviridae (adeno-associated virus, aav -gfp) [ ] retroviridae (vector; momlv-derived vsv-g-pseudotyped retroviral vector) ac fractogel emd heparin (s) [ ] filtration, uf, df, ac, sec fractogel emd heparin (s) sepharose cl- b retroviridae (vector; momlv-derived vsv-g-pseudotyped retroviral vector) ac fractogel emd heparin (s) [ ] retroviridae (vector; momlv-derived rd -pseudotyped retroviral vector) ac fractogel emd heparin (s) [ ] retroviridae (vector; vrx a vsv-g pseudotyped human immunodeficiency virus type -derived vector) aec mustang q [ ] sec sephacryl s - retroviridae (vector; vsv-g-pseudotyped retroviral vector) sec sepharose cl- b [ ] uf, sec, uf/df sepharose cl- b retroviridae (vector; his -tagged retroviral vector) ni-nta agarose [ ] retroviridae (vector; biotin-tagged retroviral vector) ac fractogel emd streptavidin ‡ ‡ [ ] steptavidin-monolith § § [ ] retroviridae ( review be considered as very thin slices of monoliths. pores between membrane layers are connected by a void space between individual membrane slices, leading potentially to eddy formations. flow distributions in hollow-fiber and radial-flow membrane adsorbers are less well controlled compared with either monoliths or packedbed columns. in addition to eddy formations this could result in reduced dynamic binding capacities and dispersions. the latter does not only affect negative-mode adsorption chromatography, but also reduce the resolution of positive-mode applications. the majority of membrane adsorbers are designed for singleuse applications, supporting current efforts in the pharmaceutical industry to reduce costs for validation, cleaning and sanitization. monoliths are currently following this trend. for beads, however, it remains to be seen if disposable approaches can be realized economically. nevertheless, a wider diversity of adsorptive surface chemistries is currently available for resins than for convective alternatives, but the product range for membrane-based separations constantly expands. currently, five different modes are used for the chromatographic separation of virus particles: size-exclusion chromatography (sec), ion exchange chromatography (iec), affinity chromatography (ac), hydrophobic-interaction chromatography (hic) and mixed-mode chromatography (table ). the action principles and possibilities for process optimization are limited by the stability of the individual viruses. extreme running conditions in terms of ph, osmolarity, ion compositions or certain organic solvents might influence the efficacy of vaccines or gene therapy vectors. hence, normal or reversed-phase chromatography requiring organic solvents is usually not applied for whole-virus separations. however, for split vaccines or subunit vaccines, where virus membrane integrity does not necessarily have to be conserved, organic solvents and thus reversed-or normal-phase chromatography might be used depending on the solvent effects on the target proteins. partition chromatography such as sec can only be achieved by porous particles but not by membrane adsorbers or monoliths. for sec, virus particles are generally recovered in the column void volume allowing the separation of bulk proteins and small molecular contaminants without buffer changes under gentle operating conditions, thereby maintaining virus infectivity and immunogenicity. owing to the large difference in hydrodynamic volume of intact virus particles and soluble contaminants, the sec exclusion limit can easily be optimized not to distinguish between morphologically similar viruses, allowing robust process conditions and broad applicability where different strains are routinely required (e.g., for influenza vaccines). the main disadvantages of sec are the low capacity, product dilution and the poor pressure resistance of the matrix, requiring low flow rates. despite these disadvantages, sec represents a valuable method in addition to adsorptive chromatography, but high-throughput options for ultrafiltration processes will challenge the application of this unit operation for the separation of virus particles in the future. industrial chromatography processes are usually optimized for high dynamic binding capacities, particularly for the primary capturing step, and the elimination of residual contaminants during the polishing step (figure ). high capacities are attained by large accessible surface areas, optimized ligand densities and high selectivity. the latter is important if highly contaminated loads (i.e., feedstocks) are applied. in addition to the dynamic binding capacity, the ligand density of adsorption matrices such as iec, ac, and hic also contributes to the overall adsorption strength. this is mainly due to the multivalent interactions of virus particles with the matrix ligands. thus, ligand density needs to be carefully selected taking into account both of these aspects. furthermore, the desorption conditions need to be considered, selecting optimal ligands and ligand densities, and allowing elution conditions that do not affect the virus immunogenicity or transfection efficiency of vectors. strong adsorptions, requiring harsh desorption conditions for the target virus particles, are often observed for iec or hic [ ] . adsorption matrices are commonly used both in positive mode (i.e., virus particles adsorbing to the chromatography medium) and negative mode (i.e., contaminants adsorbing to the matrix and virus particles eluting). positive-mode applications are usually used for capturing or intermediate purification and negative modes for the final polishing. ion exchange chromatography exploits the charge-to-charge interaction between the virus surface and immobilized ion exchange groups on the matrix. depending on the overall isoelectric point (table ) of the individual virus particles, cation-or anion exchange chromatography (aec) matrixes are utilized. different variations of iec have been applied for the purification of a variety of viruses (table ) . adsorbed virus particles are in general displaced by an increasing ionic strength. alternatively, they are desorbed by a change in buffer ph, leading to an unfavorable overall charge of the virus surface or the matrix, at which the virus particles do not bind anymore. iec has a relatively low specificity resulting in a reduced dynamic binding capacity for the target virus and often results in co-elutions of contaminants (e.g., host cell dna for aec). differential elution between the target virus and contaminants can sometimes be achieved by linear-or stepgradient elutions. if high salt concentrations or nonphysiological ph conditions are required for purification, it is crucial to monitor immunogenicity or transfection efficiency of virus particles [ , ] . hence, iec matrices have to be carefully selected in terms of the type of charged groups, ligand density, and applied buffer conditions with respect to the virus type, product application and utilized assays for process characterization. affinity chromatography of virus particles relies on a specific and reversible adsorption and subsequent recovery of the active target virus from a ligand immobilized onto the chromatography matrix. the specific interactions between ligand and virus are based on individual structural properties, and the virus particles are commonly eluted by an altered conformation via a changed buffer ph or ionic strength or competitive displacement. however, the latter is generally not economic for large-scale production processes, but is frequently applied at smaller scales. applied ligands or matrices for the purification of virus particles are immobilized metal ion affinity [ , ] , sulfated carbohydrates (e.g., heparin) [ , [ ] [ ] [ ] , specific antibodies [ ] or antibody fragments, biotin streptavidine system for recombinant viral vectors [ ] , lectins [ , ] , dna aptamers [ ] and peptides (table ). for the majority of viruses, no specific ligands are known, resulting in the application of lessspecific ligands such as lectins or sulfated carbohydrates (i.e., heparin). lectins interact with accessible carbohydrate moieties of the surface glycoproteins according to their specificity. heparin is primarily a weak and strong cation exchanger containing sulfo and carboxyl groups as well as numerous hydroxyl groups [ ] . desorption of the virus particles from heparin matrices is done by increasing the ionic strength. however, it often requires a higher ionic strength than for classical cation exchangers owing to the multivalent interactions of the branched linear structure of heparin and the formation of hydrogen bonding between the target virus and heparin [ ] . an alternative to laborious ligand screens or the application of less specific pseudo-affinity ligands are engineered viral vectors containing affinity tags on the virus surface [ , ] . crucial for specific affinity ligands is the consideration of their dissociation constants. for recombinant proteins, dissociation constants of - - - m are usually considered a good working range [ ] . optimal affinity ligands allow fast binding kinetics for high flow rates but mild elution conditions to avoid reduction in immunogenicity or activity. therefore, it is essential to consider the overall binding strength of large virus particles, which is due to the already discussed multivalent interactions. if proteins or larger biomolecules are used as ligands, their potential toxicity or immunogenicity has to be considered and, if applied, assays have to be developed for their detection and quantification. furthermore, for many proteins, high costs are associated and the low stability of these ligands towards sanitizing agents in some cases prohibits their application for large-scale processes [ ] . in some cases, additional drawbacks of specific ligands are their restricted application as platform technology and limited process robustness for vaccines with frequently varying subtypes (e.g., influenza vaccines). furthermore, for viruses with varying progenies (e.g., vaccinia virus) [ ] , some virus particles might be excluded, thereby affecting the overall yield. on the other hand, the applied specificity allows the separation of specific progenies. ligands targeting post-translational modifications on the virus surface are heavily susceptible to the cultivation conditions and host cells [ ] . hence, target epitopes for ac matrices on the virus surface have to be carefully selected, considering their stability during the entire process. nevertheless, matrices with highly specific affinity ligands and high dynamic binding capacities are very well suited to capture virus particles after clarification from low concentrated cultivation broths. hydrophobic-interaction chromatography separates biomolecules dependent on the differential interaction of these compounds with hydrophobic ligands on the surface of the stationary phase. it is routinely used for bioseparations of proteins [ ] [ ] [ ] [ ] , and has also been applied for virus purification [ , ] since it is an orthogonal separation technique to purification methods based on ionic interactions. however, many factors such as type of salt and ionic strength of buffer, ph, temperature, ligand and ligand density influence the performance of hic, making its development and optimization a difficult and time-consuming endeavor. the two most commonly used hic ligands are butyl and phenyl [ ] . interactions with these ligands are relatively strong and have been frequently cited to denature labile proteins, potentially reducing their efficacy. however, often proteins subjected to these methods revert upon elution to their native structure [ ] . even so, if used for purification of gene transfer vectors or vaccines, their transfection efficiency or immunogenicity has to be closely monitored, just as for the high salt applications for the iec methods. stronger hic ligands such as hexyl and octyl are commonly destructive to proteins, and most likely also to virus particles or gene therapy vectors if applied in positive mode. furthermore, hic requires the application of high concentrations of kosmotropic salts, which potentially also reduces virus efficacies. also, the application of high salt concentrations influences virus aggregation in the majority of cases, affecting further downstream processes and often process analytics. in addition, disaggregation of the formed aggregates usually impacts virus transfection efficiency and immunogenicity. nevertheless, it was recently shown that hic can be successfully applied to remove residual dna after ac for purification of active vaccinia virus particles (mva-bn ® ; bavarian nordic a/s, kvistgaard, denmark) (table ) [ , ] . mixed-mode chromatography exploits a multimodal functionality that allows virus particles or contaminants to adsorb to the stationary phase by a combination of ionic interactions, hydrogen bonds and/or hydrophobic interactions. a typical example for a mixed-mode chromatography media is hydroxyapatite, which supports metal affinity interactions through its hydroxyapatite calcium groups, and cation exchange interactions through its hydroxyapatite phosphate groups [ ] . successful applications of mixed-mode matrices for the purification of gene therapy vectors have been described for adenoviruses [ ] , aav [ ] , moloney murine leukemia viruses [ ] and retroviruses (table ) [ ] . continuous chromatographic processes, such as the simulated moving bed (smb) technology is today well established for the separation of binary mixtures of petrochemicals, sugars and small molecule pharmaceuticals [ , ] . smb significantly improves the volumetric throughput as well as the purity and concentration factor relative to batch chromatography. however, purification of complex biological mixtures is still challenging because most systems are operated as a binary fractionator, providing only two exit streams [ ] . however, new operation modes such as the threefraction smb [ , ] , solvent gradient smb, and cleaning in-place smb are important steps leading the way for its successful application in bioseparations. finally, the complexity of the equipment and the experimental setup -that is, the large number of valves required -poses a serious challenge for process validation. the design of chromatographic purification processes is challenging owing to the multitude of unit operations and process variables, that is, a constantly growing number of available matrices, different chromatography modes and diverse operating conditions. as a result, an extremely large amount of design freedom exists. so far, the selection of unit operation and the choice of process conditions relies mainly on practical experiences or on trial and error approaches. in order to save valuable time and resources, miniaturized parallel screening tools have been developed [ ] , that can be combined with additional kinetic studies (e.g., surface plasmon resonance [spr] spectroscopy) [ , ] , thermodynamic measurements and dynamic light-scattering ana lysis [ ] to guide the early stage process development and optimization. for the selection of the most appropriate experiments and parameters, the screens are commonly planned via experimental design software tools (e.g., design-expert, modde, statistica), implementing the concepts of 'quality by design'. furthermore, software tools for computer-aided process design, production scheduling and process debottlenecking is often used [ , ] . to date, high-throughput process developments have been mainly applied to production processes for recombinant proteins. however, these methods can also be applied for the dsp of viral vaccines and gene therapy vectors. here, progress will largely depend on the establishment of reliable and robust analytical assays, which can cope with the high number of samples. a particular problem is the determination of product titers, which requires provision of suitable standards, biological and technical replicates, and assay validation on a logarithmic range. the latter typically results in comparatively large errors. typically, standard deviation of methods for virus titer assays in the range of . to . log have to be accepted. screening and ana lysis of well-proven operating conditions are one point, to further facilitate efficient downstream development, good modeling tools have to be established and applied. for instance, vicente et al. combined the steric mass action model of ion exchange [ ] with a standard chromatographic column model to simulate and predict adsorption and elution conditions for virus-like particles on anion-exchange (aex) membrane adsorbers [ ] . the same research group recently described the effect of ligand density on aex membrane adsorbers for the purification of recombinant baculoviruses by comparing model strategies based on spr spectroscopy with diethylaminoethyl (deae) ligand densities of membrane adsorbers. both methods indicated that a lower ligand density increased the overall yields by over % [ ] , demonstrating that spr technology can be successfully used to model membrane adsorption processes. their studies were further aided by a theoretical model to predict process conditions for dsp improvement [ ] . overall these studies demonstrate how chromatographic models combined with parallel screening techniques and rationally designed experiments can streamline the design and optimization of viral dsp in the future. single-use components for individual unit operations in dsp have been used for years in the pharmaceutical industry, mainly for filtration and buffer/media storage. over the last decade, the relevance of disposable concepts has been extended to other unit operations including chromatographic applications for which matrices such as membrane adsorbers, monoliths and more recently resins are used. the driving force for the increasing interest in single-use concepts for the production of viral vaccines is the fast and highly flexible set up of production processes, which is expected to improve the response time for increasing vaccine demands such as for pandemic preparations. in addition, disposable concepts for limited production campaigns are economically attractive considering that under current good manufacturing practice (cgmp) standards, raw material and equipment in direct contact with the product have to be dedicated to the particular production process [ ] . furthermore, single-use concepts commonly reduce or eliminate time-consuming and cost-intensive process steps such as cleaning and sanitization and their validation. however, the economic application depends heavily on the scale and requirements of the individual product, in addition to the available production facilities. hence, the use of disposable concepts always has to be considered on a by-case basis, taking into account investment costs, the entire variable (running) costs of the particular manufacturing facility and individual processes, and the product requirements [ ] . during recent years, kits for small-and medium-scale purifications and concentrations were introduced for adenoviruses (adenoviridae), aav (parvoviridae) and lentiviruses (retroviridae). in the ease of operation they are comparable to plasmid purification kits and require approximately h for the entire process. the majority of manufacturers rely on adsorption membrane technology based on centrifugal devices (small scale), syringe capsules (small scale) or larger capsules for fast protein liquid chromatography systems. purification of lentivirus and aav particles is mainly achieved by the use of aex and cation-exchange (cex) membrane adsorbers. according to the manufacturer's handbooks, the maximum capacities of the largest units currently available are approximately × and × total virus particles for adenovirus and aav, respectively, and × infectious particles for lentivirus. the required culture volumes range from to ml, allowing recoveries of approximately - % of the total particles depending on the culture conditions and virus. for the best results, it is important to consider the virus type for which the respective kit has been optimized. however, the kits can be used for other virus species after adapting the operating conditions. importantly, nucleic acids (i.e., host cell dna and proteoglycans) will also bind to aex membrane adsorbers and potentially co-elute. hence, kit procedures often include nuclease treatment of the harvest to reduce nucleic acid contaminations and to improve the flow rates downstream processing of cell culture-derived virus particles and thus process time (i.e., kits for adenovirus purification). in addition, a digestion step using chondroitinase abc, an enzyme that degrades glycosaminoglycan side chains of chondroitin sulfate proteoglycans [ ] , is sometimes included to remove proteoglycan contaminations. certainly, the purity of the resulting virus fractions heavily depends on culture conditions, virus species, type and strain, as well as the applied kit. therefore, no detailed information on virus purity can be provided by the manufacturers. it should also be pointed out, however, that viruses purified by this method can only be used for research purposes and are not intended for clinical studies or medical applications. current downstream processes aim to combine individual unit operations to an overall purification train taking into account the virus type (morphology, specific surface characteristics), the final product type (e.g., subunits vaccines, split vaccines, inactivated or active virus particles), application requirements (contamination levels), product yields and size of the production batch. for the development of new purification schemes it will be equally important to develop and optimize new technologies, as to design efficient dsp trains based on optimal combinations of these new technologies with currently used methods. unfortunately, very few complete purification schemes, from culture supernatant to clinical grade virus product, have been described in detail with overall recoveries and degree of contaminations in the literature. hence, a precise evaluation of the respective methods is nearly impossible. in general, however, overall viral recoveries of greater than % are currently considered acceptable for human pharmaceutical products. a particularly challenging issue for the dsp of human vaccines and the purification of gene therapy vectors is the removal of host cell dna. current guidelines for newly licensed human inactive vaccine products from continuous cell lines stipulate that residual dna levels exceeding ng per dose are not acceptable [ , ] . levels of contaminating host cell dna for gene therapy applications are even lower. here, levels of - pg of residual host cell dna per parenterally administered dose are considered acceptable by most medical agencies [ ] . for many applications these levels can only be achieved via nuclease treatments such as benzonase. the clear advantage of such a treatment is the elimination of potential oncogenes or other functional dna sequences [ ] even in very low amounts of host cell dna and the cleavage of nucleic acids adsorbed to the target virus. in addition, nuclease and condroitinase treatment frequently results in improved efficacies for gene therapy vectors. for the purification of gene therapy vectors, the removal of empty capsids or virus particles containing nucleic acid sequences other than the intended genome is crucial for their efficacy. however, owing to the small differences in specific surface characteristics or in virus morphology this is fairly difficult. successful separations were described via density centrifugation (adenovirus) [ ] , iec (aav) [ , ] , and partial depletions by hic and immobilized zinc-chelating interaction chromatography (adenovirus) [ ] . however, the possible application of the methods and their optimization has to be considered on a by-case basis. a further substantial challenge for dsp of viral vaccines, gene therapy vectors and other biotechnological products is batch-tobatch variations from the upstream processes. cell culture harvests can vary considerably not only in product concentration and quality but also in the type and amount of contaminations. for efficient and successful process development, all the aforementioned issues have to be addressed within a tightly coupled up-and down-stream process. therefore, critical parameters of the cultivation process effecting the dsp should be closely monitored and the subsequent purification methods adapted accordingly [ ] . in the following paragraphs, we summarize unit operations and purification trains for different virus types that have been described in the last years (table ). a thorough review has been published by lusky et al. on the gmp production of adenoviral vectors for clinical trials [ ] . another overview by burova and loffe deals with the chromatographic purification of recombinant adenoviral and aav vectors [ ] . in general, aec is the driving force for the described processes for adenoviruses [ , ] . eglon described a purification via aec in combination with sec from clarified and benzonase-treated cell lysate, which yielded total virus recoveries of % compared with . % via the classical cscl-purification method [ ] . duffy et al. compared a novel membrane-based technology for the purification of adenovirus and aav to conventional techniques [ ] and peixoto et al. described a downstream process for adenoviral vectors based on membrane technology [ ] . this purification train comprising clarification, ultrafiltration, aex membrane adsorber, ultrafiltration and sterile filtration resulted in a recovery of % of the infectious particles [ ] . another very important aspect, namely the virus aggregation and association of dna to virus particles, was addressed by konz et al. [ ] . wright reviewed, among other issues, vector quality characteristics such as aav-related impurities (e.g., aav-encapsidated dna impurities) and their pharmacological impact as well as gmp considerations for clinical aav vector production methods [ ] . a chromatographic purification train for aav using a combination of hydroxyapatite, deae-sepharose and cellufine ® (chisso america incorporated, rye, ny, usa) sulfate with an overall yield of % has been described by o'riordan et al. [ ] . more recently okada et al. described the use of a combination of cex and aex membrane adsorbers for the purification of aav serotype and with average recoveries of . and %, respectively [ ] . downstream processing strategies for purification of retroviral vectors are summarized in at least two excellent reviews from segura et al. [ ] and andreadis et al. [ ] , the latter being published in , ahead of the former. rodrigues et al. described the purification of murine leukemia virus-derived gene therapy vectors by means of membrane separation and aec with overall review recoveries of % and a purity of greater than % relative to the protein concentration [ ] . moreover, lesch et al. illustrated a scalable capture step for lentiviral vectors generated in t cells with baculoviral vectors [ ] . capturing based on deae monolithic columns reached a % recovery. ausubel et al. described the cgmp large-scale production of an oncolytic recombinant vesicular stomatitis virus based on a downstream process composed of a primary clarification step (filtration), benzonase treatment, aec, dia-/ultra-filtration and sterile filtration. the obtained yields of individual unit operations for infectious virus particles ranged from % to approximately % after sterile filtration [ ] . the fact that full recovery was obtained after the complete purification of some batches suggests that some of the titers, which were estimated via plaque forming assays, might be overestimated. however, the overall process is described comprehensively. working et al. discussed critical points affecting the development and production of clinical grade oncolytic adenoviruses, describing the up-and downstream process in detail [ ] . they illustrated a downstream process composed of cell lysis (triton), clarification, benzonase treatment, aec capture, ultra-/dia-filtration, sec, ultra-/dia-filtration (final formulation), followed by a final sterile filtration. however, the achievable purities and yields were not discussed in detail. downstream processes for purification of influenza virus particles were recently reviewed, summarizing applied purification trains for egg-and cell culture-derived influenza virus particles by wolff and reichl [ ] . motivated by the recent pandemic threats of influenza, numerous research laboratories worked on the optimization of influenza vaccine production processes. for the downstream side the center of attention was on specific chromatography media to substitute the frequently applied cellufine sulfate for the capturing of influenza virus particles [ , ] and on the use of chromatographic matrices with improved volumetric throughputs (i.e., monoliths [ , ] ) and membrane adsorbers [ , ] . wolff et al. described a downstream process for vaccinia virus based on a combination of pseudo-affinity and ion-exchange membrane adsorbers as well as pseudo-affinity and hic matrices [ , ] . the most promising downstream train resulted in an overall yield of % (infectious virus particles). furthermore, depletion of total dna to . % of the starting material and a total protein amount of less than µg per dose was achieved [ ] . however, batch variations of the starting material resulted in significant variations in the product yield and purity, and need to be further addressed. the success story of vaccination against major infectious diseases rests on a -year-old history [ ] . gene therapy in contrast is a relatively new but promising technology with the revolution of molecular genetics in the s paving the way into successful medical applications [ ] . vaccines currently comprise a rapidly growing market within the biopharmaceutical industry and gene therapy vectors have the potential to follow this line. nevertheless, for some vaccines, manufacturing capacities are still limited (i.e., for pandemic influenza vaccines). in general these problems are related to both, upsteam processing and dsp, and can only be efficiently solved if both are considered together. in the last years, upstream production processes of some vaccines moved away from their respective classical systems to the usage of diploid and continuous cell lines. however, downstream purification trains were in many cases only adapted to the new requirements but still lack further optimization. main improvements for vaccine purifications have to be governed by enhanced throughputs, capacities and potentially specificities of individual unit operations, as well as an enhanced linking of applied unit operations and the reduction of the number of purification steps. process development for gene therapy vectors could significantly benefit from the experience accumulated in vaccine production, especially with vectors derived from vaccines. nevertheless, there are still several technical problems to be solved for both product classes such as the benzonase-free removal of host cell nucleic acids. specific difficulties for the purification of gene therapy vectors are: • high final vector titers; • removal of transduction inhibitors from producer cells (proteoglycans, glycosaminoglycans [ ] [ ] [ ] ); • elimination of free envelope proteins, defective-virus particles and empty capsids. removal of host cell proteins, medium proteins and peptides is usually less problematic but equally important owing to potential allergic reactions. of note, the reduced starting levels of process-related protein contaminations are mainly due to increased usage of serum-free, protein-free or even fully defined media for upstream cultivations -an excellent example for improved overall process economics via a tighter coupling of up-and down-stream methods. in summary, for the downstream processes described in table , overall yields of virus particles of greater than % for production of human vaccines or gene therapy applications should be considered satisfactory. for other purposes, higher yields can be achieved depending on the requirements on product purity, the type and the amount of contaminants in the starting material, and the exertion. constant pressure towards improved product quality and safety as well as tighter timelines for the development of bio pharmaceuticals will certainly push for more efficient process development. current advances in the field indicate that increased application of highthroughput automated scaled-down models in combination with good mathematical models for experimental design and process development will support these efforts. certainly, these advances will also require the establishment of high-throughput assay • the application of parallel screening techniques, rationally designed experiments and the development of chromatographic models needs to be implemented to support downstream processing (dsp) development. • development and application of dsp platform technologies to improve the flexibility and response time of production processes is also required. • overall process economics need to be enhanced via a tighter coupling of upstream and downstream methods. • specific capturing of virus particles from bioreactor harvests via affinity chromatography can optimize the overall dsp by combining the primary concentration and purification step. • utilization of modern chromatography matrices such as membrane adsorbers and monoliths in place of classical resins enables an improved productivity in numerous dsp for viral particles. platforms for precise and reliable process characterization. as a result, unit operations should be available that have not only higher specificity and increased volumetric throughput but also enable design of purification trains with a reduced number of steps. advances in dsp will be most likely involve a broader use of filtration techniques (e.g., tangential flow filtration and diafiltration), the availability of new chromatography matrices such as membrane adsorbers, monoliths or bifunctional beads, and the identification of new, specific ligands. furthermore, single use concepts and the introduction of continuous dsp methods will play an important role as well as tighter process integration in terms of the up-and downstream process. furthermore, the establishment of platform technologies will have high priority whereas a large step towards platform technologies has already been accomplished by the development of downstream purification kits for gene therapy vectors. for additional readings, pedro et al. recently published a detailed summary dealing with the purification of bionanoparticles [ ] , gagnon published a comprehensive description on the chromatographic purification of virus particles [ ] and segura et al. published an overview of current scalable methods for purification of viral vectors [ ] . hammar l, gilljam g. extraction of hiv- in aqueous -phase systems to obtain a high-yield of gp aids res. hum. retroviruses ( ), - ( ). morenweiser r. downstream processing of viral vectors and vaccines. gene ther. , s -s ( ). been highlighted as: • of interest •• of considerable interest the use of aqueous -phase systems to concentrate and purify bovine leukemia-virus outer envelope protein gp concentration and purification of feline leukemia-virus (felv) and its outer envelope protein gp by aqueous phase systems purification of simian immunodeficiency virus, sivmac , and of its external envelope glycoprotein, gp chromatographic purification of recombinant adenoviral and adeno-associated viral vectors: methods and implications extensive overview on methods and implications on the chromatographic purification of adenoviral and adeno-associated viral vectors adenovirus vector production using low-multiplicity infection of cells construction of the vero cell culture system that can produce infectious hcv particles purification of a herpes-like virus from abalone (haliotis spp.) with ganglioneuritis and detection by transmission electron microscopy production and concentration of pseudotyped hiv- -based gene transfer vectors overview of current scalable methods for purification of viral vectors purification and characterization of retrovirus vector particles by rate zonal ultracentrifugation a rapid method to produce high yields of purified rotavirus particles comparative proteomics of human monkeypox and vaccinia intracellular mature and extracellular enveloped virions downstream processing of oncoretroviral and lentiviral gene therapy vectors summarizes downstream processing of oncoretroviral and lentiviral vectors simian sarcoma and feline leukemia virus antigens: isolation of species-and interspeciesspecific proteins the endogenous reverse transcriptase activity of gibbon ape lymphoma virus: characterization of the dna product determination of antibody to hepatitis b core antigen by means of immune adherence hemagglutination comparative study of the biological properties if influenza virus preparations purified by methods of centrifugation and chromatography purification of japanese encephalitis virus vaccine by zonal centrifugation murine type-c virus groupspecific antigens -interstrain immunochemical, biophysical, and amino-acid sequence differences isolation of the mouse mammary tumor virus sequences not transmitted as germinal provirus in the c h and riii mouse strains physical and chemical properties of an oncornavirus associated with a murine adrenal carcinoma cell line large-scale purification of animal viruses in the rk-model zonal ultracentrifuge: iii. semliki forest virus and vaccinia virus biochemical characterization of the amphotropic group of murine leukemia viruses preparation of a highly purified vaccine against tick-borne encephalitis by continuous flow zonal ultracentrifugation purification, potency and immunogenicity analysis of vero cell culture-derived rabies vaccine: a comparative study of single-step column chromatography and zonal centrifuge purification infectious poxvirus vectors have capacity for at least base pairs of foreign dna new method for large-scale growth; and concentration of the epstein-barr viruses structure of the hepatitis a virion: peptide mapping of the capsid region antigenic differentiation of m-msv(o) from mouse, hamster, and cat c-type viruses purification of retroviral vectors for clinical application: biological implications and technological challenges •• comprehensive review on the purification of retroviral vectors clinical retroviral vector production: step filtration using clinically approved filters improves titers a novel purification strategy for retrovirus gene therapy vectors using heparin affinity chromatography harvesting and concentration of human influenza a virus produced in serum-free mammalian cell culture for the production of vaccines antibody production virus removal by filtration: points to consider virus removal from factor ix by filtration: validation of the integrity test and effect of manufacturing process conditions high-performance tangential flow filtration using charged membranes tangential flow filtration for virus purification electrostatic and electrokinetic interactions during protein-transport through narrow pore membranes effect of solution ph and ionic-strength on the separation of albumin from immunoglobulins (igg) by selective filtration protein fractionation using electrostatic interactions in membrane filtration bioprocess membrane technology high performance tangential flow filtration ultrafiltration of parvovirus polymethacrylate monoliths for preparative and industrial separation of biomolecular assemblies •• compares conventional chromatography media and monoliths for industrial bioseparations chromatographic media for bioseparation •• detailed overview on chromatographic media used for bioseparations hydroxyapatite for biomolecule purification development of a purification process for adenovirus: controlling virus aggregation to improve the clearance of host cell dna describes the development of a purification process for adenoviruses (ad ) screening anion-exchange chromatographic matrices for isolation of onco-retroviral vectors capture and release of viruses using amino-functionalized silica particles the rapid concentration and purification of influenza virus from allantoic fluid controlled-pore glass as a stationary phase in chromatography direct visualisation of plasmid dna in individual chromatography adsorbent particles by confocal scanning laser microscopy chromatographic approaches in the purification of plasmid dna for therapy and vaccination noninvasive methods for characterization of largevolume monolithic chromatographic columns dispersion effects in preparative polymethacrylate monoliths operated in radial-flow columns large volume monolithic stationary phases: preparation, properties, and applications macroporous elastic polyacrylamide monolith columns: processing under compression and scale-up flow-dependent entrapment of large bioparticles in porous process media monoliths for fast bioseparation and bioconversion and their applications in biotechnology review: purification of proteins by membrane chromatography membrane processes in biotechnology: an overview • comprehensive review on membrane processes in biotechnology protein separation using membrane chromatography: opportunities and challenges • detailed overview on protein separations via membrane adsorbers impact of ligand density on the optimization of ion-exchange membrane chromatography for viral vector purification the emerging generation of chromatography tools for virus purification outlines the advantages of monoliths as chromatography tools for virus purification purification of cell culturederived modified vaccinia ankara virus by pseudo-affinity membrane adsorbers and hydrophobic interaction chromatography monoliths emerge as key purification methodology direct capture of influenza a virus from cell culture supernatant with sartobind anion-exchange membrane adsorbers engineering adeno-associated virus for one-step purification via immobilized metal affinity chromatography purification of cell culture-derived influenza virus a/puerto rico/ / by membrane-based immobilized metal affinity chromatography sulfated membrane adsorbers for economic pseudo-affinity capture of influenza virus particles exploiting heparin-binding properties of momlv-based retroviral vectors for affinity chromatography purification of porcine reproductive and respiratory syndrome virus from cell culture using ultrafiltration and heparin affinity chromatography removal of poliovirus type from a protein mixture using an immunoaffinity chromatography column affinity capture of a biotinylated retrovirus on macroporous monolithic adsorbents: towards a rapid single-step purification process concanavalin a affinity chromatography for efficient baculovirus purification lectin-affinity chromatography for downstream processing of mdck cell culture derived human influenza a viruses one-step selection of vaccinia virusbinding dna aptamers by monolex chromatographic purification of virus particles tagging retrovirus vectors with a metal binding peptide and one-step purification by immobilized metal affinity chromatography protein adsorption dissociation constants in various types of biochromatography large-scale processing of recombinant retroviruses for gene therapy capture of cell culture-derived influenza virus by lectins: strain independent, but host cell dependent development and scale up of preparative hic for the purification of a recombinant therapeutic protein high-throughput screening of chromatographic separations: ii. hydrophobic interaction recent advancement in application of hydrophobic interaction chromatography for aggregate removal in industrial purification process new method for preparation of hydrophobic interaction chromatographic stationary phases based on polymer-grafted silica and their chromatographic properties scaleable chromatographic purification process for recombinant adeno-associated virus (raav) purification of a functional gene therapy vector derived from moloney murine leukaemia virus using membrane filtration and ceramic hydroxyapatite chromatography step gradients in -zone simulated moving bed chromatography: application to the purification of antibodies and bone morphogenetic protein- simulated moving-bed chromatography and its application to chirotechnology theoretical study of multicomponent continuous countercurrent chromatography based on connected -zone units two-fraction and threefraction continuous simulated moving bed separation of nucleosides analysis of a simulated moving bed operation for three-fraction separations ( f-smb) high-throughput screening of chromatographic separations: i. method development and column modeling one article of a series (four publications) covering in detail high-throughput screening of chromatographic separations modeling protein binding and elution over a chromatographic surface probed by surface plasmon resonance modeling electrostatic interactions of baculovirus vectors for ion-exchange process development the role of process simulation in pharmaceutical process development and product commercialization • describes the application of batch process simulators to expedite the development of pharmaceutical products advanced production scheduling for batch plants in process industries steric mass-action ion exchange: displacement profiles and induced salt gradients anionexchange membrane chromatography for purification of rotavirus-like particles disposables in downstream processing chondroitinase abc promotes corticospinal axon growth in organotypic cocultures issues associated with residual cellsubstrate dna in viral vaccines dna removal from a purification process of recombinant hepatitis b surface antigen who study group on cell substrates for production of biologicals removal of empty capsids from type adeno-associated virus vector stocks by anion-exchange chromatography potentiates transgene expression empty capsids in column-purified recombinant adenovirus preparations. hum rapid highperformance liquid chromatographic analysis of adenovirus type particles with a prototype anion-exchange analytical monolith column good manufacturing practice production of adenoviral vectors for clinical trials • overview on gmp production processes for adenoviral vectors including a section focusing on gmp regulations in the usa and europe towards purification of adenoviral vectors based on membrane technology • describes the purification of adenoviral vectors based on a combination of filtration techniques and membrane adsorbers production and formulation of adenovirus vectors purification of adenoviral vectors by combined anion exchange and gel filtration chromatography purification of adenovirus and adeno-associated virus: comparison of novel membrane-based technology to conventional techniques scalable purification of adenoassociated virus serotype (aav ) and aav vectors, using dual ion-exchange adsorptive membranes. hum scateabte purification process for gene therapy retroviral vectors production and purification of lentiviral vectors generated in t suspension cells with baculoviral vectors meeting product development challenges in manufacturing clinical grade oncolytic adenoviruses • describes the up-and down-stream process in detail of clinical grade oncolytic adenoviruses downstream processing: from egg to cell culture-derived influenza virus particles summary on downstream processing methods and purification trains for egg-derived and cell culture-derived influenza viruses development of a strategy of influenza virus separation based on pseudoaffinity chromatography on short monolithic columns comptes rendus de l academie des sciences serie iii-sciences de la vie from the molecular genetics revolution to gene therapy: translating basic research into medicine removal of proteoglycans increases efficiency of retroviral gene transfer proteoglycans secreted by packaging cell lines inhibit retrovirus infection analysis of a envelope levels in retroviral preparations and effect on target cell transduction efficiency purification of bionanoparticles purification of densonucleosis virus by tangential flow ultrafiltration gene therapy: promises and problems isoelectric-focusing of parvoviruses the structure of nuclear polyhedrosis viruses: ii. the virus particle the complete dna-sequence of autographa-californica nuclear polyhedrosis-virus polyethylenimine coating to produce serum-resistant baculoviral vectors for in vivo gene delivery bromovirus isolation and rna extraction in: methods in molecular biology: plant virology protocols: from virus isolation to transgenic resistance fields virology properties and classification of hepatitis-a virus vaccine isoelectric focusing of hepatitis a virus in sucrose gradients hepatitis b: the virus and disease the hepatitis c virus: overview electron cryotomography reveals the portal in the herpesvirus capsid the genome sequence of an h n avian influenza virus from a thick-billed murre (uria lomvia) shows marine-specific and regional patterns of relationships to other viruses les virus influenza chez l'homme et les animaux the rab pathway is required for influenza a virus budding and filament formation isolation of a -singapore- influenza virus v and s antigens by isoelectric focusing studies on the adaptation of influenza-virus replication at low temperature. . isoelectric-focusing studies study of the electrophoretic mobility of a (leningrad) and a (leningrad) influenza-viruses electrophoretic studies on pr influenza virus overview: japanese encephalitis morphological, chemical, and biological characterization of japanese encephalitis virus virion and its hemagglutinin gene delivery by lentivirus vectors an overview the interferon-inducible rna helicase, mda- , is involved in measles virusinduced expression of antiviral cytokines high-throughput analysis of mumps virus and the virus-specific monoclonal antibody on the arrays of a cationic polyelectrolyte with a spectral spr biosensor functions of the ´ leader of murine leukemia virus genomic rna in virion structure, viral replication and pathogenesis, and mlv-derived vectors human papillomavirus (hpv) type distribution in females with abnormal cervical cytology. a correlation with histological study the ph stability of the papilloma virus protein the removal of phages t and pp , and poliovirus from fluids with hollow-fiber ultrafilters with molecular-weight cutoffs of , , and behaviour of a guanidinedependent strain of poliovirus in sucrose density and ph gradients characterization of type poliovirus by electrophoretic analysis electrofocusing of viruses and sensitivity to desinfection viral aggregationeffects of salts on aggregation of poliovirus and reovirus at low-ph epidemiological survay of different rabies virus strains in iran morphological characteristics of rubella virus mapping the rubella virus subgenomic promoter biology and application of alphaviruses in gene therapy alphaviruses: semliki forest virus and sindbis virus vectors for gene transfer into neurons the complete genomic sequence of the modified vaccinia ankara strain: comparison with other orthopoxviruses cryo-x-ray tomography of vaccinia virus membranes and inner compartments identification of orthopox virus by isoelectrofocusing in a granulated gel micro-electrophoresis of pox viruses in molar sucrose micro-electrophoresis of cowpox and vaccinia viruses in molar sucrose measurement of the electrokinetic properties of vaccinia and reovirus by laser-illuminated whole-particle micro-electrophoresis isoelectric points of viruses • detailed collection on isoelectric points of viruses development of a novel adenovirus purification process utilizing selective precipitation of cellular dna purification of infectious adenovirus in two hours by ultracentrifugation and tangential flow filtration pilot-scale adenovirus seed production through concurrent virus release and concentration by hollow fiber filtration efficient purification of bromoviruses by ultrafiltration purification of anatid herpesvirus particles by tangential-flow ultrafiltration and sucrose gradient ultracentrifugation a simple method for purifying the white spot syndrome virus using ultrafiltration downstream processing of mdck cell-derived equine influenza virus tangential flow microfiltration and ultrafiltration for human influenza a virus concentration and purification purification of the densonucleosis virus by tangential flow ultrafiltration and by ion exchange membranes purification of minute virus of mice using high performance tangential flow filtration increased viral titer through concentration of viral harvests from retroviral packaging lines improved methods of retrovrral vector transduction and production for genetherapy concentration of live retrovirus with a regenerated cellulose hollow-fiber optimisation of a multipartite human immunodeficiency virus based vector system; control of virus infectivity and large-scale production development and optimization of an adenovirus production process purification of adenoviral vectors using expanded bed chromatography high yield purification of functional baculovirus vectors by size exclusion chromatography ion-exchange membrane chromatography method for rapid and efficient purification of recombinant baculovirus and baculovirus gp protein hepatitis c virus and hepatitis b virus bind to heparin: purification of largely igg-free virions from infected plasma by heparin chromatography purification of cell culturederived human influenza a virus by size-exclusion and anion-exchange chromatography impact of adsorbents selection on capture efficiency of cell culture derived human influenza viruses binding aedes aegypti densonucleosis virus to ion exchange membranes densonucleosis virus purification by ion exchange membranes a simplified baculovirus-aav expression vector system coupled with one-step affinity purification yields high-titer raav stocks from insect cells serum-free production and column purification of adeno-associated virus type purification of recombinant adenoassociated virus type vectors by ion exchange chromatography generates clinical grade vector stock large-scale purification of a lentiviral vector by size exclusion chromatography or mustang q ion exchange capsule size-exclusion chromatography purification of high-titer vesicular stomatitis virus g glycoproteinpseudotyped retrovectors for cell and gene therapy applications. hum affinity recovery of moloney murine leukaemia virus purification of rabies virus produced on vero cells using chromatography techniques the authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.no writing assistance was utilized in the production of this manuscript. key: cord- -aesiff f authors: romero-brey, inés; bartenschlager, ralf title: membranous replication factories induced by plus-strand rna viruses date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: aesiff f in this review, we summarize the current knowledge about the membranous replication factories of members of plus-strand (+) rna viruses. we discuss primarily the architecture of these complex membrane rearrangements, because this topic emerged in the last few years as electron tomography has become more widely available. a general denominator is that two “morphotypes” of membrane alterations can be found that are exemplified by flaviviruses and hepaciviruses: membrane invaginations towards the lumen of the endoplasmatic reticulum (er) and double membrane vesicles, representing extrusions also originating from the er, respectively. we hypothesize that either morphotype might reflect common pathways and principles that are used by these viruses to form their membranous replication compartments. members of the family flaviviridae are enveloped viruses with a single stranded rna genome of positive polarity. this family contains four different genera: hepacivirus (from the greek hepar, hepatos, -liver‖), flavivirus (from the latin flavus, -yellow‖), pestivirus (from the latin pestis, -plague‖) and the recently included genus pegivirus [ , ] (figure ). hepatitis c virus (hcv) is the prototype species of the genus hepacivirus. it was first discovered by choo et al. [ ] in the serum and tissues of a chimpanzee experimentally inoculated with serum from an individual with chronic, non-a, non-b hepatitis. this virus was associated with a mild form of chronic hepatitis frequently observed in recipients of blood transfusions [ ] and was called hcv. a second species within the genus hepacivirus is gbv-b which was first identified in tamarins that developed hepatitis following inoculation with the serum from a surgeon (initials g.b.) with acute hepatitis. additional gb-like viruses were discovered later on and have been assigned to the new genus pegivirus (an acronym derived from pe, persistent; g, gb or g) within the family flaviviridae [ ] . and cms may represent the site of denv replication and rna translation/polyprotein processing, respectively [ ] . the most complete characterization of denv-induced intracellular membrane rearrangements elucidated their d architecture as well as their spatial connection with viral assembly sites [ ] . tem of resin-embedded infected cells revealed a complex collection of convoluted and vesicular structures, including cms that were usually surrounded by multiple vesicles, often appearing as longitudinal vesicle arrays. by using electron tomography (et), the latter were found to correspond to er tubules containing - nm single-membrane vesicles (ve) that result from the invagination of the er membrane into the er lumen. by conventional em, these vesicles appeared as double membrane vesicles, likely corresponding to the vps described earlier [ ] . immuno-em confirmed that the vesicles visible in resin-embedded cells were induced by denv infection and contained all ns proteins. however, only ns was detectable within the cms, which could be due to lower affinity of the antibodies or poor accessibility of the other ns proteins in the cms. double-stranded rna (dsrna) detected by immunostaining appeared as discrete electron-dense structures inside or on the cytosolic surface of a subset of vesicles, suggesting that dsrna might be present only in some of the vesicles at a given time point. furthermore, the vesicles contain rather uniform pores of ~ nm diameter towards the cytosol ( figure a ). thus, both the topology of the vesicles and the immunolabeling results support the idea that the vesicles might be the site of rna replication. moreover, these results showed that replication factories are a continuous membrane network that provides a platform for the transport of viral proteins and genomes between sites of rna replication, ribosome-containing compartments (rna translation) and virus assembly sites. in fact, virus budding sites were found in close proximity to the pores of the replication vesicles. this topological link may ensure efficient production and delivery of viral rna for the assembly of infectious virus progeny. consistent with these findings, a very recent publication using et showed that these virally modified structures were also observed in denv-infected mosquito cells, with one exception: cms were absent from denv-infected c / mosquito cells [ ] . in addition, after multiple rounds of virus replication, tubular structures were also observed in the vicinity of vps. these structures might represent a hallmark of chronically infected insect cells, since these structures are also induced by tbev in tick cells (see below). the first reports on wnv-infected cells described the visualization of virions [ ] . an extensive characterization of kunjin virus-the australian variant of wnv (wnv kun )-infected cells has been carried out more recently [ ] [ ] [ ] . three well-defined structures were found, corresponding to large cms, paracrystalline arrays (pcs) and vps that appeared as membrane sacs containing small vesicles (ve) [ , ] . based on immunolocalization studies, a distinct redistribution of the trans-golgi network (tgn) and colocalization of tgn markers with dsrna has been observed, suggesting that the replication factories of wnv kun were derived from the tgn [ ] . three-dimensional reconstructions of the wnv kun replication sites revealed an intimate association of the rough er (rer) with the bounding membrane of the vps [ ] (figure b ), resembling the vesicles observed in denv-infected cells. these results argue for an additional role of the rer in the formation of the wnv kun replication factories. similar to denv, individual necks were observed in the vesicles as well as the majority of the viral rna, as detected by immunolabeling with a dsrna-specific antibody, resided within these vesicles [ , , [ ] [ ] [ ] . in most cases, viral rna spanned the breadth of the vesicles and was juxtaposed to the necks open to the cytoplasm [ ] . . slices through tomograms of infected cells (on the left) and d top and lateral ( ° rotation) views of the same tomograms (on the right) are depicted, showing the characteristic virus-induced structures. the replication vesicles (ve) of denv, wnv and tbev (genus flavivirus) correspond to invaginations of er membranes that remain connected to the cytosol via nm-pores (highlighted with white arrows in the d lateral views), forming vesicle packets (vps). the replication factory of hcv (genus hepacivirus) is primarily composed of double membrane vesicles (dmvs) that seem to be formed aser protusions connected to er membranes via neck-like structures (highlighted with white arrows in the d lateral view). the er is shown in yellow (denv, tbev and hcv) or in red (wnv) and the replication organelles in brown (denv, tbev and hcv) or in white (wnv). the outer and inner membranes of dmvs are depicted in different shades of brown (outer membrane in dark brown and inner membrane in light brown). figure b is reproduced with permission from [ ] . in cells infected with tbev, one of the most important tick-transmitted viruses in europe and asia, virus particles and membrane-connected vesicles were also observed inside the er [ ] , similar to what was described for denv and wnv kun . the viral dsrna was only detected inside the vesicular structures within rer, suggesting that tbev rearranges internal cell membranes to generate a compartment that protects viral rna from detection by cytoplasmic pathogen recognition receptors (prrs) [ ] [ ] [ ] . this localization of dsrna might suffice to delay the onset of the ifn response [ ] . for tbev [ ] and wnv kun [ ] it was shown that treatment with brefeldin a (bfa), a drug which disrupts the golgi apparatus, did not interfere with viral replication. however, this treatment rendered wnv kun sensitive to the antiviral action of the ifn-induced protein mxa, indicating that bfa might have disrupted the membranous wnv kun replication compartments, thus leading to exposure of dsrna and its detection by prrs. in contrast, treatment of tbev-infected cells with bfa neither affected viral replication, nor the level of ifn production. these findings indicate that tbev dsrna might be stored inside bfa-resistant membrane vesicles that robustly protect the viral rna from recognition by cellular sensors. vector-borne flaviviruses like denv and tbev must replicate in both mammalian and arthropod cells. a few comparative studies have been published describing virus-induced structures such as cytoplasmic membrane proliferations and vesicle formation, also in insect cells [ , , , , ,] . a detailed comparative ultrastructural analysis of tbev-induced modifications revealed that the extent of membrane expansion and the abundance of vesicles were lower in insect cells [ ] . single-membrane vesicles, ranging in diameter from - nm were frequently found within proliferated er areas, often occurring in large groups contained within er cisternae. pore-like openings connected these vesicles to the cytoplasm and to other vesicles. apart from these vesicles, in tick-infected cells elongated vesicles or tubules were found that were much more prevalent in persistently than in acutely infected cells. these tubules were only occasionally noted in infected mammalian cells, similar to what was found with denv-infected cells [ ] . the tubular structures had a cross-sectional diameter of - nm, similar to the one of vesicles, reached up to nm in length, were closed at the ends and often arranged in fascicle-like bundles, shrouded with the er membrane. however, no pores between the tubules or towards the cytoplasm were observed [ ] . the function of these tubules is unclear and it is not known whether they represent bona fide features of replication factories, aberrant structures as a result of incorrect membrane remodeling, or the result of a cellular process to restrict infection [ ] . in any case, the tubules might be a feature of persistent infection, eventually linked to the high number of defective virus particles, because the lack of pores could prevent proper replication or packaging of the viral genome [ ] [ ] [ ] . further studies are required to shed light on the biogenesis and biological significance of these membranous tubular structures. a recent study identified nm-diameter vesicles within the er lumen of tbev-infected bhk- cells and in cells transfected with a tbev replicon [ ] . et revealed that these vesicles are invaginations of the er within a highly organized network of interconnected membranes with half of vesicles containing pore-like connections to the cytoplasm ( figure c ). however, no pore-like openings were observed between adjacent/neighboring vesicles, in contrast to what has been described for cells infected with langat virus (lgtv), a naturally attenuated tick-borne flavivirus [ ] or in wnv kun -infected cells [ ] . interestingly, in tbev replicon cells, the number of pore-containing vesicles was slightly larger (~ %) and they were found in much more fragmented er tubules as compared to tbev-infected cells. however, despite more extensive er rearrangements in replicon cells, they contained fewer vesicles, consistent with the lower level of viral replication [ ] [ ] [ ] . conventional em analysis of neurons infected with murray valley encephalitis virus (mvev) revealed several ultrastructural features, including proliferation of er and golgi complex membranes as well as the appearance of membrane-bound spherical vesicles ( - nm diameter) [ ] , similar to those observed for the related flaviviruses japanese encephalitis virus (jev) [ , ] and st. louis encephalitis virus (slev) [ ] . in the latter case, cylindrical membranous structures (or tubules) were also observed [ ] . the presence of vesicles was also detected in monkey liver cells infected with yellow fever virus (yfv) [ ] . these findings indicate that all members of the genus flavivirus utilize the er as a source of membranes for the formation of their replication factories, whereas assembly of new virions seems to occur at er sacs in close proximity to the replication sites [ , ] , thus creating an optimized membranous environment to support efficient viral replication and assembly. maturation of the newly synthesized virions takes place in the golgi apparatus, where flavirirus virions are often observed [ ] . in stark contrast to flaviviruses, hcv, the prototype of the genus hepacivirus, provokes an alternative rearrangement of intracellular membranes, originally designated -membranous web‖ (mw). this term referred to compact vesicle accumulations embedded into a membranous matrix [ ] as detected in cells inducibly expressing the hcv polyprotein. by using different em methods, we and others have recently found that the mw is primarily composed of double membrane vesicles (dmvs) [ ] [ ] [ ] . the fact that the kinetics of their appearance correlates with hcv replication suggests that these structures play an important role for viral rna amplification [ ] . indeed, immunolabeling of purified dmvs revealed an enrichment for viral proteins as well as dsrna [ , ] . importantly, dmvs contain enzymatically active viral replicase [ ] and they originate from er membranes, similar to what has been found for other members of the family flaviviridae. et analysis showed that most of the dmvs remain connected to the er via their outer membrane [ ] ( figure d ). although dmvs are primarily closed structures, ~ % of them have an opening towards the cytosol. late in infection, multi-membrane vesicles (mmvs) with an average diameter of nm are generated, likely originating from dmvs by secondary enwrapping events [ ] . by using huh . cells infected with the highly replicative hcv strain jfh- , ferraris and coworkers observed three different types of membrane alterations: vesicles in clusters (vics), contiguous vesicles (cvs) and dmvs [ ] . the vics were small single-membrane vesicles of variable size ( - nm) , grouped together in well-delimited areas. most of them had an internal invagination. the cvs were small single-membrane vesicles, present in large numbers and widely distributed throughout the cytoplasm, with a more homogeneous size (around nm). they were tightly associated to each other and tended to form a collar around lipid droplets (lds). dmvs were heterogeneous in size ( - nm) and had a thick, electron-dense membrane consisting of two closely apposed membranes. the increase of cvs' number correlated with an increase of intracellular hcv rna levels, arguing for a possible role of cvs in the early stages of viral replication. the presence of ns a in cvs, as demonstrated by immunogold staining, is consistent with this hypothesis. alternatively, cvs might constitute the membranous platform for viral assembly. in fact, the core protein is present in these structures ( %) as well as on the ld surface ( %). however, so far visualization of virus particles in infected cells has not been possible, making this hypothesis difficult to prove. while most of the dsrna signal was located within dmvs or at dmv membranes, vics were free of viral components and rna and these structures as well as cvs were very rarely observed in cells with a subgenomic jfh- replicon [ ] or absent in cells infected with a jfh- variant designated jc [ ] . the first d reconstruction of a complete hcv-infected cell revealed that all these three membrane structures were tightly connected and closely associated with ld clusters [ ] . taken together, these findings indicate a fundamental role of dmvs in hcv replication. an in-depth comparison of the study by ferraris and coworkers [ ] and our publication [ ] suggests that cvs might be also dmvs for several reasons: first, cvs have electron dense tightly apposed membranes; second, by using correlative light and electron microscopy, we also detected dmv accumulations around lds, reminiscent of the cvs described by ferraris and coworkers [ ] ; third, taking into consideration the density of content and morphology, some of the structures described as dmvs by ferraris and colleagues might correspond to mmvs according to our nomenclature. this might account for the differences in size between the dmvs reported in both studies (up to nm versus nm, respectively). alternatively, the difference might be due to the use of distinct virus strains (jfh- and jc ) that differ in their capacity to produce infectious virus particles by ~ orders of magnitude [ ] , which might also explain the presence of vics only in jfh- infected cells. much less about membranous replication factories is known for pestiviruses. tem-based studies from the times in which the genus pestivirus was still belonging to the family togaviridae reported that pestivirus-infected cells exhibited ultrastructural modifications of rer and contained small numbers of virus-like particles (vlps) [ , ] . gray and nettleton ( ) reported that border disease virus (bdv)-infected cells contained several profiles of er and many dense lamellar bodies, which when transversely sectioned appeared as multiple rows of tubules, nm in diameter [ ] . these lamellae were often found in association with rer and in one occasion vlps appeared to be budding within them. bovine viral diarrhea virus (bvdv)-infected cells contained rer modified into tubules, in which electron-dense vlps were present. more recent studies on bvdv-infected cells revealed cytoplasmic vacuolization and vlps in dilated er cisternae [ , ] . in addition, membrane structures consisting of vesicles of various sizes enclosed in much larger vesicles have been reported [ ] . these structures that morphologically resemble multivesicular bodies (mvbs) are distinct from the hcv-induced membranous web and more reminiscent of the flavivirus-induced vps. studies on the morphogenesis of pestiviral particles were hampered by a low rate of virion production. in a recent study, schmeiser and colleagues have overcome this problem by using high multiplicity of infection in mdbk cells with a distinct virus strain, the giraffe- strain [ ] . obtained results define the er as the site of pestivirus particle assembly, where budding of virions was observed. virus particles were also found inside the lumen of the golgi and in vesicles associated with the golgi compartment, suggesting that virus egress occurs via the conventional secretory pathway. interestingly, replication kinetics of pestiviral rna did not correlate with distinct membrane rearrangements and only slight dilatation of the er lumen was noticed. the absence of significant membrane rearrangements argues for a major difference between pestiviruses and other members of the flaviviridae family. interestingly, the authors detected the capsid protein and dsrna, the marker for viral replication intermediates, mainly in mvbs, indicating that pestiviruses are either using this compartment for replication or that viral rna and proteins are transferred to this compartment for degradation. similar assumptions have been made for hiv [ ] and marburg virus, a member of the filoviridae family [ , ] . alternatively, pestiviral rna and protein in mvbs might be intermediates of the entry process, prior to fusion of the envelope with the endosomal membrane. indeed, particles inside mvbs matching the morphological criteria of pestivirus virions were detected [ ] . however, mvbs of non-infected cells also contain vesicles for lysosomal degradation termed intraluminal vesicles (ilvs) that display a very similar morphology to pestiviral virions. thus, unambiguous discrimination between ilvs and pestivirus particles will require detailed immunolabeling approaches. the first visualization of the d architecture of a (+) strand rna virus replication factory was reported for flock house virus (fhv), a member of the family nodaviridae [ ] . this insect nodavirus induces the formation of invaginations at the outer mitochondrial membrane (omm) with an average diameter of ~ nm [ , ] ( figure a ). the interior of these vesicles (called spherules) is connected to the cytoplasm by a necked channel of ~ nm diameter, which is wide enough to allow import of ribonucleotides and export of synthesized rna (diameter < nm) [ ] . furthermore, metabolically labeled fhv rna localized between inner and outer mitochondrial membranes inside these spherules, thus validating the spherules as bona fide fhv-induced compartments for viral rna synthesis [ ] . conventional tem analyses of coronavirus-infected cells identified large numbers of isolated dmvs [ ] . at least in case of the severe acute respiratory syndrome (sars)-coronavirus, these dmvs are part of an elaborate reticulovesicular network (rvn) of modified er that consists of convoluted membranes, numerous dmvs (diameter - nm) ( figure b ) [ ] , and -vesicle packets‖ apparently arising from merging of dmvs. the cms were most intensively immunolabeled for viral replicase subunits whereas dmvs labeled abundantly for dsrna. while this result argues that dmvs might be the site of viral rna synthesis, et analyses failed to detect dmv connections to the cytoplasm to allow transport of nascent rna. instead, dmvs are connected to each other, to cms and to the er via their outer membranes. also, in case of another coronavirus, the mouse hepatitis virus (mhv), er membranes are thought to be the lipid donor of the membranous replication compartment [ , ] . qualitative and quantitative analyses by (immuno)-electron microscopy of mhv-induced membrane rearrangements revealed the appearance, in strict order, of dmvs (diameter - nm), cms, large virion-containing vacuoles, tubular bodies and cubic membrane structures [ ] . the recently identified coronavirus middle east respiratory syndrome coronavirus (mers-cov) induces extensive membrane rearrangements in the perinuclear region, including the formation of dmvs and cms [ ] . the diameter of mers-cov induced dmvs ranged from - nm, comparable to the sars-cov induced dmvs. in addition, cms were always surrounded by dmv clusters and were only observed in cells that appeared to be more advanced in infection. this observation strengthens the notion that dmv formation precedes the development of cms, as postulated previously for sars-cov [ ] . in addition to the betacoronaviruses (sars-cov, mhv and mers-cov), er-derived dmvs with a diameter of ~ nm have been observed in primary avian and mammalian cells infected with infectious bronchitis virus (ibv), an important poultry pathogen belonging to the genus gammacoronavirus [ ] . however, the most striking structures induced by ibv are zippered er membranes ( figure c ). the zippered er was associated to - nm diameter spherules, structures that are not present in cells infected with betacoronaviruses. et showed that these ibv-induced spherules are tethered to the zippered er and contain a . nm long channel connecting their interior to the cytoplasm of the cell, making them the ideal candidates for the site of ibv rna synthesis. er membranes are also targeted by nidovirales that belong to the family arteriviridae. cells infected with the prototypic arterivirus, equine arterivirus (eav), also contain dmvs associated to er tubules. these dmvs are ~ - times smaller (~ nm) as compared to coronaviruses [ ] . a recent in-depth ultrastructural analysis revealed that the outer membranes of eav-induced dmvs are interconnected with each other and with the er (figure d ), thus forming a reticulovesicular network (rvn) resembling the one previously described for the distantly related sars-cov [ ] . despite significant morphological differences, a striking parallel between the two virus groups, and possibly all members of the order nidovirales, is the accumulation of dsrna, the presumed intermediate of viral rna synthesis, in the dmv interior. along these lines, dmvs visualized by means of electron spectroscopy imaging contained phosphorus in amounts corresponding on average to a few dozen copies of the eav rna genome. like in sars-coronavirus infected cells, connections between dmv interior and cytosol could not be unambiguously identified, suggesting that dsrna is compartmentalized by the dmv membranes. in addition, et revealed a network of nucleocapsid protein-containing protein tubules, intertwined with the rvn. this potential intermediate in nucleocapsid formation, which was not observed in coronavirus-infected cells, suggests that arterivirus rna synthesis and assembly are spatially coordinated. membrane remodeling in picornavirus-infected cells has been studied for more than years. massive virus-induced membrane modifications have been reported already in [ ] , but the origin of these membranes is still a matter of controversy. several lines of evidence, including biochemical and structural data, suggest that the er must play a major role in the formation of those structures [ ] [ ] [ ] . early in infection membranous replication factories contain markers of the golgi [ , ] , whereas markers of the er, golgi and lysosomes were all found to be associated with poliovirus replication sites late in infection [ ] . initial reports identified membrane rearrangements as u-bodies because of their horseshoe-like shape [ ] . later, bienz et al. described rearranged membranes as clusters of single-membrane vesicles [ , ] , while other reports [ , ] noticed the double-membrane morphology of picornavirus-induced vesicles. the single-versus double-membrane morphology of the vesicles was first interpreted as two different models of their formation. however, several recent publications suggest that picornavirus-induced membrane rearrangements might occur in a consecutive manner. thus, early in poliovirus infection small clusters of single-membrane vesicles predominate that are transformed into bigger irregularly shaped single-membrane structures ( figure g ) and, late in infection, replaced by either round or irregularly shaped dmvs [ ] . interestingly, the small clusters of single-membrane vesicles of ~ - nm diameter contain gm , a cis-golgi marker, but did not stain positive for calnexin, an er marker. however, this does not exclude a role of the er for biogenesis of these vesicles, because er-resident proteins might be sorted out as these membranes are transformed. although dsrna and metabolically labeled viral rna were detected in single-membrane vesicles and dmvs, the exponential phase of viral rna synthesis correlates with the appearance of single-membrane and intermediate structures [ ] arguing that these structures are most relevant for high level poliovirus rna synthesis. similar results have been obtained with another member of the family picornaviridae, coxsackie b virus (cvb ) that also induces the formation of single-and double-membrane compartments, whose relative abundance correlates with the stage of the replication cycle [ ] (figure f ). based on the observation that the golgi apparatus disappears in cvb -infected cells, the membrane rearrangements might originate from this organelle (montserrat bá rcena, personal communication). similar to poliovirus, single-membrane tubular clusters occur predominantly early in infection, whereas the number of dmvs increases as infection progresses. a budding event could account for the formation of the tubules, depicting an average length of ± nm and an average diameter of ± nm. a subsequent enwrapping of these single-membrane tubules via an -autophagy-like‖ mechanism could then lead to the formation of dmvs that have an average diameter of nm ± nm. this transformation may require several steps: (i) membrane pairing; (ii) induction of curvature; and (iii) membrane fusion [ ] . this scenario would be consistent with the membrane surface of dmvs as an average-sized dmv with a diameter of nm would be equivalent to a tubule with a length of nm and a diameter of nm. er membranes were found near dmv clusters. however, in contrast to previous observations in nidovirus-infected cells, these dmvs were not connected to neighboring structures. in addition to these compartments, a third type of modification was detected in cvb -infected cells: multilamellar structures, which are typical for the late phase of infection and that correspond to enwrapped dmvs: despite their various shapes and degrees of complexity, in all instances they contained one dmv, surrounded by one or several layers of curved cisternae. in conclusion, these results, and similar observations made for foot and mouth disease virus (fmdv) (genus aphthovirus) [ ] suggest that members of the picornaviridae family induce singleand double-membrane vesicles. they appear in a time-dependent manner and seem to evolve from each other, possibly in coordination with the progression of the viral replication cycle [ ] . these membrane rearrangements occur independently from the used virus strain and cell line. rubivirus (family togaviridae) [ ] . rubv anchors its rna synthesis machinery to membranes of a cell organelle known as -cytopathic vacuoles‖ (cpvs) that is derived from modified endosomes or lysosomes and has an average diameter of - nm [ ] [ ] [ ] . freeze-fracture and et analysis of rubv-infected cells revealed a high complexity of cpvs that are composed of stacked membranes, rigid sheets, small vesicles and large vacuoles ( figure i ) [ ] . the cpvs are interconnected and linked to the endocytic pathway, as deduced from labeling experiments with endocytosed bsa-gold. furthermore, rer cisternae, mitochondria and golgi stacks are recruited around cpvs to build up rubv factories. cpvs have several contacts with cellular organelles: they are coupled to the rer through protein bridges of ~ - nm and closely apposed membranes and they are attached to golgi vesicles, whereas contacts with mitochondria were not detected [ ] . it has been proposed that rna synthesis occurs on vesicular membranes within the cpvs, which are linked to the cytosol and that the viral replicase molecules are associated with vesicles that transform with time into large vacuoles and straight elements [ ] . this is supported by immunogold labeling revealing replicase components and dsrna within the cpvs [ , , ] . the modification of late endosomes and lysosomes is a feature that rubv shares with alphaviruses, the other genus of the family togaviridae [ , ] . cells infected with alphaviruses like semliki forest virus (sfv), sindbis virus and western equine encephalitis virus (weev) contain large cpvs with a diameter ranging between and nm. the inner surface of these cpvs is covered with small invaginations or spherules that originate at the plasma membrane [ ] [ ] [ ] ( figure j ). these spherules are comprised of a single membrane forming a vesicle with a diameter of ~ nm. in addition, the spherules were shown to be the site of viral rna synthesis as deduced from metabolic labeling and detection by em [ , , ] . importantly, the inside of the spherule is connected to the cytoplasm by a pore with a diameter of - nm. the spherules are formed at the plasma membrane by the concerted action of the viral nonstructural proteins (nsp -nsp ) and genomic viral rna [ ] . furthermore, froshauer et al. [ ] showed that the cpvs that contain the spherules possess endosomal and lysosomal markers. time course studies revealed that the spherules of sfv undergo an unprecedented large-scale movement between cellular compartments [ ] . the spherules first form as blebs (exvaginations) at the plasma membrane. then, they are internalized by an endocytic process requiring a functional actin-myosin network. the spherules therefore represent an unusual type of endocytic cargo. after endocytosis, spherule-containing vesicles, namely cpvs-i fuse with acidic endosomes and move along microtubules. this leads to the formation of a very stable compartment, where the spherules accumulate as invaginations on the outer surface of unusually large, acidic vacuoles localized in the pericentriolar region [ ] . members of the genus norovirus (novs, family caliciviridae) are major agents of acute gastroenteritis [ ] . ultrastructural examination of murine norovirus (mnv- )-infected cells revealed a striking change in their overall morphology and intracellular organization [ ] . structures resembling virus particles were observed within or next to single-or double-membrane vesicles in the cytoplasm. the vesiculated areas increase in size with time and by hpi, large numbers of these vesicles and viral particles occupy most of the cytoplasm and displace the nucleus ( figure h ). in addition, a complete rearrangement of the er and loss of intact golgi apparatus was observed. both dsrna and mnv- nonstructural protein , the rna dependent rna polymerase, localize to the limiting membrane of individual vesicle clusters by immuno-em [ ] . immunofluorescence-based double-labeling showed that mnv- appears to recruit membranes derived from multiple cellular organelles and/or compartments: the er, trans-golgi apparatus and endosomes. however, despite extensive efforts, human norovirus cannot be grown in cultured cells [ ] . thus, detailed studies have not been possible, but it is assumed that replication structures are similar to those of mnv- . feline calicivirus (fcv), a member of the genus vesivirus within this family, is a major agent of respiratory disease in cats, which replication originates also membranous rearrangements and vesicles [ ] . brome mosaic virus (bmv, family bromoviridae) generates its replication factory by hijacking er membranes, similar to what has been described for other plant viruses like tobacco mosaic virus (tmv, family virgaviridae) [ ] , tobacco echt virus (tev, family potyviridae) [ ] and red clover necrosis mosaic virus (family tombusviridae) [ ] . however, other plant viruses such as alfalfa mosaic virus (amv) [ ] and cucumber mosaic virus (cmv), both belonging to the family bromoviridae, and turnip yellow mosaic virus (tymv, family tymoviridae) [ ] anchor their replication sites on chloroplasts. cucumber necrosis virus (cnv), family tombusviridae, utilizes peroxisomal membranes as replication platforms [ ] , while other plant viruses replicate on the surface of mitochondria [ ] . although the d architecture of these membranous replication sites remains largely unknown, their characteristics are strikingly similar to those for fhv (family nodaviridae). best studied is bmv that induces spherules, of similar size as the insect nodavirus fhv, in the er close to the nucleus, where viral rna synthesis and viral replication proteins are localized [ ] [ ] [ ] . in the case of beet yellows closterovirus (byv, family closteroviridae), tem of infected plant cells revealed the formation of ~ nm-diameter dmvs and multivesicular complexes (single-membrane vesicles surrounded by a common membrane) ( figure e ) [ ] . these multivesicular complexes often reside next to stacks of aligned filamentous byv particles [ , ] and resemble the dmvs and vps produced by nidoviruses and flaviviruses. several byv replication-associated proteins (l-pcp, mtr and hel) colocalize with dmv and vp membranes, supporting the role of these structures as replications platforms [ , ] . the membranes in closterovirus dmvs and vps are likely to be derived from er for members of the genus crinivirus [ ] or mitochondria in case of ampelovirus [ , ] . whether these structures are -closed‖ or -necked‖ remain unknown. based on amino acid sequence homologies of their rna-dependent rna polymerases, (+) rna viruses have been classified into three large supergroups [ , , ] : supergroup i (picornavirata or picorna-like group), including picorna-, corona-, arteri-and nodaviruses; supergroup ii (flavivirata or the flavi-like group), including tombus-, diantho-, pesti-, hepaci-and flaviviruses as well as single-strand rna bacteriophages; supergroup iii (rubivirata or the alpha-like group), including tobamo-, hordei-, alpha-and rubiviruses as well as hepatitis e virus (hev). these higher-order taxonomic units encompass diverse viruses infecting different hosts from almost all kingdoms of life. as discussed earlier [ ] , amongst these viruses two main architectures of remodeled membranes (morphotypes) can be found that may reflect two alternative strategies to induce the membranous microenvironments required to allow virus replication (summarized in table ). the first morphotype involves the formation of negatively curved membranes, initiated by invaginations of the pre-existing membrane bilayer and giving rise to spherules, vesicles or vacuoles towards the lumen of the targeted cell organelle. these structures have been identified in a broad range of mammalian, plant and insect cells infected with viruses belonging to supergroups ii and iii. the second strategy involves the formation of membranes with positive curvature, giving rise to double-membrane structures that are the predominant characteristic of the replication factories of the picorna-like virus supergroup. the conservation of these two sorts of morphotypes in distantly related viruses supports the assumption of an evolutionary conserved mechanism. a striking finding in this regard was the observation that hcv, despite belonging to the flavi-like supergroup, induces dmvs whereas flaviviruses induce the formation of negatively curved membranes. to our current knowledge, hcv is the only member of the family flaviviridae inducing the formation of membrane structures with positive curvature, suggesting that hcv might share common host cell pathways to induce membranous replication compartments with distantly related viruses such as corona-, arteri-, picorna-, calivi-or closteroviruses (table ) . however, it still remains to be elucidated whether other members of the family flaviviridae, belonging to the genera pestivirus and pegivirus, also utilize a picorna-like membrane remodeling strategy. a common feature associated with the spherule/vesicle/vacuole/-type of rearranged membranes is their size ( - nm diameter) and the presence of a pore connecting the interior of the vesicle with the cytoplasm [ , , , ] . since rna replication occurs in the vesicle interior, the pore allows exchange of nucleotides and rna products with the cytoplasm. the size of the pore is variable, ranging from . nm in case of ibv-induced spherules to ~ nm in case of membrane invaginations induced by flaviviruses. in contrast, in the majority of dmvs no such channel or pore has been detected. nevertheless, as exemplified with nidoviruses, the inner compartments enclosed by interconnected dmvs contain the bulk of dsrna [ ] , and in some cases they depict an electron-dense core assumed to correspond to viral rna (ibv and eav) [ , ] . although this represents a functional enigma in terms of rna synthesis and transport, the presence of dsrna in the dmv interior does not necessarily indicate active rna replication. assuming a temporal regulation, it is possible that dmvs might be sites of rna synthesis as long as they are linked to the cytoplasm, but replication would stop upon closure of the vesicles. yet, another strategy appears to be used by enteroviruses, where active rna replication has been detected on the cytosolic side of the membranous structures [ , ] , consistent with the membrane topology of the nonstructural proteins catalyzing rna replication [ ] [ ] [ ] . as described above, studies conducted with picornaviruses revealed that the exponential phase of viral rna synthesis coincides with the accumulation of single-membrane tubules [ , ] . importantly, pulse-radiolabeling experiments localized sites of active rna replication to the outer surface of single-membrane tubules [ ] and isolation of the membranous replication factories and their subsequent visualization by em revealed that they form rosette-like structures composed of virus-induced cytoplasmic vesicles [ ] . rna replication is thought to occur at sites where the vesicles cluster, whereas rna translation probably takes place on the exposed periphery of the vesicles. this raises the question, what the role of dmvs in the replication cycle of picornaviruses might be. it is possible that dmvs either support rna synthesis or serve as rna storage sites (especially in case of closed dmvs). in this manner, dmvs might be involved in regulating viral rna replication: by complete sealing of the viral replicase inside the vesicle, it would be inactive, thus regulating overall rna copy number in the infected cell. alternatively, dmvs might be an epiphenomenon, resulting from the over-expression of membrane-active proteins that accumulate especially during the late stages of infection. the mechanism responsible for dmv formation is not clear. in case of picornaviruses, it is thought that single-membrane structures are the precursors of dmvs [ , ] . nevertheless, dmv formation might also involve the autophagy machinery, or at least several components thereof, by a process analogous to the formation of autophagic vacuoles [ ] . this hypothesis is supported by the morphological resemblance of dmvs and autophagosomes. it has been shown that the inhibition or stimulation of autophagy results in a modest inhibition or stimulation of poliovirus and coxsackie b virus yield, respectively, and there are also data supporting the involvement of autophagy in the replication of rhinovirus and [ , ] . however, brabec-zaruba et al. [ ] reported that replication of rhinovirus was insensitive to pharmacological manipulation of autophagy and did not induce detectable modification of lc . this discrepancy might be due to the use of different cell types and experimental conditions. the mechanism of dmv formation in case of hcv and coronaviruses is also unclear. biochemical analysis of isolated host cell membranes associated with hcv rna and proteins identified markers of the autophagy machinery, including lc -ii, the lipidated form of lc (lc -ii) that is generated upon activation of the autophagy machinery [ ] . however, the role of autophagy in the hcv replication cycle is also a matter of controversy. for instance, immunolabeling did not identify lc -ii at those sites where nonstructural proteins accumulate [ ] . moreover, different roles of autophagy for the hcv replication cycle have been proposed. these include a role of autophagy in hcv rna translation [ ] , initiation of rna replication [ , ] , production of infectious virus particles [ ] or suppression of the innate antiviral defense [ , ] . to clarify these discrepancies, future studies should combine biochemical and cell biological approaches with ultrastructural analyses. the autophagy machinery might also be involved in the formation of virus-induced membrane invaginations/spherules. for instance, lee and coworkers provided evidence that denv infection enhances autophagolysosome formation and that inhibition of the autophagy machinery by -methyladenine ( -ma) reduces denv particle production [ ] . however, the effects were moderate, arguing that this pathway may contribute to denv replication to only a minor extent. moreover, autophagy includes membrane wrapping, leading to double-membrane compartments involved in lysosomal degradation whereas denv-induced vesicles are invaginations. finally, immunolabeling experiments failed to detect lamp- at these vesicles [ ] , arguing against the involvement of lysosomes in the formation of the denv replication vesicles. it remains to be determined whether autophagy is actively induced by these viruses to provide a compartment favoring replication or induced as a bystander defense against infection leading to degradation of the replicase proteins [ ] . another membrane compartment frequently induced by (+) rna viruses are convoluted membranes (cms) that were observed e.g., in sars-cov-, mhv-, wnv-or denv-infected cells [ , , , , , , ] . morphologically, cms resemble smooth er membranes, lack ribosomes and in case of denv are induced by the sole expression of ns a [ , ] . cms are often associated with late stages of infection, suggesting that dmv formation might precede the development of cms. in sars-cov-infected cells, dmvs appear to be connected with cms [ ] , while in mhv-infected cells no such connections have been observed [ ] . the role of cms for the viral replication cycle is not well understood. in case of wnv kun , cms are supposed to be the site of polyprotein processing [ , ] . this conclusion is based primarily on the strong immunolabeling for ns b and ns and the absence of ns and ns b. since polyprotein cleavage occurs co-translationally and thus, should happen at the rer, this model would require the formation of rather stable processing intermediates that are transferred from the rer to the cms where further cleavage would occur. alternatively, at least in case of denv, cms might represent a storage site for proteins and lipids involved in viral replication that can be recruited to vesicles upon demand. the fact that cms are physically linked with er-containing invaginations and contain ns would be consistent with this assumption [ ] . along these lines, the fact that insects are cholesterol auxotrophs and lack several enzymes in the cholesterol biosynthesis pathway [ ] , suggests that cholesterol might be a key component of cm structures, which would explain their absence in infected insect cells [ ] . viral replication complexes are targeted to the respective membranous organelle primarily by nonstructural (ns) proteins rather than viral rna [ ] . these ns proteins seem to have some specificity in recognizing organelle subpopulations and often contain multiple hydrophobic domains implicated in membrane targeting and rearrangement. the molecular mechanisms orchestrating the formation of these complex structures are still poorly understood, but it is clear that ns proteins, often working in a concerted action, are key players in replication factory biogenesis. one well-studied example among the single-membrane vesicle inducers is bmv where it was shown that the sole expression of the ns protein a is sufficient to induce the formation of single-membrane spherules resembling the ones observed in infected cells. these spherules had a diameter of - nm, resided in the er lumen and were shown to be the site of viral rna synthesis [ ] . in case of the insect nodavirus fhv, protein a and replication competent rna were required for induction of the spherules. expression of protein a alone induced only -zippering‖ of the surfaces of adjacent mitochondria, but did not induce spherules. thus, protein a is necessary, but not sufficient for spherule formation. moreover, spherules were not formed when replication-competent fhv rna templates were expressed with a protein a mutant lacking polymerase activity or when wild-type protein a was expressed with a replication-incompetent fhv rna template. thus, the membranous fhv replication compartment requires both a viral protein and active rna synthesis [ ] . feline calicivirus (fcv) infection results in rearrangement of intracellular membranes and production of numerous membrane-bound vesicular structures on which viral genome replication is thought to occur. expression of individual fcv nonstructural proteins revealed that p induces significant reorganization of the er into large, fenestrated membrane networks, resembling the structures found in infected cells [ ] . moreover, expression of p and p , two additional fcv ns proteins, induced extensive reorganization of the er and the nuclear envelope suggesting that the er is the primary source of the membranous replication factory [ ] . flavivirus membrane rearrangements are mainly induced by ns a, as suggested by recent studies with wnv kun and denv [ , ] , but it is unknown whether the same applies to ns a of tbev. in case of denv, ns a is thought to contain a central peripheral membrane domain that intercalates into the luminal leaflet of the er membrane [ ] . it is tempting to speculate that ns a oligomers [ ] might dilate the luminal leaflet, resulting in membrane invaginations towards the er lumen. however, the sole expression of ns a is not sufficient to induce er membrane invaginations that have been detected in infected cells. instead, expression of ns a lacking the c-terminal k fragment (corresponding to fully processed ns a) induced er membrane rearrangements reminiscent of cms, whereas unprocessed ns a/ k did not induce membrane alterations [ ] . these results provide strong evidence that processing at the ns a- k site is required for the induction of membrane alterations. the critical role of polyprotein cleavage for induction of membrane rearrangements is supported by studies conducted with wnv kun . there it was shown that a regulated cleavage of a ns a/ k/ b precursor by the viral ns b/ protease is needed for induction of membrane rearrangements [ ] . however, the same study reported that expression of full-length (uncleaved) wnv kun ns a/ k led to membrane alterations similar to those induced in infected cells whereas the k fragment impaired the ability of ns a to induce membrane rearrangements. whether this reflects a biological difference between wnv kun and denv ns a or is due to the use of alternative experimental approaches remains to be determined. one of the most fascinating mechanisms employed by (+) rna viruses to induce their replication factories is used by sfv. it was shown that the spherules of sfv arise by blebbing at the surface of the plasma membrane [ ] . these blebs are internalized and after fusion with lysosomes give rise to large cytoplasmic vacuoles. formation of these membrane alterations requires the viral protein nsp , which has several functions. it has guanine- -methyltransferase and guanylyltransferase activities and thus is critically involved in capping of the viral rnas [ ] [ ] [ ] , but at the same time has affinity to lipids [ ] . in fact, of the four ns proteins of sfv, only nsp has affinity for membranes, and when expressed alone, it is specifically targeted to the inner surface of the plasma membrane [ ] . nsp is a monotopic membrane protein and its affinity for membranes is dictated by an amphipathic α-helix, located in the central region of the protein [ , ] . nsp has a specific affinity for negatively charged phospholipids, which might account for its prevalent localization to the plasma membrane, where such lipids are enriched. membrane binding of nsp via its amphipathic α-helix is essential for alphavirus replication [ ] . however, nsp is not sufficient for cytoplasmic vacuole formation. for instance, it was found that nsp contributes to the transport of the replicase polyprotein from the plasma membrane to the surface of endosomes [ ] . these results indicate that nsp has to cooperate with other viral and cellular factors to allow formation of the cytoplasmic vacuoles. furthermore, in a recent study kallio et al. [ ] have shown that the size of the spherule is dependent on the length of the rna template, in contrast to what has been observed for fhv, another spherule-inducer [ ] . these results indicate that in addition to the ns proteins, the viral rna template itself critically determines the morphology of the membranous vesicles. in order to induce the variety of membrane alterations observed in cvb -infected cells ( figure f ), several membrane-remodeling mechanisms are required: induction of membrane curvature, membrane fusion and membrane-membrane interactions [ ] . these rearrangements require the enteroviral proteins bc and a; their coexpression generates er membrane-derived structures mimicking those observed during viral infection [ ] . importantly, b and c both contain an amphipathic α-helix [ ] [ ] [ ] , a well-known curvature-inducing motif [ ] . along the same lines, fmdv b and bc locate to the er when expressed on their own and cause a swelling of er cisternae [ ] . in case of hcv, we recently found that a concerted action of ns / a, ns b, ns a and ns b is required to generate the membranous web. furthermore, all these replicase components were capable of inducing membrane vesiculation with ns a having the highest potential to trigger membrane curvature. importantly, some of these ns a-induced structures corresponded to dmvs [ ] . in addition, ns b also plays an important role in triggering rearrangements of intracellular membranes [ ] . ns b is an integral membrane protein containing two n-terminal amphipathic α-helices, a highly hydrophobic central core domain composed of four putative transmembrane segments, and a highly conserved c-terminal domain that is thought to harbor two α-helices (reviewed in [ ] ). a recent study has demonstrated that ns b oligomerizes through multiple conserved determinants and that oligomerization appears to be required for membranous web induction [ ] . indeed, mutations affecting the highly conserved c-terminal domain impairing ns b self-interaction resulted in the formation of aberrant dmvs arguing for a central role of ns b in formation of functional replication compartments [ ] . studies on arteriviruses revealed that the sole expression of eav nsp and nsp is sufficient to induce membrane structures similar to those generated during eav infection [ ] . mutations within nsp , which is a tetra-spanning integral membrane protein, alter membrane rearrangements, highlighting the importance of this protein for the biogenesis of eav-induced dmvs [ ] . in case of sars-cov, nsp , nsp and nsp were found to be sufficient to induce the formation of dmvs that are similar to those observed in sars-cov-infected cells [ ] . these dmvs were, however, smaller in diameter, suggesting a role for other viral proteins or the presence of viral rna in determining the dmv morphology. importantly, em analysis of nsp mutants that are impaired in rna replication and virus growth, revealed an aberrant morphology of dmvs as well as an increased prevalence of cms [ ] . another important viral protein involved in inducing the sars-cov membranous replication factory is nsp , which is predicted to contain seven transmembrane segments and a hydrophilic cytoplasmic domain [ ] . nsp was shown to induce vesicles containing atg and lc -ii as well as phosphatidylinositol- -phosphate, thus sharing many features with omegasomes, which are omega-shaped membrane compartments that are formed during activation of autophagy [ ] . this result suggests that autophagy might contribute to the formation of the membranous replication site of sars-cov. in the last couple of years, our knowledge of the architecture of the replication factories induced by (+) rna viruses has increased substantially. this is primarily due to the more widespread use of et and other high-resolution imaging methods. nevertheless, our knowledge is still rather restricted to descriptions of the morphologies of these complex structures, whereas our understanding of their biogenesis in most cases is very rudimentary. more efforts are required to elucidate the role of the viral proteins in the formation of the replication vesicles, to identify the involved cellular components and the mechanisms used by these proteins to subvert and exploit cellular pathways to establish membranous replication factories. this includes determination of the d structure of involved (viral) proteins as well as evaluation of host cell factors and lipids contributing to biogenesis and activity of the replication compartment. in addition, further studies are needed to understand how viruses utilize these compartments to coordinate the different steps of their life cycle (replication, assembly and release) in space and time to achieve efficient replication. work in the authors' laboratory was supported by the deutsche forschungsgemeinschaft (sonder-forschungsbereich the gb viruses: a review and proposed classification of gbv-a, gbv-c (hgv), and gbv-d in 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nsp proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate the authors are very grateful to erik j. snijder (leiden university medical center, leiden, the netherlands) and kè vin knoops (european molecular biology laboratory, grenoble, france) for providing the unpublished pictures depicted in figure b and d and to montserrat bá rcena (leiden university medical center, leiden, the netherlands) and tero ahola (university of helsinki, helsinki, finland) for providing the unpublished pictures depicted in figure f and j, respectively. figures a, c , e, g, h and i are reproduced with permission from [ ] , [ ] , [ ] , [ ] , [ ] and [ ] , respectively.we also would like to thank jason m. mackenzie the authors declare no conflict of interest. key: cord- -pamxy av authors: teissier, elodie; zandomeneghi, giorgia; loquet, antoine; lavillette, dimitri; lavergne, jean-pierre; montserret, roland; cosset, françois-loïc; böckmann, anja; meier, beat h.; penin, françois; pécheur, eve-isabelle title: mechanism of inhibition of enveloped virus membrane fusion by the antiviral drug arbidol date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: pamxy av the broad-spectrum antiviral arbidol (arb) inhibits cell entry of enveloped viruses by blocking viral fusion with host cell membrane. to better understand arb mechanism of action, we investigated its interactions with phospholipids and membrane peptides. we demonstrate that arb associates with phospholipids in the micromolar range. nmr reveals that arb interacts with the polar head-group of phospholipid at the membrane interface. fluorescence studies of interactions between arb and either tryptophan derivatives or membrane peptides reconstituted into liposomes show that arb interacts with tryptophan in the micromolar range. interestingly, apparent binding affinities between lipids and tryptophan residues are comparable with those of arb ic of the hepatitis c virus (hcv) membrane fusion. since tryptophan residues of membrane proteins are known to bind preferentially at the membrane interface, these data suggest that arb could increase the strength of virus glycoprotein's interactions with the membrane, due to a dual binding mode involving aromatic residues and phospholipids. the resulting complexation would inhibit the expected viral glycoprotein conformational changes required during the fusion process. our findings pave the way towards the design of new drugs exhibiting arb-like interfacial membrane binding properties to inhibit early steps of virus entry, i.e., attractive targets to combat viral infection. distinct from specific antiviral compounds that target key viral functions are a group of broad-spectrum medicinal drugs that were originally designed for other treatments [ ] [ ] [ ] or targeted toward a number of viruses ( [ ] ; reviewed in [ ] ). the advantage of this group of antivirals is that they have already met the pharmacological criteria for medicinal drugs and are already approved for clinical use in some countries. among these molecules, antiviral agents targeting viral entry of enveloped viruses are of major interest since they seize an early step in the viral life cycle, before damages have occurred to cells (recently reviewed in [ , ] ), and since they can be incorporated into combinations of multiple drugs with different targets. one of these compounds, arbidol [arb; hindole- -carboxylic acid, -bromo- -[(dimethylamino)-methyl]- hydroxy- -methyl- -[(phenylthio)methyl]-, ethyl ester, monohydrochloride; cas registry number - - ( figure )], is already licensed in russia and china, and is described as an antiinfluenza drug with immunostimulant properties. arb is in use for several years as prophylaxis and treatment for influenza a and b infections. it inhibits influenza virus-induced membrane fusion and may have the capacity to induce serum interferon [ ] . recent studies extended its inhibitory activity to other human viruses such as the respiratory syncytial virus, parainfluenza virus , rhinovirus , and hepatitis b virus (reviewed in [ , ] ). we demonstrated that it also inhibits hepatitis c virus (hcv) infection in vitro, and hcv replication [ ] , hcv cell entry and membrane fusion using hcv pseudoparticles (hcvpp) and hcv grown in cell culture (hcvcc) [ , ] . most recently, ciliberto and coworkers demonstrated the efficacy of arb derivatives at inhibiting hcv entry and replication into hepatoma cells in the low micromolar range [ ] . hcv infection is a leading cause of liver diseases, including hepatocellular carcinoma, and therapeutic options are still limited (for recent reviews, see [ ] and refs therein). there is thus an urgent need to develop efficient and well tolerated drugs to combat this virus. arb demonstrated a propensity to enter into hydrophobic interactions with membranes, and with membrane-like environments such as detergent micelles [ ] . here we further characterize the mechanism of action of arbidol, and analyze at the molecular and atomic level the interactions of arb with membranes, tryptophan-rich derivatives and peptides. we first examined how arb inhibits hcv entry and membrane fusion using hcvpp of different genotypes, and found that arb inhibition was genotype-independent. by combining surface plasmon resonance, fluorescence and nmr spectroscopy approaches, we showed that arb directly interacts with the phospholipid membrane interface, with an affinity in the micromolar range, comparable to the concentration inhibiting hcvpp membrane fusion by % (ic ). arb also displayed micromolar affinity toward aromatic components of proteins such as tryptophan and derivatives, and toward peptides containing tryptophans and derived from hcv envelope glycoproteins. altogether our results demonstrate that arb interacts with the polar head of phospholipid membranes and protein motifs enriched in aromatic residues, suggesting that the inhibitory activity of arb on hcv entry and fusion could involve both types of interactions. phosphatidylcholine from egg yolk (pc, % pure), dimyristoylphosphatidylcholine (dmpc, % pure), cholesterol (chol, % pure), lyso-phosphatidylcholine (lysopc), dodecyl-phosphocholine (dpc), triton x- , tryptophan octyl ester hydrochloride (toe) and n-acetyl-l-tryptophanamide (nata) were purchased from sigma. octadecyl rhodamine b chloride (r ) was from molecular probes. the peptides used were part of the sequence of structural or non structural (ns) proteins of hcv and of the bovine viral diarrheal virus (bvdv). the amphiphilic helix of bvdv ns a [ ] and the transmembrane domain of hcv ns a [ ] were obtained as described previously. the peptides identified as important for hcv fusion [ ] were purchased from clonstar biotech ( % purity) or sigma genosys ( % purity), respectively, and dissolved in dmso before preparation of lipid:peptide mixtures. arbidol [arb, h-indole- -carboxylic acid, -bromo- -[(dimethylamino)methyl]- -hydroxy- -methyl- -[(phenylthio)methyl]-, ethyl ester, monohydrochloride ( figure )] was a kind gift from stephen j. polyak. arb was readily soluble in ethanol, and soluble in the mm range in water. ethanol stock solutions of arb were diluted to a . mm final concentration in milliq water (the final stock solution contained % ethanol). for spr experiments, one mg of arb was resuspended in water, followed by centrifugation ( g, min, uc). arb concentration in solution was measured at nm in the supernatant (arb extinction coefficient = m .cm ). after solvent evaporation, samples were resuspended in phosphate-buffered saline (pbs, ph . ) or water, and underwent freeze/thaw cycles (liquid nitrogen and uc, respectively). liposomes were prepared by extrusion over a stack of avestin polycarbonate filters ( nm), as described [ ] . huh- cells [ ] were maintained in dmem containing . g/ l d-glucose and mm l-glutamine (invitrogen, cergy-pontoise, france), supplemented with u/ml penicillin, mg/ml streptomycin and % fcs (lonza). productions of pseudotyped viruses were obtained by the transient transfection of t cells by the calcium-phosphate method. for the genotype study, hcvpp of genotypes a (h ; af ), b (con ; aj ), a (jfh ; ab ), b (ukn b . , ay ), a (ukn a . , ay ), a (ukn . , ay ), a (ukn . . , ay ) and a (ukn . . , ay ) were produced as described previously [ ] from t cells co-transfected with a murine leukemia virus (mlv) gag-pol packaging construct, an mlv-based transfer vector encoding gfp as a marker protein, and the e -e expression constructs. supernatants were collected h post-transfection and filtered on . mm. for genotypes a and a, pseudoparticles were concentrated -fold after ultracentrifugation through a % sucrose cushion at , g for h at uc. pellets were resuspended in the regular medium of huh- cells. for infection experiments, huh- cells were seeded at cells/well in -well plates. the following day, cells were infected in the presence of increasing arb concentrations for h. arbidol effect on viral infectivity was evaluated by assaying gfp activity hours after infection using flow cytometry (facscalibur). pseudoparticles harbouring at their surface the influenza hemagglutinin (happ) and the envelope glycoprotein of the rd feline oncovirus (rd pp) were prepared as described in [ ] and [ ] , respectively. lipid mixing between pseudoparticles and pc:chol:r liposomes was monitored by fluorescent spectroscopy, as the dequenching of r [ ] . in brief, r note that numbering in panel a refers to proton numbers, as identified in nmr (cf figure and table ). doi: . /journal.pone. .g ay ( a) . e ; ay ( a) . e ; ay ( a) . e ; ha . e ; rd . e ], and incubated min. fusion was initiated by acidification to ph with hcl, and recorded on an slm aminco spectrofluorimeter over a -min time period, at excitation and emission wavelengths of nm and nm, respectively. maximal r dequenching was measured after the addition of . % triton x- (final concentration) to the cuvette to lyse liposomes. the same procedure was used to follow pseudoparticle fusion in the presence of arb; in this case, after a -min incubation of pseudoparticles with liposomes, arb ( . mm final concentration) was added and incubated for min, and fusion initiated by acidification. indole emission fluorescence spectra of tryptophan derivatives were recorded at excitation wavelength of nm (spectral zone of lowest absorption of arb), under various conditions: nata ( mm final) in pbs at ph . or . ; toe at mm in lyso-pc micelles (toe:lysopc molar ratio : ), and in pc and pc:chol liposomes (toe:lipid molar ratio : ); peptides at mm in pc:chol liposomes (peptide:lipid molar ratio : ). spectra were obtained in the absence or presence of increasing concentrations of arb ( to mm). samples were incubated min at uc prior to recording. emission spectra were collected in the - nm region (with nm-increments), with blanks substracted, using a black flat-bottom, low-binding -well microplate (greiner bioone). measurements were recorded on a tecan infiniteh m spectrofluorimeter. k d app values were calculated from the difference between the areas under the spectra in the absence or presence of arb (da), at various arb concentrations, by nonlinear fitting using the equation da = da max c/(k d +c). fluorescence measurements were repeated three times to obtain averaged values of k d app. guvs were made by the electroformation method [ ] . the flow chamber (warner instruments, connecticut, usa) used for vesicle preparation was equipped with two glass coverslips, each coated with optically transparent and electrically conductive indium tin oxide (ito) (philips, eindhoven, nl). mixtures of lipids [pc:chol:r ( : : , molar ratio)] were prepared at . mm in chloroform. the lipid mixture ( nmoles) was spread into a thin and uniform film on the conductive face of ito-coated slide. after chloroform evaporation, the dried lipid film was hydrated by adding water into the chamber (, ml) and an alternative electrical field ( hz and . v) was applied at room temperature for hours. guvs in the absence or presence of increasing amounts of arb solubilized in water ( to nmoles), were observed by epifluorescence microscopy. interaction of arb with dmpc layers was investigated with a biacore h using a l sensor chip at uc. the sensor chip surface was washed with a mixture of mm naoh and isopropanol ( : , v:v) , at a flow rate of ml/min for min. the running buffer was milliq water. the influence of liposome concentrations on the final spr signal was tested; we assayed lipid concentrations from . to mm and measured the resulting resonance units (ru). we obtained a well detectable, reproducible and stable signal from mm, and further increasing this concentration did not improve the signal. we therefore chose the mm concentration for our experiments. dmpc liposomes were resuspended in milliq water and captured on sensor chip at ml/min for min. the flow rate was increased to ml/min and the liposome surface was then washed with mm naoh for min. liposomes immobilized on the chip surface gave approx. a ru signal. to calculate arb affinity for lipids, its association to and dissociation from dmpc layers were studied at different arb concentrations in water, from . to mm, at a flow rate of ml/min. after each binding cycle, the sensor surface was regenerated to the original matrix by injecting mm naoh/ isopropanol ( : , v:v). the sensor surface was then coated with a fresh liposome suspension for the next binding cycle. k d values were calculated from the equilibrium resonance signal (r eq ) as a function of the analyte concentration. r eq values were estimated by extrapolation to infinite time using plots of resonance signal as a function of the reciprocal of time. apparent k d were then calculated by nonlinear fitting to the expression r eq = r max c/ (k d +c), where r max is the maximum binding capacity of the surface and c is the analyte concentration, using the sigmaplot software. for the nmr studies, the bicellar system was prepared by another sample with similar lipid concentration and higher arb content (molar ratio / ) was also prepared. the amount of free arb in arb:dmpc mixture was estimated after rapid separation of lipids on ultrafiltration membrane (cutoff da) and measure of arb concentration in the ultrafiltrate at nm. for arb:dmpc molar ratio of : at neutral ph, free arb was found to be lower that . %. we thus concluded that the amount of free arb in nmr samples was negligible. h nmr experiments were performed on a bruker dmx spectrometer operating at a proton frequency of mhz. spectra were recorded with a mm triple resonance inverse txi probe equipped with z-gradient. the p/ pulse was . ms, the recycle delay was s and solvent suppression with presaturation was used. d h spectra were measured acquiring scans. these spectra were used for the assignment of the drug signals together with d noesy and h/ c hsqc spectra (data not shown). the assignment of the lipid resonances was derived from the comparison with data in the literature [ ] . to times were measured by using inversion recovery experiments with an interpulse delay ranging between ms and s. each measurement was repeated times, adding scans with a delay time between scans of s. all the spectra were processed using matnmr [ ] . the h frequency scale is given in terms of chemical shift relative to the acetone signal used as an external reference ( . ppm). we have previously shown that arb could inhibit cell entry and membrane fusion of hcvpp of genotypes a, b and a [ , ] , and hcvcc of genotype a [ ] . here we sought to investigate the effect of arb on other major hcvpp genotypes as well. hcvpp infectivity toward huh- cells, objectifying hcvpp entry, was assayed by counting cells positive for gfp (as the marker protein), incubated with or without increasing arb concentrations for h (see materials and methods). a representative data set is shown in figure s a , and inhibition obtained for the highest concentration of arb ( . mm) is presented in figure a . the inhibitory effect of arb on hcvpp cell entry depends on hcvpp genotype. indeed, within biological intrinsic variability of hcvpp preparation and samples, three cases could be distinguished: entry of hcvpp of genotypes a and a was inhibited by ca. %, while a, b and b exhibited a %-inhibition, but entry of genotypes a and a was weakly affected by the presence of arb ( fig. a) . the influence of arb on hcvpp-mediated lipid mixing was assayed by fluorescence spectroscopy using fluorescent liposomes, as previously described [ ] . lipid mixing between hcvpp and liposomes was only observed at low ph and optimal at ph . [ ] . in the presence of increasing arb concentrations, lipid mixing was inhibited in an arb dose-dependent manner ( figure s b for hcvpp genotype a). in contrast to what was observed for hcvpp infectivity, the effect of . mm arb on hcvppmediated membrane fusion ( figure b ) was similar for all tested genotypes, with about % inhibition of membrane fusion activity. this indicates that membrane fusion inhibition by arb is not genotype-dependent. these data suggest that the differential inhibitory effect of arb on hcvpp infectivity of various genotypes is likely due to a genotype-dependent modulation of hcv glycoproteins interaction with the cellular proteins (e.g. hcv receptors) involved in hcv cell entry. conversely, arb inhibition of hcvpp membrane fusion, as assessed by a in vitro model system where the only proteins present are the viral glycoproteins, could merely reflect the interaction of arb on lipids and/or on motifs present in hcv glycoproteins of any genotype. to test these hypotheses, we further investigated arb interaction properties with lipids and protein fragments using the approaches described in the following. we previously showed that arb could interact with liposomes and membrane-like environments such as detergent micelles [ ] . we further investigated this feature by studying the interactions of arb with giant unilamellar liposomes (guv) by optical microscopy ( figure ). guv are pure lipid bilayers, intrinsically flexible and unstable due to their very large size (in the range of tens of mm) [ ] . increasing arb concentrations were added to the chamber where guv composed of pc:chol were electroformed (see methods section), with arb-to-lipid molar ratios of : , : , : , : , : and : . the guv bilayer was unaffected by the presence of arb up to a : arb-to-lipid ratio, with occasional membrane flickerings ( fig. c and asterisk in fig. e ). at higher ratios, membrane inhomogeneities and invaginations appeared (fig. f , asterisks in fig. d ), and a major overall membrane reorganization was observed at a : arb-to-lipid ratio (fig. g ). note that no lysis or membrane dislocation of guv was observed happ are presented as control pseudoparticles sensitive to arbidol (cf also [ ] ), and rd pp insensitive to arbidol (cf also [ ] ). * , the mutant hcvpp w a (cf [ ] ) are presented as a negative control of entry, displaying very low infectivity. b, membrane fusion between hcvpp and r -labeled liposomes was measured by recording the kinetics of lipid mixing by fluorescence spectroscopy (excitation and emission wavelengths were and nm, respectively), as described in the materials and methods section. values of the last s of fusion kinetics (final extent of fusion) were used to calculate the percentage of fusion in the presence of arb, relative to fusion kinetics without arb ( %). results are the mean +/ sem of separate experiments. happ and mutant hcvpp w a were taken as controls. * : no fusion was observed for rd pp. doi: . /journal.pone. .g for any condition, even at the highest ratio (data not shown). these results reveal that only very high concentrations of arb with respect to lipids could significantly perturb the lipid organization of these bilayers. this also indicates that the direct interaction of arb to lipid bilayers at the concentrations used to inhibit hcvpp infectivity and membrane fusion (panel e) do not perturb lipid organization. in addition, hcvpp pre-incubated at neutral or acidic ph with arb, even at very high concentrations ( mm), displayed similar morphology (visualized by transmission electron microscopy) as those observed in the absence of the drug (data not shown). indeed we counted over hcvpp for each condition, and no difference in hcvpp morphology could be observed between the parameters assessed. this indicates that arb inhibition of hcvpp fusion is not due to viral particle disruption/damage. to gain insight into the molecular details of the interaction of arb with lipid membranes, we next investigated the lipid binding properties of arb by using surface plasmon resonance (spr, biacoreh technology). we used a biacore's l sensor chip to capture dmpc liposomes. this sensor chip displays lipophilic groups attached on the surface of a carboxymethylated dextran layer, and was shown to provide a quick and reproducible method for the preparation of bilayer-mimetic systems [ ] . we first tested whether arbidol per se could bind or not to the chip. arb at . mm (the highest concentration relevant in the biological context) was injected onto the chip devoid of liposomes. this led to approx. resonance units (ru, see methods section). dmpc liposomes ( mm) captured onto the sensor chip reached about ru, and a further , ru was seen when arb was pulsed onto the liposome-coated chip. the binding of arbidol alone on the l chip remains therefore negligible. measures of arb/dmpc association and dissociation were performed with various arb concentrations ranging from . to . mm. after passage over the surface of the sensor chip, arb bound to immobilized dmpc in a concentration-dependent manner ( figure ). arb initial binding was fast, but then slowed down without reaching saturation equilibrium (from to s). after stopping the arb flow onto the sensor chip (from s), bound arb was rapidly but incompletely dissociated from dmpc membranes. indeed, for all arb concentrations tested, about % of arb remained bound to dmpc. this demonstrates that arb is capable of interacting with lipid membranes, in a stable association between arb and dmpc. however the behaviour of arb binding to membranes rendered difficult the fitting of a kinetic model to the data, and hence the determination of reliable on-and off-rates. indeed using global fitting, binding curves could not be fitted properly with the different models included in the biaevalution . software ( : langmuir binding, bivalent analyte, heteroge- neous ligand, heterogeneous analyte, conformational change), with or without mass transport. furthermore, because equilibrium was not reached during the association phase, the direct use of scatchard analysis to calculate the apparent equilibrium dissociation constant was not allowed. instead, the apparent equilibrium dissociation constant k d was calculated from the equilibrium resonance signal (r eq ) as a function of analyte concentration, r eq values being estimated by extrapolation to infinite time using plots of resonance signal as a function of the reciprocal of time [ , ] . apparent k d was then calculated by nonlinear fitting to the expression r eq = r max c/(k d +c), where r max is the maximum binding capacity of the surface and c is the analyte concentration, using sigmaplot software. this calculation, performed on separate experiments, gave an apparent k d of . . mm (see inset to figure for a representative experiment). this dissociation constant is in the same order as the ic of hcvpp fusion ( . mm) . this result indicates that the inhibitory effect of arb on hcvpp membrane fusion is at least in part deriving from arb association to lipid membranes. nmr spectroscopy was used to characterize the arb insertion in a model membrane system. the h nmr spectrum of arb recorded at k in deuterated water is shown in figure a (black spectrum), and the assignment of the proton signals deduced from d spectra analysis (data not shown) are reported in table . in order to study arb in a membrane-mimetic environment, we used isotropic phospholipid bicelles consisting of a mixture of dmpc/dhpc in water. long-chain phospholipid molecules of dmpc self-assemble into planar bilayers, while the short-chain molecules of dhpc segregate to edge regions of high curvature [ ] . bicelles with [dmpc]/[dhpc] molar ratio , form fast and isotropically tumbling aggregates, amenable to solution nmr studies. still, isotropic bicelle systems are used as a phospholipid bilayer mimetic, since dmpc has been shown to form a flat bilayered surface [ ] [ ] [ ] . the h nmr spectrum of arb in this bicellar phase is shown in figure a (red spectrum). in this system, the spectral crowding due to the presence of phospholipid resonances allowed only the observation of protons denoted , , , , and of the arbidol molecule (see fig. a ) where only signals can be distinguished for the three protons , , . additional h resonances could be resolved from d h noesy spectra and h/ c hsqc spectra ( figure s ) and the corresponding chemical shifts are shown in table together with the assignment of the proton lines for arb in water. arbidol interaction with lipids induce chemical-shift changes in the arb resonances when compared to that observed in water. in order to investigate the immersion depth of arb in the membrane, we monitored the proton longitudinal relaxation rate of arb protons upon the addition of the soluble paramagnetic agent gadolinium-diethylenetriamine pentaacetic acid-bismethylamide gd(dtpa-bma) [ ] . this paramagnetic contrast agent stays soluble in the water surrounding the membrane and induces a paramagnetic relaxation enhancement (pre) on the spin of the atoms close to the surface of the membrane. recently, pre effects due to gd(dtpa-bma) were used to probe the immersion depth and orientation of an anti-microbial peptide [ , ] . here we measured the proton t relaxation times of both arb and phospholipid protons to probe the immersion depth of arb in the membrane, using the pre values of the phospholipids as an approximated yardstick. a titration of arb with gd(dtpa-bma) was performed at increasing concentrations of the paramagnetic agent, from . to . mm. at each step of the titration, the proton t relaxation times for the protons , , , , and of arb, and for the protons alpha, beta, gamma, g , g , g , c , c and omega of the phospholipids, were measured. the plot in figure b shows the relaxation times as a function of the gd(dtpa-bma) concentration. this titration leads to a curve for each proton whose slope corresponds to the pre values. as shown in fig. c , pre measured on the arb-bicelle system range from . sec to . sec for the phospholipid protons and from . sec to . sec for arb protons. the pre observed for each spin can be described as an overall relaxation enhancement [ ] , due to all the paramagnetic agents in solution. for a planar membrane surrounded by a buffer containing a non-interacting paramagnetic probe, the total pre of a nucleus with immersion depth d is given by the equation [ , ] : pre = z/d , where d is the immersion depth of a specific nucleus within the membrane plus the radius of the magnetic probe, and where the constant z is a combination of various parameters, among them a correlation time, itself a combination of the electron relaxation time, the lifetime of the intermolecular adduct bicelle-gd(dtpa-bma), and the rotational correlation time. in order to determine the immersion depth of arb, instead of determining z, we used the phospholipids as a yardstick by comparing their pre with the one of arb. the pres of the resolved signals of arb and phospholipids are reported and compared in figure c . this procedure is based on two assumptions: (i) the amount of free arb in solution in the presence of lipids is negligible (see nmr sample preparation in experimental procedure section), and it does therefore not affect significantly the pre of arb in the membrane ; ii) the constant z is the same for lipids and arb. figure c shows that methyl protons of arb are at the lipid/ water interface as the gamma-proton of the hydrophilic headgroup of the lipids. protons and of arb are at the level of respectively protons alpha and g of the phospholipids. the aromatic protons and are the most buried protons of arb, close to the beginning of the hydrophobic chain (protons c ). the validity of assumption (ii) is supported by the noe crosspeaks detected between proton of arb and the glycerol moiety of phospholipids. in addition, the maximum pre measured for arb (protons ) and phospholipids (c protons) are about the same, suggesting that the corresponding molecule regions are the most exposed to water. this estimation of the immersion depth of arb protons relative to the phospholipids protons enables us to propose a model for the positioning of arb in the membrane, shown in figure d . these nmr data clearly demonstrate that arb interacts at the membrane interface, mainly at the level of phospholipid polar head. this result supports the assumption that the arb inhibitory effect on hcvpp membrane fusion is dependent, at least in part, from this interaction. a second possibility regarding arb activity is that arb might interact with key motifs present in viral proteins, thereby impeding their structural reorganization at the onset of fusion and thus leading to fusion inhibition. a first set of experiments was designed to investigate whether the order of addition of fusing partners would affect arb-induced fusion inhibition. for this purpose, we measured fusion after preincubation of hcvpp or liposomes or both in the absence or presence of . mm arb. as shown in table , when arb was pre-incubated with both partners before fusion was initiated by lowering the ph, fusion inhibition was ca. %. in contrast, only ca. % fusion inhibition was observed when arb was preincubated with either hcvpp or liposomes. the greater inhibitory effect of arb when it has simultaneously access to both viral and target membranes suggests that arb could also act by interacting with selective residues of the hcv glycoprotein sequences. this assumption was tested by studying the interaction behaviour of arb with tryptophan (trp) derivatives, as tryptophan is a constituent of proteins often found in regions located close to membrane interfaces, such as stem regions in several viral fusion proteins (e.g. hiv gp [ ] ). we also reasoned that arb, being an indole derivative, might interact with tryptophan and tyrosine residues through aromatic ring stacking. for this purpose, we tested the effect of increasing concentrations of arb on the fluorescence of n-acetyl tryptophanamide (nata) as a water-soluble trp derivative, and tryptophan octyl-ester (toe) as a membranotropic molecule ( figure b-c) . the fluorescence of nata and toe was recorded between and nm, using an excitation wavelength of nm, which corresponds to an absorption minimum of arb [ ] . results are presented in table and figure . (table ) . similarly, the k d of arb for toe in lyso-pc micelles was in the mm range at both ph. this indicates that arb is able to interact with indole rings, but with a relatively low affinity. in contrast, when arb was added to toe associated to liposomes ( : , toe/lipid molar ratio), a marked increase in affinity was observed (table , compare toe/micelles and toe/ liposomes), reaching k d values in the mm range. note that toe fluorescence could not be measured in dpc micelles, due to a great intrinsic fluorescence of the dpc used for our experiments. interestingly, these k d values are comparable to arb ic inhibition of hcvpp fusion (see above and discussion section). indole fluorescence decreased when arb concentration increased, with virtually no measurable fluorescence for mm arb ( figure ). this further confirms that arb interacts with indole rings, but with a higher affinity when indole is incorporated into lipid membranes. this affinity was higher for pc than for pc:chol liposomes (table ) , and at neutral than at acidic ph (table and figure ). at acidic ph, arb is most likely protonated in the lipid environment [ ] , as is probably toe as well. arb affinity for toe under these conditions might then be lower than that of uncharged arb at neutral ph, because of repulsive electrostatic interactions. a third set of experiments was designed to assess the behaviour of arb in the presence of aromatic residues into protein sequences, more specifically toward trp present in peptides. for this purpose, we used synthetic membrane-binding peptides of known structure and containing only one tryptophan residue, expected to be localized at the membrane interface: the transmembrane helix of hcv ns a protein [ ] and the n-terminal amphipathic helix of bvdv ns a protein interacting in-plane of the membrane interface [ ] . intrinsic tryptophan fluorescence of both peptides was monitored in the presence of increasing concentrations of arb; this is illustrated in figure for hcv ns a peptide inserted into pc:chol liposomes (and in figure s for bvdv ns a, : peptide-to-lipid molar ratio). the arb dose-dependent quenching of tryptophan fluorescence at both neutral and acidic ph clearly indicates arb interaction with both peptides (insets in figure and fig. s ). for the ns a peptide at both ph, a red shift of the spectral maximum, proportional to arb concentration, accompanied the fluorescence quenching; this effect was more pronounced at acidic ph ( nm at ph . for mm arb, and nm at ph . for mm arb). this red shift suggests that arb, when interacting with ns a peptide, relocates its tryptophan residue to a more shallow zone of the membrane, where the trp environment would be more hydrophilic. the measure of the apparent affinity of arb for these peptides inserted into pc:chol liposomes was performed as described above. interestingly arb displayed an apparent k d toward peptide trp between . and . mm (table ), twice lower than that observed for toe in pc:chol liposomes. since both peptides contain one or two tyrosine residues (hcv ns a and bvdv ns a, respectively) in addition to the trp, interaction of arb molecules with these aromatic residues might account for a higher affinity of arb for peptides than for a small molecule such as toe. since arb is an inhibitor of hcv membrane fusion, we reasoned that it might interact with the regions of e and e described as important for hcv fusion [ , ] . these peptides were described as membranotropic on model membranes [ ] and contain aromatic residues. we therefore analyzed the effect of increasing concentrations of arb on the fluorescence quenching of two peptide sequences derived from hcv e (positions - and - , see aa sequences in table ), and inserted into pc:chol liposomes ( : peptide-to-lipid molar ratio). note that we also tested a third peptide located at position - of e , containing only one tyr; but its fluorescence quantum yield was too low to monitor any interpretable fluorescence signal (data not shown). we then calculated the k d values as described above. as shown in table , e - contains only one trp, whereas e - contains one trp and tyr. the apparent affinity was in the mm range at ph . for both peptides, reminiscent to arb ic of hcvpp fusion. this indicates that arb is able to interact with the aromatic residues of both peptides in the membrane, and lends further support to our hypothesis that arb could interact with key residues/motifs in viral fusion proteins, which would constitute a possible (partial) explanation to its inhibition of hcvpp fusion. strikingly this affinity decreased at acidic ph for both peptides, and even drastically to mm for e - . interestingly, this relatively high k d value is reminiscent of that observed for arb interaction with nata in solution (table ) . this suggests that the interaction between the e peptide and the membrane would be weak at acidic ph, and that most of the peptide could be in solution. moreover an histidine residue, located in the immediate vicinity of trp in the sequence of both peptides, is expected to be charged at ph . . since arb is also protonated at that ph value, this could create repulsive forces affecting the interaction between trp and arb. moreover, as protonation of the histidine cycle is expected to decrease the free energy of partition from lipids to water, the peptide could be released from the membrane at acidic ph, possibly in relation with peptide conformational change(s). this behavior in not in favor with their direct role as fusion peptides of hcv, a virus dependent on low ph for its membrane fusion activity. however, due to their membranotropism [ ] , and since our and other studies showed their involvement in hcv membrane fusion [ , ] , it is possible that the conformational changes they might undergo at low ph would lead to a proper relocation of the actual fusion peptide/loop toward the target membrane [ ] (and see discussion section). this study aimed at further investigating the molecular mechanism of action by which arbidol (arb) inhibits virus cell entry and membrane fusion, using hcvpp as a model of an enveloped virus. we showed that arb displayed a dual binding capacity, on lipid membranes interface on one hand and on the aromatic residue table ). doi: . /journal.pone. .g tryptophan of proteins on the other hand. it therefore appears plausible that the observed inhibitory effect of arb on viral entry and membrane fusion might result from a combined effect of binding of arb on membranes and on (fusion) proteins. from a physico-chemical point of view, arb displayed tropism for membranes or membrane-like environments such as detergent micelles, particularly prominent at low ph [ ] . by combining several biochemical approaches, we show here that arb has the propensity to bind to and incorporate into lipid bilayers, with calculated apparent affinities in a similar range as the ic value for fusion, i.e. ca. mm. our nmr studies of arb interaction with dmpc leads to a model where arb binds at the membrane interface and establishes contacts mainly with the polar heads of phospholipids (fig. d ). altogether these data suggest that at least part of arb inhibitory activity could be explained by its membranotropism. this physicochemical property has been further emphasized in a recent work by villalain [ ] , using fourier-transform infrared spectroscopy. arb interaction with phospholipids would disturb membrane fluidity crucial to the fusion process, thereby rendering the lipid bilayer less prone to fusion. such a model is consistent with the behavior of other indole derivatives, that were shown to exhibit a preference for membrane interfaces [ , ] , due to the flat rigid structure of these molecules and to their aromaticity, which allows them to establish cation-p interactions with the positively charged quaternary ammonium lipid headgroups [ , ] . at low ph, the optimal ph for hcv fusion, these interactions would be favored due to the protonation of the amino groups. as was described for other substituted indoles [ ] , it is possible that protonation of the carbon bearing the ester group of arb could displace this group out of the indole plane, and place it in a better position to bond with neighboring molecules. this could in turn lead to a better membrane association. arb might therefore have the propensity to intercalate into lipids of the viral and target membranes while adopting a consistent orientation by filling the gaps between lipid molecules. the interfacial region of the lipid bilayer provides a suitable environment for a wide range of chemical groups, as long as they possess a large enough hydrophobic moiety and a group capable of forming hydrogen bonds with the lipid carbonyl groups. several compounds with antiviral pharmacological properties belong to this category, in particular adamantanes active against influenza a viruses [ , ] and against some hcv clones but not all [ , ] , the natural triterpene glycyrrhizin efficient in the treatment of chronic viral hepatites [ ] and the flavonolignan molecules composing silymarin, an herbal extract with potent anti-hcv activities [ ] [ ] [ ] , ] . in a previous study, we noticed that arb inhibition of cell entry concerned hcvpp and pseudoparticles bearing the influenza hemagglutinin (happ), but not pseudoparticles bearing the envelope glycoprotein of a feline oncogenic retrovirus (rd pp) [ ] . these data suggest that arb might display selectivity for the recognition of key motifs inside envelope proteins. this hypothesis was tested by assessing the influence of arb on the fluorescence properties of aromatic compounds derived from tryptophan (trp) and of peptides containing trp. trp is a component of proteins with interfacial properties [ , ] , often located at the lipid/water interface and grouped into so-called tryptophan-rich motifs crucial to protein/membrane association [ ] , and found in the envelope (fusion) proteins of the sars coronavirus or hiv- [ , , ] . trp is also enriched at protein/protein binding interfaces of the small envelope protein of the hepatitis b virus [ ] and of membrane proteins in general [ ] . we demonstrated here that arb was able to alter/quench the fluorescence properties of small trp derivatives in solution (nata), in detergent micelles and in liposomes (toe, [ ] ), in a dose-dependent manner. this occurred most likely through stacking of the aromatic rings of both molecules which is often involved in stabilization of inter-cations. interestingly the apparent affinity of the arb/trp derivative interaction was in the order: lipid bilayers.micelles.solution, indicating that arb binding strength for trp could increase in membrane environments where both molecules accomodate and get packed. indeed arb apparent affinity for toe in liposomal membranes was in the mm range, a value comparable to the ic of fusion. arb affinity was even greater for membrane peptides containing trp and tyrosine (tyr) residues (ca. mm). due to its indole group, it is conceivable that arb might display selectivity not only for indole rings (trp) but more generally for aromatic groups, as the phenol ring of tyr. a greater number of arb molecules could therefore interact with aromatic residues in peptide sequences, leading to some cooperativity in the quenching effect and to an overall larger apparent affinity. the nmr structures of synthetic peptides hcv ns a * and bvdv ns a peptides have been reported in references [ ] and [ ] , respectively. the solubilization tags kkgg and ggkk at the n-and c-terminal ends, are indicated in italic. aromatic residues trp and tyr are indicated in bold, his is underlined. b peptide-to-lipid molar ratio was : . although hcv entry inhibition by arb was found genotypedependent, hcv membrane fusion was inhibited by arb in a genotype-independent manner. hcv entry and fusion are early steps in the life cycle of the virus [ , ] . hcv first interacts through its envelope glycoproteins with a set of coreceptors at the plasma membrane level (recently reviewed in [ , ] ) and eventually becomes endocytosed [ ] [ ] [ ] . due to a combined action of acidification in the endosome and particular lipids like cholesterol and sphingomyelin [ , ] , viral fusion occurs over a broad spectrum of ph's ranging from . to . [ , , ] . hcv binding to the hepatocyte membrane followed by endocytosis therefore requires several cellular proteins, and most likely involves several levels of interactions (interactions between viral proteins, between cellular and viral proteins, between viral/cellular proteins and lipids). these features might explain the differential effect exerted by arb on entry of various hcv genotypes: indeed subtle differences in protein sequences could translate into modified interactions with several partners and/or at several levels. conversely some common principles of action apply to all fusion reactions, viral fusion and cellular fusion processes alike [ ] . indeed all fusion processes involve two partners: lipids and the fusion protein(s). this might account for the similar inhibitory effect of arb on hcv fusion observed for all genotypes. this is in line with the observations that arb displayed potent antiviral activity against some antigenic serotypes of influenza viruses, but not against all [ ] . previously we noticed that arb inhibition of primary infection of huh- . . cells with hcv (clone jfh- ) was efficient only when cells were preincubated with arb or h before infection [ ] ; in addition, inhibition of hcvpp and happ cell entry was most efficient when arb was pre-incubated with both viral and cell membranes [ ] . here, using our in vitro fusion assay, we observed that arb inhibition of hcvpp fusion was maximal when both viral and target membranes were incubated with arb, before fusion was initiated. this suggests that a certain level of membrane impregnation and/or saturation with arb must be achieved to efficiently inhibit viral infection. membranes might therefore act as ''concentrators'' of arbidol, and high concentrations of the molecule might be locally achieved. this could explain why arb, exhibiting an apparent (medium to low) affinity for membranes in the mm range, exerts a relevant antiviral activity without noticeable membrane damages. along these lines, in spite of its marked membranotropism, arb displays only low toxicity [ , ] . arb exhibited a comparable micromolar apparent affinity for aromatic residues present in membrane peptides in a membrane environment. altogether, these observations lead us to propose a mechanistic model of the way arb would inhibit hcv entry and fusion. through its membranotropism, arb is able to freely interact with viral and target membranes, and could locally get highly concentrated. arb is also able to interact with aromatic residues within viral proteins involved in membrane interactions and membrane destabilization necessary for fusion. through this dual binding capacity, arb could then locally impede the structural rearrangements required for the fusion protein to adopt its fusion conformation. the fact that arb is active in the mm range suggests that arb would act by reducing the overall speed of the fusion reaction rather than by blocking a specific protein conformation. this could therefore explain the broad antiviral spectrum of arb, and the genotype independence of its inhibitory effect on hcv fusion, since hcv envelope proteins contain well-conserved aromatic residues in all genotypes. mechanistically, the key point is the relative accessibility of these residues to arb at the membrane interface. a cooperative effect between arb and several aromatic residues might therefore occur. also the local environment of these aromatic aa is important, since the presence of residues such as histidines (his) in their vicinity could modify their accessibility with respect to ph. interestingly enough, in the sequence of both hcv e peptides studied here (table ) and shown to be involved in hcv fusion [ ] , his is contiguous to trp, and in the - peptide, his is surrounded by three tyrosines. the concept of his as a critical ph sensor at a key intramolecular domain interface in a viral fusion protein has recently emerged [ , ] . indeed, the protonation of a sole his in the e protein of the tick-borne encephalitis flavivirus (tbev) triggers large-scale conformational changes leading to viral fusion. concerning hcv, rey and coworkers recently proposed a model of the d arrangement of the e ectodomain [ ] . in this model, the fusion loop/peptide would lie within the poorly structured domain ii, and the e - peptide would be found in the globally unstructured domain iii, where a critical his residue is disposed at the interface with domain i. the putative fusion loop contains a phenylalanine and a tyrosine [ ] . at low ph, the optimal ph for hcv membrane fusion, key histidine(s) could become protonated. this could result in conformational rearrangements and, in the context of arb fusion inhibition, aromatic residues might consequently become more or less accessible to arb molecules present in their vicinity. we noted that the apparent affinity of arb for hcv peptides was weaker at ph . than at ph . . at low ph, arb is also protonated, and this protonated form could exhibit a greater preference for the interfacial region of the lipid bilayer than the deprotonated form, as demonstrated for adamantanes [ ] . combined with the notion that key aromatic and his residues would also display interfacial (re)localization at low ph, this would in turn explain the higher efficiency of arb at inhibiting fusion at acidic ph [ ] . in conclusion our data reveal that arb directly interacts with the lipid membrane-water interface, and is able to bind to aromatic residues present in hcv glycoproteins, in their membraneassociated form. through a subtle binding interplay between arb, lipids, viral and cellular proteins, arb might efficiently block hcv entry and membrane fusion interacting with the main actors of the early steps of viral entry. most interestingly, arb inhibition of these processes demonstrated an affinity in the mm range, although the membranotropic properties of arb suggest that it could become locally more concentrated in membranes. together, these findings suggest that arb could increase the strength of viral glycoprotein's interactions with the membrane due to a dual binding mode, involving aromatic residues and phospholipids. the resulting complexation would inhibit the expected viral glycoprotein conformational changes required during the membrane fusion process. the antiviral mechanism of arb therefore opens promising perspectives for the development of small membranotropic low affinity molecules, that would become locally concentrated in membranes and would mainly act on the kinetics of the conformational rearrangements of the viral fusion protein. figure s arb inhibits infectivity and membrane fusion in a dose-dependent manner. a, infectivity. results are the mean sem of separate experiments. black, no arb; blue, . mm; green, . mm and red, . mm arb, respectively. b, membrane fusion between hcvpp of genotype ( . . ) and r -labeled liposomes. the lipid mixing kinetic was followed by fluorescence spectroscopy using excitation and emission at and nm, respectively. fluorescent liposomes ( . mm final lipid concentration) were added to ml of hcvpp in pbs ph . at uc, in the absence or presence of the indicated concentrations of arb. after a min-equilibration, lipid mixing was initiated by decreasing the ph to . with diluted hcl, and r dequenching was recorded. maximal fluorescence was obtained after addition of . % final triton x- . average value of the last s of fusion (i.e. final extent of fusion) was used to calculate the percentage of fusion in the presence of arb, relative to % fusion without arb ( figure ) . black, no arb; blue, . mm green, . mm and red, . mm arb, respectively. (tif) of increasing concentrations of arb ( , , , and mm from top to bottom). trp emission fluorescence was measured between and nm, with excitation at nm. the apparent affinity of arb toward trp was calculated from the plot of the difference da between areas under the curve (auc) of peptide without arb (da = auc no arb auc with arb ) as a function of arb concentration (insert). 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temperature-dependent activation of hepatitis c virus for low-ph-triggered entry hepatitis c virus entry requires a critical postinternalization step and delivery to early endosomes via clathrincoated vesicles rna interference and single particle tracking analysis of hepatitis c virus endocytosis functional analysis of hepatitis c virus envelope proteins, using a cell-cell fusion assay viral and developmental cell fusion mechanisms: conservation and divergence identification of specific histidines as ph sensors in flavivirus membrane fusion the ph sensor for flavivirus membrane fusion the xplor-nih nmr molecular structure determination package this work was presented at the th international symposium on hepatitis c and related viruses, (nice, france, october - , ).the authors gratefully acknowledge sylvie ricard-blum for spr analysis expertise. fluorescence experiments were performed on the platform ''production et analyse des protéines'' of the ifr biosciences gerland-lyon sud. key: cord- -u hfkjv authors: xu, mengchang; zhou, wenhu; chen, xuncai; zhou, ying; he, binsheng; tan, songwen title: analysis of the biodegradation performance and biofouling in a halophilic mbbr-mbr to improve the treatment of disinfected saline wastewater date: - - journal: chemosphere doi: . /j.chemosphere. . sha: doc_id: cord_uid: u hfkjv disinfectant-containing wastewaters have been generated from many places, including marine industries. the synthetic naclo-containing wastewaters have been effectively treated in a saline mbbr-mbr (moving bed biofilm reactor & membrane bioreactor) system containing marine microorganisms. a low concentration of nacl (below mg/l) is not enough to kill the microorganisms, but can affect their bioactivity and induce membrane biofouling. a linear relationship has been obtained for the half-life of membrane biofouling as a function of the naclo concentration ( - mg/l): [half-life] = － . ×[naclo concentration]. the cod and nh( )-n removals are the highest at a salinity of g/l for the marine bioreactors. the behaviour of the typical biofoulants, measured real-timely by fluorescence spectroscopy, can indicate the levels of membrane biofouling and microbial activity, responding to the naclo and nacl influences. based on the behavior of biofoulants, this work has also proposed a novel strategy of biofoulants monitoring for membrane antifouling, where antifouling responses can be carried out when the concentration of biofoulants significantly increases. eps and smp are key biofoulants that require to be monitored for membrane antifouling. degraded to be smaller molecules with less biofouling tendency (tan and li, ) . following the above antifouling strategies, however, a proper monitoring system is required to warn us j o u r n a l p r e -p r o o f about any biofouling situations in mbr for timely antifouling responses (flemming, ) . transmembrane pressure (tmp), read from a pressure gauge, has been widely used as an indicator in practice for real-time monitoring, where a high tmp over kpa means a significant membrane fouling (liang et al., ) . however, the increase of tmp lags behind the changes in respectively, as shown in figure a . as a novel strategy of biofoulants monitoring for membrane antifouling, antifouling responses could be carried out when the detected fluorescent intensity significantly increased. as shown in figure b , the intensity of biofoulants significantly increased at the th day and the th day responding to the environmental shocks. as soon as the warnings observed, the naclo and nacl shocks were removed for antifouling when the novel strategy was used. subsequently, the intensity of biofoulants gradually decreased. the tmp then had a slower raising rate, showing a smaller tendency of membrane fouling. the antifouling response was carried out when the intensity of biofoulants suddenly increased. the recovery of bioactivity of bioreactors took about days (from th day to th day; from th day to th day). overall, using the novel strategy of biofoulants monitoring the membrane biofouling rate was approximately half of the rate when the conventional strategy of tmp monitoring was used. it is suggested that the spectroscopic monitoring of membrane biofoulants has anti-biofouling advantage in the saline mbbr-mbr system for the naclo-containing wastewater treatment. the naclo-containing saline wastewaters were effectively treated. the mbr was not sensitive to low naclo concentrations under mg/l. a linear relationship was noticed for the half-life of membrane biofouling as a function of the naclo concentration. the synergistic detrimental effect from naclo and nacl was found to be higher than their toxicities simply acting alone. the behaviour of the typical biofoulants, measured real-timely by fluorescence spectroscopy, manual of diagnostic tests for aquatic animals . methods for disinfection of aquaculture establishments correlation of eps content in activated sludge at different sludge retention times with membrane fouling phenomena enhanced membrane biofouling potential by on-line chemical cleaning in membrane bioreactor cleaning and disinfection for community facilities: interim recommendations for u.s. community facilities with suspected/confirmed coronavirus disease anaerobic membrane bioreactors for antibiotic wastewater treatment: performance and membrane fouling issues gb - : safety and hygienic standard for disinfection by sodium hypochlorite generator water and wastewater monitoring methods membrane fouling reduction and improvement of sludge characteristics by bioflocculant addition in submerged membrane bioreactor water sprinkler transforms into "disinfection vehicle long-term operation of submerged membrane bioreactor (mbr) for the treatment of synthetic wastewater containing styrene as volatile organic compound (voc): effect of hydraulic retention time (hrt) role and levels of real-time monitoring for successful anti-fouling strategies -an overview 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extracellular polymeric substances (epss) in membrane bioreactors: characteristics, roles in membrane fouling and control strategies observations of fouling biofilm formation applicability of an integrated moving sponge biocarrier-osmotic membrane bioreactor md system for saline wastewater treatment using highly salt-tolerant microorganisms effect of high salinity on activated sludge characteristics and membrane permeability in an immersed membrane bioreactor effect of high salinity on activated sludge characteristics and membrane permeability in an immersed membrane bioreactor novel external extractive membrane bioreactor (embr) using electrospun polydimethylsiloxane/polymethyl methacrylate membrane for phenol-laden saline wastewater desalination and reuse of high-salinity shale gas produced water: drivers, technologies, and future directions protein, cell and bacterial fouling resistance of polypeptoid-modified surfaces: effect of side-chain chemistry cultivation of activated sludge using sea mud as seed to treat industrial phenolic wastewater with high salinity real-time monitoring of biofoulants in a membrane bioreactor during saline wastewater treatment for anti-fouling strategies behaviour of fouling-related components in an enhanced membrane bioreactor using marine activated sludge a critical review on saline wastewater treatment by membrane bioreactor (mbr) from a microbial perspective membrane fouling from ammonia recovery analyzed by atr-ftir imaging review on the state of science on membrane bioreactors for municipal wastewater treatment concentration and detection of sars coronavirus in sewage from xiao tang shan hospital and the th hospital of the chinese people's liberation army impact of temperature seasonal change on sludge characteristics understanding ultrafiltration membrane fouling by soluble microbial product and effluent organic matter using fluorescence excitation-emission matrix coupled with parallel factor analysis removal and degradation mechanisms of sulfonamide antibiotics in a new integrated aerobic submerged membrane bioreactor system probing the radical chemistry in uv/persulfate-based saline wastewater treatment: kinetics modeling and byproducts identification key: cord- -kjzkk t authors: zou, guijin; liu, yue; gao, huajian title: eml webinar overview: simulation-assisted discovery of membrane targeting nanomedicine date: - - journal: extreme mech lett doi: . /j.eml. . sha: doc_id: cord_uid: kjzkk t the covid- pandemic has brought infectious diseases again to the forefront of global public health concerns. in this eml webinar (gao, ), we discuss some recent work on simulation-assisted discovery of membrane targeting nanomedicine to counter increasing antimicrobial resistance and potential application of similar ideas to the current pandemic. a recent report led by the world health organization (who) warned that million people worldwide could die of bacterial infections each year by . to avert the crisis, membrane targeting antibiotics are drawing increasing attention due to their intrinsic advantage of low resistance development. in collaboration with a number of experimental groups, we show examples of simulation-assisted discovery of molecular agents capable of selectively penetrating and aggregating in bacterial lipid membranes, causing membrane permeability/rupture. through systematic all-atom molecular dynamics simulations and free energy analysis, we demonstrate that the membrane activity of the molecular agents correlates with their ability to enter, perturb and permeabilize the lipid bilayers. further study on different cell membranes demonstrates that the selectivity results from the presence of cholesterol in mammalian but not in bacterial membranes, as the cholesterol can condense the hydrophobic region of membrane, preventing the penetration of the molecular agents. following the molecular penetration, we establish a continuum theory and derive the energetic driving force for the domain aggregation and pore growth on lipid membrane. we show that the energy barrier to membrane pore formation can be significantly lowered through molecular aggregation on a large domain with intrinsic curvature and a sharp interface. the theory is consistent with experimental observations and validated with coarse-grained molecular dynamics simulations of molecular domain aggregation leading to pore formation in a lipid membrane. the mechanistic modelling and simulation provide some fundamental principles on how molecular antimicrobials interact with bacterial membranes and damage them through domain aggregation and pore formation. for treating viral infections and cancer therapy, we discuss potential size- and lipid-type-based selectivity principles for developing membrane active nanomedicine. these studies suggest a general simulation-assisted platform to accelerate discovery and innovation in nanomedicine against infectious diseases. eml webinar speakers are updated at https://imechanica.org/node/ one of the most important public health issues that the world faces today is the threat of global infectious disease outbreaks. in the last several decades, newly identified and re-emerging virus related infectious diseases including human influenza, hiv/aids, severe acute respiratory syndrome (sars), dengue, malaria, ebola, middle east respiratory syndrome (mers) have demonstrated serious epidemic potential with devastating loss of life and wealth. the covid- pandemic has brought infectious diseases again to the forefront of global public health concerns. as of jun. , , more than million people had been infected and hundreds of thousands had died worldwide [ ] . in addition to the virus, bacterium is another major human pathogen that are responsible for a wide range of lifethreatening infections [ ] . antibiotics are widely prescribed and can effectively kill or inhibit the growth of metabolically active bacteria [ , ] . however, in recent years, the increasing prevalence of antibiotic resistant strains and the ability of bacterial cells to convert to a quiescent subpopulation (often referred to as "persisters" [ ] ) that exhibits a high level of tolerance to most antibiotics [ ] [ ] [ ] [ ] [ ] have caused clinical challenges that may eventually lead to a crisis level shortage of effective antibiotics for treating bacterial infections. a recent report led by the world health organization (who) warned that million people worldwide could die of bacterial infections each year by [ ] . consequently, there is an urgent societal need to develop novel antibiotics with unconventional antimicrobial strategies. antimicrobial chemotherapy seeks to eradicate the infecting pathogen from its host in the shortest possible treatment period. most conventional antibiotics targeting biosynthetic processes that occur in actively growing bacteria, including the biosynthesis of proteins, rna, dna, peptidoglycan and folic acid [ ] , fail to meet the challenges related to the mutation, resistance and persisters [ ] [ ] [ ] [ ] [ ] . on the other hand, the bacterial membrane, as an essential structural and functional component of biological organisms, providing selective permeability for cellular homeostasis and metabolic energy transduction even if the bacteria are in the dormant state, has attracted increasing attention as targets for new generation antibiotics, as membrane-active agents are inherently effective on sleeping bacterial persisters and also difficult for the bacterial to acquire resistance [ ] . viruses, especially the rna viruses such as the influenza and coronaviruses, are highly susceptible to mutations [ ] . since the outbreak of sars-cov- in january, abundant mutations have been reported in just a few months [ ] [ ] [ ] [ ] , and membrane active nanomedicine might be a promising but underexploited approach to effectively counter viral mutations. in this webinar, we discuss some examples of simulation-assisted discovery of molecular agents which are promising candidates for a new generation of membrane-active antibiotics and potential application of similar ideas to the current pandemic. as shown in fig. , the webinar covered the antimicrobial mechanisms of synthetic antibiotics in attachment, penetration and aggregation in the cell membrane, leading to membrane distortion, pore formation and rupture. some derived theory and physical insights can also be applied to some antiviral agents that seem effective for a broad range of viruses by disrupting the lipid envelop [ ] , which may help discover, design and fabricate membrane targeting antiviral agents to combat covid- . the study suggested a simulation-assisted platform to accelerate discovery and innovation in nanomedicine. j o u r n a l p r e -p r o o f fig. interactions between membrane targeting antibiotic agents and bacterial membrane, including membrane attachment and penetration, diffusion and aggregation within the membrane, and membrane deformation, distortion, pore formation and rupture. staphylococcus aureus is an opportunistic human pathogen carried by approximately one third of human populations, and infections by s. aureus remain a major cause of death [ ] . to demonstrate how simulation can assist the discovery of membrane-active antibiotics, we started from all-atom molecular dynamics (md) simulations of the interactions between a class of retinoids (fig. a ) and the lipid bilayer membrane of s. aureus. a previously established lipid bilayer composed of dopc/dopg at a : ratio was adopted to model the negatively charged s. aureus membrane [ ] . four synthetic retinoids (fig. a) were systematically investigated, among which cd and cd were identified as potential antibiotics through a biological screening assay [ ] , while adarotene and adapalene were selected because of their similar structures as cd and cd . the simulations showed that the carboxylic acid and the phenolic groups of cd , cd and adarotene help anchor these retinoids to the surface of the membrane bilayer through binding with hydrophilic lipid heads, followed by penetration into the bilayer and becoming embedded in the outer membrane leaflet, inducing substantial perturbations to the membrane. in comparison, adapalene, a molecule of similar structure, cannot penetrate into the bilayer as the hydrophobic methoxy group does not bind to the lipid heads ( fig. b ). free energy profiles associated with the membrane penetration of these molecular agents were calculated based on steered molecular dynamics and umbrella sampling [ ] [ ] [ ] . the free energy mapping shows that membrane penetration by cd , cd and adarotene is energetically favourable with barriers on the order of thermal fluctuation while adapalene exhibits a high energy barrier and an unfavourable transfer energy against membrane penetration (fig. c ). in comparison with cd and cd , adarotene shows higher energy barrier and smaller transfer energy, hence less favourable for membrane penetration. these results are consistent with the biomembrane-mimicking giant unilamellar vesicles (guvs) experiments (fig. d) , where domain aggregation and pore development are observed on the surfaces of the guvs exposed to cd , cd and adarotene, while the guvs exposed to adapalene remain intact. further in vivo and in vitro experiments indicated that cd and cd are potential new membrane-active antibiotics with fast bacteria killing rate and low resistant development, while adarotene and adapalene exhibit lower and no antimicrobial activities, respectively [ ] . the discovery of this class of retinoids as new antibiotics demonstrated the powerful roles of molecular simulations. the simulations and free energy mapping revealed atomistic insights on how molecular agents attach on and penetrate into the bacterial membranes, and the excellent agreement with experiments indicated that modelling and simulation can effectively capture whether a given compound is a potential candidate for membrane-active antibiotics. one of the major challenges in developing membrane targeting agents is to ensure their selectivity for pathogen since those of low membrane selectivity are typically toxic to mammals [ ] . despite many common features among all biological membranes, the lipid compositions of bacterial and mammalian cell membranes are quite different. specifically, unlike the negatively charged s. aureus membrane, the outer leaflet of mammalian cell membrane is neutrally charged, with cholesterol ranging from to mol% [ ] [ ] [ ] . this intrinsic difference between bacterial and mammalian cell membranes provides a basis for developing membrane targeting antibiotics. taking a clinically approved anthelmintic drug bithionol (fig. a) as an example, we found that the simulations can also help capture membrane selectivity. all-atom md simulations show that bithionol can be recruited to the bacterial membrane surface, penetrates into the membrane and embeds in the outer leaflet of the lipid bilayer. in contrast, bithionol fails to penetrate mammalian mimetic lipid bilayers with : popc/cholesterol ratio [ , ] (fig. b) . the free energy mapping confirms that penetration of bithionol into the mammalian mimetic membrane is energetically unfavourable (fig. c) . moreover, the presence of cholesterol leads to a more ordered alignment of the membrane lipids, condenses the hydrophobic region of the membrane and decreases membrane fluidity. as a result, the energy barrier and transfer energy associated with membrane penetration increases monotonically with the percentages of cholesterol, suggesting that cholesterol plays a key role in membrane selectivity [ ] . the guvs experiments on the effects of bithionol on different lipid bilayers are in agreement with the simulations: bacterial mimetic guvs exposed to biothionol undergo domain aggregation, pore development and rupture, while no such effects are observed on mammalian mimetic guvs. cell-based assays further demonstrate that bithionol indeed exhibits bactericidal activity and low levels of toxicity to mammalian cells [ ] . our modelling and simulation demonstrated that the selectivity of bithionol for bacterial membranes correlates with its ability to penetrate and embed in bacterial-mimic lipid bilayers, but not in cholesterolrich mammalian-mimic lipid bilayers. this then provided a fundamental insight into the molecular mechanisms by which membrane-active molecular agents selectively disrupt bacterial over mammalian membranes . the above studies demonstrated that modelling and simulation can predict the ability of molecular agents to selectively penetrate into lipid membranes of pathogen, providing a theoretical basis to understand membrane activity and selectivity of membrane active nanomedicine. however, we also found that, while some molecular agents (such as pq ) can penetrate into the mammalian-mimetic membrane, they do not disrupt mammalian-mimetic guvs and red blood cells [ ] . this observation suggests that penetration is only the first and necessary step, but not sufficient for the final rupture of the lipid membrane. more detailed experimental observations on the evolutions of guvs exposed to membrane-active compounds indicate that the entire interaction process leading to the final rupture of lipid membranes include further intricately coupled physical processes such as diffusion, domain aggregation, deformation, pore formation and growth (figs. , d, d) , which raise a number of open questions. for example, what is the driving mechanism for domain aggregation? why is there a characteristic domain size? why does domain aggregation facilitate pore formation? what are the effects of homogeneous vs interfacial nucleation of pores? how does the spontaneous curvature of domain affect pore formation? how does the vesicle size affect pore formation? how to predict the energy barrier and time scales for pore formation? in this webinar, we highlighted some selected questions related to the characteristic domain size and pore formation and growth. regarding domain aggregation, consider an inclusion domain of inserted molecular agents with area in an infinite membrane . the domain adopts an axisymmetric shape while inducing an intrinsic curvature . the membrane has bending modulus and is under remote tension Σ . when the aggregated domain incorporates dispersed molecules, a mixing energy per unit area Δ is induced, which includes the interaction energy between the dispersed molecules and lipids as well as the entropy change during the aggregation process. for simplicity we ignore the line tension on the domain interface. we find that if − / < Δ < , the domain has an optimal size. specifically, when − / ≪ Δ < , the domain size can be written as * ≈ − Δ Σ . thus, we conclude that a negative mixing energy drives domain growth while the intrinsic curvature and surface tension limits its size. we also modeled the pore (denoted by ) growth in the presence of an inclusion domain. apart from the bending energy and in the inclusion and the lipid membrane, the system energy also includes three line tensions, namely − on the lipid-inclusion interface, − on the lipid-pore interface and − on the inclusion-pore interface. we find that both the bending energy and the lipid-inclusion line tension contribute a negative energy variation during the pore growth process, thereby facilitating the pore growth. following this discovery, it is also shown that smaller vesicles have lower energy barrier for rupture since their high curvature lead to higher bending energy, a conclusion in line with the experimental observations [ ] . such size effect can potentially be utilized to understand the recently developed antiviral agent by cho et al. [ ] with a strong size-based selectivity. they showed experimentally that this antiviral agent can selectively rupture smaller liposomes and have a broadspectrum efficacy against a wide range of viruses. given that sars-cov- possesses a similar size, this antiviral agent may also be a potential candidate for treating covid- . we also probed the influence of domain size on pore formation. with numerical solutions, we find that larger domains exhibit lower energy barrier for pore growth than smaller ones. as the interfacial pore grows, the domain shape remains almost unperturbed in large domains. yet in smaller domains, either the shape of the pore or the domain can be distorted with substantial increase in the energy cost. in this webinar, we summarized some recent studies with a long term goal to establish a simulationassisted discovery platform for membrane active nanomedicine capable of selectively penetrating and aggregating in bacterial/viral lipid membranes, causing membrane permeability/rupture. our multiscale modelling and simulation studies demonstrated that the membrane activity of molecular agents correlates with their ability to enter, perturb and permeabilize the lipid bilayers. further study on different cell membranes revealed the molecular mechanism of cholesterol-based selectivity. following the molecular penetration, we established a continuum theory and derived the energetic driving force for domain aggregation and pore growth on lipid membranes. we showed that there exists an optimal domain size and that the energy barrier to membrane pore formation can be significantly lowered through molecular aggregation on a large domain with intrinsic curvature and a sharp interface. the curvature effect of lipid membrane derived from the theory may have important implications to develop membrane targeting antiviral agents with size-based selectivity. the current drug discovery process for antibiotics using biological screening methods (such as c. elegans survival [ ] ) are usually expensive, time consuming and inefficient. moreover, it has been difficult to identify the underlying mechanisms of positive hits in the screening and improve them subsequently. here, we showed that mechanistic modelling and simulations can help reveal some fundamental principles on how molecular antimicrobials interact with bacterial membranes and damage them through domain aggregation and pore formation. our study highlights a promising approach to establishing a simulation-assisted platform to accelerate the screening, assessment, design and optimization of membrane targeting nanomedicine at reduced cost against infectious diseases and, more importantly, to effectively counter increasing challenges with pathogen resistance/mutation. this work was supported by the u.s. national science foundation (grant cmmi- ) and a start-up grant from the nanyang technological university and institute of high performance computing, a*star, singapore. targeting non-multiplying organisms as a way to develop novel antimicrobials non-inherited antibiotic resistance targeting bacterial membrane function: an underexploited mechanism for treating persistent infections persister cells clinical practice guidelines by the infectious diseases society of america for the treatment of methicillin-resistant staphylococcus aureus infections in adults and children metabolite-enabled eradication of bacterial persisters by aminoglycosides activated clpp kills persisters and eradicates a chronic biofilm infection novel antibody-antibiotic conjugate eliminates intracellular s. aureus secretary-general, o.f. the, u. nations, no time to wait: securing the future from drug-resistant infections report to the secretary-general of the united nations april summary of recommendations and key messages summary of iacg recommendations a exploiting current understanding of antibiotic action for discovery of new drugs growth of mycobacterium tuberculosis biofilms containing free mycolic acids and harbouring drug-tolerant bacteria the evolution and emergence of rna viruses global spread of sars-cov- subtype with spike protein mutation d g is shaped by human genomic variations that regulate expression of tmprss and mx genes phylogenetic network analysis of sars-cov- genomes we shouldn't worry when a virus mutates during disease outbreaks a genomic perspective on the origin and emergence of sars-cov- therapeutic treatment of zika virus infection using a brain-penetrating antiviral peptide methicillin-resistant staphylococcus aureus process of inducing pores in membranes by melittin whole animal automated platform for drug discovery against multi-drug resistant staphylococcus aureus steered molecular dynamics and mechanical functions of proteins the weighted histogram analysis method for free-energy calculations on biomolecules. i. the method free weighted histogram analysis implementation including robust error and autocorrelation estimates a new class of synthetic retinoid antibiotics effective against bacterial persisters complementary biophysical tools to investigate lipid specificity in the interaction between bioactive molecules and the plasma membrane: a review microsecond molecular dynamics simulations of lipid mixing understanding the diversity of membrane lipid composition a selective membrane-targeting repurposed antibiotic with activity against persistent methicillin-resistant staphylococcus aureus the neutrally charged diarylurea compound pq kills antibiotic resistant and antibiotic tolerant staphylococcus aureus, mbio key: cord- -b zev zb authors: sobocińska, justyna; roszczenko-jasińska, paula; ciesielska, anna; kwiatkowska, katarzyna title: protein palmitoylation and its role in bacterial and viral infections date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: b zev zb s-palmitoylation is a reversible, enzymatic posttranslational modification of proteins in which palmitoyl chain is attached to a cysteine residue via a thioester linkage. s-palmitoylation determines the functioning of proteins by affecting their association with membranes, compartmentalization in membrane domains, trafficking, and stability. in this review, we focus on s-palmitoylation of proteins, which are crucial for the interactions of pathogenic bacteria and viruses with the host. we discuss the role of palmitoylated proteins in the invasion of host cells by bacteria and viruses, and those involved in the host responses to the infection. we highlight recent data on protein s-palmitoylation in pathogens and their hosts obtained owing to the development of methods based on click chemistry and acyl-biotin exchange allowing proteomic analysis of protein lipidation. the role of the palmitoyl moiety present in bacterial lipopolysaccharide and lipoproteins, contributing to infectivity and affecting recognition of bacteria by innate immune receptors, is also discussed. geranylgeranyl cysteine thioether h-and n-ras ( ) rab proteins ( ) attachment of glycosylphosphatidylinositol anchor or phosphatidylethanolamine ( ) to the c-terminus of proteins is also a form of lipidation but is not shown here. a n-myristoylation is in most cases co-translational, but during apoptosis caspases can cleave some proteins, such as bid, exposing their n-terminal glycine residue, which is then modified by attachment of myristate ( ) . b hedgehog proteins are additionally modified by covalent attachment of cholesterol to their c-terminus ( ) . c o-acylation of wnt proteins is reversed by notum of the α/β hydrolase superfamily ( ) . exceeds the consumption of other saturated fatty acids and, in the usa it accounts for about % of the total intake of saturated fatty acids ( ) . a growing body of experimental and clinical evidence points to a link between a westernized diet, including a high intake of saturated fatty acids, and chronic inflammatory diseases ( ) ( ) ( ) . as dietary saturated and unsaturated fatty acids apparently modulate activity of immune cells, their influence on the immune responses triggered upon infection is also beginning to be investigated ( ) . these facts drive the interest in palmitic acid with an aim of elucidating the molecular mechanisms of its immunomodulatory properties. in this review, we focus on s-palmitoylation of proteins crucial for the interactions of pathogenic bacteria and viruses with the host. we emphasize novel data on the role of s-palmitoylated proteins in the invasion of host cells by pathogens and those involved in the host innate immune responses to the infection, which have been obtained thanks to the application of new technical approaches. recently, substantial progress in the understanding of protein palmitoylation was made possible by the development of methods allowing high-throughput analysis of cellular/tissue palmitoyl proteomes. we begin, however, by showing how unique protein s-palmitoylation is among other protein lipidations. s-palmitoylation is a posttranslational modification of proteins consisting in a potentially reversible covalent attachment of palmitoyl chain to a cysteine residue(s) of proteins through a thioester bond ( table ) . thus, s-palmitoylation resembles other reversible regulatory posttranslational protein modifications, including phosphorylation or acetylation, well-established factors affecting protein structure and functions. in particular, s-palmitoylation modifies cellular localization of proteins and their stability. the most dramatic changes of localization concern cytosolic proteins which upon s-palmitoylation acquire a hydrophobic anchor facilitating their docking into membranes (figure ) . however, several integral membrane proteins also undergo s-palmitoylation. it often occurs on cysteine residue(s) located in the proximity of the junction of the transmembrane and cytoplasmic domains of the protein. s-palmitoylated transmembrane proteins occupy various cellular compartments, such as endoplasmic reticulum, golgi apparatus, and the plasma membrane. in accordance, for some proteins, such as transmembrane adaptor proteins in leukocytes, s-palmitoylation was found secondary to the length and hydrophobicity of the transmembrane domain as a determinant of plasma membrane destination ( ) . s-palmitoylation also contributes to the compartmentalization of proteins to distinct domains of membranes-rafts and tetraspanin-rich microdomains. in fact, the interest in s-palmi toylation was boosted when it was found to be required for the targeting of some signaling proteins to rafts. rafts are nanodomains of the plasma membrane and some intracellular membranes, mainly of the trans-golgi apparatus, rich in sphingolipids, glycerophospholipids with saturated fatty acid chains, and cholesterol ( ) . the plasma membrane nanodomains are sites of signal transduction by distinct receptors of immune cells involved in both acquired immune reactions, such as t cell receptor (tcr), fcε receptor i, fcγ receptor ii, and in innate immune responses, such as toll-like receptor (tlr ) ( , ) . rafts are also sites of virion assembly and budding, as established, e.g., for influenza a virus and human immunodeficiency virus- (hiv- ) ( , ) . peripheral membrane proteins acylated with saturated fatty acids are likely to anchor preferentially between the ordered saturated lipids of rafts rather than between the disordered lipids of the surrounding membrane. it has been shown that, owing to their raft localization, s-palmitoylated kinases of the src family interact with raft-associating plasma membrane immunoreceptors and initiate signaling cascades fundamental to acquired immunity ( , , ) . it is worth noting that also the acyl chains attached to proteins can affect the membrane structure. studies on model membranes have revealed that palmitic and myristic acids facilitate formation of ordered lamellar membrane regions ( , ) . in accordance, s-palmitoylation of erythrocyte peripheral membrane protein called membrane-palmitoylated protein (mpp ) was found to be required for the proper lateral organization and fluidity of erythrocyte membrane. in the absence of mpp s-palmitoylation, raft assembly was disturbed and erythrocyte functioning compromised leading to hemolytic anemia in patients deficient in the enzyme catalyzing this reaction ( , ) . preferential raft association is a feature of some s-palmitoylated transmembrane proteins, e.g., adaptor proteins pag, lat, and ntal, which collaborate with the abovementioned immunoreceptors. in fact, palmitoylation is required for the raft association of most integral raft proteins ( , , ) . on the other hand, s-palmitoylation does not obligatorily confer raft localization on transmembrane proteins. certain s-palmitoylated proteins, such as transferrin receptor, glycoprotein g of vesicular stomatitis virus (vsv), and anthrax toxin receptor, tumor endothelial marker (tem ), are actually excluded from rafts. apparently, a combination of s-palmitoylation and the properties of the transmembrane domain of the protein contribute to its destination to the raft or non-raft environment ( , ) . it has also been proposed that the attachment of a fatty acyl chain at the juxtamembrane cysteine(s) of a protein can induce tilting of its transmembrane fragment, determining in which part of the membrane it will accommodate to avoid a hydrophobic mismatch potentially caused by the thickness of the bilayer ( ) . that not all s-palmitoylated proteins associate with rafts has been shown convincingly for macrophage-like raw cells, where only about half of those proteins were found in the triton x- -resistant membrane fraction enriched in rafts ( , ) . in accordance, proteomic data on the distribution of s-palmitoylated proteins in prostate cancer cells have revealed that several such proteins are recovered in the non-raft (triton x- -soluble) fraction and are likely localized to microdomains enriched in scaffold proteins called tetraspanins ( ) . the tetraspanins are small integral membrane proteins found in the plasma membrane and other cellular membranes, having four transmembrane helices and undergoing s-palmitoylation at several conserved cysteine residues. the tetraspanins interact with each other and with various transmembrane and cytosolic partners, often also s-palmitoylated, forming microdomains ("tetraspanin web") ( ) . it has been suggested that the amino acid composition of the s-palmitoylation site in some transmembrane proteins, such as the adaptor proteins involved in acquired immune responses, determines the association of those s-palmitoylated proteins with rafts or with the tetraspanin-enriched microdomains ( ) . an intriguing and still poorly addressed question concerns the relation between rafts and the tetraspanin-enriched microdomains, apparently of functional significance, e.g., during virus budding from host cells ( ) . this uncertainty stems partially from the fact that s-palmitoylation of tetraspanins governs their interactions with cholesterol and gangliosides leading at certain conditions to the recovery of tetraspanins in detergent-resistant membrane fractions enriched in rafts ( , ) . besides its involvement in targeting proteins to rafts or tetraspanin-enriched microdomain, s-palmitoylation has been found to govern accumulation of the transmembrane chaperone protein calnexin in the perinuclear domain of endoplasmic reticulum ( ) . s-palmitoylation also affects protein stability through its interplay with ubiquitination or phosphorylation, as found for the anthrax toxin receptor tem , antiviral interferon-induced transmembrane protein ifitm , calnexin, and zdhhc , one of palmitoyl acyltransferases described below ( ) ( ) ( ) ( ) . possibly the most intriguing is the reversible character of s-palmitoylation. enzymes catalyzing palmitoylation and depalmitoylation of proteins have been characterized ( , ) . palmitate is transferred onto the thiol group of cysteine from cytosolic palmitoyl-coa by palmitoyl acyltransferases, enzymes containing the zinc finger dhhc domain named after the highly conserved asp-his-his-cys peptide (figure ). this is a twostep reaction comprising transient autoacylation of zdhhc enzymes and transfer of the fatty acyl chain from this intermediate to a protein substrate ( ) . in mammals, the zdhhc enzyme family consists of proteins, and zdhhc proteins are also found in other eukaryotes but not in bacteria nor are they encoded by viral genomes. mammalian zdhhc enzymes, each having at least four transmembrane helices, are located in the plasma membrane, endoplasmic reticulum, and golgi apparatus ( ) . they display some specificity toward their protein substrates and also selectivity toward fatty acyl moieties other than palmitate, which contributes to the heterogeneity of lipids attached to proteins, such as viral glycoproteins described below ( ) . in the opposite process, the thioester bond is cleaved by acyl-protein thioesterases (apts) (apt and apt ) and palmitoyl protein thioesterases (ppts) (ppt and ppt ), which are localized in the cytosol and in lysosomes, respectively. apt and apt likely govern the dynamic functional changes of s-acylation of proteins ( ) while ppt and ppt depalmitoylate proteins during their degradation ( , ) . recently, serine hydrolases of the abhd family have also been identified as depalmitoylating enzymes, and their specific substrate proteins determined ( , ) . of note, the zdhhcs, apt /apt , and abhd proteins are s-palmitoylated themselves, and palmitoylation of zdhhcs and depalmitoylation of apt / can occur in a cascade manner ( , ) . the dynamic cycles of palmitoylation/depalmitoylation detected for several peripheral membrane proteins are often synchronized with intracellular trafficking of those proteins. they circulate between the plasma membrane and the golgi apparatus or endosomes, as exemplified by n-and h-ras, r -regulator of g protein and apts. in fact, it is proposed that palmitoylationdependent anchoring of apt in the plasma membrane allows it to depalmitoylate h-ras at this location, while subsequent autodepalmitoylation releases apt guiding it, alongside h-ras, for another round of palmitoylation at the golgi apparatus ( , ( ) ( ) ( ) . cycles of palmitoylation/depalmitoylation are crucial for signaling by distinct plasma membrane receptors and for their distribution ( ) ( ) ( ) . activation of tcr receptor or fas receptor in t cells was found to trigger quick and transient palmitoylation of lck kinase of the src family ( , ) , but the exact meaning of the dynamic protein s-palmitoylation for processes triggered during the host-pathogen interaction awaits elucidation. it is worth mentioning that although the zddhc enzymes catalyze bulk protein palmitoylation in eukaryotic cells ( ) , some proteins have a unique autopalmitoylation activity. these include bet , a component of a multisubunit transport protein particle complex involved in vesicular trafficking, tea domain transcription factors, and also bacterial evf protein ( ) ( ) ( ) ( ) . the palmitic acid residue is attached constitutively to a specific cysteine residue of those proteins, remains buried inside a hydrophobic pocked in their core thereby affecting the tertiary structure and, thus, interactions with other proteins ( , ) . an exhaustive discussion on the physiology of s-palmitoylated proteins in eukaryotic cells can be found in several recent reviews ( , , ) . it has been established that, in addition to palmitate, various other fatty acyl moieties, such as saturated stearate (c : ) or protein palmitoylation in host-pathogen interactions frontiers in immunology | www.frontiersin.org january | volume | article monounsaturated palmitoleate (c : ), and oleate (c : ) can be attached via the thioester linkage to proteins. the early reports on the heterogeneity of the fatty acyl moieties attached to cysteines obtained by analysis of selected immunoprecipitated proteins ( , , ( ) ( ) ( ) have recently been complemented by a comprehensive proteomic analysis of fatty-acylated proteins of macrophage-like raw cells ( ) . the latter study showed that an enrichment of culture medium of cells with monounsaturated fatty acids leads to their incorporation into a similar set of proteins as those normally modified with palmitate. among them, several proteins relevant to innate immune responses were found. all these data justify the use of a broader term s-acylation rather than s-palmitoylation ( table ). the physiological consequences of s-acylation of proteins with individual fatty acids are slowly being revealed. modification of fyn kinase with polyunsaturated fatty acid residue, such as arachidonate (c : ), disturbed its raft localization and, thereby, tcr signaling ( ) . a heterogeneity of s-acylation was also found in viral spike proteins, such as hemagglutinin (ha) of influenza a virus, and e and e of semliki forest virus, which are modified in host eukaryotic cells by attachment of both palmitate and stearate ( ) . in ha, stearate is attached at the transmembrane cysteine while palmitate is attached to two cysteine residues in a membrane-proximal region of the protein. the stearoyl chain seems to accommodate into a groove formed by amino acids of the transmembrane helix shaping the domain in a way that facilitates its fitting into rafts ( ) . s-stearoylation of human transferrin receptor at the juxtamembrane cysteine residues(s) is a key factor of the signaling cascade controlling mitochondrial morphology and functioning ( ) . of interest, the latter study also showed that dietary supplementation of stearic acid reversed the deleterious effects of a genetically determined mitochondria dysfunction in drosophila. taking into account that unsaturated fatty acids affect the profile of s-acylation of proteins in vitro ( , ) , it is of outmost interest whether a similar effect of unsaturated and saturated (palmitic) fatty acids could be achieved in vivo with respect to proteins of immune cells. beside s-acylation, less frequently palmitate can also be attached to the amine group of various amino acids (glycine, cysteine, and lysine) giving n-palmitoylation or to the hydroxyl group of serine or threonine in a process called o-palmitoylation ( table ) . as during s-palmitoylation, also other fatty acids can be utilized in these processes named then n-and o-acylation. thus, a type of protein n-acylation is n-myristoylation, a frequent modification contributing to membrane anchoring of peripheral proteins. the saturated myristate (c : ) is transferred to the protein from myristoyl-coa by n-myristoyl transferase (two isozymes in mammals). in a vast majority of cases, myristate is attached cotranslationally to the n-terminal glycine residue (after removal of the initiator methionine) via an amide linkage ( table ) . like most lipidations, this modification is irreversible. several viral proteins are n-myristoylated, such as gag of hiv- crucial for budding of newly formed virions from plasma membrane rafts of host cells, and proteins of parasitic protozoa plasmodium falciparum, trypanosoma brucei, and leishmania donovani (causing malaria, african sleeping sickness, and leishmaniosis, respectively). for this reason, n-myristoyl transferase is considered a potential drug target in the therapy of these diseases ( - , , ) . data on the n-and o-palmitoylation of proteins involved in the host-pathogen interactions are limited, but interesting conclusions can be drawn from the information concerning proteins taking part in other processes. n-palmitoylation of the n-terminal glycine of the α-subunit of a heterotrimeric g protein (gαs) has been described ( ) ( table ) besides the wellknown s-palmitoylation of this pivotal signaling protein. the n-palmitoylation of gαs is irreversible, and the enzyme responsible for this modification is unknown. it has been speculated that s-to n-palmitoyl migration can occur both in vivo and also in vitro during mass spectrometry analysis ( , ) . this suggests that caution is needed in interpreting results of this methodological approach, which is used with increasing frequency to study fatty acylation of proteins in immune cells (see next sections). probably the best-characterized is the n-palmitoylation of the n-terminal cysteine residue of hedgehog proteins (sonic, indian, and desert in mammals). it determines secretion of these proteins, which regulate embryonic patterning ( table ) . secreted wnt and ghrelin proteins are examples of o-acylation of serine residues with unusual fatty acid residues such as palmitoleate (c : ) and octaonoate (c : ) ( table ). the fatty acylation of hedgehog, wnt, and ghrelin is catalyzed by enzymes from the multipass membrane-bound o-acyl transferases family ( ) . besides these unusual fatty acid residues, attachment of palmitate to serine and threonine residues is found in secreted venom toxins of the spider plectreurys tristis, which selectively target neuronal ion channels ( ) . also histone h is o-palmitoylated at a serine residue in the nucleus by acyl-coa:lysophosphatidylcholine acyltransferase ( ) ( table ). the latter is of special interest in the context of innate immune responses since histone h o-palmitoylation regulates transcriptional activity, which is the final outcome of the pro-inflammatory signaling pathways triggered by receptors of the innate immune system. special attention should be devoted to ε-n-acylation consisting in the attachment of a fatty acid residue to the side chain of lysine by amide linkage ( table ) . ε-n-myristoylated are interleukin α (il- α) and tumor necrosis factor α (tnfα), the pro-inflammatory cytokines crucial in combating bacterial infections ( ) . the enzyme(s) catalyzing this reaction is unknown, but it has been established that sirtuins reverse this modification ( ) . the ε-n-acylation affects the release of tnfα by immune cells ( , ) . surprisingly, this rare modification is also found in toxins of so-called rtx (repeats-in-toxin) class released by some pathogenic gram-negative bacteria ( , ) . we describe these cases in more detail in the following sections. besides s-palmitoylation and n-myristoylation, s-prenylation is another common lipidation that endows proteins with a hydrophobic moiety and contributes to their association with membranes. this modification relies on the posttranslational and irreversible attachment of either farnesyl or geranylgeranyl chains to a cysteine residue in the c-terminal caax box (alternatively also cc and cxc motifs) via a thioether linkage. the process is catalyzed by protein prenyl transferases that use polyprenylpyrophosphate as the donor of the isoprenoid group ( table ) . in peripheral membrane proteins, the s-palmitoylation site is often located in proximity of n-myristoylation or s-prenylation sites or a polybasic motif, which all are likely to mediate initial weak binding of a protein to a membrane and thereby facilitate subsequent attachment of palmitate to the protein by the integral membrane zdhhc enzymes ( ) . in contrast to s-palmitoylation, data on the role of s-prenylation of proteins key to the host-pathogen interactions are scarce ( ) . however, since s-prenylation is typical for the ubiquitous small gtpases of ras superfamily, it is vital for proper functioning of b and t cells ( , ) . a glance at table indicates that palmitate can be covalently bound via oxyester, amide, and thioester linkages to respective amino acid residues creating an array of possible modifications. o-and n-palmitoylation of proteins seems to be stable, resembling in this regard the other common protein lipidations, n-myristoylation and s-prenylation. by contrast, there exist enzymes cleaving the thioester bond formed during s-palmitoylation. for a long time, our understanding of protein s-palmitoylation and its dynamics was poor in comparison with other reversible protein modifications due to technical difficulties. only recently have these difficulties been overcome with the introduction of methods allowing high-throughput identification of palmitoylated proteins, also those involved in the immune response to microbial pathogens, as discussed in the next sections. one of the basic problems hindering studies on protein s-palmitoylation lies in the fact that there is no identifiable consensus sequence for the palmitoylation site that could facilitate its prediction. from the technical point of view, the progress in a comprehensive survey of protein s-palmitoylation was also hampered by a lack of antibodies detecting this modification, with the sole exception of an antibody specific to palmitoylated psd- ( ) . a classical method used to demonstrate protein palmitoylation is based on metabolic labeling of living cells with [ h]-palmitic acid, subsequent immunoprecipitation of a selected protein and detection of the incorporated tritiated fatty acid by autoradiography ( ) . a major disadvantage of this method is its low sensitivity. only a minute fraction of the radioactive palmitate is bound to proteins, the majority being incorporated into lipids, which requires lengthy film exposure (counting in days). a methodological breakthrough in the identification of palmitoylated proteins came with the development of two nonradioactive methods based on so-called click chemistry ( ) ( ) ( ) and acyl-biotin exchange (abe) ( , ) . these techniques have paved the way for high-throughput mass spectrometry-based proteomic analysis of protein palmitoylation in various cells and tissues and facilitated identification of new palmitoylated proteins of both pathogens and host cells involved in the innate immune responses. in the click chemistry-based method, cells are metabolically labeled with a palmitic acid analog bearing an alkyne group at the ω carbon of the fatty acyl chain, such as -octadecynoic acid ( odya) or alk- (figure a) , and this step resembles the classic labeling of cells with [ h]-palmitic acid. however, in the click chemistry-based assay, the labeling and lysis of cells is followed by in vitro coupling of the function group of the palmitic acid analog to a reporter tag, which greatly enhances the sensitivity of detection of labeled proteins ( , ) . thus, after cell lysis, the labeled proteins are subjected to cu (i) -catalyzed cycloaddition known as "click" reaction with an azide-bearing detection tag. in this step, a triazol is formed between the alkyne group in the palmitic acid analog and the azide of the tag (figure a) . the azide-bearing tags can be either fluorescent, such as tetramethylrhodamine or dyes with infrared fluorescence, or carry a biotin moiety. depending on the tag used, subsequent sds-page separation of proteins allows global visualization of palmitoylated proteins by simple in-gel fluorescence or by blotting with a streptavidin-conjugated reporter ( , , ) . notably, proteins biotinylated via the click reaction can also be enriched on streptavidin-coated beads and then subjected to on-bead tryptic digestion (or in-gel digestion if eluted from the beads) followed by identification by mass spectrometry. such comprehensive click chemistry-based proteomic analysis has brought about identification of an array of palmitoylated proteins in dendritic cells ( , ) , macrophage-like raw cells ( ) , and t cells ( , , ) . some of the s-palmitoylated proteins newly identified in those studies, such as ifitm and tlr , are involved in the host-pathogen interactions regulating innate immune responses ( , ) , while many others are known to contribute to adaptive immunity ( , ) , as described below. recently, global profiling of toxoplasma gondii (the causative agent of toxoplasmosis) has been performed revealing that many components of the parasite's motility complex are palmitoylated ( ) . similar studies on cryptococcus neoformans (the fungus causing cryptococcal meningitis) have revealed a contribution of specific zdhhc palmitoyl acyltransferase, called pfa , to its virulence ( ) . moreover, application of analogs of various saturated and unsaturated fatty acids confirmed the heterogeneous nature of the fatty acylation of proteins in raw cells and suggested that dietary unsaturated fatty acids, after incorporation to proteins, can change their properties and thereby affect the functioning of immune cells ( ) . the major advantage of the click chemistry-based method is that it can reveal the time course of protein s-palmitoylation. by using click chemistry-based labeling in the pulse-chase mode, one can follow the dynamics of protein palmitoylation. with such an approach, it was found that the palmitate turnover on lck, an src-family tyrosine kinase, is accelerated by t cell activation ( ) . additional introduction of stable isotope labeling by amino acids in cells (silac) has provided quantitative proteomic data , and after cell lysis, the click reaction is conducted with azido-tagged biotin or fluorescent probes allowing enrichment and detection of labeled proteins in various ways. biotinylated proteins can be bound on a streptavidin resin and then released using, e.g., high concentrations of urea and sds ( ) . when a cleavable derivative of biotin, azido-azo-biotin, is used the labeled proteins are eluted from streptavidin beads with sodium dithionite, which cleaves the diazobenzene moiety in the linker arm of azido-azo-biotin, and analyzed by mass spectrometry or immunoblotting ( ) . (b) abe method. cells or tissues are lysed, free thiol groups of proteins are blocked by alkylation, and palmitoyl moieties are released with hydroxylamine. the newly exposed protein thiol groups are subjected to labeling with biotin-hpdp allowing selective binding, elution, and analysis of the originally s-palmitoylated proteins. the proteins can also be captured without biotinylation through a direct interaction of their thiol residues with a thiol-reactive resin (acyl-rac technique). on the dynamics of protein palmitoylation in the cell ( , ) . this approach revealed, rather unexpectedly, that in unstimulated t cell hybridoma, the palmitoylation of most protein species does not undergo turnover ( ) . another advantage of the click chemistry-based assay is its high specificity, because the alkyne group introduced in the analog of palmitic acid is not normally found in cells ( , ) . the click chemistry-based methods can also be used to follow the cellular localization of palmitoylated proteins by immunofluorescence when combined with the proximity ligation technique ( , ) . palmitoylation of individual proteins can also be studied after their immunoprecipitation ( , , , ). despite its unquestionable success, the click chemistry-based methods have limitations. they will detect only those proteins that undergo palmitoylation during the period of the metabolic labeling of cells. one should also bear in mind that the palmitic acid analog can be incorporated at s-, n-, and o-palmitoylation sites alike ( , ) . in addition, although odya (alk- ) is preferentially used to mimic palmitoylation of proteins, it can also be incorporated with low efficiency at n-myristoylation sites of proteins ( , ) . another group of proteins that will be labeled with the palmitic acid analog but are not s-palmitoylated are those bearing the glycosylphosphatidylinositol (gpi) anchor ( , ) . most of these limitations can be overcome using various fatty acid reporters, inhibitors, and by exploiting the sensitivity of the thioester bond to hydroxylamine treatment. given the large variety of chemical reporters preferentially mimicking distinct fatty acids, recent years have witnessed a plethora of chemistry-based proteomic studies not only on palmitoylated but also myristoylated proteins and proteins bearing the gpi anchor, including those of pathogens and immune cells ( , , , , ) the abe method reveals protein the abe method can be used as a complement to the click chemistry-based approach in cell studies but unlike the latter it is uniquely suitable for studying whole tissues. abe does not require metabolic labeling of proteins in living cells, thus some of the abovementioned limitations and difficulties do not apply. the abe method relies on in vitro exchange of thioester-linked palmitate to a derivative of biotin which allows subsequent affinity purification of the resulting biotin-labeled proteins on streptavidin-coated beads ( figure b) . the first step of the abe involves lysis of cells or tissues followed by irreversible blockage of free thiol groups in the solubilized proteins by alkylation, most often with n-ethylmaleimide. subsequently, the thioester bonds existing in s-palmitoylated proteins are broken with hydroxylamine, releasing palmitoyl moieties. the newly exposed thiol groups can now be tagged with sulfhydryl-reactive derivatives, such as biotin-hpdp, forming disulfide bonds with thiols. the biotinylated proteins are subsequently captured on streptavidin-coated beads and eluted with agents that reduce the disulfide bond between the protein and biotin-hpdp, such as β-mercapthoethanol, dtt, or tcep ( , , , ) . as an alternative to biotinylation, in the so-called acyl-rac technique, the newly exposed protein thiol groups in hydroxylamine-treated cell lysates are captured on a resin containing sulfhydryl-reactive groups ( ) . in both abe and acyl-rac, the eluted proteins can be separated by sds-page and visualized by gel staining or immunoblotting, or identified by mass spectrometry. furthermore, when the hydroxylaminereleased palmitoyl moieties are exchanged for a polyethylene glycol-maleimide derivative of a distinct molecular weight, a shift in-gel migration of tagged proteins is observed reflecting the number of fatty acyl residues originally s-bound to the protein ( , ) . the abe method has so far been used successfully for proteomic profiling of s-acylated proteins in immune cells, such as raw cells ( ), several types of blood cells, such as platelets, primary t cells, and immortalized b cells ( ) ( ) ( ) , pathogenic microorganisms such as t. brucei and t. gondii ( , ) , and tissues ( , ) . to quantify the aberrations in protein palmitoylation in a mouse model of huntington's disease, whole animal stable isotope labeling of mammals (silam) was applied followed by tissue isolation and abe procedure ( ) . in another approach, for quantitative analysis of the t-cell palmitoylome, abe was combined with labeling of proteins with various oxygen isotopes during their digestion with trypsin before mass spectrometry analysis ( ) . in addition, preselection of tryptic peptides obtained by abe on streptavidin-coated or sulfhydrylreactive resins greatly facilitates the identification of s-acylation sites by mass spectrometry ( , , ) . some aspects of the abe method deserve a comment. since the assay relies on the sensitivity of thioester bonds to hydroxylamine, abe detects all s-acylation without distinguishing between s-palmitoylation and the other cases. furthermore, there is a possibility of false-positive detection of proteins bearing a thioester linkage with compounds other than fatty acyl residues, such as ubiquitin in the e ubiquitin conjugase ubc ( ) . another source of false-positives is proteins in which free thiol groups were not completely alkylated before biotinylation. on the other hand, insufficient deacylation of bonafide fatty-acylated proteins with hydroxylamine results in their absence in the final sample ( ) . in summary, the click chemistry-based method relies on metabolic labeling of cells with a palmitic acid analog which incorporates into proteins and next tagging it with reporter molecules greatly enhancing the sensitivity of detection. it only reveals proteins undergoing s-palmitoylation during metabolic labeling of cells and allows revealing turnover of this modification. by contrast, the abe method is based on direct binding of sulfhydryl-reactive derivatives to thiol groups of cysteines unraveled by hydroxylamine treatment after lysis of cells or tissues. it allows the investigation of the whole but static palmitoylome. a comparative proteomic study of protein palmitoylation in p. falciparum found that the sets of proteins identified using these two approaches overlapped in . % ( ) , indicating that they provide complementary data on the cellular palmitoyl proteome. thanks to the application of the click chemistry-and abe-based methods numerous new palmitoylated proteins have been identified. in , a swisspalm database was launched, ( ) which provides an excellent, manually curated resource of information on palmitoylated proteins, palmitoylation sites, etc., available at http://swisspalm.epfl.ch/. all these efforts have greatly furthered our knowledge on molecular mechanisms regulating diverse aspects of cell functioning, including host-pathogen interactions and progress of infectious diseases, as highlighted below. bacteria lack protein palmitoyl acyltransferases of the zdhhc family and, therefore, are essentially devoid of s-palmitoylated proteins. yet, they have developed unique mechanisms utilizing fatty acids, such as palmitic acid, to modify their glycolipids and proteins. these modifications augment infectivity and help bacteria evade recognition by the host innate immune system. for example, the vast majority of gram-negative bacteria produce lipopolysaccharide (lps) as a part of their outer membrane. lps is composed of the variable polysaccharide o-antigen and more-conserved lipid a containing two glucosamine residues hexa-acylated with hydroxymyristic, myristic, and lauric acid. lipid a is recognized by cd protein and tlr receptor complexed with md protein on the plasma membrane of the host immune and some non-immune cells. activation of tlr triggers strong pro-inflammatory reactions aiming at eradication of the bacteria, but when exaggerated, eventually leading to sepsis ( ) . incorporation of an additional palmitoyl chain into lipid a markedly diminishes its ability to activate tlr and to induce the host pro-inflammatory responses, which is correlated with an increased survival of bacteria forming a biofilm ( , ) . this strategy is utilized among others by salmonella typhimurium, a causative agent of gastroenteritis, by bordetella bronchiseptica, a respiratory pathogen of human and other mammals, and by protein palmitoylation in host-pathogen interactions frontiers in immunology | www.frontiersin.org january | volume | article yersinia pestis causing plague ( , ) . the formation of the extra-acylated lps relies on the transfer of palmitate from phospholipids onto the hydroxymyristate chain at position of glucosamine of lipid a. the reaction is catalyzed by lipid a palmitoyltransferases (pagp in salmonella and its homologs in other bacteria) localized in the outer membrane of these pathogens ( , ) . in addition to causing steric hindrance preventing the binding to the tlr /md complex, the hepta-acylation of lps also protects bacteria from the lytic activity of cationic antimicrobial peptides, most likely by reducing the fluidity of the bacterial outer membrane ( , ) . apart from being incorporated into lps in diverse bacteria, palmitate has also been found to modify a virulence factor of gram-negative erwinia carotovora, the evf protein. the palmitoyl chain is linked via a thioester bond to the cys residue at the center of evf, plausibly by a self-palmitoylating activity of the protein. e. carotovora is a phytopatogen using insects such as drosophila as vectors for dissemination between plants. the palmitoylation of evf is required for infectivity of e. carotovora and its persistence in the insect gut, however, its mode of action of unknown. it has been speculated to be linked with an ability of evf to associate with lipid bilayers, but the lack of similarities between evf and any other bacterial protein of known function makes prediction on this subject difficult ( ) . a number of bacterial toxins of so-called rtx class released during infection of mammals by pathogenic gram-negative bacteria undergo ε-n-acylation of the side chain of internal lysines. these toxins include adenylate cyclase of bordetella pertussis, acylated with palmitic acid, and α-hemolysin of extraintestinal (uropathogenic) escherichia coli, acylated with myristic acid and also -and -carbon fatty acids. the acylation is catalyzed by an endogenous bacterial acyltransferase which, unlike its eukaryotic counterparts, transfers the acyl chain not from acyl-coa but from acyl-carrier protein. the acylated toxins secreted by the bacteria bind to the plasma membrane of the host cells, oligomerize and form pores causing cell lysis. in the case of the toxin of b. pertussis, essential is also the delivery of the adenylate cyclase moiety to the cell interior. acylation is required for virulence possibly being involved in oligomerization of the toxins ( , , ) . although lacking s-palmitoylated proteins (with the single known exception of evf), bacteria express a wide range of membrane-bound proteins modified by a complex lipidation at the n-terminus, with palmitate frequently being a component of the lipid moiety ( , ) . the bacterial lipoproteins are synthesized in a multistep process catalyzed by a unique set of lipoprotein processing enzymes, lgt, lspa, and lnt, absent in eukaryotic cells. the formation of these lipoproteins begins with the attachment of a diacylglycerol via a thioester bond to a cysteine residue located in the so-called lipobox motif of the signal sequence of the transmembrane lipoprotein precursor. the signal sequence is then cleaved next to the lipid-modified cysteine leaving it at the n-terminus of the mature protein ( ) . in gramnegative and less frequently also gram-positive bacteria, a third fatty acid residue is additionally attached via an amide linkage to the amino group of the cysteine in a reaction analogous to the n-acylation of hedgehog proteins (see table ). this di-and tri-lipidation ensures membrane anchoring of the lipoproteins. all such lipoproteins of gram-positive bacteria are exposed to the milieu while in gram-negative bacteria some face the periplasm. the lipoproteins of gram-positive bacteria, e.g., streptococcus pneumoniae (causing pneumonia), mycobacterium tuberculosis (tuberculosis), and gram-negative bacteria, such as neisseria meningitidis (meningitis), y. pestis (plague), the spirochaete borrelia burgdorferi (lyme disease) and treponema pallidum (syphilis) are crucial for their virulence. they control several aspects of the host-pathogen interactions, like adhesion and entry to host cells, protection against proteolysis and oxidative stress in the host cell, and regulation of expression of genes encoding cytokines both during initiation and progress of the disease ( ) ( ) ( ) . the surface exposure of the lipoproteins allows their involvement in the host cell invasion while on the other hand forming the so-called pattern signal recognized by the tlr receptor, which triggers the pro-inflammatory responses helping to combat the bacteria ( ) . of interest, tlr is s-palmitoylated, as discussed below. the involvement of lipoproteins in pathogenesis fuels studies on their properties. one such recent work employing click chemistry to profile the lipoproteins of e. coli identified lipoproteins with high/medium confidence, % of them predicted before by bioinformatics analysis ( ) . notably, in that study a -carbon alkynyl fatty acid analog alk- rather than alk- was preferentially incorporated into the lipoproteins, contradicting earlier studies using gas chromatography and tlc, which found that palmitate was predominantly used for bacterial protein modification ( ) . further studies are required to establish whether the fatty acid found in lipoproteins varies depending on culture conditions or is species specific. for example, odya labeling for click reaction confirmed incorporation of palmitate into pallilysin (tp ), a lipoprotein of t. pallidum. pallilysin is a metalloprotease that degrades human fibrinogen and laminin. it is suggested that its exposure on the bacteria surface enables degradation of host structural proteins to facilitate rapid dissemination of this highly invasive pathogen ( ) . bacteria occasionally high-jack the palmitoylation machinery of host cells to modify the environment so as to favor their internalization, survival, and replication inside the cells. bacillus anthracis (the causative agent of anthrax) is an example of such bacteria that modify s-palmitoylation of host proteins to their ends. the anthrax toxin produced by this pathogen binds to the tem and cmg (capillary morphogenesis protein- ) proteins which, under physiological conditions, are involved in cell-cell and cell-extracellular matrix interactions. they are s-palmitoylated at multiple (two to four) cysteines ( ) . the s-palmitoylation of tem was found to inhibit its association with plasma membrane rafts preventing its ubiquitination by the raft-associated e ubiquitin ligase cbl. the binding of anthrax toxin drives association of the receptor-toxin complexes with rafts possibly correlated with depalmitoylation of the receptor. this allows subsequent ubiquitination of the receptor, an uptake of the receptor/toxin complexes in a clathrin-dependent manner and eventual delivery of the toxin to endosomes. these events are facilitated by s-palmitoylation of partner(s) of the receptors, most likely including kinases of the src family ( , , ) . while b. anthracis utilizes palmitoylated host proteins to induce its internalization, a growing body of data suggests that protein palmitoylation in host-pathogen interactions frontiers in immunology | www.frontiersin.org january | volume | article also bacterial proteins can undergo s-palmitoylation inside the host cells. this type of modification concerns so-called effectors, bacterial proteins that are injected into the host cell cytoplasm either across the plasma membrane or the membrane of vesicles enclosing internalized pathogens, with the help of their secretion systems. these are secretion systems type iii and type iv, homologs of which have been described for pathogens and symbionts of mammals, insects, and plants ( , ) . the bacterial effectors can be s-palmitoylated to reach host cell membranes and thereby accumulate at a location most suitable for their activity. application of the click chemistry-based method utilizing an analog of palmitic acid (alk- ) for cell labeling has revealed s-palmitoylation of two effector proteins of salmonella enterica, such as ssph and ssei ( ) . s. enterica invades gut endothelial cells and is a leading cause of gastroenteritis and typhoid fever. ssph carries an e ubiquitin ligase domain while ssei shows sequence homology to bacterial proteins that have a deamidase activity, and inhibits migration of salmonella-infected cells. the latter activity requires s-palmitoylation of ssei. both proteins are stably s-palmitoylated, most likely by zdhh and zdhh of the host and bind to the plasma membrane in a palmitoylationdependent manner ( ) . also two effector proteins of the ipah family of shigella spp. were found to be s-palmitoylated in that study, suggesting that this modification can control the activity of effector proteins of other pathogens as well ( ) . indeed, gobx and lpda, effector proteins of legionella pneumophila, the causative agent of legionnaires' disease invading macrophages and lung endothelial cells, are s-palmitoylated as was found recently using click chemistry. lpda is a phospholipase hydrolyzing various phosphatidylinositols while gobx is an e ubiquitin ligase. gobx is targeted in a palmitoylation-dependent manner to the golgi apparatus, and lpda to the plasma membrane and a subset of intracellular vesicles ( , ) . thus, the diversified subcellular localization of bacterial effector proteins reflects that of eukaryotic proteins. it is worth noting that global profiling of acylated proteins with the application of click chemistry and an alkyne-functionalized analog of myristic acid, alk- , for cell labeling was effective in reveling the mechanism of action of shigella flexneri effector protein ipaj of type iii secretion system. this is a unique protease that cleaves off the n-terminal myristoylated glycine. this proteolytic demyristoylation activity of ipaj is specific toward golgi-associated arf/arl family of gtpases regulating cargo transport through the golgi apparatus, inhibition of which is apparently pivotal for virulence of the bacteria causing diarrhea in humans ( ) . in addition to the s-palmitoylation of the effectors of pathogenic bacteria of mammals mentioned earlier, double acylation, n-myristoylation and s-palmitoylation, has been reported of the so-called avirulence (avr) proteins (effectors of type iii secretion system) of pseudomonas syringae, a causative agent of diverse plant diseases. among them, avrrpm and arvb are n-myristoylated and s-palmitoylated by host acyltransferases at neighboring glycine and cysteine residues localized at the n-terminus of the proteins (similarly to eukaryotic kinases of the src family), while in avrpphb and two avrpphb-like effectors-orf and nopt, the double acylation motif is exposed after auto-cleavage of the proteins (similarly to some eukaryotic proteins cleaved by caspases). the acylation of the avr proteins ensures their anchoring in the host plasma membrane, which is required for their functioning. in disease-susceptible plants avr proteins contribute to successful infection; however, in plants expressing host resistance (r) genes they trigger plant defense signals, in both cases engaging plasma membrane-associated host proteins ( , ) . the importance of palmitoylation of bacterial effector proteins for their infectivity is only beginning to be uncovered, in no small part owing to the development of the click chemistry-based method for detection of this protein modification. however, the strategy of high-jacking the host palmitoylation machinery to modify own proteins seems to be much more commonly employed by viruses. viruses do not encode palmitoyl acyltransferases but exploit extensively the host palmitoylation machinery to modify their proteins essential for infection of host cells and own replication. in fact, s-palmitoylation of proteins was discovered in as a modification of envelope glycoproteins of sindbis virus and vsv. in those studies [ h]-palmitic acid was used for metabolic labeling of virus-infected cells and labeled proteins were identified by autoradiography ( , ) . subsequently, a number of other viral proteins have been found to be palmitoylated using this approach. the most-studied group of viral palmitoylated proteins is those found in enveloped viruses, i.e., viruses covered by a lipid bilayer obtained during their replication from a membrane of the host cell, such as the plasma membrane or endoplasmic reticulum. influenza virus, hiv- , hepatitis c virus (hcv), and herpes simplex virus (hsv) are the best known enveloped viruses. the envelope is rich in transmembrane, often s-palmitoylated, glycoproteins called spikes, which can bind to cognate receptors on the host cell plasma membrane triggering endocytosis of the virion, mediate subsequent fusion of the viral and cellular membranes allowing entry of the viral genome to the cytoplasm, and are also involved in the budding of newly formed virus particles from the cell. an example of such multifunctional palmitoylated transmembrane glycoproteins is ha present in the envelope of influenza virus together with another palmitoylated transmembrane protein, the matrix protein m , which forms a proton channel earning the protein the name viroporin. as mentioned earlier, ha of influenza a virus is s-stearoylated and s-palmitoylated, respectively, at one cysteine residue located in the transmembrane domain of ha and two cysteines found in the cytoplasmic (intraviral) tail in close proximity to the membrane ( ) . on the other hand, m is s-palmitoylated on the amphiphilic helix located in the cytoplasmic part of the protein. due to the s-palmitoylation and the presence of a cholesterol-binding motif the helix bends toward and associates with membranes ( , ) . during infection, ha binds to sialic acid residues of glycans localized on the surface of airway and alveolar epithelial cells. the bound virions are endocytosed and next the viral and endosome membranes fuse. the membrane fusion is driven by ha, which undergoes conformational changes induced by low ph of endosomes. acidification of endosomes activates also the m proton channel activity, protons entering viral core facilitate dissociation of the viral genome which then moves to the nucleus where rna replication occurs. the s-palmitoylation of ha is required for the fusion of the viral and endosome membranes at least in some subtypes of the virus while the ion channel activity of m is not dependent on its s-palmitoylation ( ) . newly synthesized viral proteins and rna are assembled into virions in the plasma membrane rafts which merge into lager platforms crucial for the virion assembly and budding off. the triple fatty acylation of ha is required for its targeting to plasma membrane rafts ( , ) . besides s-palmitoylation, also the amino acid sequence of the transmembrane domain of ha determines its association with rafts ( ) . on the other hand, among the amino acids of the cytoplasmic tail of ha no other than the two s-palmitoylated cysteines are required for viral assembly and replication, although it is still not clear whether raft targeting (in cooperation with the transmembrane fragment) is the only mechanism of their participation. it is proposed that they affect conformation of the ha tail controlling its interaction with structural matrix protein m lying beneath the viral envelope ( , ) . the budding off of the virion is facilitated by m which localizes at the edges of rafts as a result of a combination of its s-palmitoylation, cholesterol binding, and properties of the transmembrane fragment. m protein can create a "wedge" altering membrane curvature thereby facilitating membrane scission and release of the virion ( , ) . the influenza virus s-palmitoylated proteins are the archetype for many other viral proteins. thus, s-palmitoylated spike glycoproteins include s-protein of coronaviruses (e.g., severe acute respiratory syndrome virus), the fusion (f) protein of paramyxoviruses (e.g., measles virus), env of retroviruses [e.g., hiv- , feline immunodeficiency virus (fiv)], and filoviruses (e.g., ebola). other viral proteins modified with palmitate are viroporins, such as e protein of coronaviruses, and also peripheral membrane proteins or nucleocapsid proteins absent in influenza virus. it has been found that s-palmitoylation of f l, a peripheral protein of the envelope of vaccinia virus, controls the association of the protein with intracellular membranes, thereby the formation of the envelope ( ) . the core protein of the nucleocapsid of hcv resides on the surface of lipid droplets and binds in a palmitoylation-dependent manner to membranes of the droplet-associated endoplasmic reticulum. subsequently, it recruits viral proteins and newly synthesized rna for viral particle formation ( ) . besides the interest in the role of viral protein s-palmitoylation for infectivity and possible use of host zdhhc enzymes as targets of anti-influenza drugs ( ) , viral proteins often serve as a model to study the consequences of fatty acylation for protein functioning and localization in distinct membrane domains (see s-palmitoylation of proteins and its influence on protein localization, trafficking, and stability of this review). readers are referred to recent exhaustive reviews that consider these topics ( , , ) while we will focus here on the recent advances in the field of viral protein palmitoylation brought about mainly by proteomic studies. the click chemistry-based approach has led to the identification of s-palmitoylation in the cytoplasmic domain of the transmembrane spike protein env of fiv, considered to be the cat equivalent of hiv- . env comprises three transmembrane gp glycoproteins and three associated gp which bind to cd receptor and coreceptors on the surface of t lymphocytes allowing fusion of the viral envelope and the plasma membrane and entry of viral capsid. four cysteines in fiv env are s-palmitoylated vis-a-vis two found in the env of hiv- . the two most membrane-proximal cysteines, and , are required for the fiv membrane-fusion activity and incorporation of env into virions ( ) , in agreement with the importance of env s-palmitoylation for virion assembly of some hiv- strains ( ) ( ) ( ) . the assembly of hiv- virions takes place in plasma membrane rafts and is driven by n-myristoylated gag protein which anchors and oligomerizes preferentially in these plasma membrane domains due to the presence of the fatty acyl chain ( ) . the development of click chemistry-based methods allowed for the first time global profiling of acylated proteins in virusinfected cells. in addition to identifying acylated viral proteins this approach has also revealed how the viral infection modulates the acylation pattern of the host cell proteins. thus far, click chemistry has been used to study protein myristoylation and palmitoylation in cells infected with hiv- and with hsv. in the latter case, the standard metabolic labeling with alkynefunctionalized myristic and palmitic acid analogs followed by click chemistry and mass spectrometry was combined with silac to discern between the changes in the extent of protein acylation and those in their abundance following viral infection. this approach allowed an elaborate quantitative analysis of host protein acylation and has revealed an overall downregulation of the level of both host protein modifications in infected cells. while the decreased content of myristoylated proteins resulted mainly from suppression of host protein synthesis, the drop in several s-palmitoylated proteins ensued from the inhibition of their palmitoylation in infected cells. the affected proteins were localized mainly to the plasma membrane and the golgi apparatus and were involved in vesicle-mediated transport and ion transport. in addition, the study has expanded the list of hsv-encoded acylated (mostly palmitoylated) proteins that play different functions in the viral cycle, such as ge, gi, gk, us , and us ( ) . similar results pointing to global changes of host protein acylation were obtained upon analysis of protein myristoylation and palmitoylation in cells infected with hiv- . in that study, the cells were labeled with analogs of palmitic or myristic acid tagged with an azide moiety for click chemistry reaction; however, the following mass spectrometry analysis did not address the relation between changes of protein acylation vs. alteration of protein level. the study identified palmitoylated and myristoylated proteins significantly differing in abundance between hiv- infected and uninfected cells. several of the proteins affected by the infection were of host origin. the abundance of myristoylated proteins was in general increased while that of the palmitoylated ones-decreased in infected cells ( ) . in other words, the two studies have revealed that hsv and hiv- not only encode proteins that are acylated in the host cell but protein palmitoylation in host-pathogen interactions frontiers in immunology | www.frontiersin.org january | volume | article also alter the palmitoylation of host proteins, likely to adapt the cellular environment to favor their replication and budding. the majority of the acylated proteins affected by hiv- or hsv infection had not been described earlier in this context; therefore, further studies on these proteins could be crucial for better understanding of viral infection. thus, the click chemistry-based approach has been highly effective in revealing changes of the host protein palmitoylation and opening new possibilities for the identification of novel antiviral drug targets. the innate immune responses are the first line of active defense against microbial infections. the application of click chemistrybased and abe methods and their use for large-scale analysis of protein palmitoylation in murine dendritic cd . cells ( , ) , and murine macrophage-like raw cells ( , ) complemented by proteomic analysis of the raft fraction of those cells ( ) have contributed significantly to the understanding of the role of palmitoylation of host receptors and signaling proteins involved in innate immune responses. thus, the palmitoyl proteome analysis of murine dendritic cells unraveled s-palmitoylation of tlr , a receptor expressed in cells of myeloidal lineage, which heterodimerizes with tlr or tlr to bind bacterial tri-or diacylated lipoproteins, respectively, and also other microbial components, such as glycolipids (e.g., lipoarabinomannan) of mycobacterium and yeast zymosan ( ) . besides tlr , two other human tlrs out of ectopically expressed in hek cell, flagellin receptor tlr , and tlr , a unique tlr negatively regulating the pro-inflammatory activity of tlr , were also found to be palmitoylated. the s-palmitoylation site of human tlr was mapped to cys adjacent to its transmembrane domain. the modification was present in unstimulated cells and was linked with up-regulation of the cell surface localization of tlr . mutation of cys abolished the ability of the receptor to induce pro-inflammatory signaling in response to microbial ligands of tlr ( ) . further studies are needed to reveal whether s-palmitoylation of tlr controls its association with rafts as sites of tlr activation ( ) and/or affects endocytosis of the receptor, as found for the anthrax toxin receptor ( ) . one of the most extensively studied tlrs, tlr activated by bacterial lps, is not palmitoylated. yet, saturated fatty acids have been indicated to trigger pro-inflammatory signaling of tlr . thus, the tlr /md receptor complex is involved in the pro-inflammatory outcome of a diet rich in palmitic acid, as was found when analyzing markers of inflammation in the heart and adipose tissue of high fat diet-fed mice ( , ) . the molecular mechanisms underlying the pro-inflammatory properties of palmitic acid can involve its influence on the plasma membrane lipid order, hence raft organization, in a way that facilitates translocation of tlr (and tlr ) toward rafts ( , ) . palmitic acid also directly binds to the tlr -associated md protein ( , ) . an influence of palmitic acid on sphingomyelin/ceramide metabolism, which enhances the lps-induced responses, has also been considered ( ) . recent proteomic studies based on odya labeling of raw macrophage-like cells followed by click chemistry have revealed that stimulation of cells with lps induces profound changes of the abundance of palmitoylated proteins ( ) . the data are in agreement with earlier findings showing that lps induces accumulation of s-palmitoylated lyn kinase in the raft-enriched fraction of cells, allowing it to downregulate tlr signaling ( ) . one of the upregulated s-palmitoylated proteins was type ii phosphatidylinositol -kinase iiβ, which phosphorylates phosphatidylinositol to phosphatidylinositol -monophosphate. it was shown that palmitoylation determines the involvement of the kinase in lps-induced signaling ( ) . these data suggest that s-palmitoylated proteins, including enzymes catalyzing phosphatidylinositol synthesis and turnover, are important factors affecting the pro-inflammatory responses triggered by lps. notably lps induces production of tnfα, a pro-inflammatory cytokine that is s-palmitoylated itself. tnfα is synthesized as a transmembrane -kda precursor (tmtnfα) transported from the endoplasmic reticulum to the plasma membrane through the golgi apparatus and recycling endosomes ( ) . human tmtnfα is s-palmitoylated at cys located at the boundary between its transmembrane and cytosolic fragments, as was found independently by radiolabeling and by labeling with odya followed by click chemistry ( , ) . poggi et al. ( ) arrived at a complex model explaining how the s-palmitoylation of tnfα affects its activity ( figure a) . the modification was shown to favor the association of tmtnfα with rafts. upon cell activation, the extracellular domain of tmtnf is cleaved by adam metalloproteinase whereupon the soluble tnfα (stnfα) is released to the extracellular milieu and activates tnf receptor (tnfr) and tnfr . as adam localizes to both non-raft and raft regions of the plasma membrane, the s-palmitoylation of tmtnfα does not affect its cleavage and production of the soluble cytokine. however, s-palmitoylated tmtnfα interacts with tnfr in rafts thereby reducing the binding of stnfα and consequently reducing the sensitivity of the cell to this cytokine. in addition, the fragment of tmtnfα which remains after the release of stnfα in rafts if further processed by intramembrane sppl a and b proteases giving rise to icd (intracellular domain) of an own biological activity. by contrast, the non-raft fragment of the adam -cleaved tmtnfα is rapidly degraded ( ) . the transport and maturation of tnfα are also regulated by another posttranslational acylation, ε-n-myristoylation ( ) . as shown in figure b , myristic acid residues are attached to two lysines (lys and ) of human tmtnfα. this modification is reversed by sirtuin catalyzing the demyristoylation. depletion of sirtuin decreases the release of stnfα since the ε-n-acylated tnfα precursor is redirected to and accumulates in lysosomes ( , ) . it is worth noting that exogenous palmitic acid stimulates the ε-n-myristoylation of tmtnfα, thereby reducing the release of stnfα in favor of accumulation of tmtnfα in lysosomes ( , ) . this somehow surprising anti-inflammatory effect of palmitic acid can be explained by competitive binding between long-chain fatty acids (in this case, palmitic) and myristoylated substrates of sirtuin found in vitro- ( ) and adds a new dimension to the potential effects of palmitic acid. (a) non-palmitoylated tmtnfα is localized outside rafts while that s-palmitoylated on cys -in rafts of the plasma membrane. tmtnfα is cleaved by adam protease in both these plasma membrane environments giving rise to stnfα, which subsequently activates tnf receptor (tnfr) receptor leading to activation of nfκb and erk / . however, only the raft-residing tmtnfα is further processed by sppl b protease to yield icd, which activates the promoter of interleukin (il)- β and expression of il- . on the other hand, a pool of s-palmitoylated tmtnfα interacts in rafts with tnfr preventing its activation by stnfα. (b) tmtnfα is transported from the endoplasmic reticulum via golgi apparatus and recycling endosomes [ , ] to the plasma membrane [ ] . in the plasma membrane, tnfα is cleaved by adam giving rise to stnfα [ ] or is internalized [ ] and either returns from the endosomes to the plasma membrane [ , ] or is directed to lysosomes for degradation [ ] . ε-n-myristoylation of tmtnfα at lys and lys facilitates its degradation [ , ] at the expense of processing to stnfα [ ] . oligomerization of tmtnfα and tnfr is not shown. s-palmitoylation of host proteins is also vital in antiviral defense. viral nucleic acids, which are recognized by several tlrs and also cytoplasmic pattern-recognition receptors, induce robust production of type i interferons (ifns), mainly infα and ifnβ. the ifnα and ifnβ released from cells which first encounter viruses, e.g., dendritic cells, induce an antiviral reaction in an autocrine and paracrine manner upon binding to plasma membrane ifnα/β receptor (ifnar) consisting of subunits and . both human ifnar subunits are s-palmitoylated, as has been found by classical radiolabeling. the s-palmitoylation of ifnar on cys , localized near the cytoplasmic end of the transmembrane domain, is required for downstream activation of stat and stat and the following transcription of ifnα-activated genes ( ) . among the ifn-induced proteins, some have been shown to be palmitoylated, using click chemistry and abe. they include the immunity-related gtpase irgm , bst also known as tetherin, and ifitm and ( , ) . ifitms are potent restriction factors against a wide range of enveloped viruses, e.g., influenza, west nile, dengue, and zika viruses ( , ) . ifitms localize primarily to endolysosomal membranes where they inhibit viral replication by blocking their fusion with these membranes and also facilitate virus degradation ( ) . the exact mechanism of this antiviral activity is not clear, but it seems to rely on a perturbation of the organization of endolysosomal membranes. this can be linked with the intramembrane topology of ifitms and their s-palmitoylation. ifitm and likely possess two loops embedded in but not spanning the membrane with both the n-and c-termini facing the cytoplasm ( , ) . s-palmitoylation of conserved cysteine residues adjacent to these loops, cys , , and in murine ifitm , contributes to the membrane binding, similarly as found earlier for caveolins ( , ) . the s-palmitoylation also facilitates clustering of ifitm in the membranes, which is of potential significance for its antiviral activity ( ) . in support of the latter, the antiviral capacity was markedly reduced for non-palmitoylated mutant forms of ifitm ( , ) . however, s-palmitoylation did not affect the endolysosomal localization or stability of ifitm . subsequent studies have revealed that the localization and degradation of murine ifitm , both shaping its antiviral capacity, are orchestrated by numerous posttranslational modifications comprising polyubiquitination, tyrosine phosphorylation by the src-family kinase fyn, and methylation ( , ) . by contrast, s-palmitoylation alone of the closely related murine ifitm endowed it with an antiviral activity and enhanced stability by preventing proteasomal degradation ( ) , which indicates diverse effects of this modification on individual ifitm isoforms. the presented data are only beginning to fill the gap which existed in our understanding of the role of protein palmitoylation in innate immune responses. for a long time, it was lagging behind that on acquired immune responses, in which a plethora of s-palmitoylated proteins have long been known to be involved. they include receptors (cd and cd ), tyrosine kinases of the src family, transmembrane adaptor proteins (e.g., lat, ntal, and pag/cbp), and α subunits of heterotrimeric g proteins. their s-palmitoylation in most cases targets them to rafts and is a prerequisite for their involvement in the signaling pathways triggered by immunoreceptors [tcr, b cell receptor (bcr), and fcγ and fcε receptors] crucial for the acquired immune responses. an association of some components of these signaling pathways with tetraspanin-enriched domains has also been considered. these topics are discussed in several earlier reviews ( , , , ) . it is worth noting that large-scale proteomic analyses of fatty-acylated proteins of t cells ( , , , ) and b cells ( ) , identifying numerous new palmitoylated proteins, have been published recently. further studies will shed light on the possible engagement of those proteins in acquired immune responses and/or in the cross talk between the innate and the acquired immune system, in which phagocytic cells, such as macrophages and dendritic cells, are essential ( ) . protein s-palmitoylation affects their localization, trafficking, and stability. it has long been known as an important factor controlling signal transduction by the bcr and tcr receptors involved in acquired immune responses. it is now becoming evident that palmitic acid is also a key lipid affecting the diverse processes at the host-pathogen encounter. palmitate is a component of bacterial lps and lipoproteins; s-palmitoylation of viral, some bacterial, and numerous host proteins is recognized as a crucial factor affecting both the virulence of pathogens and the innate immune reactions of the host. our understanding of the latter has benefited greatly from the development of novel methods of detection of this protein modification. their application has led to the identification of numerous proteins involved in the host-pathogen interaction. the methods have also allowed highthroughput proteomic analysis of palmitoylation of proteins in infected cells, showing widespread changes of the host cell palmitoylome. future studies will tell whether complex feedback loops comprising palmitoyl acyltransferases and acylthioesterases, similar to those of kinases and phosphatases carrying out protein phosphorylation/dephosphorylation, are involved in controlling protein s-palmitoylation in infected cells. revealing how the s-palmitoylation of particular proteins is regulated during the host-pathogen interactions should allow its modulation to favor the host defense. all authors contributed to writing and critically revised the paper. the authors thank prof. andrzej sobota from the laboratory of molecular membrane biology of the nencki institute (warsaw, poland) and dr. jan fronk from the faculty of biology, university of warsaw for helpful comments and critical discussion. the work was supported by the national science centre, poland, grant number dec- / /a/nz / to kk. mechanisms of nutritional and hormonal regulation of lipogenesis atherothrombosis and coronary artery disease microbial induction of immunity, inflammation, and cancer nutritional modulation of metabolic inflammation the impact of western diet and nutrients on the microbiota and immune response at mucosal interfaces 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mediated by clathrin, actin and unconventional adaptors anthrax toxin triggers the activation of src-like kinases to mediate its own uptake type iii secretion systems and disease exploitation of eukaryotic subcellular targeting mechanisms by bacterial effectors subcellular targeting of salmonella virulence proteins by host-mediated s-palmitoylation host cell-catalyzed s-palmitoylation mediates golgi targeting of the legionella ubiquitin ligase gobx legionella pneumophila effector lpda is a palmitoylated phospholipase d virulence factor myristoylome profiling reveals a concerted mechanism of arf gtpase deacylation by the bacterial protease ipaj eukaryotic fatty acylation drives plasma membrane targeting and enhances function of several type iii effector proteins from pseudomonas syringae a family of bacterial cysteine protease type iii effectors utilizes acylation-dependent and -independent strategies to localize to plasma membranes evidence for covalent attachment of fatty acids to sindbis virus glycoproteins s acylation of the hemagglutinin of influenza viruses: mass spectrometry reveals site-specific attachment of stearic acid to a transmembrane cysteine the influenza virus ion channel and maturation cofactor m is a cholesterol-binding protein intrinsic membrane association of the cytoplasmic tail of influenza virus m protein and lateral membrane sorting regulated by cholesterol binding and palmitoylation acylation-mediated membrane anchoring of avian influenza virus hemagglutinin is essential for fusion pore formation and virus infectivity role of lipid modifications in targeting proteins to detergent-resistant membrane rafts. many raft proteins are acylated, while few are prenylated quantitative analysis of the lipidomes of the influenza virus envelope and mdck cell apical membrane influenza virus hemagglutinin (h subtype) requires palmitoylation of its cytoplasmic tail for assembly: m proteins of two subtypes differ in their ability to support assembly two cytoplasmic acylation sites and an adjacent hydrophobic residue, but no other conserved amino acids in the cytoplasmic tail of ha from influenza a virus are crucial for virus replication influenza virus m protein mediates escrt-independent membrane scission palmitylation of the vaccinia virus -kda major envelope antigen. identification of a conserved acceptor motif and biological relevance palmitoylation of hepatitis c virus core protein is important for virion production s-acylation of influenza virus proteins: are enzymes for fatty acid attachment promising drug targets? palmitoylation, pathogens and their host palmitoylation of the feline immunodeficiency virus envelope glycoprotein and its effect on fusion activity and envelope incorporation into virions palmitoylation of the hiv- envelope glycoprotein is critical for viral infectivity human immunodeficiency virus type envelope glycoproteins that lack cytoplasmic domain cysteines: impact on association with membrane lipid rafts and incorporation onto budding virus particles the cytoplasmic domain of human immunodeficiency virus type transmembrane protein gp harbors lipid raft association determinants bioorthogonal mimetics of palmitoyl-coa and myristoyl-coa and their subsequent isolation by click chemistry and characterization by mass spectrometry reveal novel acylated host-proteins modified by hiv- infection toll-like receptors and their crosstalk with other innate receptors in infection and immunity lipoteichoic acid and toll-like receptor internalization and targeting to the golgi are lipid raft-dependent dietary saturated fatty acids prime the nlrp inflammasome via tlr in dendritic cells-implications for diet-induced insulin resistance saturated palmitic acid induces myocardial inflammatory injuries through direct binding to tlr accessory protein md modification of pro-inflammatory signaling by dietary components: the plasma membrane as a target mechanisms for the activation of toll-like receptor / by saturated fatty acids and inhibition by docosahexaenoic acid palmitic acid is a toll-like receptor ligand that induces human dendritic cell secretion of il- β acid sphingomyelinase plays a key role in palmitic acid-amplified inflammatory signaling triggered by lipopolysaccharide at low concentrations in macrophages lps upregulates palmitoylated enzymes of the phosphatidylinositol cycle. an insight from proteomic studies cytokine secretion in macrophages and other cells: pathways and mediators transmembrane tnf (pro-tnf) is palmitoylated palmitoylation of tnf alpha is involved in the regulation of tnf receptor signalling palmitoylation of interferon-α (ifn-α) receptor subunit ifnar is required for the activation of stat and stat by ifn-α ifitm-family proteins: the cell's first line of antiviral defense the ifitms inhibit zika virus replication s-palmitoylation and ubiquitination differentially regulate interferon-induced transmembrane protein (ifitm )-mediated resistance to influenza virus phosphorylation of the antiviral protein interferon-inducible transmembrane protein (ifitm ) dually regulates its endocytosis and ubiquitination greasing their way: lipid modifications determine protein association with membrane rafts protein acylation and localization in t cell signaling (review) patterns, receptors, and signals: regulation of phagosome maturation key: cord- -w sqy authors: crone, niek s. a.; kros, alexander; boyle, aimee l. title: modulation of coiled-coil binding strength and fusogenicity through peptide stapling date: - - journal: bioconjug chem doi: . /acs.bioconjchem. c sha: doc_id: cord_uid: w sqy [image: see text] peptide stapling is a technique which has been widely employed to constrain the conformation of peptides. one of the effects of such a constraint can be to modulate the interaction of the peptide with a binding partner. here, a cysteine bis-alkylation stapling technique was applied to generate structurally isomeric peptide variants of a heterodimeric coiled-coil forming peptide. these stapled variants differed in the position and size of the formed macrocycle. c-terminal stapling showed the most significant changes in peptide structure and stability, with calorimetric binding analysis showing a significant reduction of binding entropy for stapled variants. this entropy reduction was dependent on cross-linker size and was accompanied by a change in binding enthalpy, illustrating the effects of preorganization. the stapled peptide, along with its binding partner, were subsequently employed as fusogens in a liposome model system. an increase in both lipid- and content-mixing was observed for one of the stapled peptide variants: this increased fusogenicity was attributed to increased coiled-coil binding but not to membrane affinity, an interaction theorized to be a primary driving force in this fusion system. intramolecular cross-linking of peptides, commonly referred to as peptide stapling, is often employed to change or constrain the secondary structure of small peptides and to induce unstructured peptides to mimic complex protein folds and protein−protein interactions (ppis). − stapling also contributes to an increased resistance to denaturation and proteolytic degradation, making it a useful technique for the modification of peptide-based therapeutics. hydrocarbon stapling, a technique which is based on catalyzed olefin metathesis, has seen widespread application with multiple compounds being investigated in academic, preclinical, and clinical studies. − peptide stapling techniques can be broadly divided into two categories: single-and two-component strategies. singlecomponent strategies incorporate amino acids that can be cross-linked selectively, or protection strategies are chosen that allow selective cross-linking. common single-component stapling strategies include disulfide bonding, lactam bridges, , and olefin metathesis. two-component stapling adds a bifunctional cross-linker to bridge two amino-acid side chains; the most common techniques are based on cysteine cross-linking and triazole linkages. − two-component strategies are in principle more complex than singlecomponent strategies, but they allow for a more flexible cross-linker design, as it does not need to be compatible with solid-phase peptide synthesis. although most stapling techniques are used to increase or constrain peptide helicity, systems that compare different methods are often based around short peptide sequences, and although multiple comparisons have been made, , the ideal cross-linking technique is still open to debate. the α-helix secondary structure motif has been mimicked using stapled peptides due to its common occurrence in proteins and therefore its potential as a ppi mimic. coiled coils, which are protein-folding motifs comprising two or more α-helices, are intrinsically helical, and therefore, techniques commonly used for the stapling of helices should permit modulation of coiled-coil interactions. indeed, rao et al. have shown lactam bridges can be used to generate short, helical, cfos binding peptides, and haney and horne have used oxime cross-linking to generate stapled variants of the gcn -p coiled-coil domain. more recently, wu et al. used a bistriazole stapling technique to increase peptide binding to the polymerase α accessory factor ctf , and lathbridge and mason showed that lactam-bridged heptapeptides can be used for the de novo design of a coiled-coil binding peptide. together, these studies provide methods for the crosslinking of coiled-coil or coiled-coil-binding peptides, but it is unclear which method would prove to be the most effective when applied to a different coiled-coil system. the size of the macrocycle formed varies significantly between the different cross-linking techniques, as do the polarity and hydrophobicity of the cross-linkers in question. interactions of the asymmetric oxime moiety with different amino acid side chains resulted in different binding strengths when the cross-linker was reversed in haney and horne's method. this necessitated the preparation and evaluation of both variants, and indicates oxime cross-linking effectiveness is dependent on amino acid composition. our lab has developed a model system for membrane fusion, inspired by naturally occurring snare (soluble nsf attachment protein receptors) proteins. this system consists of a pair of complementary peptides dubbed e and k, which form a heterodimeric coiled-coil that can be attached to lipid membranes via a peg spacer and lipid anchor. like snare proteins, this model system promotes the fusion of lipid membranes, and it can be facilely modified to study the process of membrane fusion via structure−activity relationships. it has recently been discovered that these two peptides play different roles in the fusion process. the interactions of the k peptide with lipid membranes have been hypothesized as an important factor in membrane fusion efficiency. membrane interactions can occur simultaneously with the formation of the coiled-coil domain in a membrane fusion interface (as visualized in scheme c); therefore, a fine balance between the two must be achieved. in addition, both membrane binding and coiled-coil formation depend on the peptides adopting a helical structure; we believe stapling should allow for the generation of peptides with varied helical structures, which will in turn affect coiled-coil formation and membrane binding interactions. studying the effects of modulating the membrane interactions and coiled-coil binding affinity will generate insights into the importance of both factors in membrane fusion. when attempting to modulate the behavior of the heterodimeric coiled coil used in our group, the choice of cross-linking technique was not obvious, due to the differences observed in previously employed cross-linking techniques (vide supra). the position of the cross-linker and the macrocycle size were deemed the most influential characteristics in the previously mentioned cross-linking strategies; therefore, we wanted to evaluate both of these criteria independently for our system. the most favorable candidates could then be used to test the effect of structural changes on coiled-coil-based membrane fusion. one stapling strategy that attracted our attention was developed by the degrado lab, and it is based on the alkylation of cysteine using dibromoxylenes. the advantage of this system lies in the rigidity provided by the aromatic ring, allowing precise spacing between the two thiol moieties by selecting one of the three different structural isomers of dibromoxylene: ortho, meta, and para; scheme . in the original study, meta-xylene showed the most promise as a cross-linker, and further investigations in the same group have therefore focused on this variant. , other recent investigations have also predominantly used the meta derivative, , and when a comparison was made between the isomers, only short or unstructured peptides were used. this means the question of whether, for a helical or coiled-coil peptide, metaxylene is indeed the best cross-linking moiety is unanswered. therefore, to probe the effect of stapling on coiled-coil peptides, we elected to investigate dibromoxylene cross-linking of cysteines, employing all three structural isomers in order to elucidate the role of cross-linker size and its effect on structure and activity. in this study, a library of nine stapled peptides was prepared by modifying peptide k via cysteine alkylation. these stapled k-peptide derivatives exhibited systematic variations in helicity and thermal stability, as observed by circular dichroism (cd) spectroscopy. the coiled-coil binding thermodynamics were studied using isothermal titration calorimetry (itc), and it was discovered that increased coiled-coil binding is based on a preorganization effect. these observed changes in structure and binding dynamics were heavily dependent on the location of the staple and the choice of cross-linker. in lipid-and content-mixing experiments, a significant change in fusogenicity was measured for selected stapled peptides, which was attributed to the altered coiled-coil interactions. stapled peptide design. the starting point for structural modification is one peptide of a three-heptad heterodimeric coiled-coil pair first reported by litowski and hodges. the two peptides are named after the abundance of either glutamic acid (glu, e) or lysine (lys, k), and each peptide contains a c-terminal glycine and either tyrosine or tryptophan as a fluorescent reporter, giving rise to e gy and k gw. to facilitate stapling, two amino acids in peptide k gw were modified to cysteine, spaced i to i + to best match a single αhelical turn. amino acids that are involved in electrostatic (positions e and g) or hydrophobic (positions a or d) interactions were not varied to ensure the stapled peptides retained the same stabilizing coiled-coil interactions as the parent peptides. three different variants were generated each with the cysteines and therefore the staple, in a different heptad, table . each of these positional variants was stapled with ortho-, meta-, and para-dibromoxylene, generating a library of nine stapled peptides. when referring to these stapled peptide variants, a notation which reflects the position and type of cross-linker is used, for example, k gw- m signifies the cross-linker is in the first heptad and the meta variant has been employed. secondary structure analysis. cd spectroscopy was employed to determine the secondary structure of the stapled peptide variants; the effects of both the stapling location and the size of the cross-linker can be clearly observed, figures and s . peptide stapling close to the c-terminus (k gw- variants) showed the largest increase in α-helicity for all three xylenes, whereas modification in the second heptad (k gw- variants) showed the lowest increase. notably, when paraxylene was used as the cross-linker in the second heptad, the overall peptide helicity was reduced, figure b , showing paraxylene is too large to form an ideal α-helix. the n-terminal positions (k gw- variants) all show a moderate increase in helicity, largely independent of staple size, confirming the previously observed trend for hydrocarbon stapling to be most effective at peptide termini. using temperature-dependent cd spectroscopy, an increase in melting temperature (t m ) could be determined for the stapled peptide variants, as shown in figures and s , with the change in t m closely following the observed changes in helicity. c-terminal modification showed the largest increase in melting temperature, with the ortho-xylene cross-linker yielding the most stable peptides over all three peptide variants, followed by the meta-xylene crosslinker. all stapled peptides interacted with e gy, showing typical coiled-coil spectra as is evident in figures c and s . c-terminal stapling showed the highest helicity, while the nterminal stapled peptides did not have increased coiled-coil helicity compared to the staples located in the central heptad. in contrast to the stapled peptides in isolation, meta-xylenemodified peptides show the most α-helical structure as a coiled coil. ortho-xylene stapled peptides had the largest increase in t m for all three positions (figure ), and the trends in coiledcoil stability are similar to those observed for the single peptides, with an average increase in t m of . °c for the stapled peptides (table s ) and . °c for their coiled coils (table s ) . meta-xylene was previously shown to have the largest increase in helicity in small unstructured peptides, but in the e/k system ortho-xylene stapled variants yielded the highest single-peptide helicity and largest increase in t m for both the peptides and their respective coiled-coils. because it is possible that stapling affects coiled-coil interactions without changing peptide helicity as observed via the thermal unfolding experiments, the effect of peptide stapling on coiled-coil binding was further investigated using isothermal titration calorimetry (itc). binding thermodynamics of stapled coiled-coils. direct determination of the dissociation constant (k d ) and enthalpy of binding (Δh b ) and therefore calculation of the free energy (Δg b ) and entropy of binding (Δs b ) is possible using itc ( figure s ), allowing investigation of peptide interactions independent of peptide structure. the results shown in figure and table s show that, in general, coiled-coil binding of peptides k gw and e gy is strongly enthalpically favored but entropically unfavored. the effect of enthalpy can be explained via the formation of amide hydrogen bonds and electrostatic interactions upon folding of the peptide. when the c-terminally stapled variants of peptide k gw are analyzed, the k d is decreased from to and nm for the o and m variants, respectively, and to nm for the p variant. a large decrease in Δs b was observed and was directly related to the size of the implemented staple. ortho-xylene stapling at the c-terminus reduced the effect of entropy upon binding from to kj/mol, a reduction of %. at the same time, an increase in the Δh b from − to − kj/mol was observed, counteracting the observed entropic effects and leading to the conclusion that the mechanism of peptide stapling relies on a preorganization effect: through conformational restriction, the peptide is preorganized in a helical conformation which reduces the entropic effects of binding, but some of the energy that is gained upon formation of an αhelix is also lost. although the k d for the c-terminal ortho-and meta-xylene stapled peptides is comparable, the Δs b is more favorable for the ortho variant, explaining the large differences in t m observed for these two peptides. at all three stapling positions, the ortho variants show a reduced effect of entropy upon binding compared to the meta variants, which is likely caused by the smaller size of the ortho cross-linker. a smaller cross-linker restricts the maximum distance between the two helical turns and therefore limits the number of possible conformations that the peptide can assume. recently, miles et al. screened hydrocarbon-stapled peptides as protein−protein interaction (ppi) mimics against bcl-x l / mcl- and observed similar changes in the Δh b and Δs b for their stapled peptides; however, they observed an overall increase in Δg b . binding kinetics determined via a surface plasmon resonance (spr) assay showed that the binding of their ppi mimic could best be explained via an induced fit mechanism, where the ppi can interact via multiple binding modes. restricting the potential conformations of the peptide through the introduction of a staple reduced the number of possible binding modes and therefore increased the overall k d bioconjugate chemistry pubs.acs.org/bc article of the system. the e/k peptides used in this paper are designed and experimentally confirmed to form heterodimeric coiled-coils exclusively. as there is only one binding mode, the observed changes in structure and stability, as determined via cd, show a direct correlation with the binding thermodynamics in itc: c-terminal stapling using orthoand meta-xylene is the most effective way to increase the binding strength of coiled-coil peptides. membrane interactions of peptide k gw are perturbed by peptide stapling. the effectiveness of e/ k-based membrane fusion is partially attributed to the membrane interactions of peptide k, which are theorized to induce membrane curvature and therefore accelerate the transition from membrane docking to hemifusion. the interactions of peptide k with lipid membranes are based on a lysine snorkeling mechanism, which describes the hydrophobic amino acids in the "a" and "d" position inserting in a lipid membrane, helped by the favorable electrostatic interactions between lysines and the phosphate groups of the lipid membrane. this is a reversible process that can only happen when the peptide folds into an amphipathic helix and all the hydrophobic amino acids are positioned on the same face. peptide stapling, which changes the overall peptide conformation, is therefore theorized to have an effect on membrane binding. the membrane partition coefficient (k p ) of the stapled k variants was assayed via tryptophan fluorescence titration experiments, and the results are shown in figure . membrane binding was either comparable to that of unmodified k gw or was increased up to a factor of and did not show any correlation to the location of the staple or to the overall helicity of the peptide ( figure s ). the difference in partition coefficient between k gw- o and k gw- m is striking, as the value is almost half for the ortho variant despite the helicity of the two being very similar. this shows that the and are shown in figure s . bioconjugate chemistry pubs.acs.org/bc article addition of a hydrophobic cross-linking moiety between the "b" and "f" position does not increase the membrane affinity of amphiphilic α-helical peptides in a structure-dependent manner and leads to the hypothesis that peptide k gw does not bind to liposomal membranes as a highly structured αhelix. cd experiments were performed with the c-terminal stapled peptides in the presence of liposomes, and this data showed a reduced ellipticity at nm and a high / nm ratio (see figure s ). this indicates that the peptides are less α-helical in the presence of liposomes, which supports this hypothesis. if partitioning from the aqueous phase into the membrane is assumed to require partial unfolding of the peptide helix, the difference in binding strength between the ortho and meta variants can also be explained by the smaller size of the ortho cross-linker, which restricts the ability of the peptide to unfold. lipid-and content-mixing is increased for c-terminal stapled peptides. complete fusion of two lipidmembrane-enclosed spaces will result in homogeneous mixing of the lipids in the inner and outer leaflets, as well as mixing of the inner contents. in a liposomal system, this process can be studied via the incorporation of chromophores into the lipid bilayer or on the inside of the liposomes. fusion of these liposomes with nonlabeled liposomes will result in a fluorescence change which can be quantified to compare the peptide fusogenicity. lipopeptides were prepared which contained cholesterol and a polyethyleneglycol (peg ) spacer at the n-terminus, facilitating membrane anchoring. stapled peptides k gw- o and k gw- m were selected for fusion studies because these gave rise to the largest structural and thermodynamic changes. moreover, their binding strength is comparable, but their partition coefficient differs by a factor of ; therefore, by testing both and comparing them to unmodified k gw, the effect of both coiled-coil binding strength and membrane binding on fusogenicity can be determined. the lipopeptides were prepared using a novel on-resin stapling technique enabled by the use of methoxytrityl (mtt) protected cysteine; full details are available in the experimental section. these peptides were tested for fusogenicity together with the lipidated variant of e gy (structures can be found in scheme s ). lipid mixing was quantified using a forster resonance energy transfer (fret) pair incorporated in the lipid membrane, and the results are shown in figure a . the amount of lipid mixing observed was comparable for k gw and k gw- m at % peptide concentration, while the k gw- o variant showed increased lipid mixing min after the start of the experiment. this indicates that docking of the liposomes occurs at the same speed but more lipid mixing occurs for the k gw- o variant. as the absolute amount of lipid mixing was low, the same experiment was also performed with % of the lipopeptides, which doubled the amount of lipid mixing observed while retaining the same trends ( figure s ). content mixing experiments when performed properly are the best measure for complete fusion of two lipid membranes. the membrane-impermeable sulforhodamine b (srb) dye was employed as a fluorescent reporter and showed significant increases in fusion for both stapled peptide variants, figure b , with the k gw- m variant doubling the amount of content mixing compared to k gw (from . % to . %). the k gw- o variant produced an even larger increase; up to % content mixing was observed after min, with an average of . % . table s and figure s . bioconjugate chemistry pubs.acs.org/bc article this is surprising, since there was no observed difference between k gw and the k gw- m variant during lipid mixing experiments. an immediate difference between the three peptides is observed at the start of the experiment, which is not the case for lipid mixing, raising the concern that the stapled peptide variants might be destabilizing the liposomes and causing leakage of srb across the lipid membranes. plain liposomes and liposomes modified with % lipidated e gy were tested for leakage but did not show significant differences ( figure s ), indicating that the stapled peptides do not destabilize the liposomal membranes. insights into the mechanism of coiled-coil based membrane fusion. membrane fusion occurs in multiple stages, starting with the docking of two membranes to create a membrane fusion interface, followed by hemifusion which results in the mixing of the outer lipid leaflets, and proceeding via the formation of a fusion pore to complete fusion of the two liposomes, meaning their contents are exchanged. both lipid-and content-mixing experiments showed increased fusiogenicity for the lipidated k gw- o peptide, with increased content mixing also observed for the k gw- m variant. differences in the lipid-mixing amount are obvious after min, indicating that the rates of initial docking and outer leaflet mixing are comparable for the three peptides. because complete fusion of the liposomes, as judged by content mixing, is increased significantly for the k gw- o variant, the observed difference in lipid mixing is most likely caused by an increased mixing of the inner leaflet lipids. the increased coiled-coil binding strength observed via itc,could explain the increase in fusion except for the fact that k gw- o and k gw- m are dissimilar in their fusogenicity, yet they have a comparable k d . the k gw- o and k gw- m stapled peptides differ in their effects of entropy on coiled-coil binding and the strength of their membrane interactions, which are both increased for k gw- m. the k d of coiled-coil formation is dependent on the association and dissociation rate constants, which show different behavior in temperature dependent stopped-flow experiments of coiled-coil peptides. the dissociation rate was shown to be more dependent on temperature and therefore had a much larger entropic component then the rate of association. the stapled peptide variants tested have a decreased entropic binding component and should therefore also show a lower rate of dissociation. at a membrane fusion interface, dissociation of the coiled-coil is most likely followed either by another peptide binding event or by the insertion of peptide k into the lipid membrane. a decrease in the dissociation rate should therefore result in an increase in the rate of fusion, although the total amount of fusion observed is not expected to change. for snare-mediated membrane fusion, it is known that multiple protein complexes are required to drive fusion of a single vesicle, and the likelihood of fusion occurring is dependent on the number of protein complexes at the fusion interface. , this cooperativity is likely also necessary for our coiled-coil based system, and any interactions that influence the amount of coiled-coils that can be coassembled around a fusion interface will influence the amount of fusion observed. in this case, both k gw- o and k gw- m show increased binding and lowered binding entropy and therefore increased fusion via a lower dissociation rate. for k gw- m, this difference is less significant and is likely to be partially counteracted by the increased membrane affinity of the peptide. this is a competitive interaction in the formation of the coiled-coil complex, and an interaction which can provide a pathway for dissipation of the free peptide after dissociation of the coiled coil. in this manner, the total number of peptide complexes that are formed around a membrane fusion interface is reduced, and no increase in membrane fusion is observed. this reasoning can also be applied to homomeric peptide interactions, which could provide a pathway for dissipation of the lipopeptide away from the fusion interface. cd titration was performed with k gw and the k gw- o and k gw- m analogues to test for homodimerization ( figure s and table s ), but the dimerization constant was found to be comparable for all variants and weak enough that this should not be bioconjugate chemistry pubs.acs.org/bc article considered an important part of the fusion mechanism. this mechanistic understanding derived from the observed differences between the two stapled peptide variants will require further confirmation in different systems and experiments. we have employed a cysteine bisalkylation stapling technique to generate of a series of nine structurally isomeric α-helical peptides that can form a heterodimeric coiled-coil when mixed with their binding partner. cd and itc experiments showed that both stapling location and choice of staple affected the properties of the resulting peptides and coiled-coil complexes, with the largest increase in structure, binding, and stability observed for peptides stapled close to the c-terminus with ortho-xylene. binding strength is increased via a preorganization mechanism, which consists of a large reduction of the unfavored entropic binding component, combined with a negative change in binding enthalpy. ortho-and meta-xylene cross-linkers resulted in similar coiled-coil binding strengths, although ortho-xylene reduced the effect of entropy the most. this effect was true for all three stapling sites and is due to the smaller size of the ortho-xylene cross-linker. although there may be some dependence on amino acid composition, we conclude that ortho-xylene is the best cross-linker to stabilize helical peptides, despite meta-xylene being more widely employed to date. the effect of stapling on peptide-membrane partitioning was determined and showed a -fold difference between stapled peptide variants, although no direct correlation to location or staple type could be made. lipopeptides of k gw- m and k gw- o were prepared via a novel on-resin stapling method. these peptides were tested in lipid-and content-mixing experiments, and large increases in fusogenicity for the k gw- o variant were observed. k gw- m also showed significantly increased content mixing, but it exhibited a similar amount of lipid mixing to the parent peptide. we theorize that these differences in fusogenicity can be explained via reduced dissociation; increasing coiled-coil interactions without increasing lipid membrane interactions allows accumulation of more coiled-coil pairs at the fusion interface and therefore increases membrane fusion. tentagel resin was purchased from rapp polymere. dimethylformamide (dmf), piperidine, pyridine, acetic anhydride, trifluoroacetic acid (tfa), and acetonitrile (mecn) were supplied from biosolve. n,n-diisopropylethylamine (dipea) and oxyma were purchased from carl roth. dichloromethane (dcm) and diethyl ether were supplied by honeywell. hbtu and all protected amino acids except fmoc-cys(mtt)−oh were purchased from novabiochem. all other chemicals were purchased from sigma alrdrich. ultrapure water was obtained from a milli-q water purification system. peptide concentration was established via absorption at nm, determined using a cary- uv−vis spectrophotometer. peptide synthesis and purification. all peptides were synthesized on solid phase using a cem liberty blue automated, microwave-assisted, peptide synthesizer. peptides were prepared on a . mmol scale using tentagel hl ram resin with a loading of . mmol/g. fmoc deprotection was performed using % piperidine in dmf at °c for s. amide coupling was achieved using equiv of protected amino acid, equiv of dic as the activator, and equiv of oxyma as the activator base, heated at °c for s. acetylation of the peptide n-terminus after automated synthesis was performed using an excess of acetic anhydride and pyridine in dmf. lipidated peptides were made on resin via the coupling of . equiv of n -peg -cooh (see supporting information methods for synthesis details), with . equiv of hbtu, and equiv of dipea in dmf for h at room temperature. after washing the resin with dmf, the azide was reduced using equiv of pme ( m in toluene), with : dioxane/water as solvent for . h. after the reaction was finished, the resin was washed thoroughly with : dioxane/water, meoh, and dmf. lipidation was achieved using equiv of cholesteryl hemisuccinate, equiv of hbtu, and equiv of dipea in : dmf/dcm, and this lipidation step was performed twice to achieve complete conversion. after the final coupling, the resin was washed with dmf, meoh, and dcm and dried under vacuum, and the peptide was cleaved using a . : . : . : . mixture of tfa/tips/eddt/water for h, after which the peptide was precipitated in cold diethyl ether, collected via centrifugation, and lyophilized. all peptides were purified by hplc on a shimadzu system consisting of two kc- ar pumps and an spd- a or spd-m a detector equipped with a kinetix evo c column. eluents consisted of . % tfa in water (a) and . % tfa in mecn (b), with all peptides eluted using a gradient of − % b over min, with a flow rate of ml/min. collected fractions were checked for purity via lcms, with the pure fractions being pooled and lyophilized. lc/ms spectra were recorded using a thermo scientific tsq quantum access max mass detector connected to a ultimate liquid chromatography system fitted with a × . mm phenomenex gemini μm c column. lc/ms spectra of the purified peptides can be found in the supporting information. peptide stapling. intramolecular cross-linking was achieved by dissolving the peptide in a : mixture of mecn/h o containing mm nh hco up to a peptide concentration of μm. tcep, equiv, was added as a mm stock solution, and the reaction was stirred for h, followed by addition of . equiv of the dibromoxylene crosslinker ( mm in dmf) and reacted for h. the reaction was quenched by the addition of % acetic acid and purified using preparative hplc. for the lipidated peptides, the cross-linking was performed on the solid phase. in short, cysteines protected with mtt were incorporated into the peptide, and after automated synthesis these protecting groups were removed by incubating the resin with % tfa, % tis in dcm for min, followed by washing the resin with dcm twice. this was repeated until no more color appeared when a small amount of the resin was mixed with tfa. cross-linking was achieved by addition of . equiv of the cross-linker and . equiv of dipea in : dmf/tfe and incubating this reaction for h. on-resin stapling was usually performed before lipidation. circular dichroism measurements. cd spectra were recorded on a jasco j- cd spectrometer fitted with a peltier temperature controller. unless otherwise specified, samples were measured at °c in a quartz cuvette with a mm path length. spectra were recorded from to at nm intervals, with a bandwidth of nm, with the final spectrum consisting of the average of sequentially recorded ( ) with [θ] obs representing the observed ellipticity in mdeg, c being the peptide concentration in mm, n being the number of peptide bonds, and l being the path length of the cuvette in cm. the fraction of the α-helical peptide could be calculated from the mean residue molar ellipticity using eq : tryptophan fluorescence titration. fluorescence was measured in -well plates using a tecan infinite m pro microplate reader. liposomes of the composition : : dopc/dope/cholesterol were prepared at a mm concentration via extrusion in pbs buffer, using an avanti mini extruder with nm polycarbonate membranes. titration series of liposomes in pbs buffer were prepared with concentrations between and μm, with the peptide concentration held constant at . μm. samples were prepared in -well plates, and after min of incubation a fluorescence spectrum was taken between and nm. the maximum fluorescence of each sample was plotted as a fold increase of the fluorescence of the peptide without liposomes present and fitted against eq to determine the partition constant: where the normalized fluorescence, f, is dependent on the maximum fluorescence when all peptide is bound to the membrane f max , the molar partition coefficient k p , the lipid concentration x, and the concentration of water which is assumed to be constant at . m. experimental data representing three separate experiments was fitted to eq using the least-squares method to yield the partition coefficient and the standard error of fitting. isothermal titration calorimetry. itc measurements were performed on a malvern microcal peaq-itc automated calorimeter. in a standard experiment, the measurement cell contained μl of μm peptide k and the syringe was filled with e gy at μm concentration, with both peptides dissolved in pbs. the syringe content was added in injections of . μl at s intervals, except the first injection which was . μl. the reference power was set at . μcal/s, and experiments were performed at °c. the data was analyzed with the microcal peaq-itc analysis software and fitted to a single binding site model to generate the thermodynamic binding parameters. the experiment was repeated on three separate occasions, and the experimental results with the lowest reduced χ value are represented in this paper. lipid and content mixing experiments. liposomes with the lipid composition : : dopc/dope/cholesterol were used at a μm concentration, where % of the lipids was substituted with the respective lipopeptide. lipid films were prepared via evaporation of lipid and lipopeptide stock solutions in : chcl /meoh under a stream of nitrogen, followed by high vacuum for at least h. the lipid films were rehydrated via vortex mixing with pbs buffer and sonication for min at °c in a branson bath sonicator. the liposomes were checked for size and polydispersity (pdi) via dynamic light scattering (malvern zetaszier nano s) and then sonicated for a second time if the pdi was larger than . . lipid mixing was assayed via the incorporation of . % dope-nbd ( , -dioleoyl-sn-glycero- -phosphoethanolamine-n-( nitro- - , -benzoxadiazol- -yl)) and . % dope-lr ( , dioleoyl-sn-glycero- -phosphoethanolamine-n-(lissamine rhodamine b sulfonyl)) in the lipid membranes of the cpkcontaining liposomes. a volume of μl of fluorescent cpkcontaining liposomes was mixed with μl of nonfluorescent cpe-decorated liposomes, and the emission of nbd at nm was followed over time. each experiment included a positive control consisting of liposomes at a μm concentration and . % of both dope-lr and dope-nbd, and a negative control where the fluorescent liposomes were combined with liposomes without cpe. the standard deviation was calculated on the average of four separate measurement samples, and the experiment was repeated at least three times. content mixing was assayed via the incorporation of mm sulforhodamine b in the hydration buffer of cpe-decorated liposomes. after sonication, the unincorporated rhodamine was removed using an illustra nap- size-exclusion column. for each experiment, μl of sulforhodamine-containing cpe-liposomes was mixed with μl of cpk-containing liposomes, and the fluorescence of sulforhodamine followed over time at nm. the value was normalized via referencing a positive control consisting of liposomes containing mm sulforhodamine b prepared in the same manner and a negative control where the fluorescent cpe liposomes were combined with plain liposomes. the standard deviation was calculated on the average of four separate measurement samples, and the experiment was repeated at least three times. change in fluorescence was measured in -well plates using a tecan infinite m pro microplate reader. the percentage of lipid and content mixing was calculated using the following formula (eq ): where f t is the fluorescence at time t and f and f max are the fluorescence of the negative and positive controls at the same time point, respectively. processing of fluorescence data and one-way anova analysis were performed in graphpad prism . structure-based design of inhibitors of protein-protein interactions: mimicking peptide binding epitopes stabilized helical peptides: overview of the 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solution structure of a highly stable de novo heterodimeric coiled-coil a coiled-coil peptide shaping lipid bilayers upon fusion spontaneous adsorption of coiled-coil model peptides k and e to a mixed lipid bilayer situ modification of plain liposomes with lipidated coiled coil forming peptides induces membrane fusion energetics of coiled coil folding: the nature of the transition states snare-mediated membrane fusion is a two-stage process driven by entropic forces entropic forces drive self-organization and membrane fusion by snare proteins the authors declare no competing financial interest. the authors gratefully acknowledge professor nathaniel i. martin and ioli kotsogianni from the institute of biology (ibl) at leiden university for access to, and technical assistance with, the itc measurements. key: cord- - dwhgj authors: knoops, kèvin; kikkert, marjolein; van den worm, sjoerd h. e.; zevenhoven-dobbe, jessika c; van der meer, yvonne; koster, abraham j; mommaas, a. mieke; snijder, eric j title: sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum date: - - journal: plos biol doi: . /journal.pbio. sha: doc_id: cord_uid: dwhgj positive-strand rna viruses, a large group including human pathogens such as sars-coronavirus (sars-cov), replicate in the cytoplasm of infected host cells. their replication complexes are commonly associated with modified host cell membranes. membrane structures supporting viral rna synthesis range from distinct spherular membrane invaginations to more elaborate webs of packed membranes and vesicles. generally, their ultrastructure, morphogenesis, and exact role in viral replication remain to be defined. poorly characterized double-membrane vesicles (dmvs) were previously implicated in sars-cov rna synthesis. we have now applied electron tomography of cryofixed infected cells for the three-dimensional imaging of coronavirus-induced membrane alterations at high resolution. our analysis defines a unique reticulovesicular network of modified endoplasmic reticulum that integrates convoluted membranes, numerous interconnected dmvs (diameter – nm), and “vesicle packets” apparently arising from dmv merger. the convoluted membranes were most abundantly immunolabeled for viral replicase subunits. however, double-stranded rna, presumably revealing the site of viral rna synthesis, mainly localized to the dmv interior. since we could not discern a connection between dmv interior and cytosol, our analysis raises several questions about the mechanism of dmv formation and the actual site of sars-cov rna synthesis. our data document the extensive virus-induced reorganization of host cell membranes into a network that is used to organize viral replication and possibly hide replicating rna from antiviral defense mechanisms. together with biochemical studies of the viral enzyme complex, our ultrastructural description of this “replication network” will aid to further dissect the early stages of the coronavirus life cycle and its virus-host interactions. viruses rely on the host cell's infrastructure and metabolism during essentially all stages of their replication cycle and have therefore adopted strategies to coordinate a variety of molecular interactions in both time and intracellular space. the fact that the replication complexes of positive-strand rna (þrna) viruses of eukaryotes are invariably associated with (modified) intracellular membranes appears to be a striking example of such a strategy [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . specific þrna virus replicase subunits are targeted to the membranes of particular cell organelles that are subsequently modified into characteristic structures with which viral rna synthesis is associated. the morphogenesis, ultrastructure, and function of these complexes, sometimes referred to as ''viral factories,'' are only beginning to be understood. they may facilitate the concentration of viral macromolecules and provide a membrane-based structural framework for rna synthesis. other potential benefits include the possibility to coordinate different steps in the viral life cycle and to delay the induction of host defense mechanisms that can be triggered by the double-stranded rna (dsrna) intermediates of þrna virus replication [ , , ] . defining the structure-function relationships that govern the membrane-associated replication of þrna viruses, a large virus cluster including many important pathogens, will enhance our general understand-ing of their molecular biology and may have important implications for the development of novel antiviral control strategies. following the outbreak of severe acute respiratory syndrome (sars; for a review, see [ ] ), the coronavirus family of þrna viruses received worldwide attention. in addition to sars-coronavirus (sars-cov), several other novel family members were identified, including two that also infect humans [ ] . coronaviruses, and other members of the nidovirus group, have a polycistronic genome and employ various transcriptional and (post)translational mechanisms to regulate its expression [ , ] ). the gene encoding the replicase/transcriptase (commonly referred to as ''replicase'') comprises about two-thirds of the coronavirus genome, which-at - kb-is the largest rna genome known to date. the replicase gene consists of open reading frames (orfs) a and b, of which the latter is expressed by a ribosomal frameshift near the end of orf a. thus, sars-cov genome translation yields two polyproteins (pp a and pp ab) that are autoproteolytically cleaved into nonstructural proteins (nsp to ; figure ) by proteases residing in nsp and nsp [ ] [ ] [ ] . several of the replicative enzymes of coronaviruses, like an rna-dependent rna polymerase (rdrp) and a helicase, are common among þrna viruses, but they also contain a variety of functions that are rare or absent in other þrna viruses, including a set of intriguing proteins that are distantly related to cellular rna processing enzymes [ , , ] . the complexity of coronavirus rna synthesis is further highlighted by the fact that it entails not only the production of new genome molecules from fulllength negative-strand rna (''replication''), but also a unique mechanism of discontinuous rna synthesis to generate subgenome-length negative-strand rna templates for subgenomic mrna production (''transcription'') [ , ] . the resulting set of subgenomic transcripts (eight in the case of sars-cov) serves to express structural and accessory protein genes in the -proximal domain of the genome. ultimately, new coronavirions are assembled by budding of nucleocapsids into the lumen of pre-golgi membrane compartments [ , ] . the nidovirus replicase includes several (presumed) multispanning transmembrane proteins that are thought to physically anchor the replication/transcription complex (rtc) to intracellular membranes. in the case of coronaviruses, these domains reside in nsp , nsp , and nsp ( figure ) [ , ] . in the cytoplasm of infected cells, nidoviruses induce the formation of typical paired membranes and double-membrane structures that have commonly been referred to as ''double-membrane vesicles'' (dmvs) [ ] [ ] [ ] [ ] . these structures are mainly found in the perinuclear area of the cell, where-according to immunofluorescence (if) microscopy studies-de novo-made viral rna and various replicase subunits colocalize, presumably in the viral rtc [ , , , ] . immunoelectron microscopy (iem) previously revealed that sars-cov nsp and nsp localize to the outside of dmvs and/or the region between dmvs. although these proteins also colocalized in part with endoplasmic reticulum (er) marker proteins [ , , ] , the origin of dmv membranes has remained undecided since other studies have implicated other organelles in the formation of rtcs and dmvs, e.g., late endosomes, autophagosomes, and most recently, the early secretory pathway and potentially also mitochondria [ ] [ ] [ ] [ ] [ ] . previous ultrastructural studies may have been hampered by the technical challenge of dmv preservation [ ] . in particular, the dmv inner structure is fragile, and loss or collapse of dmv contents likely was a complicating factor. although the use of cryofixation methods dramatically improved dmv preservation [ ] , our understanding of the three-dimensional ( -d) organization and origin of dmvs was hampered by the inherent limitations of analyzing ''conventional'' thin sections ( nm) by electron microscopy (em), in particular since the diameter of dmvs was estimated to be between and nm [ ] . to develop a -d ultrastructural model for the rtc-related the domain organization and proteolytic processing map of the sars-cov replicase polyprotein pp ab. the replicase cleavage products (nsp - ) are numbered, and conserved domains are highlighted (blue, conserved across nidoviruses; grey, conserved in coronaviruses). these include transmembrane domains (tm), protease domains (plp and mp), and (putative) rna primase (p), helicase (hel), exonuclease (exo), endoribonuclease (n), and methyl transferase (mt) activities. for more details, see [ , ] . the delineation of amino acids encoded in orf a and orf b is indicated as rfs (ribosomal frameshift), and arrows represent sites in pp ab that are cleaved by the nsp papain-like protease (in blue) or the nsp main protease (in red viruses with a positive-stranded rna genome replicate in the cytoplasm of infected host cells. their replication is driven by a membrane-bound viral enzyme complex that is commonly associated with modified intracellular membranes. little is understood about the formation and architecture of these replication structures and their exact role in viral rna synthesis. we used electron microscopy and tomography for the three-dimensional imaging of the membrane alterations induced by severe acute respiratory syndrome (sars)-coronavirus, a member of the virus group with the largest rna genome known to date. previously, coronaviruses were reported to induce large numbers of isolated ''double-membrane vesicles'' (dmvs). however, our present studies reveal an elaborate reticulovesicular network of modified endoplasmic reticulum membranes with which sars-coronavirus replicative proteins are associated. the lumen of this unique membrane network contains numerous large (diameter - nm) ''inner vesicles,'' which were formerly thought to reside in isolated dmvs. intriguingly, although the interior of these vesicles does not appear to be connected to the cytosol, it labels abundantly for double-stranded rna, which presumably is present at the site of viral rna synthesis. the ultrastructural dissection of this elaborate ''replication network'' shows how coronaviruses extensively reorganize the host cell's membrane infrastructure, to coordinate their replication cycle, and possibly also hide replicating rna from antiviral defense mechanisms. membrane alterations in sars-cov-infected cells, we have now employed electron tomography (et; for reviews, see [ , ] ). this technique uses a set of two-dimensional ( -d) transmission em images, recorded at different specimen tilt angles with respect to the primary beam, for calculating a -d image (tomogram). typically, the specimen is tilted over a range of in small tilt increments ( ) , and an image is recorded at each tilt angle. the tomograms of infected cells allowed us to trace dmv membranes and establish previously unnoticed structural connections. in particular, et revealed that coronavirus dmvs are not isolated vesicles, but instead are integrated into a unique reticulovesicular network of modified er membranes, which also includes convoluted membranes that were not previously implicated in viral rna synthesis. strikingly, the latter structure-and not the dmvs-were primarily immunolabeled using antibodies recognizing viral replicase subunits. in contrast, immunolabeling with an antibody recognizing (presumably viral) dsrna abundantly labeled the dmv interior. since we could not discern a connection between the dmv interior and cytosol, our analysis raises several questions about the mechanism of dmv formation and the actual site of sars-cov rna synthesis. the virus-induced ''replication network'' documented here places the early stages of the viral lifecycle and accompanying virus-host interactions in a new perspective. previously, we experienced that, compared to more traditional chemical fixation protocols, the preservation of the fragile coronavirus dmv structures could be significantly improved by using a combination of cryofixation and freeze substitution (fs) [ ] . we now further refined the fs protocol, in particular by improving membrane contrast by adding % water to the fs medium [ ] . using these optimized conditions to prepare thin sections ( nm) of sars-cov-infected vero e cells, we could detect the first dmvs at h postinfection (h p.i.) and were able to monitor the subsequent development of virusinduced membrane alterations. early dmvs had sizes ranging from to nm, were distributed throughout the cytoplasm, and were sometimes located in the proximity of small reticular membranes with which, occasionally, they appeared to be connected ( figure a ). from h p.i. on, the number of dmvs increased dramatically, and dmv clusters were observed throughout the cell, again frequently accompanied by and sometimes clearly connected to reticular membrane structures ( figure b , arrow). as infection progressed, dmvs became increasingly concentrated in the perinuclear area of the cell ( figure c ), in accordance with the available if microscopy data for various sars-cov replicase subunits [ , , ] . at h p.i., a -nm-thick slice through the center of an infected vero e cell generally contained between and dmvs. initially, the dmv inner and outer membranes were generally tightly apposed, but occasionally, some luminal space between the two lipid bilayers could be discerned ( figure b , arrowhead). although similar observations were previously made for different nidoviruses using a variety of chemical and cryofixation protocols, and despite the generally excellent preservation of cellular membranes, the documented fragility of coronavirus dmvs makes it clear that we cannot formally exclude the possibility that these local separations could result from preparation damage. from h p.i. on, we also observed large assemblies of convoluted membranes (cm), often in close proximity to dmv clusters ( figure d ). these structures, with diameters ranging from . to lm, are probably identical to the ''reticular inclusions'' that were first observed in cells infected with mouse hepatitis coronavirus (mhv) more than y ago [ ] and were later referred to as 'clusters of tubular cisternal elements,' which may have a connection to the er-golgi intermediate compartment (ergic) [ ] . we noticed that the sars-cov-induced cm resembled one of the replicationrelated membrane alterations induced by flaviviruses, which were proposed to be the site of viral genome translation and polyprotein processing [ , , ] . in some of our images, the sars-cov-induced cm appeared to be continuous with both dmv outer membranes ( figure d ; inset) and er cisternae, suggesting a link to the viral rtc also in coronaviruses. especially at later stages of sars-cov infection (generally beyond h p.i.), we observed packets of single-membrane vesicles surrounded by a common outer membrane, as previously described by goldsmith et al. [ ] . the diameter of these vesicle packets (vps) ranged from to lm, and they sometimes included more than inner vesicles ( figure e ). in terms of size, morphology, electron density, and immunolabeling properties (see below), the vesicles contained in vps strongly resembled the inner vesicles of dmvs, as seen at earlier time points. during these later stages of infection, the clustered single dmvs ( figure c ) gradually disappeared, suggesting their merger into the vps. the average outer diameter of dmv inner vesicles at h p.i. was nm (n ¼ ), whereas later in infection, their average diameter (dmvs and vps combined) increased to about nm ( nm at h p.i., lm at h p.i.). our observations define vps as a third distinct modification of intracellular membranes that is induced by sars-cov infection. by h p.i., vps appeared to have merged into even larger cytoplasmic vacuoles, containing both vesicles as well as significant numbers of budding and completed virions ( figure e ). dmvs, cm, and vps were not observed in mockinfected vero e cells. although, occasionally, the analysis of ''conventional'' thin sections suggested cm and dmv outer membranes to be continuous and connected to er cisternae, a more accurate assessment required an analysis in three dimensions. we therefore employed et of semi-thick ( nm) sections of cryofixed, sars-cov-infected vero e cells. by using a specimen holder that could also be tilted around a second axis, perpendicular to both electron beam and first tilt axis, we obtained datasets, each consisting of differently tilted -d images, which were used to produce a high-resolution reconstruction in three dimensions. such ''dual-axis'' tomograms allowed us to visualize and analyze membrane continuities between the respective structures defined in the previous paragraph (as illustrated by videos s -s and abundantly present in the absence of advanced cytopathology. nevertheless, in some cells, infection had progressed more than in others, allowing the visualization of both advanced and earlier stages of infection in the same specimen. two major conclusions from this et analysis were ( ) that most or-likely-all coronavirus dmvs are interconnected by their outer membrane and ( ) that they are part of an elaborate network that is continuous with the rough er. as illustrated by the -d reconstruction in figure , for most dmvs, we observed one or multiple thin (; nm in diameter), ''neck-like'' connections of their outer membrane to the outer membranes of other dmvs, to cm, and to cisternae of the rough er ( figure ; insets). for example, in the two tomograms used for videos s and s , at least one such connection was visible for out of dmvs analyzed, strongly suggesting that for the remaining dmvs, such outer membrane connections existed but fell outside the volume reconstructed using these particular tomograms. of the dmvs for which at least one outer membrane connection was detected, had a single connection, whereas , nine, and three dmvs had two, three, and four connections, respectively. of these connections, approximately one-half were between the outer membranes of dmvs and the other half were connections to er or cm membranes, a ratio that was more or less stable when dmvs were differentiated in groups having one, two, or three connections. consequently, the original concept of ''free floating'' coronavirus-induced dmvs (i.e., structures surrounded by two, fully detached unit membranes) should be adjusted, and it would appear more appropriate to describe dmvs as single-membrane vesicles confined in the lumen of an er-connected membrane network. the vps (figures and ) and the tightly apposed membranes of the cm ( figure c ) were found to be integral parts of the same reticulovesicular network. the et analysis further suggested the presence of fibrous material inside dmv inner vesicles (figures - ). although ribosomes were clearly visible on rough er cisternae and dmv/vp outer membranes (figure , arrowheads; video s ), they were not detected on the membranes or in the interior space of the inner vesicles. by h p.i., in part of the cells, the formation of vps had begun (figures and ), for which we could distinguish two different morphologies in our tomograms. in the first type ( figure ; video s ), the membranes of the adjacent inner vesicles were tightly apposed but intact, and there was little luminal space between the inner vesicles and the surrounding outer membrane. in contrast, the outer membrane of the second type of vp appeared more relaxed and generally contained multiple inner vesicles ( figure a ; video s ). strikingly, instead of the intact inner membranes observed in dmvs and the first type of vp, we observed inner membrane discontinuities for many of the vesicles present in the second type of vp ( figure a ), de facto resulting in the fusion of vesicles or in apparent connections with the lumen of the membrane compartment. interestingly, we also observed virus budding from the outer membranes of the second type of vps ( figure a and b, arrowheads), suggesting the ultimate convergence of rtc-associated membrane structures with compartments involved in virus assembly. in order to assess the association of replicase subunits with the various coronavirus-induced membrane structures, we performed iem experiments on sars-cov-infected vero e cells. in view of previously experienced problems to preserve dmv ultrastructure for iem [ ] , the fs protocol was further optimized, and samples were embedded in lowicryl hm . when using this fixation and embedding protocol, several of our antisera unfortunately no longer recognized their target, restricting our analysis-for the moment-to a relatively small number of replicase subunits. on the other hand, the various sars-cov-induced membrane alterations documented in the previous paragraphs could now readily be recognized in iem samples ( figure ). furthermore, dmv inner structure was preserved, which had proven impossible in previous iem studies [ ] . for samples fixed at h pi, highly specific immunogold labeling results were obtained with antisera [ ] recognizing the large nsp subunit, which contains one of the viral proteases and is also a presumed transmembrane protein [ , ] , the viral main protease nsp [ ] , and the nsp putative rna primase, which has been postulated to be a subunit of the core rdrp complex [ , ] . protein contrast was enhanced in these fs samples, due to the absence of stained membranes, revealing electron-dense areas between dmvs that were strikingly similar, both in size and localization, to the cm structures documented above ( figure ). remarkably, using all three reactive sars-cov antisera, cm were the most abundantly labeled structures. for nsp and nsp , small numbers of gold particles were also found on dmv membranes, but the interior of dmvs (and vps) was essentially devoid of label ( figure ). in the case of nsp , some labeling of the dmv interior was observed, but again the majority of the label localized between dmvs on the cm structures. in combination with our data from previous if studies, documenting the colocalization of several key replicative enzymes [ ] , our iem data suggest that the cm structures are the major site of sars-cov nsp accumulation. a critical step in the replication of þrna viruses is the production of a negative-stranded copy of the genome, which (b) from h p.i. on, clusters of dmvs began to form. occasionally, connections between dmv outer membranes and reticular membrane structures were observed (arrow). locally, luminal spacing between the dmv outer and inner membranes could be discerned (arrowhead). (c) as infection progressed, dmvs were concentrated in the perinuclear area (nucleus; n), often with mitochondria (m) lying in between. (d) example of a cluster of cm, which were often surrounded by groups of dmvs. the structure seems to be continuous with the dmv outer membrane (inset). (e) during the later stages of infection, dmvs appeared to merge into vps, which developed into large cytoplasmic vacuoles (asterisk) that contained not only single-membrane vesicles (arrowhead pointing to an example), but also (budding) virus particles. scale bars represent nm (a), nm (b and d), or lm (c and e). doi: . /journal.pbio. .g is used as a template for genome replication by the viral rdrp. coronaviruses also generate a set of subgenome-length negative-strand rnas, which serve as templates for subgenomic mrna synthesis [ , ] . it is widely assumed that viral negative-strand rna synthesis leads to the formation of partially and/or completely dsrna structures, commonly referred to as replicative intermediates (ris) and replicative forms (rfs) and, in the case of coronavirus subgenomic mrna production, transcriptive intermediates (tis) and transcriptive forms (tfs) [ , ] . whereas rfs/tfs are (nearly) completely double stranded, and may accumulate, e.g., when rna synthesis ceases and the last positive strand is not released from the negative strand, ris/tis are viewed as dynamic multistranded intermediates engaged in positive strand synthesis. they are thought to be only partially double stranded and contain multiple tails of nascent single-stranded rna produced by the successive rdrp complexes engaged in copying the negative-strand template (see [ ] and references therein). for a variety of þrna viruses, the (presumed) dsrna intermediates of replication have been visualized in situ by using antibodies recognizing dsrna [ , [ ] [ ] [ ] . in particular, monoclonal antibody j [ ] , recognizing rna duplexes larger than base pairs, was reported to be a useful tool in recent if studies [ , ] . we here used the j antibody in if and em studies, resulting in a highly specific labeling of sars-cov-infected cells, whereas mock-infected cells were essentially devoid of signal ( figure a ). even before immunodetection of nsps was feasible, the first if signal for dsrna could already be detected (at - h p.i.) as small but very bright foci throughout the cell ( figure a ). by h p.i., the distribution of dsrna-containing foci generally mirrored that of nsp , nsp (unpublished data), and nsp ( figure b ). however, high-resolution confocal microscopy ( figure c and d) revealed that the overlap was far from complete, and frequently, multiple dsrna foci appeared to surround an area that labeled for replicase. later in infection, the labeling for both dsrna and nsps was mainly concentrated in the perinuclear region ( figure e ). whereas different nsps colocalized to a large extent ( figure e , bottom row), this was less obvious when the labeling for dsrna and replicase subunits was compared. in subsequent iem experiments, the j antibody was found to retain its reactivity for dsrna in sections of cells that had been embedded in lowicryl, following the fs procedure described above. an abundant and highly specific labeling for dsrna was observed on the interior of sars-cov-induced dmvs (figure ) , with some additional label being present in the vicinity of dmvs where cm were frequently observed during our studies ( figure b) . also, type and type vps were positive for dsrna ( figure c ), whereas (budding) virions present in these structures were always negative. thus, figure . immunogold em of the sars-cov replicase in infected cells sars-cov-infected vero e cells were cryofixed at h p.i. and processed for fs and iem using rabbit antisera (see materials and methods). in all images, -nm colloidal gold particles conjugated to protein a were used for detection of primary antibodies. (a and b) labeling for sars-cov nsp was mainly found on the electron-dense areas between dmvs, presumably representing cm as most clearly visible in (b). (c) immunolabeling for sars-cov nsp (the viral main protease), which was essentially similar to that for nsp . (d) when using an antiserum recognizing sars-cov nsp (the putative viral primase), the majority of label was again present on cm. however, a small fraction of the nsp signal was reproducibly found on the interior of dmvs. scale bars represent nm. doi: . /journal.pbio. .g our data revealed the accumulation of dsrna, presumably of viral origin (see discussion), in the interior vesicles of dmvs and vps, and also suggested that the fibrous material observed in our et analysis (figures - and videos s and s ) may consist (in part) of viral nucleic acids. the functional dissection of the multienzyme complexes that drive þrna virus replication and transcription is membrane vesicles (for a recent review, see [ ] ). the first -d ultrastructural analysis of a replication structure of the spherular type was recently reported by kopek et al. [ ] . in an et-based study, the replicase and rna synthesis of flock house virus were found to be confined to spherular invaginations of the mitochondrial outer membrane. this ''viral mini-organelle'' was reported to be connected to the cytosol by a neck-like channel with a diameter of about nm. this connection is assumed to be both sufficient and essential for the import of, e.g., nucleotides into replication spherules and for export of viral rna products, which need to be released into the cytosol for translation and packaging. we have now employed et to analyze the replication structures of sars-cov, a prominent member of the coronavirus group which-at about , amino acidsencodes the largest known þrna virus replicase [ ] . dmvs had previously been observed in cells infected with coronaviruses and related nidoviruses [ ] [ ] [ ] [ ] ] , and the precise origin of their membranes had remained debated. they were generally assumed to be ''free floating'' vesicles associated with viral rna synthesis. however, our present et analysis has revealed that they form a unique reticulovesicular membrane network (figure ) with which both viral replicase subunits and dsrna are associated (figures - ) . the network is continuous with the rough er and contains in its lumen numerous ''inner vesicles,'' which stand out for their relatively large size ( - -nm diameter), their number (several hundred, possibly more than , vesicles per cell), and for the fact that they label abundantly for dsrna. remarkably, however, their interior does not appear to be connected to the cytosol (see also below). the fact that the average diameter of the sars-cov-induced dmvs ( - nm) exceeds the maximal thickness (; nm) of the sections that could be used for et made it generally impossible to visualize their entire perimeter. however, the number of inter-dmv connections that could be observed in the reconstructed volume (at least one for % of the dmvs analyzed, with more than half of those having multiple connections) justifies the conclusion that they form an integrated network and makes it highly unlikely that free dmvs exist (figures - ) . vps and cm structures are also an integral part of the network (figures - and ) , and in particular, the latter structures appear to be a major site of immunolabeling for sars-cov nsps. essentially similar observations were made for cells infected with a second coronavirus, mhv (unpublished data). our studies identify the er as the source for a virus-induced membrane network that integrates cm, dmvs, and vps, although the (additional) involvement of the ergic remains a possibility [ ] . in combination with biochemical studies, the ultrastructural description of this network is important to take our understanding of coronavirus rtc structure and function to the next level. finally, our analysis of cells at more advanced stages of sars-cov infection ( figure ) opens the intriguing possibility that the membrane network involved in virus replication is continuous (or merges) with membranes involved in virus assembly. for mhv, based on if microscopy studies using the nsp helicase and viral membrane (m) protein as markers for rtcs and virus assembly sites [ ] , respectively, such a connection was previously proposed [ ] , but could not be corroborated in our studies using the same protein markers in sars-cov-infected cells [ ] . according to the data presented in this study, the bulk of the labeling for nsps is found on the cm structures ( figure ) , not on dmvs, which would explain the minimal overlap between the nsp labeling and that for the m protein [ ] . furthermore, it cannot be excluded that merger of type vps and compartments involved in virus budding is a relatively rare event that could result from general cytopathology and/or fusion of different membrane compartments. notably, in some of the larger vps (e.g., see figure e ), a kind of polarity was observed, with budding and mature virions mostly on one side and the inner vesicles of (former) dmvs on the other, as if two previously ''dedicated compartments'' recently merged into a larger vesicle. although the juxtaposition and functional connection of compartments involved in genome replication, encapsidation, and assembly remains a fascinating idea, a thorough quantitative analysis of sars-cov assembly is beyond the scope of this paper and would require the collection of extensive datasets, in particular around the peak time of virus assembly ( - h p.i.). by analogy with the replication-associated ''membrane spherules'' of several other þrna viruses [ , ] , it was anticipated that the dmv interior would be connected to the cytosol, thus allowing import of (macro)molecules required for rna synthesis and export of rna products, e.g., for translation and packaging. however, our tomograms revealed a sealed dmv inner membrane and an uninterrupted outer membrane that was clearly continuous with other membrane structures. the two tomograms that were the basis for figures and and videos s and s were scrutinized for discontinuities of dmv inner and/or outer membranes, with the expectation of finding at least one such connection per vesicle when dmv interior and cytosol would indeed be continuous. neck-like connections between the outer membranes of different dmvs were readily discerned (see above; figure ) , and the quality of our images also allowed the highresolution visualization of, e.g., membrane necks of budding virions ( figure ). however, for the vast majority of dmvs, an extensive search for a repetitive pattern showing a neck, channel, or other type of structure connecting dmv interior and cytosol remained negative. for only one out of dmvs visible in videos s and s , an aligned gap of both inner and outer membrane could be detected ( figure s a ). furthermore, in three other dmv profiles, the inner membrane was locally disrupted (figure s b-s d), but since these sites also showed local separation of the two leaflets of the bilayer, we consider it likely that these interruptions were fixation or processing artifacts. given the previously documented fragility of the dmv inner membrane in particular, this would not be surprising, and this property may also be related to the puzzling inner membrane discontinuities observed for type vps late in infection ( figure and video s ). however, for the vast majority of dmvs in our images, the inner membrane was found to be uninterrupted, and thus, the dmv interior appears not connected to the cytosol. in view of the resolution provided by our tomograms, we are confident that we would have readily detected connections to the cytosol with a diameter ( - nm) in the range previously described for other þrna virus replication structures [ , ] . thus, our data suggest that-at least at the moment of fixation-dmv inner membranes form closed vesicles and their morphogenesis has now become one of the major unresolved issues. it should be stressed that we cannot exclude the possibility that proteinaceous pores or transporters may be present in dmv membranes, since similar complexes (e.g., the translocon) have not been recognized in situ in em/et studies yet. however, despite the fact that the large coronavirus proteome was recently found to include several unexpected and unprecedented functions, proposing the existence of such a channel would seem highly speculative at this moment. three coronavirus replicase subunits (nsp , nsp , and nsp ) contain hydrophobic domains that are each predicted to traverse the membrane multiple times [ , , ] . their properties have not been characterized in detail, but the recent phenotypic characterization of a temperaturesensitive mhv mutant with a lesion in nsp revealed a dramatic reduction of dmv formation at the restrictive temperature, thus clearly implicating this protein in the formation of the reticulovesicular network documented in this study [ ] . still, apart from the question whether the transmembrane nsps are able to form membrane-spanning channels or recruit host proteins capable of forming such a connection, other conceptual problems would remain. for example, the alignment of the channels spanning the inner and outer membranes would appear to be a requirement, much like it has been proposed for the sophisticated tom and tim complexes engaged in import across mitochondrial outer and inner membranes [ , ] . moreover, the transport across membranes of a large, negatively charged rna molecule like the approximately -kb coronavirus genome poses a challenge that in biology appears to be met only by the nuclear pore complex. in addition to the recent data on mhv nsp [ ] , results obtained with the distantly related arteriviruses indicate that the (predicted) membrane-spanning nsps of nidoviruses are likely to play a critical role in inducing membrane alterations. it was shown that the expression of two such arterivirus nsps sufficed to induce paired membranes and dmvs similar to those found upon virus infection [ , , ] . most likely, these subunits are first inserted into ''regular'' er membranes, which may thus also be the site of early viral rna synthesis. when replication leads to a rapid increase of replicase expression, the accumulating transmembrane nsps may induce membrane pairing and curvature, due to, e.g., their specific structural features, oligomerization, or recruitment of cellular factors involved in membrane bending. the notion that inner and outer bilayer may be ''physically associated,'' due, for example, to interacting luminal domains of protein partners present in the two membranes, is supported by the fact that the two membranes remain tightly associated just up to the point where narrow neck-like connections protrude to the outer membrane of other vesicles or compartments ( figure ) . apparently, at later time points after infection when dmvs merge into the larger vesicle packets, the inner membranes are able to more and more detach from the outer membrane. interestingly, during our recent (unpublished) studies using the drug brefeldin a, which interferes with vesicular transport and de facto results in fusion of golgi complex and er into one large, dilated compartment, similar observations could be made much earlier in infection. this would suggest that the interaction between the two membranes eventually weakens, possibly in particular when the outer membrane network becomes dilated due to cytopathology and/or merger of multiple vesicles. presumably, membrane pairing is followed by the wrapping of membrane cisternae around cytosolic constituents and leads to the membrane fission event that is needed to explain the sealed dmv inner membrane. however, despite the presence of several hundred dmvs in infected cells and despite the extensive em analysis of hundreds of cells in the course of this study, we were unable to find morphological profiles that seemed obvious examples of an actual dmvforming fission event. although some smaller dmvs were sometimes observed (video s ), the average dimensions of their inner compartments ( - nm in diameter) should have made the detection of nascent dmv structures straightforward. arguably, dmv formation might be very rapid, and thus rarely captured, or obscured in, e.g., the complex architecture of the cm structure, where smaller dmvs were sometimes apparent (figure and video s ). alternatively, the conspicuous absence of ribosomes from dmv inner membranes lends some credibility to a scenario involving a preformed inner vesicle derived from another membrane source. the observed narrow neck-like connections in the network ( figure ) and the fact that many dmvs were found to have multiple (up to four) of such outer membrane connections with other dmvs, cm, or er also leaves the possibility that additional fusion and fission events may occur during the formation or maturation of the network, which would obviously hamper the analysis of the initial dmv forming event. the future identification of inhibitory drugs or dominant-negative mutants of viral or host proteins involved in this step may facilitate the visualization of this crucial intermediate stage in dmv morphogenesis. in infected cells, several other groups of þrna viruses induce membrane alterations that differ from the spherular membrane invaginations described, e.g., for nodaviruses [ ] and alphaviruses [ ] . in the case of picornaviruses (for a recent review, see [ ] ), the pioneering work of the bienz laboratory demonstrated that poliovirus rna replication occurs on the cytosolic surface of er-derived vesicles [ ] , which aggregate into rosette-like structures [ ] . however, the first detectable negative-stranded rna of poliovirus is associated with regular er cisternae, which may thus be the initial site of rna synthesis [ ] . other studies revealed that poliovirus-induced vesicles may have a double membrane [ ] and implicated the autophagic pathway in their formation [ ] . a similar hypothesis was launched to explain mhv dmv formation [ ] . despite a convincing link between overall mhv replication and the expression of a host protein with a critical function in autophagy (apg ), the ''autophagy hypothesis'' was contradicted by if studies using autophagosomal marker proteins [ , ] . our studies may in fact have uncovered a closer parallel to the membranes with which the replication complexes of flaviviruses are associated. ultrastructural studies of cells infected with kunjin virus have defined various characteristic membrane structures, which were implicated in viral rna synthesis on the basis of immunolabeling and biochemical studies ( [ ] ; for reviews, see [ , ] ). these structures include ''convoluted membranes'' and ''vesicle packets,'' terms that we have chosen to adopt in our study, without wanting to imply a direct ultrastructural or functional similarity. whereas the flavivirus cm have been implicated in replicase polyprotein synthesis and processing, the vps were proposed to be the site of viral rna synthesis, in particular because they could be immunolabeled for replicase subunits, dsrna, and de novo-synthesized viral rna that had been metabolically labeled by bromouridine (bru) incorporation [ ] . a key premise, however, in the current model proposed for flaviviruses [ , , ] is the idea that-as in the case of viruses employing spherular replication compartments (see above; [ ] )-the interior of the vesicles enclosed in the vps are connected to the cytosol. for the dmvs induced by coronaviruses and other nidoviruses, a similar hypothesis was among the previously formulated models [ ] , but-as explained above-in our sars-cov tomograms, an open connection between dmv interior and cytosol could not be discerned. recent biochemical studies on the in vitro activity of sars-cov rtcs, which were associated with membrane fractions prepared from infected cells, revealed that a detergent treatment is required to render the viral rna synthesizing complex susceptible to digestion with proteases or nucleases [ ] . thus, the isolated rtc appears to be protected by at least one membrane, a conclusion also drawn from similar biochemical studies on flavivirus rtcs, leading to an alternative model [ ] in which flavivirus vps would be ''topologically similar'' to coronavirus vps and consist of a closed inner vesicle surrounded by an outer membrane that is continuous with cm and er. if a future et analysis of flavivirus replication structures were to confirm this similarity, we would essentially be faced with the same question for both virus groups [ ] : if rna synthesis would indeed occur inside closed dmvs or vps, how then are import and export across the double membrane achieved? the presence of both viral nsps and dsrna on the sars-cov-induced membrane network strongly suggests its involvement in viral replication and transcription. however, the apparent separation in immunolabeling studies between the bulk of the nsps and most of the dsrna emphasizes the need to pinpoint the active coronavirus rtc. in particular the exact role in viral rna synthesis of the dmv inner vesicle, its ''fibrous content,'' and its abundant labeling for dsrna are intriguing. extensive proteolytic processing of replicase polyproteins pp a and pp ab (figure ) is assumed to be a critical posttranslational step in the activation of coronavirus replicative enzyme functions. it is also a complicating factor in immunolabeling studies since antibodies will commonly recognize both mature cleavage products and larger processing intermediates. moreover, immunolabeling will merely reveal the site of accumulation of specific antigens, not necessarily their site of synthesis. the fact that most of the label for sars-cov nsps was present on cm may seem incompatible with the presence of most of the dsrna signal on dmvs. if, however, as proposed for flaviviruses, the coronavirus cm would be the site of polyprotein synthesis and processing, abundant labeling of this region could be expected, in particular for the two viral proteases, nsp and nsp , that were detected on the cm in this study. the labeling observed for the putative nsp primase differed slightly, with some label consistently being present on the dmv interior ( figure d ). the nsp subunit possesses a secondary rna polymerase activity and has been postulated to be part of the core enzyme complex of the virus [ , ] . additional antisera, in particular targeting the viral key enzymes encoded in orf b (figure ), are currently being generated to increase our possibilities for detection of subunits of the multicomponent sars-cov rtc. also, the search for suitable antibodies against cellular marker proteins continues, which could aid in defining the interaction with the host cell's secretory pathway in more detail. as recently concluded for hepatitis c virus [ ] , a huge excess of nonstructural proteins may be produced in virus-infected cells, with only a fraction of these molecules actively participating in viral rna synthesis at any point in time. likewise, the labeling for dsrna, although widely considered a marker for þrna virus rtcs [ , , ] , does not formally pinpoint rtc activity. clearly, molecules inside active rtcs may be among the dsrna strands recognized, as is strongly suggested by colocalization of dsrna and newly made viral rna following bru pulse labeling [ ] . on the other hand, however, it is likely that part of the signal, a part that may in fact vary between different viruses, represents dsrna molecules that are no longer actively engaged in viral rna synthesis. in this context, it is noteworthy that the calculations on the number of active replication complexes in hepatitis c virus replicon cell lines (less than ; [ ] ) are not easily reconciled with the much larger number of discrete foci detected in such cells when labeling with the j anti-dsrna monoclonal antibody [ ] . given these considerations, it would be most straightforward to localize the site of activity of the sars-cov rtc early in infection, using ultrastructural studies that are combined with pulse labeling of viral rna synthesis using bru [ ] or radioisotope-labeled nucleosides [ ] , or by transfecting the corresponding nucleoside triphosphates. experiments to explore whether it is technically feasible to combine such an approach with the cryo-em and fs fixation protocols required for sars-cov dmv preservation are in progress. in our opinion, previous iem studies using bru labeling of mhvinfected cells [ ] cannot be considered conclusive in view of the obvious loss during fixation of the dmv inner vesicles, and possibly also the cm. nevertheless, the bru labeling detected by these authors on dmv outer membranes and surrounding structures suggests that at least part of the newly made rna was cytosolic after a -h labeling interval. in conclusion, a scenario in which part, or even most, of the sars-cov dsrna signal represents molecules that are not present in active rtcs (figures and ) cannot be ruled out at present. in this alternative scenario, the active complex might, for example, localize to the cm, where small amounts of dsrna labeling and the bulk of the viral nsps were detected. the subsequent formation of dmvs could then even be postulated to constitute an elegant mechanism to conceal viral rna and aid in the evasion of dsrna-triggered antiviral host responses. a variety of recent studies have made clear that coronaviruses are capable of interacting and interfering with the innate immune system at multiple levels, likely also depending on the cell type involved (for a recent review, see [ ] ). both sars-cov and mhv [ ] [ ] [ ] were found to counteract the induction of interferon via cytoplasmic pattern recognition receptors that can sense the presence of viral dsrna [ , ] and possibly also viral negative-strand rnas carrying uncapped -triphosphates [ ] . further analysis of the structure, interactions, and function of the coronavirus rtc may reveal to which extent this property should be attributed to the unusual network of modified membranes with which coronavirus rna synthesis appears to be associated. virus, cells, and antisera. sars-cov strain frankfurt- (kindly provided by dr. h. f. rabenau and dr. h. w. doerr [johann-wolfgang-goethe-universitä t, frankfurt am main, germany]; [ ] ) was used to infect vero e cells. all work with live sars-cov was performed inside biosafety cabinets in the biosafety level facility at leiden university medical center. a multiplicity of infection of was used in all experiments, and infection rates were routinely confirmed in if assays. a panel of rabbit antisera against the sars-cov replicase, including the nsp , nsp , and nsp subunits, was described previously [ ] . a mouse monoclonal antibody j [ ] , which is specific for dsrna, was purchased from scicons. electron microscopy. for ultrastructural morphological investigations, sars-cov-infected vero e cells were prefixed (for biosafety reasons) overnight with % paraformaldehyde in . m phem buffer ( mm piperazide- , -bis[ -ethanesulfonic acid], mm hepes, mm mgcl , mm egta) at various time points after infection. for cryofixation, cell monolayers adhered to thermanox coverslips (nunc) were plunged into liquid ethane. freeze substitution was performed at À c in an automated freeze-substitution system (leica) using an fs medium consisting of % acetone and % water, containing % osmium tetroxide and . % uranyl acetate. after washing with pure acetone at room temperature, the samples were embedded in epoxy lx- resin. thin sections were contrasted with uranyl acetate and lead hydroxide, and subsequently viewed at kv with a philips cm- transmission electron microscope. for iem, infected cell monolayers were cryofixed by either plunging them into liquid ethane or by high-pressure freezing using a leica em pact . the freeze substitution was performed using anhydrous acetone containing . % glutaraldehyde and . % uranyl acetate. after washing with ethanol, samples were infiltrated with lowicryl hm and polymerized under uv light at À c. thin sections were labeled with specific antisera [ ] , which were detected with protein a-gold particles ( or nm). a bridging rabbit-antimouse igg antibody (dakocytomation) was used for mouse monoclonal antibodies. grids were contrasted with uranyl acetate and lead hydroxide, and subsequently viewed with a philips cm- transmission electron microscope. when quantifying dmvs per infected cell, thin sections were cut in the direction parallel to the substrate, and the slice producing the largest nuclear diameter was analyzed, since this plane was generally found to contain the largest number of dmvs. electron micrographs (between and ) covering the entire cross-section of the cell were recorded, and to facilitate counting, these were digitally merged to produce a single image representing a -nm-thick plane through the center of the infected cell. merged images were analyzed with zoomify software. only dmvs for which the surrounding bilayers could be readily distinguished were counted, and their diameter was measured using imagej software (http://rsb.info.nih.gov/ij/). electron tomography. freeze-substituted infected cell samples, processed for morphological investigation as described above, were used to cut -nm-thick sections. to facilitate the image alignment that is required for the subsequent image reconstruction step, a suspension of -nm gold particles was layered on top of the sections as fiducial markers. for dual-axis tomography, two single-axis tilt series were recorded of the specimens with an fei t transmission electron microscope operating at an acceleration voltage of kv. per single-axis tilt series, images were recorded at tilt increments between À c and c. automated tomography acquisition software was used (xplore d; fei company). images were acquired with a cooled slow-scan charge-coupled device (ccd) camera ( k eagle; fei company) with , , pixels and were recorded by binning . the electron microscope magnification was , , corresponding to a pixel size of . nm at the specimen level. to enable dual-axis tomography, the specimens were rotated around the z-axis using a dual-axis tilt tomography holder (fishione; model ). to compute the electron tomogram, the dual-axis tilt series were aligned by means of the fiducial markers using the imod software package [ ] . the size of the voxels in the tomograms corresponds to . nm. full datasets have been deposited in the cell centered database (http://ccdb.ucsd.edu; [ ] ) under accession numbers - , respectively, containing the datasets of the tomograms shown in videos s , s , and s , and a zoomify image showing a high-resolution cross-section of an entire sars-cov-infected cell. the -d surface-rendered reconstructions of viral structures and adjacent cellular features were processed using amira visualization package (tsg europe) by surface rendering and thresholding. during this process, some volumes were denoised using the nonlinear anisotropic diffusion filtering [ ] . denoised volumes were used only for producing the surface-rendered masks. final analyses and representations were done using undenoised data (either masked or unmasked). immunofluorescence microscopy. infected cells on glass coverslips were fixed with % paraformaldehyde in pbs at various time points after infection and were processed for if microscopy essentially as described previously [ ] . following permeabilization, single-or duallabeling if assays were carried out with rabbit antisera and/or mouse monoclonal antibodies, which were detected using indocarbocyanine (cy )-conjugated donkey anti-rabbit immunoglobulin (ig) and alexa fluor -conjugated goat anti-mouse ig secondary antibodies, respectively (molecular probes). for dual-labeling experiments with two rabbit antisera recognizing different sars-cov nonstructural proteins, the anti-nsp antibodies were directly coupled to alexa fluor , as described previously [ ] . samples were examined with a zeiss axioskop fluorescence microscope (equipped with the appropriate filter sets, a digital axiocam hrc camera, and zeiss axiovision . software) (carl zeis, microimaging) or with a leica sp confocal laser scanning microscope, using a pinhole size of airy unit (for both channels) to give optical sections with a theoretical thickness of nm. images were minimally optimized for contrast and brightness using adobe photoshop cs . figure s . in-depth analysis of discontinuities in the membranes of sars-cov-induced dmvs see the legend to figure for details. a total of dmvs in the two tomograms that were the basis for figure a and b and videos s and s were scrutinized for discontinuities of dmv inner and/or outer membranes that might reveal a connection between the dmv interior and the cytoplasm. however, an extensive search for a repetitive pattern showing a neck, channel, or other type of structure connecting the dmv interior and cytoplasm remained negative. one out of dmvs ([a]; arrow) showed a small, aligned gap of both inner and outer membrane. in three other dmv profiles ([b-d]; arrows), the inner membrane was locally disrupted, but the separation of the two leaflets of the bilayer made it likely that these discontinuities were artifacts that had occurred during fixation and processing of the fragile dmv inner structure. the scale bar represents nm. figures c and d anaglyph images were produced and superimposed to provide a stereoscopic -d effect when viewed with spectacles with red (left) and green (right) glasses. found at doi: . /journal.pbio. .sg ( . mb jpg). video s . animation through a z-series of -nm-thick digital slices (total depth nm) of a dual-axis electron tomogram of a sars-cov-infected vero e cell at h p.i. the video shows a group of interconnected dmvs and also shows the connections of dmv outer membranes with the er. the tightly apposed double membranes and fibrous material inside the dmvs are clearly visible. to facilitate image alignment during image reconstruction, a suspension of -nm gold particles was layered on top of the sections as fiducial markers. found at doi: . /journal.pbio. .sv ( . mb wmv). video s . animation illustrating the derivation of the model presented in figure d structural and functional characterization of the poliovirus replication complex parallels among positive-strand rna viruses, reversetranscribing viruses and double-stranded rna viruses wrapping things up about virus rna replication virus factories: associations of cell organelles for viral replication and morphogenesis a guide to viral inclusions, membrane rearrangements, factories, and viroplasm produced during virus replication topology of double-membraned vesicles and the opportunity for non-lytic release of cytoplasm threedimensional analysis of a viral rna replication complex reveals a virusinduced mini-organelle modification of 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group coronaviruses prevent immediate early interferon induction by protection of viral rna from host cell recognition mouse hepatitis virus does not induce beta interferon synthesis and does not inhibit its induction by double-stranded rna inhibition of the alpha/beta interferon response by mouse hepatitis virus at multiple levels -triphosphate rna is the ligand for rig-i computer visualization of three-dimensional image data using imod the cell centered database project: an update on building community resources for managing and sharing d imaging data noise reduction in electron tomographic reconstructions using nonlinear anisotropic diffusion orf a-encoded replicase subunits are involved in the membrane association of the arterivirus replication complex for helpful discussions and support, we thank many of our colleagues at lumc, in particular, montserrat bá rcena, martijn van hemert, henk koerten, roman koning, hans van leeuwen, danny nedialkova, willy spaan, cindy swett tapia, and aartjan te velthuis. we gratefully acknowledge the skillful assistance of ronald limpens and jos onderwater in protocol development. competing interests. the authors have declared that no competing interests exist. key: cord- - kbnyqt authors: nathan, lakshmi; daniel, susan title: single virion tracking microscopy for the study of virus entry processes in live cells and biomimetic platforms date: - - journal: physical virology doi: . / - - - - _ sha: doc_id: cord_uid: kbnyqt the most widely-used assays for studying viral entry, including infectivity, cofloatation, and cell-cell fusion assays, yield functional information but provide low resolution of individual entry steps. structural characterization provides high-resolution conformational information, but on its own is unable to address the functional significance of these conformations. single virion tracking microscopy techniques provide more detail on the intermediate entry steps than infection assays and more functional information than structural methods, bridging the gap between these methods. in addition, single virion approaches also provide dynamic information about the kinetics of entry processes. this chapter reviews single virion tracking techniques and describes how they can be applied to study specific virus entry steps. these techniques provide information complementary to traditional ensemble approaches. single virion techniques may either probe virion behavior in live cells or in biomimetic platforms. synthesizing information from ensemble, structural, and single virion techniques ultimately yields a more complete understanding of the viral entry process than can be achieved by any single method alone. called spikes. the coat protein of non-enveloped viruses and spike proteins of enveloped viruses facilitate the virus's entry into the cell. these viral proteins engage receptors on host cells to promote attachment to the host cell surface, but after that initial binding event, non-enveloped and enveloped viruses breach host cell membranes through different pathways [ ] . non-enveloped viruses typically enter cells by endocytosis and then either lyse the intracellular compartment, or release genetic material through a pore created in the membrane of the intracellular compartment [ , ] . enveloped viruses, on the other hand, must undergo membrane fusion with a cellular membrane to release their genetic material into the host cell ( fig. . ) . some enveloped viruses, like coronaviruses or human immunodeficiency virus (hiv), can undergo fusion at the plasma membrane (a), while others, like influenza, fuse within endosomes (b) [ ] . binding and uncoating of nonenveloped viruses is mediated by the capsid surface or capsid proteins projecting from it. for enveloped viruses, the fusion or spike proteins embedded in the envelope are responsible for both receptor binding and membrane fusion. membrane fusion proteins can be subdivided into three categories based on their structure [ ] . class i fusion proteins are primarily α-helical, class ii fusion proteins contain mainly β-sheets and β-strands, and class iii fusion proteins contain a mix of both α-helices and β-sheets. class i and ii fusion proteins require a proteolytic priming event to convert them from a fusion-incompetent state to a fusioncompetent one. once the fusion protein is in a fusion-competent state, the structural the entry process begins with the virus binding to a receptor on the surface of the cell. this binding may result in (a) release of the viral genome via fusion at the plasma membrane or (b) uptake by endocytosis or macropinocytosis (not shown) followed by fusion within the endosome. the location and mechanism of fusion can be impacted by environmental conditions, including ph, ions, protease activity, and lipid composition. once the genome has been released into the cytosol, it can access cellular machinery, leading to replication of the viral genome and synthesis of new viral proteins. newly synthesized virions can then exit the cell by budding rearrangement necessary for exposure of the fusion peptide and its burial into the target membrane is often triggered by some change in environmental conditions. fusion may be triggered by receptor binding itself, lowering of ph, protease action [ ] , or a combination of these [ , ] . viruses that fuse at the plasma membrane may be triggered by receptor binding or protease activation, while those that fuse within the endosome generally require low ph and may require other additional cues, such as changes in other ions than protons, or endosome-specific protease exposure. despite the diversity in structure and triggering mechanisms, fusion proteins typically follow a common pathway for merging the viral envelope with the host membrane ( fig. . ), as described in white et al. [ ] and harrison [ ] . after fusion is triggered, the protein rearranges into a prehairpin intermediate ( ) with the fusion peptide inserted into the host membrane. while the fusion protein may be found as dimers or trimers on the surface of the virus ( ), the prehairpin intermediate consists of a trimer. it is thought that several prehairpin intermediates cluster together then fold back on themselves to bring the membranes close to each other ( ) . as the prehairpins fold into a six-helix bundle, the membranes are brought close enough that lipids from their outer leaflets are able to mix, creating a structure often referred to as a "stalk" ( ) . this step is known as hemifusion. lastly, a pore forms ( ) enabling the viral genome to pass through this opening as the fusion proteins form into a stable trimer of hairpins. although all viral fusion proteins studied to date follow this common pathway, much remains unknown about viral entry itself. for example, the dynamics of these fusion steps are not fully understood, the fusion trigger of some viruses remains ambiguous, and developing antivirals to halt virus entry requires further characterization of all of the steps between initial viral contact with the host cell and viral genome release. techniques for studying virus entry can be broadly classified as those that examine the entry of a collective group or ensemble of virions and those that track individual virion entry. ensemble methods provide information on the broader context of entry steps, while single virion imaging provides detailed information on the dynamics of those steps. ensemble methods include many of the most wellestablished techniques for studying viral entry and may probe overall infection, receptor binding, fusion, or cargo transfer. single virion imaging techniques can provide mechanistic detail and kinetics for steps that are masked in ensemble methods. in addition, for hiv [ ] , influenza [ ] , and ebola virus [ ] , most virions that encounter a cell are not involved in productive entry so the ability of single virion techniques to distinguish entry-competent particles from non-competent ones and characterize their individual behavior provides valuable data on heterogeneity in viral populations and its ultimate impact on infection. the remaining part of the chapter will focus on single particle tracking microscopy techniques compatible with dynamic/temporal data acquisition, their salient features, and how the data generated complement ensemble methods for studying viral entry processes and their intermediate steps and mechanisms. over the past years, microscopy optics, fluorescent labeling strategies, computing power and image processing have enabled the dynamic tracking of individual virions throughout the entry process and the decomposition of their intermediate steps. for some examples, see the following references: [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in particular, charge coupled device (ccd) cameras combined with advanced microscopy enable single particle resolution [ ] . the frame rates of today's cameras dictate temporal resolution of dynamic studies, which can range from a few to hundreds of milliseconds [ , ] . in the following sections, the main advances enabling single virion tracking techniques are summarized. three microscopy configurations are commonly employed in virology research: epifluorescence, total internal reflection fluorescence (tirf), and confocal microscopy [ , ] as illustrated in fig. . . the epifluorescence configuration ( fig. . a, d) illuminates everything within the field of view. so, unbound virions in the bulk phase or cells with high background autofluorescence result in high noise that obscures signals from individual virions. as a result, epifluorescence is not typically used for single virion tracking. in the tirf configuration ( fig. . b, e), only particles within a shallow field are illuminated, eliminating noise from the bulk solution and thereby enhancing the signal-to-noise. this configuration is achieved by setting the incidence angle of the laser to be higher than the critical angle for total internal reflection of the laser light at the glass/water or glass/cell interface, resulting in an exponentially decaying evanescent field that penetrates roughly nm into the sample [ , ] . the advantage with this configuration is that a two-dimensional plane can be monitored precisely, enabling the distinction between binding and fusion events of the virions, with appropriate virion labeling (described next). however, no three dimensional tracking information can be obtained; data collection is confined to the interfacial region of interest. this configuration is most suitable for examination of virus behavior at biomimetic surfaces like supported lipid bilayers or live cell plasma membranes close to the glass surface. in the confocal microscopy configuration (fig. . c), a pinhole optic assists in filtering out background noise while scanning across a sample one z-plane at a time. cross sections may be stacked to yield a three-dimensional image, making this configuration suitable for imaging live or fixed cells and observing trafficking of viruses in the cytosol. the disadvantage of this configuration is the difficulty in temporally tracking phenomena in real-time, while simultaneously scanning across slices of the sample and reconstructing the image. furthermore, exposure to strong light can be toxic to living cells [ ] . as technology advances, these drawbacks should subside. in all of these microscopy techniques, virions must be labeled to visualize them. virions are commonly labeled by attaching fluorescent proteins to viral proteins, by incorporating chemical dyes into the virion, or by incorporating quantum dots [ ] [ ] [ ] . here, we focus on fluorescent and chemical labels; for a review of quantum dot techniques we refer the reader to liu et al. [ ] . both fluorescent proteins and chemical labels have been used to label viral coatings as well as contents. fluorescent viral protein constructs can be prepared for enveloped [ ] or non-enveloped viruses [ , ] and virions are labeled as they are synthesized. chemical labels, including alexa dyes, octadecylrhodamine b (r ), and carbocyanine perchlorates (did, dii, or dio) may be added during virion synthesis or post-production. some chemical labels, like lipophilic dyes, are only suitable for labeling enveloped viruses while others, like amino-reactive dyes, may be employed with either enveloped or non-enveloped virions [ ] . membrane hemifusion is typically monitored with lipophilic dyes that undergo dequenching when the virion fuses with the target membrane, while pore formation is typically tracked with a dye that can partition into the capsid and then dissipate upon release from the virion [ , ] . it is also possible to incorporate ph sensors into the envelope of virions to measure the ph of the fusion environment [ ] . in all cases, care must be taken to ensure that labeling does not adversely impact viral infectivity. this can be easily assessed by conducting cell infectivity assays with labeled virions. in general, these labeling approaches can be optimized to mitigate any significant negative impact. once the microscopy configuration and virion labeling method have been chosen, image processing assists in extracting information from the images. today's fast computers and image processing algorithms assist in noise filtering, virion tracking, and trajectory mapping, as described in the references that follow. in single virion imaging, noise is always a primary concern, regardless of the microscope configuration. to combat this, several particle detection and image restoration techniques have been developed specifically for single virion tracking [ , ] . coordinates of the particles are obtained by scanning filtered images for areas of fluorescence intensity that exceed a certain threshold or fit a particular intensity profile [ , ] . the next challenge is obtaining accurate virion tracking from one frame to the next (temporal trajectories). particle trajectories can be calculated from nearest-neighbor associations [ , ] . measurements of virion movement and mean squared displacements are then used to determine whether the virions exhibit directed, normal, or anomalous diffusion [ ] . the diffusion type can indicate the type of interactions the virion is having with the surface of the cell or extracellular environment. tracking frame-to-frame is important in measuring binding residence times as well, and by extension, binding strength characteristics [ ] . once bound, the progression of membrane fusion can be tracked from frame-to-frame using strategies like fluorescence dequenching, where the evolution of the fluorescence signal reports on the merging of membranes, the rate of membrane mixing, and the release of viral genome. finally, obtaining good statistical data from virion tracking experiments requires collecting data on hundreds of individual virions. single virion tracking may follow virions through the infection process in live cells using confocal microscopy or within an in vitro biomimetic platform using tirf microscopy. cell-based virion imaging techniques enable direct visualization of the viral entry pathway and interactions between virions and host cell machinery within the native complexity of the cellular environment. on the other hand, biomimetic platforms utilizing the tirf configuration enable observation of membrane surface phenomena and can be integrated with tools like microfluidics that allow the user to define and test a tightly controlled environment. this control enables decoupling of factors that may be hard to detangle in vivo. sections . . . and . . . provide overviews of these two approaches while sect. . provides examples of specific implementations of single virion tracking to the investigation of viral entry and contrasts them with ensemble approaches. in current cell-based fluorescence assays, both virions and cellular components are fluorescently labeled so their interactions can be tracked with multicolor real-time microscopy. live cell virion tracking can be used to observe cell-cell spread, receptor binding, intracellular trafficking and membrane fusion. virions may be labeled with fluorescent proteins, chemical dyes, or quantum dots while intracellular components are typically labeled using fluorescent proteins. epifluorescence, tirf, and confocal microscopy have all been used in live cell single virion tracking. virions may be tracked in two or three dimensions depending on the microscope configuration, as described above. certain cell types may be easier to image due to autofluorescence of cellular components [ ] . as early as the s, fusion of individual virions at the plasma membrane surface was observed. virions were labeled with r , which self-quenches at high concentrations, and dequenching of the r upon fusion of the virion with a membrane was detected by a simple fluorescence microscope. early assays monitored fusion at the plasma membrane of erythrocytes, where fusion of influenza could be triggered by lowering the ph [ , ] . an important aspect of live cell approaches is that it is possible to inhibit cellular machinery by drug treatment or gene knockout. such cells can be used in combination with live cell imaging to identify key cellular components that are necessary for virus entry. a review of implementations and insights gained from live cell single virion imaging will be provided in sect. . . we also recommend the reviews by brandenburg and zhuang [ ] , ewers and schelhaas [ ] , otterstrom and van oijen [ ] , sun et al. [ ] , and wang et al. [ ] . in early work, perhaps the first, observations of membrane fusion of individual virions to biomimetic membranes was monitored with video fluorescence microscopy [ , ] . in this work, planar bilayers were suspended over a small hole in a teflon sheet, a so-called black lipid membrane (blm), to serve as the host cell membrane mimic. however, blms are fragile and prone to rupture and continued progress using this approach was slow. in recent years, supported lipid bilayers, which are more robust than blms, have become more widely used in in vitro single virion tracking experiments. because these slb platforms are becoming the backbone of many types of single virion tracking experiments, it is worth describing them in some detail here. a supported lipid bilayer is a planar, single bilayer, typically self-assembled by rupturing liposomes at the surface of hydrophilic silica surfaces like glass microscope slides. for reviews of supported lipid bilayer technologies, we encourage the reader to consult sackmann [ ] , tanaka and sackmann [ ] , and castellana and cremer [ ] . these supported lipid bilayers act as mimics for cellular membranes and the compatibility of these bilayers with flat glass surfaces makes them ideal for coupling to microscopy techniques. additionally, supported bilayers may be assembled within a flow cell or microfluidic device, which enables the exchange of buffers and, consequently, the precise control over the binding and fusion environment during single virion tracking microscopy. the composition of these bilayers is highly tunable, ranging from the simplest lipid components, to recapitulating the complexity of the plasma cell membrane of specific cell types ( fig. . ). initial studies employed purely lipid bilayers with glycolipid viral receptors [ , , , ] . more recent work has used reconstituted proteoliposomes that contain protein receptors [ ] . there has also been progress in using composite membranes made from cell-derived membrane components mixed with pegylated liposomes to engender the slb with both biologically relevant material (receptors, etc.) and a built-in cushion to maintain constituent mobility [ , ] . other work has demonstrated the ability to form bilayers from cell plasma membrane blebs as a way to incorporate transmembrane proteinaceous receptors and complete native cell materials into planar geometries [ , , ] . the incorporation of transmembrane proteins greatly expands the range of viruses that can be studied with single virions tracking techniques [ ] . notably, the simplest lipid-only bilayer on glass is roughly nm thick and sits above a thin layer of water on the order of a nanometer thick [ , ] . this feature enables the two-dimensional mobility of lipids within the bilayer necessary for membrane fusion, but mobility is also a key property for allowing receptors to rearrange to permit multivalent binding interactions, as they do in live cell membranes. mobility of protein receptors, particularly those with transmembrane domains, can be a challenge in these platforms if the water gap is insufficient for limiting interaction between the protein and the glass support. to overcome this challenge, various cushions, such as polyethylene glycol (peg) [ ] , bovine serum albumin, dextran [ , ] , or polyelectrolyte brushes [ ] , have been placed between the bilayer and the supporting surface to improve protein mobility [ ] . it is also possible to form supported lipid bilayers that mimic the viral membrane instead of the host membrane and conduct experiments in the opposite configuration. here, binding and fusion is studied by monitoring liposomes decorated with host cell receptors interacting with the planar virus-like bilayer containing embedded viral proteins [ ] [ ] [ ] . such an arrangement could be used for screening applications of antivirals that target entry processes, without the need for live virus or pseudotyped particles. in summary, biomimetic platforms enable a level of environmental control that cannot be attained in live cell particle tracking techniques. first, there is a degree of control over the host cell membrane mimic's composition that is difficult to alter in live cells. second, in these platforms, the buffers in contact with the virus can have a defined composition and the experimenter controls the timing and order of exposure to proteases, ph, or any other component of interest to the virus. but perhaps the most salient feature of this experimental approach is that these platforms allow detailed examination of the binding and membrane fusion process and gathering of dynamic data from these processes. however, the two-dimensional, in vitro nature of these platforms make them unsuitable for measuring cytoskeletal involvement in entry. thus, to obtain the most complete information about the infection process, combining data from complementary approaches using live cells and biomimetic platforms is an excellent strategy. in the following sections we describe how single virion tracking has been applied to investigate different steps in virus entry. we also include overviews of a selection of ensemble methods to appreciate the synergy between the data collected by the different techniques in providing a complete description of virus entry. table . provides a quick reference of techniques and the data that can be obtained in each approach for each entry stage. there are two scales of transport to be observed during virus spread and infection. on the larger scale is the transport and spread of virions between neighboring cells. also of interest is the smaller-scale tracking of an individual virion on a cell plasma surface before it is internalized by that particular cell. in the following sections, experiments at each scale will be described with selected examples and references. tracking virion movement in the in vivo environment has revealed various avenues of virus spread to surrounding cells. the predominant transport mechanisms of virus spread between cells are: ( ) virions freely diffusing through the extracellular environment to neighboring cell surfaces, or ( ) spreading to neighboring cells through direct transmission across adjoining membranes. for the first mechanism, the mean-squared displacement of virions over time is used to classify their motion as diffusive or sub-diffusive through the extracellular environment. for example, live cell single virion tracking of adeno-associated viruses [ ] and simian virus virus-like particles [ ] indicates that particles undergo normal diffusion in the extracellular environment. adeno-associated viruses slow down when in the vicinity of a cell, and touch the cell membrane multiple times before penetrating the cell [ ] . in contrast, hiv follows the second mechanism and preferentially transmits directly from one neighboring cell to another through virological synapses rather than transmission by extracellular diffusion [ ] [ ] [ ] some viruses exploit cytoskeletal components to facilitate transport from one cell to another. vaccinia virus, for • cytoskeletal interaction • extra-and intra-nuclear movement n/a ifa acronyms: qcmd quartz crystal microbalance with dissipation, elisa enzyme-linked immunosorbent assay, spr surface plasmon resonance, tem transmission electron microscopy, ifa immunofluoresence assay, blam beta lactamase example, induces the formation of actin protrusions from the cell surface and is transported along these to spread from cell to cell [ ] . looking at viral transport over a longer distance scale, single virion imaging has shown that pseudorabies virus is able to spread from the site of infection to the peripheral nervous system ganglia for replication then back along axons to reseed the initial infection site [ ] . in vitro experiments have been used to study how respiratory mucosa hinders the ability of pseudorabies virus to cross into the epithelium, revealing that both size and charge interactions are important [ ] . there are multiple strategies for observing lateral viral movement along the plasma membrane of a live cell. one method is labeling the core of virions and cellular actin (to delineate the border of cells) and track virion movement along the periphery of the cell [ ] . another method is to label the viral membrane and look at diffusion on top of membrane until the virion overlaps with an endocytosis site [ ] . a few examples of virion movement that have been studied this way are as follows. simian virus undergoes actin independent diffusive movement on the cell membrane until it reaches caveolae [ ] . dengue virus similarly freely diffuses on the membrane until it reaches an existing clathrin-coated pit [ ] . murine polyoma virus-like particles also freely diffuse at first, then become confined and follow actin-directed trajectories afterwards [ ] . vaccinia bound to filopodia have been observed to move towards the cell body along the plasma membrane [ ] . some viruses employ a mix of two mechanisms, free diffusion and directed motion. some examples of this type of movement are as follows. after binding to filopodia, murine leukemia virus, avian leucosis virus, vesicular stomatitis virus, and rabies virus appear to "surf" on top of the membrane via actin and myosin ii driven transport towards entry sites [ , ] . similarly, adenovirus binds its receptor then drifts along the cell surface with the assistance of actin and myosin ii [ , ] . coxsackievirus has been shown to bind on the apical surface of epithelia, which then triggers cytoskeletal rearrangement that transports the virus to the site of viral entry, the tight junctions [ ] . finally, influenza virus appears to undergo actindirected motion in the area near its initial binding site on the cell surface [ , ] . in the in vitro environment of supported lipid bilayer platforms, virions diffusing along the bilayer follow a continuous trajectory, whereas those that bind, detach, and rebind elsewhere on the surface "appear" and "disappear" under tirf microscopy. these modes of transport are easily distinguished from each other using this microscopy approach [ ] . supported lipid bilayers have an advantage over live cells for certain types of studies because of the ability to tightly control composition, receptor density and mobility, and surface geometry and heterogeneity. for example, supported lipid bilayers have been used to observe sliding and tumbling of quantum dot-labeled simian virus virus-like particles on bilayers containing low concentrations of gm and back-and-forth rocking at high receptor concentrations [ ] . however, because lipid bilayers do not contain the full cytoskeletal network of cells, it is possible that virion motion in these platforms could be different from their motion in vivo. studies of virus binding kinetics and determination of binding strength are often carried out in in vitro assays, either by monitoring many single virion events or following ensemble behavior of a population. in this section, we describe these approaches and highlight some selected studies that illustrate their utility. direct observation of many individual virion interactions with supported lipid bilayers can be used to measure receptor binding and detachment rates, multivalent avidity characteristics, and equilibrium binding constants. tirf microscopy is the most common method used in monitoring individual virions binding to biomimetic membranes because it clearly distinguishes labeled virions that are bound to the lipid bilayer from unbound virions in the bulk solution ( fig. . ). in this experimental approach, the residence time of bound virions can be collected for each individual residence times can be determined by the number of frames the virion remains observable, as shown in the images below the cartoons, before the virion unbinds virion. as experimental conditions change, shifts in residence times can be monitored. from this data, binding characteristics can be quantified. initial work monitoring nanoparticle binding to supported bilayers and surfacetethered vesicles demonstrated the advantage of using tirf microscopy in this application. here, a useful approach, equilibrium-fluctuation-analysis, was developed to quantify apparent kinetic rate constants of carbohydrate-bearing particles with carbohydrate presenting slbs [ ] . this analysis was then extended to studies of virus-like particles [ , ] and live virions [ ] interacting with glycosylated bilayers to provide new insight into virus attachment to cell surfaces and applications in biosensing [ ] . the kinetics of particle detachment [ ] can also be studied in these platforms, as can multivalent binding behavior [ , ] . such studies can reveal changes in binding behavior that promote viral attachment. for example, both influenza and canine parvovirus undergo "adhesion-strengthening" where the longer a virion is bound, the more strongly it adheres to the bilayer [ , ] . overall, these platforms are convenient for gathering insight on this critical virus entry step and how it depends on the host cell surface. one advantage of this platform is the tunability of the bilayer compositions and heterogeneity. for example, single particle binding studies have revealed the importance of microdomains in binding of norovirus-like particles, which preferentially bind the edges of glycosphingolipid-enriched domains [ ] . later it was also shown that hiv particles prefer to bind at the edges of cholesterol-rich lipid domains that were reconstituted in supported bilayers [ , ] ; however, observing this preference in live cells due to the small scale and dynamism of lipid rafts is difficult, illustrating the power of using an in vitro system for such studies. focusing further on the receptors themselves, tirf microscopy has also been used to measure the affinity of hiv glycoprotein for the glycosphingolipids galactosyl ceramide, glucosylceramide, lactosylceramide and α-hydroxy glucosylceramide in slbs [ , ] . the affinity of glycoprotein for these lipids is roughly times lower than its affinity for cd [ ] . the importance of studying the binding step is that the tropism of a virus is strongly tied to its ability to bind various receptors. a nice example, illustrating the power of combining single virion binding measurements with cell infectivity studies, clearly showed that a single mutation in the canine parvovirus capsid is able to alter binding to dog and raccoon transferrin receptors and completely change the tropism of the virus [ ] . binding can be monitored using ensemble approaches that monitor the overall change in an aggregate signal from many virions interacting with a target surface. the simplest manner to measure virion binding to a particular host cell receptor is the cofloatation assay. in these assays, viral particles or purified fusion proteins are labeled with a probe and mixed with liposomes containing the receptor for the virus. after the virus has bound, the mixture can then be added to a sucrose gradient and fractionated. the fractions are analyzed with page or western blot to determine under which conditions the virus and liposomes comigrate or cofloat [ , ] . altering the composition of the liposomes can be used to determine what lipids and proteins the virus binds to. this assay is able to probe virus-receptor interactions but does not provide information on binding kinetics. an additional limitation is that it may be difficult to purify or prepare liposomes containing the receptor, particularly if it is a transmembrane protein. some ensemble viral binding assays, like enzyme-linked immunosorbent assay (elisa) or glycan arrays, immobilize proteins, carbohydrates, or glycans on a rigid surface. these arrays do not preserve the structure, complexity, or two-dimensional fluidity of cellular membranes, which may limit accurate assessment of binding avidity. however, they can be useful for rapidly identifying binding partners in screening, for example, potential tropism changes [ ] . measurements of real-time binding and desorption can be assessed using techniques like surface plasmon resonance (spr) [ ] and quartz crystal microbalance with dissipation (qcm-d) [ , ] . spr uses changes in refractive index to report the binding behavior, while qcm-d uses a shift in resonance frequency to report virus interaction. in both approaches, biomimetic membranes can be used to preserve many properties of the host cell surface, including integration of the receptor. kinetic analysis of binding with these techniques requires two experimental phases to decouple binding and unbinding of virions. in the first phase, virions are added to a biomimetic surface and bind to the receptors. in this way, an "on" rate can be obtained. in the second phase, a virus-free buffer is added and the dissociation of the virus from the receptor is monitored. in this arrangement, an "off" rate can be obtained. it should be noted that averaged data from many single virion tracking binding/ unbinding events should match the ensemble results generated with spr or qcm-d. however, direct imaging with single virion tracking allows collection of a richer set of data for on and off rates simultaneously because each particle trajectory is captured [ ] . furthermore, by having the signature of each individual virion's binding behavior, heterogeneities in the virus population or membrane surface can be identified, which can then be compared to infection trends to understand how population dispersity impacts infection [ ] . internalization, or the uptake of the virus particle into the cytosol, can be assessed either with live cell imaging or in fixed cells through immunofluorescence imaging. in live cell virion tracking, cellular components, like clathrin and caveolin, can be fluorescently labeled and colocalization of these components with virions enables determination of whether the primary means of viral entry is through clathrin-dependent endocytosis, caveolin-dependent endocytosis, or a clathrin/caveolin independent uptake mechanism. when tracking labeled virions on live cells, rapid unidirectional motion indicates that particles have been internalized [ , , ] . live-cell tracking has revealed myriad information about virus internalization, including identifying viral dependence on clathrin or caveolin for uptake as well as the ability of viruses to promote their own uptake. for example, reovirus induces the formation of clathrin-coated pits for uptake [ ] . simian virus has the ability to induce actin rearrangement to further promote its own internalization whereas echovirus does not [ , ] . clathrin-mediated endocytosis is utilized for the uptake of many viruses including australian bat lyssavirus [ ] , hiv [ ] , infectious hematopoietic necrosis virus [ ] , and rabiesvirus [ ] . other viruses are caveolinindependent or use macropinocytosis. for example, mouse polyomavirus is also delivered to early endosomes by a caveolin independent pathway [ ] . adenovirus enters the cell viaboth clathrin dependent and independent endocytosis and triggers macropinocytosis [ ] [ ] [ ] [ ] . still other viruses have been shown to be agile in their internalization route, for example, in the absence of caveolin, simian virus can exploit a clathrin/caveolin independent pathway [ ] . studies have shown that bound influenza virions have the ability to induce the formation of clathrin-coated pits, but can also enter in a clathrin/caveolin independent manner [ ] . influenza is then preferentially sorted into a population of early endosomes that quickly matures [ ] . to monitor virus entry without live cell particle tracking, infected cells can be fixed and imaged with an immunofluorescence assay. permeablized cells can be probed with antibodies against viral proteins, and cytoskeletal elements, or endocytosis markers. colocalization of viral particles and cellular components can then reveal the general entry pathway of the virus [ ] [ ] [ ] . cells can be fixed at various time points after infection to determine the general time course of entry [ , ] . this method provides snapshots of the viral entry process because the cells are fixed before imaging, but it requires less specialized microscopes and cameras than livecell imaging. for viruses that are internalized, live-cell fluorescent imaging can provide insight into how viruses use cellular machinery, such as the cytoskeleton or microtubules, to propel their movement within the cell. one common approach to determining if cytoskeletal elements are involved is to compare the speed and shape of virion trajectories in the presence and absence of cytoskeletal inhibitors such as nocodazole, and cytochalasin d [ ] . this strategy has been employed to determine that reovirus movement after endocytosis is microtubule-directed [ ] . microtubules are involved in the cytosolic movement of adeno-associated virus type [ ] , hiv [ , ] infectious hematopoietic necrosis virus [ ] , rabies virus [ ] , in contrast, polio movement inside cells is actin dependent, but microtubule independent [ ] . adenovirus interacts with minus-end dynein and a plus-end directed factor to traffic along cytoplasmic microtubules [ ] . influenza in endosomes undergoes unidirectional dynein-directed translocation to the perinuclear region followed by intermittent back-and-forth microtubule-dependent motion within the perinuclear region prior to virion fusion with endosomes [ ] . in addition to investigating movement towards the nucleus, live-cell single virion tracking has been used to monitor movement within the nuclear region. adenovirus utilizes microtubules for movement within the cell, but detaches from them when the virions reach close proximity to the nucleus [ ] . hiv cores move toward the nucleus with a microtuble-and actin-dependent motion; within the nucleus, the motion is slow and diffuse [ , , ] . hiv rna alone moves through the cytoplasm by diffusion [ ] . after reaching the nucleus, hiv pre-integration complexes target areas of decondensed chromatin [ ] . for influenza, genes are transported to and within the nucleus by diffusion [ ] . intracellular movement of virions is not necessarily mediated by spike or capsid proteins alone; for herpes simplex virus, the inner tegument proteins promote movement along microtubules and are necessary for movement away from the cell body along axons [ ] [ ] [ ] . an alternative method is to use immunofluorescence assays of fixed cells to obtain snapshots of intracellular virion trafficking, including what cytoskeletal components are involved [ , ] . these do not allow for the tracking of individual virion trajectories and are more limited in temporal resolution than live cell virion tracking, but require less specialized equipment. membrane enveloped viruses must fuse their membrane with the host membrane to deliver their genome to the cytosol for replication. fusion can occur either at the plasma membrane surface or in endosomes after the virion is internalized. fusion can be monitored in both live cells and in in vitro platforms. each approach has advantages and disadvantages, and some examples of how these experiments are conducted follow next. viral fusion at the plasma membrane can be differentiated from fusion within endosomes in live cells. to do so, the viral contents can be labeled with a diffusible content marker while the envelope is labeled with an acid-stable lipophilic dye. if fusion occurs at the plasma membrane, both fluorescent markers will seem to disappear because they will be diluted into the plasma membrane and cytosol. if fusion occurs within an endosome, the viral content marker will disappear as it is released into the cytosol and diluted, but the lipid marker will continue to appear bright as the dye mixes into the relatively small endosomal membrane [ ] . this approach has been used to show that hiv can undergo full fusion inside endosomes while fusion at plasma membrane is halted at the lipid mixing step. however, this may be cell-type dependent; hiv content release from plasma membrane fusion was observed for u cells but not jc . or hos cells [ ] . the ability to detect viral fusion in live cells within the endocytic pathway often relies on labeling virions with quenched amounts of dye. colocalization of dequenching virions with labeled cellular components, such as clathrin or rab proteins, can reveal whether fusion occurs in early or late stage endosomes. this approach has been employed in the study of influenza virus, which fuses in early endosomes, revealing that the virus is able to enter a clathrin-dependent manner as well as a clathrin and caveolin independent fashion, and viral fusion can occur in both pathways [ ] . in macrophages, hiv undergoes fusion in rab a positive endosomes [ ] . fusion of dengue virus [ ] and ebola virus [ ] colocalizes with rab , a marker of late stage endosomes. alternatively, endosomal fusion may be mimicked by binding virions to the plasma membrane then lowering the extracellular ph. this method of acid-induced endosomal bypass has been employed to characterize avian sarcoma and leukosis virus fusion behavior [ ] . viral fusion can also be monitored by observing the release of fluorescent viral cargo. avian sarcoma and leukosis virions labeled with fluorescent cargo and an envelope ph sensor showed little correlation between endosomal ph and fusion lag time [ ] . however fusion lag time and pore size did vary with surface receptor and endosome type, indicating that endosomal composition may modulate fusion [ , , ] . in the case of vesicular stomatitis virus, cytoplasmic nucleocapsid release does not necessarily immediately follow fusion. vsv has been shown to undergo fusion between the early and late endosome stage [ ] . with the lipid bilayer platform, hemifusion and pore formation can be observed within the same particle by dual-labeling the viral envelope and contents with different colors (fig. . ). by using a flow cell or microfluidic device, this platform also enables control over the timing of fusion trigger(s) or other environmental changes within the viral environment. a common fusion trigger is a drop in ph. to trigger fusion, acidic buffer can be passed over the bilayer inside a microfluidic device. incorporation of ph sensitive dye into the bilayer enables detection of the time at which bound virions encounter the acid [ ] . alternatively, ultraviolet light can be used to "uncage" protons from onitrobenzaldehyde in solution to rapidly acidify the bilayer and virions [ ] . both of these methods enable measurement of the lag time between exposure to a fusion trigger and the onset of hemifusion. by analyzing this lag time for hundreds of individual fusion events, the rate constants of hemifusion can be calculated. pore formation can be monitored by particles also containing internal capsid dyes. monitoring the drop in signal of this co-localized fluorophore reports the opening of the fusion pore. if there is a single rate liming step, the distribution of lag times will follow a simple exponential decay, with the decay constant corresponding to the rate constant of that limiting step. if there are multiple rate liming steps, the lag time distribution will rise and decay [ ] . a simple approach to quantifying the kinetics of hemifusion and pore formation is fitting the cumulative distribution of fusion events to a convolution of poisson processes with the equation: where p is the probability of a virion being in that state (hemifused or pore forming) at time t after the fusion trigger has been added and Γ(t) is the gamma distribution function. this fit yields k, the rate constant of that step, and n, the number of stochastic rate limiting events governing the kinetics of that step [ ] . if there is only one rate-limiting step (n ¼ ), the above equation simplifies to a single exponential decay. as the number of rate-limiting steps increases, more events must be observed to accurately calculate n. for processes with three steps or fewer, events is sufficient to calculate n with a standard deviation of [ ] . there are other more complex methods for fitting lag time distributions that may capture more of the details of hemifusion [ ] . viruses with class i, class ii and class iii fusion proteins have been studied at the single virion level with slbs. the kinetic analysis described here has indicated that influenza virus hemifusion is controlled by three rate-limiting steps while the kinetics of pore formation is governed by a single step [ ] . chikungunya virus also has three-rate limiting steps governing hemifusion [ ] . for west nile virus, hemifusion has one or two rate-limiting steps, depending on the ph [ ] . vesicular stomatitis virus hemifusion also has multiple rate-limiting steps at higher ph and a single rate-limiting step at low ph [ ] . while it cannot be assumed that the kinetics obtained from this method reflect the actual reaction rates within a cell, comparison of these rates under different conditions, such as different bilayer compositions, ph, or viral strains, can yield valuable insight. the ability to manipulate the composition of supported lipid bilayers has been used to determine that anionic lipids are essential for vesicular stomatitis virus fusion and that the lag between hemifusion and pore formation is shortened in the presence of bis(monoacylglycero)phosphate, a lipid found in late endosomes [ ] . precisely controlling the triggering ph has revealed that influenza virus requires a lower ph to trigger fusion than sindbis virus [ ] , suggesting that it fuses later in the endocytic pathway. while the rate of influenza x- virus hemifusion increases at lower ph [ ] , the rate of brisbane is much less ph dependent [ ] . the rate of pore formation for influenza virus is ph independent. multiple influenza hemagglutinin trimers are involved in fusion [ ] , although not all are active participants [ , ] . this platform is able to detect differences in the acid stability and fusion behavior of different strains and reassortants of influenza, which is tied to their tropism [ , ] . this platform can also be used to measure the number of antibodies required to neutralize fusion of a single virion [ ] . figure . provides a summary of the flexibility, the biomimetic fusion assay and the kinds of insight that has been gained from these tunable assays. with the advances in supported bilayer complexity and fabrication, these platforms can also be used to study viruses that bind proteinaceous receptors, such as feline coronavirus [ ] . finally, given the highly-defined nature of these platforms, viruses that do not have a known fusion trigger or receptor might be difficult to study, because each component must be intentionally added to the platform. however, because slbs can be formed in a microfluidic device, it should be possible to formulate a high-throughput screen for possible fusion triggers or binding partners with this configuration. a common method to study fusion behavior is cell-cell fusion assays, where fusion between target cells expressing the receptor for the virus and effector cells expressing the viral fusion protein is monitored. when the viral fusion protein is triggered, for example by lowering the ph, the cells fuse to each other, leading to clusters of multiple nuclei surrounded by a single cell membrane [ ] . these clusters are known as syncytia. lipophilic and aqueous dyes can be used to label the membrane and contents of the effector cell, providing readouts for lipid mixing or pore formation. to isolate the hemifusion step, fusion can be triggered then arrested by quickly cooling the cells to c [ , ] . this rapid-cooling approach has been used to determine that hemifusion of avian sarcoma and leukosis virus requires low ph, but expansion of the fusion pore does not [ ] . as an alternative to monitoring pore formation by observing the transfer of intracellular fluorescent cargo from effector cells to target cells [ ] , whole cell patch-clamp electrical measurements can be used [ ] . in this configuration, a pipette connected to an electrode and amplifier pierces the membrane of the effector cell. the voltage across the membrane can be manipulated and the resulting current measured. during cellcell fusion, the cell surface area increases as the membranes merge, resulting in an increase in membrane capacitance. such measurements can also allow for estimation of fusion pore size [ ] . while syncytia assays do not require viral particles or specialized equipment, making them easy to carry out, there are some limitations. syncytia form over the course of hours, so this technique is not suitable for obtaining kinetic data features that can be easily controlled in biomimetic fusion assays. in combination with a high throughput approach, these conditions (left) can be evaluated to provide new insight into virus fusion. (right) examples of the kinds of insight that can be gained with a biomimetic assay that isolates the fusion reaction from other virus entry steps [ ] . furthermore, the extracellular environment can differ substantially from the endosomal environment, which may create artifacts when using this technique to study viruses that typically fuse within the endocytic pathway. in this approach, any impact of the virion size or shape on fusion is not captured. in bulk fusion assays, viral particles are fluorescently labeled, then they are mixed with liposomes in a cuvette and the fluorescent signal of the solution is measured by fluorimetry as fusion triggers are added. in one labeling approach, particles and/or liposome membranes can be labeled with fluorescence energy resonance transfer (fret) pairs. if fusion occurs, fret will stop as the pairs become separated, and the fluorescence signal will change accordingly [ ] . in the second labeling approach, particle membranes are labeled with a single quenched fluorescent marker [ ] . upon fusion with a liposome, the quenched fluorophores will become diluted and dequench, causing a marked increase in fluorescence. in a third approach, the release of viral cargo can be detected by measuring β-lactamase (blam) activity. in this type of assay, target cells are loaded with a dye containing a β-lactam ring and a fret pair. chimeras of viral core proteins and blam are incorporated into virions. if virions are able to release their cargo during infection, blam will cleave the lactam ring, interrupting fret and causing the cells to fluoresce a different color [ ] . infected and uninfected cells can then be sorted by flow cytometry for further analysis. this technique requires the ability to make chimeric proteins but otherwise does not require specialized equipment. blam activity assays have been combined with single virion imaging to indicate that hiv releases cargo by fusion inside endosomes and not fusion at the plasma membrane surface [ ] . bulk fusion provides information on the kinetics of the fusion of the overall population in the solution, including the lag time between when the viruses are exposed to a fusion trigger and when dequenching begins, with a resolution of less than a second [ ] . varying the composition of the liposomes can reveal how lipids impact viral fusion [ ] . however, liposomes containing the viral receptor may be hard to prepare if the receptor is a transmembrane protein. these assays cannot resolve intermediate steps like hemifusion, or resolve fusion of individual virions, but can be used to determine conditions interesting for further investigation with single virion tracking with slbs [ ] . it is worth noting that single virion fusion tracking experiments agree with data obtained from bulk fusion experiments. in a study directly comparing the two methods a combination of bulk fusion and single virion tracking showed that cholesterol enhances the extent of sindbis virus fusion, 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uptake the first step of adenovirus type disassembly occurs at the cell surface, independently of endocytosis and escape to the cytosol clathrin-and caveolin- -independent endocytosis ligands for clathrin-mediated endocytosis are differentially sorted into distinct populations of early endosomes the human polyomavirus, jcv, uses serotonin receptors to infect cells actin-dependent receptor colocalization required for human immunodeficiency virus entry into host cells role of protein kinase c betaii in influenza virus entry via late endosomes cellular and viral requirements for rapid endocytic entry of herpes simplex virus rab associates with early endosomes to mediate sorting and transport of semliki forest virus to late endosomes detection of individual fluorescently labeled reovirions in living cells cytoplasmic trafficking, endosomal escape, and perinuclear accumulation of adeno-associated virus type particles are facilitated by microtubule network quantitative four-dimensional tracking of cytoplasmic and nuclear hiv- complexes rapid actin-dependent viral motility in live cells adenovirus transport via direct interaction of cytoplasmic dynein with the viral capsid hexon subunit the nuclear export factor crm controls juxta-nuclear microtubule-dependent virus transport direct visualization of hiv- with correlative live-cell microscopy and cryo-electron tomography cytoplasmic hiv- rna is mainly transported by diffusion in the presence or absence of gag protein hiv- pre-integration complexes selectively target decondensed chromatin in the nuclear periphery using single-particle tracking to study nuclear trafficking of viral genes the herpesvirus capsid surface protein, vp , and the majority of the tegument proteins are dispensable for capsid transport toward the nucleus targeting of herpesvirus capsid transport in axons is coupled to association with specific sets of tegument proteins the inner tegument promotes herpes simplex virus capsid motility along microtubules in vitro eclipse phase of herpes simplex virus type infection: efficient dynein-mediated capsid transport without the small capsid protein vp nuclear targeting of adenovirus type requires crm -mediated nuclear export hiv enters cells via endocytosis and dynamindependent fusion with endosomes time-resolved imaging of hiv- env-mediated lipid and content mixing between a single virion and cell membrane imaging individual retroviral fusion events: from hemifusion to pore formation and growth imaging single retrovirus entry through alternative receptor isoforms and intermediates of virus-endosome fusion synchronized retrovirus fusion in cells expressing alternative receptor isoforms releases the viral core into distinct sub-cellular compartments endosome-to-cytosol transport of viral nucleocapsids mechanisms of influenza viral membrane fusion statistical mechanics of viral entry chikungunya virus fusion properties elucidated by single-particle and bulk approaches sequential conformational rearrangements in flavivirus membrane fusion mechanism of membrane fusion induced by vesicular stomatitis virus g protein variations in ph sensitivity, acid stability, and fusogenicity of three influenza virus h subtypes influenza-virus membrane fusion by cooperative fold-back of stochastically induced hemagglutinin intermediates distinct functional determinants of influenza hemagglutininmediated membrane fusion. elife :e stochastic fusion simulations and experiments suggest passive and active roles of hemagglutinin during membrane fusion viral fusion efficacy of specific h n influenza virus reassortant combinations at single-particle level relating influenza virus membrane fusion kinetics to stoichiometry of neutralizing antibodies at the single-particle level cell fusion by semliki forest, influenza, and vesicular stomatitis viruses a study of low ph-induced refolding of env of avian sarcoma and leukosis virus into a six-helix bundle low ph is required for avian sarcoma and leukosis virus env-induced hemifusion and fusion pore formation but not for pore growth dilation of the influenza hemagglutinin fusion pore revealed by the kinetics of individual cell-cell fusion events patch clamp studies of single cell-fusion events mediated by a viral fusion protein the first milliseconds of the pore formed by a fusogenic viral envelope protein during membrane fusion methodologies in the study of cell-cell fusion use of resonance energy transfer to monitor membrane fusion fluorescence method for measuring the kinetics of fusion between biological membranes a sensitive and specific enzyme-based assay detecting hiv- virion fusion in primary t lymphocytes inhibition of hiv- endocytosis allows lipid mixing at the plasma membrane, but not complete fusion delay time for influenza virus hemagglutinininduced membrane fusion depends on hemagglutinin surface density membrane fusion of semliki forest virus requires sphingolipids in the target membrane key: cord- -w spqqt authors: huan, yuchen; kong, qing; mou, haijin; yi, huaxi title: antimicrobial peptides: classification, design, application and research progress in multiple fields date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: w spqqt antimicrobial peptides (amps) are a class of small peptides that widely exist in nature and they are an important part of the innate immune system of different organisms. amps have a wide range of inhibitory effects against bacteria, fungi, parasites and viruses. the emergence of antibiotic-resistant microorganisms and the increasing of concerns about the use of antibiotics resulted in the development of amps, which have a good application prospect in medicine, food, animal husbandry, agriculture and aquaculture. this review introduces the progress of research on amps comprehensively and systematically, including their classification, mechanism of action, design methods, environmental factors affecting their activity, application status, prospects in various fields and problems to be solved. the research progress on antivirus peptides, especially anti-coronavirus (covid- ) peptides, has been introduced given the covid- pandemic worldwide in . alexander fleming discovered lysozyme in , and this discovery marked the birth of modern innate immunity. since then, antibiotics and antimicrobial peptides (amps) have been discovered. a total of , amps have been reported in the antimicrobial peptide database (apd ) updated on august , . different types of amps have the following commonalities: their number of amino acid residues is between and (average: . ), and almost all amps are cationic (average net charge: . ). however, several anionic amps also exist, and they have several acidic amino acids like aspartic acid and glutamic acid (malkoski et al., ; schittek et al., ; lai et al., ) . the anti-microbial resistance of microorganisms is becoming increasingly serious with the abuse of antibiotics in medicine, agriculture and animal husbandry, especially in developing countries. research from kenya has detected substantial amounts of antibiotic residues in edible meat (ayukekbong et al., ) . the prevalence of vancomycin-resistant enterococcus (vre) and methicillin-resistant staphylococcus aureus (mrsa) is increasing in clinical medicine, so the countermeasures are urgently needed to address these bacterial infections. however, from the http://aps.unmc.edu/ap/ perspective of pharmaceutical companies, the development of new antibiotic drugs results in low profit. thus, replacing antibiotics has become a consideration in the pharmaceutical, agricultural, animal husbandry, and food industries. research on amps is continuously developing and considerable amounts of data on amps have been stored in amp databases. however, the mechanism of amps remains incompletely understood, and further work needs to be performed to determine the relationship between different physicochemical properties to obtain low-cost and highly safe amps with remarkable antimicrobial effects and the specificity and a high capacity for synergies of amps should also be further developed (lazzaro et al., ) . the diversity of natural amps causes difficulty in their classification. amps are classified based on ( ) source, ( ) activity, ( ) structural characteristics, and ( ) amino acid-rich species (figure ). the sources of amps can be divided into mammals (human host defense peptides account for a large proportion), amphibians, microorganisms, and insects according to statistical data in apd . the amps found in oceans have also attracted widespread attention. mammalian antimicrobial peptides are found in human, sheep, cattle, and other vertebrates. cathelicidins and defensins are the main families of amps. defensins can be divided into α-, β-, and θ-defensins depending on the position of disulfide bonds (reddy et al., ) . human host defense peptides (hdps) can protect human from microbial infections but show different expressions in every stage of human growth. for example, cathelicidin ll- , a famous amp derived from the human body, is usually detected in the skin of newborn infants, whereas human betadefensin (hbd- ) is often expressed in the elderly instead of the young (gschwandtner et al., ) . hdps can be identified in many parts of the body such as skin, eyes, ears, mouth, respiratory tract, lung, intestine, and urethra. besides, amps in human breast milk also play an important role in breastfeeding because it can decrease the morbidity and mortality of breastfeeding infants (field, ) . what's interesting is that casein (peptide derived from β-casein - aa), identified in colostrum, shows different levels in preterm human colostrum and term human colostrums . dairy is an important source of amps, which are generated through milk enzymatic hydrolysis. several amps have been identified from α-lactalbumin, β-lactoglobulin, lactoferrin, and casein fractions, and the most famous peptide obtained is lactoferricin b (lfcinb) (sibel akalın, ) . furthermore, whether the amps derived from dairy products can be used for dairy preservation is also an interesting subject to develop. in addition to antimicrobial activity, hdps, such as cathelicidins and defensins, also affect immune regulation, apoptosis, and wound healing (wang, ) . antimicrobial peptides from amphibians play an important role in the protection of amphibians from the pathogens that have induced the global amphibian population decline (rollins-smith, ). frogs are the main source of amphibian amps and the most famous amp from frogs is magainin; the skin secretions of frogs from genera xenopus, silurana, hymenochirus, and pseudhymenochirus under the pipidae family are rich in amps (conlon and mechkarska, ) . furthermore, cancrin, which has an amino acid sequence of gsaqpykqlhkvvnwdpyg, has been reported as the first amp from the sea amphibian rana cancrivora (lu et al., ) . this marks a broader source of amps of amphibians. figure | classification of antimicrobial peptides. (semreen et al., ) . myticusin-beta is an immune-related amp of mytilus coruscus and a promising alternative to antibiotics (oh et al., ) . moreover, ge , known as pardaxin, is a marine amp and the ge -based vaccine has shown the ability to enhance antitumor immunity in mice (huang et al., ) . the activity of amps can be divided into categories according to the statistics of the adp database. these categories can be summarized as antibacterial, antiviral, antifungal, antiparasitic, anti-human immunodeficiency virus (hiv), and anti-tumor peptides (figure ). antibacterial peptides account for a large part of amps and have a broad inhibitory effect on common pathogenic bacteria, such as vre, acinetobacter baumannii, and mrsa in clinical medicine and s. aureus, listeria monocytogenes, e. coli in food and salmonella, vibrio parahaemolyticus in aquatic products. many natural and synthetic amps like nisin, cecropins and defensins have shown good inhibition activity to gram-positive bacteria and gram-negative bacteria. in recent research, amps p (yirkirrffkklkkilkk-nh ) and p (syerkinrhfktlkknlkkk-nh ), which are designed based on aristicluthys nobilia interferon-i, inhibit mrsa and show a low cytotoxicity . antifungal peptides are a subclass of amps that address fungal infections with enhanced drug resistance. many afps have shown excellent anti-fungal activities against common pathogenic fungi, such as aspergillus and candida albicans in clinical medicine, yeast, filamentous fungi (e.g., aspergillus flavus), mold in food and agriculture. except for brevinin, ranatuerin, cecropins, many synthetic peptides also show good antifungal activity. for example, aurh , derived from aurein . , can effectively treat c. albicans infection, which has a lethal rate up to % (madanchi et al., ) . aflatoxin, which is a carcinogen produced by a. flavus, is harmful to the human body. many afps can inhibit the growth of a. flavus. for example, an afp with a sequence of fpshtgmsvppp can inhibit the growth of a. flavus md . a total of antifungal peptides isolated from lactobacillus plantarum te and their mixture can reduce a. flavus spore formation in fresh maize seeds (muhialdin et al., ) . moreover, two chemically synthesized radish amps show a good inhibitory effect against different yeast species, such as zygosaccharomyces bailii and zygosaccharomyces rouxii (shwaiki et al., ) . viruses cause serious harm to human life and huge economic losses to the animal husbandry. the covid- , which is the recent outbreak, has caused great loss of lives and properties. furthermore, foot-and-mouth disease virus, avian influenza virus (aiv), and hiv are long-term threats to human life. so, it is extremely urgent to solve these problems, and antiviral peptides provide new ways. antiviral peptides show a strong killing effect on viruses mainly by ( ) inhibiting virus attachment and virus cell membrane fusion, ( ) destroying the virus envelope, or ( ) inhibiting virus replication (jung et al., ) (shown in figure ). a recent report has shown that amp epi- mediates the inactivation of virus particles and has good inhibitory activity against foot-and-mouth disease virus . moreover, infectious bronchitis virus (ibv) is the pathogen of infectious bronchitis and the inoculation of swine intestinal amp (siamp)-ibv mixed solution remarkably reduced the mortality of chicken embryos compared with the ibv infection group, showing the good inhibitory activity of siamp on ibv (sun et al., ) . anti-hiv peptides are a subclass of anti-viral peptides. the most important examples of these peptides include defensins (including αand β-defensins, which have different mechanisms), ll- , gramicidin d, caerin , maximin , magainin , dermaseptin-s , dermaseptin-s , siamycin-i, siamycin-ii, and rp (madanchi et al., ) and antiviral peptide fuzeon tm (enfuvirtide) has been commercialized as an anti-hiv drug (ashkenazi et al., ) . due to the global spread of the covid- (figure a ), the antiviral peptides against the coronavirus will be discussed in more detail. coronaviruses (covs) belong to the family coronaviridae; they are enveloped viruses with a positive-sense single-stranded rna genome and have a helical symmetry (franks and galvin, ) . covs, including severe acute respiratory syndrome cov (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) (mustafa et al., ) , and the recent outbreak of covid- have caused serious threats to human life and property. covs can cause lifethreatening respiratory diseases and the viral particle is formed by spike glycoprotein (s), the envelope (e), the membrane (m), and the nucleocapsid (n) (vilas boas et al., ) . it should be noted that their infectivity requires viral spike (s) protein. fusion inhibitor peptides combine with the s protein to interfere with its folding and prevent infection. besides, the s domain of the sars-cov s protein contains heptad repeat hr and hr sequences. peptide hr (hr : sltqinttlldltyemlslqqvvkalnesyidlkel) and its lipid-binding peptide is highly similar or even identical to the near-membrane portion of s protein ferredoxin, which interferes with refolding into post-fusion fusion-catalyzing domains (fds) (du et al., ; park and gallagher, ) . according to recent research, the lipopeptide ek c , derived from ek (sldqinvtfldleyemkkleeaikkleesyidlkel), is the most effective fusion inhibitor against covid- s proteinmediated membrane fusion . homology modeling and protein-peptide docking showed that temporin has potential therapeutic applications against mers-cov (marimuthu et al., ) . two amps from the non-structural protein nsp of sars-cov, k , and k , can inhibit sars-cov replication (ke et al., ) . furthermore, rhesus thetadefensin (rtd- ) treated animals have a marked reduction in mortality in the presence of sars-cov while the peptide alone shows airway inflammation and the one possible mechanism of action for rtd- is immunomodulatory (wohlford-lenane et al., ) . in general, amps against coronavirus can be roughly classified as i) peptides derived from hr , hr and rbd subunits of the spike protein, ii) peptides derived from other amps, iii) peptides derived from non-structural protein (mustafa et al., ) . furthermore, molecular docking analysis indicated that peptides were employed to disrupt the interaction between covid- and ace (angiotensin-converting enzyme ) to inhibit covid- entrance in cells ( figure b ) (souza et al., ) . finally, it should be noted that this therapy lacks clinical trials and the main method of animal experiments is an intranasal administration. this reminds us that nasal drug delivery (ndd) is a potential therapy for amps as anti-coronavirus drugs. besides, the antiviral database avpdb includes numerous antiviral peptides. parasitic protozoa can cause diseases in human and animals through a variety of routes, including animal-to-person or person-to-person contact, water, soil, and food (chalmers et al., ) . and with the increase in parasite drug resistance, the need for new treatments has increased. antiparasitic peptides show their killing effect on parasites which cause diseases such as malaria and leishmaniasis (mangoni et al., ; rhaiem and houimel, ) and amps like cathelicidin, temporins-shd show high inhibition activity against parasites (abbassi et al., ) . in recent research, epi- , a marine synthetic amp, can remarkably inhibit trichomonas vaginalis by destroying its membrane (neshani et al., ) . the peptide jellein derived from bee royal jelly which has introduced above and -amino acid amp kdel (lysine, aspartic acid, glutamic acid, and leucine) has shown a significant effect on the leishmania parasite (cao et al., ; zahedifard et al., ) . however, it should be noted that their mechanisms are not the same. cyanobacterial peptides differ from higher-eukaryote amps because their antiparasitic action depends on specific protein targets. thus, these target parasites can be distinguished accurately even though they belong to the same family or genus (rivas and rojas, ) . the acps show anticancer mechanisms by ( ) recruiting immune cells (such as dendritic cells) to kill tumor cells, ( ) inducing the necrosis or apoptosis of cancer cells, ( ) inhibiting angiogenesis to eliminate tumor nutrition and prevent metastasis, and ( ) activating certain regulatory functional proteins to interfere with the gene transcription and translation of tumor cells (wu d. et al., ; ma et al., ) . tritrpticin and its analogs induce considerable toxicity toward jurkat cells in vitro, whereas indolicidin and puroindoline a can also act as acps (arias et al., ) . it should be noted that both net charge and hydrophobicity play important roles in optimizing the anticancer activity of acps and they can constrain and influence each other. thus, achieving a balance between net charge and hydrophobicity is important for better anticancer activity. besides the peptide mentioned above, anti-inflammatory, anti-diabetic peptides, spermicidal peptides etc. have been noticed, but they are not the same as antimicrobial peptides. simply put, anti-inflammatory peptides decrease the release of inflammatory mediators and inflammatory cytokines (nitric oxide, interleukin- , and interleukin- β) and some of them also inhibit inflammatory signals like nf-κb, mapk, and jak-stat pathways (meram and wu, ; gao et al., ) . anti-diabetic peptides play their function by modulating the g proteincoupled receptor kinase (grk / ) or activating glucagonlike peptide- (glp- ), glucagon receptors (marya et al., ; graham et al., ) . however, it is not accurate to classify these types of peptides as amps and bioactive peptides may be more convincing. proline is a typical non-polar amino acid. pramps behave differently from other amps, that is, they enter bacterial cytoplasm by the inner membrane transporter sbma instead of killing bacteria through membrane destruction (mattiuzzo et al., ) . once in the cytoplasm, pramps target ribosomes and block the binding of aminoacyl-trna to peptidyltransferase center or trap decoding release factors on the ribosome during the termination of translation to interfere with protein synthesis (seefeldt et al., ) . for instance, tur a, which is an orthologous amp of bovine pramp bac discovered from tursiops truncatus, interferes with the transition from the initial phase to the extension phase of protein synthesis by binding to ribosomes. in addition, different pramps lack a high sequence similarity but have short motifs containing repeating proline and arginine (arg) residues (e.g., -ppxr-in bac and -prpxin bac ) (mardirossian et al., (mardirossian et al., , . although pramps mainly kill gram-positive bacteria, ppr-amp , a proline-rich amp identified from crab (scylla paramamosain), exhibits antimicrobial activity against gram-positive and gram-negative bacteria (imjongjirak et al., ) . besides, pieces of research have shown that pramps have immunostimulation activity (li w. et al., ) . tryptophan (trp), as a non-polar amino acid, has a remarkable effect on the interface region of the lipid bilayer, whereas arg, as a basic amino acid, confers peptide charge and hydrogen bond interactions, which are essential properties to combine with the bacterial membrane's abundant anionic component. and it seems that trp residues play the role of natural aromatic activators of arg-rich amps by ion-pair-π interactions (walrant et al., ) , thereby promoting enhanced peptide-membrane interactions (chan et al., ) . in addition to indolicidin and triptrpticin which both are famous amps that rich in arg and trp residues. octa (rrwwrwwr) is also a typical trp-and arg-rich amp that inhibits gram-negative e. coli and pseudomonas aeruginosa and gram-positive s. aureus. and short trp-and arg-rich amps designed based on bovine and murine lactoferricin have also shown strong inhibitory action against bacteria (strøm et al., ; bacalum et al., ) . histidine is a common basic amino acid, and histidine-rich amps show good membrane permeation activity. hv is a histidinerich amp designed based on rr(xh) xdpgx(yh) rr-nh (where x represents i, w, v, and f). this peptide increases the permeability of bacterial cell membranes to cause cell membrane rupture and death. in addition, hv inhibits bacterial movement in a concentration-dependent manner and shows a strong anti-inflammatory effect by inhibiting the production of tumor necrosis factor α (tnf-α) ). an amp designed based on octa has shown good therapeutic potential by replacing its arg residues with histidine (bacalum et al., ) . furthermore, l h , which is designed based on the linear cationic amphiphilic peptide magainin, also shows good antibacterial activity and cell penetration properties by inserting four histidine sequences in leucine and alanine (lointier et al., ) . the r group of glycine is generally classified as a non-polar amino acid in biology. glycine-rich amps, such as attacins and diptericins, widely exist in nature (lee et al., ; kwon et al., ) . these peptides contain % to % glycine residues, which have an important effect on the tertiary structure of the peptide chain. a glycine-rich amp derived from salmonid cathelicidins activates phagocyte-mediated microbicidal mechanisms, which differ from the mechanism of conventional amps (d'este et al., ) . furthermore, the glycine-rich central-symmetrical gg is an ideal commercial drug candidate against clinical gramnegative bacteria . antimicrobial peptides can be divided into four categories based on their structures including linear α-helical peptides, β-sheet peptides, linear extension structure, and both α-helix and β-sheet peptides ( figure ) (lei et al., ) . moreover, progressively cyclic peptides and amps with more complex topologies (including lasso peptides and thioether bridged structures) are reported (koehbach and craik, ) . the membrane-targeting mechanisms of amps can be described through models, including the pole and carpet models and the pole model can be further divided into the toroidal pore and barrel-stave models (figure ). the toroidal pore model is also known as the wormhole model. in this model, amps vertically embedded in the cell membrane accumulate and then bend to form a ring hole with a diameter of - nm (matsuzaki et al., (matsuzaki et al., , . the typical examples of this model are magainin , lacticin q, and arenicin. furthermore, cationic peptides, including tc , tc , and bp , compromise the membrane barrier by creating fluid domains (omardien et al., ) . antimicrobial peptides aggregate with each other, penetrate the bilayer of the cell membrane in the form of multimers, and form channels that result in the cytoplasmic outflow. in severe cases, amps can induce cell membrane collapse and lead to cell death (lohner and prossnigg, ). for instance, alamethicin performs its pore-forming activity by using this model. besides, hairpin amp protegrin- can form stable octameric β-barrels and tetrameric arcs (half barrels) in implicit and explicit membranes by simulations (lipkin and lazaridis, ) . antimicrobial peptides are arranged parallel to the cell membrane. their hydrophilic end faces the solution, and their hydrophobic end faces the phospholipid bilayer. amps will cover the membrane surface that similar to a carpet and destroy the cell membrane in a 'detergent'-like manner (oren and shai, ) . however, this pore-forming mechanism requires a certain concentration threshold and the required concentration of amps is high. human cathelicidin ll- exhibits its activity through this mechanism, and amps with β-sheet structure also play a role in this model (shenkarev et al., ; corrêa et al., ) . polarized light-attenuated total reflection fourier transform infrared spectroscopy (atr-ftir) was used to study the effect of amp cecropin p on the bacterial cell membrane and found that it was an applied flat on the surface of the pathogen's cell membrane to destabilize and eventually destroy the cell membrane . membrane targeting mechanisms (the cell membrane composition differences of bacteria and fungi shown in figure ) can be further refined to address the large differences in the lipid composition of the cell membranes of bacteria, fungi, and mammals. the main lipids in cell membranes include glycerophospholipids (gpls), lysolipids, sphingolipids, and sterols. phosphatidylethanolamine (pe), phosphatidylglycerol (pg), and cardiolipin (cl) are the most common anionic lipids in bacteria, whereas phosphatidylcholine (pc), phosphatidylinositol (pi), pe, and phosphatidic acid (pa) are the main gpls in fungal cell membranes (ejsing et al., ; singh and prasad, ; li et al., ) . furthermore, fungal cell membranes are more anionic than mammalian cell membranes and have higher pc content. meanwhile, ergosterol is the sterol found in the plasma membrane of lower eukaryotes, such as fungi, whereas that of animals contains cholesterol (faruck et al., ) . many amps take advantage of differences in membrane components to exert their effects. antimicrobial peptides are promising to be anti-biofilm agents but it should be noticed that they are different from the cell penetrating peptides (cpps) which typically comprise - amino acids and can translocate across the cell membrane. cpps could be categorized according to physicochemical properties into three classes: cationic, amphipathic, and hydrophobic, but anti-biofilm peptides have stricter requirements for these physicochemical properties. anti-biofilm peptides target the biofilms by different mechanisms including ( ) degradation of signals within biofilms; ( ) permeabilize within cytoplasmic membrane/eps; ( ) modulating eps production etc. and then can address chronic multi-resistant bacterial infections (pletzer et al., ; ribeiro et al., ; guidotti et al., ; derakhshankhah and jafari, ; rajput and kumar, ) . for instance, saap- , synthesized based on ll- , showed activity to prevent biofilm formation by s. aureus and a. baumannii (crunkhorn, ) . the way of amps entering cells is direct penetration or endocytosis. after entering the cytoplasm, amps will identify and act on the target. depending on the target, amps can be divided into the following categories. antimicrobial peptides affect transcription, translation, and assembly into functional peptides through molecular chaperone folding by interfering with related enzymes and effector molecules. for example, bac - targets ribosomes to inhibit protein translation (mardirossian et al., ) , whereas tur a inhibits protein synthesis in e. coli and thermus thermophilus by inhibiting the transition from the initial phase to the extension phase. however, the differences between tur a and bac also lead to various ways of binding to ribosomes and interacting with the ribosomal peptide exit tunnel (mardirossian et al., ) . but some amps' have different targets. for instance, genome-wide transcription shows that the amp dm can affect many important intracellular pathways of protein biosynthesis . chaperones are key proteins for correctly folding and assembling newly synthesized proteins and make them have stereoisomerism, which makes amps have cell selectivity and can prevent cytotoxicity. according to a previous review: both pyrhocoricin and drosocin can prevent dnak from refolding misfolded proteins by inducing a permanent closure of the dnak peptide-binding cavity (kragol et al., ; le et al., ; wrońska and boguś, ) . antimicrobial peptides can affect key enzymes or induce the degradation of nucleic acid molecules to inhibit nucleic acid biosynthesis. indolicidin, a c-terminal-amidated cationic trprich amp with amino acids, specifically targets the abasic site of dna to crosslink single-or double-stranded dna and it can also inhibit dna topoisomerase i (subbalakshmi and sitaram, ) . tfp (tissue factor pathway inhibitor) - tc , which is an amp from tongues, enters the cytoplasm of target cells after the rupture of cell membrane and then degrades dna and rna (he et al., ) . many amps can inhibit various metabolic activities by inhibiting protease activity. for example, histatin has a strong inhibitory effect on the proteases secreted by the host and bacteria. amps enap- and indolicidin inhibit microbial serine proteases, elastase, and chymotrypsin (le et al., ) . cathelicidin-bf is a peptide isolated from the venom of bungarus fasciatus, it can effectively inhibit thrombin-induced platelet aggregation and further block protease-activated receptor (shu et al., ) . antimicrobial peptides inhibit cell division by inhibiting dna replication and dna damage response (sos response), blocking the cell cycle or causing the failure of chromosome separation (lutkenhaus, ) . for instance, app (glaraltrllrqltrqltra), which is an amp with amino acid residues, can efficiently kill c. albicans because of its cell-penetrating efficiency, strong dna-binding affinity, and ability to induce s-phase arrest in intracellular environment . mciz, which has amino acid residues, is an effective inhibitor of bacterial cell division, z-ring formation, and localization (cruz et al., ) . moreover, it has been reported that several afps have damaging effects on the organelles of fungi. for example, histintin can interact with mitochondria, causing the production of ros, and inducing cell death (helmerhorst et al., ) . in addition to intracellular targets, differences in cell wall composition, such as lipopolysaccharide (lps), lipid a and mannoproteins, are potential targets for amps. specifically, gram-positive and gram-negative bacteria are classified based on their bacterial cell wall structure. gram-positive bacteria have a layer of cross-linked peptidoglycan, whereas gram-negative bacteria have an additional outer membrane with an inner leaflet containing only phosphatidic acid and an outer leaflet made of lps. lps has numerous negatively charged phosphate groups, which combine with a salt bridge with a divalent cation (e.g., ca + and mg + ) to form an electrostatic network (nikaido, ) . this electrostatic zone is the main barrier against hydrophobic antibiotics and causes the low permeability of gram-negative bacteria. the main components of the fungal cell wall are mannoprotein, β-glucans and chitin (polymers of , -β-n-acetylglucosamine) and the mutations in the relevant genes of the lps pathway and phospholipid trafficking provide resistance to the amps (cabib, ; spohn et al., ) . mannoproteins in fungal cell walls include a variety of proteins, including structural proteins, cell adhesion proteins (floccrin and lectin) and enzymes involved in cell wall synthesis and remodeling (hydrolytic enzymes and transglycosylase). these proteins differ from human cell membrane proteins and are potential targets of afps (rautenbach et al., ) . furthermore, teichoic acid and lipoteichoic acid in the cell wall are also potential targets of amps and these theories could support the design of amps with low cytotoxicity. antimicrobial peptides have good application prospects. however, amps have the following problems. ( ) amps damage the cell membrane of eukaryotes and cause hemolytic side effects; ( ) rising production costs and technical problems limit their manufacture; ( ) their stability is limited at certain ph; ( ) amps have reduced activity under the presence of iron and certain serum; ( ) amps are easily hydrolyzed by proteases. therefore, the ideal amp should meet the following characteristics: (i) high antimicrobial activity; (ii) low toxicity to mammalian membranes; (iii) high protease and environment stability; (iv) low serum binding capacity and (v) ease of access and low cost production . therefore, designing amps to achieve the desired effect has attracted increasing attention. the rational design of antibacterial peptides should focus on the following five aspects: chain length, secondary structure, net charge, hydrophobicity, and amphiphilicity and these have been mentioned in many studies and this review will focus more on several specific methods of antimicrobial peptide design. site-directed mutation refers to the redesign of natural antimicrobial peptides by adding, deleting or replacing one, or several amino acid residues (torres et al., ) . the de novo design of peptides attaches importance to the design of amphiphilic amps (guha et al., ) . for example, gala is a well-known de novo-designed amp. amphipathic α-helical peptide gala is created by placing protonatable glutamic acid residues in most positions with the spacing of i to i + (goormaghtigh et al., ) . the repeated sequence (xxyy)n, where x and x are hydrophobic amino acids, y and y are cationic amino acids, and n is the number of repeat units, is designed based on the hydrophobicity cycle that mimics natural α-helical amps and successfully designs broadspectrum α-helical amps. sequences (lkkl) and (wkkw) . have the highest selectivity (khara et al., ) . moreover, l l k m w model peptides are also de novo-designed peptides. amphipathic helical properties were conferred by using leucines and lysines, and two tryptophan residues were positioned at the amphipathic interface between the hydrophilic ending side and the hydrophobic starting side. among the model peptides, l k w has good anti-mrsa activity (lee et al., ) . sequence templates can be obtained by comparing a large number of structurally homologous fragments of natural amps (such as hdps) and extracting conservative patterns based on the type of residue (such as charged, polar, hydrophobic, etc.) (zelezetsky and tossi, ) . based on the modification, the parameters, such as helix formation tendency, cationic, amphiphilicity and overall hydrophobicity, can be systematically changed. for instance, cecropin, magainin, protegrin, and lactoferrin have all been used as amp templates (fjell et al., ) . peptides can form nanostructures, such as micelles, vesicles, nanotubes, nanoparticle nanobelt, and nanofibre nanotube, and can increase or impart antibacterial activity to amps during the self-assembly of peptides. for example, kld- (kld) is a self-assembling peptide with amino acid residues that can adopt nanostructures and are known for their tissue engineering properties. the addition of arg residues in kld shows no remarkable change in its β-sheet secondary structure and the self-assembly characteristics of the forming nanostructures (tripathi et al., ) . dimer structure can also be used to enhance the antimicrobial activity of amps and reduce toxicity, but membrane-destabilizing effects are reduced after dimer formation (malekkhaiat häffner and malmsten, ). various chemical modifications of amps, including residue phosphorylation, the addition of d-amino acids or unnatural amino acids (homoarginine), cyclization, halogenation, acetylation, and peptidomimetics, have been used to improve the stability of peptides against proteases. given that the enzyme is stereospecific, the incorporation of unnatural d-amino acids into the amp sequence can reverse the stereochemistry and prevent protease degradation (zhong et al., ) . the so-called peptidomimetics, whose main elements mimic the structure of peptides, are usually produced by modifications, such as chain extension or heteroatom incorporation of existing peptides (patch and barron, ) . ornine, which is an unnatural residue with a positive charge and has a high resistance to protease activity, is also used in non-chemical modification. replacing trp residues with family residues, such as β-dihydrophenylalanine, can stabilize secondary structures and improve antibacterial properties (maurya et al., ) . halogenation is highly related to the activity, specificity, and stability of amps. in the latest report, halogen is introduced into jelleine-i which is a short peptide isolated from the royal jelly of honeybees (apis mellifera) by replacing phenylalanine with a halogenated phenylalanine analog, increasing the antibacterial activity in vitro and anti-biofilm activity. in addition, the proteolytic stability of jelleine- is increased by - times by halogenation (jia et al., ) . the halogenated peptidomimetic α,α-disubstituted β-amino amides are also promising bacteriostatic drugs that have inhibitory effects on more than multi-resistant clinical isolates of gram-positive and gram-negative bacteria (paulsen et al., ) . halogenation is also related to the specificity of amps. the o-fluorine substitution in phenylalanine residues maintains the activity of temporin l on e. coli but leads to the loss of activity on s. aureus and p. aeruginosa (setty et al., ) . three modes of cyclisation, including cyclisation via disulfide bonds, head-to-tail cyclisation and internal bonding between side chains, have been found in natural amps. the synthesis of disulfide bonds often complicates the development of synthetic peptides. the circularisation of the main chain of arenicin- molecule resulted in increased activity against drug-resistant clinical isolates but caused no substantial effect on cytotoxicity (orlov et al., ) . the hdps tachyplesins i, ii, and iii and their cyclic analogs cti, ctii, and ctiii, respectively, have similar structures and activities and can resist bacterial and cancer cells. the cyclisation of the backbone reduces the hemolytic activity and improves the stability of the peptides whilst maintaining effective anticancer and antibacterial activities (vernen et al., ) . capping refers to the addition of specific motifs or modifications, such as amidation at the c-terminus and acetylation at the n-terminus, rendering amps with more natural peptide characteristics. post-translational modifications play an important role in the function of amps and are the most commonly used in peptide design. the c-terminal rana box (consisting of a c-terminal cyclic heptapeptide with a conservative disulfide bond) and amide group are important c-terminal capping methods. for example, the c-terminal amide group of maximin h can enhance antibacterial efficacy without increasing lytic ability (dennison et al., ) . the n-terminal lipidated analog c vg krkp shows enhanced antibacterial activity against various gram-negative bacteria. the functions of n-terminal lipidation include (i) increasing lps neutralization, (ii) increasing stability to proteases and peptidases, and (iii) reducing cytotoxicity (datta et al., ) . furthermore, hydrophobic end labeling is a commonly used method to increase the activity of antimicrobial peptides. acyl lipid peptides have a linear or cyclic structure in which one or more hydrocarbon tails are connected to the n-terminus of a short oligopeptide (chu-kung et al., ) . lipopeptides have covalently attached hydrophobic moieties, such as sterols or fatty acids. aromatic amino acid terminal labeling is also the main hydrophobic terminal labeling method. tryptophan (w) and phenylalanine (f) are the commonly used aromatic amino acids. their large and polarisable residues have an affinity for the interface, and the w/f tag is also sensitive to the differences between ergosterol and cholesterol and can prevent self-assembly. this condition results in low aggregation numbers and high critical aggregation concentrations (schmidtchen et al., ) . peptide conjugation has been the goal of most research in recent years to produce active and stable amps with high selectivity. different side chains or amp fragments can be used aside from the repetition of the same amino acid motifs. for example, conjugating fatty acids with a length of - carbon atoms to the th or th side chain of the d-amino acids of ano-d , improves antibacterial selectivity and anti-biofilm activity. in addition, the new peptide exhibits high stability against trypsin, serum, salt, and different ph environments (zhong et al., ) . the conjugation of different amps can also be performed. for example, the hybrid peptide (pa -gnu ) constructed by the addition of pa to gnu has a high activity and specificity to p. aeruginosa . smamps include a broad family of molecular entities based on the structure and function of amps. however, their backbones are not entirely based on α-amino acids, including β-amino acid oligomers, arylamide oligomers, and phenylene ethynylenes (michael henderson and lee, ) . for instance, smamp , which is a potential drug for intravenous treatment, causes no drug resistance and has a strong inhibitory effect on mrsa and vancomycin-resistant enterococcus faecium (tew et al., ) . peptoids are peptide isomers, in which the side chain is bonded to the main chain nitrogen instead of α-carbon or poly-nsubstituted glycine in which the side chain is connected to amide nitrogen instead of the α-carbon on the main chain (andreev et al., ) . for example, the cationic peptide sa (iowagolfolfo-nh ) and its poly-n-substituted glycine homolog spo (ninonwnangnonlnfnonlnfno-nh ) inhibit the planktonic and biofilm formation of a. baumannii strains, which are susceptible to multi-drug resistance (sharma et al., ) . motifs with specific functions have been reported increasingly. these motifs can be repeated units for combining into new antimicrobial peptides, or specific amino acid combination units appearing at the end (such as capping) of or even in the peptide chain. this motif includes two tripeptide structures, including gly-gly-his or val-ile-his, which are added at the end of the peptide chain. atcun-containing amps in the presence of hydrogen peroxide and ascorbic acid combine with cu + to induce the valence of copper ions between + and + oxidation states and form an atcun-cu (ii) complex, generating ros by fenton-like reactions. extracellular polymeric substances (eps) are important for biofilms and can enhance the resistance of cells to antibacterial agents (flemming, ) . atcun-amps have been used to degrade environmental dna, which is one of the major components of eps. several related practical applications have been reported. for example, the biological activity against carbapenem-resistant enterobacteriaceae is increased by adding this motif to the n-terminus of an alpha-helical amp (such as cm ). besides, the cu-atcun derivative of ov- containing a c-terminal ggc sequence showed high levels of membrane permeation and lipid peroxidation. the concept of catalytic metal drugs has attracted widespread attention although the concept is still in its infancy because of the role of metal ions (alexander et al., ; agbale et al., ) . rana box: rana box is a heptapeptide motif (cglxglc) from the nigrocin family. rana box consists of two cysteine residues that are separated by four or five other residues on the side and can form a cyclic disulfide bond. rana box peptide has shown structural analogies with polymyxin (colistin), and the primary structure of the rana box motif is important in determining bacteriostatic activity (kozić et al., ) . the deletion of the 'rana box' motif will cause the amp antibacterial effect to disappear, but replacing the natural 'rana box' sequence of amps with amidated phenylalanine can expand its efficacy against antibiotic-resistant microorganisms, including mrsa and p. aeruginosa, and reduce cytotoxicity. this phenomenon also shows that the effect of the motif on amps needs to be determined based on the specific situation and is not completely beneficial (bao et al., ) . the lps binding motif (g-wkrkrf-g) can produce a broad spectrum of antibacterial activity when introduced into the c-terminus of temporin- ta and temporin- tb (close isoforms of temporin) (mohanram and bhattacharjya, ) . antifungal peptides have a conserved gxc(x − ) c γ-core motif (residues - , gkcykkdnic; d-isomer) at its n-terminus, which is a cation part of the ring. this conserved motif interferes with the integrity of the plasma membrane of the cell (yount and yeaman, ) . conserved γ-core motifs are directly involved in protein-membrane interactions and strongly contribute to membrane binding (utesch et al., ) . if replace d-phe -pro sequence in peptide chain with d-phe- -abz turn motif ( -abz is an abbreviation of -aminobenzoic acid d-amino acid) in amp tyrc a, and nuclear magnetic resonance shows that this change retains the β-hairpin structure. unlike the traditional β-turn motif, the d-phe- -abz motif can be used as a tool for β-hairpin libraries. the hydrophobic peptide can be formed into the nucleated β-hairpin formation by adding the d-phe- -abz motif. moreover, the inclusion of this part in two designed cationic amphiphilic peptides can produce broadspectrum antibacterial activity and low hemolysis rate (cameron et al., ; cameron et al., ) . the ngr motif is composed of asn-gly-arg, and amps with this structure have strong cytotoxicity ( table ). the data indicate that the new amps containing ngr may bind to cd + or αvβ + tumor cells by binding to cd or αvβ , respectively, to exert anti-tumor activity, especially on cd + tumor cells . the central gxxxg motif can induce strong self-assembly and have been already used in the design of amps (brosig and langosch, ; krauson et al., ) . bovine lactoferrin b is an amp composed of amino acid residues and has antibacterial, antifungal, and antiparasitic activities. the multivalent molecules lfcinb ( - ) and lfcinb ( - ) contain the lfcinb ( - ) motif (rrwqwr) and show inhibition activity against e. coli, p. aeruginosa, and s. aureus. chimeric peptide chimera containing two motifs, namely, the rrwqwr of lfcinb ( - ) and the rllr of bfii computer design includes simple statistical modeling, structureactivity relationships study (abdel monaim et al., ) , neural networks (müller et al., ) , deep learning (veltri et al., ) , word embedding (hamid and friedberg, ) and machine learning. for example, a machine learning method by matlab is proposed based on the concept of scoring the contribution of each amino acid's antibacterial activity (wu x. et al., ) . the genetic algorithm was used to design the amphiphilic α-helical peptide guavalin , which has an uncommon amino acid composition (three tyrosine and three glutamine residues) and interestingly causes membrane hyperpolarization, which is a different mechanism from those of other amps (porto et al., ) . two research methods have been developed based on the research background of quantitative structure-activity relationships: prediction method based on amp therapeutic index and the identification of novel potential amps from the expressed sequence tag database based on the principles of the highly conserved signal peptide subclasses related to amps (juretić et al., ) . in this way, a variety of amp variants can be obtained. if combined with high-throughput screening, it can effectively obtain the desired amp. for instance, some new amps are designed by the combinatorial peptide library of melittin and show higher activity and lower cytotoxicity (krauson et al., ) . cations, such as na + and mg + , may affect amp activity . however, the different valences of metal ions have varied effects on amps. for example, divalent cations show stronger antagonism to bacteria than monovalent cations with thanatin and s-thanatin, which are insect amps (wu et al., ) . in the presence of nacl, the signal response during the association phase remarkably decreased in single-cycle and multi-cycle kinetic experiments, resulting in a decreased association rate. this occurrence may be caused by the shielding effect of nacl between the cationic peptide and the zwitterionic membrane. another possible reason is that na + can bind to the phospholipid bilayer, where the ions interact with the phosphate and the carbonyl oxygen of lipid head groups (sabapathy et al., ) . the reduced activity of synthetic peptide [rllr] under high salt concentration is possibly caused by the destruction of its α-helix structure. table shows that several amps, including histatin, myxinidin, and hepcidin, contain atcun motifs (amino terminal copper and nickel with xxh sequence). iron is the most abundant metal ion in human saliva, but the combination with this metal ion results in the loss of the α-helix of histatin and greatly reduces its antifungal activity (puri et al., ) . however, the coordination of copper (ii) and nickel (ii) ions can induce the formation of ros, which is essential for bactericidal activity (jeżowska-bojczuk and stokowa-sołtys, ). anionic amps have a large number of negatively charged aspartic and glutamic acid residues (lakshmaiah narayana and chen, ) . they require zinc as a functional cofactor and the zinc complex shows stronger antibacterial activity (jiang et al., ) . several of these amps use metal ions to form cationic salt bridges with the negatively charged components of the microbial membrane to penetrate the membrane. anionic amps may attach to ribosomes or inhibit ribonuclease activity when in the cytoplasm (jeżowska-bojczuk and stokowa-sołtys, ). metal ions also affect the self-assembly of peptides. these ions can recognize specific amino acids, such as lysine and glutamic acid, and may form salt bridges between peptide molecules to induce peptide self-assembly. for example, zn + can stabilize the aggregation of peptides on the cell membrane, which results in the enhanced antibacterial effect of dcd- l in the presence of zn + (tian et al., ) . ph many amps are stable and retain their antimicrobial activity in a wide ph range. amps have enhanced activity at low ph because of their basic properties. this condition is related to the protonation of histidine at acidic ph, which promotes electrostatic interactions with anionic surfaces, including lps and the anions of phospholipids, and subsequently enhances antibacterial properties. the effect of ph on the antibacterial activity of amps varies. for example, thanatin's activity at neutral ph is slightly higher than that under acidic conditions. by contrast, the activity of xylan on e. coli, listeria, and c. albicans is remarkably higher at ph . than at ph . (holdbrook et al., ) . the inactivation of the histidinecontaining amp c g-his under low ph conditions involves ph-dependent changes in the state of the aggregates in the solution, because the aggregates, which are sensitive to ph and lipid composition, may be affected by binding and conformation. peptides can also enhance bacterial membrane permeability at low ph (hitchner et al., ) . thrombin-derived c-terminal peptides (tcps) will also change the mode of cd (a protein that is abundant in human plasma) from anti-inflammatory mode to bacterial elimination mode from ph . to ph . (holdbrook et al., ) . a dimer (e.g., p- ) can be created to provide amps with resistance to a higher ph range. the sensitivity of this ph-sensitive amp can be used to achieve a certain targeting effect in practical applications. in addition, charge interaction is one of the most important factors in peptide self-assembly. ph affects the charge state of amino acid and substituent functional groups. therefore, adjusting the ph is the most common method for controlling peptide assembly and disassembly (tian et al., ) . proteases have a strong destructive effect on amps. for instance, ll- , which has the strongest inhibitory effect on chlamydial infection, is inhibited by the protease chlamydial protease-like activity factor (cpaf) secreted by chlamydia (tang et al., ) . studies have been focused on the design of amp carriers to solve this problem (lewies et al., ; nordström et al., ) . the presence of chitosan-silica solid support of kr- peptide can protect it be hydrolyzed by α-trypsin, and the degree of protection is increased by % compared with the free kr- (diosa et al., ) . however, several enzymes, such as protease , esterase and phosphatase , cut the blocking group of the peptide and trigger the self-assembly of the peptide, which positively affects amps (tian et al., ) . antimicrobial peptides can regulate pro-inflammatory reactions, recruit cells, stimulate the proliferation of cells, promote wound healing, modify gene expression and kill cancer cells to participate in the immune regulation of human skin, respiratory infections, and inflammatory diseases (de la fuente-núñez et al., ) . for example, α-defensins hnp- , hnp- , and hnp- showed effective antibacterial activity against adenovirus, human papilloma virus, herpes virus, influenza virus and cytomegalovirus. pulmonary diseases, such as idiopathic pulmonary fibrosis, alveolar proteinosis, and acute respiratory distress syndrome, show elevated levels of amps (guaní-guerra et al., ) . likewise, amps secreted by the paneth cells in the mammalian gut are important to shape the gut microbiota (bevins and salzman, ) . the application of amps in medicine, such as dental, surgical infection, wound healing and ophthalmology is developing now. but there are only three amps that have been approved by fda including gramicidin, daptomycin, and colistin. dental caries, endodontic infections, candidiasis, and periodontal disease are common diseases in the human oral cavity. dental caries is a prevalent oral disease and some acidogenic bacteria like streptococcus sp. are the main cariesassociated pathogens (izadi et al., ) . several amps have good application potential. for instance, peptide zxr- (fkiggfikklwrslla) has shown potent activities against pathogenic bacteria of dental caries, streptococcus mutans, streptococcus sobrinus, and porphyromonas gingivalis and peptide pac- (clinical trial identifier: nct ) that has been sold over the counter in taiwan for treating oral candidiasis (chen l. et al., ) . in surgical infection and wound healing: surgical infection occurs after surgery, burns, accidental injury, skin disease, and chronic wound infections have a serious hazard to human life (thapa et al., ) . several amps have shown the therapeutic potential of these diseases. for example, amp pxl shows pronounced efficacy as an anti-infective agent in burn wounds in mice and amp d a has been in the third phase of clinical trials for treating burn wound infections (björn et al., ) . in ophthalmology: human eyes are prone to be infected by several organisms including bacteria and fungi in which s. aureus, streptococcus pneumoniae, p. aeruginosa, aspergillus spp., and c. albicans are the most relevant pathogens (silva et al., ) . although amps such as lactoferricin b, protegrin- exhibited antimicrobial activity against these pathogenic bacteria, their application in the field of ophthalmology is only at the theoretical stage. with the popularity of contact lenses and the increase in cases of related eye infections, antimicrobial peptides have shown good application prospects in ophthalmology (khan and lee, ) . additional methods need to be performed for the application of amps as drugs in medicine. the main strategies include ( ) constructing precursors to reduce cytotoxicity and improve protease stability, ( ) using amps in combination with existing antibacterial agents, ( ) inducing the correct expression of amps with appropriate drugs and using engineering probiotics as vectors to express amps. for example, in the field of wound repair, different formulation strategies, such as loading amps in nanoparticles, hydrogels, creams, gels, ointments, or glutinous rice paper capsules, have been developed to effectively deliver amps to the wound (borro et al., ; thapa et al., ) . in recent research, the sponges developed from modified starch and hs-peg-sh are covalently immobilized with amp showed effective antibacterial activity (yang et al., ) . more technical means, including pheromone-labeled amps, local environment-triggered amps (enzyme precursor drug release system, ph-activated amps, etc.), have been developed to improve the targeting mechanism of amps. furthermore, nanotubes, quantum dots, graphene, and metal nanoparticles have been proposed to be a potential method to enhance drug delivery of amps (magana et al., ) . hybrid peptides have also been used to build targeting peptides. for example, pa , which is a p. aeruginosa-targeting peptide, was combined with gnu (a broad-spectrum amp) to construct a hybrid peptide (pa -gnu ) that targets oprf protein and has good bactericidal activity and specificity . furthermore, some antibiotics, for instance, daptomycin (a lipopeptide), lugdunin which is a -membered cyclic peptide consists of amino acid residues plus a thiazolidine moiety and telavancin (a glycopeptide) have been widely used for the clinic (durand et al., ; lampejo, ) . although they are antibiotics, they have provided broader ideas for the design of amps. food preservatives have potential harm to the human body. therefore, natural preservatives are being advocated by more people. amps have a good inhibitory effect on common bacteria and fungi in food, and many amps are resistant to acids, alkalis, and high temperatures are easily hydrolyzed by proteases in the human body. thus, amps are a promising alternative to preservatives. nisin is a bacteriocin produced by l. lactis subspecies. lactic acid bacteria have been widely used as food preservatives. nisin is categorized as generally recognized as safe (gras) by the us food and drug administration (fda) and is used as a food preservative in other countries (khan and oh, ) . however, only nisin and polylysine are currently approved by the fda as food additives (santos et al., ) . pedocin pa- , a bacteriocin consisting of amino acids produced by a diplococcus, is also used as a food preservative and is sold on the market under the trade name alta . pedocin pa- is used as a food additive to inhibit the growth of l. monocytogenes, which can cause meat deterioration (settanni and corsetti, ) . enterocin as- is an amp used to preserve cider, fruit and vegetable juices, and enterocin ccm is used to preserve soy milk (rai et al., ; santos et al., ) . encapsulating bacteriocins into liposomes is a new method used to overcome the problems of amps in food applications (such as proteolytic degradation or interaction with food ingredients) (da silva malheiros et al., ) . moreover, active packaging by adding amps is a novel packaging method that has great potential in the food industry. for instance, ε-poly-l-lysine is used in conjunction with starch biofilms to show good inhibitory effects on aspergillus parasiticus (aflatoxin producer) and penicillium expansum and nisin have the potential to be dairy preservative because it is a highly surface-active molecule (luz et al., ) . the european union banned the use of animal growth promoters in animal feed in . thus, a new antibacterial strategy is needed. many amps are the potential to be used in poultry, swine, and ruminants breeding and aquaculture because they can improve production performance (liu et al., ; bao et al., ) , immunity and promote intestinal health and some of them have a stronger inhibitory effect on bacterial inflammation if used with antibiotics cote et al., ) . for example, siamp has a good effect on the treatment of ibv in chicken (sun et al., ) . by adding swine gut intestinal antimicrobial peptides (sgamp), broilers showed higher average daily gain and feed efficiency under chronic heat stress conditions (hu et al., ) . frog caerin . , european sea bass dicentracin and nk-lysine peptides (nklps) have good inhibitory effects on nodavirus, septicaemia haemorrhagic virus, infectious pancreatic necrosis virus and spring viremia carp virus, which are devastating to fish farming (león et al., ) . the amp in soybean meal fermented by b. subtilis e effectively inhibits v. parahaemolyticus and vibrio alginolyticus and enhances the resistance level of litopenaeus vannamei against v. parahaemolyticus when added to feeds (cheng et al., ) . for agriculture, the plant pathogenic infection of bacteria and fungi causes the loss of economy, for instance, aspergillus flavus infection of corn and peanuts, citrus green mold caused by penicillium digitatum, gray mold disease caused by botrytis cinerea on strawberries and geotrichum citriaurantii infection of citrus fruit all cause great harm to the growth and post-harvest of agricultural products (liu et al., ; liu et al., ) . several afps have shown prospect to control these problems. however, the practical application of antimicrobial peptides in the transportation and preservation of agricultural products is still lacking, because the use of antimicrobial peptides will greatly increase the cost in the transportation of fruits and vegetables (application examples of amps in these four fields are shown in table ). antimicrobial peptides constitute a global research hotspot, but many key issues in design and application need to be solved urgently. several restrictive factors hinder the application of amps. the interaction of multidisciplinary subjects, such as biology, materials science, chemistry, bioinformatics, molecular informatics and pharmacy can further develop prospective amps. computer molecular dynamics simulation, cell membrane simulation, and more methods are being applied to study the mechanism of amps. how to further understand the correlation between amps and various targets instead of conducting one-sided experimental research might improve experimental designs to obtain stronger systemic and scientific demonstrations. on this basis, further animal experiments are required instead of simple cell-level experiments to test the effect of amps under complex physiological conditions. several complicated methods, such as the chemical method of peptidomimetics and non-natural amino acid modifications, have been applied in designing amps to solve the problem of protease hydrolysis. most methods use chemical substrates, but the cost of these methods cannot be ignored in practice. in addition, chemical synthesis and the use of engineered bacteria are currently the mainstream for such procedures. finding a better biological preparation method, reducing the cost and increasing the yield is important problems in practical application. furthermore, studying the amp expression of the organism itself and finding a better expression vector are necessary for mass production in the future as more amps in nature are discovered. further research is needed on the reported amps to solve the problem on structure-function relationship. as a branch of peptide drugs, amps need to progress with the advancement of medical science against the background of the current low success rate of the clinical application of amps. more attention can be 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targeting its spike protein that harbors a high capacity to mediate membrane fusion fabricating antimicrobial peptide-immobilized starch sponges for hemorrhage control and antibacterial treatment multidimensional signatures in antimicrobial peptides secretory production of antimicrobial peptides in escherichia coli using the catalytic domain of a cellulase as fusion partner comparative study of different forms of jellein antimicrobial peptide on leishmania parasite identification and biochemical characterization of a new antibacterial and antifungal peptide derived from the insect sphodromantis viridis alpha-helical antimicrobial peptides-using a sequence template to guide structure-activity relationship studies antimicrobial activity and mechanism of the human milk-sourced peptide casein an antimicrobial peptide containing ngr motif has potent antitumor activity against cd + and cd -tumor cells antimicrobial peptides conjugated with fatty acids on the side chain of d-amino acid promises antimicrobial potency against multidrug-resistant bacteria isolation and structure of corticostatin peptides from rabbit fetal and adult lung characterization of antimicrobial activity and mechanisms of low amphipathic peptides with different α-helical propensity we thank the national key r&d program of china ( yfd ). key: cord- - rnkotqy authors: vieira, flávia sarmento; corrêa, gladys; einicker‐lamas, marcelo; coutinho‐silva, robson title: host‐cell lipid rafts: a safe door for micro‐organisms? date: - - journal: biol cell doi: . /bc sha: doc_id: cord_uid: rnkotqy the lipid raft hypothesis proposed that these microdomains are small ( – nm), highly dynamic and enriched in cholesterol, glycosphingolipids and signalling phospholipids, which compartmentalize cellular processes. these membrane regions play crucial roles in signal transduction, phagocytosis and secretion, as well as pathogen adhesion/interaction. throughout evolution, many pathogens have developed mechanisms to escape from the host immune system, some of which are based on the host membrane microdomain machinery. thus lipid rafts might be exploited by pathogens as signalling and entry platforms. in this review, we summarize the role of lipid rafts as players in the overall invasion process used by different pathogens to escape from the host immune system. since it was first described as a simple barrier between extra and intracellular compartments, different plasma membrane structural and composition models have been proposed (pike, ). among these different models, the 'fluid mosaic' model proposed by singer and nicolson ( ) helps to provide answers about the different roles of the plasma membrane in cell biology, including transport processes, cell protection, cell-cell contact and, principally, cell signalling. the plasma membrane harbours many molecules involved in different cell signalling cascades, such as glycero-and sphingo-lipids, proteins (receptord, kinases, etc.), which interacts upon different stimuli triggering different cell responses. over the last decade, several works have provided evidence that the plasma membrane is really more mosaic than fluid (pike, ; engelman, ) . it was shown that lipids are not randomly distributed, leading to the proposal of a new model for understanding the biological membrane architecture (pike, ; garcía-marcos et al., ) . the new model suggests that the plasma membrane is patchy, with segregated portions that are distinct in structure and function and that can also vary in thickness and composition. from the first reports in the s provided by palade ( ) and yamada ( ) showing stable flask-like invaginations of the plasma membrane, to the current view of multiple membrane domains allowing different protein-lipid and protein-protein interactions that compartmentalize and temporarily order the membrane moiety, many different reports still bring some controversy to the theme. the concept of lipid microdomains arose in the s in the reports of van meer and simons ( ) and klausner and colleagues ( ) . it is now widely accepted that lipid rafts are not pre-existing domains in which proteins dynamically partition, but rather that the formation and disassembly of raft domains is a dynamic process (see, for example, plowman et al., ) . later, rafts were visually observed by simons and ikonen ( ) , who described them as lipid rafts, imagining these microdomains as floating islands in the membrane (triantafilou et al., ; luo et al., ) . further studies provided evidence that lipid rafts are mainly composed of cholesterol, (glyco)sphingolipids and (glycero)phospholipids, with a high degree of saturation in their fatty-acid chains ( figure a ). the tight interaction between these components provides the basis of their packing and rigidity, which leads to a phase separation (simons and ikonen, ) . this tight-packing organization of lipid rafts confers their resistance to solubilization by non-ionic detergents, which allows their separation and isolation from the rest of the plasma membrane using sucrose-density gradients (brown and rose, ) . the development of this method is widely used for the isolation and analysis of lipid rafts and their associated proteins in different cell types (triantafilou et al., ; bouillon et al., ; chazal and gerlier, ; laughlin et al., ; tortelote et al., ; vacca et al., ; olsson and sundler, ) . however, new considerations about the term 'detergent-resistant membranes' (drms) had already been reviewed. drm no longer defines the bona fide rafts (lai, ) , because this technique can create several artifacts, such as the loss of membrane components during extraction (edidin, ) . however, many proteins functionally involved with microdomains had already been described via this method, as discussed by lai ( ) . another efficient tool used to study lipid rafts is cholesterol depletion, by the use of cholesterol-removing and -binding agents, such as cyclodextrins and filipin (schnitzer et al., ; luker et al., ) . this method is based on the theory that cholesterol acts as a dynamic 'glue', holding lipid rafts together, and its removal from the review membrane results in the dispersion of raft-associated lipids and proteins (mañes et al., ; hawkes and mak, ) . considering that cholesterol is a relatively rigid molecule, it was postulated that cholesterol-rich microdomains have a slower mobility in the membrane than non-raft regions (shaw, ) . nowadays, more accurate methods are employed in order to improve the research into lipid rafts. fluorescent biosensors that are capable of tracking signalling events in live cells are powerful tools to aid the understanding of dynamic cellular signalling (gao and zhang, ) . the proposals regarding microdomains postulated in the late s can be now supported by, for example, fret (fluorescence resonance energy transfer) microscopy and afm (atomic force microscopy). given the importance of the akt pathway in cell proliferation and survival, and thus its involvement in cancer, this pathway has been increasingly studied. gao and zhang ( ) developed a genetically encodable akt activity reporter and analysed the spatiotemporal dynamics of akt activity within plasma membrane microdomains by fret. using this tool, it was observed that akt activity is differentially regulated between raft and non-raft regions of the plasma membrane (gao and zhang, ) . in addition, afm has been described as an essential tool to follow the motion of lipid-raft microdomains and proteins that might be interacting with them (henderson et al., ) . several works have demonstrated the importance of afm in experimental approaches to study lipid rafts. poole et al. ( ) , studying mdck (madin-darby canine kidney) cell microvilli, suggested that lipid rafts might be involved in the maintenance of these structures. these authors used a combination of afm and laser-scanning confocal microscopy in order to attest their hypothesis. cecchi et al. ( ) interestingly observed an increase in specific amyloid ligands when raft components, such as cholesterol, are depleted. this group suggested that cholesterol can reduce membrane modifications triggered by amyloid residues at the lipid-raft level, possibly involving physicochemical features. the techniques of afm and confocal microscopy in combination with inhibitors of cholesterol synthesis and an agent that chelates cholesterol were allied in the study of lipid rafts in this context, as well as the pursuit of flotillin- and the ganglioside g m , which are known markers of microdomains. the lipid raft size in vivo has been estimated to be - nm by using fret and single-moleculetracking microscopy. the size, arrangement and movement of lipid rafts are dynamic and can be found in localized cellular structures, such as filipodia and cell adhesion points (chazal and gerlier, ; hawkes and mak, ) . it is accepted that membrane rafts in basal conditions are small regions of the lipid membranes, which tend to cluster after certain stimuli to form larger structures that are also called platforms (liu and anderson, ; holopainen et al., ; kusumi et al., ; bollinger et al., ; rao and mayor, ) . it is important to mention that the assembly of those small regions (pre-rafts) seems to be dependent on the presence of ceramide (liu and anderson, ; bollinger et al., ) . in addition to the lipid components, a variety of cell receptors and signalling proteins are known to be associated with membrane rafts. they include the gpi (glycosylphosphatidylinositol)-anchored proteins, kinases and adaptor molecules that act as intermediate transducers for many receptors, including tcrs (t-cell receptors) and bcrs (b-cell receptors) (montixi et al., ; cheng et al., ; chazal and gerlier, ; hawkes and mak, ) . the role of lipid rafts in different cell types has been the subject of numerous studies, and their physiological significance for cell biology has recently become clear. these membrane regions play an important role in a variety of cellular functions, including polarization, signal transduction, endocytosis, secretion, cell-cell and cell-pathogen adhesion (mañes et al., ; martin-belmonte et al., ; harris et al., ; grimmer et al., ; ha et al., ; pierini et al., ; jacobson et al., ) . one of the most widely appreciated roles of lipid rafts is the recruitment and concentration of molecules involved in cellular signalling. the formation of a molecular cluster and their signal transduction machinery in membrane rafts leads to enhanced signalling efficiency (triantafilou et al., ) . anderson et al. ( ) reported that mhc class ii molecules are located in lipid rafts of murine and human b-cell lines. such a localization seems to be critical for t-cell activation. the binding of tcrs to mhc class ii molecules of apcs (antigen-presenting cells) occurs in lipid rafts. the raft aggregation promotes tyrosine phosphorylation and recruitment of signalling proteins, but excludes certain proteins, such as the tyrosine phosphatases cd and cd , which leads to the formation of a supramolecular activation cluster (monks et al., ) . however, in vivo, tcr does not constitutively reside in membrane lipid rafts. after t-cell activation, the tcr moves into the rafts (monks et al., ; anderson et al., ; luo et al., ) . this characteristic was also observed for other molecules that migrate to the lipid rafts after specific stimulation in several physiological events. there is a specific subtype of microdomain called caveolae. these structures are small membranesurface invaginations, which were initially described as cave-like invaginations of the plasma membrane, - nm in size and found in many cell types (figure b) . although they were identified by electron microscopy more than years ago as an invagination in the plasma membrane with a flask-shape morphology that can be singular or found in detached grape-like clusters, caveolin caveolae have remained enigmatic structures (palade, ; yamada, ; razani et al., ; lajoie et al., ) . forty years after the description of caveolae, their structure could be more closely studied upon the discovery of caveolin, the signature protein present in calveolae (rothberg et al., ) . it has been suggested that caveolae can be stabilized by caveolin and, additionally, may further become immobilized by filamin, which binds to caveolin, as well as to the actin cytoskeleton (stahlhut and van deurs, ; hommelgaard et al., ) . since that time, caveolae have been implicated and demonstrated to be important in a variety of cellular functions, including endocytic processes, cholesterol and lipid homoeostasis, signal transduction and tumour suppression (razani et al., ; duncan et al., ; mañes et al., ; cohen et al., ; tortelote et al., ) . the protein caveolin- has itself been implicated in signal transduction, because of its direct interaction with a multitude of signalling molecules through the caveolin-scaffolding domain. in addition, caveolin- has been shown to be phosphorylated on tyrosine residues during some signalling events (mastick and saltiel, ; okamoto et al., ; ushio-fukai et al., ; duncan et al., ) . vesicular transport is one of the most important roles of caveolae, including endocytosis when caveolae are, indeed, functional endocytic vesicles (lajoie and nabi, ) , and promotes transcytosis of specific macromolecules in endothelial cells (minshall et al., ) . previous studies proposed that, as well as caveolae, caveolins also have an important involvement in signal transduction, principally due to its scaffold domain, which acts as a harbour for different cytosolic proteins involved in different signalling cascades (sargiacomo et al., ; lisanti et al., ; razani et al., ; cohen et al., ) . it is well known that intracellular parasites have many mechanisms to avoid the host defence response. the inhibition of lysosomal fusion, a classical escape mechanism, was observed after infection by mycobacterium, chlamydia, toxoplasma (coutinho-silva et al., ) and trypanosoma cruzi (hall and joiner, ) , for example. another way that pathogens can prolong their survival inside the host is by prevention of host-cell apoptosis and by the modulation of reactive oxygen and nitrogen species generation (coutinho-silva et al., ). an interesting manner that allows pathogens to evade the immune system is through membrane microdomains. as signalling for the innate and adaptative immune responses is initiated in rafts, some pathogens have evolved mechanisms to subvert this signalling by co-opting raft-associated pathways (mañes et al., ) . different pathogens, such as viruses, bacteria and protozoa, can use the host-cell lipid rafts to secure their entrance and maintenance caveola: a special type of lipid raft, comprising small ( - nanometre) invaginations of the plasma membrane, found in many vertebrate cell types. a family of proteins involved in receptor-independent endocytosis. the caveolin gene family has three members in vertebrates, caveolin- , - and - . the term pathogen is most commonly used to refer to infectious organisms. these include bacteria, viruses, protozoa and fungi. several pathogens can directly interact with different target cells through membrane microdomains in different manners. it is known that the entry via lipid rafts can avoid lysosomal fusion and therefore allow pathogen survival. in addition, parasites might modulate signalling pathways, including lipid-raft-associated protein kinases [srfk (src family kinases)]. especially viruses, which do not have their own protein synthesis machinery, target the er after subverting the lysosomal pathway. lys, lysosome; pv, parasitophorous vacuole. inside target cells ( figure ). the benefit provided by interaction with lipid rafts can vary from one pathogen to another. the list of pathogens that hijack rafts also includes the non-classical infectious agent, the scrapie prp (prion protein). here, we provide an update of how different pathogens modulate host immune response by using their lipid rafts. different viruses have evolved strategies to subvert raft-associated signalling, enabling their efficient replication in immune cells, and at the same time blocking the immune response that is elicited by the target cells (hawkes and mak, ) . virus entry into a host cell involves the binding of the virus to one or more cell-surface receptors, followed by entry into the cell. many animal viruses exploit the endocytic machinery of their host cell for infection, and lipid rafts are often a site for entry, assembly and budding of microbial pathogens, as confirmed by biochemical approaches and microscopy evidence (kovbasnjuk et al., ; suomalainen, ; lu et al., ) . for non-enveloped viruses, after the attachment to cell-surface receptors, the bound capsids are internalized, mostly by invagination of the plasma membrane and intracytoplasmic vesiculation. the involvement of lipid rafts in mediating this process has been described for several viruses, as reviewed by chazal and gerlier ( ) . the most thoroughly studied of these is sv (simian virus ). sv initiates infection by binding to the mhc class i molecules (stang et al., ; anderson et al., ; duncan et al., ; chazal and gerlier, ) . sv directly associates with caveolae, leading to a loss of actin stress fibres and the appearance of actin tails budding: in virology, budding is a form of viral shedding by which enveloped viruses acquire their external envelope from the host-cell membrane, which bulges outwards and encloses the virion. emanating from the virus containing caveolae (duncan et al., ; pelkmans et al., ) . moreover, caveolae transport sv particles to the er (endoplasmic reticulum), where the virus is disassembled (norkin et al., ; chazal and gerlier, ) . both the polyoma virus and ev- (echovirus type ) also directly associate with caveolae (richterová et al., ; marjomaki et al., ) . the polyoma virus associates with caveolin- after entry, a possible association with 'caveosomes' and trafficking to the er (richterová et al., ) . ev- is internalized into caveolae using the integrin α β as cellular receptor. studies have shown that ev- , α β integrin and caveolin- were internalized together in vesicular structures and accumulated in a perinuclear compartment (marjomaki et al., ) . interestingly, it was shown (richterová et al., ; marjomaki et al., ) that the entry of the virus does not occur by endocytosis through the classic clathrin-coated vesicles. however, these authors observed that virus particles had merged with caveolin- , and incubation with methyl-β-cyclodextrin inhibited the virus entry. enveloped viruses also use rafts during the internalization and fusion process. the entry of enveloped virus involves virus attachment, followed by close apposition of the virus and plasma membranes. then the two membranes fuse to deliver the virus' genomic rna into the host cells, which requires conversion of the virus-encoded envelope glycoprotein (env) from its native state to its fusion-activated form (fantini et al., ; chazal and gerlier, ; mañes et al., ) . the glycoproteins of several viruses, including influenza virus, hiv, mlv (murine leukaemia virus), measles virus and ebola virus, are associated with host-cell membrane rafts (scheiffele et al., ; manie et al., ; vincent et al., ; pickl et al., ; bavari et al., ) . additionally, there is biochemical evidence showing that cholesterol and possibly cholesterol-rich lipid rafts are required for efficient porcine pseudorabies virus entry (desplanques et al., ) . another recent study reported that the sars-cov (severe acute respiratory syndrome coronavirus) receptor is located in lipid rafts and the productive entry of the sars-cov pseudovirus into the host cell requires the presence of intact and functional lipid rafts (lu et al., ) . fusion of sfv (semliki forest virus) and sin (sind-bis virus), as well as other alphaviruses, depends on the presence of cholesterol and sphingolipid in the target membrane (bron et al., ; lu et al., ; smit et al., ; chazal and gerlier, ) , which are known to be abundant in lipid rafts (pike, ) . other enveloped viruses enter the host cell using a ph-independent fusion process, as found for hiv. the hiv- env is composed of two associated glycoprotein subunits, gp and gp . the external gp is responsible for the attachment to the cellular receptors and co-receptors (chemokine receptor family member ccr and/or cxcr ), whereas the transmembrane protein gp is responsible for the fusion of viral envelope with the plasma membrane of the target cd + t-cells (popik et al., ) . rafts are proposed to be the specific cell membrane regions in which these clustering events occur. it is important to point out that the entry of hiv- through rafts may direct the virus complex into a favourable compartment for a productive infection (fantini et al., ; chazal and gerlier, ; mañes et al., ; luo et al., ) . a recent study showed that hiv entry into macrophages is sensitive to membrane cholesterol depletion, which favours the hypothesis for a role of macrophage lipid rafts in the hiv- entry process (carter et al., ) . the cellular receptor for the mlv, cat (cationic amino acid transporter ), is physically associated with caveolin in membrane rafts, and the disruption of rafts inhibits the early step of mlv infection, suggesting that the localization of the receptor within rafts is crucial for the virus entry (lu and silver, ) . it was already demonstrated that the penetration of filoviruses, such as ebola virus and marburg virus, is inhibited after cholesterol depletion of the host cell, and, after internalization, viral proteins co-localized with caveolin (bavari et al., ; empig and goldsmith, ; chazal and gerlier, ) . the late stages of the viral life cycle are the assembly of viral components into virions, maturation into infectious particles, and, in the case of enveloped viruses, release from the cell via a budding process (ivanchenko et al., ) . assembly and budding are the last, but critical, steps in the virus life cycle for the survival of the virus and its disease-producing ability in the host (chen et al., ; wang et al., ). an explanation as to why viruses use lipid rafts review is that these structures offer an efficient system for concentrating all the virus proteins that are required for the assembly of new virions, as reviewed by nayak et al. ( ) . hiv- is enclosed in a lipid envelope enriched in cholesterol and sphingolipids, suggesting specific membrane localization for assembly (aloia et al., ; campbell et al., ; raulin, ) . recent studies have reported that rafts represent a necessary step during hiv- assembly. with similar methods of both assembly and budding within membrane rafts, many other viruses, including influenza virus, measles virus, ebola virus and possibly sendai virus, also use lipid rafts as assembly platforms (luo et al., ) . in this regard, it was also suggested that the rsv (respiratory syncytial virus) assembled within lipid rafts where viral proteins co-localize with caveolin- (brown et al., a (brown et al., , b . although rafts are involved in virus assembly, we have to keep in mind that only a fraction of viral proteins are found associated with rafts; this could be due to the poor biochemical characterization of raft subsets or to the transient nature of the association. whereas non-enveloped viruses are released from the infected cell by disruption of the plasma membrane, enveloped viruses contain a host-cell-derived lipid bilayer, which is acquired during budding (garoff et al., ; chazal and gerlier, ) . membrane lipids are not randomly incorporated into the viral envelope. in addition, some authors suggest that viral glycoproteins determine the site of virus assembly and budding (garoff and simons ; allison et al., ; vennema et al., ; bruss, ) . on the other hand, in polarized epithelial cells, the viral glycoproteins contain sorting signals or motifs and are directed to the specific site where assembly and budding will occur (nayak et al., ) . lipid rafts function as microdomains for concentrating viral glycoproteins and may serve as a platform for virus budding. the structures have the ability to regulate budding; however, the mechanism by which the lipid raft can favour the budding and/or fission process is as yet unknown (nayak et al., ; luo et al., ) . the budding of new virions from the raft allows the exclusion or inclusion of specific host-cell membrane proteins in the virus particle, which could disrupt cellular and/or humoral immune responses to the virus (vanderplasschen et al., ; peterlin and trono, ) . the lipid composition of the influenza virus family is due to affinity of the haemagglutinin and neuraminidase glycoproteins for these lipids, and some authors suggest that the influenza virus buds from raft domains (chazal and gerlier, ; nayak et al., ) . several reports suggest that hiv- buds from lipid rafts (campbell et al., ; fantini et al., ) . hiv incorporates raft-associated complement regulatory proteins, which remain functionally active on the surface of the virus and down-regulate the complement cascade (mañes et al., ; peterlin and trono, ) . after budding from the host cell, viruses are released into the surrounding medium to infect other cells. the mechanism of this bud completion is as yet unclear and a number of both viral and host factors may affect this process (nayak et al., ; luo et al., ) . studies have been suggested that several bacteria interact with host lipid rafts to enter and survive inside the cell (mañes et al., ; hawkes and mak, ) . the mechanisms that underlie this interaction are starting to be unravelled. activation of secretion, binding, perforation of the host-cell membrane and signalling to trigger bacterial phagocytosis are involved with components of membrane microdomains (lafont and van der goot, ) . it was found that the polarity of epithelial cells and the involvement of cd are important in the interaction of bacteria with lipid rafts (peiffer et al., ) . two advantages in bacteria invasion were postulated: ( ) avoidance of the intracellular degradative pathway and ( ) triggering of the cell signalling cascades that lead to membrane ruffling and cytoskeleton rearrangement (mañes et al., ) . the avoidance of the host immune pathway after phagocytosis was developed by several microorganisms, mainly bacterial cells. subversion of phagosome fusion with lysosome and presentation to immune system was observed in pathogens such as mycobacterium and chlamydia. gatfield and pieters ( ) showed for the first time the relevant role of cholesterol for mycobacteria entry into macrophages. in addition, some bacteria hidden inside the cell took advantage of host lipids to generate phagosomes and survive inside them, such as brucella spp. and legionella pneumophila (naroeni and porte, ; watarai et al., watarai et al., , . besides, these parasites can hijack rafts, altering host-cell signalling, for example shigella flexneri (lafont et al., ) . the interaction between pathogens and the host cell can also modulate other host features, such as cytoskeletal dynamics. in the case of s. flexneri, cholesterol removal decreased the binding of an effector protein, called ipab, with host cd , which is known to be involved in cytoskeleton-dependent signalling events (hirao et al., ) . seveau et al. ( ) demonstrated for the first time that the cell adhesion molecule, e-cadherin, and hgf-r (hepatocyte growth factor receptor) require host lipid rafts to mediate listeria monocytogenes entry. it had already been reported by the same group that, in l. monocytogenes, two major proteins, internalin and inib, mediate bacterial invasion into host and bind to e-cadherin and hgf-r respectively (cossart et al., ) . salmonella, shigella and the entheropathogenic escherichia coli have a common requirement for a t ss (type iii secretion system), which is a multicomponent molecular syringe that allows the translocation of so-called effector proteins from bacterial cytoplasm, through the inner and outer bacterial membrane, as well as the host plasma membrane, directly into cytoplasm (van der goot et al., ; lafont and van der goot, ) . activation of this system requires contact with the host cell, and has effector proteins, named sipb and sipc for salmonella, ipab and ipac for shigella and popb and popc for pseudomonas. hayward et al. ( ) showed a new requirement for cholesterol, for which the main binding determinant was sipb/ipab to host cells, and lafont and van der goot ( ) suggest that this must occur downstream of the t ss activation. the pathogenic bacterium brucella, which causes brucellosis, can avoid bactericidal activity of macrophages triggering the camp/pka (protein kinase a) pathway. this process occurs immediately after the first contact with the target cell (jimenez de bagues et al., ) . lipid-raft-associated molecules, such as gpi-anchored proteins, g m gangliosides and cholesterol, were found selectively incorporated into macropinosomes containing brucella. in contrast, the lysosomal glycoprotein lamp- (lysosomeassociated membrane protein ) and the host-cell transmembrane protein cd were excluded from these macropinosomes (watarai et al., ) . interestingly, it had already been demonstrated that brucella abortus infection is related with prp c (cellular prp), one of the lipid raft-associated molecules on the plasma membrane of different cell types. in addition, watarai et al. ( watarai et al. ( , postulated that the signal transduction induced by the interaction between bacterial hsp (heat-shock protein ) and prp c on macrophages contributes to the establishment of b. abortus infection. coxiella burnetti, the causative agent of human acute and chronic q fever, can be found in cholesterol-rich vacuoles with lipid-raft proteins, and also can modulate the cholesterol metabolism from the host cell (howe and heinzen, ) . the importance of cholesterol in c. burnetti infection can be directly associated with its pathophysiology (howe and heinzen, ) . it was also suggested that pathogens and particles that bind to lipid-raft components may trigger the macrophage autophagic machinery (amer et al., ) . l. pneumophila and a uropathogenic e. coli can stimulate autophagosome formation, which contains both lipid rafts and autophagy-involved cell molecules. in addition, it was observed that internalization of pathogen and the autophagy stimulation are cholesterol sensitive and the pathogens harbouring in autophagosomes could avoid immediate killing (amer et al., ) . components of lipid rafts do not appear to be essential for assembly of autophagosomes, but instead may affect a signal transduction pathway dedicated to host recognition of microbes, as suggested by amer et al. ( ) . the specific subtype of microdomain, caveolae, also appeared to be directly involved in the interaction with bacteria (duncan et al., ) , such as chlamydia trachomatis (norkin et al., ) , e. coli (shin et al., ) and campylobacter jejuni (wooldridge et al., ) . among the chlamydiae, depending on the serovar, or the species, one or both of the caveolin proteins ( or ) may play important roles in the developmental cycles (stuart et al., ; webley et al., ) . porphyromonas gingivalis capitalizes on the lipidraft structure to down-modulate innate defence mechanisms. remarkably, this novel mechanism employs host-cell signalling pathways through cross-talk between tlr (toll-like receptor- )/ chemokine receptor to attenuate the protective and bactericidal response to p. gingivalis infection (darveau, ) . campylobacter enteritis, regardless of its own invasiveness, promotes the translocation of the noninvasive bacteria e. coli across the intestinal epithelium via a lipid-raft-mediated transcellular process (kalischuk et al., ) . furthermore, bacterial toxins, such as cholera toxin, listeriolysin o and anthrax toxin, also target lipid domains (orlandi and fishman, ; coconnier et al., ; abrami et al., ) . more recently, several pathogenic bacteria have been associated with lipid rafts, such as francisella tularensis (tamilselvam and daefler, ) , helicobacter pylori (lai et al., ) , p.s gingivalis (hajishengallis et al., ; wang and hajishengallis, ) and m. tuberculosis (shin et al., ) , which are members of an ever increasing list as the years progress. recently, caserta et al. ( ) described evidence for the first time that clostridium perfringens enterotoxin acts independently of lipid microdomains. the involvement of fungal infection with lipid rafts is not yet well explored; however, for mycopathogens, a modulation in host-cell signalling pathways has been reported, as described below. the invasion process of candida albicans or paracoccidioides brasiliensis had already been associated with activation of host-cell tyrosine kinases (belanger et al., ; monteiro da silva et al., ) . the manipulation of signalling pathways, which involve the host-cell kinases, can lead to an efficient way to enter, proliferate and exit the host cell during the infectious cycle (münter et al., ) . recently, maza et al. ( ) investigated yeast forms of p. brasiliensis in the context of kinase signalling. it was observed that this pathogen promotes the aggregation of lipid rafts in epithelial cells, which is an important step to fungal adhesion and src kinase family activation. thereby, for the first time, it was shown that a pathogenic fungus can interact with host-cell membrane rafts to establish infection. encephalitozoon cuniculi, a microsporidiam parasite that affects the nervous system, as well as the respiratory and digestive tracts, resides in a parasitophorous vacuole surrounded by host-cell lipids, was labelled with dilc ( , -dihexadecyl- , , , -tetramethylindocarbocyanine), a marker for lipid rafts, and dio ( , -dilinoleyloxacarbocyanine), a marker for non-raft membrane domains, which suggests that both contribute to the formation of the vacuole membrane (rönnebäumer et al., ) . this report points to an alternative method of fungal infection in host cells that is raft-independent. the protozoan invasion in the host cell occurs during specific stages of the pathogen life cycle. intracellular entrance of these parasites does not depend on the endocytic machinery of the host cell, as in the case of bacteria and viruses. this can be explained due to the protozoan's larger size ( - μm). as shown for other pathogens, the exploitation of host membrane microdomains by protozoa constitutes a crucial step for its maintenance, survival and modulation of host immune response (mañes et al., ) . members of the apicomplexa group, such as toxoplasma gondii and plasmodium falciparum, the aetiological agents of toxoplasmosis and malaria disease respectively, are obligatory intracellular parasites and actively enter their target cells (aikawa et al., ; suss-toby et al., ) . previous studies related that these parasites can interact with lipid rafts during the infection process, because parasitophorous vacuole membranes contain host raft lipids and proteins (aikawa et al., ; mordue et al., ; lauer et al., ) . this event shows that parasites might hijack or recruit these microdomains during infection. furthermore, gpi-anchored proteins, such as cd and cd , that are major inhibitors of membrane complement, are progressively depleted from the infected cell surface (haldar et al., ) . it was further demonstrated that host raft cholesterol is important to vacuolar parasites because, when cholesterol was depleted from plasmodium-infected erythrocytes, the expulsion of non-infective parasites occurred (lauer et al., ) . in addition, cholesterol depletion from red blood cells prevents p. falciparum infection (samuel et al., ) . theileria parva, another member of apicomplexa, also interacts with host-cell rafts, with further regulation of host protein kinases (dobbelaere et al., ; baumgartner et al., ) . studies indicated that raft association might not be sufficient to shuttle membrane molecules past the moving junctions (tight constrictions formed between parasite and host cell), for example, caveolin- is excluded from the t. gondii parasitophorous vacuole (mordue et al., ; coppens and joiner, ) . flotillin- , a raft protein anchored in plasma membrane by acylation, was also discarded from its parasitophorous vacuole (charron and sibley, ) . it was demonstrated during t. gondii infection that selective portioning at the host-parasite interface is a highly complex process and that the raft interaction can benefit the parasite inclusion into parasitophorous vacuoles (charron and sibley, ) . on the other hand, it was observed that the association with membrane microdomains is not necessary to direct insertion of host-cell membrane molecules into t. gondii parasitophorous vacuole (charron and sibley, ) . murphy et al. ( ) related that different remodelling and sorting may occur in distinct endo-vacuoles. in this case, primaquine was used to disturb red blood cell membranes and induce detergent-free vesicles, which are enriched in cholesterol, raft proteins (flotillin and stomatin) and pip (phosphatidylinositol , bisphosphate). however, pip was abrogated of plasmodium parasitophorous vacuoles and another lipid was found, ps (phosphatidylserine). so, interestingly, erythrocyte raft lipid recruited to the site of invasion can be remodelled by malaria parasites to establish blood-stage infection. protein and lipid distribution in the erythrocyte membrane may be more ordered than previously expected (murphy et al., ) . it is known that there is a unique relationship between cholesterol and caveolae (caveolins), which is involved in cholesterol homoeostasis. therefore, caveolae become sensitive to cholesterol depletion and a cross-link between cholesterol and caveolin has been demonstrated (murata et al., ; razani et al., ) . cholesterol also appeared to be important in the infection by the trypanosomatide, leishmania (pucadyil et al., ) . this parasite can enter, survive and proliferate inside macrophages (alexander and russell, ) . leishmania donovani is responsible for the visceral leishmaniasis (parson et al., ) , which is characterized by defective cellmediated immunity (basak et al., ; saha et al., ; sen et al., ) . l. donovani lpg (lipophosphoglycan) requires intact membrane rafts to control host-cell functions. it was reported that lpg associates with membrane rafts in the host cell and exerts its actions on host-cell actin and phagosomal maturation through subversion of raft function (winberg et al., ) . macrophages infected with leishmania are unable to present, even processing-independent peptide sequences, to t-cells, and this event is not due to mhc expression (prina et al., ) . in this context, lipid rafts are also involved in the interaction between mhc and apc (poloso and roche, ) . l. donovani can affect antigen presentation of macrophages due to the increase in membrane fluidity, which leads to a lipid-raft disruption. although the number of mhc complexes in infected cell surfaces was sufficient, there was no possibility of forming an aggregate and stimulating t-cells (chakraborty et al., ) . over millions of years, hosts and pathogens coevolved to improve their mechanisms of parasite elimination and maintenance of the infection respectively. as described above, pathogens can take advantage of lipid rafts for their own benefit. in the same way, the host membrane microdomains can trigger and enhance the immune response against microorganisms. studies of lipid rafts of mammals have described the interaction between tlrs and rafts. this family of receptors, which might be activated by pamps (pathogen-associated molecular patterns), such as gram-negative bacteria lps (lipopolysaccharide), is important to pathogen recognition. however, flotillin: flotillins belong to a family of lipid-raft-associated integral membrane proteins. flotillin members are ubiquitously expressed and located to non-caveolar microdomains on the cell plasma membrane. two flotillin members have been described, flotillin- and flotillin- . stomatin: stomatin is a kda integral and lipid-raft-associated membrane protein that was first characterized in human red blood cells. stomatin might play a fundamental role in the control of the surface expression of membrane proteins. tlrs: toll-like receptors are a class of proteins that play a key role in the innate immune system. they are single membrane-spanning non-catalytic receptors that recognize structurally conserved molecules derived from microbes. pamps: pathogen-associated molecular patterns are the molecules associated with groups of pathogens, which are recognized by cells of the innate immune system. pamps are recognized by tlrs. review only a few members constitutively co-localize with membrane microdomains (triantafilou et al., ) . besides, other tlrs can migrate to this specific region after activation. in the macrophage-like cell line raw . , for example, lps stimulation induces translocation of cd , erk- (extracellular-signalregulated kinase ) and p to lipid rafts, but other proteins also involved in the lps signalling response do not migrate within these microdomains (triantafilou et al., ; olsson and sundler, ) . in addition, cuschieri et al. ( ) observed that when the human monocytic cell line thp- was stimulated with lps, there was a mobilization of tlr and hsp into the lipid raft. taken together, these examples show the importance of the aggregation of specific receptor molecules within lipid rafts facilitating the lps signalling to favour the clearance of intracellular pathogens (triantafilou et al., ) . other molecules are able to induce receptor migration into lipid rafts, for example, damps (damageassociated molecular patterns). extracellular atp is a nucleotide which works as an important damp (di virgilio, ) . p purinergic receptors, a family of nucleotide receptors, are involved with inflammatory responses (burnstock and knight, ; bours et al., ; burnstock, ) and the clearance of intracellular pathogens (coutinho-silva et al., . several motifs in p x receptors have been identified that are homologous with those known to be involved in protein-protein interactions and lps binding. it was suggested that the c-terminal region of the p x receptor may directly associate with proteins and/or lipids that are important for regulating macrophage function (denlinger et al., ) . in addition, the activation of p x receptors can induce ceramide generation and accumulation in macrophages (raymond and le strunff, ) , which is a sphingolipid implicated to be involved with the formation of larger rafts, so called signalling platforms (gulbins et al., ) . previous studies have already related that specific p x and p y receptors can also be recruited towards membrane microdomain regions (vacca et al., ; bannas et al., ; vial and evans, ; garcía-marcos et al., ; barth et al., ; norambuena et al., ) . recently, we observed that the disruption of lipid rafts can reduce p x -activated pore formation on dendritic cells and macrophages from humans and mice (f.s. vieira and r. coutinho-silva, unpublished data). thus it is possible to suggest that the integrated signals from p receptors and tlrs located on rafts might explain the synergic effects of these sensors on stimulation of the immune response (hu et al., ; perregaux et al., ; garcía-marcos et al., ) (figure ). lipid rafts as targets for chemotherapy: two sides of the coin nowadays, several groups are studying and developing new treatment strategies for less harmful chemotherapeutic agents, especially those against viral infections. one of these strategies could be to block hiv- entry and its replication using natural dietary and plant-derived compounds that target lipid rafts, principally due to its affinity for cholesterol (verma, ) . sakamoto et al. ( ) showed that a secondary fungal metabolite (na ) acts as a new anti-hcv (hepatitis c virus) replication inhibitor that targets host lipid rafts, suggesting that the inhibition of sphingolipid metabolism may provide a new therapeutic strategy for treatment of hcv infection. in addition, a novel therapeutic strategy, considering the biochemistry of raft-pathogen interaction, called glycolipidomimetics, was proposed by taïeb et al. ( ) . new concepts in the chemotherapy field interestingly reported the ability to specifically deliver therapeutic agents or drugs to selected cell types, thus minimizing systemic toxicity. this is the principal goal of nanoparticle-based drug-delivery approaches. it was reported by partlow et al. ( ) that the predominant mechanism of direct delivery of lipophilic substances to the target cell plasma membrane acts via lipid mixing and subsequent intracellular trafficking through lipid-raft-dependent processes. damps: damage-associated molecular pattern molecules can initiate and perpetuate the immune response in the non-infectious inflammatory response. they serve as a start signal. a family of cation-permeable ligand-gated ion channels that open in response to the binding of atp. p y receptors: p y receptors are a family of purinergic receptors, and are g-protein-coupled receptors stimulated by nucleotides, such as atp, adp, utp, udp and udp-glucose. (a) p x receptor (p x r) and/or tlr activation, by atp or pamps respectively, in non-raft membrane regions. (b) these receptors, when stimulated, migrate to lipid-raft domains. in addition, the p x r is involved with inflammatory immune response, as well as tlrs which are related to the initial signal to the immune response. thus it is suggested that the action of both receptors, together within lipid rafts, can lead to a more intense immune response. myd , myeloid differentiation primary response gene . initially, the special regions of plasma membrane, socalled lipid rafts, were identified by their relative resistance to detergent extraction, namely drm. however, several articles still refer to membrane microdomains as a drm; this denomination should be used carefully, considering that this separation method is not completely reliable in unveiling these structures. there is an ongoing controversy regarding the nature and function of lipid rafts, because different experimental approaches have yielded different results. meanwhile, these substantial experimental data in the literature have provided not only biochemical but also microscopical evidence for the close relationship between different pathogens and lipid rafts. another important consideration is the now widely accepted view that lipid rafts may not be pre-existing domains, but dynamic membrane regions that can fuse to each other to form larger rafts or signalling plat-forms enriched in ceramide (liu and anderson, ; holopainen et al., ; bollinger et al., ; plowman et al., ) . this fusion of small pre-rafts may be induced in different cell types upon infection by different pathogens. thus the association of a protein with lipid rafts during cell infection may be a unique event concerning this protein and rafts, which may mean that this association never occurs naturally. so upon the parasite adhesion, this protein is directed to the raft. similarly, cholesterol is known to be involved in many different cellular functions, and not only the assembly and maintenance of lipid rafts, which leads us to propose that other cellular processes, rather than the disruption or disturbance of lipid rafts, may explain how some pathogens enter and survive within their hosts. taken together, the explanations above show us that each pathogen has developed its own strategies to maintain virulence and disseminate the disease. signalling platforms: lipid rafts have been demonstrated to be aggregated in response to different stimuli. in addition, they play an important role in transmembrane signalling. at present, lipid rafts have emerged as a safe entrance door to pathogens. furthermore, host-cell raft lipids were seen to be recruited to the invasion loci which may be remodelled by parasites to help in the establishment of the infection. there are at least two major mechanisms involving the host lipid raft by which parasites gain entry to the host cytoplasm and are able to survive: (i) the avoidance of lysosomal fusion and posterior degradation; and (ii) the modulation of host-cell signalling pathways to its own benefits. each year the number of reports implicating new pathogens and their interactions with lipid rafts rapidly increases. new methods and techniques are also helping the researchers to identify, in a more precise way, the interaction of pathogens and lipid rafts. in this regard, biophysical approaches have been increasingly employed to better understand the aspects that are not yet fully elucidated regarding microdomains and pathogen association. in parallel, development of new drus targeting host lipid rafts has been extensively studied, especially for viruses. however, many questions about the immune evasion and infection control yet remain to be answered and further studies are necessary to minimize the historical controversy about the nature and cell biology of the membrane lipid rafts. this work was supported by the conselho nacional de desenvolvimento científico e tecnológico do brasil (cnpq); the fundação de amparoà pesquisa do estado do rio de janeiro-programa de apoio 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endothelial cells cholesterol depletion inhibits epidermal growth factor receptor transactivation by angiotensin ii in vascular smooth muscle cells: role of cholesterol-rich microdomains and focal adhesions in angiotensin ii signaling p x receptor localizes into lipid rafts in neuronal cells rafts can trigger contact-mediated secretion of bacterial effectors via a lipid-based mechanism extracellular enveloped vaccinia virus is resistant to complement control proteins into its envelope an efficient method for introducing defined lipids into the plasma membrane of mammalian cells nucleocapsidindependent assembly of coronavirus-like particles by coexpression of viral envelope protein genes hiv: a raft-targeting approach for prevention and therapy using plant-derived compounds disruption of lipid rafts inhibits p x receptor-mediated currents and arterial vasoconstriction measles virus assembly within membrane rafts interaction of the coronavirus infectious bronchitis virus membrane protein with β-actin and its implication in virion assembly and budding lipid raft-dependent uptake, signalling and intracellular fate of porphyromonas gingivalis in mouse macrophages legionella pneumophila is internalized by a macropinocytotic uptake pathway controlled by the dot/icm system and the mouse lgn locus macrophage plasma membrane cholesterol contributes to brucella abortus infection of mice interaction between brucella abortus and cellular prion protein in lipid raft microdomains caveolin- associates with intracellular chlamydial inclusions independently of caveolin- leishmania donovani lipophosphoglycan inhibits phagosomal maturation via action on membrane rafts host signal transduction and endocytosis of campylobacter jejuni the fine structure of the gall bladder epithelium of the mouse key: cord- -i gpxcyo authors: zhang, jianguo; chen, deyu; liang, guoxin; xu, wenrong; tao, zhimin title: biosynthetic polymalic acid as a delivery nanoplatform for translational cancer medicine date: - - journal: trends biochem sci doi: . /j.tibs. . . sha: doc_id: cord_uid: i gpxcyo poly(β-l-malic acid) (pmla) is a natural polyester produced by numerous microorganisms. regarding its biosynthetic machinery, a nonribosomal peptide synthetase (nrps) is proposed to direct polymerization of l-malic acid in vivo. chemically versatile and biologically compatible, pmla can be used as an ideal carrier for several molecules, including nucleotides, proteins, chemotherapeutic drugs, and imaging agents, and can deliver multimodal theranostics through biological barriers such as the blood–brain barrier. we focus on pmla biosynthesis in microorganisms, summarize the physicochemical and physiochemical characteristics of pmla as a naturally derived polymeric delivery platform at nanoscale, and highlight the attachment of functional groups to enhance cancer detection and treatment. high-grade production of pmla from fermentation by fungi or myxomycetes enables increasing applications of this biodegradable polymer in medical research. pmla-based nanoconjugates can successfully penetrate the blood-brain barrier in rodent models, thus delivering imaging and/or therapeutic reagents to intrabrain targets and showing great potential for treating neurological disorders in human. with unmatched compatibility and resorbability, biosynthetic pmlas are good examples of future macromolecular compounds generated by a green and sustainable approach, eventually benefiting human health. given its great sustainability because it is produced from natural sources, pmla serves as a platform for potential multimodal conjugates with unmatched bioavailability, biocompatibility, and biodegradability. for this reason, pmla has been intensively and extensively used in biomedical and medicinal research, particularly in drug delivery and in bioimaging for cancer theranostics [ , ] . following its discovery in p. cyclopium as an acidic substance containing no nitrogen (molar mass m =~ g/mol), pmla was also found in the myxomycete p. polycephalum (m = - g/mol) as an inhibitor to dna polymerases, and in the yeast-like fungi aureobasidium sp. (m = - g/mol) and a. pullulan (m = - g/mol) as an extracellular secretion in culture broth [ , ] . bacteria producing pmla have not yet been identified. a study that examined pmla bioproduction from strains of a. pullulan glossary antisense oligonucleotides (aons): synthetic, short, single-stranded nucleotides that target dna or mrna. crispr/cas : clustered regularly interspaced short palindromic repeats, dna fragments originally derived from bacteriophages, whereas crisprassociated protein (cas ) is an endonuclease that specifically recognizes and cleaves doublestranded dna at a sequence complementary to the crispr sequence. endosome escape: release of entrapped molecules by endosomes to avoid further degradation. ester bond: a chemical bond between an acid and an alcohol via the elimination of water. first-order kinetics: a reaction that proceeds at a rate proportional to the reactant concentration. glioblastoma (gbm): one of the most aggressive brain tumors; has a poor prognosis. hyperosmolarity: fluid of abnormally high osmolarity. immunogenicity: the ability of a molecule to provoke an immune response in the host. ld : lethal dose %, a dose that kills a half the group of animals studied. malyl unit: the repeating unit in polymalic acid. mitochondrial pyruvate carrier (mpc): a protein gatekeeper that governs the transport of pyruvate into mitochondria to provide oxidative fuel. molecular chaperone: a class of proteins that assist in the folding and translocation of newly synthesized polypeptides. myxomycetes: mostly known as slime molds, these are fungus-like microorganisms whose life cycle includes spore, plasmodium, and fruiting body stages. nonribosomal peptide synthetase (nrps): microbial enzymes that synthesize peptides independently of ribosomes and mrnas. nucleophilic: a substance that can provide an electron pair to generate a chemical bond. oligomers: intermediate products generated by polymer breakdown or by condensation that contain only a few repeating units. phylogenetic clade: a group of lineages that share a common ancestor in their phylogenetic tree. (a) structures of α, β, and α,β types of polymalic acid. (b) pmla hydrolases break down the first ester bond from oh-terminus while binding to the th malyl unit along the polymer chain towards the c-terminus [ ] . (c) intracellular trafficking of pmla hydrolases and their extracellular activation. in typical eukaryotes, pmla hydrolase binds to pmla and remains inactive, carrying nuclear proteins into the nucleus while pmla hydrolase remains at the surface of nuclear envelope. cargo-discharged pmla exits from the nucleus into the cytoplasm via nuclear pores; it binds to pmla hydrolases that then translocate across the cell membrane where they are phosphorylated by membrane-bound tyrosine kinases, restoring their activity to degrade pmlas extracellularly [ , ] . of a diversity of phylogenetic clades indicated high productivity but low molar mass ( - g/mol), where pmla was bound to polysaccharides of varying molar mass depending on the exact strain type [ ] . by screening various aureobasidium spp. and optimizing the culture conditions, efforts have been made to obtain efficient biosynthesis of pmla with high molar mass (up to g/mol) [ ] . using a highly productive aureobasidium sp. strain isolated from mangrove systems, purified pmla with m = g/mol was reported [ ] . by contrast, pmla produced from the plasmodial stage of p. polycephalum sustains a much elongated linear chain in its pure form, with m = - g/mol [ , ] . using d-glucose as the most efficient carbon source, and relying on nutrients available in the extracellular culture medium, independent extramitochondrial and intramitochondrial metabolic routes for generating l-malate (the immediate precursor to pmla) in vivo were unraveled [ ] ( figure ). on the one hand, in the presence of exogenous carbonates (e.g., caco or na co ), l-malate is produced via a reductive pathway in the cytoplasm; because of the high level of co in the cytoplasm, pyruvate is prioritized for carboxylation to oxaloacetate by pyruvate carboxylase, and this is further reduced by malate dehydrogenase to malate [ ] . alternatively, in a similar manner, phosphoenolpyruvic acid is directly converted by phosphoenolpyruvate carboxylase to oxaloacetate, forming malate [ ] . therefore, phosphoenolpyruvic acid and pyruvate from the glycolysis pathway are indispensable molecules for malate synthesis in vivo, whereas their carboxylases govern carbon flux for malate biosynthesis because regulated carboxylase activity influences the ultimate level of pmla bioproduction [ , ] . on the other hand, if exogenous carbonates are absent, malate formation via the oxidative pathway is mostly achieved in the mitochondrial matrix through either the tricarboxylic acid (tca) cycle or the glyoxylate bypass [ ] . glucose or another sugar is transformed into pyruvate through glycolysis, followed by import into mitochondria by mitochondrial pyruvate carrier (mpc) proteins and subsequent decarboxylation by pyruvate decarboxylase complex to produce acetyl coenzyme a (acetyl-coa), thus entering the tca cycle and being converted to four-carbon oxaloacetate and subsequently to six-carbon citrate [ , ] . the tca cycle continues as a series of biochemical transformations that take place in an enzyme-mediated cascade, generating products including cis-aconitate, isocitrate, α-ketoglutarate, succinate, fumarate, malate, and plasmodial: related to the plasmodium, a single cell that contains multiple nuclei but a single cytoplasm. polyhydroxyalkanoates (phas): a group of plastic polyesters produced by various microorganisms in nature. polyhydroxybutyrate (phb): a bacteria-generated plastic that is biodegradable and water-insoluble. protein homeostasis: biological pathways for balanced protein synthesis, modification, trafficking, and degradation in living organisms. renal tubular reabsorption: a process in which functional units within the kidney remove water and solutes from pre-urine tubular fluid, and these are reabsorbed into the bloodstream for reuse. serine protease: a proteolytic enzyme that contains a catalytic serine residue within its active site t / : half-life, the time needed to reduce the concentration of a drug administered to half its original level. tyrosine kinase: an enzyme that covalently adds a phosphate group to tyrosine residues of proteins to modify their conformation and function. pmla hydrolases (also called pmla depolymerases or polymalatases) have been purified and characterized from both eukaryotic and prokaryotic microorganisms [ , ] . in eukaryotes, particularly pmla-producing myxomycetes, soluble pmla hydrolase serves as a molecular chaperone of pmla and binds to its hydroxyl terminus at the penultimate malyl residue for catalytic cleavage, although another binding site malyl residues down the polymeric chain was also proposed to govern stability and carriage function ( figure b ) [ ] . intracellular pmla hydrolase remains inactive and forms complexes with pmla that may carry other nuclear proteins, assisting the translocation of pmla into the nucleus while leaving pmla hydrolase on the surface of nuclear envelope, possibly because of either its binding to envelope proteins and/or the outer nuclear membrane, or disruptive variation in local ionic strength ( figure c ) [ ] . after discharging its nuclear protein cargo, pmla exits through nuclear pores and to join newly synthesized pmla in the cytoplasm for the next deliveries [ ] . pmla thus shuttles between the nucleus and the cytoplasm to maintain homeostasis in different phases of the cell cycle. excess pmlas bound to hydrolases are excreted through the cell membrane, where a membrane-bound tyrosine kinase then phosphorylates pmla hydrolases and activates their hydrolytic activity, leading to degradation of concomitantly released pmlas in the culture medium (peak activity at ph - ) [ ] . however, the hydrolases may differ across different eukaryotes such as pmla-producing myxomycetes and fungi: pmla isolated from p. polycephalum had higher molar mass and polydispersity than that from a. pullulan [ ] . in prokaryotes such as bacteria that are devoid of pmla, insoluble pmla hydrolases are confined to the outer cell membrane where they hydrolyze outsourced pmla into malic acid monomers that are further taken up for cellular metabolism [ ] . unlike phb depolymerase, pmla hydrolase does not exhibit serine esterase activity in which a nucleophilic serine in the active site initiates substrate hydrolysis, and is not inhibited by serine protease inhibitors [ ] . eventually oxaloacetate again. in parallel, taking a shortcut in the tca cycle, a process known as the glyoxylate shunt, isocitrate is directly converted into glyoxylate and succinate; glyoxylate then reacts with acetyl-coa to produce malate, and succinate generates malate catalyzed by succinate dehydrogenase and fumarase [ ] . the glyoxylate pathway could thus be driven to produce malate and pmla if key enzymes (e.g., fumarase, succinate dehydrogenase) in the tca cycle are blocked [ ] , or if malate synthase is upregulated by exogenous ethanol stress [ ] . because the inner membrane of mitochondria remains a barrier to most molecules, malate and oxaloacetate from the oxidative tca cycle or glyoxylate shunt interchange with their counterparts in the cytosolic reductive pathway via the malate/aspartate shuttle, where oxaloacetate is reduced to malate both extramitohchondrially and intramitochondrially to permit transportation and avoid oxaloacetate accumulation in the cytoplasm [ ] . in cultures of p. polycephalum and a. pullulan, the addition of exogenous carbonates augments co fixation and pyruvate carboxylation into oxaloacetate by pyruvate carboxylase in the cytoplasm, abolishing the intramitochondrial pathways for l-malate production and ensuing pmla synthesis ( figure ) [ , ] . under these conditions, the figure . generic biosynthetic pathways of poly(β-l-malic acids) (pmlas) in eukaryotic cells. glucose is transported into the cytoplasm where it is metabolized by glycolysis to produce pyruvate (green). excess co activates the reductive extramitochondrial pathway to generate malate (blue). when exogenous carbonates are absent, intramitochondrial malate formation via the oxidative pathway takes place in the mitochondrial matrix through either the tricarboxylic acid (tca) cycle or the glyoxylate shunt (orange). through either pathway, malates are polymerized to form pmla in the cytoplasm (gray). a pmla synthetase has been proposed that possibly combines the activities of a malyl-amp ligase and an unknown pmla polymerase, in conjunction with an auxiliary peptide or enzyme (e.g., spherulin b in p. polycephalum) [ , ] . addition of tca cycle metabolites into the cell culture might not promote pmla production [ ] . in other words, environmental carbonate works as a switch between oxidative and reductive pathways to produce malate. furthermore, carbonate adjusts the acidity of the culture medium, noting that low ph values accelerate pmla hydrolysis. in contrast to p. polycephalum, that merely uses d-glucose as a carbon source in malate and pmla bioproduction, a. pullulan takes up a broad spectrum of saccharides (e.g., sucrose, fructose, and maltose) because it expresses a remarkable diversity of polysaccharide lyases, glycoside hydrolases, carbohydrate esterases, glycosyltransferases, and sugar transporters, enabling its efficient utilization of diverse carbohydrates to produce malate [ ] [ ] [ ] . by depleting the nitrogen supply or augmenting the carbon/nitrogen ratio in the cell medium, pmla bioproduction is significantly enhanced per unit of cell mass because nitrogen starvation upregulates the expression of key enzymes in pmla biosynthetic pathways, uncoupling cell growth from pmla production [ , ] . at the cost of atp probably derived from glycolysis, malates are polymerized to form pmla in the cytoplasm [ ] . the acellular slime mold p. polycephalum only synthesizes pmla in its multinucleated plasmodia, whereas the yeast-like fungus a. pullulan spends its entire life cycle (except hyphae form) producing pmla [ , ] . although the search for pmla synthetase is still underway, a nonribosomal peptide synthetase (nrps) machinery was proposed that first forms malyl-amp via the action of malyl-amp ligase, and this is then assembled into a polymeric chain by an unidentified polymerase [ ] . in addition, a plasmodium-specific polypeptide spherulin b (i.e., nka ) was found to assist pmla synthesis, although similar enzymes have not been identified in fungi [ ] . alternatively, in a. pullulan, it was proposed that cytosolic malate may be polymerized into pmla using malyl-coa as the precursor, followed by the action of malate-coa ligase and pmla synthetase [ ] . in one variety of a. pullulan, namely aureobasidium melanogenum, a characteristic nrsp was recently reported to be a putative pmla synthetase that contains an adenylation domain for atp binding and malyl-amp formation, an activatable thiolation domain for phosphopantetheine attachment and polymerization of malyl-amp into pmla, and a hexatransmembrane region for transport of pmla out of the cytoplasm [ ] . because the whole-genome sequences of several pmla-producing fungal strains have been determined, a conserved pmla synthetase across species is expected to be unmasked in the near future [ , ] . biosynthetic pmla in aqueous solution ( % w/v) has a ph of . but a pk a of . - . (average m = - g/mol) [ ] . at acidic ph less than the pk a (e.g., ph - ), pmla remains protonated, promoting the formation of intramolecular double hydrogen bonding between side-chain carboxylic acids and the construction of dense, inflexible, double-stranded segments [ ] . in phosphate buffer (ph . ) at °c, pmla with fully ionized carboxylic groups retains an opencoil conformation owing to the negatively charged neighboring side-chains, and undergoes hydrolysis with a half-life of h, initially following first-order kinetics, whereas elevated temperature and acidic ph dramatically accelerate its hydrolytic degradation [ , , ] . moreover, random hydrolysis prioritizes the breakdown of intrachain ester bonds over those at the ends of the molecule, producing oligomers instead of malates until hydrolysis proceeds to completion, whereas hydrophobic substitution (e.g., alkylation) of pmla side-chains delays this hydrolytic degradation, possibly because of limited water access to ester bonds in the backbone as the polymer conformation alters [ , ] . in addition, a larger substituent group or a higher degree of substitution in the sidechain that increases hydrophobicity, leads to the slower degradation in aqueous solutions [ , ] . in a pilot study, repeated intraperitoneal injection of synthetic pmla into rabbits returned no detectable immune response, demonstrating non-immunogenicity, whereas the same injection into trends in biochemical sciences mice revealed nearly no acute toxicity (ld = . g/kg body weight) [ ] . intravenous (i.v.) injection of pmla sodium salt into mouse tail vein, with repetitive administration at low dosage, showed no mortality or adverse effects at doses up to g/kg body weight; however, mice only tolerated a one-time i.v. high-dose injection (up to~ g/kg body weight or otherwise) toxicity was due to the hyperosmolarity of the concentrated polymer solution injected rather than to the polymer concentration per se [ ] . the elimination of injected pmla from blood was very fast, with a t / of min or much less, and injection might not even be complete before the polymer is exported into urine [ , ] . indeed, % of injected pmla was excreted after h, and % after h, although there was low but persistent liver accumulation h after injection, yet no substantial accumulation in other organs, including kidney, lung, intestine, spleen, heart, muscle, and brain [ , ] . notably, these early studies on the pharmacokinetics and biodistribution of synthetic pmla laid a solid foundation for recent preclinical research using natural pmla as a pharmaceutical carrier [ ] . bioproduced pmlas possess a similar or even superior biocompatibility profile to synthetic pmlas, and their end-product is only l-malic acid. intriguingly, l-malate administered i.v. into the tail vein in mice had a half-life of only min, and one third of injected dose ended up in exhaled co , whereas the majority of the remainder accumulated in tissues via renal tubular reabsorption, participating in the tca cycle [ ] . pmla is negatively charged at ph . , and hence poses no disruptive threat to phospholipid membranes because they have the same charge. given that there is no specific cell-surface receptor of pmla, water-soluble pmla is transported into the cytoplasm through the invagination of cell membrane via a process of non-specific endocytosis, although the efficiency of transmembrane transport can be very low. in contrast to pmla, its copolymers with lipophilic ligands undergo hydrophobic interactions in aqueous solution and assemble into lipophilic patches, and these can interact with the cell membrane, leading to anchoring of lipophilic patches in close proximity to lipid bilayers [ ] . depending on the attached hydrophobic ligands, three distinct mechanisms have been proposed by which different pmla-conjugated copolymers can induce membrane permeation ( figure ); these are discussed in the following text: (i) the carpet model is typified by pmla leucine ethyl ester (pmla-loet x h −x ) [ ] . at physiological ph, esterification of carboxylic acid groups in pmla side chains leads to permanent charge neutralization, excluding further protonation even when the environmental ph drops. upon binding to the cell membrane, pmla-loet x h −x orients itself to insert the hydrophobic loet side chain into the phospholipid layer, leaving the outer membrane surface expansively covered by the hydrophilic backbone of the polymeric chain. the hydrophobic binding energy is sufficient to strongly bend the plasma membrane into a curved structure, creating a transient pore that enables membrane permeation [ , ] . this process is independent of ph and its membranolytic efficiency varies according to the ratio of hydrophobic/hydrophilic moieties (box ) in the polymer (i.e., x −x ). (ii) the belt model is typified by pmla tritryptophan (pmla-www x h −x ) [ ] . tritryptophan contains three side-chain indoles and one terminal α-carboxylic acid, constituting a nonpolar hydrophobic tripeptide. at ph . , the terminal α-carboxylic acid in the side chain is deprotonated and ionized; this would be repelled from the cell membrane, but, because of strong hydrophobic interactions, indole in the side chain can attract and intercalate into phospholipids, generating pmla tritryptophan-lipid complexes and releasing binding energy to stabilize the structure. in another scenario, ph reduction from neutral to acidic may protonate (and neutralize) the end-group carboxylate in the side chain, whereas protonation of the indole moieties is constrained because this would lead to loss of aromatic stabilization. under both circumstances, the pmla backbone is sandwiched between two layers of phospholipids inserted with outflanking tryptophans, forming a 'belt-like' or 'dental brace' configuration [ ] . the ph-dependent charge neutralization of the carboxylic acid endgroups does not hamper strong hydrophobic interactions between indole and membrane lipids, thereby leading to ph-independent membrane permeation. this permeation thus resembles the 'boomerang model' that was proposed to mediate viral membrane fusion with the host cell [ ] . a highly conserved tryptophan-rich domain has been found in many human viruses, including coronavirus, influenza virus, and hiv, and has been proposed to be a key determinant of viral entry through strong interactions between the aromatic rings of the tryptophan-rich domain and lipids in the target membrane lipid, thus perturbing the lipid bilayer and mediating membrane fusion [ ] . (iii) the barrel-stave model typified by pmla trileucine (pmla-lll x h −x ) [ ] . trileucine in the side chain of pmla-lll x h −x has three hydrophobic isobutyl groups and one α-carboxylic acid end-group that is subject to ph-dependent protonation. at neutral ph, ionized pmla-lll x h −x is likely to generate a random-coil conformation, similarly to pmla, and is largely unable to penetrate the cell membrane owing to its negative charge. as the ph was decreased below , pmla trileucine was found to form aggregates via oligomerization that vertically pierce the membrane core and tentatively form a transmembrane pore to allow entry. in this manner, an increase in the fraction of hydrophobic substituents or the molar mass of the amphiphilic polymer could augment its membranolytic activity [ , ] . importantly, acidic ph-triggered membranolysis would enable selective disruption of intracellular membranes by ionizable polymeric carriers, including endosomes that are of particular interest for drug delivery (ph~ . ), thus escaping endosomal capture and lysosomal degradation, leading to release of drug payloads in the cytosol, thereby promoting intracellular drug trafficking and targeting. in addition to pmla conjugation with hydrophobic ligands through covalent coupling, methods to regulate its membrane permeability have been developed by promoting non-covalent interactions between the pendant carboxylic acids of pmla and attaching moieties to the polymer. given that protonated pmla can only form hydrogen bonds with functional groups containing electronegative atoms at acidic ph [ ] , and these are much weaker than covalent or ionic bonds, pmla indole moieties in the side chain and membrane phospholipids induces the pmla backbone to be sandwiched between two layers of phospholipids, resulting in a 'belt-like' configuration. (c) the barrel-stave model: pmla polymers first self-aggregate to form oligomers and then vertically penetrate the membrane core, generating a transmembrane pore. (d) endocytosis: cellular engulfment of pmla. once pmla comes into contact with the cell surface, the cell membrane forms vesicles that wrap around the random-coil polymers to generate early endosomes; these can mediate secretion from the cell or develop into late endosomes. in view of its hydrophilicity and negative charge at physiological ph, pmla is a polyanion that has little affinity for negatively charged lipid bilayers and does not translocate through cell membranes. to increase the interaction between the biopolymer and the plasma membrane, methylation of carboxylic acid groups with different levels of diazomethane was used to generate a pmla-me x h −x copolymer (where x is the percentage of methyl units) [ , ] . as x increases, the hydrophobicity of the molecule increases in the order pmla < pmla-me h < pmla-me h < pmla-me h < pmla-me. both pmla-me h and pmla-me were completely insoluble in water, similarly to pmla benzyl esterification. the same order was observed for hydrolysis in saline and plasma, rupture of liposome membranes, and cytotoxicity [ ] . in addition, hydrophobic amino acids or peptides can be conjugated to pmla side-chain carboxylic acids to modulate its hydrophilicity and net charge, thus tuning the interplay with cellular/subcellular membranes in a ph-responsive manner. adjacent carboxylic acid pendants on the pmla backbone are five atoms apart, equal to their distance in poly(aspartic acid) that has a similar membranolytic profile [ ] . importantly, this spacing dictates an optimized combination of physicochemical parameters for side-chain substituents, including their individual hydrophobicity, charge-neutralizing capacity, ligand length, and density, thus determining the optimal molecular geometry and charge distribution for effective membrane disruption [ , ] . complexes generated by hydrogen bonding are unsuitable for membranolytic modification or pharmaceutical loading. nonetheless, the generation of pmla ionic complexes via electrostatic interactions offers an alternative strategy for enhanced cellular uptake. at neutral ph, pmla is a polyanion in which negatively charged carboxylates can form stable complexes with positively charged compounds [ ] . depending on the stoichiometry and chemistry of the attached cations, the polyelectrolyte complexes generated can have a variety of sizes, surface charges, water solubilities, molecular structures, and morphologies, each modulating their membrane penetration. in a scenario where anionic pmla segments are preferentially situated on the surface of polyelectrolyte complexes, they are internalized via non-specific endocytosis. conversely, cationized pmla complexes translocate into cells in a similar manner to polycations, which first adsorb onto the hydrophilic outer surface of the cell membrane and then induce the formation of aqueous pores or defects in the membrane hydrophobic core, and subsequently integrate into or permeabilize the cell membrane [ ] . once transported into the cytoplasm, ph reduction or, to a lesser extent an increase in ionic strength, would accelerate hydrolytic degradation of the pmla backbone and dissociation of ionic bonds, liberating the complexed moiety for intracellular utility [ ] . a novel type of pmla-based nanoconjugate has been developed, termed polycefin [ ] . through step-by-step chemical synthesis, pmla was activated by n-hydroxysuccinimidyl ester to enable direct conjugation or further modification by linker molecules, permitting linkage with a variety of chemical and biological ligands, including polyethylene glycol (peg), monoclonal antibody against transferrin receptor (tfr), an endosome escape unit (lll or loet), and two antisense oligonucleotides (aons) [ ] , to synergistically inhibit the α and β chains of laminin- that are overexpressed by human glioblastoma (glioblastoma multiforme, gbm); a fluorescent reporter was also covalently bonded with the pmla backbone to visualize and localize the biodistribution of polycefin (figure ) [ ] . the attachment of pendant functionality groups established a new hydrophobic-hydrophilic balance (box ) in the macromolecular structure, as the sizes of pure pmla and variant polycefins were determined to be < and~ nm, respectively [ ] . these pmla-based nanoconjugates provide a multifunctional delivery system that can effectively pass through the blood-brain and blood-tumor barriers, leading to enhanced accumulation in brain tumors following i.v. injection into the mouse tail, thus allowing visualization of cancerous lesions and liberating medicinal agents to inhibit tumor angiogenesis or growth [ ] . since this invention, variant polycefin biopolymers have been made by combining them with different therapeutic antibodies [ , ] , penetrating peptides [ , ] , and fluorescent or magnetic contrast agents [ ] [ ] [ ] that are linked to the pmla chain through direct or indirect conjugation chemistry, and these have demonstrated improved targeting and accumulation in specific tumors such as breast and brain cancers. notably, by targeting and inhibiting the laminin α and β subunits, treatment of mice bearing intracranial human gbm xenografts with pmla nanoconjugates led to significantly prolonged survival and reduced tumor sizes relative to mice bearing gbms in which laminins had been knocked out using crispr/cas [ ] . the minute physical size, potential for multifunctional conjugation, and outstanding water solubility of these pmla conjugates give them enormous advantages over many other drug delivery systems in terms of finding their way through a labyrinth of cancers, highlighting their huge potential for clinical translation. microbial production of pmla currently remains a challenge because of its low yield, high cost, and the difficulty in defining the length of the biopolymer produced (see outstanding questions). novel gene-editing techniques are urgently needed that would allow efficient delivery of given the complications of scalability from bench to industrial pilot plant production, how best to scale up pmla microbial production and further functionalization for medicinal purposes? what is the best way to uncover the genes that govern pmla synthesis in relevant microorganisms such that they can be engineered in industrial bacteria or yeasts for large-scale pmla bioproduction? how can we tune the length of biosynthesized pmla? what is the precise relationship between the molar mass of pmla and its suitability as a delivery platform for cancer medicine? what are the potential applications of pmla-based biopolymers other than as delivery platforms? trends in biochemical sciences exogenous gene sequences into physarum plasmodia or filament fungal cells to upregulate pmla production. crispr/cas -mediated gene modification was recently successfully applied in a. pullulan, leading to a mutation rate nearly ten-fold higher than that obtained by traditional homologous recombination [ ] . it is likely that further genes involved in pmla biopolymerization will soon be identified, with the prospect of producing pmla in a controlled manner by using novel gene-editing tools that preselect high-yielding strains. indeed, pmla per se could be used as an effective transmembrane gene-delivery shuttle. future genetic modifications will be necessary not only to increase pmla yield but also to predetermine the molecular weight of the polymer. furthermore, suppression of polymalatase to inhibit the decomposition of pmla could also be achieved through gene editing, thus increasing control over both polymer yield and length. therefore, to harness the potential of biopolymer production, further elucidation of the genes and molecular machinery that mediate the biosynthesis of pmla and its decomposition will be essential. because pmlas represent a versatile platform for both disease diagnosis and treatment, further research is required into the pmla biosynthetic machinery, the biosafety of bioproduced pmla, and clinical translation of pmla for the detection and therapy of diseases (particularly at early stage) including cancers. there is also an urgent need to scale-up pmla production from the laboratory to the industrial pilot plant. in addition, multiple new uses of biodegradable pmla in daily life can be envisaged, and this would not only stimulate industrial interest in pmla bioproduction and increase the popularity of pmla as a natural biomaterial, but would also promote wider medical applications of these remarkable biopolymers. poly-(l)-malic acid; a new protease inhibitor from penicilliumcyclopium poly(beta-l-malic acid) (pmla) from aureobasidium spp. and its current proceedings microbial polyhydroxyalkanoates and nonnatural polyesters screening for beta-poly(l-malate) binding proteins by affinity chromatography her -positive breast cancer targeting and treatment by a peptide-conjugated mini nanodrug enhanced endocytic and ph-sensitive poly (malic acid) micelles 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of nanoparticle cellular endocytosis we thank jiangsu university for financial support. key: cord- -kxxy x authors: kumari, sudha; mg, swetha; mayor, satyajit title: endocytosis unplugged: multiple ways to enter the cell date: - - journal: cell res doi: . /cr. . sha: doc_id: cord_uid: kxxy x endocytosis occurs at the cell surface and involves internalization of the plasma membrane (pm) along with its constituent membrane proteins and lipids. endocytosis is involved in sampling of the extracellular milieu and also serves to regulate various processes initiated at the cell surface. these include nutrient uptake, signaling from cell-surface receptors, and many other processes essential for cell and tissue functioning in metazoans. it is also central to the maintenance of pm lipid and protein homeostasis. there are multiple means of internalization that operate concurrently, at the cell surface. with advancement in high-resolution visualization techniques, it is now possible to track multiple endocytic cargo at the same time, revealing a remarkable diversity of endocytic processes in a single cell. a combination of live cell imaging and efficient genetic manipulations has also aided in understanding the functional hierarchy of molecular players in these mechanisms of internalization. here we provide an account of various endocytic routes, their mechanisms of operation and occurrence across phyla. over the years, numerous modes of vesicular endocytic trafficking have been discovered that coexist in the same cell type and operate concurrently. the operation of an internalization route and its cargo specificity is determined by factors that vary in a context-dependent manner. these factors include one or more underlying principle in cargo enrichment, necessitating specific coat and coat-associated protein assembly, a scission mechanism, and a means to integrate these steps; several molecules and membrane parameters can influence and diversify an endocytic process. in this review, with the intention of summarizing present understanding of principles of endocytosis, we have surveyed different types of endocytic processes, focusing on their molecular attributes and mechanism of vesicle formation at the cell surface. the physical and chemical properties of cargo molecules usually dictate the nature of the primary vesicular structures that participate in their internalization; the physical process of membrane deformation at initial entry is likely to dictate the mechanism necessary for endocytosis. at first glance, the scale of this initial membrane invagination provides a natural means of classifying different pathways of endocytosis (figures and ) . ingestion of particles larger than nm size typically occurs via triggered processes called 'phagocytosis or macropinocytosis' (figure ) whereas cargo below this size limit is often internalized by any of the other diverse endocytic processes available at the cell surface ( figure ). these include the well-characterized receptor-mediated clathrin-dependent as well as the less understood panoply of clathrin-independent 'pinocytic' pathways. there are endocytic processes that involve the internalization of large-sized particles or a large volume fraction of the extracellular bulk phase relative to the cell volume. these are termed phagocytosis and macropinocytosis, respectively ( figure ). these processes are tightly regulated, distinct internalization events often resulting in phase-lucent structures inside cells when viewed in a light microscope [ ] . they involve longrange remodeling of membrane and the cytoskeleton lying beneath it, and have been focus of some recent re-a cup-shaped membrane distortion around the particle, culminating in a phagosome. two well-described types of phagocytic entry are: (i) ig receptor, fcr-mediated engulfment of immunoglobulin g-opsonized particles and (ii) complement receptor cr -mediated ingestion of c bi-coated particles. both the means of phagocytic entry form morphologically distinct phagocytic cups and represent different types of phagocytic mechanisms ( figure a ). thus the morphology of forming invagination and resultant compartment is a crucial parameter for classifying these processes. in fcr-mediated phagocytosis (termed 'zipper like'), bound fcr receptors elicit local signaling responses mediated by cytosolic immunoreceptor tyrosine-based activation motifs. this leads to localized actin rearrangement, membrane extension around ligand, and thereby further engagement of receptors with ligand in a protruding cup with zipper-lock arrangement. in cr -type phagocytosis (termed 'trigger-like'), on the other hand, the process is triggered by treatment with either phorbol esters or extracellular agonists [ , ] and the particle is loosely encased in a large membrane vesicle. the site of phagocytosis has discrete patch-like distribution of f-actin, and occurs by a regulated and triggered depression in the cell surface. there are instances, for example, engulfment of necrotic cells, where the morphology is in between the two [ ] . in either case formation of phagosomes is achieved by spatiotemporally controlled sequential action of various small actin-remodeling gt-pases [ ] . the two modes also differ in their requirements for rho-gtpases, in that fcr-mediated phagocytosis relies on rac and cdc activities, whereas cr mediated entry is specifically dependent on rhoa [ , ] . the host phagocytic machinery can also be manipulated by bacterial pathogens to facilitate their own internalization. here too, two general models have been described that outline bacterial entry into cells: the 'trigger' or 'zipper' models. salmonella and shigella are examples of 'triggering' bacteria; salmonella injects its effector, sopb, into cells, which activates an exchange factor for rhog, leading to actin remodeling that causes internalization of the bacterium [ ] . the 'zippering' model of entry of the bacteria is similar to fcr-mediated internalization, as it involves engagement of cell-surface receptors all around the particle being internalized. a good example of this 'zippering' kind of bacterium is listeria monocytogenes, where the bacterium interacts with host epithelial cells through its adhesins, inla and inlb, that bind host cell receptors e-cadherin and met, respectively [ ] . live cell imaging using raw . macrophages, a phagocyte model system, and forster's resonance en-ergy transfer sensor for the small, activated gtpases, adp ribosylation factors (arfs) showed that fcr-mediated phagocytosis accompanies dynamic and stepwise activation of arf and arf [ ] . this correlated with activation of cdc and rac [ ] . activity of the rho gtpases is presumably required for regulation of actin polymerization during phagocytic cup formation; simultaneous inhibition of both cdc and rac abolishes this. inhibition of these gtpases individually however only partially inhibits phagocytosis, suggesting they act in tandem during the process. these rho gtpases upon activation, recruit wasp and thereby the arp / complex for actin nucleation and polymerization [ ] . the action of gtpases is probably regulated by the composition of polyphosphoinositides (pips) of the phagosomal membrane. pips are also intimately linked to actin dynamics [ ] . during early stages of cup formation, pi , p is enriched at the inner leaflet of the cup; during phagosome closure, rapid disappearance of f-actin at the base of the cup is correlated with the reduction in local concentration of pi , p . consistent with this, overexpression of the pi-kinase (pipki), which converts pi p into pi , p or inhibition of plc and phosphatidylinositol -kinase (pi k, enzymes that produce ip , dag, and pi , , p , respectively) abrogates phagosome closure [ , ] . the shape of the phagocytic cup is usually dictated by the nature of ligand. macrophages, when subjected to an opsonized flat surface, exhibit pseudopod extension and abortive attempts to phagocytose the surface [ ] . since the phagocytic cargo is large, sometimes as large as an apoptotic cell, internalization involves vesicular addition of membrane in the vicinity of cup. although the origin of this membrane remains controversial, the delivery of new membrane is dependent on the adaptor protein complex, ap , and small gtpase, arf [ ] , suggesting a secretory origin. the composition of phagosomal membranes has been extensively characterized. a proteomics approach helped identify more than molecules present in or on latex bead-induced phagosomes in j cells [ ] . these include, lysosomal proteins, lamp , lamp , lgp , limpii; mitochondrial vdac ; several members of the ras superfamily of small gtpases, rabs ( , c, , , , and ) ; as well as proteins found in late-endosomes, alix and flotillin- . once formed, phagosomes are gradually acidified and the cargo is destined for degradation. this feature of phagosomes is sometimes manipulated by pathogens, prominently by bacteria [ ] to evade degradation and gain intracellular access. further knowledge of the molecular machinery involved in phagosome formation will increase our understanding of the process as well as how www.cell-research.com | cell research sudha kumari et al. npg it is manipulated by pathogens in a given context. macropinocytosis is another process whereby a relatively large amount of the fluid phase is engulfed with respect to the cell volume, in some instances along with particles such as bacteria. first described by lewis [ , ] as large phase-bright organelles originating from plasma membrane (pm) ruffles, macropinosomes were initially thought to occur via non-selective membrane uptake. this view has changed with increased understanding of this process. owing to its specific characteristics such as its inhibition with na + /h + exchanger inhibitor, amiloride [ ] , and dependence on growth factor (gf) receptor (gfr) signaling, macropinocytosis is now defined as a highly coordinated, triggered process with distinct molecular regulators. in a study performed using soil ameba, dictyostelium discoideum, an active phagocytic and macropinocytic organism, it was shown that during macropinosome formation, some membrane proteins were selectively depleted from the site at the pm [ ] . it is now clear that there is sorting of membrane components during the formation of macropinosomes. macropinosomes are variable in size and can range from . to µm in diameter [ ] . the size and morphology of macropinosomes are typically independent of ligand, although their formation can be stimulated by agents such as phorbol esters, some pathogens, and gfs [ , [ ] [ ] [ ] . in professional antigen-presenting cells, macropinocytosis operates constitutively in quiescent circulating cells such as immature dendritic cells and is downregulated after these cells begin to mature [ , ] , suggesting a role in immune surveillance. as noted above, intracellular pathogens also exploit macropinocytosis as a means of entry into cells. pathogens such as shigella and salmonella inject virulence factors that modulate the cytoskeleton, induce ruffling and subsequently, macropinocytosis. infection rates of bacteria such as sphingomona [ ] and viruses such as hiv type i [ ] are reduced upon inhibiting macropinocytosis using amiloride. a common feature of macropinocytosis and phagocytosis is the dependence on actin-polymerization machinery. an archetypal example of gf-induced macropinocytosis is the upregulated fluid uptake in cells stimulated with epidermal growth factor (egf) [ ] ( figure b) . activation of egfr signaling leads to an increase in general actin polymerization and cell ruffling. egf binding results in activation of rac gtpase as well as generation of the phosphoinositide, pi , p , both of which together activate wasp/scar proteins. these in turn bind to the arp / complex, hence modulating actin po-lymerization. most instances of macropinocytosis, with few exceptions (for example, bone marrow dendritic cells, where cdc is implicated [ ] ), are dependent on rac activation). another molecular regulator of macropinosome formation is pak (p -activated kinase); manipulation of pak activity correlates with macropinocytic extent in nih t cells [ ] . while pak is capable of phosphorylating and therefore activating cdc /rac , it also phosphorylates ctbp /bars proteins. ctbp /bars (c-terminal-binding protein- /brefeldin a ribosylation substrate) proteins were originally demonstrated to regulate dynamin-independent fluid uptake in a variety of cell lines, and were later reported to localize to the site of and affect macropinosome membrane closure in a phosphorylation-dependent manner [ ] . it is possible that bars proteins represent a link between dynamin-independent fluid endocytosis and macropinocytosis (see below). the nature of fission machinery involved in macropinosome scission from the pm is not yet clear. there is evidence that implicates isoforms of dynamin in specific contexts. although egf or serum-repletion-induced macropinocytosis in epithelial cell lines is insensitive to dynamin inhibition [ ] , macropinocytosis is severely impaired in pdgf-stimulated dynamin-null embryonic stem cells [ ] . in these embryonic stem cells, fluidphase tracer uptake, as an indicator of stimulated macropinocytosis, was rescued by reintroduction of dynamin- or [ ] , suggesting that dynamin is crucial for this form of macropinocytosis. however, at this stage it is not clear whether dynamin could serve as a scissoring protein for specific cases of macropinocytosis, or has other regulatory functions for endocytosis. dynamin is known to regulate cellular distribution of rac [ ] , and therefore can indirectly influence macropinocytosis by regulating the availability of rac , necessary for actin remodeling. even though phagocytosis and macropinocytosis differ in their nature of induction and detailed mechanisms, these processes share multiple operational similarities, pointing toward similar membrane remodeling pathways during internalization. both have slow kinetics, which incidentally enables the study of the formation of these vesicles in live cells. multiple studies have demonstrated localization of regulatory proteins to phagocytic cups and macropinosomes [ , ] as well as the local and distinctive membrane composition during their formation [ ] . both processes employ molecules such as the pi k and other actin polymerization effectors, and are typically initiated by phosphoinositide-dependent processes. since dependence for endosome formation on the actin machinery is also exhibited by many other endocytic pathways, the distinctive feature of phagocytosis and endocytosis at a scale smaller than nm poses specific demands on membrane machinery needed to bend membrane at this scale [ ] . the eukaryotic cell appears to have resolved this in many ways. there are several modes of endocytic processes, distinguished by specific sets of molecular regulators and functional modules that are associated with their optimal operation. molecules involved in the generation of a vesicle can be part of a module assisting coat assembly, or involved in the pinching process, or may represent specific membrane lipids and lipid-modifying proteins. here we discuss these attributes and the endocytic processes that have come to be defined by the combinations of them. a convenient way of classifying some of the modes of microscale endocytosis is by the nature of the coat proteins that are associated with a specific process (table , figure ). clathrin-mediated endocytosis initially identified in electron micrographs used to study yolk protein uptake in mosquito aedes aegypti, clathrin was one of the first endocytic coat proteins to be discovered as a major component of 'bristled vesicles' [ , ] . studies on clathrinmediated endocytosis have dominated and defined the paradigm for endocytosis. morphological studies with clathrin indicated that it is capable of coating vesicles - nm in diameter. clathrin functions as a trimer of heterodimers, each unit consisting of one heavy and one light chain forming a triskeleton [ ] . these triskelia can assemble into a lattice-like structure around the vesicles. adaptor proteins function to link up specific cargo with the clathrin coat [ ] . simply put, clathrin-pit-mediated endocytosis involves cargo recognition and coat assembly, followed by membrane invagination, and finally pinching off of the dimpled deformation. although it was believed for a long time that clathrinmediated endocytosis is a cargo-induced process, it is now known that clathrin coats can spontaneously assemble at the pm and are stabilized by interactions with cargo [ ] ; the presence of sorting motifs yxxf, dexxx-lli, fxnpxy, and polyubiquitination in transmembrane proteins that can bind to adaptor proteins often couple productive coat formation to capture of cargo [ ] . clathrin-coated vesicle (ccv ) formation and its regulation have been reported to be associated with ~ proteins, however, at this stage, with the exception of a few, their precise sequence of action during ccv formation remains unexplored. in a study carried out in live [ ] uncovered the chronology of a few effector protein dynamics during the formation of a mature coat. more of such investigations will shed light on dynamics of critical molecular events that occur during ccv formation. imaging of clathrin-coat nucleation at pm suggests that it assembles randomly, mediated by cargo protein, ap, and accessory proteins [ ] . in neurons, aps direct lattice formation to specific sites at pm via interaction with pi , p and synaptotagmin i and clathrin. this assembly can be further stabilized by epsin and ap / calm, which bind to ap , pip , and clathrin [ ] . subsequent to this, stabilization of the clathrin-coated pit (ccp) requires cargo acquisition via subunits of ap and clathrin [ , ] . this step proceeds via the recruitment of various ap -binding partners that facilitate cargo binding, optimal clathrin polymerization, and membrane bending. the process of membrane bending is brought about in part by bar (bin amphiphysin rvs) domaincontaining proteins [ ] , such as endophilin and amphiphysin, and is further facilitated by epsin and inherent curvature properties of assembled clathrin structures [ , ] . mature clathrin-coated vesicle, while still attached to the pm, contains amphiphysin and recruits the pinching module that contains the protein dynamin. dynamin eventually promotes the scission of the vesicle (see later section for details). the prevalent view of clathrin assembly is of a homogenous, symmetric polygonal lattice [ ] , the size of individual vesicle varying depending on the cell type of origin. ccps at the cell surface are of diverse composition with respect to the usage of adaptor and associated proteins [ , ] , and offer distinct microenvironments for the regulated entry of specific combinations of cargoes [ ] . interestingly, ligand concentration also affects the mode of this endocytic route: for example, egf, at low concentrations, is internalized by a clathrin-dependent endocytic route [ ] , but at higher concentrations, seems to enter cells through clathrin-independent routes with consequences for signaling via the egfr [ ] . how such fine-tuning and discrimination are regulated is a subject of current investigation [ ] . several viral pathogens such as ebola virus, sars coronavirus, and some nonenveloped mammalian reoviruses enter cells by receptor-mediated endocytosis, targeting receptors internalized by the clathrin-dependent pathway [ ] . for some viruses this route is obligatory, and for others the clathrin pathway is one of a few that support infection. studies on the entry of influenza virus in epithelial cells have indicated that a large fraction is endocytosed into ccps; viral particles are also endocytosed by non-clathrin pathway operating in parallel [ ] . viruses that take a clathrin-dependent route more often induce formation of clathrin pit at the site of binding at the pm rather than entering a preassembled clathrin structure [ ] . this suggests a flexibility in regulation of clathrin-coated vesicle formation. similarly, in case of bacterial uptake, the entry of the bacterium, l. monocytogenes, into hela cells was sensitive to depletion of clathrin heavy chain and other endocytic proteins such as dynamin, eps , but not the adaptor ap . this was unexpected because it suggests that cargo times larger than a normal endocytic cargo could use the clathrinbased machinery to enter a non-phagocytic mammalian cell, reiterating the flexibility in clathrin-mediated endocytic processes, both in terms of size and associated machinery [ ] . caveolin and the caveolar endocytic process another membrane coat at the cell surface is caveolin. caveolae were originally described as flask-shaped structures in ultrastructure of blood capillaries by palade in [ ] , but it was not until much later that the coat component, caveolin, was identified [ ] . caveolae are formed by assembly of caveolins, integral membrane proteins that bind directly to membrane cholesterol. there are three subtypes of caveolin proteins, caveolin- , caveolin- , and caveolin- , the former two being responsible for caveolae formation in non-muscle cells and latter in muscle cells [ ] . a recent study demonstrated that while morphological features of caveolae are widely conserved, the functions of individual caveolin proteins may be diverse [ ] . many mammalian cell lines such as human colorectal adenocarcinoma cells, caco [ ] , or tissues (e.g. many blood cell lineages) do not have detectable levels of caveolin, pointing toward the specialized tissuespecific function of caveolins. overexpression of caveolins in the cells lacking them induces de novo formation of caveolae [ , ] . the precise function and regulation of caveolar assembly have been a matter of intense investigation. caveolins have a predicted hydrophobic stretch of potential hairpin structure composed of α-helices. insertion of transmembrane amphipathic helices in a wedge-like manner such that inner leaflet lipids are displaced more than outer leaflet lipids could induce membrane curvature [ ] . moreover, caveolins are capable of oligomerization (caveolin- and caveolin- ) on membranes, which could further potentiate membrane curvature. consistent with this idea, the density and number of caveolae generally correlate with cellular caveolin expression [ ] . caveolae formation is cholesterol dependent and loss in membrane cholesterol leads to disassembly of the caveolar structures. recent investigations have identified additional factors for the coat component of caveolae, polymerase i and transcript release factor (ptrf), and serum deprivation protein response (sdpr) [ , ] . these molecules appear to be both part of the coat and necessary for the assembly of caveolae from distributed caveolins at the pm. loss of ptrf in mammalian cells as well as in zebrafish correlates with lack of caveolar structures [ ] ; ptrf possibly acts by modulating relative amounts of caveolae-bound caveolin to free caveolin in the pm [ ] , whereas sdpr is likely to be a protein that is required at the sites of caveolar assembly. the caveolar pathway is responsible for the endocytosis of ligands such as albumin [ ] , autocrine motility factor [ ] , tetanus toxin [ ] , cholera toxin [ ] , and www.cell-research.com | cell research sudha kumari et al. npg viruses such as polyoma and simian (sv ) [ , ] ; to name a few. in many of these cases the receptors for specific cargoes have also been identified but the mechanism by which cargo is concentrated in caveolae is not understood. in addition, endocytosis via caveolae also remains poorly characterized. it is not clear if caveolar endocytosis is constitutive, albeit occurring at a low rate, and is massively up-regulated on specific induction. fluorescence recovery after photobleaching studies has indicated caveolae to be relatively stable structures on pm [ ] , which can however be made to bud off by the interaction of pathogens such as sv virus [ ] or can also be induced by incorporation of fluorescent analogues of sphingolipids, namely, lactosyl ceramide [ ] [ ] [ ] . during integrin-dependent cell adhesion, deadherence induces removal of caveolin-coated membrane from the cell surface, and it has been shown that caveolin is required for this process [ ] . this results in altered pm lipid composition, due to the loss of many lipids and proteins that partition into detergent-resistant membrane (drm) domains. this has significant functional consequences foremost of which is loss of rac activity at the cell surface, and consequently remodeling of the cortical cytoskeleton, and activation of anoikisis, a form of apoptosis induced by the loss of cell anchorage [ , ] . caveolin knockout mice have proven to be useful genetic tools for studying the function of caveolin and ascertaining caveolar cargo specificity [ ] . in mouse embryonic fibroblasts (mefs) obtained from caveolin- knockout mice, albumin uptake was found to be defective [ ] , but the entry of sv [ ] and cholera toxin b subunit (ctxb) [ ] , two typical cargoes of caveolae, was not compromised. this could be explained by the possibility that caveolin, when present, induces formation of caveolae, but is dispensable for internalization of some caveolar cargoes. in addition, there is evidence that caveolins can negatively regulate entry of certain cellsurface proteins, including ctxb [ , ] , where overexpression of caveolins prevents their internalization, suggesting that apart from their direct role in endocytosis, caveolins can indirectly influence other endocytic pathways. a novel role for caveolin- has been reported, in regulation of cdc activity during membrane trafficking events. caveolin- binds preferentially to cdc in its gdp-bound form and functions as a guanine nucleotide dissociation inhibitor (gdi) for cdc . thus, by maintaining cdc in an inactive state, caveolin- regulates basal secretion in the absence of stimuli in pancreatic β-cells [ ] . it is possible that caveolin- has similar indirect regulatory roles at pm via inhibitory interactions with mediators of other endocytic pathways. endocytosis of sv virus has been reported to increase in cells lacking caveolin- [ ] , indicating an inhibitory role for caveolin- in this process. however in this study it was not clear whether this effect is due to squelching of effector molecules or due to sequestration of a specific kind of membrane components. studies exploring redundant endocytic roles of caveolin will be invaluable in understanding the regulatory cross-talk between caveolar and non-caveolar internalization pathways. once a coated patch of membrane has been generated, the formation of an endocytic vesicle requires fission of the budded membrane from the parent membrane. a large gtpase, dynamin, has been implicated in multiple fission processes in eukaryotic cells [ ] [ ] [ ] . there are three dynamin genes identified in mammalian cells, each gene expressing at least four multiply spliced forms (isoforms). dyn is restricted to neuronal cells; dyn is ubiquitously expressed; and dyn may be limited to brain, lung, and testis tissues [ ] . a wide variety of endocytic processes, including clathrin-dependent receptor-mediated endocytosis, require dynamin for the scission of endocytic intermediates to generate vesicles (figure a) . originally, dynamin was identified as a microtubule-binding protein and was found to be homologous to the drosophila shibire gene [ , ] , mutations in which were shown to cause a temperature-sensitive paralysis. insights into dynamin function came primarily from morphological analysis of synapses in the shibire ts mutant. cycling of mutants through restrictive and permissive temperatures, koenig and ikeda observed a reversible loss of synaptic vesicles at neuromuscular synaptic junctions, implicating dynamin in synaptic vesicle endocytosis [ ] . overexpression of dominant negative, gtp-binding mutants of dynamin also blocked receptor-mediated endocytosis in various cells, suggesting a role for the gtpase activity of dynamin in the clathrin-dependent endocytic process outside the nervous system. dynamin was found to localize to ccps at the pm and in isolated synaptosomal preparation [ , ] , providing further evidence of a direct role for dynamin in endocytosis. dynamin is likely to be recruited during clathrin-pit assembly, a process where n-bar domain-containing proteins, such as amphiphysin, which can directly interact with dynamin [ ] , are also recruited. at the molecular level, dynamins contain a pleckstrin homology (ph) domain, a gtpase effector domain (ged), and a cterminal proline/arginine-rich domain (prd). ged can interact with the gtpase domain of an adjacent dynamin molecule thus causing oligomerization of the protein [ ] , and concomitant activation of the gtpase activity. the prd of dynamin can interact with a variety of proteins, including the ones that contain src homology sh domain, and ph domain binds to pi , p phosphoinositides. wide range of interaction partners for dynamin enables it to recruit and bind proteins during coated vesicle formation. at high local concentrations, dynamin is also capable of forming multimers and spirals on membranes. dynamin oligomerization and dynamics lead to constriction and eventually budding of the vesicle. the exact function of dynamin at the level of vesicle fission has been a subject of controversy. there are various hypotheses regarding the gtp-dependent mechanism of dynamin action for vesicle pinching. first is the conformational change in dynamin brought about by gtp loading (switch model), which can cause scission by recruiting other molecules that will fuse membranes and acts essentially as a timer for this reaction, whereas the other relies on the actual mechanical force generated at the vesicle neck (pinchase model). while there is evidence for both the gtp switch and mechanoenzyme functions of dynamin [ ] , recent studies support a mechanoenzyme or motor-like function of dynamin. the in vitro demonstration of dynamin-coated lipid tubules twisting in response to supply of gtp was a direct evidence supporting a mechano-enzyme behavior where the authors argue that in addition to membrane tension, torsional strain generated at the neck of the vesicle can lead to pinching [ ] . two other studies demonstrated that it is the kinetics of dynamin depolymerization and polymerization on 'tension-free' membranes that brings about changes in membrane curvature, necessary for fission [ , ] . at this point both these possibilities have distinct implications on the dynamics of dynamin action during pinching as discussed [ ] . apart from acting as a part of the scission machinery for the clathrin-dependent pathway, dynamin function is also implicated in vesicle formation in a set of clathrinindependent pathways, including caveolar endocytosis [ ] and a class of macropinocytosis [ , ] . other clathrin-independent pathways that utilize dynamin are the rhoa-dependent il- receptor endocytic route [ ] and app endocytosis in primary neurons [ ] . although ultrastructural studies indicate that il- receptors are not recruited to ccps, they were internalized by small uniform-sized invaginations. the uniform and small size ( - nm) of the forming endocytic pit at the surface that appears to concentrate the cargo suggests the presence of a protein coat that could aid in the recruitment of these proteins to the site of endocytosis. a lot more information is necessary to uncover the mechanism of operation of this type of endocytosis. similarly, there exists an unusual mode of endocytic clearance of egfr from dorsal surface of migratory epithelial cells. here, cells on stimulation with egf generate circular wave-like ruffles that on concentric closure internalize ~ % of cell-surface egfr. this process employs dynamin but is independent of clathrin or caveolins [ ] . overall, the function of dynamin seems neither to be strictly dependent on the presence of clathrin on buds, nor is limited by the size of invaginations. as indicated above, the mechanism by which dynamin is recruited to these endocytic sites at pm is unclear, and ought to be the subject of further investigations. however, a general theme that emerges is that most coat-dependent mechanisms of endocytosis use dynamin to assist in scission of the membrane deformation from the cell surface. on the other hand there are a number of coatindependent endocytic processes that appear to function in the absence of dynamin (table ) . at this time there are a number of endocytic pathways where neither specific coat proteins nor a particular pinching system have been identified ( figure b ). in fact, the existence of a coat-independent route for endocytic uptake has taken a long time to be well accepted; careful experimentation coupled with genetic manipulations provided the necessary evidence. for example, the acceptance of clathrin-independent endocytosis was built on a number of studies: (i) injection of african green monkey kidney cells (cv ) with anti-clathrin antibodies abolished receptor-mediated uptake completely, but inhibited the uptake of a bulk phase marker (lucifer yellow) by only % [ ] , and (ii) clathrin-dependent endocytosis could also be inhibited by hypotonic shock, cytosolic potassium depletion, or acidification of cytosol while under all these conditions, endocytosis of the plant toxin ricin continued to take place [ , ] . together, these and other experiments paved the way to the idea that cells could exhibit one or more clathrin-independent endocytic routes. in some instances, ultrastructural visualization of endocytic intermediates did not reveal electron-dense coats [ ] , suggesting that either these pathways do not have a proteinaceous coat or the coat assembly, if any, is too transient to be captured by current methodologies of cell fixation and processing. it is also possible, however, that lipid or protein accretions could initiate membrane deformation (vesicle formation) without defined coat proteins. while the pathways that require coats also have their specific lipid requirements [ , ] , in some instances, lipid organization alone has proven to be sufficient for membrane invagination. shiga toxin entry is one such example where binding of toxin to the ganglioside, gb , www.cell-research.com | cell research sudha kumari et al. npg induces invaginations in cells as well as model membranes [ ] . this was specific to toxin binding, since simply cross-linking gb with antibodies failed to result in invaginations. these studies provide evidence for the requirement of a specific lipid-binding architecture for this process. a lipid-compaction model was proposed for the segregation and membrane budding induced by the toxin. this process was inhibited by increasing membrane tension, indicating that membrane properties, tension, and bending rigidity might play a crucial role in the regulation of lipid-based invaginations. endocytosis of the lipid-anchored proteins such as glycosylphosphatidylinositol-anchored proteins (gpi-aps) also does not appear to require any of the well-characterized coat proteins [ , , , ] . this pathway could be another example where the foundation for membrane deformation is laid by membrane lipids (see the clic/geec pathway below). an example where a protein platform suffices to support endocytosis is the process of human papillomavirus (hpv ) virus infection, where tetraspanin-enriched domains are required for virus entry [ ] . it was widely accepted that dynamin would be indispensable for all endocytic activities in the cell. in part this notion had been fuelled by the existence of a number of dynamin genes, and isoforms/splice variants [ , , ] . however, this notion was difficult to reconcile with a few key observations: (i) overexpression of dominantnegative mutant form of dynamin- in hela cells [ ] inhibited clathrin-dependent endocytosis but could not completely block fluid phase uptake, and (ii) hemocytes derived from the temperature-sensitive shibire mutants of drosophila exhibited a complete block in clathrindependent endocytosis of scavenger receptors, whereas fluid-phase and gpi-ap uptake remained unaffected [ ] . the dynamin gene mutation in drosophila renders all dynamin splice forms mutant since the mutation lies in the common exon encoding the gtpase domain [ ] . to assess a role for dynamin in a pathway, most studies have used dominant-negative inhibition of dynamin to test concomitant quantitative defects in endocytic cargo uptake. interpretation of such experiments is however, complex. it is difficult to ascertain whether lack of defect is due to lack of involvement of dynamin, or due to an alternative internalization pathway that is induced by inhibition of dynamin. alternatively, the ability of dynamin mutants to interfere with the activity of rac [ ] suggests that inhibition of a particular pathway may not reflect a direct role for dynamin. from the existing examples under the canopy of dynamin-independent pathways, there are also specific requirements for small gtpases such as the arf family proteins, and the rho family proteins (table ) . these gtpases could function as timers for regulation of the assembly and disassembly of actin-based endocytic mechanism or as scaffold to recruit other regulatory proteins. however, it is evident that while these gtpases can be utilized in combination, they have a diversity of functions not solely restricted to endocytosis [ , ] . there is a growing understanding of the molecular mechanism involved in the operation of different endocytic pathways and the role for the actin cytoskeleton in its regulation [ ] (see table and the following sections). this necessitates a new understanding of the role that actin and its associated molecular network may play in different aspects of the endocytic process. actinmeshwork could function as a coat and/or help in the recruitment of adaptor proteins, thus allowing for cargo concentration and membrane deformation, leading to the formation of membrane buds. in addition, coupled with motor protein activity and dynamic polymerization, it could play a role in the generation of a scission force. in this context, extensive genetic, biochemical, and morphological studies in budding yeast have provided valuable insights. in a mutant screen for endocytic defects in yeast, a large number of identified genes were related to actin and its regulators [ ] ; subsequently, the recruitment of actin to the sites of endocytosis was elegantly demonstrated in live cells [ ] . one of the first identified genes involved in α-factor uptake was act (end ), the yeast actin gene. nearly a third of endocytic proteins identified in yeast bind actin or regulate its assembly. a specific kind of actin structure, called 'cortical actin patches' has been observed to be associated with endocytic sites. using real-time fluorescence microscopy and particle tracking, drubin and colleagues [ ] demonstrated that there are different cortical actin patcheswith distinct protein composition and motility properties; and these were intermediates in the endocytic process. subsequently, using a live imaging screen of deletion mutants, where they dissected defects in coat dynamics and actin assembly, they provided evidence for the existence of distinct protein modules for coat formation, membrane invagination, actin-meshwork assembly, and vesicle scission during clathrin-or actin-mediated endocytosis [ ] . in the same study, they also demonstrated that, in yeast, while clathrin facilitates endocytic site assembly, it is not required for membrane invagination or vesicle formation. based on these studies, a fission mechanism for such a pathway has been recently proposed [ ] . this model suggests that endocytosis can be initiated at a site where the membrane invaginates, and then elongates into a tube through the encasement by ac- tive polymerization of actin, culminating in the scission of a membrane bud of different membrane and protein compositions via traction by myosin motors [ ] . considering that clathrin and its effectors also associate with these vesicles, it is not yet established if actin alone acts as the coat [ ] . in mammalian cells, specifically nih t cells, actin and cortactin are asymmetrically associated with highly dynamic early endocytic structures and move correlatively with these vesicles, supporting the notion that actin could generate the force required for vesicle movement and thus fission [ ] . disruption of the actin cytoskeleton also causes a reduction in endocytosis in a number of clathrin-independent pathways [ , ] . large vesicles formed as a result of compensatory endocytosis in xenopus oocytes also have been shown to have actin coats around them, although the precise function of actin there has not been addressed [ ] . it should be, however, noted that actin is a multifunctional structural protein and perturbation in actin and actin nanomachinery can also result in non-specific and indirect defects in endocytosis; nevertheless colocalization of f-actin with endosomes indicates a more direct role in endocytosis. in the following sections we discuss some of the pathways that utilize actin and its regulators, in further detail. the clic/geec pathway as discussed above, endocytosis of gpi-aps represents a clathrin-and dynamin-independent internalization route. gpi-aps are outer leaflet membrane proteins that lack cytosolic extensions and thus are unable to directly bind cytosolic adaptors. these proteins belong to a family of proteins with diverse functions and have been shown to be internalized by a dynamin-independent endocytic route, into a specialized early endosomal compartment, where a majority of the internalized fluid phase is also detected in many cell types [ ] . endocytic structures containing nascent endocytosed gpi-aps are named gpi-ap enriched early endosomal compartments (geecs). geecs are proposed to result from fusion of uncoated tubulovesicular clics (clathrin-independent carriers) directly derived from the cell surface [ ] . clics/geecs are selectively enriched for gpi-aps [ ] although at this stage the sorting determinants at pm are not clear. it could potentially be lipid-based sorting, as alterations in cholesterol and sphingolipid levels as well as perturbation of the nanocluster state of the gpi-ap affect endocytosis through this pathway [ , , ] . however, a recent study shows the size of extracellular moiety attached to the lipid tail as an additional determinant for inclusion into geecs [ ] . it will be interesting to investigate the size range of proteins that allows them to be endocytosed via geecs and also, how the size-based regulation crosstalks to lipidmediated sorting into the geec pathway. another feature of endocytosis via clics/geecs is its dependence on the actin polymerization machinery [ ] ; maintenance of dynamic cortical actin architecture appears to be required for geec endocytosis. this is brought about by the cycling of the rho family gtpase cdc and downstream activation of the wasp protein, since locking of cdc in gdp or the gtp state leads to inhibition of the geec pathway [ ] . cdc dynamics on pm is regulated by arf ; activation of arf leads to recruitment of a rho-gap domain-containing protein, arhgap , and inactivates cdc , thus maintaining its cycling [ ] . another recently reported effector of geec formation is a bar domain-containing protein, gtpase regulator associated with focal adhesion kinase- (graf ). endogenous graf localizes to clic/geec carriers in hela cells and silencing its expression results in reduced geec marker uptake [ ] . graf colocalizes with active cdc and contains a rho-gap domain, so it may be involved in regulating cdc dynamics apart from its other functions. graf also contains a bar domain, which could aid in membrane curvature and an sh domain, capable of interacting with dynamin gtpase. it is interesting to note that while geec formation is dynamin-independent, dynamin appears to be recruited to geecs post internalization [ ] ; this could potentially be mediated by geec-associated proteins such as graf , which has been shown to interact with dynamin. although there is increasing information about the molecular regulation of geecs, the functional relevance of this pathway remains unexplored. since a variety of gpi-aps are internalized by the geec pathway, and in some cases endocytosis via geecs specifically aids in accomplishing their cellular function [ ] , this pathway could be of vital importance for this whole family of proteins. considering its constitutive nature, fast kinetics and heterogeneous resultant vesicle size, the geec pathway could mediate the regulation of pm homeostasis. there are an increasing number of proteins internalized by pathways that bear a resemblance to the geecs. endocytosis of cholera toxin [ ] , internalization of vaca toxin, and sorting nexin (snx )-regulated gpi-ap internalization routes are examples similar to geecs [ , ] . another cargo for a geec-like pathway is dysferlin, a muscle repair protein, mutations in which are associated with several myopathies. in mefs devoid of caveolin- , dysferlin is endocytosed into compartments containing gpi-aps and its cellular entry is independent of dynamin activity, indicating that internalization could potentially be carried out via the geec pathway [ ] . arf -dependent pathway cell-surface proteins such www.cell-research.com | cell research sudha kumari et al. npg as mhc i and tac (the il- receptor α-subunit) are internalized in a dynamin (and clathrin)-independent manner into an arf -positive tubular endosomal system. these are distinct from the clathrin-dependent cargo-containing endosomes [ ] . overexpression of constitutively active form of arf was shown to increase endocytosis of tac; however, inhibition of arf via overexpression of a gtp-exchange defective form does not perturb endocytosis [ ] . there have been several additions to the cargoes and effectors of this pathway, recently reviewed [ ] . endosomes-containing cargo internalized via this pathway, remain associated with activated arf [ ] ; hyperactivation of arf traps a pip -enriched early endosomes of a vacuolated morphology, while its constitutive inactivation blocks the process of recycling back to pm from tubular compartments. recently, candidates for cargoes endocytosed via the arf -pathway were identified using hyperactivated arf -trapped endosomes [ ] . while arf associates with endosomes containing a specific set of cargoes, it is not clear whether it is involved directly at the internalization level or the recycling step or both. the localization of arf on endosomes can result both from stable association during endocytosis and acquisition on endosomal membrane post-internalization. while arf regulates dynamindependent endocytosis of the herpes simplex protein vp [ ] , it has also been demonstrated to control the recycling of membrane [ ] . it will of interest to visualize the localization of arf in the context of cargo endocytosis at pma. although the arf pathway bears a resemblance to the geec pathway, it is likely that these two processes are distinct modes of endocytic trafficking. perturbation of graf in hela cells causes inhibition of fluid uptake (a geec marker which is internalized into tubular endosomes as well), but it neither affects mhc i internalization nor does it associate with mhc i-positive structures [ ] . an important function of this pathway may be to regulate membrane availability for migratory cells, and recycle activated small gtpases to the pm [ ] . it is also responsible for recycling of a prohormone sorting receptor, carboxypeptidase e, to the golgi [ ] . role of flotillins flotillins appear to outline a dynaminand clathrin-independent endocytic process, since cargoes including fluid phase and a gpi-ap, cd , in hela cells are endocytosed via this pathway [ ] . in the cells depleted of flotillin- , there is a defect in uptake of gpi anchor protein cd , and ctxb internalization is shifted to a dynamin-dependent mode. however, the relationship of this pathway to the geec/clic pathway in other cell types is not yet established; a role for cholesterol and specific rhogtpases remains as yet unaddressed. there are two flotillin genes, flotillin- and flotillin- (identical to reggie- and , respectively) [ ] . in live cells, flotillins are localized to small puncta. flotillin- and are both required for induction of membrane invaginations in a dose-dependent manner [ ] . similar to the caveolins, flotillins are associated with drms [ ] . while flotillins were shown to form heteromeric complexes with caveolins, as demonstrated by co-immunoprecipitation experiments, it is yet unknown if this interaction is functional in the context of caveolae formation [ ] . in mefs lacking caveolin- , while majority of flask-shaped invaginations are absent, there are some structures that remain. this suggests a role for other molecules in the formation of caveolae (flask)-shaped invaginations [ ] . by em and immunogold labeling, flotillin appears to decorate small membrane invaginations distinct from clathrin-or caveolin- -labeled pits. however, in a recent study in fibroblasts lacking caveolin- , overexpression of flotillin isoforms, flotillin- and could not induce flask-shaped invaginations, suggesting that factors more than flotillins may be necessary for the generation of the flotillin-decorated invaginations [ ] . a recent study has implicated flotillin- in cxcr recruitment to drms and signaling [ ] . it is possible that flotillins generate specialized surface platforms that aid in the regulation of signaling of immunoreceptors and their endocytosis. the trigger (if any) for flotillin-mediated endocytosis has recently been shown to be initiated by fyn-mediated phosphorylation, suggesting that this endocytic process is regulated by non-receptor tyrosine kinase activation [ ] . in summary, while flotillins may define a dynamin-independent endocytic process, the studies thus far raise interesting questions regarding the function of endogenous flotillins, their means of interacting with specific cargo and the mechanism of vesicle generation. role of tetraspanins another protein platform that supports endocytosis is the family of tetraspanins. dynamin-independent endocytosis of hpv is localized to tetraspanin domains (cd , cd ) of the pm and cd knockdown in cells blocked hpv entry [ ] . this mode of endocytosis is distinct from other dynaminindependent routes in that, it is not inhibited by perturbation of cholesterol levels in the cell [ ] , nor does it rely on cellular levels of clathrin, caveolin, and flotillin. however, exact steps in this endocytic mechanism, as well as the associated molecular players, are still unclear. recently, it was demonstrated that endocytosis of a tetraspanin, cd , is dynamin-independent and occurs via a cdc -dependent geec-like pinocytic pathway [ ] . it raises an interesting possibility that while tetraspanins regulate a distinct cholesterol-independent internalization route, they may utilize a different endocytic pathway for their own cellular entry [ ] . another such example, where inhibition of dynamin and cholesterol depletion does not affect uptake is the antagonist-induced internalization of nicotinic acetylcholine receptor [ ] . binding of the antagonist α bungarotoxin causes slow internalization of nachr, which proceeds via activation of rac gtpase; inhibition of rac activity or actin polymerization impairs the process [ ] . these observations suggest that specialized protein-centric molecular platforms could assemble in the membrane to aid in this form of endocytosis. there are now numerous examples of dynamin-independent endocytic systems [ , , , ] that offer ample variety in terms of molecules employed and cargo endocytosed. further study will enrich our understanding of principles behind mechanisms of endocytosis. undoubtedly, there are a variety of endocytic pathways available at the pm. based on the environmental adaptations and molecular divergence, different kingdoms seem to have evolved specializations in endocytosis. in prokaryotes, evidence for a well-defined endocytic system is missing. a recent report using cryoelectron tomography showed various stages of the potential formation of membrane buds, including magnetosomes with inner membrane in magnetospirillum magneticum [ ] . these structures are reminiscent of endocytic membrane buds in higher organisms [ ] . in unicellular eukaryotes, some subgroups have been shown to have spatially defined distribution of endocytic proteins, and specialized structures for endocytosis. for example, in paramecium, endocytosis is confined to the cytopharynx, in trypanosoma, assembly of endocytic machinery is restricted to flagellar pocket and in euglenoids, the ingestion tubule serves as the site of endocytosis [ ] . in unicellular eukaryotes such as fungi, owing to the availability of genetic, pharmacological and biochemical tools, endocytosis (especially in the budding yeast, saccharomyces cerevisiae) has been dissected in great detail [ ] , as discussed earlier. riezman and colleagues had identified several endocytic mutants (end mutants) that were defective in internalization of the α-factor [ ] . emr and colleagues had isolated dim (defective in internalization) mutants, defective in internalizing fm dye [ ] . a quantitative genome-wide screen performed in yeast recently, examined the internalization of the vamp homologue, snc [ ] . the screen uncovered novel regulators of endocytosis. it also uncovered phenotypes for predicted endocytosis genes for which no defects had previously been reported. apart from these fairly extensive studies in s. cerevisiae, initial ultrastructural studies in neurospora and membrane dye uptake assays in pisolithus tinctorius yielded no recognizable endocytic structures [ ] . however, different species of ustilago were found to contain homologs of endocytic proteins in their genome and could internalize lucifer yellow and the membrane marker fm - , suggesting the operation of endocytic pathways. these studies were carried out in cells after removal of the cell wall, and were therefore subject to validation in hyphae with an intact cell wall [ ] . similarly in aspergillus nidulans, a cluster of vesicular structures at the tip of hyphae called spitzenkörper were identified [ ] . there seem to be differences in endocytic machinery in filamentous fungi and budding yeast. for example, internalization relies on arf activity in a. nidulans, whereas in yeast, arf is involved in polarity and bud growth but not in endocytosis [ , ] . in invertebrates, drosophila has been most extensively used to study endocytosis. a limited variety of endocytic routes are reported to operate in drosophila, typically demonstrated in cultured hemocytes, isolated wing disc, oocyte nurse cells, and cultured neurons and pericardial cells for selected cell-surface proteins, primarily signaling receptors and bulk volume components [ , , ] . recently, increased usage of cultured cell lines derived from drosophila, and advancement in techniques of genetic manipulation using dsrna, have made it feasible to study endocytosis and other membrane-trafficking processes on a genome-wide scale [ , ] . in nematodes, typified by c. elegans, a plethora of genes involved in yolk protein endocytosis and trafficking were uncovered in a genome-wide screen [ ] . the function of these genes was further validated in mammalian cells, where mutation in some of these genes replicated transport defects as were observed in c. elegans. these studies also indicated that components of the molecular machinery underlying membrane trafficking may be conserved across systems. in a study that carried out extensive phylogenetic analysis of caveolin genes in metazoan (which provided evidence of extensive gene duplication), it was observed that vertebrate caveolin- and caveolin- isoforms and an invertebrate caveolin (apis mellifera) were able to form morphologically identical caveolae in caveolin- null mouse cells, while the c. elegans caveolin could not. this indicates diversity of function in the caveolin gene family [ ] . primary and immortalized mammalian cell lines have proven to be an invaluable system to study endocytosis. while some internalization routes seem to be conserved in primary and immortalized cell lines, it is not certain whether their kinetics is conserved and if immortalization has consequences on overall endocytic activity of a cell type. genetic knockout of known endocytic proteins in mice allows for the derivation of primary fibroblasts that are now routinely used to dissect the process [ ] . these fibroblasts serve as excellent tools to study the involvement of candidate proteins in endocytosis -this approach not only avoids complexities associated with overexpression of their dominant-negative isoforms and off-target effects, but also can reflect on the functional redundancy of a given protein in the system. in plants, taking into account the cell wall as a physical barrier to pm accessibility, and the high intracellular vacuolar pressure that builds up turgor pressure, the very existence of endocytosis was debated for a long time. in a study using electron microscopy, pm-associated invaginations containing electron-dense tracer and clathrin coats were visualized [ ] . more recently, uptake of fm dyes was demonstrated in plant cells [ ] , following which many studies reported endocytosis of membrane markers and specific proteins in plant root hair cells, pollen tube, and cultured tobacco cells [ ] . now there is accumulating evidence about the diversity of endosomal pathways and endocytic compartments in plants. although most of the studies used fm dyes to monitor internalization in plants, advancements in probing techniques are revealing a diversity of endocytic routes in this kingdom. apart from clathrin-dependent receptor-mediated endocytosis, actin-sensitive fluidphase endocytosis in inner cortex cells of maize root [ ] and pi k-dependent bulk phase sucrose endocytosis in suspension culture cells of sycamore have been reported [ ] . blockade of clathrin-mediated endocytosis using ikarugamycin (ika) did not inhibit uptake of positively charged nanogold particles, suggesting the operation of a clathrin-independent pathway [ ] . however, in another study, dhonukshe et al., showed that total membrane uptake in by- tobacco cells could be inhibited by inhibiting clathrin using clathrin hub dominant negative form, suggesting that clathrin is absolutely required for any form of endocytosis [ ] . it is possible that a diversity of endocytic portals are conserved in plant and animal systems, and several modes of endocytosis operate in plant cells in a cell type-and tissue-specific manner. given the diversity and complexity of endocytic routes available in different cellular context, we provide here only a simplified overview of these pathways. detailed understanding of the functioning of an individual pathway is complex in terms of its associated molecules and mechanism, and will require an individual focus. it is likely that there are many different biochemical and physical principles behind the formation of vesicles in cell membrane. for example, cell-surface proteins such as the gpi-aps, which lack cytosolic extensions, cannot directly associate with cytosolic coat and adaptor proteins. these proteins therefore need to utilize radically different principles for sorting, membrane deformation, and vesicle generation at pm. similarly, much less is known about endocytosis of membrane lipids. dissection of the endocytic process of lipids will give insights into a novel way of vesicle formation. also considering the multiplicity of functions of proteins, it will be interesting to investigate the overlapping functions of molecules known to be associated with specific trafficking pathways. the identification of an endocytic pathway as distinct has been primarily based on associated cargo proteins or lipids, and molecular regulators; the contribution of kinetics and detailed physical mechanism to such categorization is not generally available except in some wellcharacterized situations, namely clathrin-pit endocytosis or endocytosis by actin-dependent forces in yeast. considering the limited knowledge and vast diversity in molecular players that are employed for efficient functioning of endocytic trafficking pathways, it is difficult to clearly demarcate these mechanisms from one another. on a cautionary note, it should be recognized that numerous cell lines have been utilized to study different types of endocytosis. these studies have helped uncover complex endocytic networks in a given cell system. the extent to which observations in one cell system may be extrapolated to another, or whether basic components of specific endocytic pathway are conserved across cell lines, is not completely clear. the possibility that modes of immortalization of cell lines itself could have consequences on how the endocytic pathways are configured in these cells has not been addressed. extensive work has been carried out to understand and elucidate the mechanism of endocytosis in some eukaryotes, and a considerable amount of information is available in some free-living eukaryotes and pathogens [ ] . the integration of known information from complex endocytic systems in metazoan and comparison with uptake mechanisms in other eukaryotes could provide a means of understanding the scope of evolution, conservation, and redundancy in the internalization process across phyla. furthermore, whether specific endocytic modes are conserved in their entirety across phyla is also a subject of lively debate [ ] . what is clear is that cells have many ways to endocytose material from the extracellular milieu, and perhaps as many reasons to do it by a particular mode. until we understand the basic principles behind cargo concentration, membrane deformation, and scission in each of these modes it will be difficult to provide any rationale for the proliferation of the mechanisms of entry. regardless, the endocytic process, in addition to its role in uptake of nutrients and fluid, influences diverse key processes in metazoans, such as establishment of cellular asymmetry and modulation of signaling. in addition to its physiological roles, the endocytic process is also exploited by various pathogens. insights into the underlying mechanisms will reveal new targets for drug design, and selective employment of these entry routes for drug delivery will also allow for the manipulation of cellular endocytic pathways to alleviate effects of specific diseases. shaping cups into phagosomes and macropinosomes molecular mechanisms of phagocytic uptake in mammalian cells phagosome maturation: going through the acid test fusion, fission, and secretion during phagocytosis the phagosome: compartment with a license to kill defining macropinocytosis macropinocytosis: regulated coordination of endocytic and exocytic membrane traffic events sdpr induces membrane curvature and functions in the formation of caveolae mechanisms of endocytosis studies of the macrophage complement receptor. alteration of receptor function upon macrophage activation phorbol esters cause sequential activation and deactivation of complement receptors on polymorphonuclear leukocytes identification of two distinct mechanisms of phagocytosis controlled by different rho gtpases molecular definition of distinct cytoskeletal structures involved in complement-and fc receptor-mediated phagocytosis in macrophages differential activation and function of rho gtpases during salmonella-host cell interactions subversion of cellular functions by listeria monocytogenes a phosphatidylinositol- -kinase-dependent signal transition regulates arf and arf during fcgamma receptor-mediated phagocytosis cdc , rac , and rac display distinct patterns of activation during phagocytosis cdc regulates fc{gamma} receptor-mediated phagocytosis through the activation and phosphorylation of wasp and n-wasp phosphatidylinositol- , -bisphosphate: actin dynamics and the regulation of atp-dependent and -independent secretion phosphatidylinositol- , -bisphosphate hydrolysis directs actin remodeling during phagocytosis a requirement for phosphatidylinositol -kinase in pseudopod extension ap- and arf control endosomal dynamics at sites of fcr mediated phagocytosis the phagosome proteome: insight into phagosome functions distinct endocytotic pathways in epidermal growth factor-stimulated human carcinoma a cells selective membrane exclusion in phagocytic and macropinocytic cups phorbol esters stimulate macropinocytosis and solute flow through macrophages diacylglycerols and pma are particularly effective stimulators of fluid pinocytosis in human neutrophils virus entry by macropinocytosis dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class ii compartment: downregulation by cytokines and bacterial products a sphingomonas bacterium interacting with epithelial cells human immunodeficiency virus type enters brain microvascular endothelia by www.cell-research.com | cell research sudha kumari et al. npg macropinocytosis dependent on lipid rafts and the mitogenactivated protein kinase signaling pathway the coated pit and macropinocytic pathways serve distinct endosome populations developmental control of endocytosis in dendritic cells by cdc regulation of macropinocytosis by p -activated kinase- the closure of pak -dependent macropinosomes requires the phosphorylation of ctbp /bars dynamin mediates fluid-phase micropinocytosis in epithelial cells isoform and splice-variant specific functions of dynamin- revealed by analysis of conditional knock-out cells modulation of rac localization and function by dynamin membrane phosphatidylserine regulates surface charge and protein localization chirality-induced budding: a raft-mediated mechanism for endocytosis and morphology of caveolae? clathrin: a unique protein associated with intracellular transfer of membrane by coated vesicles yolk protein uptake in the oocyte of the mosquito aedes aegypti the structural era of endocytosis endocytosis by random initiation and stabilization of clathrin-coated pits endocytosis: clathrin-mediated membrane budding coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells structural insights into the clathrin coat adaptors for clathrin-mediated traffic the bar-domain family of proteins: a case of bending and binding? direct observation of bin/amphiphysin/ rvs (bar) domain-induced membrane curvature by means of molecular dynamics simulations the 'vesicle in a basket'. a morphological study of the coated vesicle isolated from the nerve endings of the guinea pig brain, with special reference to the mechanism of membrane movements cargo regulates clathrincoated pit dynamics clathrin-dependent mechanisms of g protein-coupled receptor endocytosis clathrin-coated pits: vive la difference? epidermal growth factor clathrin-independent endocytosis of ubiquitinated cargos virus entry: open sesame assembly of endocytic machinery around individual influenza viruses during viral entry listeria hijacks the clathrin-dependent endocytic machinery to invade mammalian cells fine structure of blood capillaries caveolin, a protein component of caveolae membrane coats cholesterol-sensitive cdc activation regulates actin polymerization for endocytosis via the geec pathway ptrf-cavin, a conserved cytoplasmic protein required for caveola formation and function detergent-resistant membrane microdomains from caco- cells do not contain caveolin expression of caveolin- and polarized formation of invaginated caveolae in caco- and mdck ii cells de novo formation of caveolae in lymphocytes by expression of vip -caveolin membrane curvature and mechanisms of dynamic cell membrane remodelling arf -independent gpi-anchored protein-enriched early endosomal compartments fuse with sorting endosomes via a rab / phosphatidylinositol- '-kinase-dependent machinery ptrf triggers a cave in filipin-sensitive caveolae-mediated transport in endothelium: reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules localization of autocrine motility factor receptor to caveolae and clathrin-independent internalization of its ligand to smooth endoplasmic reticulum lipid rafts act as specialized domains for tetanus toxin binding and internalization into neurons cholera toxin is found in detergent-insoluble rafts/domains at the cell surface of hippocampal neurons but is internalized via a raft-independent mechanism bound simian virus translocates to caveolin-enriched membrane domains, and its entry is inhibited by drugs that selectively disrupt caveolae endocytosis via caveolae caveolae are highly immobile plasma membrane microdomains, which are not involved in constitutive endocytic trafficking caveolar endocytosis of simian virus reveals a new two-step vesicular-transport pathway to the er selective caveolin- -dependent endocytosis of glycosphingolipids the glycosphingolipid, lactosylceramide, regulates beta -integrin clustering and endocytosis selective stimulation of caveolar endocytosis by glycosphingolipids and cholesterol phospho-caveolin- mediates integrin-regulated membrane domain internalization integrins regulate rac targeting by internalization of membrane domains genome-wide rnai screen for host factors required for intracellular bacterial infection caveolin- null mice are viable but show evidence of hyperproliferative and vascular abnormalities clathrin-and caveolin- -independent endocytosis: entry of simian virus into cells devoid of caveolae ultrastructural identification of uncoated caveolin-independent early endocytic vehicles caveolin regulates endocytosis of the muscle repair protein, dysferlin caveolin- regulation of dynamin-dependent, raft-mediated endocytosis of cholera toxin b-subunit occurs independently of caveolae caveolin- functions as a novel cdc guanine nucleotide dissociation inhibitor in pancreatic beta-cells disappearance and reformation of synaptic vesicle membrane upon transmitter release observed under reversible blockage of membrane retrieval reversible blockage of membrane retrieval and endocytosis in the garland cell of the temperature-sensitive mutant of drosophila melanogaster, shibire ts mutations in human dynamin block an intermediate stage in coated vesicle formation differential distribution of dynamin isoforms in mammalian cells identification of dynamin, a novel mechanochemical enzyme that mediates interactions between microtubules multiple forms of dynamin are encoded by shibire, a drosophila gene involved in endocytosis induction of mutant dynamin specifically blocks endocytic coated vesicle formation tubular membrane invaginations coated by dynamin rings are induced by gtp-gamma s in nerve terminals tandem arrangement of the clathrin and ap- binding domains in amphiphysin and disruption of clathrin coat function by amphiphysin fragments comprising these sites regulatory mechanisms of dynamin-dependent endocytosis dynamin: switch or pinchase? gtp-dependent twisting of dynamin implicates constriction and tension in membrane fission real-time visualization of dynamincatalyzed membrane fission and vesicle release gtpase cycle of dynamin is coupled to membrane squeeze and release, leading to spontaneous fission the long and short of membrane fission dynaminmediated internalization of caveolae negative guidance factorinduced macropinocytosis in the growth cone plays a critical role in repulsive axon turning interleukin receptors and detergent-resistant membrane domains define a clathrin-independent endocytic pathway internalization of beta-amyloid peptide by primary neurons in the absence of apolipoprotein e a novel endocytic mechanism of epidermal growth factor receptor sequestration and internalization inhibition of endocytosis by anti-clathrin antibodies inhibition of coated pit formation in hep cells blocks the cytotoxicity of diphtheria toxin but not that of ricin toxin regulated portals of entry into the cell distinct mechanisms of clathrin-independent endocytosis have unique sphingolipid requirements loss of endocytic clathrin-coated pits upon acute depletion of phosphatidylinositol , -bisphosphate shiga toxin induces tubular membrane invaginations for its uptake into cells folate receptor endocytosis and trafficking gpi-anchored proteins are delivered to recycling endosomes via a distinct cdc -regulated, clathrin-independent pinocytic pathway clathrin-and caveolin-independent entry of human papillomavirus type -involvement of tetraspanin-enriched microdomains (tems) analysis of dynamin isoforms in mammalian brain: dynamin- expression is spatially and temporally regulated during postnatal development three dynamin-encoding genes are differentially expressed in developing rat brain shibire mutations reveal distinct dynamin-independent and -dependent endocytic pathways in primary cultures of drosophila hemocytes probable mechanisms underlying interallelic complementation and temperature-sensitivity of mutations at the shibire locus of drosophila melanogaster clathrin-independent endocytosis used by the il- receptor is regulated by rac , pak and pak actin and endocytosis: mechanisms and phylogeny end , end , and end : mutations that cause actin delocalization and block the internalization step of endocytosis in saccharomyces cerevisiae the budding yeast endocytic pathway a pathway for association of receptors, adaptors, and actin during endocytic internalization a modular design for the clathrin-and actin-mediated endocytosis machinery endocytic vesicle scission by lipid phase boundary forces role of type i myosins in receptor-mediated endocytosis in yeast actin in the endocytic pathway: from yeast to mammals association of cortactin with dynamic actin in lamellipodia and on endosomal vesicles nicotinic acetylcholine receptor is internalized via a rac-dependent, dynaminindependent endocytic pathway cdc -dependent actin polymerization during compensatory endocytosis in xenopus eggs nanoscale organization of multiple gpi-anchored proteins in living cell membranes steric and not structure-specific factors dictate the endocytic mechanism of glycosylphosphatidylinositol-anchored proteins the gtpase-activating protein graf regulates the clic/geec endocytic pathway folate receptors targeted to clathrin-coated pits cannot regulate vitamin uptake helicobacter pylori vaca cytotoxin: a probe for a clathrinindependent and cdc -dependent pinocytic pathway routed to late endosomes snx couples actin assembly to phosphoinositide signals and is required for membrane remodeling during endocytosis convergence of nonclathrin-and clathrin-derived endosomes involves arf inactivation and changes in phosphoinositides clathrin-independent endocytosis: a unique platform for cell signaling and pm remodeling adp-ribosylation factor regulates a novel plasma membrane recycling pathway discovery of new cargo proteins that enter cells through clathrin-independent endocytosis cellular internalization of green fluorescent protein fused with herpes simplex virus protein vp via a lipid raft-mediated endocytic pathway independent of caveolae and rho family gtpases but dependent on dynamin and arf arf and microtubules in adhesion-dependent trafficking of lipid rafts activation of arf by arno stimulates epithelial cell migration through downstream activation of both rac and phospholipase d recycling of raft-associated prohormone sorting receptor carboxypeptidase e requires interaction with arf flotillin- defines a clathrin-independent endocytic pathway in mammalian cells dissecting the molecular function of reggie/flotillin proteins coassembly of flotillins induces formation of membrane microdomains, membrane curvature, and vesicle budding flotillins/cavatellins are differentially expressed in cells and tissues and form a hetero-oligomeric complex with caveolins in vivo. characterization and epitope-mapping of a novel flotillin- monoclonal antibody probe molecular mechanisms of clathrinindependent endocytosis evolutionary analysis and molecular dissection of caveola biogenesis cxcl -induced partitioning of flotillin- with lipid rafts plays a role in cxcr function endocytosis of flotillin- and flotillin- is regulated by fyn kinase cd endocytosis and cholesterol-dependent reorganization of tetraspanin webs and lipid rafts cholesterol depletion activates rapid internalization of submicron-sized acetylcholine receptor domains at the cell membrane pathways of clathrin-independent endocytosis clathrin-independent endocytosis: from nonexisting to an extreme degree of complexity magnetosomes are cell membrane invaginations organized by the actin-like protein mamk reconstructing the evolution of the endocytic system: insights from genomics and molecular cell biology protein and lipid requirements for endocytosis a novel fluorescence-activated cell sorter-based screen for yeast endocytosis mutants identifies a yeast homologue of mammalian eps regulators of yeast endocytosis identified by systematic quantitative analysis endocytosis in the plant-pathogenic fungus ustilago maydis kinesin from the plant pathogenic fungus ustilago maydis is involved in vacuole formation and cytoplasmic migration aspergillus nidulans arfb plays a role in endocytosis and polarized growth role for arf p in development of polarity, but not endocytosis, in saccharomyces cerevisiae endocytosis, endosome trafficking, and the regulation of drosophila development macromolecular uptake in drosophila pericardial cells requires rudhira function phagocytosis of candida albicans by rnai-treated drosophila s cells genome-wide analysis identifies a general requirement for polarity proteins in endocytic traffic receptor-mediated endocytosis in plants is energetically possible uptake of a fluorescent marker in plant cells is sensitive to brefeldin a and wortmannin molecular dissection of endosomal compartments in plants actin-dependent fluid-phase endocytosis in inner cortex cells of maize root apices sucrose-inducible endocytosis as a mechanism for nutrient uptake in heterotrophic plant cells distinct endocytic pathways identified in tobacco pollen tubes using charged nanogold clathrin-mediated constitutive endocytosis of pin auxin efflux carriers in arabidopsis phylogeny of endocytic components yields insight into the process of nonendosymbiotic organelle evolution key: cord- -pw qx c authors: armstrong, john; niemann, heiner; smeekens, sjef; rottier, peter; warren, graham title: sequence and topology of a model intracellular membrane protein, e glycoprotein, from a coronavirus date: journal: nature doi: . / a sha: doc_id: cord_uid: pw qx c in the eukaryotic cell, both secreted and plasma membrane proteins are synthesized at the endoplasmic reticulum, then transported, via the golgi complex, to the cell surface( – ). each of the compartments of this transport pathway carries out particular metabolic functions( – ), and therefore presumably contains a distinct complement of membrane proteins. thus, mechanisms must exist for localizing such proteins to their respective destinations. however, a major obstacle to the study of such mechanisms is that the isolation and detailed analysis of such internal membrane proteins pose formidable technical problems. we have therefore used the e glycoprotein from coronavirus mhv-a as a viral model for this class of protein. here we present the primary structure of the protein, determined by analysis of cdna clones prepared from viral mrna. in combination with a previous study of its assembly into the endoplasmic reticulum membrane( ), the sequence reveals several unusual features of the protein which may be related to its intracellular localization. in the eukaryotic cell, both secreted and plasma membrane proteins are synthesized at the endoplasmic reticulum, then transported, via the golgi complex, to the ceu surface - . each of the compartments of this transport pathway carries out particular metabofic functions - , and therefore presumably contains a distinct complement of membrane proteins. thus, mechanisms must exist for locafizing such proteins to their respective destinations. however, a major obstacle to the study of such mechanisms is that the isolation and detailed analysis of such internal membrane proteins pose formidable technical problems. we have therefore used the el glycoprotein from coronavirus mhv • a as a viral model for this class of protein. here we present the primary structure of the protein, determined by analysis of edna clones prepared from viral mrna. in combination with a previous stu'!} of its assembly into the endoplasmic reticulum membrane , the sequence reveals several unusual features of the protein which may be related to its intracellular localization. the coronaviruses are a diverse class of enveloped rna viruses of considerable medical and agricultural significance; they also provide a model for the study of persistent viral infections (see ref. for review). in contrast to many enveloped viruses, the corona virus mouse hepatitis virus (mhv) a buds inside the cell, into the lumen of the endoplasmic reticulum the assembled virion then appears to travel, via the golgi complex, to the cell surface. of the two viral membrane proteins, the smaller one, el, is necessary for formation of the envelope, and is restricted to internal cell membranes; apparent?; it only reaches the cell surface as part of the budded virion • . thus, the el glycoprotein is potentially a convenient model for studying those features of a membrane protein that determine its arrest at a particular destination on the membrane transport pathway. the mrnas of mhv-a form a 'nested set': the seven rnas share the ' region of the positive-stranded genome, but extend to different lengths towards the ' end - • from each rna, only the ' gene is translated ' . in addition, a noncoding 'leader' sequence of approximately bases, from the ' end of the genome, is common to the mrnas • • . the e gene is second from the ' end and is therefore translated from the second smallest mrna, rna (refs , ) . the sequence of the ' -terminal gene, encoding the viral nucleocapsid protein, has been determined previously • • copy dna clones spanning the e gene were prepared by two methods - and sequenced in the vectors m mp (ref. two versions were found, in two different clones, for the sequence immediately upstream from the e initiator codon. the shorter one is shown in fig. ; in the second clone, an additional copy of the pentanucleotide a tct a was found between nucleotides and , making the sequence similar to that of the region adjacent to the nucleocapsid gene of another strain of mhv . this difference could represent a mutation; alternatively, it may reflect heterogeneity in the normal mrna population. indirect support for the latter possibility comes from the observation that a rnase-t oligonucleotide from this region of rna , corresponding to the shorter sequence, was recovered in markedly lower yield than those from the rest of the molecule • this site represents the point of fusion between the ' leader sequence and the coding portion of the rna. the fusion is thought to occur by 'jumping' of the viral rna polymerase to particular sites on its genome-length, negativestranded template; the resumption of transcription then produces each of the subgenomic mrnas " ' • thus, it seems possible that the polymerase may jump to more than one point on the template for each mrna, generating variable numbers of the repeated pentanucleotide aucua in the resulting transcript. figure shows the amino acid sequence encoded by the e gene. the predicted molecular weight of the protein is , , slightly higher than that observed by gel electrophoresis • but consistent with the unusual electrophoretic behaviour of this , and other, hydrophobic proteins. several features of the protein, when assembled into membranes in the virus , or in vitro , are reflected in the sequence. first, in contrast to the majority of membrane proteins, el is known to lack a cleaved 'signal peptide' : the n-terminal region of the sequence contains no good candidate for a cleava e site • second, the n-terminal region bears -linked sugars ' , which, uniquely among viral proteins so far studied, are the only known post-translational modification to el. assuming that the terminal met is removed , then-terminal sequence is ser-ser-thr-thr, which is identical to the -glycosylated amino terminus of m-type glycophorin a (ref. ). the -linked sugars of el are them- selves identical to those found in glycophorin . third, most of the protein is resistant to proteolysis when assembled in the membrane. only . kilodaltons of polypeptide from the nterminus are cleavable on the luminal side of the membrane (or outside the virion) and . kilodaltons from the c-terminus from the cytoplasmic (or intra-virion) side , suggesting that the protein is largely buried in the membrane. in the sequence, a run of uncharged residues from positions to represents a potential membrane-spanning region; residues - correspond to the portion removable by protease. a further sequence of uncharged residues, positions - , is sufficiently long to cross the membrane twice more. if this region is divided in two, and each half plotted as an a-helical 'wheel', all the polar side chains of both sections cluster within °. thus, a plausible conformation for this region is two hairpinned helices in the membrane, with adjacent polar faces (fig. a) . there are no other long hydrophobic sequences, implying that the region from residues to - is either folded in the membrane to neutralize charges, or, more likely, is adjacent to the membrane but resistant to proteolysis. these features are summarized in fig. b . which, if any, of these various features might be responsible for the protein's intracellular localization? we do not know, for example, whether the protein has an active 'signal' causing its arrest on the transport pathway, or, alternatively, if it lacks a signal for onward transport; nor do we know whether a sorting process might operate on one or the other side of the membrane. the availability of a edna clone for the protein presents the opportunity to investigate these questions by allowing expression of the cloned dna and in vitro mutagenesis. this approach has already been applied to two other viral glycoproteins, to investigate the importance of their cytoplasmic domains for transport to the cell surface, yielding opposite conclusions • • an intrinsic problem with the method, however, is the difficulty of distinguishing specific effects due to alterations at the site of mutagenesis, from a general structural disruption of the molecule. in this respect the e protein may be advantageous in that it provides the possibility of creating a more 'active' phenotype in the mutated molecule: specifically, particular alterations to the protein may result in its transport to the cell surface. we thank willy spaan for communicating results before publication, g. heisterberg-moutsis (g. b. f. braunschweig) for help with oligonucleotide synthesis, ben van der zeijst for discussion and annie steiner for preparing the manuscript. j.a. was supported by fellowships from the royal society and the outside (lumenl fig. a, distribution of polar side chains in the hydrophobic regions of the el sequence. residues ( ), - ( ) and - ( ) are plotted as a-helices and viewed endon. polar side chains are boxed: proposed hydrophilic faces of helices and are indicated. b, possible topologies of the el protein across the membrane. arrows indicate sites accessible to protease; broken arrows represent inefficient pro-teolysis . european molecular biology organization, h.n. by the deutsche forschungsgemeinschaft, sfb (virologie), teilprojekt b , and p.r. by a short-term embo fellowship. some of these results have been presented in preliminary form elsewhere • • proc. natn. acad. sc< u.s.a molecular biology and pathogenesis of coronaviruses molecular biology and pathogenesis of coronaviruses key: cord- -uvx si authors: chen, yaping; wang, ji; li, xiangling; hu, ning; voelcker, nicolas h.; xie, xi; elnathan, roey title: emerging roles of d vertical nanostructures in orchestrating immune cell functions date: - - journal: adv mater doi: . /adma. sha: doc_id: cord_uid: uvx si engineered nano–bio cellular interfaces driven by d vertical nanostructures ( d‐vns) are set to prompt radical progress in modulating cellular processes at the nanoscale. here, tuneable cell–vns interfacial interactions are probed and assessed, highlighting the use of d‐vns in immunomodulation, and intracellular delivery into immune cells—both crucial in fundamental and translational biomedical research. with programmable topography and adaptable surface functionalization, d‐vns provide unique biophysical and biochemical cues to orchestrate innate and adaptive immunity, both ex vivo and in vivo. the intimate nanoscale cell–vns interface leads to membrane penetration and cellular deformation, facilitating efficient intracellular delivery of diverse bioactive cargoes into hard‐to‐transfect immune cells. the unsettled interfacial mechanisms reported to be involved in vns‐mediated intracellular delivery are discussed. by identifying up‐to‐date progress and fundamental challenges of current d‐vns technology in immune‐cell manipulation, it is hoped that this report gives timely insights for further advances in developing d‐vns as a safe, universal, and highly scalable platform for cell engineering and enrichment in advanced cancer immunotherapy such as chimeric antigen receptor‐t therapy. advances in bionanotechnology have fostered significant improvements in biomedical engineering and preclinical studies. [ ] impressive progress in the nascent field of programmable nano-bio interfaces-especially in the design of sensitive, and rapid analysis of biological species and manipulation of cellular activities. for instance, silicon nanowires (sinws) with nanoscale diameter ( - nm) and high aspect ratio (< ) enable direct access into intracellular compartments of living cells, which can greatly facilitate the study of complex regulatory and signaling pathways at cellular and subcellular levels. [ ] a wide variety of mammalian cells (adherent and nonadherent, cell lines and primary cells) have been investigated to probe biosystems using d-vns platforms. significant work has recently been done on immune cells-the central players in disease fighting that hold great clinical potential. early attempts to modulate nanomaterial-immune system interactions by tuning the topographical characteristics and/ or surface chemical properties of nanomaterials focused on macrophages, an innate immune cell type involved in nonspecific host defense against pathogen infections. [ ] [ ] [ ] research interest has since widened to embrace different cell types from the vast immunity family, covering both innate immune cells such as monocyte, dendritic cell (dc), nature killer (nk), neutrophil, and adaptive immune cells such as t and b lymphocytes ( figure b ). [ ] [ ] [ ] [ ] the major advantage of d-vns materials arises from their nanoscale dimensions: enhanced interaction with immune cells, which allows bidirectional information flow to be regulated by biophysical and biochemical signals traveling both into and out of immune cells. vns-induced membrane deformation and rearrangement of local cellular components, such as actin networks and membrane-associated proteins, may lead to a cascade of immune responses. vns-displaying platforms, including planar substrates [ , , , ] and d microparticles, [ ] have demonstrated the ability to modulate in vitro and in vivo immune responses. provided with localized proengraftment cues, nanopatterned bulk metallic glasses triggered the release of inflammatory factors; [ ] micro-and nanopatterned topographical cues modulated macrophage cell shape and phenotype; [ ] pdms nanopillars amplified cytotoxic cd + t lymphocyte (ctl) response by stimulating the formation of actin-rich protrusions; [ ] and nanospike-decorated tio microparticles activated dcs by upregulating expression of costimulatory molecules like cd . [ ] recent success in vns-mediated intracellular delivery suggests that d-vns represent a promising platform for cellbased immunotherapy by introducing exogenous genetic and therapeutic materials into immune cell candidates. [ , , , ] vns allows rapid and direct intracellular access while inducing minimal cell cytotoxicity; a wide range of biomolecules, including dnas, rnas, proteins, quantum dots (qds), and impermeable drugs, have been successfully delivered into live cells. [ ] in particular, sinw arrays demonstrated high efficiency (> %) in transfecting exogenous genetic materials into primary immune cells-which are notoriously hard to transfect-while maintaining high viability and immunological competence; [ , , ] sirnas were transfected via sinws into primary b cells and cd + t cells to knock down predicted target genes, facilitating discovery of essential signaling pathways and genetic regulatory circuits for immune cell activation and differentiation; [ , ] sinws were used to administer a small-molecule inhibitor of the enzyme polo-like kinase (plk) into bone-marrow derived dendritic cells (bmdcs), confirming the essential role of plk and plk in regulating antiviral gene expression in these cell; [ ] by using vertical carbon nanosyringe arrays (cnsas) under applied centrifugal g-force, pegfp plasmids were transfected www.advmat.de www.advancedsciencenews.com into primary lymphocytes with significantly higher efficiency than conventional lipofectamine -mediated method. [ ] despite the emerging use of d-vns technology in immunology, a deeper understanding of vns-immune cell interplay is urgently needed, especially on how direct physical entanglement with vns and indirect mechanotransduction alter immune cell status, and how vns interfacing mechanisms could be the basis for immune cell engineering. such understanding will help to develop strategies and solutions to overcome side effects in the use of d-vns devices, which still represent an important challenge in biomedical research. the progress report is a snapshot of this very active and dynamic area. we summarize the most recent studies on the performance of d-vns in orchestrating immune cell functions, behaviors, and fate conversions through experimental and theoretical studies. specifically, two major aspects are examined: i) the effects of biophysical/biochemical cues from d-vns on triggering immune responses and modulating functions of different immune cell types; and ii) the mechanisms and efficacy of vns-mediated intracellular delivery for immune cells. by identifying fundamental challenges and exploring opportunities for further interdisciplinary research into vnsmediated immune-cell manipulation and interrogation, we expect the development and use of d-vns to be instrumental in meeting biomedical goals. the immune system has evolved to elicit nonspecialized innate and highly specialized adaptive immune responses (figure ) to distinguish self from nonself (e.g., viruses, bacteria, and cancer cells), eliminating nonself subjects from the body. numerous types of cells are involved in the generation of effective immune responses against antigens. [ ] a good analogy to the molecular mechanisms of immune responses is that of an orchestral performance. each musician has specialized skills and reads from a different set of music; but everyone is coordinated by the conductor. the processes that generate and b a i) iv) ii) i ii) v) figure . emerging application of d-vns platforms in immunological studies. a) sem images of five types of d-vns, including nanowires (i), nanopillars (ii), nanoneedles (iii), nanocones (iv), and nanostraws (v). scale bars: µm (i-iii) and nm (iv,v). i) reproduced with permission. [ ] copyright , the authors, published by national academy of sciences; ii) reproduced with permission. [ ] copyright , springer nature; iii) reproduced with permission. [ ] copyright , acs; iv) reproduced with permission. [ ] copyright , elsevier; v) reproduced with permission. [ ] copyright , acs. b) sem images of primary mouse bone-marrow dendritic cells (bmdcs), b cells, dendritic cells (dcs), macrophages (mΦs), natural killer (nk) cells, and t cells (false colored orange) on top of sinws (false colored blue) after h culture. reproduced with permission. [ ] copyright , acs. www.advmat.de www.advancedsciencenews.com regulate the host immunity are comparable: different arms of the immune system are coordinated by genes that encode for proteins with specific functions. innate immune cells form the front line to defend nonspecific pathogenic threats that bypass natural physical and chemical barriers of the body. neutrophils, monocytes, macrophages, basophils, eosinophils, dcs, and nk cells are key components of the innate system. they eliminate invading pathogens and cancer cells by various mechanisms, including inflammation, reactive oxygen species release, complement activation, receptor-mediated killing, phagocytosis, and autophagy. [ ] inflammatory mediators-such as histamine, defensins, lysozyme, chemokines, and cytokines like interferon (ifn) and tumor necrosis factor (tnf)-α-are often secreted during acute inflammation to clear the initial cause of cell injury and tissue damage; classic symptoms accompanying inflammation include heat, pain, redness, swelling, and loss of function. but persistent acute inflammation may lead to a progressive shift in the local microenvironment, where destruction and healing of the tissue occur simultaneously; this results in chronic inflammation, which is associated with diseases such as asthma, atherosclerosis, and osteoarthritis. [ ] when an innate immune response is insufficient to eradicate an infection, the adaptive immune system is informed by antigen presenting cells (apcs) such as dcs. once mobilized, they recruit an army of immune cells-mainly t and b lymphoctyes-specifically designed to attack that antigen. cd + helper t cells are pivotal in adaptive immunity, since they are required for almost all adaptive immune responses, comprising cellular and humoral immunity. [ , ] in cell-mediated immunity, cd + t cells release a spectrum of cytokines to activate cytotoxic cd + t (ctl) cells, promoting their differentiation and maturation to recognize, bind, and lyse infected target cells. [ ] [ ] [ ] humoral immunity is mediated by antibodies generated by b cells. with the assistance from cd + t cells, b cells undergo clonal expansion, class switching, and somatic hypermutation to differentiate into plasma b cells that can produce high-affinity antibodies against a specific antigen; these antibodies bind to antigens, neutralizing them, or causing lysis or phagocytosis. [ , ] importantly, adaptive immunity forms a "memory"-sometimes life-long-that makes it more efficient to evoke future responses against the same antigen. immunomodulation is the core of multiple therapeutic interventions, including but not limited to immunotherapies for schematic of the complex network of host immune system, comprising innate and adaptive immunity, to recognize and target non-selfpathogens, including virus, bacteria, and cancer cell (highlighted in yellow). www.advmat.de www.advancedsciencenews.com tumors, infectious diseases, autoimmune diseases, and graftversus-host diseases. given that most applications proposed by d-vns studies were on cancer immunotherapy, the progress report is mainly focusing on this area. modulation of the immune system to enhance anticancer response holds great promise in the prevention and treatment of the reoccurrence and metastasis of cancer. numerous modalities have been involved in immunotherapeutic translation, including cancer vaccines, oncolytic viruses, and administration of such immunomodulators as cytokines, [ ] monoclonal antibodies, [ ] and checkpoint inhibitors, [ ] which either costimulate cells or block the so-called immune checkpoint pathways. [ ] immunotherapeutic regimes that use specific monoclonal antibodies to block cytotoxic t lymphocyte-associated protein (ctla- ) and programmed cell death protein (pd ) have shown exciting results for melanoma treatment. [ ] cell-based immunotherapy, such as adoptive cell transfer (act) of ex vivo activated t cells, is ideal for activating immune response, by generating effector t cells to recognize and kill tumor cells. in particular, chimeric antigen receptor (car)-t cell therapy is one of the most promising approaches in treating hematological malignancies, such as relapsed or refractory b cell acute lymphoblastic leukemia and diffuse large b cell lymphoma. [ , ] despite significant progress, clinical and commercial translation of laboratory-based immunotherapy technologies is still stymied by unpredictable toxicity and immunogenicity, limited efficiency in treating large solid tumors, costly and lengthy procedure, insufficient in vitro expansion for scaled-up production, and off-target side effects from systemic dosing. [ , ] due to the staggeringly complicated immune regulatory network, precise cues are needed to induce and fine-tune the response of a specific cell type. much work has studied how immune cells react to biochemical cues such as bacterial lipopolysaccharide (lps), cpg dna, viral rna, and toll-like receptor (tlr) agonists. [ ] [ ] [ ] but a smaller body of work has examined the role of biomechanical and biophysical cuessuch as envelope spike proteins on coronavirus, and pilus/ fimbria on bacteria [ ] [ ] [ ] [ ] -in triggering host immunity. indeed, biophysical cues have been increasingly recognized as critical regulators of cancer progression, detection biomarkers, and therapeutic targets. cell-based stiffness sensors can reveal how cells actually feel in their native environment and dynamically interrogate the mechanobiology of primary tumors and metastases. existing immune cell therapies, such as car-t cells, could be further engineered to exploit the biophysical cues of tumors to improve target specificity. with programmed topological characteristics and surface functionality, d-vns provide unique biophysical, biomechanical, and biochemical cues when interfacing with live cells; vns-featured platforms have been developed as safe tools for manipulating activities of many cell types, including immune cells. [ , , , , ] the most prominently observed phenomena induced by d-vns at cellular level are nuclear deformation, plasma membrane remodeling, and cytoskeleton reorganization. [ , , ] the mechanical stability and deformability of the cell nucleus are crucial to diverse biological processes, including migration, proliferation, and polarization. [ ] [ ] [ ] for example, mechanical forces applied to nuclei can alter epigenetic regulation networks and modulate gene expression, ultimately affecting cellular functions. [ ] neutrophil nuclear morphology has historically been used in hematology for neutrophil identification and characterization. [ ] nuclei are the key organelle for genomic information storage and transcription, which are tightly regulated by d structure and mechanical features of chromatins. [ ] in this context, d-vns were demonstrated as an effective approach for noninvasive subcellular perturbation of nuclei, the deformation of which can be well-controlled by varying vns geometry. spacing and diameter of vertical nanopillar arrays had a strong effect on nuclear deformation, where a larger pillar pitch and/ or a smaller pillar diameter drastically increased the nuclear deformation, whereas the height of pillars had only a mild effect (figure a -c). [ ] by identifying how nuclei respond to topography and inherent mechanics, it is possible to program nanopatterned pdms elastomers for directional nuclear deformation. vns platforms can also be developed to characterize early changes in nuclear mechanics and morphology associated with a number of diseases, such as hutchinson-gilford progeria syndromea premature aging disease of children where abnormal nuclear shapes can be detected, including lobulation of the nuclear envelope, thickening of the nuclear lamina, loss of peripheral heterochromatin, and clustering of nuclear pores. [ ] aside from nuclear deformation, most engineered d-vns materials interface extensively with the cell membrane, which features heterogeneous but regulated structures and composition. membrane heterogeneity plays an indispensable role in multiple cell functions, such as adhesion, migration, viability, and receptor-mediated activation. [ ] in response to mechanical stimuli, immune cells can reorganize their cytoskeleton quickly-as fast as in few minutes. [ ] the mechanosensitive ion channels on immune cells' plasma membrane can be opened through membrane tension. [ ] and mechanical force plays an important role in directly activating transmembrane receptors, such as b cell receptors (bcrs). [ ] mesoporous si nanoneedle arrays have demonstrated the ability to interact with multiple mechanoresponsive components simultaneously, inducing the reorganization of the lipid bilayer and membrane-associated proteins, as well as the actin cytoskeleton beneath the cell cortex. [ ] a gradient array of sio nanopillars with radii of - nm was fabricated to investigate the effect of pillar radius on membrane curvature (figure d ,e). [ ] tem images showed that nanopillars could induce plasma membrane invaginations with sizes close to www.advmat.de www.advancedsciencenews.com those of clathrin-coated endocytic pits (figure f ). in addition, immunostaining results confirmed the local accumulation of two key proteins for clathrin-mediated endocytosis-clathrin (ctla) and dynamin (dnm )-on highly curved membranes along pillars with radii < nm ( figure g ); such curvature progression induced by nanopillars resulted in enhanced endocytosis ( figure h ). tin oxide nanocones fabricated on a planar substrate also caused local inward membrane curvature ( figure i ) and recruitment of n-bar domain proteins-versatile membrane-associated regulatory elements that function over a wide range of cellular processes including regulation of cortical actin structures and endocytosis. [ ] confocal imaging revealed that nanocones induced the selective accumulation of full-length and isolated n-bar domains of nadrin , a regulator of actin polymerization (figure j ). tem imaging confirmed that n-bar domains of nadrin were recruited to the curved membrane around the nanocones (figure k ). this nanocone-induced recruitment was also applied to the isolated n-bar domain of amphiphysin , a regulator of clathrin-mediated endocytosis. since endocytosis is a crucial initial step for immune cells to sense and engulf pathogens, leading to cascades of innate and adaptive immune responses, manipulating membrane curvatures and endocytic activation by d-vns are promising approaches for immune modulation. aside from these d-vns tools for immune-cell modulation, custom-tailored cellular modification and subcellular-level precision control are promising future directions in research and clinical study. increasingly diverse nanotopographies have emerged in recent years. [ ] [ ] [ ] [ ] porous si nanoneedles have shown the potential to be harnessed for cellular modulation at the organelle level, since cellular compartments such as cell membrane, cytoskeleton, and nucleus generated distinct responses to topographical cues. [ ] by electrochemical oxidation/reduction, a vertical polypyrrole (ppy) array was reversibly switchable between nanotubes (highly adhesive hydrophobic) and nanotips (poorly adhesive hydrophilic). [ ] such ppy arrays provided dynamic stimuli that can alter surface adhesion, enabling controlled differentiation of mesenchymal stem cells at the nanoscale. despite their promising potential, these newly developed vns-enhanced tools have not yet been applied to probe and regulate immune cell functions. immune cells actively sense and respond to physical deformation and mechanical stress via mechanosensitive channels and the cytoskeleton, resulting in activation of immune pathways such as inflammasomes. [ ] so in addition to their fundamental role as research tools for understanding the immune system, d-vns platforms can directly modulate immune responses. arrays made of bulk metallic glasses (bmgs, diameter - nm; figure a ,i) induced a change in the size, spreading, and elongation of various cell types. [ ] fibroblasts decreased in cell area as the bmg diameter increased, and responded to bmgs of diameters as small as nm. endothelial cells detected bmgs of diameter nm, but decreased significantly in cell size and elongation with larger bmg sizes. unlike fibroblasts and endothelial cells, macrophages failed to detect bmgs of diameter ≤ nm, but responded to those of diameter nm, presenting more enlarged and elongated cell morphology (figure a ,ii). aside from influencing macrophage morphology, there is evidence that surfaces and scaffolds with nanotopographies can alter macrophage polarization between a proinflammatory m phenotype and an anti-inflammatory/prohealing m phenotype. [ ] for example, a surface with nanopatterned grooves of - nm can drive murine bone-marrow-derived macrophages (bmms) toward the m phenotype, secreting significantly higher levels of the anti-inflammatory cytokine il- , compared with bmms cultured on a flat surface. [ ] this suggests that topography at the nanoscale can be designed to manipulate immune cell differentiation; but whether this and other types of advanced cellular manipulation can be applied to d-vns topography rather than groove structures is still to be determined. vns can augment the induction of immune responses by enhancing a site-specific endocytosis, a key process for apcs to effectively engulf extracellular antigens or dead tumor cells. cd + t cells formed complex interactions with elastomer pdms pillar arrays; the dimension and flexibility of these nih- t fibroblast on nanopillars, with the nucleus deformed around nanopillars. scale bar: µm. b) nuclear deformation visualized by immunostaining of lamin a (green). scale bar: µm. c) fluorescence (left and middle) and differential interference contrast (dic, right) images of a live t cell transfected with gfp-sun , demonstrating the nuclear deformation. scale bars: µm. a-c) reproduced with permission. [ ] copyright , springer nature. d) schematic of nanopillars with different radii (gray) deforming the cell membrane to generate different membrane curvatures (red line). e) top: sem image of a gradient nanopillar array with height nm, pitch µm, and radii ranging from to nm (left to right). scale bar: µm; bottom: zoomed-in sem images of individual nanopillars as indicated in the top image. scale bars: nm. f) tem image showing a clathrin-coated pit at the membrane around a nanopillar. scale bar: nm. g) immunostaining showing the accumulation of clathrin (clta-rfp, red) and dynamin (dnm -gfp, green) at nanopillar locations. scale bar: µm. arrowheads indicate locations of nanopillars with different radii. h) schematic of proteins involved in different stages of clathrin-mediated endocytosis (cme). d-h) reproduced with permission. [ ] copyright , springer nature. i) tem images of t cells grown on nm nanocones. cells are colored in red. scale bars: µm (top) and nm (bottom). j) confocal imaging of a t cell grown on nanocones and transfected with nadrin (red) together with a membrane marker caax (green). k) left, tem image of an immunogold-stained t cell transfected with fluorescently tagged n-bar domain of nadrin ; right, statistical analysis of immunogold density measured over nanocones where membrane deformation was observed, compared with adjacent regions (no nanocones) within the same image. i-k) reproduced with permission. [ ] copyright , springer nature. www.advmat.de www.advancedsciencenews.com . a) reproduced with permission. [ ] copyright , acs. b) top: schematic of three pdms pillar arrays used to test the effect of d structure and stiffness on t cell activation. bottom: fluorescence images illustrating microtubule structure (green, β-tubulin) and cell morphology (red, cd ) for cells on pdms pillars. scale bar: µm. c) statistical analysis demonstrating increased ifn-γ secretion with increasing spring constant. *p < . . b,c) reproduced with permission. [ ] copyright , the authors, published by national academy of sciences, usa. d) schematic of the cytolytic immune synapse involving peripheral wave -dependent protrusions and central wasp-dependent protrusions. the red arrows denote force exertion. e) time-lapse montage (image collected every s) of a representative ctl cell expressing lifeact-mruby (red) and phluorin-lamp (bright blue) on pdms pillars (gray). z-projection images (top views) are shown above with sagittal views below. the white dashed line (at min s, : ) denotes the slicing plane used for the sagittal images. yellow arrowheads indicate the fusion event. scale bars: µm. f) schematic of lytic granule fusion (visualized by phluorin-lamp in (e)) on pdms pillar arrays. d-f) reproduced with permission. [ ] copyright , the authors, published by aaas. g,h) schematic (g) and false-colored sem (h) of the activation of nk cells by mica-functionalized zno nws through surface receptor nkg d engagement. i,j) fluorescence microscopy images showing nk cells on mica-functionalized flat control (i) and nws (j). cd a staining (white) indicates enhanced degranulation in nk cells cultured on nws (j) compared with that on flat control (i). g-j) reproduced with permission. [ ] copyright , wiley-vch. www.advmat.de www.advancedsciencenews.com anti-cd /anti-cd antibodies-coated pillars can affect cd + t cell activation. in particular, stiffer pillars ( µm high, u, and tapered, tap) with a larger spring constant ( . nn µm − ) delayed transport of the centrosome/microtubule-organizing center (mtoc) toward the middle of the cell-pillar interface ( figure b )-a key step in t cell activation and immunological synapse (is) formation. stiffer pillars also promoted significantly higher expression level of ifn-γ in cd + t cells, compared with that using less stiff ( µm high, u; . nn µm − ) ones ( figure c ). [ ] these results reveal a complex effect of vns-substrate mechanics on cellular responses from mtoc centralization to cytokine secretion. similarly shaped pdms pillars were used to interact with cd + ctl cells. these pillars induced the deformation of ctl plasma membrane, stimulating formation of actin-rich protrusions, assisted by two major nucleation-promoting factors-wasp and wave- ( figure d ). these protrusions were necessary for synaptic force exertion, particularly in more central regions of the is close to lytic granules; they were also required for physical deformation of target cells in bona fide cytolytic interactions. it was deduced that the engineered interface between ctl cells and pdms pillars resembled the growth of communicative is between ctls and target cells in d. this unique approach helped identify the key role of actin protrusions in promoting synaptic force exertion and ctl killing, with elevated degranulation of granzymes and perforins (cytotoxic proteins injected into target cells via is by ctls). granule fusion events were detected in single-cell imaging experiments, using a fluorescent reporter containing a ph-sensitive gfp (phluorin) fused to the granule-targeting domain of lamp (phluorin-lamp ); f-actin protrusion were visualized via lifeact-mruby or by staining with a fluorescent fab against the surface marker cd . time-lapse observations of a µm diameter synaptic domain demonstrated that f-actin accumulation increased modestly within this domain during granule fusion, indicating that cytolytic secretion and protrusion growth occurred concurrently in the same region (figure e , top). linescans of sagittal slice images revealed that f-actin did not overlap with the fusion site but accumulate underneath it (figure e, bottom), implying that granule fusion occurred in f-actin-free zones that formed transiently at the base of active f-actin-rich protrusions on pdms pillars ( figure f ). [ ] with adjustable spatial and mechanical properties, such vertically aligned elastomer micro/nanostructures give advantages over planar substrates for their ability to capture the complexity of immune cellular network at play under environment close to real physiological conditions. surface chemistry also plays an important role in immune cell stimulation. controlled surface nanotopography by gold nanoparticle immobilization combined with carboxyl acid rich coatings enhanced the secretion of matrix metalloproteinase- (mmp- )-an enzyme involved in tissue remodeling and foreign body responses-by murine primary neutrophils, without significant impact on other markers of neutrophil functionality. [ ] in regards to bmms, nanotopography modified with carboxyl acid functionality significantly impaired the release of inflammatory cytokines (including il- and il- β) after lps stimulation; but other surface modification, such as amine or methyl group modification, showed less pronounced effects. in addition to simply modifying vns surfaces with generic polymers to obtain different charges or hydrophilic properties, coating with receptors and/or ligands to activate particular target cells could further strengthen the capability of vnsmediated immune modulation. for instance, surface functionalization of zno nw arrays with a specific antigen-major histocompatibility complex class i-related chain a (mica)greatly enhanced the binding and activation of nk cells via the surface receptor nkg d (figure g ,h). [ ] mica ligands provide essential biochemical cues to evoke nk cells, since nonfunctionalized and mock-functionalized nws produced no significant nk cell activation. but once triggered, the effects of mechanical cues from nw topography overruled the effects of mica quantity on nk immune response. mica-functionalized nws stimulated four times the expression of cd a (exposed to nks' outer membranes during activation/degranulation), compared with that on mica-coated flat substrates (figure i ,j). this is despite the fact that ≈ times the amount of mica was exposed to nk cells on the flat substrates compared with the nw array substrates, where only the tips (the top %) of nws interacted with cells. such surface-functionalized nws could enable study of immune responses under multiple stimuli that are more relevant to the in vivo milieu. in vivo modulation of immune cells by nanotopography is an important direction, permitting rapid local regulation of immune responses with biophysical cues. most studies of vns-mediated immunomodulation discussed above are based on planar substrates. notably, a distinct strategy was developed to grow a corona of nanospikes on tio microparticle surface, designated as "spiky particles," to stimulate innate immune responses in vivo (figure a,b) . [ ] the spiky nanotopography on microparticle surfaces promoted activation of inflammasomes (cytosolic multiprotein oligomers responsible for the activation of inflammatory responses during innate immunity) in both bmms and dcs. further study demonstrated that spiky particles introduced mechanical stress on the cell membrane, leading to the activation of mechanosensitve potassium channels to allow k + efflux, which is likely to be driven by myosin iia-a key contractile motor protein that generates mechanical forces during phagocytosis. spiky particles also provoked downstream il- β release in a caspase- -and nlrp -dependent fashion (figure c ). inhibition of k + efflux with a high extracellular k + concentration completely abrogated the spiky-particle-induced inflammasome activation; while treatment with k + channel inhibitors, amiodarone or ruthenium red, significantly blunted the release of il- β from bmms treated with spiky particles (figure d ). spiky particles coupled with monophosphoryl lipid a (mpl)-an agonist of tlr -effectively activated tumor antigen-pulsed dcs, which was subsequently used as a cancer www.advmat.de www.advancedsciencenews.com dc vaccine to eliminated murine tumors. when injected together with mpl, spiky particles promoted dc maturation in vivo, and robustly augmented vaccine-induced adaptive immunity against influenza viral infection. [ ] importantly, in vivo activation of immune cells by nanotopography suggests a new strategy in addition to conventional biochemical stimulation, creating a basis for engineering more potent vaccines and adjuvants for immune system activation. given its potent adjuvant effects over conventional chemical adjuvants, a combination of spiky particles and immune stimulators could be used for "in situ" cancer vaccines that have already shown initial success in multiple tumor types. [ , ] intratumoral injection of this combination after local ablation of tumors by radiation or radio frequency ablation treatment may induce robust tumor-specific immune responses and prohibit recurrence or metastasis. [ ] [ ] [ ] the demonstration of spiky particles to activate immune response has confirmed the applicability of vns-mediated immunomodulation in vivo. but current spiky particles are made of nondegradable materials, which limit application in prophylactic vaccines for healthy populations. the development of biodegradable spiky particles could fully realize the potential of d-structured vns for in vivo immune modulation in vaccination and cancer immunotherapies. these elegant studies are early evidence that d-vns could modulate activities of various cell types by inducing cellular deformation and membrane curvature, mediating reorganization of cytoskeleton and opening of mechanosensitive channels. because immune cells are sensitive to mechanical forces, it is highly likely that d-vns devices have extensive immune modulation effects, not limited to cell types and immune processes so far reported. but current vns studies have worked on a finite number of immune cell types, omitting key players and their subpopulations in the induction of an immune response; one of these missing players is the b cell. b cells are an important immune cell type likely to respond to mechanical stimuli via bcr. [ , ] b cell activation requires physical interaction with professional apcs-follicular dcs (fdcs)-which display protein fragments derived from infecting pathogens on their surface. [ ] bcrs recognize these fragments and assemble into microclusters; signaling proteins and adaptors accumulate at developing microclusters, which trigger transduction pathways, initiating transcription and cell activation. [ ] ligand mobility can modulate bcr diffusion and clustering, which are important in early b cell signaling. [ , ] dna-based nanosensors were developed to examine the role of mechanical cues from is in regulating b cell responses. [ ] the results showed that b cells primarily used mechanical forces to extract antigens from is, and resorted to enzymatic liberation only if mechanical forces failed to do so. the same study also found that the stiffer fdcs promoted strong b cell pulling forces and stringent affinity discrimination, whereas the softer dcs promoted extraction of low-affinity antigens by weak pulling forces; this suggested that distinct physical properties of apcs supported different stages of b cell activation. b cells have www.advmat.de www.advancedsciencenews.com now been exploited as promising targets for nanoparticle-based vaccines against viral and bacterial infections, due to their essential role in humoral and memory immune responses. [ ] [ ] [ ] however, less work has been devoted to exploring the use of d-vns platforms in generating mechanical forces and biophysical cues for bcr activation and b cell signaling. it would be interesting to investigate how b cells behave on an antigenconjugated vns of different size, geometry, and rigidity; and how such vns-interfacing alters b cell activation, maturation, and antibody production during humoral responses-all of which would guide innovative treatment and vaccination strategies. despite evidence for vns-mediated immunomodulation effects, the underlying molecular mechanisms require further investigation, including but not limited to the identification of primary mechanical sensors and subsequent signaling transduction cascades. a computational model suggests that local membrane curvatures-which can be induced by well-designed vns-lead to the opening of mechanical sensing channels, [ ] which play an evolutionary role from fungi to mammals in sensing environmental mechanic stress. [ , ] deformation and rearrangement of cell cytoskeletons in response to mechanical forces induced by vns were shown to activate several cellular signaling pathways, such as inflammasomes. [ , ] a recent study produced insights into how membrane curvature generated by vns was transduced into aforementioned biological processes. [ ] a f-bar domain containing protein fbp was identified to be a key curvature-sensing protein. it was found that fbp could activate downstream signaling pathways to nucleate formation of branched f-actin; this subsequently affected a number of cellular processes, including endocytosis, stress fiber organization, and focal adhesion maturation. the interactions between immune cells and d-vns are complex and dynamic. taking the initial progress described above as a start, a more systematic study involving a comprehensive comparison among shapes, sizes, geometry, rigidity, and chemical modification are likely to yield important advances. there are now significant opportunities for extending knowledge of how precisely engineered d-vns can manipulate immune responses, both ex vivo and in vivo. further exploration of the underlying mechanisms will have profound implications for vns-based multifunctional metamaterials in early diagnosis and synergistic cancer immunotherapy, such as act and cancer vaccines. vns-based ex vivo activation of dcs could serve as a platform to optimally expand tumor-specific t cells that are infused back into the patient to eliminate tumor cells. on the other hand, injectable d-vns such as spiky microparticles, can not only drive ex vivo activation of immune cells, but modulate immune responses in vivo when injected into the body, serving as an effective cancer vaccine adjuvant. intracellular delivery of genetic materials into living cells regulates the transcription and translation of exogenous genes into specialized proteins using a cell's own machinery. this valuable paradigm has been widely used in molecular biology and biomedical research, such as ex vivo immuno-oncology, t cell reprogramming, and genome editing. in the burgeoning field of cell-based immunotherapy, car-t therapy as an example, the car gene (in the form of dna or mrna) is delivered into human t cells, eliciting specific anti-tumor immunity. [ ] current delivery mechanisms can be divided into two broad categories: viral transduction and nonviral transfection (including chemical and physical methods). the process of using a nonreplicating viral vector to deliver foreign dna into a cell is called transduction. primary cells such as human-induced pluripotent stem cells (ipscs) and immune cells are notoriously hard to transfect. this is particularly true for lymphocytes, which have a thinner cell membrane with lower protein content, making them more susceptible to cell death induced by electroporation and transfection agents than adherent cells. [ ] [ ] [ ] in addition, without antigen stimulation, materials delivered are less likely to localize into the nucleus of a nondividing lymphocyte than into that of a dividing one. so viruses, especially lentivirus, are commonly used to transfect immune cells, due to their innate ability to transduce nondividing/resting cells. viral transduction is robust, highly efficient, and can lead to long-term expression; but it has drawbacks such as a costly and lengthy procedure, restricted cargo packaging, the risk of insertional mutagenesis, and undesirable t cell activation-all serious hurdles to developing cell-based therapies. chemical transfection is a popular technique due to the ease, cost, and wide variety of transfection reagents available for purchase. for example, lipofection using cationic liposomes (lipidbased or nonlipid-based) is commonly used for short-term expression of a desired gene that lasts a few days. lipofection can achieve high transfection efficiency in immortalized cells, but have low efficiency on primary lymphocytes. [ ] in particular, t cells have shown resistance to common lipofection reagents; this might be due to their low expression of exostosin- , a key enzyme in the biosynthesis of heparan sulfate (hs) proteoglycans. since hs proteoglycans donate a negative charge to the cell surface, the lack of hs on lymphocytes results in a relatively high positive surface charge; this might increase the sensitivity of lymphocytes to cell lysis by cationic vectors due to excess local positive charge. it has also been shown that transfection of lymphocytes using conventional cationic reagents can lead to apoptosis, necrosis, and secretion of tnf-α. [ , ] numerous physical intracellular delivery methods have been developed to deliver a range of bioactive cargoes into a broad www.advmat.de www.advancedsciencenews.com range of cells, all of which are to some extant capable of in vitro delivery efficiency. among these, microinjection and bulk electroporation (bep) are the most common, and have been used for effective dna transfection in range of suspension cells such as primary lymphocytes. microinjection is simple and can be very efficient when optimized, but it is a time-consuming and laborious procedure; only one cell can be injected at a time, largely limiting the amount (only a few hundred) of cells to be transfected per experiment. [ ] in bep, cells are suspended between two parallel electrodes and the cell membrane is artificially ruptured under an applied electric field of up to a few thousand volts, leading to adverse effects such as joule heating, gas bubble formation, and unwanted electrochemical reactions and ph variations-all of which contribute to poor cell viability and inconsistent delivery outcomes. [ ] critically, a recent report has shown that bep can trigger an alteration in the expression of immune-associated genes related to immune cell activation and trafficking. [ ] due to the disadvantages of existing transduction/transfection methods, there is still a need for a safer nonviral and universal delivery technique that yields high transfection efficiency on primary lymphocytes; ideally, the technique should preserve cell viability and immunological competence which is critical for potential clinical intervention. delivery by d-vns has shown great promise for overcoming the limitations of existing delivery methods; the unique topological morphologies of d-vns allow rapid and direct intracellular access of a wide range of bioactive cargos-such as dnas, rnas, proteins, and their complexes-with minimal impact on cell viability and function. [ , , , , [ ] [ ] [ ] [ ] [ ] [ ] nws with controlled geometry have achieved high sirna transfection efficiency (> %) for different types of primary immune cells, including adherent (dc, macrophage, nk) and nonadherent (t and b, figure a ,b) immune cells, with minimal immune response activation. [ ] for instance, sinwfacilitated transfection of sirnas that silence the expression of lef gene-a terminal transcriptional activator and a canonical target of the wnt signaling pathway-severely impaired the survival of human b cells obtained from a chronic lymphocytic leukemia (cll) patient (figure c, right) . at the cellular level, the wnt signaling pathway contains more than members and is critical for the proliferation and cell fate determination of many cell types, including b cells. in a follow-up study, the same sinw platform was used to deliver sirnas into primary cll-b cells to assess the impact of gene knockdown on cll survival. [ ] based on the dna sequencing results of a series of cll samples, they interrogated the effects of of mutated wnt pathway members identified across clls. the research demonstrated that inhibition of the wnt pathway at different levels adversely affected cll survival. cll samples harboring gain-of-function mutations (dkk , bcl , ryk) exhibited greater dependency on wnt pathway signaling. silencing these mutated genes in cll-b cells resulted in reduced viability compared with wild-type b cells. decrease in cll cell survival was also observed when silencing csnk e, the mutation of which led to loss of wnt pathway activation. despite the readout on cell viability alone, the sinw-mediated gene delivery approach described in those two studies helped to gain insights into wnt/β-catenin regulatory molecular circuits and how mutated genes and pathways are involved in the pathogenesis of cll. genetic characterization of the wnt signaling can identify subgroups of cll patients with greater sensitivity to targeting of this pathway, affording a potentially more fruitful and less toxic approaches for effective therapeutic interventions. furthermore, since sinws demonstrated highly efficient (> %) delivery of fluorescently labeled sirnas without compromising cellular viability (> % survival), this approach overcame the poor efficiency of conventional transfection methodologies to genetically manipulate primary cll-b cells. [ ] in a mechanistic and seminal study, molecular circuits that control differentiation of naïve t cells were probed in order to understand how extra-and/or intracellular signals are processed into coherent cellular responses and determine cell fate. [ ] when combined with high-resolution transcriptional profiling and novel computational algorithms, sinw-based perturbation tools were used for effective and functional interrogation of primary immune cells. sinws mediated efficient transfection of selected sirnas into murine cd + t cells, leading to the identification of regulatory genes ( of them novel) and two mutually antagonistic regulatory circuits-all involved in the differentiation of naïve cd + t into th cells. crucially, sinw-mediated delivery did not activate immune responses or interfere with normal immune sensing mechanisms. this approach allowed pertubation of a molecular circuit's output (such as dna replication and mrna expression) via exogenous biomolecules (such as sirnas and therapeutic materials) delivered via sinws. [ ] so by compartmentalizing a sinw array, it is feasible to realize high-throughput gene silencing by delivering different sirnas into b cells at designated areas on the array. this is particularly useful for studying how silencing specific genes affects b cell responses to a drug, which can then be used to predict therapeutic outcomes for cll patients with specific genetic mutations. [ ] a separate study used sinws to administer a small-molecule inhibitor of the plk enzyme into bmdcs, demonstrating the essential role of plk and plk in regulating antiviral gene expression. [ ] using a nanostraw-electroporation system (nes) (figure d ), anti-ncor sirnas were introduced into human monocytes, reducing ncor mrna levels to % relative to the control that were transfected with scrambled rnas (figure e ). ncor silencing resulted in the enrichment of genes that were found downregulated in monocytes treated with il- ; this indicated that ncor is a key regulator for il- -induced monocyte differentiation (figure f ). [ ] vns substrates can mediate delivery of larger and more complex macromolecules, such as plasmid dnas containing therapeutic genes, to modify immune cells; this is a considerable advantage for both basic and translational biomedical research. for example, cnsas achieved significantly higher transfection efficiency (≈ %) of pegfp plasmids into primary lymphocytes than control lipofectamine (lipo. ) (≈ %) (figure g ). [ ] in a more recent study, primary mouse www.advmat.de www.advancedsciencenews.com www.advmat.de www.advancedsciencenews.com t cells-in an unstimulated status-were successfully transfected (> % efficiency) with cy -tagged gfp plasmids coated on sinws, under a forcible interfacing by applied centrifugation (figure h-j) . transfected t cells expressed gfp within h after detachment from sinws. of note, nw-treated t cells maintained high viability and proliferative capacity, with no discernible increase in key activation markers ( figure j) ; [ ] this overcame significant limitations of conventional viral and nonviral methods that may induce unexpected immunogenicity and altered cellular function. [ ] besides classic vertically aligned nw arrays, unconventional vns-featured platforms have also been developed to fit suspension immune cells. specifically, a photothermal delivery platform was generated to deliver a range of extracellular cargoes into ramos b cells. [ ] this platform was based on a uniform microwell array with d metallic sharp nanoscale tips (nanotips) at the edges of microwells (figure k) , which served as integrated local hot spots (figure l ) upon laser irradiation, and provided controllable transient membrane disruption for each cell in the array. ramos b cells settled by gravity within each microwell in direct contact with eight sharp nanotips (figure m ). subsequent laser treatment induced cavitation bubbles (phenomenon in which rapid changes of pressure in a liquid lead to the formation of small vapor-filled cavities) and created transient pores in the cell membrane, permitting efficient transfection of different-sized cargoes, with > % for calcein green ( . kda), > % for fitc-dextran ( kda), and > % for gfp-reporter plasmids. the bacterial enzyme β-lactamase ( kda) was also delivered into ramos b cells while retaining its biological activity. importantly, cells maintained high viability (> %) after transfection. the study suggests that a microwell-nanotip integrated platform can manage the number, location, and size of poration on suspension cells to achieve high transfection efficiency and high cell viability, if there is close control over experimental parameters such as cavitation bubble size (using different laser fluences), and pore number (using juxtaposed metallic nanotip configurations). the specific d topography of vns not only provides a large surface area and a high local density of ligands presentation, but enhances local intimate and dynamic interactions between the vns surface and cellular components at the nanoscale. [ ] but unlike adherent cells that anchor naturally and extensively on many vns substrates, the suspension lymphocyte cells are only in loose contact with most vns and can easily detach with subtle external perturbations. therefore, the cell-vns interface needs to be well-configured to stabilize and enhance the adhesion of suspension cells for efficient cargo delivery. research on developing smart and functional mechano-instructive surfaces for vns-mediated transfection technology is now attempting to balance multiple independent parameters and their complex interactions at play in a cell culture environment (vns topography and patterning, surface functionality and presentation, and cell and cargo types), [ , , , ] with combinatorial intracellular delivery approaches such as applied external force, [ , , ] cell squeezing, [ ] electroporation, [ , , ] oscillation, [ , ] and laser irradiation. [ , ] understanding this balance has the potential to maximize vns transfection efficiency by generating multifunctional integrated systems, which can facilitate cell attachment/detachment, strengthen cell-vns interface, enhance membrane permeability, activate cellular uptake processes, and control biomolecular release. selective vns surface functionalization has shown the capacity for a broad biophysiochemical tunability, allowing a more efficient interfacing with biological systems at play as well as the ability to facilitate vns internalization events to achieve specific and controlled transfection in immune cells. for example, an amphiphilic polymer consisting of polyethylene glycol and dodecyl alkyl units was used to coat cnsas, making the surface hydrophilic so that aqueous cargoes could be loaded to the interior of individual carbon syringes (figure a) . [ ] cationic polymers such as poly-d-lysine, polydopamine, and polyornithine, and cell matrix noncollagenous proteins such as fibronectin and laminin were variously used for nw functionalization to enhance plasmid adsorption and promote cell adhesion via electrostatic interactions. [ , , [ ] [ ] [ ] linker molecules such as -aminopropyltrimethoxysilane and -aminopropyltriethoxysilane were used to adsorb biomolecules (e.g., sirnas, dnas, and proteins) on the sinw surface, [ , ] facilitating their delivery into naïve t cells without activating significant immune response. [ ] stimuli-responsive polymers grafted on vns can modulate cell-vns interactions by controlling cellcapture and cargo-release. [ , ] in particular, si nanopillars and sinws modified with thermoresponsive poly(n-isopropylacrylamide) were developed for temperature-induced reversible capture and release of circulating tumor cells. [ , ] using multiple levels of functionalization, gold nanopillar arrays were a-c) reproduced with permission. [ ] copyright , acs. d) schematic of the field localization and biomolecule confinement at the tip of the nanostraws in nes. reproduced with permission. [ ] , acs. e) quantification of relative ncor mrna level in monocytes transfected with control scramble rna (purple) and anti-ncor sirnas (orange). f) heatmap of the top genes being most variable induced by ncor silencing, with a few genes depicted on the right side to represent upregulated and downregulated genes. e-f) reproduced under the terms of the cc-by creative commons attribution . international license (https://creativecommons.org/licenses/by/ . ). [ ] copyright , the authors, published by elsevier. g) histogram of the gfp expression within lymphocytes transfected with pegfp plasmids using control group lipo. (top) and carbon nanosyringe arrays (cnsas, bottom). reproduced with permission. [ ] copyright , wiley-vch. h) flow cytometric and statistical analysis of cy + gfp + population of primary mouse t cells h after harvesting from flat si and sinws coated with cy -tagged gfp plasmids. i) sem image of the interface of a primary mouse t cell on an array of sinws. j) heatmap of the expression of key activation markers within inactivated t cells cultured in well-plate (neg ctrl), on flat si, and sinws, as well as activated t cells. h-j) reproduced with permission. [ ] copyright , wiley-vch. k) schematic of a cell in a microwell. l) finite element method simulation showing the hot spots at a sharp nanotip as indicated in (k). m) sem image of a ramos b cell inside a microwell. n) fluorescence image of hoechst stained (blue) cells on microwells preloaded with kda fitc-dextran (green). scale bars: µm (m) and µm (n). k-n) reproduced with permission. [ ] copyright , acs. www.advmat.de www.advancedsciencenews.com produced with spatio-selective surface chemistry; the top of pillars was grafted with gold nanoparticles, whereas the bottom of pillars and the background were modified with a bioadhesive peptide. [ ] bifunctionality of the gold nanopillar arrays creates a new paradigm for biological, biosensing, and biocatalysis applications. in addition, there is now ample evidence that cell-vns interactions enhanced via the application of external force, such as centrifugation [ , , ] (figure a ) and aspiration flow, [ ] can assist in intracellular access and delivery, sampling, and cellular interrogation. [ ] for example, it was reported that with a spin at g for min, the sinw-mediated delivery efficiency of cy -tagged plasmids into suspension immune l . cells was boosted by a factor of three compared with that without spin (nonspin) (figure b,c) . [ ] a recent study reported an "all-in-one" d-vns platform for optimal cellular engineering, including immune cells. [ ] cell surface in the immune system is highly controlled and finetuned through the complex mixture of carbohydrates structures (glycans). [ ] there is increasing evidence that suspension cells such as cancer and immune cells overexpress sialic acids (sas). sas motives typically conjugated to the termini of cellular glycans and play crucial role in fundamental cellular and molecular processes that regulate both stimulatory and inhibitory immune pathways. [ ] in this context, sinw arrays modified with a sugar-responsive polymer containing phenylboronic acid (pba) groups (termed sn-phb, figure a ,i) combined three sequential functions: cell capture, intracellular delivery, and cell harvesting. [ ] first, specific recognition of pba by the glycoproteins and sas that span the lipid bilayer of the cell membrane enabled effective capture of both adherent (hela) and suspension (t and ramos, figure b ) cells. second, appropriate near-infrared irradiation (nir) was applied to exploit photothermal properties of sinws, facilitating efficient biomolecular (proteins and plasmid dnas) delivery via membrane disruption (figure a,ii) . for hard-to-transfect t cells, flow cytometry detection demonstrated the significantly higher transfection efficiency (≈ %) of gfp reporter plasmids (pgfps) using sn-phb under nir irradiation ( . w cm − , s), compared with conventional lipofection (lipo. ) (figure c ). the captured cells were harvested under mild conditions by simply adding nontoxic sugar solution, due to the dynamic reversibility of boronated ester bonds between pba and cis-diol-containing glycoproteins and sas, allowing molecular exchange with higher-affinity molecules such as fructose (figure a,iii) . [ ] [ ] [ ] the entire capturedelivery-harvesting process took < h, including a delivery time of < min. such a multifunctional integrated sn-phb system, therefore, presents a simple yet powerful platform for efficient intracellular delivery with minimal impact on cells; this www.advmat.de www.advancedsciencenews.com is highly desirable for biological research and future clinical applications. vns-based platforms can be built in microfluidic devices to achieve massively parallelized, deterministic mechanoporation (dmp) for intracellular delivery into suspension cells. [ ] sinws (or penetrators) were specially designed and fabricated in confined wells (figure d,e) , where only a single cell can be captured and porated by impingement upon the penetrator under a negative aspiration flow ( figure f ); cells were released by reversal of flow after which intracellular delivery occurs via diffusive influx of exogenous cargos (pgfp) through the single transient plasma membrane pore. moreover, high cell viability and transfection efficiency were detected (both > %) for dmp-transfected jurkat cells; mean pgfp transfection yield by the dmp device ( %) was over four times that by conventional bep methods ( %) (figure g ). efficient dmp-mediated pgfp transfection of k- ( %) and primary human t cells ( %) was also observed. this device overcomes the inherent stochasticity in many conventional membrane poration techniques, which usually results in a trade-off between delivery efficiency and cellular viability. the massive parallelization allows high-throughput cell engineering and rapid cell collection for subsequent processing, rendering a new means for addressing critical roadblocks in the manufacture and development of ex vivo cell therapies. significant effort is now devoted to increasing the consistency of intracellular delivery on the basis of high efficacy and minimal cell perturbation. at the forefront of these efforts are the nanostructural-electroporation platform-mediated transfection technologies. conventional bep is widely used to transport diverse molecular cargoes into different cell types, but it is nevertheless stymied by limited fine control of membrane disruption, high rates of cell mortality, and inconsistent delivery outcomes. [ , , ] by contrast, nano-electro-injection systems (nes, figure h -k) can focus localized electric fields at the nanostraw tips, significantly lowering the required voltage from kilovolts (typically > v) in bep to only a few volts; [ , , , ] this largely reduces damage to cells, cargoes, and electrodes during electroporation. localized electric fields created by nes can transiently open pores on cell membranes, allowing electrophoretic delivery of bioactive agents without compromising major cellular functions. [ , , ] the combinatorial platform, by performing electroporation through nanostraws, allows a precise dosage control for cargo delivery in response to electric field modulation. nes has demonstrated highly efficient delivery of dyes, plasmid dnas, mrnas, proteins, and cas ribonucleoprotein (rnp) into a range of adherent cells and primary cells with > % viability. [ , , , ] nes has also shown effective transfection on suspension jurkat cells-a popular model for hard-to-transfect primary t lymphocytes; [ ] the net nes transfection efficiency ( . %) was significantly higher than that of biochemical ( . %), viral ( . %), and electroporation ( . %) methods. [ ] analysis of cell doubling time, acute/chronic ca + stress signals, and rna transcriptomics revealed that nes resulted in lower ca + -mediated cellular stress and negligible impact on cell division time. moreover, nes exerted minimal perturbation on gene expression associated with immune cell activation and trafficking, compared with conventional viral and bep methods. the intimate nanoscale cell-nes interface allows a drastic reduction in electroporation voltage required, maintaining higher cell viability. more importantly, this combinatorial platform highlights the advantage of creating uniform contacts between every cell and the source of the electrical pulses, reducing the variability of the local electric fields, so that a larger fraction of cells can be porated simultaneously at a specific voltage. apart from intracellular delivery, the scope for applying nes platforms has lately been broadened; they have demonstrated strong potential for nondestructive and longitudinal cell monitoring, attributed to their ability to manipulate cellular extraction/sampling and probe intracellular environment without compromising cell health. [ , , ] zno/al o nanostraws were decorated with numerous nanobranches (bnss), which were conjugated with specific antibodies to facilitate effective capture of circulating tumor cells (ctcs). [ ] artificial ctc blood samples were produced with mcf- breast cancer cells spiked into healthy human whole blood. mcf- cells overexpressed [ ] copyright , acs. h-k) design and operation of the nes. h,j) schematic (h) and photo image (j) of cells cultured on the ns membrane in a well plate; the delivery reagent is placed under the bottom of the reservoir and an electric field is applied between the platinum and ito electrode. i) schematic of nes delivery mechanism. k) sem image of the ns array on the membrane. scale bar: nm. h-k) reproduced with permission. [ ] copyright , the authors, published by aaas. reprinted/modified from ref. [ ] . © the authors, some rights reserved; exclusive licensee american association for the advancement of science. distributed under a creative commons attribution noncommercial license . (cc by-nc) http://creativecommons.org/licenses/by-nc/ . /. www.advmat.de www.advancedsciencenews.com epithelial cell adhesion molecules (epcams), and were separated selectively and efficiently (≈ %) by anti-epcam-coated bnss from the blood samples. moreover, the bns-based system facilitated both efficient delivery of biomolecules (pi dye ≥ %, gfp plasmids ≈ %) into mcf- cells, and repeated extraction of intracellular enzyme caspase- from the same group of cells; this allowed for real-time and in situ regulation and monitoring of ctc intracellular activities, which is one of the most challenging aspects of cancer research. it is conceivable that this kind of technology will bring new opportunities to better trace and understand the functions of ctcs-including circulating malignant b cells in lymphoma patients-at different stages of a cancer. the exchange of cargos and signals across the cellular plasma membrane requires a collaborative interplay of diverse molecular mechanisms. [ , ] rational design of vns-mediated intracellular delivery requires a deep fundamental understanding of these mechanisms. this has been the focus of a substantial research effort but there is still intense debate with respect to whether the mechanisms involve mechanical penetration, membrane permeabilization, endocytosis, or a combination of those effects (figure a ). under specific conditions, vns access to the intracellular milieu is through spontaneous penetration of the cell membrane or alternatively under forceful nanoinjection. published proof-of-concept work describes successful delivery via spontaneous penetration (cell piercing); but ongoing experimental and theoretical studies on the underlying penetration mechanism suggest that it is a rare event. the unresolved discussion behind cell piercing and its efficacy is underpinned by simultaneous modulation in multiple independent input parameterssuch as using multiple types of interfacing scenarios, vns geometry, cell types, cell stiffness and adhesion, and material composition and surface functionalization. several groups have found that spontaneous membrane penetration occurs when vns geometry (density, length, and diameter) is optimized for a specific cell type. [ , , , ] for example, effective delivery of biomolecules into smaller nonadherent immune (naïve b and t) cells required sinws of length - µm, diameter < nm, and density . - µm − ; slightly shorter and less dense sinws ( - µm; . - . µm − ) were favored for transfecting larger adherent immune cells (dcs and macrophages). these nws were also reported to consistently penetrate cellular membranes without impacting cell health or morphology. [ ] but subsequent studies have yielded evidence against spontaneous penetration and suggested that most vns fail to penetrate cells in the absence of external forces. [ , [ ] [ ] [ ] for instance, using a transparent cuo nw-based cell impalement device and high-resolution tem, it was observed that the plasma membrane of hela cells wrapped tightly around the nws, with the absence of spontaneous penetration. [ ] in a time-resolved gfp quenching assay, hollow nanostraws of nm diameter delivered co + to quench gfp inside chinese hamster ovary (cho) cells. detection of co + -induced gfp quenching spots revealed that only % ± % of nanostraws were penetrated into cho cells. [ ] but a strengthened cell-vns interface-caused by either surface modification with adhesion promoters and/or application of active force-can significantly increase direct penetration events. for example, focused ion beam (fib)-sem imaging has produced evidence of direct and frequent sinw penetration into nonadherent l . (mouse b) cells (figure b, i) , where appropriate centrifugal force was applied ( g, ≈ . nn, min). [ ] ultimately, data from rigorous testing of the spontaneous penetration mechanism is still ambiguous, but has resulted in highlighting a unique set of combinations in which assisted vns access and delivery is more likely to occur. the bilayer is a permeability barrier that separates the cell from its exterior. local cellular deformation induced by d-vns can cause lateral diffusion of lipids and transient membrane permeabilization. recent investigations suggest that the tight interface between d-vns and cell membrane is sufficient to induce local cellular deformation and subsequent permeabilization of lipids at the cell surface, which enables the direct access to the intracellular milieu. [ , ] by coarse-grained molecular dynamics simulations of the cell membrane, researchers found that the high bending/curvature of the lipid bilayer particularly at the nano-/ micropillar tip can dramatically lower the traction force necessary to achieve membrane rupture. [ ] to verify the theoretical findings, they fabricated two types of pillars-"sharp" and "blunt"-with comparable diameter ( µm) and height ( . and µm) but different tip sharpness (radii of curvature: r sharp ≈ nm, r blunt ≈ nm). impermeable dye propidium (pi) and permeable calcein acetoxymethyl (am) dye verified membrane disruption and healthiness, respectively, of nih- t fibroblasts cultured on sharp and blunt pillars. cells remained viable on both pillars, as confirmed by the green calcein am stain. but pi entered cells on sharp pillars with a probability of ≈ %, while no internalization by cells on blunt pillars was observed (figure c ). [ ] the experimental data supported the simulation results, indicating that accumulation of traction forces from high local deformation and tight focal adhesion increases transient permeabilization, which may allow rapid delivery of exogenous biomolecules into cytosolic compartment. endocytosis is the internalization of extracellular material. endocytic pathways are an essential membrane trafficking process that use membrane-bound vesicles as transport intermediators. given the complexity of nanotopographically induced membrane curvature artefacts-in particular, deformation and reorganization of plasma membrane (i.e., dynamic change in local lipid composition and curvature-sensing proteins), the cytoskeleton remodeling, and nuclear envelope reshaping-a change in www.advmat.de www.advancedsciencenews.com figure . mechanisms behind cell-vns interactions. a) schematic of molecular mechanisms behind the response of vns-mediated intracellular access and delivery, including mechanical penetration, membrane permeabilization, and endocytosis. b) fib-sem images showing sinw-induced direct penetration into l . cells (i) and endocytic pits in gpe cells (ii). b) reproduced with permission. [ ] copyright , the author, published by wiley-vch. c) fluorescence microcopy imaging showing the staining of pi (red) and calcein am (green) to indicate membrane permeabilization and healthiness, respectively, of nih- t cells cultured on "sharp" and "blunt" pillars (edge curvature radius: r sharp ≈ nm, r blunt ≈ nm). reproduced with permission. [ ] copyright , acs. d) fib-sem images showing the accumulation of two types of endocytic vesicles, clathrin pits (orange arrows), and caveolae (green arrows), around nanoneedles. reproduced under the terms of the cc-by creative commons attribution . international license (https://creativecommons.org/licenses/by/ . ). [ ] copyright , the authors, published by wiley-vch. e) high-magnification fluorescence images showing the distributions of clta and dnm , two endocytic components involved in the clathrin-dependent endocytosis, along nanopillars. reproduced with permission. [ ] copyright , springer nature. f) schematic of multiple mechanisms involved in the nanostructure-mediated intracellular delivery. reproduced with permission. [ ] copyright , acs. www.advmat.de www.advancedsciencenews.com endocytosis behavior is likely to occur. indeed, it is now increasingly evident that programmable surface nanotopographies on which a cell resides can trigger multiple independent endocytosis pathways-for instance, clathrin-and caveolae-mediated endocytosis-accumulating caveolae and clathrin-coated vesicles at the cell-nanostructured interfaces, and in turn facilitating active biomolecular uptake without requiring direct penetration. [ ] by investigating vns of different diameters (range nm to µm), recent studies have shown that vns of diameter - nm are favored for receptor-mediated endocytosis. [ , , , ] fib-sem and tem imaging have demonstrated vns-induced membrane curvatures and endocytic pits on adherent mouse fibroblast (gpe ) cells (figure b , ii) and human mesenchymal stem cells (hmscs) (figure d ), semiadherent neuroendocrine pc m cells, and suspension jurkat cells. [ , , ] using fluorescence staining and confocal imaging, components of multiple endocytic pathways such as clathrin light chain, caveolin- , dnm , and clta have also been observed to coassemble at highly curved cell membranes along vns (figure e ). [ , , , ] an inhibition study treated gpe cells with nystatin and chloropromazine, finding that nystatin (which suppresses formation of lipid rafts, cholesterol-enriched domains, and caveolae) caused greater impairment of si nanotube-mediated mrna delivery than chloropromazine (which inhibits clathrin-mediated endocytosis and interferes with fast endophilin-mediated endocytosis). [ ] but for primary immune cells, especially for lymphocytes, endocytic pathways are often carefully gated via foreign element detectors such as tlr. [ ] it was reported that delivery of genetic agents using sinws did not activate endocytic or inflammatory pathways. [ ] though pdms pillars have been shown to promote f-actin protrusion formation and lytic granule exocytosis of cytotoxic cd + t cells, [ ] it remains an open question whether and how membrane deformations and endocytic pathways are involved during vns-lymphocyte interfacing. because of their nonadherent nature, suspension immune cells interact less vigorously with vns than do their adherent counterparts, making characterization of the interfacial interactions more difficult. due to the complexity of cellular responses toward physical, mechanical, and biochemical signals, it is highly likely that multiple pathways are invoked in vns-mediated intracellular delivery (figure f, table ). [ , , , , , , , ] a better understanding of regulation and crosstalk between different pathways would be extremely useful for designing advanced d-vns platforms to maximize transfection efficiency while inducing minimal cellular impairment. given their high programmability and biocompatibility, d-vns allow minimally invasive cellular manipulation. with their unprecedented spatial and mechanical resolution, d-vns surfaces have strong capacity to orchestrate ex vivo and in vivo immune responses. vns-based planar substrates and d microparticles stimulate innate immune cells and promote adaptive humoral and cellular immunity; d-vns demonstrate efficient intracellular delivery of a range of biomolecules into hard-to-transfect primary immune cells, overcoming the low efficiency and potential safety issues of conventional viral and nonviral methods. five critical challenges, however, remain to be explored. first, with the rapid expansion of multimodal cellular nanotechnology, developing new, cost-efficient, and easily implemented nanofabrication routes will be essential to seamlessly integrating d-vns with biomedical research. second, cellular processes at the cell-vns interface are extraordinarily complex, due to the variability of immune cell types, diverse cargo types, as well as vns surface functionalization, chemical composition, electronic properties, and physical geometry. so the crux will be to understand how such variability can be harnessed to maximize the degrees of freedom in which vnsmediated immune-cell modulation and interrogation can be conducted. third, most reports have solely focused on establishing successful delivery of bioactive cargoes, though as yet almost no progress have been made on assessing any adverse long-term effects or comprehensively profile cell functionality www.advmat.de www.advancedsciencenews.com and characteristics after vns cellular perturbation. we anticipate that a closer convergence of experimental nanotechnology and immunology in combination with high-throughput singlecell rna-seq and new computational/bioinformatics methods will likely result in fundamental advances in immuneoncology as well as for the validation of computational preclinical disease models. specifically, codelivery of multiple types of bioactive cargoes with quantitative control is necessary if we are to open new research directions for engineering clinicalgrade car-t and car-nk [ ] cells in immunotherapy. for example, delivering into t cells a cas rnp (to knock out surface antigens that might induce host rejection) together with a car gene would enable generation of a universal off-shelf car-t product; such a product would use t cells from healthy donors, avoiding reliance on low levels of effective t cells in patients. fourth, combining powerful characterization technologies-such as cryoelectron microscopy, scanning ion conductance microscopy, and electron tomography-with apposite theoretical models will reveal detailed correlations of the synergetic parameters, enabling insights into the underlying mechanisms. further advances in bioimaging will allow in vivo realtime monitoring of molecular and cellular signaling pathways regulated by cell-vns interactions. fifth, cellular processes are natively dynamic, yet engineered vns platforms are intrinsically static; so creating straightforward and efficient routes for dynamic cell manipulation, interrogation, and sensing coupled with analysing the cellular outputs in real time will have ramifications for mapping out and understating all levels of such nanobiointerfaces. a final central challenge in the field is to achieve the ability to scale these powerful nanoscale tools for clinical translation. addressing these challenges will foster optimization and evolution of smart d-vns devices, paving the way for clinical and commercial translation of advanced cell-based immunotherapy. proc. natl. acad. sci semiconducting silicon nanowires for biomedical applications proc. natl. acad. sci academic cell molecular pathology encyclopedia of life sciences proc. natl. acad. sci proc. natl. acad. sci hepatology proc. natl. acad. sci challenges in delivery of therapeutic genomics and proteomics proc. natl. acad. sci proc. natl. acad. sci key: cord- -oj d ail authors: gorgun, d.; lihan, m.; kapoor, k.; tajkhorshid, e. title: binding mode of sars-cov fusion peptide to human cellular membrane date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: oj d ail infection of human cells by the sars-cov relies on its binding to a specific receptor and subsequent fusion of the viral and host cell membranes. the fusion peptide (fp), a short peptide segment in the spike protein, plays a central role in the initial penetration of the virus into the host cell membrane, followed by the fusion of the two membranes. here, we use an array of molecular dynamics (md) simulations taking advantage of the highly mobile membrane mimetic (hmmm) model, to investigate the interaction of the sars-cov fp with a lipid bilayer representing mammalian cellular membranes at an atomic level, and to characterize the membrane-bound form of the peptide. six independent systems were generated by changing the initial positioning and orientation of the fp with respect to the membrane, and each system was simulated in five independent replicas. in % of the simulations, the fp reaches a stable, membrane-bound configuration where the peptide deeply penetrated into the membrane. clustering of the results reveals two major membrane binding modes, the helix-binding mode and the loop-binding mode. taken into account the sequence conservation among the viral fps and the results of mutagenesis studies establishing the role of specific residues in the helical portion of the fp in membrane association, we propose that the helix-binding mode represents more closely the biologically relevant form. in the helix-binding mode, the helix is stabilized in an oblique angle with respect to the membrane with its n-terminus tilted towards the membrane core. analysis of the fp-lipid interactions shows the involvement of specific residues of the helix in membrane binding previously described as the fusion active core residues. taken together, the results shed light on a key step involved in sars-cov infection with potential implications in designing novel inhibitors. significance a key step in cellular infection by the sars-cov virus is its attachment to and penetration into the plasma membrane of human cells. these processes hinge upon the membrane interaction of the viral fusion peptide, a segment exposed by the spike protein upon its conformational changes after encountering the host cell. in this study, using molecular dynamics simulations, we describe how the fusion peptide from the sars-cov virus binds human cellular membranes and characterize, at an atomic level, lipid-protein interactions important for the stability of the bound state. coronavirus disease (covid- ) emerged in late as a significant threat to human health. it became a global pandemic by march ( , ) , and it continues to claim lives and to significantly impact all aspects of people's lives across the globe. covid- is caused by severe acute respiratory syndrome coronavirus (sars-cov ), a positive-strand rna virus that causes severe respiratory complications, among other symptoms, in humans ( ) . sars-cov recognizes and infects human cells that express a cell surface receptor termed angiotensin-converting enzyme (ace ) ( ) , which is specifically recognized by the viral spike glycoprotein (s-protein). binding of the two proteins is a prerequisite for the fusion of the viral and cellular membranes ( ) , one of the first and required steps in viral infection facilitating the release of the viral genome into the infected cell ( ) ( ) ( ) ( ) . the binding of the virus to the surface cae receptor on the host cell is mediated by the s domain in the s-protein on the viral surface. the next key step, namly, virus-host membrane fusion is mediated by the s domain of the s-protein ( ) , the domain, which consists of multiple proteolytic cleavage sites, namely, one at the boundary of s /s and one at the s ' site, which are cleaved as part of the fusion process. ( ) ( ) ( ) cleavages at the s /s boundary and the s ' sites, downstream to the two heptad repeat regions (hr and hr ), induce the dissociation of the s subunits from the s-protein, followed by a series of conformational changes that trigger membrane fusion between the host cell membrane and the viral envelope ( , ) . the remaining s trimer, a post-fusion structural motif, is shared among all the class i viral fusion proteins ( , ) . a critical part of any viral fusion protein in the coronavirus family is the relatively apolar fusion peptide (fp), present in the s domain, which is responsible for directly inserting into and interacting with the host cell membrane, thereby initiating the fusion process ( , , ) . there are several characteristics that viral fps have in common and help locating the fp sequence. the sequences of the fps are highly conserved within each family of viruses (but not between families), the frequency of glycines and alanines in the sequence is high, and bulky hydrophobic residues as well as hydrophilic residues usually flank the cleavage sites ( , ) . some fps have a central kink via a proline and a helix-turn-helix structure, as observed in influenza virus hemagglutinin (ha). in such cases, proteolytic cleavage occurs directly at the n-terminus to the fp, and the peptides are thus called external or n-terminal fps. in other cases, the proteolytic cleavage site resides upstream from the fp which is relatively longer ( - amino acids) and contains a prolonged -helix in the fusion-active state such as in the cases of ebola virus or avian leukosis sarcoma virus ( , , ) . such fps are referred to as internal fps as in the case of sars-cov . to this date, three main fp regions have been suggested in sars-cov s-protein ( , ) , which are located in between hr and n-terminus of the s domain: ( ) at the n-terminus of hr , ( ) near the s /s cleavage site, and, ( ) at the c-terminus of the cleavage site s ' ( ) . based on the criteria stated above as well as experimental studies, most recent data suggest that immediately downstream of the s ' cleavage site of the sars-cov is the leading segment involved in the fusion process ( , ( ) ( ) ( ) ( ) . mutagenesis experiments showed the significance of the fp in this region, specifically carrying a region termed the fusion active core ( ) . the outer leaflet of human cell membranes is composed of a mixture of lipids including phosphatidylcholine (pc), phosphatidylethanolamine (pe), and cholesterol (chl) ( ) . a fluorescence spectroscopy study has shown that chl plays an important role in modulating the binding affinity and organization of the sars-cov fp in the membranes ( ) . therefore, taking into account the natural lipid composition of a mammalian cell in simulation studies such as the present one is important. characterizing how the fp binds to the membrane and how it interacts with specific lipids has been challenging experimentally. computational methods, particularly atomistic molecular dynamics (md) simulation, offer an alternative strategy to capture the membrane binding process of the fp and to probe the interface between the fp and the lipid membrane. one of the major challenges in simulating such processes lies in sufficient sampling of all possible fp membrane-binding poses. due to the slow dynamics of membrane lipids, they are often insufficiently sampled on the timescales which atomistic md simulations currently can access, causing the membrane binding and insertion of proteins to be heavily biased by the initial lipid distribution and protein placement. in this context, an alternative membrane model, termed the highly mobile membrane-mimetic (hmmm) model, has been developed to enhance lipid diffusion without compromising the atomistic description of lipid head groups ( ) ( ) ( ) ( ) . the hmmm model is based on the combination of a biphasic solvent system ( ) with short-tailed lipids at the interface ( ) . owing to its significantly enhanced lipid mobility the model has proven extremely efficient in describing mixed lipid bilayers, reproducibly capturing spontaneous (unbiased) membrane binding and insertion of a wide spectrum of peripheral proteins ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , and collecting significantly improved sampling of lipid-protein interactions. of particular interest to the present study, this method has also been successfully used to capture spontaneous membrane association of the influenza virus hemagglutinin fp ( ) . in this study, we perform an extensive set of hmmm simulations, to investigate membrane binding of the sars-cov fp, also taking into account several initial, different positioning of the peptide with respect to the membrane to further improve sampling. the results provide a detailed mechanistic picture of the initial step in the fusion process, focusing on the biophysical aspects of the virus-lipid bilayer interactions taking place during this process. characterizing the mechanism of the fusion-driving, fp-host membrane interactions is key to our understanding critical steps involved in viral infection, and might pave the way for development of novel therapeutic intervention strategies against the virus. as the first step for modeling the sars-cov fp, multiple sequence alignment was carried out for the s-proteins from different human coronaviruses (hcovs). there are a total of seven known hcovs, namely, e and nl belonging to the alpha subfamily of coronaviruses, and oc , hku , middle east respiratory syndrome (mers), severe acute respiratory syndrome (sars) and sars-cov , belonging to the beta subfamily of coronaviruses. out of these, s-protein structures are available for hku , mers, sars, as well as sars-cov . additionally, bat coronavirus ratg , a closely related homolog of sars-cov , was also included in the sequence alignment. multiple sequence alignment for the above eight sequences was carried out using the mafft program with the l-ins-i method ( ) and visualized using jalview ( ) (fig. s ). the available cryoem structure of the sars-cov s-protein at the time (pdb: vsb) ( ) contained only a partial structure ( residues) of the fp, making it unsuitable for developing the initial sars-cov fp model (fig. a) . since the s domain of the s-protein containing the fp is well-conserved among the sars coronaviruses, we used the s-protein from sars-cov as a guide for modeling the fp of the sars-cov . accordingly, the cryoem structure of the s-protein from sars-cov (pdb: xlr) ( ) containing the fp structure was used as a template for constructing the initial sars-cov fp model. our sars-cov fp model also contained the loop connecting the fp to the neighboring proximal region, suggested to be important in the fusion process ( - ), but not modeled fully in the current study due to lack of a suitable template structure at the time. the only two different residues between the sequences of sars-cov and sars-cov fps (i /m and d /e ) were mutated to the sars-cov residues (fig. b) . the initial fp model was then solvated and ionized with . m nacl in vmd ( ) . energy minimization was carried out using the steepest descent method for timesteps followed by solution equilibrium md for ns, in order to obtain a fully equilibrated sars-cov fp in an aqueous environment. the resulting equilibrated fp was used in the subsequent simulations investigating the membrane binding characteristics of the peptide. s-protein with the highlighted fp (blue). each monomer in the trimer is drawn in a different color (grey, purple, and orange). b) the sars-cov fp. the missing residues of the sars-cov fp structure were modeled using sars-cov fp (pdb xlr), with the mutations of i /m and d /e . residues - are in an alpha helical conformation and residues - form a loop structure. c) hmmm membrane binding simulation setup. after simulating the modeled fp in solution for ns, we placed the equilibrated peptide above the hmmm lipid bilayers in several different orientations. the membrane lipid composition is pc/pe/chl ( / / mol% which is representative of the outer leaflet of the human plasma membrane (carbon as tan, oxygen as red, phosphorus as blue, and nitrogen as orange). we use six different initial orientations rotating the peptide around its x axis with respect the parallel orientation: parallel (p, x= • ), antiparallel (a, x= • ), nosedive (n, x= • ), standing (s, x=− • ), inclined (i, x=− • ), and reclined (r, x= • )), for the fp placement above the membrane to randomize the initial interactions. lipid bilayers were randomly and individually built. each orientation was simulated in five independent replicas, with the total replica number of for membrane-binding simulations. we developed membrane-bound models of the sars-cov fp, utilizing multiple simulation replicas using the hmmm model ( fig. c) ( , , ) . symmetric conventional (full) membranes were first constructed using charmm-gui ( ) with a lipid composition of pc/pe/chl ( / / mol%), resembling the outer leaflet of human plasma membrane ( ) . the membranes were then converted to hmmm membranes by removing the atoms after the fifth carbon in the phospholipid acyl chains while keeping the cholesterol molecules intact. to mimic the membrane core, a previously developed in silico solvent, termed scse (including two carbon-like interaction centers) ( ) , was used to match the number of heavy atoms removed from the lipid tails in the previous step. the resulting hmmm membranes contained , scse molecules ( ) and lipids in each leaflet. in order to further expand the sampling of the phase space, we varied the initial placement and orientation of the fp, i.e., six rotated orientations (p, a, n, s, i, r) (fig. c) , to further reduce the initial bias. we solvated the systems using the solvate plugin in vmd ( ), with . m nacl resulting in total system sizes of , to , atoms and box sizes of × × to × × Å , respectively. multiple replicas were simulated for each fp orientation, as the diffusion and mixing of lipids and the process of membrane binding and insertion of the fp can be slow even when using hmmm membranes, and more sampling would ensure the meaningfulness of the obtained membrane-bound configurations. five independent hmmm membranes with each specified fp orientation were generated using a monte carlo based lipid mixing protocol developed in our group to further enhance variation of initial membrane configurations for each fp orientation. the systems were energy minimized for , steps and simulated for ns with the c atoms of the peptide harmonically restrained (k = . kcal mol − Å − ), followed by a production run of ns where c restraints were removed. a harmonic restraint along the z-axis, with a force constant k = . kcal mol − Å − , was applied to the c , c , and c atoms of the phospholipids and to the o atoms of cholesterol to mimic the atomic distributions of a full lipid bilayer more closely, and to prevent the occasional diffusion of short-tailed lipids into the aqueous phase, which is expected for these surfactant-like molecules. to prevent scse molecules from diffusing out of the core of the membrane, we subjected them to a grid-based restraining potential, applied using the gridforce ( ) feature of namd ( , ) . five replicas, each with an independently generated hmmm membrane and a starting orientation of the fp, were simulated, resulting in a total of independent membrane binding simulations. we define stable membrane binding with the criteria described below (see fig. ). first, a contact between the fp and the membrane is defined for any heavy atom of an fp residue that is within . Å of any lipid heavy atoms. any contiguous segment of the simulation trajectory with a length of at least ns during which at least one contact between the lipids and the fp existed was considered stable binding. the mid-points of these -ns segments are marked as a red point in fig. . to characterize the binding orientation of the fp with respect to the membrane, the first of the three principal axes (pa) of the fp helical segment (residues - ) was computed (fig. s ) . the angle between the first helical pa and the membrane normal, pa , was used to describe the tilting of the helical segment. two auxiliary angles, f and f , were further defined by the angle between the membrane normal and the vector component of the respective selected phenylalanine c -c vector perpendicular to the first pa, which together describe the rotational degrees of freedom of the fp about the helical first pa. k-medoids clustering algorithm ( ) was performed to categorize the membrane-bound poses of the fp, resulting in two major membrane binding modes. for all the frames identified as membrane-bound (see above for definition), a vector composed of the z-distances between the com of individual fp residues and the lipid phosphate plane was used as the dissimilarity metric for clustering. the cluster approximate centers, i.e., medoids, were viewed as the representative structures for each fp membrane binding mode identified. to better visualize the distribution of membrane-bound poses as well as the clustering results, principal component analysis (pca) was performed to reduce the dimensionality of the original data set. in pca, the covariance matrix of the z-distances between the com of individual residues and the phosphate plane was computed and diagonalized. the resulting eigenvectors, i.e., the principal components (pcs), represents the coordinates that maximize the variance of projected data. the first two pcs were selected to project the original z-distance data of membrane-bound frames along with the clustering result onto the reduced dimension. the pca was performed using the scikit-learn package ( ) . the sars-cov attachment to and its penetration into the human plasma membrane are key steps in viral infection that rely on the interaction of the viral fp with the host cellular membrane. this aspect is the central phenomenon that we study here using atomistic simulations, in order to characterize the binding pose and conformation of the sars-cov fp when bound to the membrane. in the following sections we first describe the results of our spontaneous membrane binding simulations in terms of the overall binding of the fp using some coarse parameters. then, we use the interaction of individual residues from the fp in clustering and examine them in a reduced dimension to classify better the different microstates that arise during the binding process and discuss their biological significance. in order to characterize the membrane binding mode of the sars-cov fp to a lipid bilayer representing human cellular membranes, we performed independent copies of hmmm, membrane-binding simulations. spontaneous diffusion and membrane binding of the fp in each simulation replica can be monitored by tracking the center of mass (com) of the peptide. the component of the com was tracked with respect to the phosphate layer (the average position of all the phosphate groups) of each leaflet (blue and red lines, respectively, in fig. ). due to the applied periodic boundary conditions, and the free diffusion of the fp in the solution, the fp was able to diffuse towards either the upper or the lower leaflet of the membrane (fig. ) , which both include a lipid composition resembling the outer leaflet of human plasma membrane ( ) . among the performed membrane-binding simulations, instances of both stable (majority) and transient binding events, as well as cases with no membrane binding, were observed. because the lipid molecules in the membrane are neutral, there are no dominating electrostatic driving forces between the fp and the lipid bilayer. therefore, the major driving forces between the two are hydrophobic effects, and the fp diffusion in the solution can make the peptide take a longer time to make the initial encounter with the membrane. for further analysis of the membrane interactions, we selected only the portions of the trajectories where "stable membrane binding" or "membrane-bound state" was defined (see methods for details). residue contacts between any heavy atom of the fp and any heavy atom of the lipid bilayer are shown in fig. where the red segments of the graphs represent stable binding. consisting of six independent systems and five replicas of each, % of the replicas contain stably bound configurations (fig. ) . we observe stable binding in, e.g., parallel replicas p , p , p , and in antiparallel replicas a , a , a (fig. ) . in some simulations, nearly the entire length of the fp was observed to be engaged with lipids (e.g., replicas p , p , n , s , and r ), while in other cases, only a specific part of the peptide makes contact with the membrane, specifically via either the loop or the helical part. biasing the initial placement of the peptides allowed the first interaction of the fp to diversify. some peptides residues (y-axis) with the lipid bilayer over time (x-axis) in sampling step size of ns. if the fp is in contact with the membrane (heavy atom distance of less than . Å) for at least ns, the midpoint of that segment of the trajectory is marked with a black dot. short-term, transient contacts are labeled with black dots. we observe stable binding in % of the replicas. bound to the lipid bilayer as soon as in ns, while others diffused and tumbled longer prior to interacting with the membrane, naturally allowing the peptide to unbias itself more from the effect of initial placement. examples include nosedive-placed peptides resulting in membrane-interacting fp via the loop part, or standing-placed peptides buried in the bilayer via their helical segment (fig. ) . using the stable membrane-bound states defined above, we further examined the average position and lipid interaction of individual fp residues. we calculated the z-distance of the side chain com to the phosphate layer of the membrane (fig ) . each ensemble average is calculated from the membrane-bound portions of the individual trajectory. the average depth of insertion of individual residues reaches as deep as Å below the phosphate layer, indicating interaction with the hydrophobic core of the membrane. we observe two major classes of membrane-bound states where either the alpha helical part (approximately residues - ) or the loop part (the rest of the residues at c-terminus) of the fp interact with the lipid bilayer. for example, replicas p , p , and n , the fp are bound to the membrane primarily through its alpha helical segment whereas in systems p , p , a , a , and a , the c-terminal end (the loop) is mostly engaged with the lipids. in order to better classify these binding modes in a reduced space, we will next do clustering of the obtained membrane-bound configurations. to analyze the ensemble of fp membrane binding configurations captured in the hmmm simulations where stable membrane binding was observed, clustering was performed using the z-distances between the the side chain com and the membrane lipid phosphate plane. despite the large variance in membrane-bound poses among md snapshots from different simulation replicas, two major clusters can be identified, which represent the two distinct binding patterns mentioned briefly above. we call the first cluster where the n-terminal, helical part of the fp interacts with the membrane, the "helix-binding" mode. in the other major cluster, the fp interacts with the bilayer primarily from the c-terminal loop, hereby referred to as the "loop-binding" mode. for better visualization, we performed principal component analysis (pca) and reduced the dimension to the first two principal components (pcs), which together explain % of the variance. the nature of these two pcs, pc and pc , was examined by fig. ). transparent green areas represent the standard deviation of the distance. the eigenvectors, which quantitatively evaluate the contribution of membrane insertion by each residue (fig. s ) . from the eigenvectors, pc measures the contrast of membrane insertion by the helix and the c-terminal loop, whereas pc measures mostly how much the c-terminal loop is inserted. all membrane binding configurations were then projected and visualized in the reduced dimension defined by pc and pc (fig. ) . examination of the projections and selected md snapshots are consistent with the presence of two major membrane-binding modes as introduced earlier, i.e., either the helix (fig. a,d) or the loop (fig. c,f) , with a few involving both segments only at the membrane surface (fig. b,e) . the flexibility of the c-terminal loop resulted in a variety of scenarios in terms of its membrane interaction, including its deep membrane insertion (e.g., d and f in fig. ), tangential interaction (e.g., c and e in fig. ), or no interaction (e.g., a and b in fig. ). the two major clusters of membrane binding poses are also visualized in the reduced dimension (fig. a) , along with the two cluster centers showing the representative membrane binding modes (fig. b,c) . as expected, the cluster corresponding to the helix-binding mode demonstrated a larger variance of the loop involvement in the membrane binding compared to the cluster representing the loop-binding mode. to further analyze the membrane binding configurations of the fp in our simulations, we analyzed the orientation of the helical segment of the fp (residues - ), which remains alpha helical throughout all the simulation replicas (fig. s ) . we traced the internal angles pa , f , and f defined to describe the fp helix orientation (see methods for the definition of the angles) over the simulation trajectories in fig. a , for replicas with stably bound configurations. in the simulations where the fp binds the membrane in a helix-binding mode (p , p , n , n , s , s , r , r ), pa stabilizes at ∼ • which corresponds to an oblique position relative to the membrane where the n-terminus is tilted towards the membrane core. since the loop is more flexible and the reference internal angle is defined at the -helix moving in the solution, it is more difficult to provide an equally accurate description for the loop-binding modes (p , a , a , a , n , s , i , i , i , i ). in most of the replicas towards the end of the trajectory, pa is less than • . this means that the n-terminal of the helix is facing up, and as the angle decreases the helix becomes more orthogonal to the membrane. in the rest of the loop-binding modes, pa is larger than • , meaning while the loop is inserted into the membrane, the helix is also interacting with the membrane but not fully inserted. the f angles in the helix-binding mode averaged around ∼ • where f is facing towards the lipid bilayer. this preference can be clearly seen in fig. b . since f is nearly located on the opposite side of the helix, this residue is mostly facing up in the membrane with f averaging around • . however, there is no specific preference for f and f angles in the loop-binding mode (fig. b ). within each family of viruses, the sequence of the fps is highly conserved. in addition, the alpha helical part of the sars-cov fp (sfiedllfnkv) is highly conserved among the coronaviridae family ( , ) , as indicated by the multiple sequence alignment (fig. s ), highlighting its importance in the viral life cycle. in our simulations we observed two membrane binding modes for the sars-cov fp, the helix-binding mode in which the helical segment of the fp engages with the membrane, and the loop-binding mode where the c-terminal loop of the peptide is the part primarily interacting with the lipids (fig. ). mutagenesis experiments have clearly shown that residues l , l , and f play a major role in viral fusion. given their importance, these residues are termed the "fusion active core" of the fp ( , ) . consistent with these results, in the helix-binding mode found in our simulations, residues l , l , and f are closely interacting with the membrane lipids with the two leucine side chains deeply inserted into the hydrophobic core of the membrane (fig. ) . notably, the same residues are found to be highly conserved among all human corononaviruses (fig. s ). given the high sequence conservation and established role of several residues in the helical segment of the fp that we simulated, we strongly believe and propose that the helix-binding mode represents the physiologically relevant membrane-bound form of the sars-cov fp. notably, from an energetic perspective, insertion of helical segments into the membrane might be less costly, since backbone amide groups in a helix satisfy their hydrogen bonds internally ( ) . based on structural data currently available, the loop-binding mode might become relevant to potential interaction of other segments of the protein with the membrane upon structural transition and cleavage of the s protein. in addition to the simulated segment in this study (which we refer to as the fp), there are other pieces proximal to the fp (named fp in sars-cov and fppr in sars-cov ) ( , ( ) ( ) ( ) ( ) , which are implicated in membrane fusion. the fusion peptide proximal region (fppr) of sars-cov downstream to the fp was later resolved and claimed to be involved in the structural rearrangement of the s protein prior to membrane fusion ( ) . an internal disulfide bond within the fppr, between c and c , was observed and is suggested to increase membrane-ordering activity ( , ) . the membrane-ordering activity of the fp, due to the fusion active core, is significantly higher than the fppr and the activity of fp/fppr together is only slightly increased compared to the activity of fp and fppr separately ( ) . the loop-binding mode might support the formation of a "fusion platform" where both fp and fppr interact with the membrane simultaneously as two subdomains ( ) . to characterize such platform axis changing with color in time. replica names are colored according to whether the fp is in a helix-(purple) or in a loop-binding mode (yellow). b) different helix orientations displayed by the two fp membrane-binding modes. the helix-binding mode prefers a pa angle between • and • , accumulated more around • whereas the loop-binding mode prefers a wider range of pa angles, spanning from • to • . unlike the loop-binding mode, the helix-binding mode demonstrates a strong preference for f rather than f to face towards the membrane. interacting with the membrane, a longer peptide including the fppr should be studied in future. , which emerged as a severe pandemic worldwide, calls for a need to accelerate the development of novel therapeutic intervention strategies. the s-protein of the sars-cov contains the key machinery necessary for the infection of human cell, including the fp, a highly conserved segment that inserts into the human cellular membrane initiating the fusion of the virus. in this study, using a large set of simulations, we describe how the sars-cov fp binds mammalian cellular membranes and characterize, at atomic details, lipid-protein interactions important for the stability of the bound state. characterizing the mechanism of the fusion driving fp-host membrane interactions is key to our understanding critical steps involved in the process of viral infection, paving way for potential development of novel therapeutics against sars-cov . these include modulation of fp-membrane binding interface through small molecules showing high specificity for this region of the s-protein, or inhibiting the key lipid-protein interactions observed. based on the suggested binding mode elucidated in our study, mutagenesis experiments can be designed to further confirm the role of the important residues implicated in membrane binding. given the close similarity of the fusion peptides in coronaviruses in general, these results can also be applicable to infections caused by other members of this life-threatening family of pathogens. dg, kk and et designed the research. dg carried out all simulations, dg and ml analyzed the data. all authors wrote the article. a familial cluster of 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interactions using an accelerated membrane model lipid specificity of the membrane binding domain of coagulation factor x differential membrane binding mechanics of synaptotagmin isoforms observed at atomic detail capturing spontaneous membrane insertion of the influenza virus hemagglutinin fusion peptide mafft: a novel method for rapid multiple sequence alignment based on fast fourier transform the jalview java alignment editor cryo-em structure of the -ncov spike in the prefusion conformation cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding vmd -visual molecular dynamics charmm-gui: a web-based graphical user interface for charmm exploring transmembrane transport through -hemolysin with grid-steered molecular dynamics scalable molecular dynamics with namd scalable molecular dynamics on cpu and gpu architectures with namd numpy/scipy recipes for data science: k-medoids clustering scikit-learn: machine learning in python sars-coronavirus spike s domain flanked by cysteine residues c and c is important for activation of membrane fusion experimentally determined hydrophobicity scale for proteins at membrane interfaces this work was supported by the national institutes of health (grants p -gm and r -gm to et). md simulations were performed using blue waters and computational resources provided by microsoft azure. key: cord- -jqm hxps authors: nan title: abstract date: - - journal: eur biophys j doi: . /s - - - sha: doc_id: cord_uid: jqm hxps nan standard proteomics techniques are unable to describe the stoichiometry, subunit interactions and organisation of assemblies since many are heterogeneous, present at low cellular abundance and frequently difficult to isolate. we have combined two existing methodologies to tackle these challenges: tandem affinity purification (tap) and nanoflow esi-ms. we use methods designed to maintain non-covalent complexes within the mass spectrometer to provide definitive evidence of interacting subunits based on the masses of complexes and subcomplexes generated by perturbation both in solution and gas phases. structural models will be presented for three oligomeric protein complexes of unknown structure: the yeast exosome and the human u snrnp and eif complexes. these models will then be examined within the context of their function. recent developments in mass spectrometry have added a further dimension to our studies of protein complexes: that of their collision cross-section. using ion mobility mass spectrometry we have been able to add spatial restraints to our models validating our models with measurements of collision cross-sections. very recently we have had a considerable breakthrough which has enabled us to preserve intact membrane complexes in the gas phase . this enables us to establish lipid and nucleotide binding and to define the stoichiometry and post translational modifications within the intact transmembrane regions of a number of complexes. in vivo molecular sensing: fluorescence beyond labeling f. beltram scuola normale superiore, i- pisa, italy fluorescent molecules are powerful reporter tools that have much extended the impact of optical microscopy, particularly thanks to the flexibility of genetically encoded tags. detection can now target single molecules even in the complex environment of intact live cells offering unprecedented insight on biological processes in real time in live cells and tissues. fluorescent labels, however, can do more that this. our increased ability to tailor molecule and, more in general, nanosystem properties allows us to design, produce and exploit intelligent tags that can actually analyze the cellular environment. today multifunctional nanosystems can be produced that provide a signal dependent on the value of a specific biochemical parameter. importantly these nanosystems can target specific subcellular domains and have the ability to be used also in the case of live organisms. recent results will be discussed that highlight the impact of nanobiotechnology in this context with a particular emphasis given to methods suitable for in vivo studies that can be transferred to the biomedical world. far-field optical nanoscopy s. w. hell max planck institute, göttingen, germany the resolution of a far-field optical microscope is usually limited to d = λ/ ( n sin α) > nm, with n sin α denoting the numerical aperture of the lens and λ the wavelength of light. we will discuss lens-based fluorescence microscopy concepts that feature a resolving power on the nanoscale. all these concepts share a common basis: exploiting selected (pairs of) states and transitions of the fluorescent marker to neutralize the limiting role of diffraction. specifically, the fluorophore is switched on and off, that is, between a bright and a dark state, to detect the emission of adjacent features sequentially in time. the first viable concept of this kind was stimulated emission depletion (sted) microscopy in which the fluorescence ability of the dye is switched off by stimulated emission. in the sted microscope, the extent of the region in which the molecule is able to fluoresce follows d ≈ λ/ n sin α + i/i s , meaning that fluorophores that are further away than d can be separated. i is the intensity that drives a fluorophore from the bright fluorescent state to the dark ground state by stimulated emission. i s depends (inversely) on the lifetime of the states. for i/i s → ∞, it follows that d → , meaning that the resolution can be molecular). altogether, far-field optical 'nanoscopy' is a fascinating development in optics with high relevance to the many areas of sciences, in particular the life sciences. since it has already been a key to answering important questions in biology, and owing to its simplicity and commercial availability, we expect far-field fluorescence 'nanoscopes' to enter most cell biology and many nanoscience laboratories in the near future. grabbing the cat by the tail: discrete steps by a dna packaging motor and the inter-subunit coordination in a ring-atpase c. bustamante, j. moffitt university of california, berkeley, california, u.s.a. as part of their infection cycle, many viruses must package their newly replicated genomes inside a protein capsid. bacteriophage ϕ packages its . mm long double-stranded dna into a nm dia. x nm high capsid using a multimeric ring motor that belongs to the asce (additional strand, conserved e) superfamily of atpases. a number of fundamental questions remain as to the coordination of the various subunits in these multimeric rings. the portal motor in bacteriophage phi is ideal to investigate these questions and is a remarkable machine that must overcome entropic, electrostatic, and dna bending energies to package its genome to near-crystalline density inside the capsid. using optical tweezers, we find that this motor can work against loads of up to ∼ piconewtons on average, making it one of the strongest molecular motors ever reported. we establish the force-velocity relationship of the motor. interestingly, the packaging rate decreases as the prohead fills, indicating that an internal pressure builds up due to dna compression attaining the value of ∼ megapascals at the end of the packaging. we show that the chemical energy of atp is converted into mechanical work during phosphate release. using ultra-high resolution optical tweezers, we determined the step size of the motor and established the coordination of the polymerases around the ring. we propose a comprehensive model of the operation of this motor. watching proteins function in real time via timeresolved x-ray diffraction and solution scattering p. a. anfinrud laboratory of chemical physics/niddk, nih, bethesda, maryland, u.s.a. to generate a deeper understanding into the relations between protein structure, dynamics, and function, we have developed x-ray methods capable of probing changes in protein structure on time scales as short as ps. this infrastructure was first developed on the id b time-resolved x-ray beamline at the european synchrotron and radiation facility, and more recently at the id b biocars beamline at the advanced photon source. in studies of ligand-binding heme proteins, a picosecond laser pulse first photolyzes co from the heme, then a suitably delayed picosecond x-ray pulse passes through the protein and the scattered x-rays are imaged on a d detector. when the sample is a protein crystal, this "pump-probe" approach recovers time-resolved diffraction "snapshots" whose corresponding electron density maps can be stitched together into movies that unveil the correlated protein motions that accompany and/or mediate ligand migration within the hydrophobic interior of the protein. when the sample is a protein solution, we recover timeresolved small-and wide-angle x-ray scattering patterns that are sensitive to changes in the size, shape, and structure of the protein. scattering studies of proteins in solution unveil structural dynamics without the constraints imposed by crystal contacts; thus, these scattering "fingerprints" complement results obtained from diffraction studies. this research was supported in part by the intramural research program of the nih, niddk dc-sign is a trans-membrane protein expressed on antigen presenting cells and recognizes pathogens like hiv- , hepatitis c virus and ebola. by electron microscopy and near-field optical nanoscopy, we demonstrated that at the plasma membrane dc-sign is organized in well-defined nanodomains of - nm in diameter. intensity-size correlation analysis revealed remarkable heterogeneity in the nanodomains molecular packing density. we constructed and characterized several dc-sign mutated forms lacking specific molecular domains. by immunogold labeling and spatial point pattern analysis, we show that the extracellular neck domain is essential for dc-sign nanoclustering. finally, we present a model that describes the probability of a cell to have a certain number of receptors joining the contact site in the initial encounter with an external object. monte carlo simulations subsequently define the parameters that are determinant in the object-cell encounter. our results show that receptor nanoclustering is of particular importance for binding objects of sizes comparable to the nanocluster size, indicating that the nanoscale spatial organization of dc-sign is optimized for binding to virus-sized objects. imaging of mobile stable lipid rafts in the live cell plasma membrane m. brameshuber , j. weghuber , v. ruprecht , h. stockinger , g. j. schuetz johannes kepler university linz, austria, medical university of vienna, austria the organization of the cellular plasma membrane at a nanoscopic length scale is believed to affect the association of distinct sets of membrane proteins for the regulation of multiple signaling pathways. based on in vitro results, conflicting models have been proposed which postulate the existence of stable or highly dynamic platforms of membrane lipids and proteins. here we directly imaged and further characterized lipid rafts in the plasma membrane of living cho cells by single molecule tirf microscopy. using a novel recording scheme for "thinning out clusters while conserving stoichiometry of labeling" , molecular homo-association of gpi-anchored mgfp was detected at • c and ascribed to specific enrichment in lipid platforms. the mobile mgfp-gpi homo-associates were found to be stable on a seconds timescale and dissolved after cholesterol depletion. having confirmed the association of mgfp-gpi to stable membrane rafts, we attempted to use an externally applied marker to test this hypothesis. we used bodipy-gm , a probe that was recently reported to be enriched in the liquid-ordered phase of plasma membrane vesicles. when applied to cho cells at different surface staining, we found that also bodipy-gm homo-associated in a cholesterol-dependent manner, thus providing further evidence for the existence of membrane rafts. [ ] appl phys lett , ( ) . synapsin knock-out mice as an in vitro model of human epilepsy studied with multi-electrode arrays d. f. boido, p. farisello, p. baldelli, f. benfenati department of neuroscience and brain technologies, italian institute of technology, genova, italy mutant mice lacking synapsins (syn), a family of synaptic vesicles (sv) proteins implicated in the regulation of neurotransmitter release and synapse formation, are epileptic. the attacks appear after the third month of age and severity increases with age. several mutations of syn genes have been found in families of patients with epilepsy. we used micro-electrode arrays (meas) to study spontaneous and chemically evoked epileptiform activities in cortico-hippocampal brain slices obtained from wild-type (wt) and synko mice. -months old synko mice show sporadic ictal (ic) events in the entorhinal cortex. a potassium channel blocker, aminopyridine ( ap), elicits ic and inter-ictal (i-ic) events in both wt and synko slices. in the hippocampus of young synko ( -days old) mice, ap induces i-ic events at higher frequencies than in wt mice. also the frequency of ic events, mainly observed in the cortex, is higher in synko. the analysis of adult ( -year old) mice, revealed a clear age-related aggravation, which paralleled the increase in the severity of the epileptic phenotype observed in vivo. many slices from adult synko mice showed an ic event, while wt slices were refractory at this age to experience ic activity. synko mice are useful to study how neuronal network hyperexcitabilty due to mutations in sv proteins leads to the development of epileptiform activity. meas proved themselves to be useful tools to characterize the epileptic signals foci and patterns of propagation. high electron mobility transistor (hemt) structures were used to bridge the gap between the analysis of biological reactions and biophysical characterization. the combination of nanotechnological measurement approaches with biological reactions provides new possibilities for living cell examinations after exposure to ionizing radiation and basically during the irradiation experiments itself. in this transdisciplinary approach experimental data and handling of biological material enables the identification and specification of systems properties of biological responses to ionizing radiation at different hierarchical levels. gan/algan-heterostructures form a hemt with a gate very sensitive to ph-value changes and potential changes in general. to record cell membrane potentials and ion fluxes during and after irradiation experiments living cells are cultivated on the functionalised biocompatible chip surface. here, we present results of x-ray stimulated cell responses grown on gan-chip surfaces. we recorded transistor signal changes of . µa within s caused by an irradiated cell monolayer. to measure cell potentials, not only after irradiation experiments but also during the irradiation itself expands the examination restrictions in an enormous way. measuring diffusion by spatial-cross-correlation e. gratton, m. a. digman laboratory for fluorescence dynamics, university of california, irvine, u.s.a. fluorescence correlation spectroscopy (fcs) has emerged as a very powerful method to study the motions of proteins both in the interior and exterior of the cell. it provides information at the single molecular level by averaging the behavior of many molecules thus achieving very good statistics. single particle tracking (spt) is also a highly sensitive technique to measure particle movement. however, the fcs method suffers in spatial resolution while the spt technique only allows for the tracking of isolated molecules. here we propose a change of paradigm in which using spatial pair cross-correlation functions we can overcome this limitation. our method measures the time a particle takes to go from one location to another by correlating the intensity fluctuations at specific points on a grid independently on how many particles are in the imaging field. therefore we can trace the average path of the particles. for example, our method could be used to detect when a protein passes the nuclear barrier and the location of the passage. this information cannot be obtained with the frap (fluorescence recovery after photobleaching) technique or the image correlation spectroscopy method. the interaction of the bax c-terminal domain with membranes j. c. gomez-fernandez, s. sanchez-bautista, a. perez-lara, s. corbalan-garcía departamento de bioquimica y biologia molecular. universidad de murcia, murcia, spain the c-terminal domain of the pro-apoptotic protein bax (bax-c) acts as a membrane anchor during the translocation to the membrane of this protein leading to programmed cell death. we have used static and mas-nmr techniques to show that the interaction of bax-c with membranes is modulated by the presence of a negatively charged phosphatidylglycerol. the width of the resonance peaks were considerably more increased by bax-c, in the presence of phosphatidylglycerol. bax-c substantially decreased the t relaxation times of phosphatidylglycerol and those of phosphatidylcholine when mixtured with phosphatidylglycerol but they were not decreased when phosphatidylcholine was the only phospholipid present in the membrane. c-mas-nmr showed that t values were decreased when bax-c was incorporated and, when phosphatidylglycerol was also present, the decrease in t affected considerably more to some carbons in the polar region. these results indicate that bax-c interacts differently with the polar part of the membrane depending on whether phosphatidylglycerol is present or not, suggesting that an electrostatic interaction of bax-c with the membrane determines the membrane disposition of this domain. fluorescence spectroscopy showed that the trp residues of bax-c were located in a microenvironment more hydrophobic when phosphatidylglycerol was present. h. g. franquelim , l. m. s. loura , n. santos , m. castanho instituto de medicina molecular, univ. lisboa, portugal, faculdade de farmácia, univ. coimbra, portugal since the efficacy of hiv fusion inhibitors was previously reported to be related to an ability to interact with membranes, we studied the interaction of the hiv fusion inhibitor sifuvirtide, a aa negatively charged peptide, with lipid vesicles. since this peptide has aromatic residues, fluorescence spectroscopy techniques were used with no need for attached probes. results showed no significant interaction with both zwitterionic fluid phase and cholesterol-enriched membranes; however extensive partition to fluid phase cationic membranes were observed. in the dppc gel phase, however, an adsorption at the surface of these membranes was detected by using a differential quenching approach with lipophilic probes, as well as by fret. moreover, the interaction with gel phase membranes seems to be specific towards pc vesicles, since no significant interaction was retrieved for membranes composed by shingomyelin and ceramide. besides fluorescence, atomic force microscopy and zeta-potential were used to further investigate this issue. our results show a selectivity and specificity of the peptide towards rigid domains, where most of the receptors are found, and help explain the importance of the interaction with membranes in the improved efficacy of sifuvirtide compared to other fusion inhibitors, by providing a local increased concentration of the peptide near the fusion site on both cellular and viral membranes. the study of molecular dynamics at the single-molecule level with fluorescence far-field optics offers new detailed insights into scientific problems, especially in living cells. unfortunately, the resolution of common far-field techniques is limited to about nm in the lateral direction by diffraction. in recent years, several concepts such as stimulated emission depletion microscopy (sted) have been successfully applied to overcome the diffraction barrier by exploiting the photophysical properties of fluorescent labels. we present the combination of high resolution sted microscopy with different fluorescence fluctuation techniques providing the unique ability to study molecular dynamics with high spatial (< nm) and temporal resolution (< ms) in living cells. using fluorescence correlation spectroscopy (fcs) and general single-molecule analysis, we were able to explore single-molecule dynamics in up to -fold reduced focal volumes on two-dimensional samples such as lipid membranes with excellent signal-to-noise ratios. special attention is drawn to inhomogeneous lipid diffusion on the plasma membrane of living cells . by extending the available spatial scale of standard single-molecule fluorescence far-field spectroscopy techniques, our experiments outline a new way of approaching scientific problems. modulation of the properties of membrane microdomains as a control mechanism in cellular physiology p. o'shea cell biophysics group, institute of biophysics, imaging & optical science, school of biology, university of nottingham, nottingham ng rd, u.k. this presentation will outline the molecular-physical rationale of how membrane microdomains may modulate the behaviour of membrane receptor systems as a controlling mechanism in cell signaling. a number of external factors that modulate these properties will be indicated that have a bearing on controlling cellular behaviour. some of key questions will be considered such as the factors that control the assembly and disassembly of microdomains, the size and numberdensity of the microdomains and the lifetime that they exist within the membrane. the technical challenges that these questions identify will also be outlined with some possible solutions. throughout this presentation, correlations will be made between theory and experiment as well as between model membrane systems and real cellular systems. structural and dynamic properties of caveolin- and - fragments at the membrane interface c. le lan , j. gallay , m. vincent , j.-m. neumann , b. de foresta , n. jamin cea, ibitecs, sb sm, & ura cnrs , gif-sur-yvette, france, ibbmc, université paris-sud, umr -cnrs, ifr , orsay, france caveolins are major protein components of caveolae, microdomains of the plasma membrane involved in a large number of biological functions, including signal transduction, cholesterol homeostasis and transport. the consensus topological model of caveolin- includes a small central intramembrane region ( - ) flanked by two cytosolic amphiphilic domains ( - and - ) which probably constitute in-plane membrane anchors. we investigated the interaction of the cav- ( - ) juxta-membrane segment with various membrane mimics, using fluorescence, cd and nmr. this segment partitioned better in dpc and in dm/anionic lipids micelles than in dm micelles and this partitioning was coupled with the formation of an amphipathic α-helix. this amphipathic helix was located in an average shallow position, in the polar head group region of the dpc micelle, as shown by fluorescence data and intermolecular noes, with the aromatic doublet w -f probably pointing towards the inside of the micelle on average. the peptide encompassing the homologous sequence of cav- was also localized to the dpc micelle polar head group region, in which it adopted a more stable helical conformation than cav- ( - ) . these data brings experimental support for the role of this segment as an interfacial membrane anchor. resveratrol (trans- , ', -trihydroxystilbene) , a phytoalexin present in grapes and its analogue piceatannol (trans- , , ', '-tetrahydroxystilbene) are biologically active compounds and possess potential chemopreventive and anticancer properties. the activity of resveratrol and piceatannol can be mediated by membrane effects since structure of lipid membrane domains may play an important role in cell signalling pathways. drugs interactions with dmpc bilayers was investigated using a combination of esr spectroscopy and differential scanning calorimetry. spin probes used in epr experiment were located in different part of lipid bilayer. study was performed at temperatures below and above phase transition temperature (t m ). epr spectra were simulated and displayed with ghost condensation method. the values of ϑ and ϕ (the main and asymmetry cone angles of wobbling spin probe, respectively) were taken for free rotational space parameter (Ω) calculation. the decrease of Ω values was observed in the presence of both compounds and the effect was more pronounced in lipid gel phase. order parameter and correlation time were also determined and presented in form of ghost patterns. using this approach the differential influence of studied compounds on membrane heterogenity was revealed. separating hydrostatic pressure from cellular strain: development of an in vitro model system r. sulley , j. whatmore , c. p. winlove , a. shore , j. tooke , r. ellis , k. gooding peninsula medical school, school of physics, university of exeter, uk endothelial cells (ecs) line blood vessels & are constantly subjected to haemodynamic and mechanical stresses and strains. these stimuli are known to influence ecs, modifying their morphology, intracellular signalling & gene expression. most reported systems exposing ec to mechanical forces in vitro alter pressure & strain simultaneously, making it impossible to distinguish the two potentially independent stimuli. this distinction is particularly relevant when examining the interaction of haemodynamic forces on microvascular ecs, which are exposed to low hydrostatic pressure but significant strains. this research aims to create an in vitro system that can independently examine the effects of pressure and strain, over a range experienced by ecs in the microvasculature. human ecs are seeded ( x ˆ cells/cmˆ ) on to the inner surface of compliant mm diameter tubing. which is mounted on a perfusion rig inside a sealed, fluid-filled chamber. a continuous sinusoidal cyclical strain of - % is created by a pump attached to the external chamber. lumenal pressure is generated using two hydrostatic pressure heads. validation experiments show that pressure & substrate strain can be independently varied & controlled over a physiological range. the system is now being used to investigate the effects of pathophysiological haemodynamic abnormalities on ec function. membrane potential dynamics of living cells in response to femtosecond laser irradiation n. i. smith , j. ando , k. fujita , s. kawata photonics advanced research center, osaka university, suita, osaka - , japan, dept of applied physics, osaka university, suita, osaka - , japan the ultrashort pulsed near-infrared femtosecond laser has had a large impact in biomedical research fields and in microscopy, where it has enabled new imaging methodologies. at high intensities, the focused beam of a femtosecond laser has been used to irradiate specific locations inside a cell, often beneath the cell membrane, exploiting the inherent penetration and localized absorption that comes from the multiphoton absorption physics. this has been applied in photobleaching, photouncaging, laser surgery and other experiments where the light is used not merely for observation but is instead an integral tool to interact with the dynamics of cells, to probe and perturb the cell condition. in this talk i will discuss biological and mechanical effects that can be generated by short exposures to femtosecond laser irradiation, such as calcium waves, membrane hyperpolarization, and cell contraction. this talk will concentrate on the changes in membrane potential that can occur when the cell is subjected to focused femtosecond laser beams. both depolarization and hyperpolarization of the membrane potential could be evoked, depending on the laser parameters and on the position of the laser focus. these results have implications for the use of laser beams in microscopy, optical gene transfection, and laser nanosurgery. in a recent study we showed that the melting behavior of supported lipid bilayers (slbs) on mica can be influenced by the solution ionic strength and by the slb preparation temperature [ ] . by changing these parameters we could control the coupling between the two bilayer leaflets obtaining a coupled or decoupled melting behavior. thus, we could provide evidence that the slb model system is also suited for the study of lipid/protein interactions which had been questioned in the past. then we investigated the mutual interactions between the membrane lipids (pope:popg : ) and the kcsa potassium ion channel by studying kcsa proteins reconstituted in slbs. in particular, we studied the melting behavior of the slb and the ion channel distribution relative to the different membrane phases by temperature controlled atomic force microscopy (afm). by decreasing the temperature we found that the proteins underwent diffusion so to be excluded from the growing solid ordered regions. further, the ion channels tended to accumulate at the domain boundaries or they aggregated in the liquid disordered phase. both effects have been suggested to affect protein function. when we started from a low temperature at which the membrane was mainly in the solid ordered phase the membrane melting processes started in the vicinity of the included ion channels. we report fluorescence recovery after photobleaching (frap) measurements performed at variable spot radius for t -egfp-hmop receptors on sh-sy y neuroblastoma cells in the presence of ligands. two different agonists, damgo and morphine, caused markedly different changes to receptor diffusion as compared to the basal state. like receptors in the absence of ligand, receptors bound to morphine exhibited diffusion confined to joint semi-permeable domains, but with smaller domain size and diffusion coefficient. this effect was inhibited by pertussis toxin, suggesting that this dynamic behaviour is associated with early steps of signaling. in the presence of damgo, half of the receptors displayed free long-range diffusion and the other half were confined to smaller isolated domains. hypertonic sucrose buffer suppressed this effect which we attribute to receptor entry into clathrin-coated pits. it is likely that the observation of distinct receptor dynamics in the presence of damgo and morphine involves the agonist-selective phosphorylation of the receptor. the alteration of the physiological transcriptional program is one of the constant features of cancer cells. however, the characterization of the chromatin changes at single-gene level requires going beyond the diffraction limit affecting conventional fluorescence microscopy. the ability of molecular biology techniques to obtain a detailed view of the chromatin status at a sub-promoter resolution has to pay instead the price of averaging over a cell population. we report here the application of an approach based on high-resolution cytometry, chromatin immuno-precipitation and transcriptional profiling (dna microarray) for the characterization of the transcriptional and chromatin changes induced by the oncogenic transcription factor pml/rarα. the presentation will focus on the imaging protocol employed to observe the effects on the chromatin status and the extent of the deregulation induced on transcriptional activity in acute promyelocytic leukemia cells. this multiple-approach examination provides a further step towards the comprehension of the hierarchy of chromatin modifications leading to the establishment of a malignant transcriptional program. dna is rigid negatively charged polymer and in solution exists in extended conformation. i n vivo, volume occupied by dna must be reduced to fit to tiny space of cell nucleus. to condense dna, dna-dna electrostatic repulsion must be cut off that is achieved by interaction with cationic ligands. in binding to dna, oligocations compete with salt cations (k + , na + , mg + ). description of salt dependence of oligocation-induced dna condensation is still lacking. we studied dna condensation by model oligocations, ε-oligo(l-lysines), with variation of charge from + to + . combination of light scattering, uv-monitored precipitation assay and isothermal titration calorimetry allowed covering wide range of dna (c dna ) and salt (c kcl ) concentrations. salt dependence of dna condensation efficiency of the ligand, ec (ligand concentration at the transition midpoint) displays two regimes: salt-independent at low c kcl and salt-dependent at higher c kcl (steep increase of ec with c kcl ). simple formula describing ec as function of ligand charge, c dna and dissociation constant of ligand-dna complex (k d ), was proposed. in the salt-independent regime ec is defined by c dna . salt-dependence of ec is rooted in the variation of k d with c kcl earlier described in ligand-dna binding studies. importance of our findings for description of chromatin is discussed. interaction between proteins from linker region of nucleosome in presence/absence of dna in solution i. b. kipenko , e. v. chikhirzhina , a. m. polyanichko faculty of physics, saint-petersburg state university, russia, laboratory of cell biochemistry, institute of cytology, ras, saint-petersburg, russia interactions in the linker region of the nucleosome play a key role in the structural organization of the chromatin. the most fascinating and least understood is the interplay between non-histone chromatin protein hmgb and a linker histone h . it is known that both h and hmgb bind the linker region of the dna in vivo. however it is still a matter of debate as to whether these proteins assist each other or compete upon binding. the main attention in this work is paid to the investigation of the interactions between the hmgb and h proteins in physiological environment. using circular dichroism (cd) spectroscopy we have studied the interactions between hmgb and h at various hmgb /h ratios (r). it has been shown that there is a cddetectable interaction between the proteins h and hmgb at r < . we have demonstrated that the interaction between these proteins results in changes of their secondary structure. cd indicates that the structural impact of the unordered fragments decreases while the net α-helicity of the proteins increases upon the interaction. we have also shown that large higher order structures are formed in solution. in this work we have also discussed the dna-binding properties of the hmgb and h proteins. the work was supported by a rfbr grant ( - - ) and the government of staint-petersburg. t. u. rodionova , a. m. polyanichko , v. i. vorob'ev faculty of physics, saint-petersburg state university, russia, institute of cytology of the russian academy of sciences, saint-petersburg, russia hmgb is a nonhistone chromosomal protein. data regarding the structure of the hmgb-proteins obtained so far are rather different. thermodynamic experiments reveal predominant α-helical structure of the proteins only at temperatures below + • c, i.e. under physiological conditions they are mainly disordered. despite a lot of experimental data biological role of hmgb still remains unclear. it is believed that the proteins perform structural functions in chromatin and participate in various regulatory processes in cell. using circular dichroism spectroscopy and dna melting analysis we have shown that hmgb changes its structure upon binding to dna. it was shown that at room temperature only about % of amino acid residues form α-helices, while in dna-hmgb complex the degree of the α-helicity of the protein increases to approximately %. based on the data obtained we estimate the size of hmgb binding site as - b.p. we have also demonstrated that despite of strong dna-bending properties of hmgb its binding to dna results in increase of the double helix termostability. the authors are grateful for the financial support from the russian foundation for basic research (grants - - , - - ) and the government of saint-petersburg. structural organization of supramolecular complexes of dna with chromosomal proteins hmgb and h a. m. polyanichko , h. wieser faculty of physics, saint-petersburg state university, russia, dept. of chemistry, university of calgary, canada a combination of uv and ir absorption and circular dichroism spectroscopy together with atomic force microscopy was applied to investigate the structure and formation of large supramolecular dna-protein complexes. this combination of techniques was used to overcome limitations of uv-cd spectroscopy due to considerable light scattering in such solutions. based on the analysis of ftir and uv circular dichroism spectra and afm data the interaction of dna with highmobility group non-histone chromatin protein hmgb and linker histone h was studied. it is believed, that hmgb-domain proteins perform both structural and regulatory functions in chromatin. however, the particular mechanisms of it functioning remain unclear/ our data show that histone h facilitated binding of hmgb to dna by interacting with the sugar-phosphate backbone and binding of asp\glu amino acid residues of hmgb . acting together, hmgb and h stimulated the assemblage of supramolecular dna-protein structures. the organization of the ternary complexes is modulated by the interactions between hmgb and h molecules. the dna-proteins interactions in the presence of metal ions were different, causing prominent dna compaction and formation of large intermolecular complexes. the work was supported by rfbr (grant - - ). biophysical properties and mechanisms of phage dna ejection t. mdzinarashvili , m. khvedelidze , a. ivanova , t. partskhaladze , n. shengelia i. javakhishvili tbilisi state university, tbilisi, georgia, institute of molecular biology and biophysics, tbilisi, georgia to determine the requirements for phage adsorption on bacterial cell and for the realizing resources of following dna ejection thermodynamic and hydrodynamic methods were employed. the temperature, bacterial membrane fragments and receptors had been chosen as such external factors. the phages with short and long tail, both contractile and noncontractile have been studied. our viscometric studies of the phage dna ejection induced by receptor by the example of t phage and its receptor fhua have shown that the minimum protein-to-phage ratio necessary for complete dna release is to . the viscometric study of ddvi phage dna ejection induced by membrane fragments obtained from its host cells has shown that the environmental conditions play significant role in ejection process. both methods show that the thermally induced phage dna ejection for all investigated by us phages have shown that this process is nonenthalpic. finally from our experimental results we conclude that the start of the dna ejection process from the phage particle occurs without additional energy from either a physical or chemical source. we thank gnsf for the financial support. nitroxides induce apoptosis through caspase- activation and collapse of mitochondrial potential k. matczak , a. koceva-chyla , k. gwozdzinski , z. jozwiak department of thermobiology, department of molecular biophysics, university of lodz, lodz, poland nitroxides are new class of antioxidants that have been proved to show high reactivity toward free radicals. they act as superoxide dismutase mimics dismutating superoxide anions, but can also exert pro-oxidative properties. in view of their possible dual activity nitroxides could be of great importance in medicine. we have investigated pro-apoptotic activity of pyrroline and pyrrolidine nitroxides pirolid (pd) and pirolin (pl) in human breast cancer cells. in cancer, it is the failure of malignant cells to undergo apoptosis that is crucial. using microplate fluorescence methods, we estimated kinetics of changes in mitochondrial transmembrane potential and caspase- activity in breast cancer cells mcf- treated with pirolin or pirolid. these features are connected with induction of apoptosis in some type of cancer cells. we observed steady-state increase in caspase- activity up to h of postincubation that was followed by a decrease in the enzyme activity at h. caspase- activation was considerably greater in cells treated with pirolid. both nitroxides also caused notable decrease in mitochondrial transmembrane potential, which suggest that they can induce apoptosis in breast cancer cells through mitochondrial pathway. o. chernyavskiy , l. vannucci , p. bianchini , f. difato , l. kubínová dept. of biomathematics, inst. of physiology, as cr, prague, czech republic, dept. of immunology, inst. of microbiology, as cr, prague, czech republic, lambs, dept. of physics, university of genoa, italy, dept. of neuroscience and brain technologies, the italian institute of technology, genoa, italy the second harmonic generation (shg) imaging along with confocal laser scanning microscopy in reflectance mode can be applied to imaging unstained tissues in vivo, so it can be considered as a fast and non-invasive tool for in vivo studies. murine b f melanoma cells after subcutaneous inoculation in syngeneic mice were let to develop into tumor up to - mm in diameter. microscopic images were taken before and after microwave hyperthermia treatment (mwht). the microscopic images were acquired by -photon imaging in reflectance mode, shg imaging and -photon imaging of tissue autofluorescence. the evaluation of changes in the images after mwht of the tumor demonstrated changes in the architecture and organization in both the tumor capsule and tumor mass. the presented study was supported by the academy of sciences of the czech republic (grant iaa , institutional research concepts no.av z , av z ), and ministry of education, youth and sports of the czech republic (research program lc ). intracellular delivery and fate of peptide-capped gold nanoparticles: towards cellular biosensors y. cesbron , v. sée , p. free , p. nativo , d. g. spiller , m. r. h. white , m. brust , b. lounis , r. lévy liverpool institute for nanoscale science, engineering and technology, liverpool, uk, université bordeaux i / cnrs, bordeaux, france gold nanoparticles (nps) have extraordinary optical properties that make them very attractive single molecule labels. although understanding their dynamic interactions with biomolecules, living cells and organisms is a prerequisite for their use as in situ sensors or actuators. while recent research has provided indications on the effect of size, shape, and surface properties of nps on their internalization by living cells, the biochemical fate of nps after internalization has been essentially unknown. here we show that peptide-capped gold nps enter mammalian cells by endocytosis. we demonstrate that the peptide layer is subsequently degraded within the endosomal compartments through peptide cleavage by the ubiquitous endosomal protease cathepsin l. preservation of the peptide layer integrity and cytosolic delivery of nps can be achieved by a combination of cathepsin inhibition and endosome disruption. this is demonstrated using a combination of distance-dependant fluorescence unquenching and photothermal heterodyne imaging. these results prove the potential of peptide-capped gold nps as cellular biosensors. current efforts focus on in-vivo labeling of nps, nanoparticle-based real-time sensing of enzyme activity in living cells, and the development of photothermal microscopy for single nanoparticle imaging in living cells. towards intravital two photon microscopy study of lymphocytes mobility in lymphonodes m. caccia , l. sironi , m. collini , i. zanoni , t. gorletta , m. di gioia , g. francesca dipartimento di fisica, università di milano bicocca, italy, dipartimento di biotecnologie e bioscienze, università di milano bicocca, italy during the last years the edge between optical fluorescence based microscopy and the world of bio-medical research has become thinner and thinner and two photon laser scanning microscopy (tplsm) is one of the most powerful tool for immunological and medical research. one of the most limiting step in intravital microscopy is the preparation of the animal model and the number of animals to be sacrificed to get good statistics. alternative routes must be searched. we employ tplsm to explanted lymphnodes. two photon excitation and a non descanned detection mode allow to increase, respectively, the excitation and detection efficiency while the explanted organs are kept very close to the condition they experience in live animals by means of an home-made temperature controlled box surrounding the microscope and a system for the flux of physiological fluids. experiments performed on explanted lymphonodes, kept under constant flux of co -o saturated buffers and at o c, agree with literature for what concern t-cell homing and motility. this seems to confirm that t-cells behavior in explanted organs mantained in physiological conditions is very similar to that observed in live animals. we then believe that our tplsm microscope would allow to study cell behaviors in in-vivo with high efficiency and a little technical effort. a generalized quantitative frap method with no restriction on the size of the photobleached area heterochromatin protein in dna damage response -recruitment or dissociation from repair sites? j. w. dobrucki division of cell biophysics, faculty of biochem. biophys. and biotechnol., jagiellonian university, kraków, poland we studied recruitment of dna repair proteins to damage sites in live cells, by microscopy approaches, using a new method of inflicting local, sublethal damage in nuclei of live cells. oxidative damage, which was inflicted by exciting dna-intercalated ethidium with focused green light, triggered recruitment of base excision repair enzymes. surprisingly, an epigenetic regulator, heterochromatin protein (hp ) (zarebski et al., ) was recruited to damage as well. hp is a constitutive component of heterochromatin, and plays an important role in transcriptional repression and regulation of euchromatic genes, however it was not known to be required for repair of oxidative damage. the finding of hp recruitment is particularly puzzling, since in another study hp was shown to dissociate from chromatin as a result of dna damage (ayoub et al. ) . technical aspects of live cell imaging that may explain these contradictory results will be discussed. in this presentation, we report a method for determining both the presence and the stoichiometry of protein complexes at pixel resolution and apply it to disassembling focal adhesions. the method is derived from fluorescence fluctuation methods that have single molecule sensitivity and is based on our previously described n&b (number and brightness) method that measures the number and brightness (aggregation state) of fluorescent molecules in every pixel of a confocal microscope image. the new method exploits the correlation of fluorescence amplitude fluctuations for two colors and detects the presence of molecular complexes and their stoichiometry. while the original n&b method was developed for one color, i.e., a single molecular species, the new method, ccn&b, extends the analysis to two colors and introduces the concept of cross-variance. this method is similar in concept to the two-color pch analysis. however, the covariancebased ccn&b method also generates pixel resolution maps of protein complexes and can be used on commercial confocal microscopes. the method is highly sensitive and has relatively high temporal resolution. we have applied this method to adhesion complexes in cells. in addition of their structural role, to link the extracellular substratum to actin filaments, they also serve as signaling centers that regulate many cellular processes including their own assembly and turnover, migration, gene expression, apoptosis, and proliferation. spatio-temporal analysis of membrane lipid remodeling during phagocytosis s. de keijzer , d. kilic , c. g. figdor , s. grinstein , a. cambi department of tumor immunology, radboud university of nijmegen, nijmegen, the netherlands, department of biochemistry, university of toronto, toronto, canada the constant threat posed by pathogens and cell debris is tackled by phagocytosis, the process through which cells engulf and destroy dangerous material. the signaling, targeting and trafficking during phagocytosis is dependent on cytoskeleton rearrangements and membrane remodeling. it is becoming increasingly evident that lipids play an important role and can affect the phagocytic response. they assemble microdomains which can act as signaling platforms and confer charge and curvature to the membrane surface promoting electrostatic attraction and retention of proteins. little is known about the mechanism(s) regulating membrane lipid remodeling during phagocytosis. here we used fluorescently labeled biosensors based on k-ras and h-ras proteins to obtain spatio-temporal information on phagosomal membrane lipid remodeling during fcreceptormediated phagocytosis. the results show that cell activation by cytokines modulates the kinetics of anionic lipids thus affecting the membrane charge and the recruitment of cytosolic proteins to the phagosomal membrane. our data emphasize the fundamental role of lipids in the generation and transduction of signals in phagocytosis, and we believe this can be extrapolated to many important processes in a cell. fluorescence correlation spectroscopy (fcs) is a useful technique for characterizing the mobility and concentration of fluorescent molecules both in vitro and in vivo. we utilize two-photon fcs to characterize the concentration and mobility of fluorescent molecules within living cells of bacillus subtilis. autocorrelation functions were measured in bacteria expressing green fluorescent protein (gfp) under the lac promoter in both nutrient rich and nutrient poor culture medium. although considerable heterogeneity was evident from cell to cell, on average, both intracellular concentration and mobility were found to be dependent upon culture medium. we also investigated bacteria expressing gfp under control of native promoters for involved in the regulation of the carbon metabolic cycle in bacillus subtilis. the gfp concentration, which should be related to promoter activity, was investigated for single cells and cell populations under different metabolic conditions. some photobleaching was observed during the course of the measurements as a decrease in the average fluorescence intensity. this is due to the small size of the bacteria (∼ fl) and low basal expression levels of gfp (∼ nm) in the absence of iptg. methods to take this into account during data analysis are discussed. a. fassio , s. congia , p. baldelli , f. benfenati dimes, university of genoa, genoa, italy, dnbt , iit, genoa, italy several mutations have been discovered in the synapsin i (syn) gene in families with epilepsy, but the mechanism inducing the epileptic phenotype is unknown. syn is a protein associated with synaptic vesicles (svs) that control sv trafficking and neurotransmitter release. the syn mutations subject of this study are a non sense (ns- ) and of two missense (ms- , ms- ) . syn knockout (ko) hippocampal neurons were transfected with the either wild type (wt) or mutated syns. all syns presented a common punctate pattern of expression at the level of axonal arborizations but, while ns- syn targeted to synapses as wt-syn, ms- and ms- syns reached the presynaptic terminal less efficiently. we next set up a live cell imaging experiment using synaptophysin-phluorin and evaluated the effects of ns- , ms- and ms- syns on the size of sv pools. restoring wt-syn in syn i ko terminals led to a increase of all sv pools. restoring ms- and ms- syns resulted in a phenotype not significatively different from syn i ko background whereas restoring ns- syn caused a decrease of all sv pools as compared either with wt-syn or syn i ko background. these data suggest an alteration in the subcellular distribution and function of syn in patients carrying ms- e ms- mutation and a more severe effect on synaptic activity in patient carrying ns- mutation. imaging dynamics of dc-sign at the plasma membrane of hiv- -stimulated dendritic cells o understanding how viruses interact with host receptors is essential for developing new antiviral strategies. dendritic cells (dcs) can efficiently capture and take up hiv- through multiple attachment factors, such as the c-type lectin dc-sign. however, the initial interactions between hiv- and this receptor on dcs are not fully understood yet. in this work, we have used single-molecule epi-tirf microscopy in combination with fluorescently labelled hiv- virus like particles (vlps) and dc-sign-specific antibodies to image the dynamic interaction between hiv- and dc-sign nanoclusters at the plasma membrane of dcs. by tracking individual trajectories of dc-sign on the cell surface of living dcs we have found heterogeneity in the modes of motion of dc-sign: some clusters are immobile whereas others move very quickly, with a few ones showing a directed motion on the cell membrane. to investigate how such motion might be correlated to dc-sign function as virus attachment factor, we have developed glass platforms functionalized with hiv- vlps to locally stimulate living dcs. these virus platforms have allowed us to measure dc-sign diffusion rates and motion modes over the cell surface of stimulated dcs and represent a powerful tool for studying the dynamic interactions between hiv- and the dc membrane. a. esposito , t. tiffert , j. mauritz , s. schlachter , j. n. skepper , v. l. lew , c. f. kaminski dept. of chemical engineering and biotechnology, univ. of cambridge, u.k., dept. of physiology, development and neuroscience, univ. of cambridge, u.k. plasmodium falciparum (pf ) causes the most lethal form of malaria in humans. early research exposed two paradoxes: ) during its intraerythrocytic cycle pf permeabilizes the host cell so much that a comparably permeabilized healthy red blood cell (rbc) would lyse prematurely, and ) pf digests far more hemoglobin than needed for its metabolism. a model of the homeostasis of a pf infected rbc suggested a common explanation of both puzzles: excess hemoglobin digestion is required to reduce the colloidosmotic pressure within the host cell thus ensuring its osmotic stability to the end of the pf asexual cycle. we investigated these predictions with direct measurements of [hb] and volumes of parasitized rbcs. reliable volume and morphological data was obtained by confocal microscopy and quantitative surface reconstruction. furthermore, we developed a new fret-based method to measure hemoglobin molecular crowding by exploiting the reduction in fluorescence lifetime of a donor fluorophore loaded in the rbc cytosol. fret imaging techniques are powerful tools for probing the biophysics of living cells. these tools provided a first validation of the colloidosmotic hypothesis and a deeper understanding of the homeostasis of the intraerythrocytic stage of pf. hiv- assembly and release occur at the plasma membrane of infected cells and are driven by the gag polyprotein. using a combination of wide-field and total internal reflection fluorescence microscopy, we have investigated assembly of fluorescently labeled hiv- at the plasma membrane of living cells with high time resolution. gag assembled into discrete clusters corresponding to single virions. after their initial appearance, assembly sites accumulated at the plasma membrane of individual cells over - hours. using a photoconvertible fluorescent protein, we determined that assembly was nucleated by membrane bound gag molecules, while both membrane-bound and cytosol derived gag polyproteins contributed to the growing bud. assembly kinetics were rapid and three phases are observed. in phase i, the number of gag molecules at a budding site increases following a saturating exponential with a rate constant of ∼ x − s − . hence, gag assembly is complete in ∼ s. in phase ii, a plateau in fluorescence intensity is observed with no exchange of gag protein. the fluorescence intensity decays in phase iii. this decay, in some cases, corresponds to the release of a virion. the time scale from the onset of assembly to release of extracellular particles was measured to be ∼ , +/- s. fast d chromatin dynamics studied in living yeasts using a novel lab on chip technology h. hajjoul , m. dilhan , i. lassadi , k. bystricky , a. bancaud laas-cnrs, université de toulouse, france, lbme, cnrs umr , toulouse, france we present a novel lab-on-chip technology for d particle tracking yeast abased on v-shaped mirrors, which are used to observe fluorescent specimens from multiple vantage points, providing stereo-images that can be recombined for d reconstruction. our technology is based on v-shaped mirrors, which are fabricated by wet etching of silicon wafers, and used as optical and fluidic components. after rigorous optical optimization of the device in vitro, the lab-on-chip is applied to study chromatin dynamics in vivo using budding yeasts as a model system. yeasts cells are visualized using gfp fused to the associated repressor protein. we confirm earlier observations that telomeric sequences, i.e. located close to chromosome ends, accumulate at the nuclear periphery, whereas genes found midway along chromosome arms are mostly present in the nuclear lumen. the dynamics of these sequences is followed in d with an unpreceded temporal resolution of ms, showing that chromosome dynamics is non linear in the short time regime and switches to a linear regime at larger timescales. notably, this behavior is reminiscent of universal responses observed in polymer solutions, and is related to the confinement and the structure of chromosomes. this technique shows a great potential for studying dynamic processes in small living organisms. retrovirus induced remodeling of the host cell actin architecture m. gladnikoff, i. rousso department of structural biology, weizmann institute of science, rehovot, israel retrovirus budding is a key step in the virus replication cycle. yet, despite substantial progress in the structural and biochemical characterization of retroviral budding, the underlying physical mechanism remains poorly understood, primarily due to technical limitations preventing visualization of bud formation in real time. using atomic force-, fluorescence-and transmission electron microscopy we find that both hiv and moloney murine leukemia virus (mlv) remodel the actin cytoskeleton of their host cells and utilize the forces it generates to drive their assembly and budding. highly dynamic actin-filamentous structures which varied in size over the duration of budding appeared to emanate from the assembled virion. these actin structures assemble simultaneously or immediately after the beginning of budding, and disappear as soon as the nascent virus is released from the cell membrane. analysis of sections of cryo-preserved virus infected cells by tem reveals similar actin filaments structures emerging from every nascent virus. substitution of the nucleocapsid domain implicated in actin binding by a leucine-zipper domain resulted in budding of virus-like particles that was not accompanied by remodeling of the cell's cytoskeleton. notably, budding of viruses carrying the modified nucleocapsid domains was an order of magnitude slower than that of the wild type. the results of this study show that retroviruses utilize the cell cytoskeleton to expedite their assembly and budding. t. gensch institute of structural biology and biophysics (isb- ) cellular biophysics, research centre jülich, germany the determination of ion concentrations in cells -in particular in neurons -is very important for understanding cell function and life. ca + is an ubiquitous messenger in almost all cell types, cl − has several roles, e.g. plays an important role in some neuronal signaling pathways including olfaction, nociception and vision. fluorescence lifetime imaging (flim) is of advantage over intensity based fluorescence microscopy, when comparisons between micro-domains of one cell or between different cells of one cell type are performed. several (organic chromophores and fluorescent protein based) ca + -and cl − -sensors have been tested in culture cells with respect to their applicability in flim studies. the ca + -and cl − concentration in rodent olfactory sensory neurons, dorsal root ganglion neurons and neurons of the retina is investigated by time-resolved flim with two-photon excitation. viscosity is one of the main factors which influence diffusion in condensed media. in a cell viscosity can play a role in several diffusion mediated processes, such as drug delivery and signalling. previously, alterations in viscosity in cells and organs have been linked to malfunction; however, mapping viscosity on a single-cell scale remains a challenge. we have imaged viscosity inside individual cells using novel fluorescent probes, called molecular rotors, in which the speed of rotation about a sterically hindered bond is viscosity-dependent. , this approach enabled us to demonstrate that viscosity distribution in a cell is highly heterogeneous and that the local microviscosity in hydrophobic cell domains can be up to × higher than that of water. we have also shown that the intracellular viscosity increases dramatically during light activated cancer treatment, called photodynamic therapy (pdt). we have demonstrated the effect of such viscosity increase on intracellular reactions by directly monitoring the rates of formation and decay of a short lived toxic intermediate, crucial in pdt, called singlet molecular oxygen, in light perturbed cells. characean algae, close relatives of higher plants, represent a convenient model for studying the effect of propagating electrical signals, action potentials (ap) on photosynthesis. illuminated chara corallina cells produce coordinated spatial patterns of chlorophyll fluorescence (chl fl) and extracellular ph. photosynthesis is higher in the cell regions adjacent to acid zones compared to alkaline zones. under physiological conditions, the electrically induced ap differentially and reversibly suppresses photosynthesis in the alkaline and acid regions. in this work we examined the effects of an artificial psi acceptor, methyl viologen (mv), on chl fl with imaging-pam technique. mv is a divalent cation and poorly permeates through biological membranes. the presence in the medium of mv had no effect on fl as well as on p + absorbance signals until the application of a single excitatory stimulus. once an ap was generated in the presence of mv, it induced irreversible inhibition of native (nadpdependent) electron flow and a strong non-photochemical fl quenching all over the cell. this indicates that ap redirects electron flow from the main pathway to the artificial acceptor. we concluded that ap generation opens access for permeation of mv from the medium to the chloroplast stroma across two membrane barriers, plasmalemma and chloroplast inner membrane. we suggested that mv might enter chara cell via plasmalemma ca + -channels activated during ap. evaluation of cell damage after photodynamic and sonodynamic treatment h. kolarova, k. tomankova, s. binder, p. kolar, r. bajgar, m. strnad department of medical biophysics, faculty of medicine and dentistry, palacky university, hnevotinska , olomouc, czech republic photodynamic therapy (pdt) and sonodynamic therapy (sdt) is a new, combined therapy for treating cancer. the basis of the therapy is to administer a small amount of photosensitizer and sonosensitizer, which are selectively taken up by cancer cells, and then expose the body to light and ultrasound to activate these sensitizers. when the sensitizers absorb light of an appropriate wavelength, it may cause their excitation with subsequent energy transfer to oxygen; the oxygen then becomes highly reactive in cancer cells and produces reactive oxygen species (ros). the resulting damage to organelles within malignant cells leads to tumor ablation. sdt uses an agent that is sensitive to ultrasound, allowing deeper penetration and destruction of abnormal cells. changes in human melanoma cells were evaluated using fluorescence microscope and atomic force microscopy.we focused on obtaining pictures of the topography and pictures involving elastic properties of cell surface. the production of ros was investigated with the molecular probe cm-h dcfda, and morphological changes in cells were evaluated using fluorescence microscope. the quantitative ros production changes in relation to phthalocyanine concentration, irradiation doses and ultrasound intensity were measured by a fluororeader. activity correlation imaging: visualizing function and structure of neuronal populations s. junek , t.-w. chen , m. alevra , d. schild department of neurophysiology and cellular biophysics, university of göttingen, germany, dfg research center for molecular physiology of the brain (cmpb), university of göttingen, germany understanding a neuronal network relies on knowledge about both the function and the structure of its neurons. while he simultaneous observation of hundreds of neurons is possible by densely staining brain tissue with functional dyes, the low contrast of these stainings does not allow the identification of neuronal processes from the raw fluorescent images. however, as neurons are known to exhibit complex temporal patterns of activity, fluctuations in signal intensity over time could be exploited to generate contrast in densely stained tissue. we demonstrate that the uniform calcium signals within individual neurons of the olfactory bulb can be exploited to visualize the morphology and projection patterns of these cells in tissue slices. as different neurons exhibit distinct time patterns, it is possible to generate a highcontrast multi-color visualization of the network's active neurons, solely based on the specificity of temporal fluctuations of a single-color calcium dye. it is thus possible to use other spectral channels for additional labelling, for example using cell-type or protein specific markers. the ability to map function and structure of neuronal populations online opens up a number of intriguing applications, such as selecting cells or cell pairs with certain functional or projection profiles for targeted recordings, ablation or stimulation. -live cell imaging - investigation of the dynamics of redox elements in live cells by using fluorescence ratio microscopy g. maulucci , g. pani , v. labate , m. mele , e. panieri , m. papi , g. arcovito , t. galeotti , m. de spirito istituto di fisica, università cattolica s. cuore, roma, italy, istituto di patologia generale, università cattolica s. cuore, roma, italy oxidative stress or signaling events can affect cellular redox environment, which act simultaneously as regulator and indicator of key cellular functions in both physiological and pathological settings. by using a redox-sensitive protein (rxyfp), employed ratiometrically, it is possible to generate high resolution redox maps of cells. the spatial distributions of oxidized and reduced elements have been discriminated in human embryonic kidney cells by the deconvolution of the histograms of redox maps. by transfecting cell with glutaredoxin v (grx-v), a significant shift towards more reduced state with respect to that recovered from non-transfected cells is observed. despite such large differences, a common behaviour in the spatial distribution of reduced and oxidized couples can still be observed: oxidized population shows a pronounced localization on the cell borders, near the plasma membrane, while reduced population appears as a collection of well separated spots homogeneuosly distributed thoroughly the inner part of the cell with a mean dimension of um. furthermore we observe that the role of grx-v consists in causing a shift towards reduced values of the highly reduced region, while leaving unaltered the redox-balance of the intracellular side of the plasma membrane. fluorescence resonant energy transfer (fret) is a popular approach to studying molecular interactions. fluorescence lifetime imaging (flim) provides a robust read-out of fret but has largely been restricted to fixed cells owing to relatively long acquisition times. we present a highspeed wide-field multidimensional fluorescence microscope for flim fret in live cells on second timescales for high content analysis and studying fast dynamics. the system uses a nipkow disc for optical sectioning and a time-gated image intensifier for wide-field flim. a spectrally selected supercontinuum excitation source facilitates versatile realtime flim of live cells transfected with fluorescent proteins. for cell signalling studies we have developed a multiplexed fret approach. ras activation at the plasma membrane is demonstrated using flim to read out tagrfp-raf rbd interaction with mplum-h-ras. simultaneously, a spectral ratiometric read-out is used for a second fret (cfp/yfp cameleon) probe to monitor the downstream calcium flux. to further develop multiplexed fret as a tool for imaging multiple components of cell signalling networks, we are working to include polarisation-resolved imaging for steady-state and time resolved fluorescence anisotropy measurements. monitoring gene expression via novel nucleic acid and delivery methods k. lymperopoulos , c. spassova , a. seefeld , h. s. parekh , d. p. herten bioquant institute,heidelberg university, germany, school of pharmacy, university of queensland, australia application of single-molecule and high-resolution fluorescence methods to monitor gene expression in living cells increase the demand on novel probes and delivery methods.they require fluorophores with high photostability and quantum yield and highly-efficient delivery methods that ensure the minimum interference with cell processes such as metabolism and signal transduction. we used a novel class of dendrimers with varying generations and branching factors and different number of positive charges due to different moieties and functional groups.these different properties were tested for their efficiency to transfect eukaryotic cells with fluorescent oligonucleotides (odns).different parameters (temperature,concentration of dendrimers, ratio of dendrimers and odns) were evaluated and optimised.we utilised these established optimal conditions to deliver a modified concept of smartprobes to mammalian cells targeting mrnas involved in signal pathways.we tested different mrna targets and we optimised the fluorescence signal by varying a range of parameters,namely the fluorescent label and the intrinsic properties of the smartprobe (length of the loop and stem, conformation and number of guanosines).in the near future,we plan to use these probes for monitoring gene expression levels using diffusion imaging microscopy. we present a novel near-field scanning microwave microscope (smm) capable of providing surface impedance measurements of samples with nanometric resolution. the instrument is the integration of a microwave vector network analyzer (vna) and a scanning probe microscope (afm/stm). a key point is that our software, controlling and synchronizing both the instruments, creates simultaneously images of the sample at several frequency points. this can be used to extract several features of the sample depending on the frequency. moreover, close frequencies show the same features, added to random noise. exploiting this redundancy of information, we have achieved remarkable results. we have been working on the optimization of this system for biological applications, to detect functional characteristics of cells generating a variation of their dielectric properties. this instrument offers the possibility of performing local impedance measurements on a single live cell and, if correctly calibrated, it provides also quantitative information (e.g. absolute measurements of membrane permittivity). the system was demonstrated to work on saccharomyces cerevisiae. a better model for a full test of the potentialities of the new technique is given by excitable cells, characterized by a greater variability of dielectric properties. a challenging objective could be directly imaging ion channels. hiv- to efficiently complete a replication cycle has to integrate its genome into the host cellular dna.after hiv- enters target cells,neosynthesized viral dna forms along with other proteins the pre-integration complex (pic).pics are then transported into the nucleus where integration,catalyzed by the viral integrase,takes place.hiv- viral particles engineered to incorporate integrase fused to egfp have proven effective to study pics within nuclei of infected cells.in this study we report the live imaging analysis of nuclear pic dynamics obtained by time-lapse microscopy.intranuclear trajectories of in-egfp-labeled pic were collected in three dimensions and examined by both mean squared displacement (msd) and cage diameter (cd) analysis.in cd the maximum distances measured between two positions occupied by a pic in a time window of minutes were calculated while in our msd analysis -minute long trajectory segments were considered.remarkably,msd revealed the presence of an underlying active transport mechanism.to test the possible role of actin filaments,pic nuclear trafficking was analyzed in cells treated with latrunculin b (actin polymerization inhibitor).preliminary results suggest that the disruption of actin function impairs the active nuclear movement of pics. second harmonic generation microscopy reveals sarcomere contractile dynamics of cardiomyocytes n. prent , c. a. greenhalgh , r. o. cisek , j. aus der au , s. elmore , j. h. van beek , j. squier , v. barzda department of physics and institute for optical sciences, university of toronto, toronto, canada, department of physics, colorado school of mines, golden, usa, department of molecular cell physiology, vrije universiteit, amsterdam, netherlands cardiomyocytes, like other striated muscles, exhibit strong inherent second-order nonlinear optical properties that make them exemplar for live cell dynamic studies with second harmonic generation (shg) microscopy. laser scanning shg microscopy with an incident wavelength around µm, enables fast imaging for extended periods of time with negligible tissue damage. strong shg signal originates from the anisotropic (a-) bands which are comprised of regularly arranged myosin molecules, while actin molecules, the main constituent of the isotropic (i-) bands, produce negligible shg. consequently, the alternating bands along the myofibril are clearly visualized, therefore, enabling the determination of individual sarcomere lengths and the study of sarcomere length dynamics during macro-scale contractions. shg intensity is shown to positively correlate to sarcomere length, which leads to the development of real-time inherent force sensors for in vivo myocytes. rich sarcomere dynamics can be observed during myocyte contraction, which can be used for medical diagnostic purpose of muscular degenerative diseases. imaging cellular communication in insulin secretion ex vivo and in vivo d. w. piston vanderbilt university, nashville, tennessee, u.s.a. the islet of langerhans is the functional unit responsible for glucose-stimulated insulin secretion (gsis), and thus plays a key role in blood glucose homeostasis. the importance of the islet is demonstrated by the proven ability of islet transplants to reverse type i diabetes pathologies in human patients. we are interested in understanding the multicellular mechanisms of islet function, and their role in the regulation of blood glucose under normal and pathological conditions. in many ways, the islet appears to function as a syncytium, which exhibits synchronous behavior of membrane action potentials, ca + oscillations, and pulsatile insulin secretion across all β-cells in the islet. in other ways, the islet works as individual cells, especially in the regulation of gene transcription. using our unique quantitative optical imaging methods and novel microfluidic devices, the dynamics of these molecular mechanisms can be followed quantitatively in living cells within intact islets. these investigations utilize transgenic and tissue-specific knock-out mouse models with demonstrated phenotypes, as well as traditional biochemical and molecular biological approaches. do retinal rod outer segments carry out oxydative phosphorylation? i. panfoli , p. bianchini , d. calzia , s. ravera , a. morelli , a. diaspro biology dept., university of genova, italy, lambs-microscobio res.centre, university of genova, italy, neuroscience and brain technology dept., i.i.t. genova, italy visual transduction in vertebrate retinal rod outer segments (os) is very energy demanding. however, atp supply in os, that are devoid of mitochondria, is still puzzling. by a proteomic analysis we identified in purified bovine os disks, proteins involved in vision, as well as the respiratory chain complexes i to iv and f f o -atp synthase, whose activity was comparable to that of mitochondria and sensitive to specific inhibitors. rhodamine fluorescence quenching experiments showed the presence of a proton potential difference across disks. disks consumed oxygen. confocal laser scanning and transmission electron microscopy showed that cytochrome c oxydase and atp synthase are localized on disks. mitochondrial vital dyes stained os ex vivo, and disks. rhodopsin and mitotracker fluorescence co-localized in os. data, suggestive of an aerobic metabolism in os, point to the existence of "mitochondrial inner membrane-like membranes" ectopically producing atp through oxidative phosphorylation, with respect to mitochondria. new scenarios open on the patho-physiology of many retinal diseases associated to mitochondrial dysfunction. -live cell imaging -abstracts unmixing using lifetime-excitation multidimensional confocal fluorescence microscopy data s. schlachter , s. schwedler , a. esposito , a. d. elder , g. s. kaminski schierle , c. f. kaminski cambridge university, u.k., universität bielefeld, germany fluorescence imaging provides a powerful tool to probe biological systems, enabling the high resolution investigation of the localization, interaction and biochemical modification of biomolecules. multidimensional imaging, in particular, is a growing application of confocal and other forms of fluorescence microscopy. it seeks to measure not only fluorescence intensity in three spatial dimensions, but also other features of fluorescence emission, such as lifetime and fluorescence spectra. although multi-dimensional fluorescence microscopy has been demonstrated before, little attention has so far been paid to the problem of data interpretation and representation when dealing with such large datasets. we present here an instrument capable of recording fluorescence lifetime and excitation data by combining tcspc for lifetime determination and a supercontinuum excitation source for extracting excitation spectra. these technologies permit the acquisition of d and d images with lifetime and excitation spectrum information at every pixel. we demonstrate a method whereby the multidimensional datasets acquired with this instrument are processed to yield biologically relevant parameters, (e.g. unmix fluorophores in a multiply labelled sample). our method relies on a global analysis approach and uses ab plots for displaying multidimensional data. we demonstrate the instrument & processing method on dye and multiply labelled biological samples. advanced neuroimaging with diffractive optical elements p. saggau baylor college of medicine, houston, texas, usa studying neural systems is complicated by the large number and small size of its cellular elements. the traditional way of exploring neuronal function by electrical monitoring with micropipettes is increasingly replaced by molecular imaging. fluorescent molecules allow optical monitoring of neuronal signaling with cellular and often subcellular resolution. in addition to monitoring activity, analyzing the functional properties of individual neurons or neuronal populations requires their controlled activation. optical approaches are well-suited, including molecular photolysis of inert precursors and light activation of engineered proteins that control ionic membrane currents. to fully utilize optical techniques for exploring neural systems requires more than adequate spatial resolution to distinguish neuronal elements and sufficient temporal resolution to induce and/or monitor neuronal signaling. because of the non-linear and non-stationary nature of the studied system it is necessary to access many neuronal sites simultaneously. in modern imaging, wide-field illumination and imageformation are often replaced by patterned excitation and nonimaging photon collection. in many instants, diffractive illumination schemes can substitute for conventional reflective or refractive designs. in particular, the availability of programmable diffractive optical elements has made this an attractive alternative, since they permit highly versatile microscope designs and support a significant increase in the overall spatio-temporal resolution. i will present an advanced imaging approach using diffractive optical elements to analyze structure and function of live neurons in brain slices and intact cortex. efficient evaluation of fret in image cytometry with acceptor photobleaching and ratiometric methods j. roszik, d. lisboa, j. szöllősi, g. vereb department of biophysics and cell biology, university of debrecen, debrecen, hungary fluorescence resonance energy transfer (fret) is a powerful technique that can be applied to study nanoscale intra-and intermolecular events and interactions of molecules in situ in biological systems. a robust, easy to use, self-controlled fret method, independent of donor and acceptor concentration and stochiometry, is acceptor photobleaching fret, which requires only simple image mathematics. another approach with more complicated calculations is the intensitybased ratiometric method, which is not based on destroying the acceptor fluorophores, making it applicable for following molecular interactions in live cells. as the need for using fret in image cytometry for evaluating molecular interactions increases, we have undertaken to develop softwares for these methods involving the calculation and usage of all correction factors needed to obtain reliable energy transfer efficiencies. correction possibilities of the acceptor photobleaching method include unwanted photobleaching of the donor, fluorescent photoproduct of the acceptor after photobleaching, cross-talk of unbleached acceptor into the donor channel and partial photobleaching of the acceptor. in the case of intensity-based ratiometric fret, we can correct for all channel cross-talks and calibrate the fret efficiency calculations. both programs provide registration and semiautomatic processing, and they are freely available. the pendrin (slc a ) gene is responsible, when mutated, for the pendred syndrome, a recessive disorder characterized by sensorineural hearing loss often accompanied by thyroid disfunctions. pendrin is an anion exchanger. the way it works and its role in different tissues, owing to the lack of known isoforms, is matter of wide research and debate. we focused on a still unexplored pendrin function, that is most important in the inner ear: cellular volume control and cl − fluxes regulation. we used hek cells over-expressing wild type pendrin or a mutated isoform together with the eyfp. we challenged cells with hypo-osmolar solutions and followed their volume variations in time. taking advantage of the confocal optical sectioning we independently measured cell volume and fluorescence intensity. in this way, given the dependence of eyfp fluorescence from [cl − ] and ph (measured with snarf ) we could estimate at the same time cl − fluxes, volume and ph variations. the contemporary measurements of the three variables, not yet reported in living cells, allowed to assess the role of pendrin in volume regulation and evidenced its participation to cl − fluxes as compared to the mutated isoform or controls. particle tracking inside the cell largely benefits of the ability to spatially and temporally mark specific structures to follow their "signalling" over a "dark" background as made possible since the advent of the photo-activatable markers. in terms of spatial confinement of the photo-activation process, the use of multiphoton excitation provides several favourable aspects compared to single photon confocal microscopy in photomarking biological structures to be tracked: the confined excitation volumes, of the order of magnitude of subfemtoliter, due to the non-linear requirements provide a unique control of the excitation and consequently photoactivation in the d space. in this context photoactivation experiments can be used to assess quantitative information about the binding kinetics of a macromolecule expressed in different cellular compartments. in this work we extended to photoactivation procedures and models originally developed for the quantitative analysis of frap experiments and we evaluated, for different proteins of medical interest (rac-pagfp), the diffusive behaviour in the cytoplasm and the binding kinetics at the large endosomes. the results are compared with standard photobleaching experiments, in order to evidence the gained sensitivity obtained with photo-activatable proteins. motile plaques involved in stress fiber assembly revealed by high-speed spm for living cells k. tamura, t. mizutani, h. haga, k. kawabata division of biological sciences, graduate school of science, hokkaido university, kita-ku, sapporo, japan stress fibers, which are contractile actin cables aligned in a highly-ordered manner, are important for cell migration, mechanical support of plasma membranes and extracellular matrix organization. although stress fibers are dynamically disassembled and assembled again in cells, it is poorly understood how actin filaments are organized into cables with highly-ordered alignment for stress fiber assembly. for investigation of actin cytoskeletal dynamics during actin cable formation, we performed time-lapse observation of actin cytoskeleton in lamella of living fibroblasts by using scanning probe microscopy (spm). high-speed spm observation revealed that motile plaques defined front-side ends of new actin cables and that preexisting mesh-form actin networks were remodeled into new actin cables. directional order analysis of movement of plaques and pharmacological experiments clarified that plaques were driven by myosin-ii-based retrograde actin flow, indicating that plaques are associated with actin cytoskeleton. immunofluorescence experiments showed that plaques were localized on foci of vinculin, a component of cell-substratum adhesions, suggesting that plaques can bind to extracellular substrata via vinculin. based on these results, we propose a model for actin cable formation that motile plaques initiate remodeling of preexisting actin networks into actin cables aligned in a direction of actin flow by associating with extracellular substrata. abscisic acid (aba) is a plant hormone regulating fundamental physiological functions in plants, such as response to abiotic stress. recently, aba was shown to be produced and released by human granulocytes, by insulin-producing rat insulinoma cells and by human and murine pancreatic β cells. aba autocrinally stimulates the functional activities specific for each cell type through a receptor-operated signal transduction pathway, sequentially involving a pertussis toxin (ptx)-sensitive receptor/g-protein complex, cyclic amp, cd -produced cyclic adp-ribose and intracellular calcium. here, the aba receptor on human granulocytes and on rat insulinoma cells is identified as the lanthionine synthetase c-like protein lancl . co-expression of lancl and cd in the human hela cell line reproduces the aba-signaling pathway. the ptx-sensitive g protein coupled to lancl is identified as g i by transfection of cd + /lancl + hela with a chimeric g protein (gα q/i ). identification of the mammalian aba receptor will enable the screening of synthetic aba antagonists as prospective new anti-inflammatory and anti-diabetic agents. investigation of post-thaw damage of s.cerevisiae yeasts using fluorescent dyes -dab and -dab t. s. dyubko , i. a. buriak , v. d. zinchenko , i. f. kovalenko state scientific institution "institute for single crystals", national acadey of sciences of ukraine, kharkov, ukraine, institute for problems of cryobiology and cryomedicine, national academy of sciences of ukraine, kharkov, ukraine we investigate applicability of the fluorescent probes -dab and -dab available from seta biomedicals (www.setabiomedicals.com) to study the post-thaw damage of saccharomyces cerevisiae yeast cells. the leaving cells stained with these dyes have bright fluorescence in the yellow and red region, respectively. however, the freezing followed by post-thawing causes cell damage, which results in a change of fluorescence intensity. in the native living cells the dyes are preferably localized in the cell membranes and organelles which are highly fluorescent. partially damaged cells have even brighter fluorescence as compared to the living cells. however, their cell ultra-structure is not well-distinguished in the fluorescence mode. ultimately destroyed cells are almost non-fluorescent: the cell membranes are not visible in the fluorescent mode and their organelles are only weakly fluorescent. the number of intensively fluorescing cells with partially destroyed membranes, which were obtained by freezing to - • c, is lower compared to those frozen at - • c. -dab and -dab enable not only to distinguish damaged and undamaged cells, but also allow quantitative estimation of the extent of damage in membranes by cryogenic effects. the thylakoid membrane is a structured network in higher plants which is organised into stacked granal thylakoids that are interconnected by single 'stromal' lamellae. we have studied the mobility of a resident protein of the stromal lamellae, hcf , part of the tat protein translocase. hcf -green fluorescent protein fusion (gfp) was targeted into thylakoids and studied using photobleaching approaches. we show that small regions fail to recover significant levels of hcf -gfp fluorescence within minutes after photobleaching. autofluorescence from the photosystem ii light-harvesting complex (lhcii) in granal stacks likewise fails to recover over this time scale. although the thylakoid membrane is a single continuous entity, these data show that both hcf -gfp and lhcii are constrained within this network. since the hcf homologue, tatb, is highly mobile in the escherichia coli plasma membrane, we believe that the stromal lamellae take the form of distinct domains that are effectively constrained by boundaries within the thylakoid network. cytomechanical modifications induced by drugloaded carriers uptake by breast cancer cells the novel opportunities offered by the nanotechnologies have attracted great interest in the development of novel biomaterials for targeted drug delivery in cancer research. the desired features of pharmaceutical drug delivery for intravenous administration are their small size, biodegradability, high drug content, prolonged circulation in the blood and the ability to target required areas. in this work we have compared efficacy of different type of carriers having complementary properties for pharmaceutical delivery in cancer therapy. drug nano-colloids encapsulated by combination of layer by layer (lbl) techniques and ultrasonication, phytochemical encapsulated artificial oleosomes and drug-loading clay/carbon nanotubes have been used for uptake into breast cancer cells. analysis of viscoelastic response of neoplastic cells induced by cargo-carriers uptake has been carried out by a combination of high resolution optical and scanning force microscopy techniques. furthermore, the effects of drug-reservoir carriers on the cytoskeleton (re)organisation of neoplastic cells were further investigated by confocal microscopy using different fluorescent probes. mapping diffusion by raster image correlation spectroscopy (rics) with analog detection time lapse and flow cytometry data integrated in a common cell cycle proliferation model p. ubezio, v. colombo, m. lupi, f. falcetta istituto di ricerche farmacologiche "mario negri", milano, italy we present a cell cycle simulation tool, connecting the basic proliferation process to the cell population data obtained by time lapse and flow cytometry. the computer program is a general framework within which cell cycle progression can be interactively modeled at the desired level of complexity, including g /s/g m cell cycle phases with variable duration, g phase, cell subpopulations belonging to different generations or differentiation stages, distinct block and cell death parameters for each phase. in this way, we achieved a detailed rendering of cell proliferation of normal or tumor cell populations, additionally including cytostatic and cytotoxic effects of treatments. the program gives as output simulated measures, reproducing those obtained by absolute cell counting, growth inhibition tests, flow cytometric dna histograms and cell cycle percentages, pulse continuous-labeling studies, time-lapse intermitotic times and generation-wise analyses. each technique provides a piece of information related to the underlying proliferation process, catching only some of the phenomena in play, and the measures often cannot be univocally interpreted. we used the cell cycle simulator to fit together time lapse and flow cytometry time courses measures, to achieve a detailed reconstruction of the cell cycle proliferation of an ovarian cancer cell line in vitro after x-ray exposure. -live cell imaging - detection of functional modes in protein dynamics j. s. hub , b. l. de groot deptartment of cell & molecular biology, uppsala university, sweden, computational biomolecular dynamics group, max-planck-institute for biophysical chemistry, göttingen, germany proteins frequently accomplish their biological function by collective atomic motions. yet the identification of a collective motions related to a specific protein function from, e.g. a molecular dynamics trajectory, is often non-trivial. here, we propose a novel technique termed`functional mode analysis' that aims to detect the collective motion that is directly related to a particular protein function. based on an ensemble of structures, together with an arbitrary`functional quantity' that quantifies the functional state of the protein, the method detects the collective motion that is maximally correlated to the functional quantity. the functional quantity could, e.g., correspond to a geometric, electrostatic, or chemical observable, or any other variable that is relevant to the function of the protein. two different correlation measures are applied. first, the pearson correlation coefficient that measures linear correlation only; and second, the mutual information that can assess any kind of interdependence. detecting the maximally correlated motion allows one to derive a model for the functional state in terms of a single collective coordinate. the new method is illustrated using various biomolecules, including a polyalanine-helix, t lysozyme, trp-cage, and leucine-binding protein. logic estimation of the optimum source neutron energy for bnct of brain tumors boron neutron capture therapy (bnct) is a promising method for treating the highly fatal brain tumor; glioblastoma multiform. it is a binary modality; in which use is made of two components simultaneously; viz. thermal neutrons and boron- . a new concept was adopted for estimating the optimum source neutrons energy appropriate for different circumstances of bnct. four postulations on the optimum source neutrons energy were worked out, almost entirely independent of the rbe values of the different dose components. four corresponding conditions on the optimum source neutrons energy were deduced. an energy escalation study was carried out investigating different source neutron energies, between . ev and . mev. mcnp b monte carlo neutron transport code was utilized to study the behavior of these neutrons in the brain. the deduced four conditions were applied to the results. a source neutron energy range of few electron volts (ev) to about kev was estimated to be optimum for bnct of brain tumors located at different depths in brain. the results were discussed. v. l. cruz, j. ramos, j. martinez-salazar instituto de estructura de la materia. csic cannabinoid receptors are an important class of g protein coupled receptors. in particular cb and cb have received considerable interest because they mediate a variety of physiological responses in the central nervous and immune systems. their tertiary structure is still unknown. experimental information suggests the presence of both, an active and an inactive conformation. cb and cb share a common structural framework consisting in a seven transmembrane α-helix bundle connected by three extracellular and three intracellular loops. however, the knowledge of structural differences between both receptors may serve to design new ligands to activate/deactivate selectively only one of those receptors. in the present research, we report a multi-nanosecond molecular dynamics simulation of both receptors in solution, starting from a structure obtained by homology modelling using the x-ray determined bovine rhodopsin protein. we look for differences in the behaviour of cb and cb during the simulation process to shed light about those structural features which can be important for ligand selectivity. in this sense, we observed that cb tend to present a more flexible and opened helix bundle than cb . thus, it is expected than the former receptors would present less steric hindrance for ligand binding. we expect these results will be useful to design more selective ligands. self-assembly and equilibration of bola-lipids membranes studied by molecular dynamics m. bulacu, s. j. marrink groningen university, the netherlands bola-lipids consist of two monopolar, twin-tailed lipids that are held together by chemical linkage between one or both ends of the tails from one lipid and the corresponding ends from the other one. membranes formed by these lipids or by their mixtures with monopolar lipids are known to have additional mechanical stability while retaining membrane fluidity. this is traditionally attributed to the fact that, in the membrane phase, the bola-lipids have predominantly spanning configuration (the two polar heads are positioned at opposite membrane-water interfaces) in detriment to looping configuration (both head groups are located in the same membrane-water interface). we perform molecular dynamics simulations, using the coarse grained martini forcefield. we start with self-assembly simulations of bola-lipids followed by bilayer equilibration. an artificial pore is created in the membrane that significantly increases the flip-flop mobility of the lipids and hasten equilibration. the membrane properties are characterized (area per lipid, thickness, order parameter, pressure profile) with emphasis on the spanning/looping ratio. our study can help designing new artificial membranes, with higher stability under extreme conditions. -multiscale simulation - evidence for proton shuffling in a thioredoxinlike protein during catalysis d. narzi , s. w. siu , c. u. stirnimann , j. p. grimshaw , r. glockshuber , g. capitani , r. a. böckmann theoretical and computational membrane biology, center for bioinformatics, saarland university, germany, institute of molecular biology and biophysics, eth zürich, switzerland, structural and computational unit, embl, heidelberg, germany, paul scherrer institute, villigen psi, switzerland proteins of the thioredoxin (trx) superfamily catalyze disulfide-bond formation, reduction and isomerization in substrate proteins both in prokaryotic and in eukaryotic cells. all members of the trx family with thioldisulfide oxidoreductase activity contain the characteristic cys-x-x-cys motif in their active site. here, using poisson-boltzmann-based protonation-state calculations based on -ns molecular dynamics simulations, we investigated the catalytic mechanism of dsbl, the most oxidizing protein known to date. we observed several correlated transitions in the protonation states of the buried active-site cysteine and a neighboring lysine coupled to the exposure of the active-site thiolate. these results support the view of an internal proton shuffling mechanism during oxidation crucial for the uptake of two electrons from the substrate protein. intramolecular disulfide-bond formation is probably steered by the conformational switch facilitating interaction with the active-site thiolate. a consistent catalytic mechanism for dsbl, probably conferrable to other proteins of the same class, is presented. our results suggest a functional role of hydration entropy of active-site groups . the role of water in zn(ii)-abeta( - ) complexes beta-amyloid (aβ) peptides are the main component of amyloid fibrils detected in the brain of alzheimer patients. fibrils display an abnormal content of cu and zn ions whose binding to aβ-peptides has been recently studied by x-ray absorption spectroscopy [ ] and interpreted in terms of ab initio simulations. in order to perform such simulations it is of the utmost importance to find a compromise between the need of having a realistic description of the actual physical system and the difficulty of dealing with too many atoms and electrons. what is usually done is removing solvent (water molecules), thus studying the system in the so called "gasphase". in this work we investigate the relevance of water in the zn-aβ − coordination mode. relying on a combination of classical [ ] and quantum chemistry [ ] methods we find a significant difference in the zn coordination geometry depending on whether water is present or not. this information is exploited in building a full model system for subsequent car-parrinello simulations where two aβ peptides in water are in interaction in the presence of zn. [ although experiments which can directly probe the mechanical properties of proteins have only been performed recently, it is clear that the complex folds of polypeptide backbones lead to very heterogeneous mechanical behavior. this heterogeneity is likely to play an important role in protein function and interaction and it would be useful to be able to predict what mechanical (and dynamical) properties will result from a given structure. we have been using coarse-grain elastic network models to investigate this question and have found that these models are able to link specific mechanical properties to a number of functional features including enzymatic active sites, folding nuclei and changes in behavior due to point mutations. these models are also adapted to looking at complex formation between proteins and how specific recognition is achieved, while being fast enough to be applied to very large numbers of interactions. refinement of protein model structures using biasing potential replica exchange simulations s. kannan, m. zacharias school of engineering and science, jacobs university bremen, d- bremen, germany comparative protein modeling of a target protein based on sequence similarity to a protein with known structure is widely used to provide structural models of proteins. frequently, the quality of the target-template sequence alignment is non-uniform along the sequence: parts can be modeled with a high confidence, whereas other parts differ strongly from the template. in principle, molecular dynamics (md) simulations can be used to refine protein model structures but it is limited by the currently accessible simulation time scales. we have used a recently developed biasing potential replica exchange (bp-rex) md method (kannan, s. zacharias, m. proteins , , - ) to refine homology modeled protein structure at atomic resolution including explicit solvent. in standard rex-md simulations several replicas of a system are run in parallel at different temperatures allowing exchanges at preset time intervals. in a bp-rexmd simulation replicas are controlled by various levels of a biasing potential to reduce the energy barriers associated with peptide backbone dihedral transitions. the method requires much fewer replicas for efficient sampling compared with standard temperature rexmd. bp-rexmd simulations on several test cases starting from decoy structures deviating significantly from the native structure resulted in final structures in much closer agreement with experiment compared to conventional md simulations. m. s. p. sansom, p. j. stansfeld, c. l. wee, k. balali-mood department of biochemistry, university of oxford, u.k. coarse-grained molecular dynamics simulations may be used to probe the interactions of membrane proteins with bilayers and their component lipids on an extended (∼ µs) timescale ( ) . conversion to atomistic resolution allows more detailed protein/lipid interactions to be examined. this multiscale approach will be examined via three examples: (i) interactions of a small ion channel toxin (vstx ) we present a new competitive approach for the treatment of biomolecular flexibility to provide an alternative to the limitations of current methodologies such as molecular dynamics and normal mode analysis. this method, called static mode method, is based on the "induced-fit" concept and is aimed at mapping the intrinsic deformations of a biomolecule subject to any external excitations: direct mono or multi-site contact, electrical etc... the algorithm allows obtaining a set of deformations, each one corresponding to a specific interaction on a specific molecular site, in terms of force constants contained in the energy model. such a process can be used to explore the properties of single molecular intrinsic flexibility, as well as to predict molecular docking or molecule/surface interactions. from a modelling point of view, the interaction problem can be expressed in terms of reactive sites between the interacting entities, the molecular deformations being extracted from the pre-calculated static modes of each separated ones. the first applications of our method have focused on the intrinsic flexibility of biomolecules like nucleic acids and proteins. more recently, this new methodology allowed us to investigate the folding of the region - of the amyloid β-peptide, via the docking of a zinc ion on the reactive sites of the molecule. conformational study on a myelin basic protein fragment: molecular dynamics simulations in membrane e. polverini , g. harauz dipartimento di fisica, università di parma, parma, italy, department of molecular and cellular biology, university of guelph, guelph, ontario, canada myelin basic protein (mbp) is a multifunctional protein of the central nervous system whose principal role is in maintaining the compactness and integrity of the myelin sheath, the multilamellar membrane wrapped around nerve axons. however, mbp also interacts with other proteins such as cytoskeletal and signalling proteins, adapting its structure to the different roles. mbp is a candidate autoantigen in the human demyelinating disease multiple sclerosis. this study investigated at atomic detail the conformation of a highly conserved central fragment of mbp, constisting of two consecutive regions with different relevant functionalities. the first one is associated with the membrane and comprises the primary immunodominant epitope in multiple sclerosis; the second one was predicted to be a ligand for sh -domains of signalling proteins. molecular dynamics simulations were perfomed in the presence of dodecylphosphocholine micelle, starting from a structure extrapolated from experimental data (harauz and libich, curr. protein pept. sci., ). the results confirm the experimetal hypothesis, showing, in the micelle, a stable alpha-helix anchored to the membrane for the first region and, for the proline-rich second one, a poly-proline type ii helix pointing outwards, ready to interact with the signalling proteins. multiresolution modelling of drug and hormone permeability through a lipid bilayer traditional atomic-level (al) modelling of biomembranes is time-consuming, and hence limited in the range of systems and phenomena that can be simulated. to alleviate this problem, we designed a coarse-grain (cg) representation where each lipid molecule, in reality consisting of more than atoms, is modelled with only cg sites. our cg technique proves two orders of magnitude less demanding of computational resources than traditional al methodology. a unique feature of our approach is that the cg potentials are directly compatible with standard al models, thus facilitating the simulation of multiresolution systems, where the "chemically-sensitive" components (e.g., the solutes in membrane permeation studies) are modelled atomistically, while the surrounding environment is coarse-grained. in this contribution, we present a summary of our multiscale methodology, together with its application to the permeation of large molecules -drugs and steroid hormones. the calculated permeabilities are compared to the available experimental measurements and al simulation data. molecularlevel insights regarding the permeation mechanism are obtained and rationalised. -multiscale simulation - the processes of life involve a variety of events that occur on different scales, ranging from a fewÅ/ps of the triggering steps of the biochemical reactions, up to their macroscopic effects in cells and organs. intermediate steps involve the structural rearrangement of the bio-molecules (∼nm/ - ns), their aggregation, folding (∼ nm-µm/ µs-ms), internal cell diffusion and dynamics (µm-mm/ms-hours), evidently requiring a multi-scale modeling approach. here the multi-scale approaches are first briefly illustrated with a particular attention to the issue of matching the different resolutions, which is essential to achieve a coherent descripition. the focus is then fixed on the coarse grained (cg) models, typically spanning the nm-µm and µs-ms scale, and in particular on their minimalist -simplest and computationally cheapest -versions [ , ] . a parameterization strategy that combines accuracy and predictive power within these models is presented here and applications are shown to relevant cases including the proteins involved in the hiv replication, the green fluorescent proteins and examples of macromolecular complexes. [ ] controlled degradation of collagen is an important process in tissue remodeling and wound healing. collagenase cleaves fibrillar collagens about three quarters of the distance from the amino-terminus. even though the determination of the cleavage site and the collagenase structure took place decades ago, the mode of action of collagenase on collagen is not clear. to understand the mechanism of collagenase activity on collagen, the structure, stability and dynamics of collagen, its conformation around the cleavage site and the possibilities of conformational rearrangements between the two domains in collagenase was explored using md simulation. the results of principal component (pca) and normal mode (nm) analysis of the collagen and collagenase suggests that the c-terminal domain of collagenase recognizes the collagen, and then the n-terminal catalytic domain undergoes rearrangement on the substrate (with the help of linker regions). the sda software for carrying out brownian dynamics simulations of protein-protein diffusional association with the help of biochemical constraints is being used to predict the mode of interaction of collagen and collagenase. different conformations obtained from pca and nm analysis are being used for the docking. the predicted structures of the complex will help us to understand how collagenase recognizes and binds collagen specifically. acknowledgment: daad, germany, csir-srf india, and the klaus tschira foundation. membrane aoration by antimicrobial peptides combining atomistic and coarse-grained descriptions d. sengupta, a. j. rzepiela, n. goga, s. j. marrink university of groningen, the netherlands antimicrobial peptides (amps) comprise a large family of peptides that include small cationic peptides, such as magainins, which permeabilize lipid membranes. previous atomistic level simulations of magainin-h peptides show that they act by forming toroidal transmembrane pores. however, due to the atomistic level of description, these simulations were necessarily limited to small system sizes and sub-microsecond time scales. here, we study the long-time relaxation properties of these pores by evolving the systems using a coarse-grain (cg) description. the disordered nature and the topology of the atomistic pores are maintained at the cg level. the peptides sample different orientations but at any given time, only a few peptides insert into the pore. key states observed at the cg level are subsequently back-transformed to the atomistic level using a resolutionexchange protocol. the configurations sampled at the cg level are stable in the atomistic simulation. the effect of helicity on pore stability is investigated at the cg level and we find that partial helicity is required to form stable pores. we also show that the current cg scheme can be used to study spontaneous poration by magainin-h peptides. over-all, our simulations provide a multi-scale view of a fundamental biophysical membrane process involving a complex interplay between peptides and lipids. the varkud satellite (vs) ribozyme is the largest of the nucleolytic ribozymes, and the only one for which there is no crystal structure. we have determined the overall architecture of the complete vs ribozyme using small-angle x-ray scattering in solution. the substrate stem-loop docks into the tertiary fold of the ribozyme, allowing an intimate loop-loop interaction to occur. this brings two key nucleobases a and g into close proximity of the scissile phosphate, and we believe that these nucleobases are involved in general acid-base catalysis of the phosphoryl transfer reactions. this is supported by functional group substitution, and the ph dependence of the reaction rate for the natural and modified rna. although possessing totally different folds, the functional elements of the vs and hairpin ribozymes are topologically and mechanistically very similar if not identical. this has probably arisen by convergent evolution. other nucleolytic ribozymes have diversified to employ hydrated metal ions and even small molecules to participate in genera acid-base catalysis. by contrast, the larger ribozymes seem to have adopted a different, metalloenzyme, catalytic strategy. why these different groups have evolved different catalytic chemistries is an interesting challenge to our understanding of biocatalysis. comparison of dna and sirna binding and nuclease protection by non-viral vectors for gene delivery j. lam, k. witt, a. j. mason, l. kudsiova, m. j. lawrence pharmaceutical science division, king's college london, uk the biggest obstacle for the success of gene therapy is delivery. with the discovery of rnai, the use of sirna to regulate gene expression as potential therapy has attracted much attention recently. in contrast to dna, sirna delivery does not require nuclear entry. with one less barrier to overcome, the delivery of sirna seems to be easier. it is frequently assumed that comparable delivery strategy could be used for both dna and sirna. in fact the two types of nucleic acids are fundamentally different with distinct properties, which impact their delivery strategies. in the present study it was found that most non-viral delivery vectors including polymers, peptides and lipids were generally more efficient in binding with dna than sirna as shown in gel retardation assay. the inferior sirna binding is possibly due to the rigid structure of sirna, resulting in weaker electrostatic interaction with the cationic vectors. surprisingly, it was observed that all the vectors studied offered better nuclease protection for sirna than dna despite poorer sirna binding. either the nuclease protection for sirna is easier to achieve due to its small size, or the gel retardation assay did not truly reflect the binding efficiency as the weaker sirna complexes may dissociate during electrophoresis. not only the delivery strategy, the results between dna and sirna study must also be carefully adapted and interpreted. single molecule studies of spliceosomal rnas u and u z. guo, d. rueda department of chemistry, wayne state university, detroit, michigan, u.s.a. splicing is an essential step in the maturation reaction of mrna, in which intervening introns from exons. the spliceosome is a dynamic assembly of small nuclear rnas and > proteins that catalyzes splicing. u and u are the only snrnas strictly required for splicing. major conformational changes are expected to take place during spliceosomal assembly and catalysis. we have developed a single-molecule fluorescence assay to study the structural dynamics of a protein-free u -u complex from yeast. our previous data have revealed a -step large amplitude conformational change of the u -u complex. the st step is a mg + -induced conformational change where helix iii and the u -isl are in close proximity in low mg + and separated in high mg + . the nd step corresponds to the formation of the highly conserved helix ib. we now examine the role of the highly conserved bases in the folding dynamics of the u -u complex. the data show that u and the acagag loop play an important role in stabilizing the interaction between helix iii and the u -isl. we hypothesize that u flips out of the u -isl and interacts with the acagag loop to bring them in close proximity. our results raise the interesting possibility that this interaction plays an important role in bringing the ' splice site and the branched a into close proximity of u , which may be critical for catalysis. a structure-based approach for targeting hiv- genomic rna dimerization initiation site s. freisz, s. bernacchi, p. dumas, e. ennifar ibmc -cnrs, strasbourg, france all retroviral genomes consist in two homologous single stranded rnas. hiv- dimerization initiation site (dis) is a conserved hairpin in the ' non-coding region of the genomic rna and essential for viral infectivity. the dis initiates genome dimerization by forming a kissing-loop complex, further stabilized into an extended duplex upon interaction with the ncp nucleocapsid protein. x-ray structures of the dis kissing-loop and extended duplex revealed similarities with the bacterial s ribosomal rna a-site, which is the target of aminoglycoside antibiotics. as a result, aminoglycosides also bind the hiv- dis as shown by our x-ray structures of the dis kissingloop bound to aminoglycosides. using fluorescence, uvspectroscopy and microcalorimetry, we further characterized hiv- dis/aminoglycosides interactions. we found that the affinity of aminoglycosides for the dis was higher than for their natural target, the s a site. they strongly stabilize the dis kissing-loop, blocking its conversion into the duplex form. finally, we also solved x-ray structures of the dis duplex bound to aminoglycosides, revealing an important conformational change following drug binding. these structures show that it is possible to target the hiv- dis dimer before and after the ncp -assisted rna maturation with the same molecule. altogether, our studies create the basis for a rationally driven design of novel potential drugs targeting the hiv- genome. the core protein of hepatitis c virus is a multifunctional protein of aa, consisting of a hydrophilic n-terminal domain with three basic domains (bd -bd ) responsible for the interactions with rna and a hydrophobic c-terminal domain. the n-terminal domain exhibits nucleic acid chaperone properties similar to those of the nucleocapsid protein from hiv. here, we characterized the mechanism of the chaperone properties of a peptide e corresponding to the bd and bd clusters of the n-terminal domain. to this end, we monitored the promotion by this peptide of the annealing of dtar, the dna analogue of the transactivation response element to its complementary sequence, ctar dna, taken as models. the annealing involves two second-order kinetic components that are activated by at least three orders of magnitude by peptide e. this activation was correlated with the ability of peptide e to destabilize the lower half of dtar stem. using, ctar and dtar mutants, the two kinetic components were assigned to two pathways which are connected with the fast annealing of the terminal bases of ctar to dtar and slow extended duplex formation, limited kinetically by the nucleation of central segments of ctar and dtar stems. structure and conformational dynamics of a unique dead box helicase m. rudolph , m. linden , r. hartmann , d. klostermeier hoffmann-la roche, basel, switzerland, biozentrum, univ. of basel, switzerland, univ. of marburg, germany dead box helicases couple atp hydrolysis to rna structural rearrangements. t. thermophilus hera consists of a helicase core and a c-terminal extension (cte) with a putative rnase p motif. crystal structures show that the cte is bipartite, forming a highly flexible dimerization motif with a novel fold and an additional rna-binding module. we provide a first glimpse on the orientation of an rna-binding domain outside the helicase core. in a structure-based model for the complete hera dimer, the rna-binding sites of the helicase cores face each other, allowing for inter-subunit communication. the plasticity of the dimerization motif allows for drastic changes in the juxtaposition of the helicase cores within the dimer. in single molecule fret experiments we identified fragments of the s rrna and rnase p rna as substrates for hera. both substrates switch the hera core to the closed conformation and stimulate the intrinsic atpase activity. rna binding is mediated by the cte, but does not require the putative rnase p motif. atp-dependent unwinding of a short helix in s rrna suggests a specific role for hera in ribosome assembly, in analogy to the e. coli and b. subtilis helicases dbpa and yxin. in addition, the specificity of hera for rnase p rna may be required for rnase p rna folding or rnase p assembly. simultaneous action of two hera subunits on the same rna molecule may be important for efficient rna remodeling in vivo. structural basis for the encapsidation process of turnip yellow mosaic virus m. petersen , j. hansen , s. s. wijmenga nucleic acid center, department of physics and chemistry, university of southern denmark, odense, denmark, physical chemistry/biophysical chemistry, radboud university, nijmegen, the netherlands formation of hairpins with internal loops with c c and c a mismatches in the ' untranslated region is a common characteristic among the plant viruses belonging to the tymovirus genus. turnip yellow mosaic virus possesses two such hairpins, hp and hp . hp , and in particular the presence of the c c and c a mismatches in its internal loop, is important for initiation of the encapsidation of the viral genome. the encapsidation occurs under acidic conditions at the neck of invaginations of the chloroplast membrane. we have now determined the d structures of revertants involved in the evolutionary pathway using nmr spectroscopy. these structures reveal how the grooves in the hairpin become increasingly positively charged in successive generations of evolution. the similarity between the ccca mutant and the wild-type hairpin (hp ) is striking and explains why this mutant gives rise to a viable virus. in addition, a characteristic tilt of the backbone is observed upon occurrence of a protonated c c base pair. both the positively charged major groove and the kink in the rna backbone appear to be crucial for interactions with the viral capsid. at neutral ph, the structure of hp resembles the watson-crick base paired mutant which explains why encapsidation only occurs under acidic conditions. a. percot , j. vergne , m.-c. maurel , s. lecomte ladir, umr , thiais, france, lbeam, upmc, paris, france, cbmn, umr , pessac, france the existence of "rna world" as an early step in the history of life increases the interest for the characterization of these biomolecules. the studied hairpin ribozyme is a self-cleaving/ligating motif found in the minus strand of the satellite rna associated with tobacco ringspot virus. surface-enhanced raman spectroscopy (sers) was successfully used to detect sub-picomole quantities of nucleic acids. sers takes advantage of the strongly increased raman signals generated by local field enhancement near metallic (typically au and ag) nanostructures. sers spectra of dna or rna are strongly dominated by stockes modes of adenine. through an interaction of adenyl residues with silver colloid, adenyl raman signal is times increased compared to raman scattering. in controlled conditions, sers signal is proportional to the amount of free residues adsorbed on the metal surface. upon rna cleavage, residues are unpaired and free to interact with metal. in the present study, we proposed to follow the cleavage reaction of hairpin ribozyme (hpr ) using the sers signal of the liberate adenyl residues. as the sers signal is proportional to the adenyl residues, reactivity of hr was monitored by measuring the raman intensity of the fragment liberated during the cleavage of hairpin. the results obtained were compared with the electrophoresis method performed on the same sample and similar results were obtained. -rna world - eur biophys j ( ) the development of arrays for biomolecular recognition for a broad range of applications in biomedical diagnostics is receiving a constantly increasing attention. the design of efficient protein biochips, however, requires the optimization of protein immobilization protocols for improving the device sensitivity. moreover, innovative platforms for in-vitro detection in highly diluted, small volume samples need to be developed, for which standard micro-fabrication techniques are not suitable. therefore, the development of nano-scale platforms for protein and antibody detection is essential. we report here on a novel approach for the fabrication of multiple protein nanosensors using atomic force microscopy based nanografting and dna-directed immobilization (ddi), which takes advantage of the specific watson-crick hybridization of oligonucleotide-modified proteins to surfacebound complementary oligomers. using nanografting, single-stranded dna nano-structures with well defined local environments were fabricated on a flat surface. dna-protein conjugates were then anchored on the engineered binding sites by ddi in a single chemical operation and detected by the corresponding topographic height increase of the relevant patches. immunological assay were used to demonstrate the biochemical functionality of the immobilized proteins, proving the specificity of biomolecular recognition of our nanodevices, in the micro-molar to pico-molar concentration range. dna accessible surface area and indirect protein-dna recognition: study by bioinformatical approach o. p. boryskina , m. y. tkachenko , a. v. shestopalova , m. y. tolstorukov institute of radiophysics and electronics nas of ukraine, harvard-partners center for genetics and genomics, usa revealing the mechanisms of protein-dna recognition is essential for understanding the regulation of many cellular processes. there is growing evidence that recognition through sequence-specific contacts (direct readout) can be enhanced by recognition via dna sequence-dependent deformability (indirect readout). the role of changes in dna accessible surface area (asa) in distorted dna configurations in complexes with proteins is not fully understood yet, even though such changes and related changes in polarity of dna surface are among key factors of indirect readout. to fill this gap we have developed a publicly-available internet database of protein-dna complexes, which integrates the data on dna conformational parameters with information on asa of dna atoms in the minor and major grooves, protna-asa. the database has been used to analyze the effect of changes in dna backbone configuration on asa of dna atoms in major and minor grooves. we observe that sugar puckering and conformation of torsion angle γ affect the accessibility of dna atoms in both grooves to a noticeable extent. we also report sequence specific preferences of the nucleotides for structural domains of γ. these results can shed new light on the mechanisms of indirect protein-dna recognition. a. arakelyan biology dpt., yerevan state university, yerevan, armenia the influence of ligand, irreversibly binding with dna, on the isotherm of adsorption of ligand binding with dna reversibly has been modeled theoretically. the isotherm of adsorption of etbr on dna in presence of cis-ddpt has been considered at low concentrations of cis-ddpt and etbr.. the isotherm of adsorption of etbr on dna has linear form in scatchard coordinates at low degrees of occupation. the comparison with experimental isotherm permits to estimate the parameters of etbr binding with dna. with taking into account the pseudo-ring structures formation with partially molten regions we consider two types of binding regions, linear and ring at low degrees of occupation. the isotherm of adsorption of etbr on dna and also variation of the number of bounded etbr molecules are calculated with taking into account these two types of binding regions. it was shown that at low concentrations cis-ddp, bounding with dna, changes the isotherm of ligands adsorption. the linear in scathcard coordinates isotherm of adsorption transforms into non-linear isotherm. the degree of transformation depends on the fraction of dna in the ring regions, on the ratio between number of etbr binding cites in the ring and linear regions, and also on the binding constants for these regions. it was shown that low concentrations of cis-ddp affect the dependence of dispersion on the concentration of ligands, changing both the magnitude of maximum and its position. tunable nanoconfinement structures for dna manipulation e. angeli, l. repetto, g. firpo, c. boragno, u. valbusa nanomed labs, advanced biotechnology center and physics department, university of genoa, italy nanostructures, such as nanochannels [ ] or nanoslits [ ] , have been successfully used to confine and stretch dna molecules, offering interesting opportunities of investigation on conformational changes induced by confinement, physical and biological properties, etc. the integration of these nanostructures on lab-on-chip systems has shown their great potential for applications such as dna sieving or single molecule manipulation [ ] , [ ] . arrays of nanochannels fabricated, using a focused ion beam (fib) on a silicon master, are replicated using elastomeric materials, such as poly(dimethylsiloxane) (pdms), and soft-lithography techniques. the cross-section of these flexible polymeric nanoconfinement structures can be reversibly and dynamically tuned, in order to vary biomolecules transport characteristics and confinement conditions of trapped dna molecules. moreover, these nanochannels, with tunable cross-section, are used to study and exploit the sieving mechanism of "entropic recoil" [ ] for the separation of long dna chains. [ ] mannion jt, et al., ( ) biophys. j., , : - . [ ] jo k, et al., ( ) pnas, , : - . [ ] fu j, et al., ( ) nat. nanotechnol., : - [ ] huh d, et al., ( nat. mater., : - . the nucleocapsid protein ncp of hiv- is characterized by two conserved zinc fingers and plays crucial roles in the virus, through its binding to nucleic acids. ncp is required for efficient proviral dna synthesis, by promoting the initiation of reverse transcription and the two obligatory strand transfers. using fluorescence techniques as well as fcs and nmr, we investigated the chaperone properties of ncp on the primer binding sites (pbs) and transactivation response elements (tar) sequences involved in the two obligatory strand transfers. we showed that ncp binds mainly to the (-)pbs loop, which results in an extension of the loop and a destabilization of the upper base pair of the stem. these structural changes chaperone a kissing complex with the complementary (+)pbs loop and its further conversion into an extended duplex. in contrast, the ncp -promoted annealing of ctar-tar results from the destabilization of the bottom of ctar stem, which favors the invasion of the tar stem. by developing new fluorescence methodologies to site-specifically characterize these interactions, we further showed that ncp slows down the ps to ns conformational fluctuations of its nucleic acid targets and freezes the local mobility of the bases contacted by the zinc fingers. exploring dna orientation in flow e. l. gilroy, m. r. hicks, a. rodger department of chemistry, university of warwick, uk dna is one of the most important biomolecules. in order to undertake its biological role the dna needs to fold and unfold for which its structure and flexibility are crucially important. we have studied the characteristics of dna in flow to probe its structure. flow aligned linear dichroism (ld) is a technique that uses light polarised parallel and perpendicular to an orientated sample. it can be used to measure how aligned a sample is, and the orientation of any interacting molecules. to create this alignment, the sample is placed between two concentric cylinders where one is spun to create a shear flow. the longer and more rigid the molecule is the better the orientation and the signal. as the sample is in flow there are other factors than need to be considered when using ld. these include viscosity and temperature. there are many methods to measure the viscosity of a sample of dna at varying temperatures. these include viscometer, rheometer and dynamic light scattering. the effects temperature has on viscosity and the sample itself need also to be considered. the findings of all these studies have been reported and show the significance that viscosity and temperature have on dna ld measurements. possible applications of using ld to study dna have also been discussed, showing the importance of the use of ld and in the results shown. a.-m. florescu, m. joyeux laboratoire de spectrométrie physique, université joseph fourier grenoble , france we present a dynamical model for simulating non-specific dna protein interactions, which is based on the "beadspring" model for dna with elastic, bending and debye-hückel electrostatic interactions, and where the protein interacts with the dna chain through electrostatic and excluded-volume forces. we study the properties of this model using a brownian dynamics algorithm that takes hydrodynamic interactions into account and obtain results that partially agree with experiments and predictions of kinetic models. for example, we show that the protein samples dna by a combination of three-dimensional diffusion in the solvent and one-dimensional sliding along the dna chain. this model evidences the presence, in a certain range of values of the effective protein charge, of facilitated diffusion, i.e. a combination of the two types of diffusion that leads to faster than -dimensional diffusion sampling of dna. moreover, the analysis of single sliding events shows that the number of base pairs visited during sliding is comparable to those deduced from single molecule experiments. in contrast to kinetic models, which predict an increase of the number of different base pairs visited by the protein proportional to the square root of time, our model however suggests that this number increases linearly with time until it reaches a value that is close to the total number of dna base pairs in the cell (published in j. chem. phys. , ( )). use of md simulation to identify the critical radiation-induced lesions of a dna binding protein n. chalabi, s. goffinont, n. garnier, d. genest, orléans cedex , france a key step in the regulation of gene expression, dna structuring and dna repair is the binding of some proteins to specific dna sequences. we have shown previously that ionizing radiation destabilizes such dna-protein complexes mainly through damage to the protein. for the typical lactose operator -repressor system we have shown by fluorescence measurement and mass spectrometry that upon irradiation, all the four tyrosine residues of the dna binding domain (called headpiece) are oxidized into , dihydroxyphenylalanine (dopa). a circular dichroism study revealed a global conformational change and the destabilization of the headpiece. to decide which lesion is critical for the induction of these effects, a molecular dynamics simulation study was undertaken in parallel with a site-directed mutagenesis one. each tyrosine residue of the headpiece was replaced by another amino-acid that mimics the damaged tyrosine. the most common amino-acid used in site-directed mutagenesis being alanine, we have replaced one tyrosine ( , , or ) in the nmr-based structure of the headpiece from pdb databank ( lqc) by an alanine. the conformational stability of each tyr→ala mutant has been studied by molecular dynamics simulation (md) and compared to that of the native sequence and of the different tyr→dopa mutants. the mammalian high mobility group protein at-hook (hmga ) is a nuclear protein associated with mensenchymal cell development and differentiation. disruption of its normal expression pattern is directly linked to oncogenesis and obesity. our laboratory has utilized a variety of biochemical and biophysical methods to investigate the molecular mechanisms of hmga recognizing at-rich dna. using a pcr-based selex procedure, we discovered that hmga is a sequence-specific dna-binding protein and recognizes the following two sequences: '-atattcgcgawwatt- ' and 'atattgcgcawwatt- ', where w is a or t. using a double-label emsa assay, we found that hmga binds to at-rich dna as a monomer. using isothermal-titrationcalorimetry, we demonstrated that hmga binds to at-rich dna with very high binding affinity whereby the binding of hmga to a-tracts is entropy-driven and to alternate at sequences is enthalpy-driven. this is a typical example of enthalpy-entropy compensation in which the hydration difference between hmga -dna complexes is a main reason for the compensation. interestingly, the binding of hmga to different at-rich sequences is accompanied with a large negative heat capacity change indicating an important role of solvent displacement and charge-charge interaction in the linked folding/binding processes. partition of gibb´s free energy of drug-dna complexation we report a computation methodology, which leads to the ability to partition the gibb's free energy for the complexation reaction of aromatic drug molecules with dna. using this approach it is now possible to calculate the absolute values of the energy contributions of various physical factors to the dna binding process, whose summation gives a value that is reasonably close to the experimentallymeasured gibb's free energy of binding. application of the methodology to binding of various aromatic drugs with dna provides an answer to the question 'what forces are the main contributors to the stabilization of aromatic ligand-dna complexes' ? we report the effects of khz ultrasound irradiation of double-stranded dna solutions under conditions of transient cavitation. a new method was developed for studying these effects which is based on combination of two procedures: ultrasound irradiation of the solutions of double-stranded dna fragments and subsequent analysis of the high resolution denaturing gel electrophoresis data. statistical treatment of the data on the mobility of '-end-labelled restriction fragments with known sequence allowed us to discover the phenomenon of sequence specific ultrasonic cleavage of dna. our analysis results in the following conclusions: ) all steps with '-ward cytosine [ '-d(cpn)- '] have significantly higher cleavage rates than others; the intensity of cleavage diminishes in the order cpg > cpa ≈ cpt > cpc; ) the cleavage rates of all steps depend on the type of flanking base pairs; ) the cleavage rates of the complementary base pair steps are not identical. thus, subtle sequence specific conformational and physical-chemical variations modulate the reaction of sugar-phosphate single bonds on the ultrasound exposure. a theory of the mechanisms on the simultaneous binding of two aromatic drugs to dna it has long been recognized that certain combinations of dna-binding aromatic drugs, x +y, lead to synergistic biological effects. considering x as a basic ligand and y as an added ligand, the change of the integral biological response of x in the presence of y has been interpreted in terms of two mechanisms: the interceptor and protector action of y on x. this mechanisms have been characterized by two criteria, r d and a d , reflecting the removal of x from dna by y (biophys. chem., , vol. , pages - ) . in this work we develop the theory of the interceptor-protector action of a mixture of two biologically-active dna-binding aromatic drugs. the theory is based on solution of a general system of mass balance equations in the three-component system x -y -dna with respect to the two factors, r d and a d . the outcome is a set of expressions enabling estimation of the change in biological response of x on addition of y as a function of equilibrium parameters under different restrictions. the results are in good agreement with known in vitro data on caffeine+antibiotic action in leukemia cell lines. the influence of mn + ions on the structure of natural calf thymus dna was studied by raman spectroscopy. measurements were done at room temperature and ph . ± . , in the presence of the physiological concentration of mm na + ions, and in the presence of mn + concentrations that varied between and mm. no condensation of dna was observed. dna backbone conformational changes were not detected in the whole concentration range of mn + ions as judging from the raman spectra. no evidence for dna melting was identified. binding of manganese(ii) ions to the charged phosphate groups of dna, stabilizing the double helical structure, is indicated in the spectra. as judged from the marker band of dc near cm − , altered nucleoside conformations in dc residues are supposed to occur, in the mn + concentration range of - mm. binding of divalent ions to n of guanine and, possibly, in a lesser extent to adenine was observed as judging from the raman marker bands at , , and cm − . toward rapid dna sequencing -ab initio study of nucleotide sandwiched between au( ) plates c. morari, d. bogdan, i. turcu national institute for research and development of isotopic and molecular technologies, cluj-napoca, romania recently a new technique for dna sequencing based on measurement of transversal conductive properties of a single strained dna molecule has been proposed. such a method would allow single-base resolution by measuring the electrical current perpendicular to the dna backbone. until now, it is still not clear if the electrical signals obtained for the four nucleotide can be clearly distinguished by a hypothetical experimental setup. several factors -like the influence of the pentose group or the presence of water -may influence quite strongly the electrical signatures of the four bases. therefore, in order to obtain a working device, the understanding and detailed description of the conduction mechanisms through dna bases connected to a metallic electrode is essential. the goal of theoretical studies in this field is to describe the electric signatures of dna bases from a theoretical point of view. our study is focused on the detailed description of the electronic structure of dna's base pairs "squeezed" between two au plates. while such a geometrical model closely mimic the sequencing devices proposed in the literature, it allows us to compute meaningful physical properties such as density of states, charge transfer and orbital localizationby using "ab initio" methods. the results allow us to give qualitative prediction over the potential use of such a device in the dna's sequencing technology. multinuclear platinum complexes represent a new class of anticancer agents, distinct in dna binding and antitumor activity from their mononuclear counterparts. bbr as a first representative of this class has undergone phase ii clinical trials for treatment of ovarian and lung cancers. the structure of this trinuclear pt drug consists of two trans-ptcl(nh ) units bridged by a trans- the main lesions formed by multinuclear pt complexes in dna are long-range intra-and interstrand cross-links (cls) bridging two guanines separated by up to four base pairs. since interstrand cls can prevent dna strand separation interfering with critical cellular events they represent a serious obstacle in cell survival. in order to contribute to understanding the biological effects of dna interstrand cls of bbr , we analyzed the effect of the single, site-specific , -interstrand cl formed by this metallodrug between two guanine bases on opposite strands in the '- ' and '- ' direction on the thermal stability and energetics of short dna duplexes. the results demonstrate that , -interstrand cls of bbr in both '- ' and '- ' directions exist as two distinct conformers that are not interconvertible and affect thermodynamic stability of the dna differently. side-by-side and end-to-end attraction of doublestranded dna c. maffeo , b. luan , a. aksimentiev university of illinois at urbana-champaign, urbana, usa, ibm watson research center, yorktown heights, usa genomic dna is densely packed inside cell nuclei and viral capsids. such close packing suggests that electrostatic repulsion between negatively charged dna in the condensed states is balanced by counterion-induced attraction. several theoretical models have been proposed to explain dna attraction, however, specific microscopic mechanisms are not known. here, we report all-atom molecular dynamics simulations of the effective force between double-stranded dna in side-by-side and end-to-end orientations. in the side-by-side orientation, the dna molecules were found to form a bond state in the presence of magnesium ions. in the bond state, the dna molecules contact each other via their negatively charged phosphate groups, bridged by magnesium ions. the maximum attractive force in the side-by-side orientation is about pn per dna turn. in the end-to-end orientation, a strong ( > pn) attractive force was observed at short (< . nm) end-to-end distances regardless of the electrolyte concentration. the presence of a phosphate group at the 'ends of the fragments was found to direct dna end-to-end self-assembly and produce bound states resembling a continuous dna molecule. the computed potentials of the mean force suggest that the end-to-end attraction, rather than being mediated by counterions, is likely caused by hydrophobic and van der waals interactions between terminal nucleobases of the fragments. doxorubicin (dox) is an anticancer antibiotics with a four-membered ring system containing an anthraquinone chromophore, and an aminoglycoside. it has good anticancer activity against a wide spectrum of tumors and is one of the most extensively used antitumor chemotherapeutic compounds currently in clinical use. interestingly, conversion of dox to pyrrolinodoxorubicin analog (p-dox) exhibits - times higher toxic effects in human breast cancer cell line (nagy, a. et al., pnas, ( ) . molecular mechanisms responsible for this enormously enhanced cytotoxicity have not been entirely clarified. there is good evidence that a key component of the mechanism of action of dox is its intercalation into dna and the formation of dox-dna adducts. therefore, we have examined in detail, using the methods of molecular biophysics, dna interactions of p-dox in cell-free media and compared these results with the same studies focused on the parental dox. we find distinct differences between dna interactions of dox and p-dox and suggest that these differences are responsible at least in part for different biological effects of these two anticancer drugs. design of a microfluidic devices for the detection of oligonucléotides by sers designing fast and efficient analytical tools allowing the detection of free dna or rna at very low concentration within small volumes, without specific molecular labeling, remains a major issue of significance to perform diagnostic or to detect pathogen agents. our strategy is to use surface-enhanced raman scattering (sers) to probe selectively the spectral signature of each base in polynucleotides. sers takes advantage of the strongly increased raman signals of species when adsorbed on adapted silver colloids. we already demonstrated that adenyl raman signal of pa in presence of silver colloids is times enhanced compared to bulk signal. we plan to use nanoliter droplets of uniformed size that form spontaneously in microchannels when two immiscible fluid streams merge. these tiny droplets are almost ideal reactors as they create homogeneous control condition. we will design an optimized channels network platform that result in droplets production hosting both nucleotides and silver colloids: internal fluids recirculation provide fast and efficient mixing, favoring base adsorption on silver nanoparticles. sers will be used to determine the chemical composition of the droplets. nicks represent the most common damage in dna which occurs naturally in living cells. structural properties of nicked dna fragments have been an object of numerous studies due to its special role in reparation processes. here we report experimental results covering ultrasound irradiation of nicked dna solutions. several single-stranded nicks were produced into one strand of dsdna fragments by the nicking enzyme bst i. we have quantitatively estimated the ultrasonic cleavage rates in nicked dna fragments with known sequences using the polyacrylamide gel electrophoresis. computer analysis of the cleavage pattern in the '-end labeled and primarily intact strand reveal cleavage enhancement in the regions of about b. p. up and down the nicks which were initially produced into complementary strand. the intensity of cleavage near the nicks is (in average) about times higher than cleavage in the same sites of the intact dsdna fragments. at the same time, the cleavage rates in positions beyond the regions of the nick markedly grow weak even comparing to the sequence-specific cleavage of intact double-stranded dna fragments. thus, the presence of the nick serves as an expressive structural indignation, which exceeds modulation of the structure caused by the base-pair sequence and is capable of absorbing mechanical stresses applied to the nearby sites of the molecule. comparing the native and an irradiated lactose repressor-operateur complex by md simulation g. naudin, n. garnier, d. genest, rue charles sadron, orléans cedex , france the function of the e. coli lactose operon requires the binding of a protein, the tetrameric repressor, to a specific dna sequence, the operator. the formation of this complex involves the interaction of at least one protein dimer with the operator sequence. this occurs via the dna-binding domains (called headpieces) of the two constitutive monomers. we have previously shown that upon irradiation with gamma rays the complex is destabilised mainly because the repressor losses its dna binding ability. radiation-induced lesions were identified that may be responsible for this deleterious effect: all tyrosine residues of the headpieces are oxidized into , -dihydroxyphenylalanine (dopa). in order to unravel the mechanisms leading to the observed destabilization of the operator-repressor complex, we compare by md simulations two complexes: -the native complex formed by a dimer of two headpieces and a fragment of dna with the operator sequence and -the damaged complex in which all tyrosines are replaced by dopa. analysis of these trajectories shows a loss of stability and binding energy as well as changes in the structure of the damaged complex with respect to the native one. by comparing precisely the evolution of the two complexes we can explain how the oxidation of the tyrosine residues of the headpieces into dopa may trigger the destabilization of the complex. local conformation of confined dna studied using emission polarization anisotropy in nanochannels with dimensions smaller than the dna radius of gyration, dna will extend along the channel. we investigate long dna confined in nanochannels with dimensions down to * nm, using fluorescence microscopy. studies of the statics and dynamics of the dna extension or position in such confinements as a function of e.g. dna contour length, degree and shape of confinement as well as ionic strength has yielded new insight into the physical properties of dna with relevance for applications in genomics and fundamental understanding of dna packaging in e.g. viruses. our work extends the field by not only studying the location of the emitting dyes along a confined dna molecule but also monitoring the polarization of the emission. we use intercalating dyes whose emission is polarized perpendicular to the dna extension axis, and by measuring the emission polarized parallel and perpendicular to the extension axis of the stretched dna, information on the local spatial distribution of the dna backbone can be obtained. we will discuss results in shallow ( nm) and deep ( nm) channels and describe how the technique can be used to investigate non uniform stretching of a single dna molecule. raman spectroscopy of dna modified by new antitumor nonclassical platinum complexes o. vrana, v. kohoutkova, v. brabec institute of biophysics as cr, královopolská , cz- brno, czech republic platinum anticancer agents (cisplatin, carboplatin, oxaliplatin) are in widespread clinical use especially against testicular, ovarian and head and neck carcinomas. there is a large body of experimental evidence that dna is the critical target for the cytostatic activity of cisplatin. platinum complexes form several types of adducts, which occur in dna with a different frequency and differently distort the conformation of dna. clinically ineffective trans isomer of cisplatin (transplatin) also covalently binds to dna bases. the trans geometry in dichloridoplatinum(ii) complexes was activated by various ways. the replacement of at least one amine ligand by planar amine ligand represents example of such activation. raman spectroscopy is powerful technique for examining both structural and thermodynamic properties of nucleic acids in solution. interactions of cis-and trans-pt(ii) complexes having nonplanar heterocyclic amine ligand such as piperidine, piperazine and -picoline with dna have been investigated by laser raman spectroscopy. raman difference spectra reveal that the pt(ii) complexes induce great structural changes in b-dna and indicate disordering of b-dna backbone, reduction in base stacking and base pairing and specific metal interaction with acceptor sites on purine residues. acknowledgement: this work was supported by grant as cr, iqs . a. v. vargiu , a. magistrato , p. carloni , p. ruggerone cnr-infm-slacs and physics dept., university of cagliari, cagliari, italy, cnr-infm-democritos and sissa/isas, trieste, italy, iit and sissa/isas, trieste, italy the minor groove of dna is the target of several anticancer agents, which interfere with replication and translocation processes, leading to cell death. the molecular recognition event is a key step to achieve detailed knowledge of the interactions behind selectivity and affinity of a ligand towards a particular nucleic acids sequence. recognition is a multiroute process which can involve many steps before the formation of the most stable adduct. in particular, many studies have pointed out the importance of events like sliding along the groove and dissociation (which is a relevant step in the translocation among different sequences) for the affinity and the specificity of minor groove binders. despite this, no studies on the dynamics of this event were reported in the literature. in this contribution i present our recent work on the subject. umbrella sampling and metadynamics were used in the framework of classical md to characterize mechanisms andfree energy profiles of molecular recognition routes by the antitumoral agents anthramycin, duocarmycin and distamycin. our results are in very good agreement with the available experimental data, and provide insights on the influence of factors like size, charge and flexibility on the molecular recognition process. amplification of oligonucleotide sequence recognition using bioresponsive hydrogels s. tierney, b. t. stokke biophysics and medical technology, dept. of physics, the norwegian university of science and technology, ntnu, no- trondheim, norway we describe development and characterization of oligonucleotide functionalized hydrogels for amplifying the molecular recognition signal occurring on hybridization between dioligonucleotides. the µm radius hemispherical hydrogels were integrated on a high resolution interferometric fiberoptic readout platform supporting determination changes in the optical length of the hydrogel with nanometer resolution. the hydrogels were designed with hybridized dioligonucleotides grafted to the polymer network as network junctions in addition to the covalent crosslinks or oligonucleotides grafted to the network chains. the probe oligonucleotide destabilizing the junction point by displacement hybridization yielded an optical signal about five times as large as for binding within the hydrogel design with a comblike grafted oligonucleotide. the optical signal was found to be dependent on the concentration of the probe, the sequence and matching length between the probe and sensing oligonucleotide. concentration sensitivity applied as specific label-free detection of oligonucleotide is estimated to be in the nanomolar region. the current design support detection in excess of x sequences. amplification of the molecular recognition employing the developed oligonucleotide imprinted hydrogel for label-free sensing of probe oligonucleotide sequences or taking advantage of the oligonucleotide sequence designed as aptamers for determination of other types of molecules are discussed. tracing t-cells by paramagentic nanoparticles in the brain of the rat model of als d. bataveljic , g. vanhoute , g. bacic , p. andjus inst. for physiology and biochemistry, univ. of belgrade, serbia, bio imaging lab, univ. of antwerpen, belgium, school of physical chemistry, univ. of belgrade, serbia amyotrophic lateral sclerosis (als) is a devastating neurological disorder affecting upper and lower motoneurons. since immune disbalance is known to be an important manifestation of the disease we were particularly interested in following the labeled immune cells in the familial als rat model, hsod- g a . a t -or t -weighted mri protocol was used with a mini surface coil placed over the skull of the anesthetized animal in a . t wide bore magnet. in order to compare this approach to standard high field small animal imaging a . t mri system was also used. there was a congruence of images of lesions in the brainstem at the two field strengths. it was confirmed with gd-dtpa contrast that the blood brain barrier (bbb) is compromised at the interbrain level. in order to study immune cell infiltration rats were i.v. injected with magnetically labeled antibodies against helper cd + or cytolytic cd + killer t cells. by combined t , t and t * weighted imaging cd + lymphocyte infiltration was observed in the brainstem-midbrain region while the cd + cells were more confined to the brainstem region. comparison of mri of labelled cd + vs. cd + lymphocytes reveals the relevant cellular inflammatory mechanism in als. the appearance of the mri signal from the latter t cell type points to the mechanism of bbb disruption as suggested from a recent study on the role of cd + t cells in a model of multiple sclerosis. emodin uptake study in u- mg cells using optically trapped surface-enhanced raman scattering probes s. balint , s. rao , p. miskovsky , d. petrov icfo -the institute of photonic sciences, barcelona, spain, department of biophysics, university of p.j.Šafárik, košice, slovakia emodin ( , , -trihydroxy- -methylantraquinone) is a photosensitizing pigment present in plants of herbal laxatives. emodin inhibits nuclear transcription factor-κb activation and induces free radical production in human mononuclear cells resulting in its antiviral and anti-cancer activity. the uptake and distribution of emodin inside the u- mg cell line is studied by combining optical tweezers and surfaceenhanced raman spectroscopy (sers). sers greatly enhances the spectrum of an otherwise weakly scattering material which is achieved by attaching nano-sized silver colloids to micron-sized dielectric beads. the distribution of emodin in the cell is studied by simultaneously trapping and exciting the sers bead and scanning it across the membrane while recording the emitted light. secondly, the beads are statically placed inside the cell and excited at certain intervals in order to track the migration of emodin through the membrane. the results give new insight in to the metabolic pathways of emodin and demonstrate a new imaging and detection technique that is fast and less invasive than current standards. acknowledgement: miin fis - (spain), fundació cellex barcelona, nadacia spp (slovakia), apvv- - (slovakia) amphotericin b (amb) is a polyene antibiotic that has been widely used for treatment of systemic fungal infections. the main mechanism of biological mode of action of amb is considered to be associated with formation of ionic membrane pores or channels in the lipid membranes. the aim of this work was to study the influence of the k + and na + ions on the aggregation process of amb in aqueous medium. the analysis of electronic absorption and fluorescence spectra of amb shows that the increasing k + concentration have influence on the level of aggregation of the drug much more than the same amount of na + ions. this effect is especially noticed at neutral ph values. the rls technique was used to study aggregation of amb in solution, in the environment of the k + and na + ions. the application of this technique makes it possible to study the electronically coupled chromophores, especially molecular aggregates. the results of the atr-ftir and raman spectroscopic studies also support this conclusion. these results provide a better understanding of the interaction between k + and na + ions and antibiotic which has not been previously considered to be significant for biological action of amb. g-quadruplexes: combining theory with experimental spectroscopic methods guanine-rich oligonucleotides can form unique tetrameric structures with four coplanar guanine bases, known as gquadruplex motifs. the g-quadruplexes have been found in vivo in the terminal parts of telomeres and other genomic regions. ligands, specifically binding to the g-quadruplex regions inhibit telomerase activity and thus can play an important role in the cancer therapy. most recently, the potential use of these structures has been tested in biosensors and nanotechnology industry. in the present work we use the infrared spectroscopy, including the relatively novel technique of the vibrational circular dichroism (vcd), in a combination with molecular dynamics and quantum chemistry computational methods to investigate the structure and spectroscopic response of the quadruplexes formed by the d(g) and selfassociated dgmp. we obtained a good agreement between the computed and experimental spectra, confirming that the proposed geometrical models are realistic. the vcd technique appears especially convenient for the studies as it can detect the liquid-crystalline phases of the g-quadruplexes by an anomalous enhancement of the signal. j. bogdanovic pristov, a. mitrovic, k. radotic, i. spasojevic institute for multidisciplinary research, belgrade, serbia fructose, due to its high antioxidative capacity, represents a significant component of non-enzymatic defense of some plants against cold-provoked oxidative stress. in the present study, we have investigated role of fructose in seasonal adaptation of picea omorika (pančić) purkinye to cold. this endemic coniferous species is exposed to subfreezing temperatures that range from - to - • c during the autumn/winter and high temperatures exceeding • c during the summer. characteristic epr signal of free or weakly bound mn + was used as an indicator of oxidative status of needles, since coldrelated oxidative damage leads to mn + release from photosystem ii. it was observed that prooxidative conditions developed in the autumn, at the beginning of cold season, which corresponded to significant increase of fructose level. total sod, as well as mnsod activity also rose significantly higher in the autumn. observed activation of antioxidative system (non-enzymatic and enzymatic) led to adaptation of needles to cold, as oxidative status during winter was decreased and similar to the status of needles in cold-free seasons. calyx of held: sted nanoscopy of a glutamatergic terminal p. bingen , t. m. staudt , c. kempf , h. horstmann , j. engelhardt , t. kuner , s. w. hell german cancer research center / bioquant, heidelberg, germany, max planck institute for biophysical chemistry, göttingen, germany, institute for anatomy and cell biology, university of heidelberg, heidelberg, germany the calyx of held, a large glutamatergic synaptic terminal in the auditory brainstem circuit has been increasingly employed to study presynaptic mechanisms of neurotransmission in the central nervous system. a highly detailed model of the morphology and distribution of cytoskeleton, synapsin, synaptic vesicles, calcium sensors, mitochondria, the presynaptic membrane and its active zones is derived by colocalization analysis of these different key elements of synaptic transmission in the rat brain. the various cellular components are visualized with subdiffraction resolution by stimulated emission depletion (sted) microscopy. imaging individual structural elements exhibit a focal plane resolution of < nm inside µm thick tissue sections. three-dimensional shim and pem to study collagen arrangement and crimping pattern p. bianchini , m. franchi , l. leonardi , a. diaspro lambs-ifom microscobio research centre and department of physics, university of genova, genova, italy., italian institute of technology (iit), genova, italy, department of human anatomical sciences and physiopathology of the locomotorapparatus, university of bologna, italy ligaments have been generally described as multifascicular structures with collagen fibre bundles cross-connecting to each other or running straight and parallel with crimps. a different collagen array and crimping pattern in different ligaments may reflect a different mechanical role. aim of this study was to relate the d collagen arrangement and crimping pattern by backward and forward second harmonic imaging microscopy (shim) and -photon excitation microscopy ( pem). shim on a laser-scanning system is a powerful and unique tool for high-resolution, high-contrast, three-dimensional studies of tissue architecture. although it is a coherent process the multiple scattering through the tissue give us the capability to acquire signal in both backward and forward direction [ ] . shim and pem were combined in a dual-mode nonlinear microscopy to find out collagen fibre arrangement and crimping pattern. both polarization dependence and differences between forward and backward signals allowed to yield information on local structure [ ] . currently used tcspc flim systems are characterised by high counting efficiency, high time resolution, and multiwavelength capability. the systems are, however, restricted to count rates on the order of a few mhz. in the majority of applications, such as fret or autofluorescence, the photostability of the samples limits the count rate to much lower values. the limitation of the count rate is therefore no problem. however, if flim is used for ion concentration measurements or imaging of chlorophyll in plants the available count rates can exceed the counting capability of a single tcspc channel. we therefore developed a flim system that uses eight fully parallel tcspc channels. by using a polychromator for spectral dispersion and a multichannel pmt for detection we obtain multi-spectral flim data at a rate of several frames per second. we will demonstrate the application of the system to dyamic changes of the fluorescence lifetime of chlorophyll in living plants. -imaging and spectroscopy - scattering effects on non linear imaging of thick biological samples f. cella , z. lavagnino , a. diaspro lambs-ifom, microscobio research center, university of genoa, italy, iit, italian institute of technology, genoa, italy non linear optical scanning microscopy became a useful tool for living tissue imaging. biological tissues are highly scattering media and this leads to an exponential attenuation of the excitation intensity as the light travels into the sample. while performing imaging of biological scattering tissues in two photon excitation ( pe) regime, the localization of the maximum pe intensity was found to shift closer to the surface and the imaging depth limit appears strongly limited by near surface fluorescence . in this work we computed illumination and photobleaching intensity distribution in order to characterize the effects induced by scattering. simulations of pe illumination and photobleaching intensity profiles have been performed for different scattering coefficients and at different focus depth. furthermore imaging of fluorescent immobile sample (polyelectrolyte gel) allowed to perform an experimental test on thick turbid media. results confirm that under these conditions no photobleaching effects due to scattering occur close to the surface. [ ]ying et al., appl. opt. , ( ) . [ ] theer p. and denk w, j. opt. soc. am. a. ( ) [ ]mazza d. et al., appl. opt. ( ) . biospectroscopic probes for real time measurement of hydrogen-deuterium exchange p. carmona , m. molina instituto de estructura de la materia (csic), madrid, spain, escuela universitaria deÓptica, madrid, spain isotopic exchange has long been used for the analysis of biomolecular structure and dynamics. hydrogen-deuterium exchange rates depend on ph, temperature and biomolecular environment. this is due to hydrogen bonding, low solvent accessibility, and steric blocking. time resolved measurement of hydrogen-deuterium exchange for subsequent d correlation spectroscopic analysis can, then, be very useful to obtain structural information from the said viewpoint. we have developed a microdialysis quartz cell for use in conjunction with raman spectroscopy to investigate hydrogen-isotope exchange reactions of biomolecules. the system requires only µl volumes of the initial substrate and perturbing effluent solutions. we have obtained a d o efflux rate of k d = . ± . min − with the greatest mwco ( kda) used here, which involves that an exchange rate of . min − is the limiting rate that could be resolved with the said cell system. analogous results have been obtained using an infrared biospectroscopic microdialysis probe. the use of the method described here has the advantage of avoiding sample dilution (and subsequent signal loss) involved in the known stop flow methods. acknowledgements: the authors gratefully acknowledge financial support from the spanish ministerio de ciencia e innovación (project ctq - /bqu). polarized transient absorption to resolve electron transfer between tryptophans in dna photolyase photoactivation of dna photolyase comprises electron transfer through the chain fadh • -w -w -w . photo-excited fadh • abstracts an electron from the tryptophan residue w in ∼ ps (monitored by transient absorption spectroscopy). the subsequent electron transfer steps (from w to w •+ and from w to w •+ ) are difficult to resolve experimentally, because electron transfer between chemically identical species does not give rise to net absorption changes. to overcome this difficulty, we make use of the fact that polarized excitation (pulse laser) induces a preferential axis (that of the excited flavin transition) in the system (photoselection), and that w and w form different angles with that axis (known from the crystal structure). thus, polarized detection should allow distinguishing between them. using polarized "classical" transient absorption on a nanosecond time scale and the pump-probe technique on a picosecond scale, we demonstrate the feasibility of the method and provide evidence that electron transfer from w to w •+ is faster than the ps time constant of the initial electron transfer from w to excited fadh • . synchrotron based fourier transform infrared (sr-ftir) microspectroscopy was applied to investigate apoptotic death of u- mg cells induced by the photosensitizer hypericin (hyp), in using different transport systems (hyp alone vs. hyp/ldl complexes) and incubation protocols. the differences between ir spectra of non-treated and hyp treated cells are mainly manifested in the positions of amide i and amide ii vibrational bands of proteins. these vibrational shifts are attributed to the protein structure changes from dominantly alfa-helix, in the non-treated cells, to beta-sheets and random coil structures, which prevail h and h after photodynamic treatment, respectively. the observed conformational changes of proteins can be explained as the consequences of the processes leading to apoptosis as was verified by flow cytometry experiments. the results confirm suggestion that ir spectroscopy can be successfully applied for the detection of early apoptotic processes. a. k. de, d. goswami indian institute of technology kanpur, india molecular fluorescence has been an indispensable tool in modern day optical imaging. one of the state-of the-art challenges in fluorescence microscopy is having better depth resolution as embodied by the confocal and multi-photon laser-scanning microscopic techniques. however, each technique bears its own limitation in having sufficient out-of-focus signal for the former while the low non-linear photon absorption cross-section for the latter. for confocal microscopy using one-photon excitation, we have shown how the clever choice of pulsed illumination instead of continuous-wave excitation leads to a gigantic enhancement in fluorescence that also has immediate applications in microscopy. moreover, single-photon illumination with ultrafast pulses leads to a novel way of achieving axial resolution along with numerous other advantageous applications e.g. reduced photo-bleaching of the chromophore. on the other hand we have thrown new insight demonstrating that the use of mode-locked laser pulses in multi-photon microscopy induces severe solvent-induced photo-thermal damage and prescribed methods to get rid of it. besides, the use of pulse pair excitation in multi-photon microscopy leads to probe and control the dynamics of fluorophores which has crucial role in selective excitation of fluorophores from quantum control perspectives. all these cutting edge research works will be presented in addition to our recent work on application of laser pulse shaping in multi-photon microscopy. interkingdom signalling in pseudomonas aeruginosa b. davis , r. jenson , p. williams , p. o'shea institute of biophysics, imaging and optical science, university of nottingham, u.k., institute of infection, immunity and inflamation, university of nottingham, u.k. quorum sensing is the process through which some bacterial species coordinate cell-cell communication. pseudomonas aeruginosa; the pathogen responsible for over % of chronic lung infections and the leading cause of mortality in cystic fibrosis patients, expresses two major classes of quorum sensing molecules characterized as n-acyl homoserine lactones and -alkyl- -quinolones. these compounds, in addition to their signalling roles have also been found to possess virulent properties, not only against competing species of bacteria such as staphylococcus aureus but also eukaryotic cells such as t-lymphocytes. the process of bacterial quorum signalling molecules influencing eukaryotic cell activity is termed 'interkingdom signalling'. to date it has been suggested that the quorum sensing molecules outlined above act on eukaryotic cells through interactions with an as yet unidentified plasma membrane or cytosolic receptors. this project is directed towards developing an understanding of how these compounds elicit eukaryotic response through a combination of membrane based interactions at physiologically relevant concentrations. particular emphasis is placed on studies of ligand binding with membrane microdomains in and the consequent downstream signalling are also considered. this work is significant as it will not only lead to a better understanding of pseudomonas infection, but may also lead to the discovery of new classes of agents for the treatment of infective diseases. a xas study of the sulphur environment in human neuromelanin and its synthetic analogues neuromelanin is a complex molecule accumulating in the catecholaminergic neurons that undergo a degenerative process in parkinson's disease. it was shown to play an either protective or toxic role depending on whether it is present in the intraneuronal or extraneuronal milieu. in the present study x-ray absorption spectroscopy is employed to investigate the sulphur binding mode in natural human neuromelanin, synthetic neuromelanins and in certain structurally known model compounds, namely cysteine and trichochrome c. based on comparative fits of human and synthetic neuromelanin spectra in terms of those of model compounds, the occurrence of both cysteine-and trichochrome-like sulphur coordination modes is recognized and the relative abundance of these two types of structural arrangement is determined. data on the amount of cysteine-and trichochrome-like sulphur measured in this way indicate that among the synthetic neuromelanins those produced by enzymatic oxidation are the most similar ones to natural neuromelanin. c. cremer kirchhoff-institute for physics, university of heidelberg, germany here we report on "spectral precision distance/position determination microscopy (spdm) with physically modifiable fluorochromes (spdm phymod ) to analyse the spatial distribution of single nuclear proteins and dna sequences at the macromolecular optical resolution level. like other methods of "spectrally assigned localization microscopy" (salm), spdm phymod is based on labelling 'point like' objects (single molecules) with different spectral signatures, spectrally selective registration and high precision localization monitoring by far field fluorescence microscopy. the intranuclear spatial location of single molecules was determined up to a density up to ca. molecules/µm of the same type, and distances down to - nm were nanoscopically resolved. quantum dots (qds) are semiconductor nanoparticles with increasing application as fluorescent markers in biology.we investigated structure of the cell walls of different species complexed with cdse qds using fluorescence microscopy, fluorescence spectroscopy and ftir techniques. in the experiments we used the cell walls isolated from three distinct plant species: arabidopsis thaliana, acer sp. and picea omorika. we studied both unlabeled and cdse-labeled cell walls. fluorescence spectroscopy and microscopy were used for detection of qds alone or complexed to the cell walls. emission spectra were deconvolved using the nelder-mead algorithm in matlab . . we calculated approximate probability distribution (apd) for positions of spectral component maxima. there was certain difference between unlabeled cell walls and those complexed with qds. the ftir spectra also show some difference between the complexed and pure cell walls. the results show that structure was changed, but not significantly in reaction with cdse qds. these results are promising in context of use of qds as labels in cell wall studies. the characterization of the complex of cell wall structure with qds is a part of the study of nanoparticles application in investigations of plant materials. modulating the response of single neurons and neuronal networks with biophysical stimuli f. difato, a. maccione, l. berdondini, f. benfenati, a. blau italian institute of technology, department of neuroscience and brain technologies, genoa, italy during differentiation, cell processes create connections with other cells to form tissue capable of performing complex tasks. biophysical constraints provide necessary inputs for cellular organization in living organism . to better understand how biophysical conditions influence tissue development, it is necessary to bridge the gap between experiments on single cells and complex tissues , . to achieve this goal we pair optical tweezers with electrophysiology measurements . by adopting neuronal networks as a biological model, neuronal signal transmission can be recorded either by patchclamp electrophysiology or microelectrode arrays (meas). dissociated neurons will be cultured on meas to record neuronal network activity at different sites of the network while applying spatio-temporally defined biophysical stimuli to individual neurons. a. diaspro , k. cortese , p. bianchini , c. gagliani , c. tacchetti iit -italian institute of technology, morego, genova, italy, microscobio, university of genoa, italy correlative light/electron microscopy (clem) is becoming increasingly frequent in molecular and cellular biophysics. we successfully applied the method to analyze the d structure of rough and smooth russell bodies used as model systems. the major advantages of this approach are the following: (i) the ability to correlate several hundreds of events at the same time, (ii) the possibilitˆto perform d correlation, (iii) the potential to immunolabel both endogenous and recombinantly expressed proteins at the same time and (iv) the effective combination of the high data analysis capability of flm with the high precisionaccuracy of transmission electron microscopy in a clem hybrid morphometry analysis. we have identified and optimized critical steps in sample preparation, defined routines for sample analysis and retracing of regions of interest, developed software for semi/fully automatic d reconstruction and defined preliminary conditions for an hybrid light/electron microscopy morphometry approach. the relevance of the presented approach lies in two important key elements, namely: the development of optical nanoscopy methods and the potentiality for exploring different correlative frameworks like optical nanoscopy vs. optical microscopy adding scanning force microscopy techniques. multidrug resistance is a well known phenomenon which limits effectiveness in treating malignancy with chemotherapy by modifying the internalization and/or externalization flow of the drugs through the cancerous cells. combined chemotherapies, such as mvac, are therefore currently used in bladder cancer treatment. however, about % of patients do not respond this chemotherapy because of inherent or acquired drug resistance. we developed a non invasive predicative test on urinary cells to estimate the chemotherapy effectiveness before treatment, based on the fluorescence emission of mvac. we first studied the mvac photophysical properties in solution and using five cell lines: a drug sensitive cancer cell line mgh-u s, its multidrug resistant subline mgh-u r, a not tumorigenic cell line sv-huc- , its tumorigenic counterpart mc-sv-huc t- and a cell line from transitional cell carcinoma t . the results revealed a penetration and localization of the drug depending of the cell line type, allowing us to find a specific fluorescence signature for the identification of mvac resistant cells. similar data have been obtained for cytospined fixed culture cells and patients urinary cells. -imaging and spectroscopy -abstracts multilayered photoresist system as a ghost model for biological samples in confocal microscopy. l. ferrari, f. cilloco, f. r. bertani, s. selci istituto dei sistemi complessi cnr rome we have realized a bilayer photoresist system as a model to perform optical characterization in the vis-ir range of multilayered complex biological specimen. our goal is to obtain structural and spectroscopic reference parameters from the identification of reflectance pattern features within a novel project granted by miur (skintarget, ideas firb). our model is composed by a nm thick layer (shipley photoresist ) with a refractive index n= . - . , wavelength dependent, and a nm tick hsq (hydrogen silsesquioxane) layer with a refractive index n around . , both spin-coated over a glass substrate. we present the analysis of reflectance pattern that can be obtained by a confocal laser reflective system as compared to the expected values as coming from analytical calculations, using a matrix approach, or a microscopic electromagnetic finite element analysis. interfacial roughness and consequent optical scattering are analyzed using a neural network approach. translational biophysics: multimode imaging for preclinical and clinical applications d. l. farkas cedars-sinai medical center, los angeles, usa our focus is where light and patient meet, and improvements yielding better outcomes. surgery is moving towards minimally invasive intervention, where biophotonics represents a major area of hope and growth. the translation of useful laboratory-derived knowledge into clinical practice has been hampered by the difficulty of detecting, characterizing and monitoring molecules and cells in the human body, especially dynamically. advanced bi ophotonic imaging is best suited for studying such entities, but has been lagging in clinical acceptance, in spite of major advances, and a clear need for the kind of resolution (spatial, temporal, spectral) and specificity that it alone can offer. biophysics-based new strategies are needed to address this challenge. the development of biophysical methods for translational medicine will be reviewed, with emphasis on our recent advances. our approach is a multimode one -combining methods to achieve early, quantitative detection of abnormalities. with imaging fulfilling its dual role of better describing anatomy and physiology, intrasurgical histopathologyequivalent molecular and cellular imaging is achievable in vivo, as is a closer spatio-temporal connection between imaging and intervention. some application areas to be covered: cancer (early detection by spectral reflectance/autofluorescence; progression quantitation by oct; nano-and targeted chemotherapy assessment in vivo); neurobiology (imaging fast calcium transients and alzheimer's plaques); hyperspectral mie scattering imaging for in situ displasia; design and use of an advanced multimode imaging endoscope with in vivo delineation of hirschsprung's disease for better intervention; monitoring of stem cell fate in vivo. immobilization of liposomes in a sol-gel matrix: a fluorescence confocal microscopy study r. esquembre , s. n. pinto , j. a. poveda , m. prieto , c. r. mateo ibmc, universidad miguel hernández, elche, spain, cqfm, instituto superior técnico, lisbon, portugal immobilization of liposomes shows interesting applications in protein biology, membrane biophysics and biosensor technology. previous fluorescence spectroscopy works revealed that the entrappement of zwitterionic phospholipid liposomes in a silica sol-gel matrix alters the thermodynamic properties as well as the fluidity of the lipid bilayer. interactions between the polar head of phospholipids and the porous surface of the host matrix could be responsible of such behaviour. in order to get more insight into this possibility we have immobilized, for the first time, giant unilamelar vesicles (guvs) and the shape and size of these structures as well as the possible existence of lipid domains have been visualized through fluorescence confocal microscopy. this technique allows for direct observation of the effect of encapsulation on an individual liposome, in contrast to the averaged information given by macroscopic spectroscopic techniques. liposomes composed of pure popc or dopc as well as mixtures dopc/dppc were labelled with the fluorescent probes bodipy, rhd-pe and rhd-dope. preliminary results shows that only the smallest guvs ( - µm) survive to the encapsulation process but often with a slight loss of its sphericity, probably due to pressures suffered during the matrix gelation. however no change in the gel/fluid phase proportion has been observed for immobilized dopc/dppc guvs, regarding to solution. solvent fluctuations play a key role in controlling protein motions and functions. here, we have studied how the reaction catalyzed by the light-activated enzyme protochlorophyllide oxidoreductase (por) couple with solvent dynamics (g. durin et al, biophysical j. ( ) , . to simultaneously monitor the catalytic cycle of the enzyme and the solvent dynamics, we designed temperature-dependent uv-visible microspectrophotometry experiments, using flash-cooled nano-droplets of por. the temperature-dependant formation of the first two intermediates in the por reaction were measured, together with the solvent glass transition temperature (t g ) and the build-up of crystalline ice. we find that formation of the first intermediate occurs below t g and is not affected by solvent dynamics, whereas formation of the second intermediate occurs above t g and is influenced by solvent dynamics. these results suggest that internal protein motions drive the first step of the por reaction whereas solvent slaved motions control the second step. we propose that the concept of solvent slaving applies to complex enzymes such as por. amphotericin b (amb) is one of the main polyene antibiotics widely used to treat deep-seated fungal infections. the mechanism of biological action of amb is most probably directly related to the ability of the drug to form hydrophilic pores in the membrane core, thus affecting physiological transport of ions. the effects of amb-cu + complexation are demonstrated by the electronic absorption and fluorescence spectra. the absorption spectra of amb in water (ph= ) after the injection of water solution of copper(ii)sulfate display a complex structure with hypsochromic-and bathochromic-shifted bands indicative of formation of molecular aggregates of the drug. formation of the electronically coupled chromophores of amb, especially aggregates, was analyzed at different cu + concentrations by the rls (resonance light scattering) technique. intensity of the fluorescence emission spectrum (characteristic of the dimeric form of amb) decreases after the amb-cu + complex formation. this effect of the formation of the amb aggregated structures by amb-cu + are different from the spontaneous molecular aggregation process, as deduced from the spectroscopic analysis. morfo-functional asymmetry of the olfactory receptors of the honeybee apis mellifera l e. frasnelli , g. anfora , f. trona , f. tessarolo , r. antolini , g. vallortigara cimec, centre for mind/brain sciences, university of trento, italy, iasma research and innovation center, fondazione e. mach, s. michele all´adige (tn), italy, biophysics and biosignals lab., dept. of physics, university of trento, italy lateralization, i.e. the different functional specialisation of the left and right side of the brain, has been documented in many vertebrate and, recently, invertebrates species. in the honeybee apis mellifera l. olfactory memory seems to involve at first the use of the right antenna. the present study investigated physiological and anatomical differences between left and right antennae of honeybees. electroantennographic responses (eag) were recorded from the left and right antennae of honeybees to linalool, a floral volatile compound, and isoamyl acetate, an alarm pheromone, at doses (from , to µg). the number of sensilla on the left and right antennae was recorded by scanning electron microscopy (sem). each antenna segment, from insects, was observed from different viewpoints in order to image the whole antenna surface and compute the number of sensilla. the tested compounds induced higher eag responses on the right than on the left antenna at every dose. sem showed that the placoidea olfactory sensilla were slightly more abundant on the right antenna surface than on the left one. results suggest an asymmetry in the peripheral odour perception mechanism in the honeybee a. mellifera. super-resolution imaging of dna through single molecule switching of intercalating cyanine dyes c. flors, c. n. j. ravarani, d. t. f. dryden school of chemistry, university of edinburgh, u.k. a growing trend in far-field super resolution fluorescence microscopy involves the replacement of photoactivatable fluorophores by common dyes such as cy, atto or alexa [ ] . it has been shown that these dyes can blink in useful timescales for single-molecule based imaging by adding suitable buffers. this strategy greatly simplifies the sample preparation and imaging scheme, enabling its application to a wider range of biological systems. we have explored if a similar approach might be useful to study dna topology using intercalating cyanine dyes such as yoyo- . there are two main advantages of this approach: i) dna labelling with intercalating dyes is straightforward, and ii) the free dye in solution is essentially non-fluorescent, greatly reducing the fluorescence background. we show that yoyo- can blink in the absence of oxygen and in the presence of cysteamine, which allows its application to nanoscale imaging. we exemplify its use by imaging λ-dna and a puc plasmid. we also explore the compatibility of several intercalating dyes with biological systems such as enzymes or cells. our results suggest that dna intercalating dyes are a promising option for fluorescence super-resolution studies of dna topology. seeing more in total internal reflection fluorescence (tirf) microscopy r. fiolka, a. stemmer nanotechnology group, eth zürich, zürich, switzerland total internal reflection fluorescence (tirf) microscopy is an effective widefield imaging tool that selectively excites a very thin sample layer within the evanescent excitation field at the glass-water interface. lateral resolution of standard tirf microscopy is limited to approx. nm using green emission, which can be insufficient for a large class of biological investigations. additionally, the evanescent excitation field is prone to light scattering, creating out of focus blur in the final image. we present several techniques that address these mentioned shortcomings of standard tirf microscopy. using evanescent standing waves, the lateral resolution in tirf microscopy can be extended by a factor of . , reaching nm. we further show techniques to reduce the blur induced by light scattering of the evanescent field. finally, we demonstrate optical sub-sectioning capabilities in tirf microscopy by acquiring several images with different penetration depth of the evanescent field and applying suitable post processing algorithms. thereby the obtainable z-resolution exceeds the classical limit of widefield microscopy, and object structures lying within the evanescent field can be reconstructed. the natural photosentizer hypericine (hyp) exhibits potent properties for tumor diagnosis and photodynamic therapy. evidences of hyp release from ldls prior to passive diffusion within cells are addressed in this study. fluorescent properties of hyp have been used for dynamic studies of its interaction with low-density lipoproteins (ldls) and u glioma cells. subsequent non-specific staining of intracellular membranes compartment were observed by mean of colocalization fluorescent imaging studies. it was shown, that monomers of hyp are only redistributive forms. increasing of hyp concentration leads to the formation of non-fluorescent aggregates within ldls as well as within the u cells, and can preclude its photosensitizing activities. in all experiments, hydrophobic character of the molecules appears as the driving force of its redistribution process. acknowledgment: this work was supported by the slovak res. and dev. agency contracts no. lpp- - . we also kindly thank the synchrotron soleil for using the detection system of the disco beamline. ledgf/p switches from a dynamic to a tight chromatin interaction upon binding to hiv- integrase j. hendrix , z. debyser , y. engelborghs laboratory of biomolecular dynamics, katholieke universiteit leuven, belgium, laboratory of molecular virology and gene therapy, katholieke universiteit leuven, belgium human transcriptional co-activator ledgf/p is hijacked by hiv- integrase (in) during the replication of hiv. little is still known about the molecular complex of these two proteins in the living cell. in this work we first studied the cellular chromatin interaction of egfp-tagged ledgf/p with tunable-focus fluorescence correlation spectroscopy (tf-fcs) and show that ledgf/p is in equilibrium between a free brownian motion and a very slow movement on the chromatin. being dependent on the size of the laser focus, this slow movement represents a continuous associationdissociation-reassociation process that is governed by diffusion. concentration-dependent continuous photobleaching measurements (cp) furthermore revealed the existence of high-affinity chromatin binding sites. next, we co-expressed mrfp-tagged in and confirmed its intracellular interaction with ledgf/p by fluorescence cross-correlation spectroscopy (fccs). interestingly, cp and fluorescence recovery after photobleaching (frap) indicated that the affinity of this complex for chromatin is exceptionally high. by twophoton fluorescence lifetime imaging ( p-flim) we verified if the cellular stoichiometry was altered when the proteins were expressed together. we believe that this work is useful for the understanding and targeting of hiv-replication. biophysical identification of orf from clavulanic acid biosynthesis cluster as a cyp l. s. goto , c. o. hokka , j. f. lima , o. r. nascimento , a. p. u. araújo grupo de engenharia bioquímica, ufscar, br, grupo de biofísica molecular "sérgio mascarenhas", usp, br streptomyces clavuligerus produces the clinically important β-lactamase inhibitor clavulanic acid. biosynthesis related genes reside in three gene clusters, one of these, named clavulanic acid gene cluster, includes most of the known clavulanic acid biosynthetic enzymes. the penultimate step along clavulanic acid biosynthesis remains unclear. required transformation involves at least two events: oxidative deamination and double epimerization of ( s , s )-clavaminic acid into ( r, r)-clavaldehyde. downstream the known part of clavulanic acid cluster lays orf , a putative gene encoding a tentative cytochrome p -like protein which knockout has been proven deleterious to clavulanic acid biosynthesis. should such protein exist, it would be candidate to fulfill the clavulanic acid pathway missing step. in this work, orf encoded protein is characterized aiming to place it as a real p . for this task, molecular cloning and recombinant expression of orf were accomplished. purified protein was submitted to spectroscopic measurements such as circular dichroism and electron paramagnetic resonance which indicate p features, including catalytic relevant heme iron redox states and homolytic peroxide scission mechanisms. further, peroxide reaction adducts were characterized by spin trapping. this work is supported by fapesp. muscle structure and gabaergic innervations in the limbs of barnacle cyprid l. gallus , s. ferrando , c. gambardella , a. diaspro , p. bianchini , p. ramoino , v. piazza , g. tagliafierro libiom, dibio, università di genova, italy, ifom-lambs/microscobio, difi, università di genova, italy, dipteris, università di genova, italy, ismar cnr, venezia, veneto, italy balanus amphitrite is a sessile crustacean that settles at the larval stage of cyprid. in this stage we studied the gabaergic innervation of limb striated muscular fibers, by immunohistochemistry. the second harmonic generation (shg) and -photon excitation ( pe) microscopy were used to set out the muscle structure and its relationship with nerve terminal. sections were observed at a multimodal nonlinear microscope composed by the leica clsm. the laser system used is a ti:sapphire chameleon-ultra (coherent inc, santa clara, ca, usa), tunable between nm and nm and characterized by a pulse width of fs delivered at a repetition rate of mhz by means of a homebuilt set-up (bianchini p. and diaspro a. j biophotonics : - ). the z-stacks were performed in order to obtain the -dimensional distribution of the muscular fibers. in the posterior ganglion gad immunoreactive (ir) motor neurons were arranged in clusters near the emergence of the limb nerves. gaba and gababr ir neuromuscular junctions (nj) were localized in the limb muscle fibers; vgat ir cells surrounded each limb muscles. these results suggest that gaba plays a key role in the regulation of limbs movement. the shg was very useful to outline the relationship between nerve terminals and limbs muscle fibers. giant unilamellar vesicles (guvs) are very useful model membrane systems to study many aspects of lipid-lipid and lipid protein interactions, particularly employing fluorescence microscopy related techniques (bagatolli ) . the use of this model system can be particularly useful to study aspects related with the lateral structure of bacterial membranes using the aforementioned approach (i.e. fluorescence microscopy). bacterial cells have a size close to the resolution limit of optical microscopy and details about the organization of their membranes are not easy to achieve using such technique. recently a new electroformation method to prepare guvs composed of compositionally complex mixtures under physiological conditions was developed in our laboratory (montes ). in the present work we further extended this electroformation method to prepare guvs composed of bacterial lipid extracts and lipopolysaccharide (lps). in our experiments we used e.coli lipid extract to prepare small vesicles containing various lps species, from smooth strains and rough mutants. suvs were used as starting point to electroform guvs using various types of buffers with high ionic strength. the successfully obtained bacterial-guvs were used to study the interaction of these membranes with known lps-binging proteins (lung surfactant protein d, sp-d) and peptides (polymyxin b). our results remark the usefulness of this particular bilayer models to perform studies mimicking bacterial membranes. mechanical properties of polymeric membranes probed by afm m. kocun , i. mey , w. müller , m. maskos , a. janshoff georg-august universität, institute for physical chemistry, göttingen, germany, johannes gutenberg universität, institute for physical chemistry, mainz, germany many biological cell functions are dependent on the mechanical properties of the membrane. polymeric membranes that mimic native cell membranes are valuable research tools which can be used to better understand the physics of biological membranes. we have investigated free standing artificial membranes prepared from polybutadiene-b-polyethylene oxide (pb-b-peo). the membranes were prepared from vesicles ruptured on porous silicon substrates. these polymeric membranes were studied by confocal laser scanning microscopy (clsm) and atomic force microscopy (afm). force indentation curves were performed on the membranes and theoretical models were used to extract elasticity constants from the results. the study of polymeric membranes can give insight to the function of biological membranes, furthermore polymeric membranes can be used to create new hybrid systems by incorporating biological (lipids, proteins), artificial (polymers, dyes) and inorganic (nanoparticles) components. site-directed spin labeling study of the lightharvesting complex cp the topology of the long n-terminal domain of the photosynthetic light-harvesting complex cp was studied using electron spin resonance (esr). cp is a minor antenna complex of the photosystem ii (psii), a multisubunit protein complex. wild-type cp protein containing a single cysteine at position and nine single cysteine mutants were produced, allowing to label different parts of the domain with a nitroxide spin label. in all cases the apoproteins were either solubilized in detergent or they were reconstituted with their native pigments (holoproteins) in vitro. the spin label esr spectra were analyzed in terms of a multi-component spectral simulation approach, based on hybrid evolutionary optimization and solution condensation. these results permit to trace the structural organization of the long n-terminal domain of cp . we took the crystal structure of light-harvesting complex ii (lhcii), major antenna complex of psii available in pdb as a starting point and constructed a model for cp based on esr data. present opportunities and future developments in soft x-ray transmission and emission microscopy b. kaulich, a. gianoncelli, v. babin, m. kiskinova elettra -sincrotrone trieste, s.s. km . in area science park, i- trieste-basovizza, italy soft x-ray transmission and emission imaging and spectromicroscopy are bridging the gap to other microscopy techniques in terms of lateral resolution, penetration depth and chemical sensitivity. the novel soft x-ray spectromicroscopy approach of the twinmic instrument at the elettra synchrotron radiation facility is combining several imaging modes for morphology characterization, such as scanning, projection and full-field imaging with several contrast techniques including brightfield, darkfield, differential phase and interference contrast at sub- nm lateral resolution. complementary chemical information is provided by chemical imaging and micro-spectroscopy using the photon-in/photon-out xray absorption and x-ray fluorescence. unique for twinmic is the low-energy x-ray fluorescence setup operated at - ev photon energies, which allows simultaneous analysis of the morphology, and the distribution of light elements on cellular and sub-cellular level. in the presentation the principles of the methods used in the twinmic instruments the performance and potentials of the instrument will be demonstrated by several examples of applications in the field of human, animal and plant biology, biophysics and chemistry, physiology and genetics. the potential impact of microscopy techniques using free-electron x-ray lasers on biophysics will be illustrated by the diproi project at fermi@elettra. -imaging and spectroscopy - fluorescence anisotropy and afm used as tools to characterized porin´s reconstituion in luv´s s. c. lopes, i. sousa, p. eaton, p. gameiro requimte, faculdade de ciências, universidade do porto, rua do campo alegre, - porto, portugal a major requirement to perform structural studies with membrane proteins is to define efficient reconstitution protocols that assure, not only, a high incorporation degree in preformed liposomes, but also a protein directionality and topology that mimics its in vivo conditions. for this kind of studies, protein reconstitution in membranes systems via a detergent-mediated pathway is usually successfully adopted, since detergents are generally used in the initial isolation and purification of membrane proteins. in this study we report the reconstitution of ompf in preformed dmpc and e. coli liposomes using two different techniques for detergent removal: ( ) exclusion chromatography and ( ) incubation with detergent adsorbing beads. the incorporation degree was determined by bicinchoninic acid assay and fluorescence anisotropy was used to determine ompf effect on the structural order of membrane lipids. these results show that protein insertion in membranes depends both on the technique used to remove detergent and on the lipids used to prepare the liposomes. moreover more anisotropy and atomic force microscopy studies will allow a better characterization of bacterial model system membranes. the wavelength dependence of the luminescence for different sized aunp by -photon clsm k. li, m. schneider pharmaceutical nanotechnology, saarland university, campus a , d- saarbrücken, germany gold nanoparticles (aunp) with different sizes can exhibit original luminescent properties if excited with pulsed nearinfrared laser light which makes them a suitable object to be detected in biological tissues. the main obstacle is to distinguish between the object of interest (emitted light) and the autofluorescence from the sample which limits the scope of application of aunp. therefore, our aim is to characterize the luminescent properties of such nanoparticles regarding their excitation and emission. in this study, the excitation and emission spectra of aunp with different sizes below nm in an excitation range of nm to nm were investigated. our study shows the emission spectra curves of aunp are broadband spectrum and vary with the changing of excitation wavelength from nm to nm. the results also suggest the minimum laser power necessary to trap the aunp depends on particle size and excitation light wavelength and a maximum power above which the particles are destroyed. in all the experiment above, to avoid the simultaneous effects from slide and cover slip is a must. rocking, tumbling, and sliding: real-time nanomotion of a membrane-bound virus p. kukura , h. ewers , c. mueller , a. renn , a. helenius , v. sandoghdar laboratory of physical chemistry, eth zurich, ch- zurich, switzerland, institute of biochemistry, eth zurich, ch- zurich, switzerland the interaction of a virus with its receptors in the plasma membrane is decisive for its infection of cells. optical studies have revealed that after binding, virus particles move laterally on the membrane, but the complexity of the cellular environment and the drawbacks of fluorescence microscopy have prevented access to the molecular dynamics of early virus-host couplings. here, we examine a model system, in which single simian viruses (sv ) interact with their gm ganglioside receptors in supported lipid bilayers. we employed scattering interferometry and single molecule fluorescence localization to visualize the vectorial rotational motion of virions. at low receptor concentration, we observed sliding and tumbling of single virions during rapid lateral diffusion. in contrast, at increased receptor concentration the virions repeatedly underwent periods of standstill, reminiscent of their behavior prior to endocytosis. by an unprecedented combination of millisecond time and nanometer spatial resolutions, we revealed that during these immobile periods, the virions rock back and forth among nanoscopic spots separated by nm. our insights, together with the structure of the viral capsid, suggest aggregation of receptors in nanodomains and recurrent swap of binding between receptor molecules and neighboring viral protein pentamers. herein, we have developed different membrane probes binding spontaneously to the outer plasma membrane leaflet and showing sensitivity to membrane properties such as surface charge and phase state. the first two types of probes were based on -hydroxyflavone fluorophore. one of them showed a high sensitivity to the surface charge and to the phase state of lipid bilayers, while the other was only sensitive to the surface charge. the third type, which was based on nile red fluorophore, was sensitive only to the phase state. surprisingly, the probes that were sensitive only to the surface charge did not respond to apoptosis, while the other two types of probes showed significant spectroscopic response to it. moreover, the latter exhibited response to cholesterol depletion, which was similar to that observed on apoptosis. thus, according to our data, the intact living cells present a remarkable fraction of the cholesterol-rich domains, while apoptotic loss of the transmembrane asymmetry decreases it dramatically. these probes represent a new tool for quantification of surface charge and cholesterol-rich domains in cell membranes. -imaging and spectroscopy - due to its porosity and unique optical properties porous silicon (psi) is an attractive template to develop biomaterials and biosensors. porous silicon microcavity (psimc) structures were prepared then functionalized for covalent protein attachment of glucose oxidase (gox) or solubilized bacteriorhodopsin (br). functionalization and protein infiltration was monitored by specular reflectometry sensitive to change in refractive index, when a molecule is attached to the large internal surface of psi. protein infiltration into the porous scaffold was confirmed by edx spectroscopy and the structures were imaged by biphoton microscopy. second harmonic generation and enhanced two-photon excited fluorescence emission from porous silicon was observed when resonantly exciting the structures. in addition, when the microcavities were infiltrated with gox or br, the proteins acted as a very efficient internal two-photon-excited fluorescence emitter, hence protein infiltration enabled the in-depth visualization of the porous structure by taking advantage of the optical sectioning capacity inherent to the non linear optical microscopy technique. patterning of bio-molecules: methods for characterization of neuron-substrate interfaces we investigated how to use and improve micro printing techniques to obtain molecules patterns on cell culture substrates. with micro contact printing (µcp), we generated geometrically defined depositions of poly-d-lysine (pdl) using poly-dimethylsiloxane stamps and optimized neuronal culturing conditions. rat and mice hippocampal neurons grown on those patterns showed to be alive and functional. we then applied µcp to study axonal development by combining cell culture assays with atomic force microscopy (afm) to investigate in more detail the molecule deposition on the surface and to measure morphological changes in the growth cone (gc) during the early phases of neurite development. we found distinct shapes of the gcs depending on whether they were growing on l adhesion molecule patterned by indirect-µcp or on pdl coated surfaces. we also attempted to transfer such patterns on multi electrode arrays in order to constrain neuronal cell bodies on the electrode area and to improve electrophysiological recordings from neuronal networks. other patterning techniques were therefore explored using a nano-drop printing system. patterned surfaces were analyzed with afm and scanning electron microscopy to combine different approaches aimed to the improvement and characterization of the printing techniques. insights on the mechanism of electron transfer in complex i a. l. maniero , c. bergamini , m. bortolus , r. fato , s. leoni , g. lenaz università degli studi di padova, padova, italy, università degli studi di bologna, bologna, italy complex i (nadh dehydrogenase) plays a central role in cellular energy production, transferring two electrons from nadh through a series of iron-sulfur clusters (fes) to ubiquinone (coenzyme q); the electron transfer is coupled to the translocation of protons across the membrane. the fes center n is the last acceptor in the electron-transfer chain, but the mechanism through which the enzyme couples the e − reduction of the fes centers to the e − reduction of ubiquinone (q→sq→qh ) is unclear [ ] . in our experiments, submitochondrial particles were treated with different inhibitors, in the absence or in the presence of different quinone analogues, and nadh addition initiated the electron transfer. we assess by epr (electron paramagnetic resonance) spectroscopy the relative abundance of the reduced n center and of the semiquinone radical, and coupled epr data to enzymatic activity assays and to fluorescence measurements on the effect of inhibitors on reactive oxygen species (ros) production. we identify two different classes of inhibitors showing different effects on ros production. moreover the redox state of the complex has shown to depend both on the inhibitors and on the quinone analogues. a possible mechanism of the electron transfer, that can explain the experimental findings, will be presented. conventional fluorescence microscopy suffers from a resolution limit imposed by the diffraction of light. stimulated emission depletion (sted) microscopy overcomes this resolution barrier without being limited by the wavelength by taking advantage of the photophysics of the observed sample into the image formation process, and has proven to be a powerful approach for exploring relevant biological issues. the outstanding resolution of sted microscopy is achieved by drastically minimizing the spatial extent of the focal region from which fluorescent molecules can emit signal in the sample. so far, mainly complex and relatively expensive lasers systems providing pulsed beams have been used to inhibit the fluorescence in the outer area of the focal region. here we report on the development of a new setup based on a fast beam scanning confocal microscope using compact turn-key and inexpensive continuous wave lasers. the great potential of this simple configuration is demonstrated for a selection of commonly used fluorescent markers. characterization of hepatitis b antigen particles by atomic force microscopy each year, over one million people die from hepatitis b virusrelated chronic liver disease, including cirrhosis and hepatocellular carcinoma. the major surface antigen of hepatitis b virus (hbsag) is a cysteine-rich, lipid-bound protein with amino acids. recombinant hbsag produced in yeast can self-assemble into -nm immunogenic spherical particles that are used in licensed hepatitis b vaccines (protein/lipid ratio is / in mass). hbsag size and shape have been mainly investigated by transmission electron microscopy after negative staining of the particles. however, under these conditions, no details of the particle surface can be obtained because of the shadowing effect due to the uranium salts. here we describe new structural insights of hbsag particles using atomic force microscopy (afm) performed under physiological conditions. we applied atomic force microscopy to define structural details of the surface organization with a resolution in the nanometer range. as expected, the diameter of hbsag particles is , ± , nm in average. the surface of these particles clearly shows the presence of protuberances that most probably correspond to proteins. indeed, reduction and alkylation induces the disappearance of the protuberances. the number of the protuberances estimated from afm micrographs is about per spherical particles. a biophysical study of equine herpesvirus- entry into cells g. mckenzie , p. o'shea , j. kydd , c. rauch school of veterinary medicine and science, university of nottingham, uk, institute of biophysics, imaging and optical science, university of nottingham, uk equine herpesvirus- (ehv- ) can cause respiratory disease in young horses, varying degrees of paralysis and abortion during the later stages of pregnancy. furthermore, there has been a recent increase in the number of outbreaks involving paralysis of thoroughbred horses in training. virus disseminates rapidly after initial infection via cell associated viraemia. controlling this may prove crucial in combating the pathogenicity of the disease. we have investigated the cellular interactions of ehv- . initially, binding of ehv- to fluorescein phosphatidylethanolamine (fpe)-labelled phospholipid vesicles of various compositions was examined. a variety of microscopy techniques were then employed to study the events surrounding the binding of virus to equine peripheral blood mononuclear cell (pbmc) membranes. confocal microscopy images have highlighted possible colocalisation of ehv- with membrane 'rafts'. total internal reflection fluorescent microscopy was then used to identify viral binding at the membrane with high contrast images, able to observe single virus particles. establishing the viral entry pathway into pbmcs would allow drugs that target this process to be employed, reducing clinical viraemia. the interpretation of in-vivo binding rate measurements allows inferring the molecular interactions that regulate cellular functions. fluorescence recovery after photobleaching (frap) is a widely used tool to quantify binding reactions in vivo. however the lack of "golden standards" for these measurements requires the measured binding rates to be validated with other techniques. we present a new fluorescence correlation spectroscopy (fcs) method to measure the in vivo bound fractions and residence times for molecules that interact with an immobile substrate. we apply this method to measure binding of mutants of the transcription factor vbp (vitellogenin binding protein) to the dna. comparison of fcs with frap results in comparable estimates of the measured diffusion constants and bound fractions. however, fcs provides an estimate of the vbp residence time at the dna, while frap does not. this limitation in the analysis of vbp is due to the larger photobleaching volume used in frap, if compared to the observation volume of an fcs experiment. in sum, we present a method to measure binding rates with fcs. substantial agreement of this method with frap is shown. however, further validation on tightly bound molecules will be necessary to assess if frap and fcs agree in the measurement of residence times. -imaging and spectroscopy - we have investigated the changes in the mechanical properties of the zona pellucida (zp), a multilayer glycoprotein coat that surrounds mammalian eggs, that occur after the maturation and fertilization process of the bovine oocyte by using atomic force spectroscopy. the response of the zp to mechanical stress has been recovered according to a modified hertz model. zp of immature oocyte's shows a pure elastic behaviour. mature and fertilized oocyte's zps evidence, instead, a transition from a purely elastic behaviour, which occurs when low stress forces are applied, towards a plastic behaviour has been observed. the high critical force necessary to induce deformations, that well supports the non-covalent long interactions lifetimes of polymers, increase after the cortical reaction. afm images show that oocytes' zp surface appear to be composed mainly of a dense, random meshwork of nonuniformly arranged fibril bundles more wrinkled surface characterize marure oocytes with respecto to immature and fertilized oocytes from a mechanical point of view, the transition of the mature zp membrane toward fertilized zp, through the hardening process, consists in the recovery of the elasticity of the immature zp, while maintaining a plastic transition that, however, occurs with a much higher force with respect to that required in mature zp. dynamical behavior of ribose and deoxyribose supercooled water solutions s. e. pagnotta , s. cerveny , a. alegria , j. colmenero centro de fisica de materiales, centro mixto csic-upv/ehu, san sebastián, spain, departamento de fisica de materiales, universidad del pais vasco (upv/ehu), facultad de quimica, san sebastián, spain, donostia international physics center, san sebastián, spain ribose and '-deoxyribose are probably the most widespread monosaccharides in nature. they can be extensively found in ribonucleic acid (rna) and '-deoxyribonucleic acid (dna), respectively, where they form, together with a nitrogenous base and a phosphate group, a peculiar building-block structure called nucleotide. in the present work, the relaxation dynamic of ribose and deoxy-ribose water solutions at different concentrations has been studied by broadband dielectric spectroscopy and differential scanning calorimetry in the temperature range of - k. two relaxation processes are observed for all the hydration levels; the slower (process i) is related to the relaxation of the whole solution whereas the faster one (process ii) is associated with the reorientation of water molecules in the mixture. as for other polymeric water solutions, dielectric data for process ii indicate the existence of a critical water concentration above which water mobility is less restricted. moreover, according with these results, atr-ftir measurements of the same sugar solutions showed an increment in the intensity of the oh stratching sub-band close to cm − as water content increases. the regulation of the formation of cytoskeletal protein complexes by actin-binding proteins m. nyitrai, a. vig, t. kupi, z. ujfalusi, s. barkó, g. hild university of pécs, faculty of medicine, department of biophysics, pécs, hungary in living cells various groups of proteins are associated to supramolecular actin filament structures, often in a nucleation factor dependent manner. for example, actin structures associated with formins can bind tropomyosin and profilin, while those polymerised by the nucleation of the arp / complex bind cofilin and myosin i. the molecular mechanisms underlying the regulation of the formation of these protein complexes is still ambiguous. we have shown recently that formins can bind the actin filaments and change their conformational state. subsequent binding of other actin-binding proteins, such as tropomyosin and myosin, can reverse these changes. it appears that the reversal effect assumes that the actin-binding protein binds the filaments in a well-defined and specific binding site. the altered conformational state of the actin filaments observed after the binding of these proteins provides a possible explanation for the modified affinity of the filaments for other-actin binding proteins. based on the results available so far we assume that the affinities are modified differently by different nucleation factors, and the conformational changes introduced to actin by actin nucleation factors can serve as the molecular bases for the regulation of the formation of actin based intracellular protein complexes. experiments are currently in progress to test and further corroborate the existence of such regulatory mechanisms in living cells. correlation between sub-cellular distribution of photoactive drug hypericin (hyp), determined by applied delivery systems (hyp vs hyp/ldl), and mode of the cell death is addressed in this study. co-localization of hyp with mitochondria, lysosomes and golgi apparatus in u- mg glioma cells was determined by confocal laser scanning microscopy in using organelle specific fluorescent dyes as well as by time resolved fret experiments. flow cytometry experiments were realized to study a photodynamic effect of hyp ( nm/ jcm − ) on cells. significant differences in the proportional representation of live, apoptotic and/or necrotic cells were observed for different types of delivery systems of hyp hours after hyp ( x − m) photoactivation. conclusions: i) sub-cellular distribution of hyp depends on using delivery systems, ii) the mode of cell death depends more on concentration of hyp inside cells, than on different type of delivery systems (for non selective wide-field cell illumination), iii) fluorescence lifetime is sensitive parameter to study sub-cellular distribution of hyp. novel time-resolved spectroscopic methods have been used to investigate the interactions between a fluorescently-labelled mutant of the peptide melittin and supported lipid bilayers, formed by self-assembly at a silica-water interface via vesicle deposition. time-resolved evanescent wave-induced fluorescence spectroscopy (trewifs) is a surface-selective technique in which the evanescent field from a pulsed laser source is used to photoexcite fluorescent species at an interface. the resulting fluorescence decay kinetics, measured using time-correlated single-photon counting, report on the micro-domains experienced by those fluorescent species at an interface. extending trewifs to time-resolved evanescent wave-induced fluorescence anisotropy measurements (ew-trams) provides dynamic rotational information of a fluorescent species, reporting on its mobility at an interface. presented here are trewifs and ew-trams data obtained from the fluorescence of an alexa -labelled melittin mutant interacting with a dipalmitoylphosphocholine bilayer at room temperature, physiological ph and ionic strength. the results provide new insights into the conformation, location and motion of cytolytic peptides interacting with cell membranes. optical tweezers are used to controllably apply forces to red blood cells and the resulting chemical and structural changes are monitored using raman spectroscopy. the forces are applied in vivo and mimic that which the cell undergoes mechanically as it passes through vessels and smaller capillaries. the first result presented will be spectroscopic evidence of a transition between the oxygenation and deoxygenation states of hemoglobin that is caused by the stretching of the red blood cell. the transition is due to mechanically induced enhancements of hemoglobin-membrane and hemoglobin neighbor-neighbor interactions. the latter, lesser known effect is further studied by modeling the electrostatic binding of two of the protein structures using molecular dynamics methods. secondly, polarized raman spectroscopy is utilized to study the packing and ordering of the hemoglobin proteins in the red blood cell as it is stretched. depolarization ratios for a number of heme group modes change, indicating that the applied force additionally packs and orders the proteins inside the cell which further demonstrates the role of cell deformation in the oxygen transport kinetics. acknowledgements: this work was supported by miin fis - (spain) and fundació cellex barcelona fret microscopy is widely used to study protein-protein interactions in living or fixed specimens. currently, most commonly used visible fluorescent proteins for live-cell fret studies are the cerulean and venus variants of the cyan and yellow fluorescent proteins. even though this fret pair appears to be ideal for monitoring protein-protein interactions, the most commonly used fixed laser wavelengths do not excite cerulean at peak absorption. recently, we characterized an ideal donor, the monomeric teal fluorescent protein (mtfp), which is excitable using the commonly available ( ) opt. june/july, ). we used teal as a donor for various red fluorescent proteins as acceptors including tdtomato, mko , morange , mtagrfp, mkate. we have employed a "fret standard" genetic construct to minimize variability in separation distances and positioning of the fused donor and acceptor fps. using spectral fret imaging and fluorescence lifetime measurements in living cells expressing the fused proteins, we have characterized both sensitized acceptor emission and the change in the donor lifetime distribution as a result of quenching for each of the fused fp pairings. our results indicate that some red fps are better acceptors than others in terms of quenching the teal donor and sensitizing the emission of the acceptor indicating a fret event. s. m. perepelytsya, s. n. volkov bogolyubov institute for theoretical physics, nasu, -b metrologichna str., kiev, , ukraine stability of the dna double helix is determined by na + counterions, neutralizing the negatively charged phosphate groups of the macromolecule backbone. but under spatial conditions they may be replaced by much heavier ions, for example cs + . to determine the influence of heavy counterions on internal dynamics of the double helix the conformational vibrations of na-and cs-dna are studied. for this purpose the model of conformational vibrations of dna with counterions is used [perepelytsya s.m., volkov s.n., eur.phys.j. e. , , ] . as the result the frequencies and amplitudes of vibrations for b -dna with na + and cs + counerions are calculated. the frequencies of internal modes of the double helix are about , , and cm − . the frequencies of ion-phosphate modes are about and cm − for na-and cs-dna respectively. the calculated amplitudes of vibrations show that light counterions not disturb the dna internal dynamics, but heavy counterions make move all structure elements of the double helix. using the valence-optic approach the intensities of the dna vibrations in raman spectra are calculated. the calculations show that the ion-phosphate mode in cs-dna spectra is prominent, in contrast to na-dna spectra, where it has very low intensity. obtained results describe the intensity increase of the band cm − in cs-dna spectra that was observed in [bulavin l.a., et al., arxiv: . v ]. s. soria , f. quercioli , r. mercatelli , f. bianco , i. cacciari , g. righini centro studi e ricerche "e. fermi", p. del viminale , rome, italy, ifac-cnr, istituto di fisica applicata "n. carrara", via madonna del piano , sesto fiorentino (fi), italy, isc-cnr, istituto dei sistemi complessi, via madonna del piano , sesto fiorentino (fi), italy we report on the application of a simple white light source based on the supercontinuum generation from commercial photonic crystal fibres to confocal fluorescence microscopy and fluorescence lifetime imaging (flim) microscopy. the coherent white light can be tuned by varying the wavelength and intensity of the pump, a ti:sapphire laser. there are several advantages jn the use of sc sources: spatially coherent white radiation, tuning ranges of approximately nm, high brightness, a robust compact system (potentially all-fibre) and relatively low cost. being pulsed, sc sources are suitable for flim. we have used this system for measuring foerster resonance energy transfer (fret) in order to study interactions between ions channels and proteins of membrane within lives cells resolving the quaternary structure of plague f capsular antigen a. soliakov , j. r. harris , m. r. hicks , a. rodger , r. woody , a. watkinson , j. h. lakey cell and molecular biosciences inst., newcastle univ., uk, chemistry dept., univ. of warwick, coventry, uk, biochemistry and molecular biology dept., colorado state univ., usa, pharmathene uk, billingham, cleveland, uk most gram-negative pathogens express multi-subunit fibres on their surfaces that mediate host cell attachment, biofilm formation, invasion of host defenses and protection against phagocytosis. here we have studied capsular antigen fraction (or caf ), secreted through the conserved chaperone/usher pathway by the plague agent yersinia pestis [ ] . caf is highly immunogenic and is used in a recombinant subunit vaccine against plague. recent immunological studies indicated vaccines containing polymeric caf have higher protective efficacy than vaccines containing its monomeric variant. this difference in protective efficacy was attributed to the quaternary structure. however the quaternary structure of caf has not been characterized [ ] . in our study we have used transmission electron microscopy of negatively stained specimen and linear dichroism spectroscopy to determine the quaternary structure of recombinant caf . whereas electron microscopy revealed morphology of caf fibres bound to the surface of a carbon coated grid, linear dichroism gave the orientation of subunits in flow aligned caf fibres in solution. our results show recombinant caf comprises extended linear fibres and circularized fibres, both of which have high degree of conformational freedom. despite the vast application of lambert-beer law in biology, this empirical law cannot accurately describe the light absorption process of molecules with a long-lived excited state. this family of molecules includes most fluorescent molecules, which are very important in biology. lambert-beer law is in fact only valid at very low intensity of incident light and very low concentration of chromophore. if the system doesn't meet these conditions, it falls into a nonlinear regime when it is affected by a phenomenon which we call "dynamic photobleaching": the depletion of chromophores from the first layers, due to their transition to the excited state, leads to a sub-exponential propagation of light in the medium [ ] . this phenomenon leads to the necessity of a new formula for the light absorption dynamics which depends on the lifetime of the excited state of the chromophore. the predictions of the theory were successful in describing the absorption dynamics of azobenzene [ ] , but now they have been tested also on a biologically relevant molecule like chlorophyll. the results indicate that the absorbance is affected by the intensity of the incident light, and it is therefore a non reliable way of determining, for example, the concentration of the molecule. annular pupil filter to improve spatial highfrequency signal to noise ratio in linear and non linear microscopy e. ronzitti , v. caorsi , a. diaspro lambs, microscobio, department of physics, university of genoa, genoa, italy, semm, ifom-ieo, university of milan, milan, italy, neuroscience and brain technologies department, iit, genoa, italy shot-noise significantly affects and deteriorates the imaging capabilities of typical two-photon excitation and confocal laser scanning microscope, especially in biological applications where the detected signal can be remarkable slight [ ] . in particular, shot-noise substantially influences the spatial high-frequency range inducing a remarkable reduction of the optical transfer bandwidth. the insertion of an annular filter on the microscope objective lens in the illumination light pathway is here proposed to retrieve the high frequencies information loss [ ] .the electromagnetic interference effect induced by the filter insertion, gives a redistribution of the optical transfer function. in particular, the microscope frequency response in filter scheme exhibits an enhancement of signal to noise ratio at the high frequencies able to recover the high frequencies hampered by shot-noise [ ] . the goal of biomarker studies is to develop simple noninvasive tests that identify disease states. focus is beginning to shift from identification of individual biomarkers to identification of biomarker panels comprising multiple targets of different molecular species. there are, however, no current technologies available that allow for a comprehensive and simultaneous analysis of the expression levels of multiple cellular components, i.e. proteins and rna. surface plasmon resonance (spr) polaritons are surface electromagnetic waves that propagate in a direction parallel to the interface between a metal surface and an external medium e.g., liquid. these oscillations are very sensitive to any change of this boundary, a phenomenon that has been exploited to facilitate label-free, real-time detection of biological interactions, e.g. protein binding interactions. we are utilising the power of spr to develop technologies that facilitate diagnostic procedures for complex diseases such as alzheimer's disease and chronic obstructive pulmonary disease, through identification and detection of patterns of biomolecules indicative of disease. our approach will facilitate better disease characterisation, improve early detection strategies and aid drug discovery. surface generated fluorescence detection by supercritical angle confocal microscopy d. verdes, m. rabe, s. seeger institute of physical chemistry, university of zürich, winterthurerstrasse , zürich, switzerland a two channel confocal microscope for surface and simultaneously in solution fluorescence detection is presented. the microscope's core element is a parabolic mirror objective that collects the fluorescence above the critical angles for total internal reflection (tirf) of the water/glass interface. an aspheric lens incorporated into the parabolic mirror is used for diffraction limited focusing and collecting the fluorescence at low angles with respect to the optical axis. this low angles excitation approach is technically straightforward and gives an advantage over high numerical objectives that require very high angles for tirf illumination. by separating the collection of the fluorescence into supercritical and subcritical angles, two detection volumes highly differing in their axial resolution are generated at the interface. the surface selectivity of the detection volume is obtained on the basis of the dipole emission profile near a dielectric interface. its angular distribution is highly anisotropic and consists of a superposition of traveling and evanescent waves, which both are detected using the parabolic mirror objective. unlike with objective tirf microscopy, the parabolic mirror objective achieves easily diffraction limited excitation/detection volume at the water/glass interface. the objective optical performance is shown by measuring the actin cytoskeleton of cultured cells, fret energy transfer within adsorbed clustered proteins and single molecules detection. differential polarization laser scanning microscopy. anisotropy in biological samples g. steinbach , i. pomozi , g. garab biological research center, hungarian academy of sciences, szeged, hungary, pi vision bt., budapest, hungary differential polarization (dp) spectroscopy provides unique information on the anisotropic organization of biological samples [tinoco et al. ann rev biophys biophys chem : ]. however, anisotropy is often a microscopic property. the dp-lsm, constructed in our laboratory enables us to image the main dp quantities: linear and circular dichroisms (ld&cd), also in fluorescence detection ( . further applications include: periodic structure of isolated amyloids, anisotropy variations in cell walls related to drought resistance, and strong anisotropy of 'artificial chlorosomes', nanorods of synthetic porphyrins. dp-lsm might thus represent a novel tool in the better understanding of highly organized molecular macroassemblies. t. m. staudt , p. bingen , c. kempf , h. horstmann , j. engelhardt , t. kuner , s. w. hell german cancer research center/ bioquant, heidelberg, germany, max planck institute for biophysical chemistry, göttingen, germany, institute for anatomy and cell biology, university of heidelberg, heidelberg, germany the calyx of held, a large glutamatergic synaptic terminal in the auditory brainstem circuit has been increasingly employed to study presynaptic mechanisms of neurotransmission in the central nervous system. a highly detailed model of the morphology and distribution of cytoskeleton, synapsin, synaptic vesicles, calcium sensors, mitochondria, the presynaptic membrane and its active zones is derived by colocalization analysis of these different key elements of synaptic transmission in the rat brain. the various cellular components are visualized with subdiffraction resolution by stimulated emission depletion (sted) microscopy. imaging individual structural elements exhibit a focal plane resolution of < nm inside µm thick tissue sections. -imaging and spectroscopy - r. worch, t. weidemann, p. schwille biotec, biophysics group, technische universität dresden, germany interleukin- (il- ), a small soluble protein, is a principal regulatory cytokine, playing an important role during the maturation and clonal expansion of antigen specific b-cells in mammals. at the plasma membrane, il- is recognized by a receptor that consists of two single spanning transmembrane proteins: a high affinity il- r alpha chain, and a low affinity il- r gamma chain. it is still controversial by which molecular mechanism the signaling complex is fully activated: dimerization of chains, large conformational change upon il- binding or a combination of both. moreover, the influence of the lipid environment in which the activation takes place is poorly characterized. to address these issues we aim to reconstitute the receptor component in artificial membrane systems to study the various mutual interactions by means of fluorescence-based techniques, mainly fluorescence correlation spectroscopy. due to reduced background in a chemically defined system this may provide details not yet accessible in the living cell. k. wicker, s. sindbert, r. heintzmann randall division of cell and molecular biophysics, king's college london, se ul london, u.k. fluorescence confocal microscopy, an indispensable tool of modern biology, allows the imaging of live fluorescent specimen with high lateral as well as axial resolution. through the introduction of a sufficiently small confocal pinhole, the lateral resolution can be enhanced compared to that of a wide field microscope. however, this gain in resolution comes at the cost of a decrease in detection efficiency, as light blocked by the pinhole is lost. we present a method for improving the lateral resolution (extending work of sandeau et al. [ ] ) and detection efficiency of scanning microscopes by adding an interferometer with image inversion in one of its arms to the detection pathway [ ] . this surpasses the lateral resolution achievable in a conventional confocal microscope (closed pinhole) while increasing the detection efficiency substantially. point spread function measurements for a uz-interferometer (uzi) are shown. the light in this setup follows threedimensional u-and z-shaped paths and relies on reflections off planar surfaces only in order to achieve image inversion. we achieved an interference contrast of % for white light, and excellent agreement with theoretical predictions. g. vicidomini, a. schönle, j. keller, r. schmidt, c. von middendorff, s. w. hell max planck institute for biophysical chemistry, department of nanobiophotonics, göttingen, germany fluorescence far-field microscopy is a indispensable tool in modern life science. however, the resolution of its standard variants is limited by diffraction to ∼ nm in the focal plane and ∼ nm along the optical axis. to overcome this limit, a new class of super-resolution microscopy techniques has been designed. one example is pi microscopy. to obtain isotropic resolution, pi uses interference of the wave fronts from two opposing lenses. the point spread function (psf) of pi system is characterized by multiple maxima/sidelobes, which replicate the object in the image. therefore image restoration is mandatory to render unambiguous imaging. the situation is further complicated because the positions and the relative heights of the multiple maxima/sidelobes of the psf depend on the phase difference (pd) between the two wave fronts at the focus. if the refractive index of the sample varies in space, this pd becomes a function of position and pi image formation process looses its shift invariance. the pd function (pdf) may not even be known a-priori and must then be estimated from the image, leading to a blind image restoration problem. here, we propose a maximum a-posteriori based method to solve the problem. we either assumed a mathematical model for the pdf that depends on a small number of parameters or allowed for an arbitrary pdf but introduced a smoothing constraint. designer multicomponent lipoplexes have recently emerged as especially promising transfection candidates, since they are from to times more efficient than binary complexes usually employed for gene delivery purposes [ ] [ ] [ ] . here, we show, for the first time, that after internalization binary complexes of lower transfection potency remain in compact perinuclear endosomes, while multicomponent systems have intrinsic endosomal rupture properties that allow plasmid dna to escape from endosomes with extremely high efficiency [ ] . endosomal rupture results in an extraordinarily homogeneous distribution of unbound plasmid dna throughout the cytoplasm and in the nucleus [ ] . fluorescence spectroscopy and stopped-flow technique were utilized for the study of the kinetics of incorporation of hypericin (hyp), a natural photosensitizing pigment, into lowdensity lipoproteins (ldl). triphasic kinetics of hyp association with ldl was observed when solutions of hyp and ldl were mixed together. the most rapid phase of hyp incorporation is completed within tens of msec, while the slowest one lasts - min. the most of hyp molecules are incorporated into ldl in the slowest phase. the kinetics of the incorporation of hyp into ldl particles pre-loaded with hyp were also investigated. the observed decrease of the lifetime and total intensity of hyp fluorescence with the increase of the incubation time of hyp with hyp/ldl complex is a sign of the formation of aggregates and the dynamic quenching of singlet excitation state of hyp inside ldl. to study the kinetics of a transfer of hyp molecules between ldl particles, the time evolution of the stopped-flow and time-resolved fluorescence experiments were investigated after the mixing of the complex hyp/ldl= : with appropriate amounts of free ldl. for each final hyp/ldl ratio the increase of the lifetime and total intensity of hyp fluorescence was observed. the half-time of this process is similar to that one of the slowest phase of hyp incorporation into free ldl. a. bosca , r. magrassi , g. firpo , l. repetto , c. boragno , u. valbusa italian institute of technology, genova, italy, nanomed labs (difi-cba), genova, italy the technique of choice to measure the electrophysiological activity of neuronal cells is the so called patch-clamp method because of its precision and sensitivity; this procedure could play a major role also in the investigation of the behavior of a biological neuronal network and so represents an important tool for understanding its functionality and for screening the effects of drugs and compounds on it. the final aim of this project is the development of a planar patch clamp device suited to measure simultaneously the electrical activity of cultured neurons associated in a network. this device will be made of polymeric disposable material and will include a microfluidic perfusion system. in order to create a smooth micro sized pore in a thin polymeric membrane we exploited the prototyping capabilities of focused ion beam etching and we extended the air moulding technique by combining it with soft moulding, obtaining micro structured substrates with the requested features. we also subjected our substrates to a chemical treatment capable of rendering its surface stable and hydrophilic and we verified that it makes them suitable for neuron culturing. use of lipoamino acids for nasal delivery of therapeutic proteins c. bijani , s. sice , j. elezgaray , c. degert , o. broussaud , e. j. dufourc physica pharma, pessac, france, umr cbmn, cnrs-université bordeaux , iecb, pessac, france most of the therapeutic proteins are clinically administered through an intravenous injection several times a day / week. because the repeated injections are not convenient and cause pain in patients, an alternative route of administration is desirable such as oral or nasal. unfortunately, proteins are easily degraded by proteolytic enzymes in the gastrointestinal tract and, therefore, have a low bioavailability when administered via oral route. physica pharma has gained experience in forming sprayable solutions combining lipoamino acids (laa) and small therapeutic organic compounds with the aim to improve their intranasal absorption. in the present work we investigated the ability of laa to complex large molecule such as human erythropoietin, human growth hormone and salmon calcitonin in order to form easily sprayable colloids. these three proteins are used respectively to treat anaemia, growth problem and hypercalcemy. circular dichroism and dynamic light scattering were used to further characterize the laa-protein colloids. a specific molar ratio of laa versus protein was found for wich the proteins keep their secondary structure and have an overall isotropic size slightly increased. molecular dynamics show that proteins are indeed coated with laa. such a complex is shown to pass very easily through a culture of nasal cells growth at confluence. in this study we describe two systems based on soft matter designed for the drug delivery and for the replacement of synovial fluid in osteoarticular pathologies. (i) a new class of temperature sensitive hydrogels pva/poly(ma m nipaam n ) shaped as microspheres obtained with a water-in-water emulsion photocrosslinking reaction. microparticles of pva/poly(ma m nipaam n ) with m:n theoretical molar ratios equal to : ; : , : have been studied in terms of average size and responsiveness to temperature characterized by confocal laser scanning microscopy (clsm), dynamic light scattering (dls) and differential scanning microcalorimetry (dsc). (ii) a physical network based on hyaluronic acid with a small extent (degree of substitution: %) of hydrophobic moiety grafted on the backbone, hyadd , has been characterized in order to account for the influence of thermal treatment on the stability of the hydrogel. dynamic light scattering (dls) and small angle neutron scattering (sans) have been used for dynamic-structural characterization of hyadd hydrogels. diffusion of macromolecular probes has been studied by fluorescence recovery after photobleaching (frap) to study the mesoscopic texture of the hydrogel and molecular dynamic (md) simulations were used to approach the time evolution of the physical junction points and of chains clusters. multi-scale estimation of water soluble diffusivity in polysaccharide gels g. eisele , s. bertini , d. paganini , l. piazza ronzoni institute, milan, italy, distam, university of milan, italy diffusion properties in gels play important role in food and biotechnological applications. an attractive goal is to design gels in such a way that the active molecules are delivered by the material according to specific release sequences. the transport of macromolecules within polymeric gels depends: on the obstruction effects of the surrounding gel strands, on the molecular interactions between the gel and the solute, on the interactions between the solute molecules themselves and the interactions between the solute and the solvent. physical polysaccharide gels were evaluated in this work to probe diffusion over both microscopic and macroscopic distance scales. physical gels from agar and starch were investigated by high and low resolution nmr techniques in order to characterize their structures. obstruction effects of the surrounding gel strands were considered by studying diffusion of glucose. local diffusion, due to brownian motion, was quantified by low resolution nmr spectroscopy. the fickian diffusion coefficient was measured by modelling experimental concentration-distance curves obtained by means of a two-compartment diffusion-cell. diffusion coefficients depend on the viscoelastic properties of the gel matrix and on water-polysaccharide interactions. j. l. cuellar, g. koehler, m. fischlechner, e. donath medizinische institut für physik und biophysik, leipzig, germany rather than being just pathogens, from the view point of biotechnology and materials science viruses can be regarded as nanocontainers in which nucleic acids have been enclosed in a protective self assembled protein cage. in some viruses an additional lipid-protein envelope wraps the capsid shell. the capsid is constituted of several copies of one single protein subunit or just a few, arranged in a regular fashion showing icosahedral symmetry. the mechanical properties of the cage are important for understanding the infection mechanism involving the release of the encaged genome. by means of atomic force microscopy, it becomes possible to probe such material properties by nanoindentations of single viral particles. it is very interesting to learn how strong or brittle a virus can be. here we have studied the mechanical properties of empty rubella virus particles (rlps) due response of external applied forces. we found that rlps are extremely soft comparable to that of some rubber materials. a peculiarity of the rubella virus is that the capsid is considerably smaller than the surrounding shell filling only a fraction of the lumen provided by the envelope. the envelope in rubella has a distinguishable response on the material properties of the virus. deformation and fracture of the capsid requires comparatively larger forces. our results indicate that the ph is a major factor influencing on rubella particle material properties. this can be related to the infection mechanism. pentavalent antimony (pa) (glucantime, sanofi-aventis or glaxo) are the mainstream agents of choice for leishmaniasis treatment. in therapeutic doses the pa treatment has cardiac side effects like electrocardiographic (ecg) alterations, that include qt segment prolongation, t wave flattening or inversion, inversion of st segment, p, r and t waves amplitude reductions, torsade de pointes arrhythmias and sudden death by cardiac arrest. the objective of this study was to characterize the arrhythmogenic potential of pa. we used guinea-pig to assess the chronic effects of pa therapeutic dose on corrected qt interval, qt dispersion, ventricular action potential (ap) amplitude and duration at % and % of maximal repolarization and survival rate. guinea-pig received daily mg/kg pa or saline for days. eight lead ecg were recorded before and in the last treatment day. at the end the animals were killed and the left ventricle papillary muscles excised for ap recording with the intracellular microelectrode technique. our results of chronic pa treatment showed significant increase of qrs complex duration, qt interval duration, qt dispersion and incidence of t wave flattening or inversion and arrhythmias. the ap analysis demonstrated prolongation at % duration. the treatment was lethal in % of the animals. we concluded that pa is a proarrhythmic drug that upon chronic use may causes arrhythmias and mortality by disturbances in the ventricular repolarization process. m. m. khvedelidze , t. j. mdzinarashvili , t. partskhaladze , n. nafee , m. schneider ivane javakhishvili tbilisi state university, tbilisi, georgia, institute of molecular biology and biological physics, tbilisi, georgia, biopharmaceutics and pharmaceutical technology, saarbrücken, germany, pharmaceutical nanotechnology, saarbrücken, germany we have studied the thermodynamical properties of chitosan-coated nanoparticles (cnp) and non-coated nanoparticles (np) and have gained some insights about plga nanoparticles' properties using supersensitive differential microcalorimetry. the experiments show that in a wide ph interval the changes in transition temperature did not take place. it was shown that such nanoparticles could be used in acidic surrounding for drug transfer. stability and their other properties are less depended on either the particles were in bidistilled or deionized water, or the suspension of particles were located in buffer. to determine the interaction of plga nanoparticles with dna. in the case of dna presence in cnp solution the calorimetric experiments show that the heat absorption peak is constricted, what biophysically means that interaction between them takes place. for more exact determination the contribution of cnp in spectrum, we have compared the spectra of pure dna with the spectrum of the same concentration dna plus cnp. the optimal ratio for dna loading onto the cnp was found to be : . protein-based "epoxy-like" physical hydrogels for stem cell transplantation s. c. heilshorn, c. wong po foo, j. s. lee materials science and engineering, stanford univ., u.s.a. stem cell transplantation has emerged as a promising therapy for multiple injuries and diseases; however, cell survival after transplantation is often poor and unpredictable. we hypothesize that co-injection of stem cells encapsulated within an optimized physical hydrogel will enhance viability. whereas, current physical hydrogels require a shift in environmental conditions (e.g., ph, temperature) to initiate the sol-gel phase transition during encapsulation, our newly designed molecular-recognition gels do not require environmental triggers. instead, these "physical epoxy-like" gels consist of two components that undergo a sol-gel transition upon mixing due to specific hydrogen bonding. the gel viscoelasticity is predictably tuned through precise variation in the molecular-level design of the two components, created using recombinant protein techniques. the design of the two components is based on simple polymer physics considerations and utilizes bio-mimimcry. adult neural stem cells or mesenchymal stem cells are encapsulated within these gels with high viability at constant physiological conditions. the gels promote the growth and differentiation of neural progenitors into neuronal phenotypes, which adopt a d-branched morphology. the gels are further optimized for use in the central nervous system by tethering neuroprotective peptides to the gel through molecular-recognition sites. these peptides are released-on-demand by cells through the action of proteolytic enzymes. we consider the integration of the portal protein of the bacteriophage virus φ into lipid bilayers of giant unilamellar vesicles (guvs) membranes with the aim of constructing a functional cargo-device able to transport dna and later translocate it outwards. our nano-engineering plan consists of growing guvs from bilayer membranes built up from proteoliposomes previously prepared by extrusion. we have designed two alternative chemical routes for integrating the portal protein in the lipid bilayer, the first considering the native protein and the second a mutant modified with a hydrophobic belt made of histidine-tags. in our contribution we will present details on the different nano-engineering strategies and experimental evidence about the integration of the portal protein in the membrane with an orientation adequate to allow for functional dna translocation. p.-h. guelluy , m.-p. fontaine-aupart , m. hoebeke biomedical spectroscopy, ulg, belgium, laboratory of molecular photophysics, univ. of paris-sud, france ppme is a second generation photosensitizer (ps), and a promising candidate for photodynamic therapy (pdt) treatment. we have previously demonstrated that ppme can be easily and efficiently encapsulated in dmpc liposomes, used as ps-vector. we therefore compared the photophysical and photochemical properties of free and encapsulated ppme incubated with human carcinoma cells. absorption and fluorescence microspectroscopy as well as flim analysis allow evaluating the aggregation state of ppme inside the different cellular organelles and the extracellular medium. confocal microscopy established undoubtedly the colocalization of ppme, by robust probabilistic exclusion method, within mitochondrion (central siege of apoptosis). after ps activation ( h and h), the balance apoptosis-necrosis was double-estimated by facs device and fluorescence confocal microscopy. quantification of hydroxyl radical was purchased by spin trapping-esr spectroscopy and quenching technique. all these techniques required peculiar settings because of the fluorescence activity of ppme. these results allow to ascertain that the vectorization of ppme affords a better cellular penetration and a monomeric state of ppme. the presence of ppme inside mitochondrion orientated the cellular death in apoptosis h following ps-activation. but necrosis is the major actor h after treatment. active substrates to study mechanotransduction j. le digabel, p. hersen, b. ladoux laboratoire matière et systèmes complexes, université paris diderot -cnrs, paris, france cellular processes imply an important coordination of interactions with the extracellular medium. accumulating evidences demonstrate that cell functions can be modulated by physical factors such as the mechanical forces acting on the cells and the extracellular matrix, as well as the topography or rigidity of the matrix. these extracellular signals can be sensed by mechanosensors on the cell surface or in the cell interior to induce various cell responses. we have developed an original approach based on micro-fabricated substrates of polydimethylsiloxane (pdms) to study cell migration. we used a closely spaced array of flexible micropillars (diameter∼ ?m) to map the forces exerted by cells on their substrates. in this case, the micropillars act as passive force sensors. here, we propose to analyze the cell response to an external applied stress by a well-controlled actuation of the substrate. to do so, we used magnetic pillars. such substrates allow us to modify dynamic adhesion conditions of cells and to better understand the coupling phenomena between mechanical sensing and biochemical activity of a living cell. using polyacrylamide hydrogels doped with ferromagnetic iron oxide particles or ferrofluids, we can make magnetic pillars with diameters of to microns while a magnetic field can be locally applied with a magnetic needle. such a technique will be helpful to study the mechanical response of cells to an external force or to local changes in their microenvironment. glucose scavenging activities of pamam dendrimers m. labieniec , t. gabryelak , c. watala university of lodz, deptartment of general biophisics, lodz, poland, glycation is a spontaneous, non-enzymatic modification of biomacromolecules with hexoses, mainly glucose. in terms of its pathophysiological relevance, it targets predominantly proteins, but also nucleotides and phospholipids, and is of major importance to both physiology (ageing) and pathology (diseases with a metabolic background, like diabetes). earlier we demonstrated in vivo that the administration of pamam g to diabetic rats resulted in a significantly reduced blood glucose, as well as the early (hba c ) and late (ages) protein glycation products. in this study we investigated the ability of pamam g ( surface nh groups) and g ( nh ) to inhibit glycation of proteins in plasma, and a model protein -bsa. pamam g and g competed chemically with protein nh for the binding of glucose, and hampered protein glycation. in a high-glucose medium they underwent an excessive glycation themselves. this modification was more effective in pamam g , in which surface nh were more mobile and accessible. pamam modification with glucose rendered these dendrimers less polycationic in aqueous solutions. pamams neither affected bsa conformation nor formed stable complexes with a protein. we conclude that pamams are very effective glucose scavengers. thus, even less toxic pamams of lower generations, like g , may appear useful in further medical applications as the agents attenuating the detrimental effects of sever hyperglycaemia on biomacromolecules. selective drug delivery and novel drug approaches by polyelectrolytes s. krol cbm-cluster in biomedicine, area science park, s.s. , . km; trieste, italy the use of polyelectrolytes in the past was mainly related to targeted drug delivery and nanoparticle preparations for medical applications. but rarely the polyelectrolytes were investigated for the own features as a drug. in our present work we use a special physical feature of cancer cell membranes as a target for a specific polycation. we found that the polycation is selectively up-taken by cancer cells (leukemia, hepatocarcinoma, cancer stem cells) while normal cells remain unaffected. another interesting application of polyelectrolytes are in form of a multilayer coating on the surface of nanogold particles. for this topic, two new approaches were developed for a drug delivery through the blood brain barrier. in the first case, high amounts of creatine were bound to the gold particles and delivered as protective agents for ischemic stroke (viota et al., j colloid interface sci. , ( ): - ). in the second case coated multifunctional gold particles were prepared as a drug for neurodegenerative disease on the basis of protein aggregates. the use of gold as core for coated nanoparticles offers the possibility to study our systems as "theranostics", system which are modified to recognize selectively diseased cells and carry the moieties to treat or destroy the malfunctioning cells. possible treatments can be local hyperthermal therapy by using the particles as amplifier and enhancer or photodynamic therapy with gold as a drug. m. koneracka , v. zavisova , m. muckova , p. kopcansky , a. jurikova , n. tomasovicova , g. lancz , m. timko , m. fabian institute of experimental physics, slovak academy of sciences, košice, slovakia, hameln rds a.s., modra, slovakia, institute of geotechnics, slovak academy of sciences, košice, slovakia the aim of this study was to develop biodegradable and biocompatible paclitaxel loaded magnetic plga nanospheres (nps) suitable for biomedical applications. biodegradable poly(d,l-lactic-co-glycolic acid) (plga) was used as a capsulation material and the magnetic fluid was used as a magnetic carrier. incorporation of magnetic particles and drug in the plga polymer matrix was confirmed by infrared spectroscopy (ftir) and differential scanning calorimetry (dsc). the release of the drug from the prepared nps to the surroundings under the different conditions was studied also. the prepared magnetic plga nps with encapsulated paclitaxel (spherical shape, size - nm) have good stability in the presence of high nacl concentration at • c, the toxicity of prepared samples declared times higher value of lethal dose ld in comparison with pure paclitaxel (ld = mg/kg) and showed the significant response to external magnetic field which is useful from the point of view to achieve pharmaceutically acceptable drug delivery systems for tumour treatment. success of human gene therapy depends upon the development of delivery vehicles or vectors, which can selectively deliver therapeutic genes to target cells with efficiency and safety. many cationic polymers have been used to condense dna by electrostatic interaction into small particles (polyplex), for protecting the dna from degradation and enhancing uptake via endocytosis. polyethylenimine (pei) appears to be one of the most advanced delivery system that can condense dna efficiently forming pei-dna polyplex complex. the physicochemical properties of different molecular weights of pei, such as condensation ability, buffer capacity, time kinetics, ftir and surface charges of the pei-dna complexes may be important factors to obtain a higher transfection efficiency of the polycation vectors. our intent in this study was to characterize pei-dna complexes to see whether these physicochemical properties have any influence on their disposition characteristics and cellular uptake process. we found that pei-dna complexes, obtained by the k pei at n/p ratio > , were more stable in the presence of tissue culture medium & serum, and did not dissociate in nacl easily. key words: polyethylenimine, polyplex, polycations, dna, transfection, gene delivery. effect of nanopatterned substrate on neuronal growth cones activity in the last years an increasing interest has been address to explore, at the level of the single cell, the physical interaction between neurons and micro-and nano-patterned surfaces which mimic the biological environment and can induce specific biological behavior. a consistent number of substrates have been tested (nano-grooves, pillars, gap nanowires) but the effect of nano-topographical features at subcellular level e.g. branching and pathfinding of growth cones(gc)is still unexplored. using nanoimprinting lithography we fabricated gratings on glass with grooves of variable pitch, depth and width all in the nm range. embryonic stem cell derived neurons were seeded on nanostructured and on flat control glasses. we investigate gc morphology with nm resolution by afm. a significant effect on sub-cellular architecture was observed.on nanopatterned substrates % of gc were branched with a large number of long and thin filopodia(average length and height . µm and nm)while on control only %of gc were branched with an higher percentage of long and thick filopodia (average length and height , µm and nm). on the contrary we did not observe a significant influence of the nanopatterning on the alignment and elongation of neurites. in both cases the distribution of angles between axon and filopodia showed a preferential direction at • . in conclusion, the tested nanopatterns do not influence the neurite directions but do enhance the gc morphology and explorative activities. growth enhancement and adhesion control of pc on micropatterned ns-tio thin film c. lenardi , a. v. singh , p. milani c.i.ma.i.na., dip. di scienze molecolari applicate ai biosistemi, università di milano, italy, c.i.ma.i.na., semm, european school of molecular medicine, fondazione ifom, milano, italy, c.i.ma.i.na., dip. di fisica, università di milano, italy cluster assembled nanostructured titanium dioxide (ns-tio ) has been explored as novel substrate for in vitro cell culture. in this work, we report micropatterned ns-tio thin film as putative microdevice for neuron culture and growth. in additions, we show a simple scheme of molecular patterning of bovine serum albumin (bsa) as cell anti-adherent substrate complementary to ns-tio micropattern which favors a selective spatially confined adhesion of neurons. bsa was drop coated and physisorbed over glass coverslip covered with a thin conducting layer of indium tin oxide (ito) and then pmma was spin coated over it using standard protocols. later, using combinations of e-beam lithography and pulsed microplasma cluster source (pmcs), a thin layer of ns-tio was deposited over micropatterned pmma. further, lift off process enabled us to generate complementary micropatterns of hydrophobic bsa (cell repellent) and hydrophilic ns-tio (cell adhesive). cell culture studies have confirmed that pc cells like to grow on ns-tio substrate and not on bsa layer. the technique offers a novel approach for neuronal cell assay applications. gene delivery with chitosan: influence of chain length on intracellular trafficking and dissociation s. lélu , c. d. l. davies , n. reitan , s. p. strand department of physics, ntnu, trondheim, norway, department of biotechnology, ntnu, trondheim, norway chitosan, a cationic polysaccharide presenting low cytotoxicity, is a promising nonviral gene delivery vector. chitosan complexes dna into nanoparticles, and the complexation and cell transfection efficacy are function of the chitosan/dna ratio and of the intrinsic properties of chitosan, i.e. degree of polymerization dpn, charge density, and molecular structure. nanoparticles formed by shorter chitosan (dpn - ) mediated higher transgene expression than nanoparticles based on longer chitosans. the purpose of this work is to relate the dpn of linear chitosan with the cellular uptake, intracellular trafficking and dissociation of chitosan-pdna complexes, measured by confocal laser scanning microscopy and fluorescence correlation spectroscopy (fcs). cells were incubated with oligomers (dpn and ) either free or complexed with plasmid-dna. both chitosan oligomers seem to penetrate cell nucleus in association with or free from pdna. hours after incubation, accumulation of oligomers in cell nucleus is similar for free and complexed dpn , whereas it increases for complexed dpn compared to free, indicating a possible delayed dissociation of the complexes based on dpn in the nucleus and suggesting a dissociation of pdna-dpn in cytoplasm. these data are consistent with previous studies which suggested that longer chitosan chains led to tighter complexes, and hence to a delayed dissociation process and lower transfection efficiencies compared to shorter chitosans. material surface properties greatly influence dna purification and pcr yield in microsystems c. potrich , l. lunelli , l. marocchi , l. pasquardini , g. guella , d. vozzi , l. vanzetti , p. gasparini , c. pederzolli fbk, trento, italy, univ.trento, italy, univ.trieste, italy modern microchip platforms integrate dna purification, target amplification by pcr and dna detection in a single device. combination of these processes minimizes sample loss and contamination problems as well as reduces analysis time and costs. different strategies are available to perform dna extraction on a chip. here we exploited amino-coated materials as a tool for specific binding of dna through the electrostatic interaction between amine groups and nucleic acids. we analyzed the ability of different treated substrates to selectively absorb/desorb the genomic dna with the aim to purify dna from unwanted components. amino-coated substrates were characterized by afm, xps, fluorescence microscopy and absorption spectroscopy to define the surface chemical and morphological properties. the distribution of the dna adsorbed on materials was homogeneous and the eluted dna was tested for pcr. same materials were analyzed for their compatibility with pcr and the use of different enzymes and reagents or proper surface treatments was employed. we established the best conditions for dna amplification in silicon/pyrex microdevices depending on the type and the fabrication method used and on the quality of reagents more than on the passivation treatment or increment in standard taq polymerase concentration. nanoporous alumina fabricated using unconventional acids for enhanced biomolecular physisorption n. patra, m. salerno, r. losso, r. cingolani italian institute of technology, genova, italy in the last decades the available porecell sizes of porous alumina have been extended, but operating conditions to obtain some pore diameters still have to be optimized, particularly below the nm limit. in this range the use of biocompatible porous alumina thin films could find applications in biosensors, after functionalization by appropriate physisorbed biomolecules. while pore ordering and film growth rate are mainly influenced by the electrical parameters of voltage and current, respectively, the typical pore size primarily depends on the electrolyte. in the search for alternative anodization conditions, we have investigated several acids that have never been used before, namely gallic, lactic, propionic, and glycolic acid. the anodizations were carried out in galvanostatic conditions at fixed concentration and durations, by varying the current. atomic force microscopy was used to test the oxidized surface morphology. in particular, lactic and propionic acids demonstrated feasible. lactic acid gave best results for ∼ ma/cm current density, corresponding to roughly constant ∼ v potential, with resulting pore diameter in the nm range, whereas propionic acid performed best for ∼ ma/cm , corresponding to ∼ v, resulting in nm pore diameter. both kind of films looked lightgray, different from the yellowish oxalic acid porous alumina, which can be a hint of lower ion contamination. chitosan-arabic gum nanoparticles as potential vehicles for peptide delivery l. moutinho, s. rocha, m. coelho, m. c. pereira lepae, chemical engineering department, faculty of engineering, university of porto, portugal chitosan (cs) -arabic gum (ga) nanoparticles were produced by an ion-ion interaction process, using different weight ratios of polysaccharides. according to zeta potential (zp) measurements, particles are ionically stable. zp values ranged from to mv for cs:ga ratios of : . and : . , respectively. cs:ga : yielded uncharged nanoparticles and aggregates, showing that, at this ratio, the negative charges of ga neutralize the positive charges of cs. the particle diameters ranged from to nm, as measured by dynamic light scattering (dls), and displayed a slight tendency for decreasing as the ga concentration increased. transmission electron microscopy (tem) images showed numerous spherical nanoparticles. peptides were encapsulated within the nanoparticles, by mixing them with chitosan solution prior to adding ga. this system can protect short peptides from rapid metabolization, prolonging their blood half-life. physical characterization of nanocarriers for drug delivery s. motta , y. gerelli , g. sandri , e. ricci , p. brocca dept. of chem, biochem and biotech for medicine -university of milan, italy, dept. of physics -university of parma, italy, dept. of pharmaceutical chem -university of pavia, italy nanoparticles used as nanocarriers for pharmaceuticals can improve solubility and bioavailability of problematic drugs and protect labile or toxic molecules. fundamental parameters like drug encapsulation efficiency and release kinetics are tuned by the physico-chemical features of the drug/carrier complex. a challenging aspect of the pharmaceuticallyoriented issues resides in the relatively low number of molecules allowed to build up nanosystems with different properties and morphology, according to the specific drug and to the intended therapy. we have studied: ) soybeanlecithine/chitosan nanoparticles for progesterone and tamoxifen delivery; ) solid-lipid nanoparticles (compritol ato, poloxamer , tween and chitosan) for cyclosporine-a delivery via ophthalmic formulation. both void carriers and drug-loaded nanoparticles have been studied, to understand how the structure of the carriers can be modified by the active molecules. several non-invasive physical techniques have been used to achieve a detailed knowledge of the systems: a) dynamic light scattering for the dimensional distribution of the nanocarriers, b) zeta potential to determine surface charge, c) cryo-tem for morphological analysis d) x-ray scattering (in small angle configuration) and dsc to access information on the inner structure. . a. schollier , a. halperin , m. sferrazza , g. fragneto institut laue-langevin, grenoble, france, universite libre de bruxelles, belgium, cnrs-ujf grenoble, france protein adsorption on surfaces is responsible of several unwanted effects in technological and pharmaceutical applications: fouling of contact lenses, clotting in blood containing devices, inflammation around artificial organs for instance. those phenomena can be repressed with certain types of polymer brushes, in particular peg (polyethylene glycol) brushes, and a better understanding of the mechanism of adsorption could lead to improvements in the design of biomaterials. we have developed a theory describing this mechanism and carried out measurements to confirm the theoretical predictions [langmuir , , ] . three cases are predicted: primary adsorption at the grafting surface, secondary adsorption at the outer edge of the brush, and ternary adsorption within the brush itself. we prepared samples with different grafting densities and different degrees of polymerization and used different protein size and concentrations. neutron reflectivity experiments, able to determine the structure and composition of material interfaces with a fraction of nanometer resolution, were carried out and, by using deuterated proteins (anti-freeze protein, dihydrofolate reductase and myoglobin) on different peg compositions and grafting densities, it was possible to locate the proteins in the brush and to distinguish between the different kinds of adsorption : primary adsorption is dominant for short brushes and ternary adsorption for long brushes. effect of powder air polishing on nanocomposite dental materials measured by atomic force microscopy m. salerno , g. derchi , a. m. genovesi , l. giacomelli italian institute of technology, genova, italy, istituto stomatologico tirreno, lido di camaiore, italy in dentistry, airpolishing with glycine and bicarbonate powders is widely used to remove accumulated plaque. however, the resulting teeth and gingival surface roughness, which is a risk factor for further plaque accumulation, has to be considered. in this in vitro study the effect of the above mentioned technique on the surface of a novel nanocomposite dental material is evaluated by means of atomic force microscopy (afm). square specimens ( . × . cm size) were airpolished either with glycine or bicarbonate, using different application times ( , , sec) and distances ( , , mm) . four specimens were evaluated for each timedistance combination, and checked versus untreated specimens (controls). the specimens were imaged with tappingmode afm at two different scan sizes ( × and × µm ) in two different regions for each sample. the surface roughness was measured as the rms of the feature heights. for the sec mm group airpolishing resulted in an increased surface roughness as compared to the controls; however, glycine was associated with a lower roughness than bicarbonate (glycine: ± nm; bicarbonate: ± nm; controls: ± nm; p< . for treated specimens vs controls, p< . for glycine vs bicarbonate, anova test). similar results were obtained for all the other timedistance combinations. the work comprises the design of polysaccharide based nanoparticles for drug delivery. chitosan/arabic gum and arabic gum/maltodextrin nanoparticles were prepared by polyelectrolyte complexation and spray drying [ ] , respectively. dynamic light scattering characterization established that arabic gum/maltodextrin nanoparticles are more polydisperse (diameters ranging from to nm) than chitosan/arabic gum particles (diameters of approximately nm). scanning electron microscopy measurements demonstrated that the particles are spherical and have a smooth surface. the systems are highly stable to forces up to nn as observed by atomic force microscopy. due to their nature they are hydrophilic and biodegradable. the nanoparticles were used to entrap short peptide sequences and antioxidants and were proved to be efficient in maintaining the biological activity of the molecules. kyotorphin (ktp) was found in in bovine brain. despite revealing remarkable analgesic properties, analgesia was only induced after central delivery. this limited ability to cross the blood-brain barrier (bbb) combined with the unknown mechanism of action largely confines its pharmacological use. to surpass these problems, we designed new ktp derivatives: ktp-rc and ktp-rc-lipogen. biophysical studies were carried out using fluorescence methodologies to characterize the peptides' interaction with biomembrane model systems. partition coefficient quantification showed a clear preference of the derivatives towards fluid zwitterionic and anionic membranes. moreover, a relationship between anionic lipid percentage and in-depth insertion in membrane was established. additionally, studies with fluorescent probe di- -anepps revealed different membrane-interaction profiles of morphine and ktp-derivatives, suggesting distinct actions between them. the analgesic efficacy of the compounds was studied in vivo after systemic administration in models of acute and tonic pain. unlike ktp, both ktp-rc and ktp-rc-lipogen displayed high efficacy from doses as low as . and . mg/ g, respectively, indicating bbb crossing. the observed correlation between higher partition/insertion in the membrane and enhanced analgesic action proved the biophysical rationale to be a powerful strategy for early screening in cns drug development. metabolic syndrome (ms) is now regarded as a major risk factor for cardiovascular disease. as prooxidant/antioxidant balance is disturbed in the course of this disorder, it is possible that melatonin may exhibit protective effect against oxidative damage of blood cells. ms was diagnosed according to international diabetes federation definition ( ) . the investigated group consisted of patients before and after the melatonin supplementation ( mg per day for two months) and was compared to control group of healthy individuals on normal diet. our experiments show that erythrocytes from patient group exhibit significantly higher tbars and total cholesterol levels whereas the protein thiol concentration, na + k + atpase and glutathione peroxidase activities were decreased in comparison to those of healthy volunteers. after two month melatonin supplementation, tbars and cholesterol concentrations significantly decreased, whereas the na + k + atpase, catalase and glutathione peroxidase activities increased. the glutathione concentration was also higher. these results show that melatonin supplementation has a protective effect on erythrocytes of ms patients. action of ferritin, a nanoparticle model, on ros formation and glutamate uptake in synaptosomes t. waseem, a. alekseenko, s. fedorovich institute of biophysics and cell engineering, minsk, republic of belarus nanoparticles are currently used in medicine as agents for targeted drug delivery and imaging. however it has been demonstrated that nanoparticles induce neurodegeneration in vivo and kill neurons in vitro. the cellular and molecular bases of this phenomenon are still unclear. we have used the protein ferritin as a nanoparticle model. ferritin contains iron particles (fe + ) with size nm and a protein shell. we investigated how ferritin influences uptake and release of [ c]glutamate and free radical formation as monitored by fluorescent dye dcfda in rat brain synaptosomes. we found that even a high concentration of ferritin ( µg/ml) did not induce spontaneous [ c]glutamate release. in contrast the same concentration of this protein inhibited [ c]glutamate uptake two fold. furthermore ferritin induced intrasynaptosomal ros (reactive oxygen species) formation in a dose-dependent manner. this process was insensitive to µm dpi, an inhibitor of nadph oxidase and to µm cccp, a mitochondrial uncoupler. these results indicate that iron-based nanoparticles can cause ros synthesis and decrease glutamate uptake, potentially leading to neurodegeneration. functionalized carbon nanotubes (cf-cnts) have a promising future as vectors for drug delivery and for neuronal cell growth and communications . it has been demonstrated that cnts can be employed in drug delivery systems. the toxicological properties of cnts result strictly correlated with nanotube solubility . in this study we focused our attention on multi-walled cnts (mwcnts) because they are easy to manipulate and we covalently functionalize them in different ways to increase their solubility.. we grew different derivatives of a dendrimer on mwcnts surface with positive charges at the periphery, in order to study the different solubilisation properties and correlate them to the biological activity in gene therapy . slowdown of - β-peptide aggregation by addition of two synthetic biocompatible polymers p. sciortino , r. carrotta , g. cavallaro , d. bulone , p. l. san biagio ibf-cnr, palermo, italy, ctf dept., university of palermo, palermo, italy fibril deposit formation of amyloid β-protein (aβ) in the brain is a hallmark of alzheimer disease (ad). fibrils formation is triggered by molecular conformational changes and protein-protein interactions involving partially unfolded regions of different aβ peptide molecules. increasing evidence suggests that toxicity is linked to diffusible aβ oligomers, which have been found in soluble brain extracts of ad patients, rather than to the insoluble fibres. new therapeutical approach, based on searching molecules capable of regulating the peptide aggregation, is currently developing. here, we study the effects on the aggregation of aβ - peptide of two different synthetic polymers with structure similar to that of a protein (α,β-polyasparthylhydrazide -pahy and α,β-polyasparthylhydrazide-polyethyleneglycol -pahy-peg). static and dynamic light scattering measurements showed that the aggregation kinetic is slowed down by the presence of both polymers. optical microscopy revealed the presence of aggregates of different dimension in all samples. transmission electron microscopy allowed to establish that all aggregates are made of fibers, as confirmed by fluorescence spectroscopy measurements on thioflavine binding. a. smorodchenko , d. sittner , a. rupprecht , j. goyn , a. seiler , a. u. bräuer , e. e. pohl institute of cell biology and neurobiology, charité-universitaetsmedizin, berlin, germany, german federal institute for risk assessment, zebet, berlin, germany ucp is a member of the mitochondrial anion transporter family and one of the three ucps (ucp , ucp and ucp ) associated with the nervous system. in our previous work we have shown that ucp appears in brain at embryonic day (e ). we hypothesized that the ucp expression can be related to neuronal differentiation. to prove this idea we now investigated protein and mrna levels in two different systems: (i) mouse embryonic stem cells of line d (mesc) and (ii) brains from pre-and postnatal mice. ucp was not present in undifferentiated mesc. during differentiation of mesc in neurons the expression of neuronal marker map and ucp started at the same time -as early as on day in culture -and were increasing simultaneously. (ii) the comparative analysis of gene transcripts prepared from whole embryos, brains and different brain regions (neocortex, hippocampus, cerebellum) demonstrated that levels of ucp mrna were increasing from e till e , reached an expression peak between e and postnatal day (p ) and remained constant in adult animals. in contrast, expression of ucp was increasing permanently until birth, whereas ucp expression was invariant in time. our results suggest that ucp contributes to specific neuronal functions. the plant hormone abscisic acid stimulates the proliferation of human hemopoietic progenitors through the second messenger cyclic adp-ribose s. scarfì , c. fresia , c. ferraris , s. bruzzone , f. fruscione , c. usai , m. magnone , m. podestà , l. sturla , l. guida , g. damonte , a. salis , a. de flora , e. zocchi advanced biotechnology center, genova, italy, dept of experimental medicine, biochemistry section, and cebr, university of genova, italy;, institute of biophysics, cnr, genova, italy, stem cell center, s. martino hospital, genova, italy abscisic acid (aba) is a hormone involved in pivotal physiological functions in higher plants. recently, aba was demonstrated to be produced by human granulocytes, β pancreatic cells and mesenchymal stem cells (msc) and to stimulate cell-specific functions. here we show that aba expands human hemopoietic progenitors in vitro, through a cadpr-mediated increase of the intracellular calcium concentration. incubation of cd + cells with micromolar aba also induces transcriptional effects, which include nf-κb nuclear translocation and transcription of cytokines encoding genes. stimulated human msc produce and release aba at concentrations sufficient to exert growth-stimulatory effects on co-cultured cd + cells, as demonstrated by the inhibition of colony growth in the presence of an anti-aba monoclonal antibody. these results provide a remarkable example of conservation of a stress-hormone from plants to humans and identify aba as a new hemopoietic growth factor involved in the cross-talk between hp and msc. t. golovanova, g. b. belostotskaya sechenov institute of evolutionary physiology and biochemistry, russian academy of sciences, saint petersburg, russia a subpopulation of small cells (volume ± µm ) have been found in the neonatal cardiac myocytes culture whereas the same parameter of the remaining cells was ± µm on the st day. in contrast to main part of hypetrofied cardiomyocytes, the small cells were able to proliferate, form colonies and differentiate spontaneously into cardiac myocytes in the culture. on the th day there were slight, slow ( - beats/min), arrhythmic contractions in the centre of colonies. pulsing cells were not united by common contraction and had individual beating profile. on the - th days, the colonies displayed comprehensive contractile activity with the pulsation rate - beats/min and reached beats/min on the d - th day and beats/min on the - th days in the culture. it has been shown that receptors of the surface membrane and sarcoplasmic reticulum of colony cells gradually mature. by estimating the ca + transition under the specific agonists action: acetylcholine, kcl and caffeine, we have detected the activity of basic structural and functional elements of excitation-contraction coupling in contractile cells inside colonies. the small cells ability to proliferate and differentiate under the influence of near mature cardiomyocytes allows us to put forward a hypothesis, that they belong to a category of resident stem cells. changes in the gating properties of na + channels were studied in es-derived neural stem (ns) cells during in vitro neuronal differentiation with the use of the whole-cell and cell-attached variants of patch-clamp technique. ns cells represent a novel stem cell population remaining stable and highly neurogenic over multiple passages.voltage-clamp recordings during neuronal differentiation of ns cells indicated significant changes in the key properties of na + currents. a voltage-gated and tetrodotoxine-sensitive na + current,absent under self-renewal conditions, was first recorded following application of differentiative agents. current density increased with time of exposure to differentiating conditions. whole-cell and single channel analysis revealed that the observed increase in current density was due at least in part to changes in steady-state activation and inactivation properties. namely, half activation potential shifted from - mv to - mv, while half-inactivation potential shifted from - mv to - mv. furthermore, a contribution to the increase in na + current density could also be given by an enhancement in channel expression, as suggested by an augmentation in the number of single channels per patch area, with increasing neuronal differentiation. interestingly, those changes in na + channel activity well correlate with the capability of ns cells to generate action potentials during in vitro neuronal differentiation. a. viale, g. bonizzi, a. cicalese, f. de franco, c. pasi, s. pece, a. orleth, p. di fiore, p. pelicci european institute of oncology, milan, italy we are characterizing the biological differences between normal and transformed scs. scs are defined by their ability to generate more scs, termed self-renewal, and to produce cells that differentiate (asymmetric cell division). scs, however, possess the ability to expand in number (i.e. during development, in adulthood after injury/disease); this increase is not accounted by asymmetric divisions, in which only one daughter cell maintains sc identity. recent findings in invertebrates indicate that scs can also generate daughter cells destined to acquire the same fate (symmetric cell division). on the other hand, sc quiescence is critical to maintain tissue homeostasis after injury. here we show increased symmetric divisions of cscs in breast tumors (due to inactivation of the p tumor suppressor) and dependency of leukemia development on quiescent leukemia scs (due to transcriptional up-regulation of the cell cycle inhibitor p by leukemia-associated fusion proteins). we suggest that sc asymmetric divisions function as a mechanism of tumor suppression, that sc quiescence is critical to the maintenance of the transformed clone and that symmetric divisions of scs permit their geometric expansion. a. uccelli department of neurosciences ophthalmology and genetics, genoa, italy stem cells are considered as a possible source of cells for tissue repair. in this scenario damaged tissues will be reconstructed by newly formed cells with the result of a recovery of lost functions. thus, the rationale for utilizing stem cells for the treatment of neurological diseases such as multiple sclerosis (ms) stemmed from the idea that they differentiate in neural cells regenerating the damaged tissues at the basis of irreversibile disability. however, while multipotential embryonic stem cells may provide in the future an optimal source of cells competent for myelin and axons repair in ms, their use is still far from being exploitable in a clinical setting. moreover, it is widely accepted that adult stem cells may in vitro transdifferentiate in neural cells but recent experimental data have challenged the biological importance of this event in vivo. nevertheless, several experimental studies have provided new evidences supporting the use of adult stem cells derived form different human tissues for the treatment of ms. in the cases of mesenchymal stem cells and neural stem cells current experimental data support an unexpected therapeutic plasticity mediated by diverse paracrine mechanisms including neuroprotection, induction of local neurogenesis and modulation of the immune response. therefore, current experimental and clinical studies support the use of stem cells for the treatment of neurological diseases such as ms. unravelling the structure of the water splitting site of photosynthesis and implications for mechanism of catalysis j. barber imperial college london, u.k. photosystem ii (psii) is a multi-subunit membrane protein complex which catalyses the oxidation of water to molecular oxygen and reducing equivalents. the reaction occurs at a catalytic centre composed of mn ions and a ca ion, is thermodynamically demanding and generates highly oxidised species. unavoidable side reactions cause detrimental effects on the protein environment leading to the rapid turnover of the reaction centre d protein. to understand the mechanisms of water oxidation and d turnover structural information is required. initially the positioning of various protein subunits and their transmembrane helices were determined by electron microscopy. more recently a refined structure of the cyanobacterial psii unit has been elucidated by x-ray crystallography giving details of specific environments of the redox active cofactors. the implications of these structural studies will be discussed in relation to the unique facets of psii function, particularly the water splitting reaction. importantly this new knowledge is providing a blue print for the design of photochemical catalysts which can mimic the photosynthetic water splitting reaction and thus give hope that new technologies will emerge to provide humankind with a sustainable energy supply. photochemically controlled molecular devices and machines v. balzani department of chemistry "g. ciamician", university of bologna, italy the macroscopic concepts of a device and a machine can be extended to the molecular level. molecular-level devices and machines operate via electronic and/or nuclear rearrangements and, like macroscopic devices and machines, they need energy to operate and signals to communicate with the operator. the extension of the concepts of a device and a machine to the molecular level is of interest not only for basic research, but also for the growth of nanoscience and the development of nanotechnology. if a molecular device or machine has to work by inputs of chemical energy, it will need addition of fresh reactants ("fuel") at any step of its working cycle, with the concomitant formation of waste products that compromise the operation. currently there is an increasing interest in the use of light to power molecular devices and machines. the lecture will illustrate examples of recent achievements [ , , ] , which include molecular wires, switches, plug-socket systems, extension cables, antennas, and light powered nanomotors. biophysics laboratory, institute of botany, azerbaijan national academy of sciences, baku, azerbaijan it is well known that fast component of induction curves of millisecond delayed chlorophyll fluorescence (ms-df) originates via radiative recombination of reaction center with product on the donor side of psii. in view of our previous data (j. photochemistry and photobiology b: biology , ) it was shown that partners for radiative recombination of p q a − localized as a hole on the donor side of psii. depending on ph this hole might be on the camn -cluster or on y z and their recombination with p q a − can be monitored by the ms-df fast component. for analysis of site of damages, photoinhibition of psii particles from spinach at different ph was monitored by ms-df. during photoinhibition of psii ( µmol photons m − s − ) the fast component of ms-df was shown to be more stable at ph . and ratio of the fast component to the steady-state level of ms-df was approximately constant, while at ph . the fast component essentially decreased, the steady-state level increased and ratio of these components rapidly went down. it is possible concluded that strong light damaged of recombination reaction with in the presence ppbq -artificial electron acceptor -the protection of psii from photoinhibition has been observed only at acidic condition. stress factors such as heavy metals, strong light and low temperature were investigated by measurement of transient pictures of ms-df in intact plants, isolated chloroplasts and pure psii particles. the heavy metals action on psii was investigated on wheat seedlings. the targets for toxic action of al, mn and co ions were found to be a q a -q b acceptor side of psii. the damage site for cd + may be partners for recombination with p + -depend on of medium ph -either y z or camn -cluster. investigated ions (mn + , al + , cd + and co + ) have lead to reduction of chlorophyllprotein complexes pigment fund and cd + and co + destroyed also an apoproteins, especially of psii. the photoinhibition of psii was investigated in intact leaves of barley and maize seedlings at low and normal temperature. a very sharp reduction of intensity of ms-df second fast component, possibly y z * p ÿq a − radiative recombination of maize seedlings was observed even after short illumination by strong light at • c. ph dependence photoinhibition of chloroplasts and pure psii particles have shown that strong light damaged of camn -cluster in great degree than y z . hydrogenases are considered as potential energy sources. in particular, the ability of the green alga c . reinhardtii to reduce protons to hydrogen gas upon illumination by means of a [fefe]-hydrogenase is a phenomenon of great scientific interest, as it would need only light and water to generate energy. however, the catalytic activity is strongly inhibited by the o produced during photosynthesis; furthermore, the protein is expressed at very low levels, only in conditions of strict anaerobiosis. this mutually exclusive nature of o and h photoproduction represents a crucial problem in the development of h bio-production. the study of the structurefunction relationship of [fefe] hydrogenases, which would help to clarify the molecular mechanisms underlying both h production and o sensitivity, requires the characterization of purified native and modified proteins, which can be obtained by site-directed mutagenesis. we expressed the algal hydrogenase in the cyanobacterium synechocystis sp. pcc , which holds a bidirectional [nife]-hydrogenase with a well known maturation system ( ) . we obtained two constructs to stably transform synechocystis, enabling it to express the c. reinhardtii hydrogenase in an active form. this suggests that the [nife]-hydrogenase maturation pathway is able to drive the biosynthesis of functional [fefe] enzymes. these data open new perspectives about the indispensable presence of hyde, hydf and hydg auxiliary proteins ( , ) to obtain a correctly folded [fefe]-hydrogenase. the a low energy monomeric chlorophyll of cp to investigate the role of low energy spectral forms in energy transfer among chlorophyll protein complexes, we characterized one of the redmost chlorophylls of psii antenna complexes, the a of cp . pigment stoichiometry of the wild type (cp wt) and mutant lacking the a binding residue (cp a ) reconstituted complexes confirmed the loss of a chlorophyll a. to exclude the possibility of a marked excitonic interaction of the a with other chlorophylls, we quantified the decrease in dipole strength after mutagenesis as a function of the frequency and performed a second derivative analysis of the difference absorption spectrum cp wt minus cp a . these data, along with circular dichroism spectra of both complexes, support the idea that the a is a monomer in cp , in disagreement with recent suggestion of involvement in an excitonic dimer (mozzo et al., bba ) . chla a reorganization energy and inhomogeneous broadening were then determined through a thermal broadening analysis in the - k temperature range. these parameters allowed us to calculate the förster overlap integral of the homogeneously broadened a bandshape, a fundamental factor for energy transfer rate estimations between chromophores. a comparison with foi of chlorophyll in solution suggests that chla a may connect cp complex to psii core favoring energy transfer from cp towards other inner antenna low energy chlorophylls. in oxygenic photosynthetic organisms, reaction centre complexes catalyze electron transport from water to co using light energy absorbed by tetrapyrrole pigments bound to so called "antenna proteins". a major challenge in performing this type of photosynthesis consists on the difficulty of operating "one electron" transport through a multi-step pathway in the chloroplast environment which is the source of oxygen of biosphere. in fact, synthesis of ros, mainly singlet oxygen and superoxide anion and consequent photoinhibition is an intrinsically unavoidable consequence of photosynthesis that must be prevented and/or controlled in order to avoid photodestruction and death. mechanisms involved include chlorophyll (chl) triplet quenching, ros scavenging and controlled heat dissipation of excess chl singlet excited states. the latter process has been studied for over years with little success in elucidating its mechanism. reverse genomics and ultrafast-spectroscopy led to the proposal that the transient formation of carotenoid radical cations, followed by charge recombination might be the underlying mechanism of energy dissipation while three proteins belonging to the lhc superfamily could be the site hosting the reaction. probing the dark cycle to reveal regulatory networks controlling photosynthetic efficiency the food, fibre and fuel needs of an ever-increasing population are one of the challenges facing current society. higher plant photosynthetic efficiency is ∼ % whereas the theoretical maximum is thought to be ∼ - % giving potential for great improvements. it is not however clear where in the photosynthetic process the additional losses are occurring. we are adopting a systems biology approach to reveal the regulatory networks that control photosynthetic efficiency, the challenge being to make quantitative measurements of the input parameters to models of the network of photosynthetic reactions, and also to identify missing physical parameters and processes. the aim being to understand how they interact with other key physiological pathways and what impact they hold over photosynthetic efficiency. we report initial results in this poster from experiments using conventional and novel proteomic methods. e.g. an optical technique based on a non-linear dir method is being developed for proteomic and metabolomic analysis. unlike many conventional proteomic methods, which provide information on the relative levels of various proteins and metabolites, the viability of the dir method as a quantitative, sensitive and high throughput proteomics platform has recently been demonstrated. in collaboration with klug this project will employ dir as a both a proteomic and metabolomic tool. t. morosinotto , p. arnoux , g. saga , g. m. giacometti , r. bassi , d. pignol dip. di biologia, padova, italy, cea, cadarache, france, dip. di biotecnologie, verona, italy violaxanthin de-epoxidase (vde) is the enzyme responsible for zeaxanthin (z) production. the synthesis of this carotenoid in plants exposed to high light conditions is an important photoprotection mechanism, enhancing excess energy dissipation and reactive oxygen species scavenging. the inactive enzyme is normally soluble but, upon activation by low ph, it binds to the thylakoids membrane, where its substrate is found. we present here the first structural data on this enzyme obtained at both acidic and neutral ph. at neutral ph, vde is monomeric with its active site occluded in the lipocalin barrel. upon acidification, the barrel opens up resulting in a functional dimerization of the enzyme. the channel linking the two active sites of the dimer can harbour the entire carotenoid substrate and thus allow the parallel de-epoxidation of the two violaxanthin β-ionone rings, making vde an elegant example of the adaptation of an asymmetric enzyme to its symmetric substrate. structural data opened the possibility to investigate deeper this enzyme and further work allowed the identification of its active site, the protein domains responsible of its membrane association as well as key residues involved in the ph dependent conformational change. hydrogen is considered the fuel of the future if produced from sun light-driven water splitting. a hydrogen economy based on genetically modified organisms, immobilized enzymes or biomimetic synthetic catalysts requires a profound knowledge of the structure and function of the respective enzymes in nature. light-induced water splitting is performed by a tetranuclear manganese cluster located in photosystem (ps) ii of oxygenic photosynthesis. the protons generated by ps ii can be converted to molecular hydrogen by the enzyme hydrogenase, which is for example found in green algae and cyanobacteria. [nife]-and [fefe]-hydrogenases are the two main classes of this enzyme. they contain bridged binuclear transition metal cores, which are tuned by a special ligand environment to efficiently convert protons to hydrogen -or vice versa -via a heterolytic mechanism. rhodobacter sphaeroides strain a- isolated from mineral springs in armenia produces h with high rate in anaerobic conditions under light. in our previous work the inhibition of h production by n,n' -dicyclohexylcarbodiimide (dccd), the f f -atpase inhibitor, was shown, and dccd-inhibited atpase activity was determined. therefore it is possible to admit a role of this atpase in h production by r. sphaeroides; otherwise dccd inhibition of hydrogenase is not ruled out. in order to examine the mediatory role of proton-motive force (pmf) or proton atpase in this process transmembrane electrical potential (∆Ψ) and ∆ph are determined and the atpase activity is studied in r. sphaeroides grown under light. pmf was determined under anaerobic conditions in the dark. at ph . the ∆Ψ was of - mv and the reversed ∆ph was + mv, resulting in the pmf of - mv. but ∆Ψ was not affected by dccd. moreover, adenine nucleotide phosphates (anp) content is essential for cell functioning and may result with atpase activity. the percentage of atp was calculated from the total quantity of anp in whole cells, it was . %. a relatively high concentration of adp (∼ %) and amp (∼ %) and accordingly low energetic charge were noted; these might be indicative for the atpase activity, a further study is required. relationship between h production and atpase activity by rh. sphaeroides is suggested; possible mechanisms are discussed. identification of the sites of chlorophyll triplet quenching in relation to the structure of lhc-ii from higher plants. evidence the chl a molecules involved in the triplet-triplet energy transfer to the central luteins in trimeric lhc-ii are identified by time-resolved and pulse epr techniques. the concept of spin conservation during triplet-triplet energy transfer is exploited. the sites with the highest probability to form triplet states, which are quenched by the central luteins, are chl and chl . unquenched chl triplet states are also produced by photo-excitation in the lhc-ii complex. putative sites of these triplet states are chl , chl , chla and chl , since they do not contribute to the formation of the observed carotenoid triplet states. fluorescence lifetime spectrum of the plant photosystem ii core complex the photosystem ii kinetic model (diffusion or trap-limited) is still much debated. there is discussion about whether energy transfer from the core antenna (cp and cp ) to the reaction center complex (d -d -cyt b ) is rate-limiting (transfer to trap-limited). this study investigates this problem in isolated core particles by exploiting the different optical properties of the core antenna and the reaction center complex near nm, due to p and an isoenergetic pheophytin. this was used as a marker feature for the reaction center complex. if the transfer to the trap-limited model were correct, assuming excited-state thermalization, the specific reaction center fluorescence decay lifetime should be shorter near nm, where there is reaction center complex specificity, than at the other emission wavelengths. such a selective reaction center feature was not observed in fluorescence decay measurements. at the experimental resolution used here, we conclude that the traplimited energy transfer to the reaction center could, at the most, be % limiting. thus, the transfer to the trap-limited model is not supported. photosynthetic apparatus response under heat stress n. pshybytko , i. divak , l. kabashnikova , e. lysenko institute of biophysics and cell engineering, national academy of sciences of belarus, belarus, institute of plant physiology, russian academy of sciences, russia the mechanisms of psii thermoinactivation and adaptation under high temperature impact and participation of hydrogen peroxide at these processes were studied. the twice suppression of oxygen evolving activity of thylakoids with simultaneous decrease in d protein content and the release of extrinsic kda polypeptide from woc after - min heating at o c were registered. using inhibitor analysis it was shown that thermoinduced degradation of d protein after min heating occurred by proteases. the participation of ftsh protease in thermoinduced d protein degradation was observed. level of transcription of psba gene in chloroplast was raised after min heating and was decreased through h. the content of hydrogen peroxide was increased three times after min of heating and was decreased to normal level through h and was raised after . h again. it is interesting that level of peroxidation lipids products was increased after . h heating only. received data indicated that hydrogen peroxide is signal molecular at the photosynthetic apparatus under heat stress. during heating the inactivation of woc and d protein is occurred. as result the h o is generated. hydrogen peroxide as signal molecule activates transcription of psba. turnover of psii is occurred. more long heating induces degradation of proteins and lipids and h o represents as the destructive agent. a. nurisso , m. a. morando , f. j. cañada , j. j. barbero , a. imberty cermav-cnrs, grenoble, france, centro de investigaciones biológicas, madrid, spain the mechanism of symbiosis between legume plants and rhizobial bacteria is a relevant topic of interest since it is at the basis of the nitrogen fixation process. this mechanism is strictly related to the production of lipochitooligosaccharides, nod factors (nf), which allow the bacterial invasion into the legume host roots. in medicago truncatula, the recognition of nf from the symbiont sinorhizobium meliloti requires the nfp gene, able to encode a lysm-motif receptor-like kinase. the extracellular part of this protein is characterized by three lysm motifs: only one of them seems to be involved in the nf recognition. herein, we report an in silico investigation of different structural aspects of this process. first of all, the conformational behaviors of a new generation of nf analogues were elucidated by mds simulation. then, a homology model of the lysm domain from m. truncatula was proposed and compared with a model of lysm domain from pisum sativum: docking calculations of natural nf identified a common binding site in which the carbohydrate portion is the main responsible of the binding. both studies provide support for the idea that the carbohydrate part of nf plays a key role in the interaction with lysm domains, while the lipid moiety modulates ligand specificity probably interacting with a potential second receptor. interaction between frutalin with biomembrane models and its correlation with cell adhesion property frutalin is a homotetrameric lectin d-galactose (d-gal) binding that activates natural killer cells in vitro and leukocyte migration in vivo, being a potent lymphocyte stimulator. in this study we investigated the interaction of frutalin with different phospho/glyco-lipids using langmuir monolayers as biomembrane mimetic system. the results attest the specificity of the protein for the carbohydrate d-gal when attached to the biomembrane model. the adsorption kinetics for frutalin to the mixed monolayers containing glycolipids showed that the interaction depends on the presence of charged groups and on the position of the d-gal on the polar head of the glycolipid. using brewster angle microscopy (bam), we investigated the morphology of the interface for the binary mixtures containing galcer, where small domains were formed at high lipid packing, suggesting that frutalin can induce the formation of likelipid rafts domains in vitro. the results obtained with the membrane models were associated with those from fibroblast adhesion induction. the cell adhesion promoted by frutalin is in accordance with the results observed in langmuir monolayers, which probes the specificity of the interaction between the lectin and d-gal on cell-membrane surfaces. based on these results, frutalin can be considered as a promise biotechnological tool to actuate in tissue engineering regeneration. supported by fapesp, cnpq and capes aggregation and glycation processes of proteins are of peculiar interest for several scientific fields. serum albumins are widely studied proteins for their ability to self-assemble in aggregates and also to undergo to non enzymatic glycosylation in cases of diabetes. in this work we report a study on thermal aggregation of glycated bovine serum albumin (bsa) prepared with different concentrations of glucose at ph . . increasing concentration of sugar modulates the effect of different glycation levels on the protein aggregation. fluorescence spectroscopy, ftir absorption, static and dynamic light scattering are used to follow the time evolution of the aggregation process and of protein conformational changes. conformational changes of secondary and tertiary structures are measured by ftir absorption; the kinetics of amide i, amide ii and amide ii' bands are monitored. the kinetics of tryptophans fluorescence give complementary information on the tertiary structure changes and on the polarity modification of the fluorophores environment. the aggregates growth is studied by dynamic light scattering measurements and rayleigh scattering peak. the results show that the partial unfolding of the protein is not affected by the glycation, while the presence of increasing amounts of glycated molecules progressively inhibits the aggregates formation. solvent occupancy analysis in ligand structure prediction: the case of galectin- s. di lella, m. a. martí, d. a. estrin inquimae-conicet, universidad de buenos aires, argentina formation of protein ligand complexes is a fundamental phenomenum in biochemistry. during the process, significant solvent reorganization is produced along the contact surface. using md simulations in explicit solvent combined with statistical mechanics analysis, thermodynamic properties of water molecules around proteins can be computed and analyzed in a comparative view. based on this idea, we developed a set of analysis tools to link solvation with ligand binding in a key carbohydrate binding protein, human galectin- (hgal- ). specifically, we defined water sites (ws) in terms of the thermodynamic properties of water molecules strongly bound to protein surfaces. we then succesfully extended the analysis of the role of the surface associated water molecules in the ligand binding and recognition process to many other carbohydrate binding proteins. our results show that the probability of finding water molecules inside the ws, p(v), with respect to the bulk density is directly correlated to the likeliness of finding an oxhydril group of the ligand in the protein-ligand complex. this information can be used to predict possible complex structures when unavailable, and suggest addition of ohcontaining functional groups to displace water from high p(v) ws to enhance drug, specially glycomimetic-drugs, protein affinity and/or specificity. glyconanoparticles: nano-biomaterials for application in biotechnology and biomedicine s. penadés laboratory of glyconanotecnology, cicbiomagune -ciber-bbn, san sebastian, spain to study and intervene in carbohydrate interactions our laboratory has developed a chemical strategy (glyconanotechnology) to produce sugar functionalized gold nanoclusters with multivalent carbohydrate display (glyconanoparticles, gnps). by combining these tools with biophysical and analytic surface techniques (itc, afm, tem, spr) we have demonstrated and evaluated ca + -mediated carbohydratecarbohydrate interactions involved in cell adhesion processes. the gnp system complements other currently available multivalent systems incorporating carbohydrates and presents some advantages as: ) exceptionally small core size; ) high stability in water and physiological buffers; and ) multivalency and multifunctionality with control over ligands number. specific gnps have been applied to the inhibition of melanoma metastasis in mice and as inhibitors of hiv-trans infection. magnetic gnps have also been used in cellular labelling and imaging as probes for mri. in this lecture, the glyconanotechnology strategy will be presented and some application will be highlighted. -glycobiophysics - expanded ataxin- induces membrane mechanical instability: evidences from model systems and cells c. canale , s. averaimo , d. pesci , d. paulis , v. fortunati , a. gliozzi , m. mazzanti , c. jodice , a. relini iit, genoa, italy, university of milan, italy, university of tor vergata, rome, italy, university of genoa, italy spinocerebellar ataxia type is a neurodegenerative disorder involving the expansion of a polyglutamine stretch beyond a threshold in the protein ataxin- , with intracellular deposition of amyloid aggregates. ataxin- variants with polyglutamine stretches of pathological length are not associated to disease when the stretch is interrupted by histidines. mounting evidence suggests that ataxin- aggregates interact with the nuclear membrane. to get insight into the mechanisms leading to neurological disorders, we studied model lipid membranes containing an expanded pathological form of ataxin- or expanded normal forms interrupted by histidines. electrical measurements on planar bilayers showed the occurrence of current steps, much larger for the pathological form, indicating the formation of pore-like structures. atomic force microscopy measurements showed that the longer the polyglutamine tract, the smaller was the force required to penetrate the bilayer with the afm tip. the smallest penetration force, and then the strongest membrane destabilization, was observed for membranes containing the expanded pathological form. experiments on cos cells provided evidence that pathological protein aggregates damage the nuclear membrane eventually causing cell autophagy. modification by dopamine adducts links αsynuclein to oxidative stress in parkinson disease m. bisaglia , e. greggio , l. tosatto , f. munari , i. tessari , p. polverino de laureto , s. mammi , m. r. cookson , l. bubacco university of padova, italy, nih, bethesda, md, usa oxidative stress has been proposed to be involved in the pathogenesis of parkinson disease (pd). a plausible source of oxidative stress in nigral dopaminergic neurons is the redox reactions that specifically involve dopamine (da) and produce various toxic molecules, i.e., free radicals and quinone species. α-synuclein (αsyn), a small protein found in lewy bodies characteristic of pd, is also thought to be involved in the pathogenesis of pd. to investigate the possibility of a synergistic role of oxidative stress and αsyn in pd, we analyzed the modulation of da toxicity by αsyn overexpression in dopaminergic human neuroblastoma cells. our results indicate that the increased expression of αsyn enhances the cellular toxicity induced by the accumulation of intracellular da. we then correlated our results with the structural modifications induced by the oxidation products of da on αsyn by studying two potential pathways for the oxidative chemistry associated with da. the first one is the auto-oxidation reaction which leads to the concomitant formation of radical and quinone species. the second is the enzymatic oxidation, mediated by tyrosinase, which leads only to the accumulation of quinones. our data suggest a link between da and αsyn in the progression of pd and provide novel insights on both the mechanisms involved in the oxidative chemistry and the aggregation properties of αsyn. dynamic light scattering aggregation studies of aβ ( - ) peptide s. bertini, r. beretta, d. gaudesi, a. naggi, g. torri ronzoni institute, milan, italy heparan sulfate (hs) proteoglycans play a role in formation of amyloid plaques by facilitating formation of amyloid aggregates. heparins and low-molecular weight heparins which mimic hs sequences could interact with amyloid peptides putatively involved in neurodegenerative processes. the aim of this work is to study the influence of chain length and structure of heparin oligosaccharides on the aggregation of amyloid. to this purpose, the interaction with amyloid peptide aβ − with a number of heparin/heparin oligosaccharides had been investigated using dynamic light scattering (dls) which permits to determine the size and the stability of molecular aggregates in solution. as indexes of aggregation in solution, two parameters were chosen, i.e., the area under the autocorrelation function (af) and the average hydrodynamic ratio (rh). we evaluated the aggregation kinetic of different preparations of aβ and the ph of solubilization. a clear indication was obtained for the trend of aggregation of the peptide aβ − alone and in the presence of oligosaccharides. the size of the aggregate in the presence of the tetrasaccharide is definitely lower than for the peptide alone. the size of the aggregates increases with increasing size of the oligosaccharides. in fact, the octasaccharide promotes further aggregation. it is crucial for pharmaceutical protein formulations to have low aggregate content. using the monoclonal igg antibody rituximab as a model system, we have studied the mechanisms by which antibodies aggregate at physiological ph when incubated at - • c. light scattering showed two coupled stages: an initial fast stage followed by several hours of exponential growth of the scattered intensity. data analysis showed the fast formation of a large species, which subsequently increased in size. the aggregate number density had a maximum suggesting that aggregates increase in size by coagulation. the analysis also predicted the actual underlying increase in aggregate mass to be linear and reach saturation. this was confirmed by size-exclusion chromatography of incubated samples. in an arrhenius plot the activation energy of the first stage was similar to the unfolding energy of the ch domain, suggesting a pivotal role of this domain in the aggregation process. cd and fluorescence showed only minor structural changes in the temperature interval studied. we conclude that coagulation is the main mechanism driving rituximab aggregation and that aggregation is due to small structural changes. -condensed colloidal phase in biology: from proteins crystals to amyloid fibrils -abstracts solid-state nmr structural studies of alzheimer's disease amyloid β( - ) in lipid bilayers j. d. gehman , a. k. mehta , f. separovic school of chemistry, bio institute, university of melbourne, vic, australia, department of chemistry, emory university, dickey dr., atlanta, ga, usa amyloid-β peptides are believed to cause loss of nerve cell function in individuals suffering from alzheimer's disease, where evidence suggests that interaction with the cell membrane correlates strongly with cytotoxicity. previous studies report a range of different but plausible structures, which depend on the molecular environment of the peptide. this sensitivity to sample preparation suggests that the structure relevant to disease is found in lipid bilayer membranes. while model membrane vesicles are too large to be studied by conventional solution nmr, solid-state nmr is one of the few technologies available to study such systems. we present recent measurements which suggest a novel peptide structure in a lipid bilayer environment: (i) rotational-echo double-resonance (redor) distance measurements between selectively enriched c-carbonyl and n-amide positions constrain dihedral angles of intervening residues and suggest that at least part of the aβ( - ) peptide folds into a β-sheet like conformation, in contrast to the helical and coiled structures in previous reports; and (ii) double quantum filtered draws measurements indicate that the extended strand does not assemble into an in-register parallel sheet as reported for amyloid fibrils. a mutation in app gene with dominant-negative effect on amyloidogenesis: a light scattering study e. del favero , g. di fede , f. tagliavini , m. salmona , l. cantù university of milan, segrate, italy, "carlo besta" national neurological institute, milan, italy, istituto di ricerche farmacologiche "mario negri", milan, italy we studied the effect of a single-point mutation (a v) on the aggregative properties of alzheimer's peptides aβ − . by laser light scattering it was possible to follow the early stages of aggregation of aβ − wt and aβ − mut (within hours after sample preparation), when intermediates in the aggregation pathway, rather than the mature insoluble fibrils, are formed. results show that both the kinetics and the extent of aggregation are strongly enhanced by the mutation. on the reverse, coincubation of mutated and wild-type peptides slows down the aggregation process and results in aβ aggregates that are much more unstable against dilution. it is evident that the interaction between mutant and wild-type aβ − interferes with nucleation or nucleation-dependent polymerization, hindering amyloidogenesis. these results account for the highly amyloidogenic effect of the mutation in vitro, if alone, reversely reduced by the mutated-wild type mix. also a clinical case exists that constitutes the in-vivo evidence of these two opposing effects. this finding may offer grounds for the development of therapeutic strategies, based on modified aβ peptides or peptido-mimetic compounds, for the potential treatment of alzheimer's disease. the collagen i is the major structural protein of the body and it is found organized in a hierarchical manner in the extra-cellular matrix of several tissues (bone, tendon, cornea and sclera, etc..). it is responsible for their specific architecture. it is already known that collagen i is capable of forming cholesteric liquid crystalline phases that once stabilized by a ph increase, lead to collagen matrices that mimic the organization of the organic matrix found in bone [ ] . in the present work, we analyze physico-chemical ways to modulate long range organizations obtained in this liquid state. we study the effect of collagen concentration, ph ( . / . ) and acids (hydrochloride acid / acetic acid) over the liquid crystalline order. in a original approach, correlation of collagen endofluorescence and shg signals has enabled us to quantify cholesteric pitch and phase transition as a function of collagen concentration within a gradient. cholesteric pitch have proved to be very responsive to physico-chemical conditions. indeed, the results allowed us to quantify a i / cholesteric transition as a subsequent transition from cholesteric to a phase hexagonal or colummar. prion diseases are deadly neurodegenerative diseases affecting human and mammalian species. according to the 'protein-only' hypothesis, the key event in the pathogenesis is the conversion of the α-helix-rich monomer (prp c ) into a polymeric β-sheet-rich pathogenic conformer (prp sc ). we have used a combination of biophysical techniques with molecular dynamics simulations (md) to elucidate the molecular mechanisms of prp c unfolding and polymerization. under well established conditions, three β-sheet-rich soluble oligomers were generated from the partial unfolding of the monomer, which were found to form in parallel. to obtain a deeper insight into the molecular events, doublecystein mutants were designed, thus 'locking' different regions of prp. single mutations were also performed, which affected dramatically and selectively the prp oligomerization pathway. furthermore, we have now identified the minimal region that leads to the same oligomerization profile as the full-length prp, namely, h h . the existence of at least three distinct oligomerization pathways and the effect of single mutations reveal the conformational diversity of prp and a possible relationship with prion strain phenomena. the identification of domains involved in the conversion process may lead to a better understanding of the effect of mutations or gene polymorphism on the evolution of prion pathology. investigating toxic protein nanoclusters and their interactions with living cells s. nag, b. sahoo, a. bandyopadhyay, c. muralidharan, r. abhyankar, s. gurav, s. maiti tata institute of fundamental research, homi bhabha road, colaba, mumbai , india amyloid protein aggregation is responsible for many neurodegenerative diseases, such as alzheimer's and parkinson's. it appears that quasi-stable small soluble aggregates are the key to amyloid toxicity. though amyloid aggregation appears to be a nucleation mediated process, simple nucleation theory is unable to explain the stability of these intermediate species. we investigate this issue with fluorescence correlation spectroscopy in solution and on cell membranes. our initial data, varying the ph and the ionic strength of the solution, show that a charged colloid model can explain the overall stability of these species. in addition, this understanding suggests possible ways of modulating the stability of these aggregates in solution. some of these strategies successfully decimate the stable aggregate population, and also reduce the toxicity of amyloid beta to cultured neurons. afm studies of artificial amyloid fibrils and filaments p. mesquida , a. kurtossy , c. macphee department of mechanical engineering, king's college london, london, u.k., school of physics, university of edinburgh, edinburgh, u.k. amyloid fibrils are beta-sheet-rich superstructures of peptides or proteins. although these aggregates have first been found in connection with protein-misfolding diseases, such as alzheimer's or parkinson's disease, there is evidence that the ability to form fibrils is a generic property of any polypeptide rather than a result of specific, disease-related amino-acid sequences. fibrils can easily be formed in vitro from nondisease-related proteins and even from synthetic peptides. these artificial fibrils can thus serve as model systems to investigate the biophysical properties of amyloid fibrils. the system presented here is built from the artificial peptide ttr − , which contains amino-acids and which forms well-defined nanorods of ca nm diameter. we present atomic force microscopy (afm) results of their inner morphology by chemically dissecting the rods into their constituent filaments without completely breaking up the aggregates. in contrast to the stiff rods, their constituent filaments of ca - nm diameter were much more flexible. this shows that the particular way filaments are arranged around each other can massively change the mechanical rigidity of the resulting structures in amyloid fibrils, which could be one cause of the different strains in amyloid diseases. the formation of insulin fibrils is characterized by an initial apparent lag-phase, related to the formation of oligomers, protofibrils and aggregation nuclei. afterwards, the aggregation proceeds via fibril elongation, thickening and/or flocculation, and eventual gelation. here, we focus on the formation of such a gel, made of insulin amyloid fibrils, upon incubation at high temperature and low ph. by light scattering and rheological techniques, we monitor the development of the structural, dynamical and mechanical properties of fibrillar aggregates, up to the dynamic arrest of the sample and to the appearance of a non-ergodic behaviour, which marks the onset of gelation. our experiments were able to reveal the structural details hidden in the apparent lag-phase, displaying the slow fibril nucleation and elongation. this initial stage is followed by the known exponential growth of structures of different sizes. these two kinetic stages of structural growth are mirrored by the kinetics of the viscoelastic properties and, in particular, by the growth of the elastic modulus. our results show that the appearance of a noteworthy elastic network, is associated with the initial fibril nucleation and elongation more than with the formation of large structures, which causes the eventual gelation. capturing the initial events of in-cubo crystallization of membrane proteins c. v. kulkarni , o. ces , s. iwata , r. h. templer chemical biology centre and department of chemistry, imperial college london, united kingdom, division of molecular biosciences, imperial college london, united kingdom, institute für chemie, heinrichstrasse- , university of graz, austria lipid based methods for the crystallization of membrane proteins including in-cubo crystallization are becoming popular in recent times. however, a complete understanding of the basic principles behind these methodologies is still elusive. the crystallization of membrane proteins in the lipid cubic phases involves following major steps: removal of membrane proteins from the native membrane using detergentlike molecules, followed by their insertion and equilibration into a lipid bilayer. the crystallization itself commences with precipitant induced osmotic dehydration which in turn stimulates the nucleation that subsequently leads to a crystal growth. using time-resolved x-ray scattering (saxs) and ultraviolet spectroscopy we have been able to gain new insights into the mechanism behind protein insertion into these complex three dimensional lipid structures including information on the timescales of protein folding relative to crystal growth and the effect of protein insertion on the morphology of the surrounding lipid matrix. several proteins can form amyloid fibrils under given environmental or thermodynamic conditions that affect their native conformation. lysozyme forms amyloid fibrils upon incubation of solutions at acid ph and at about • c for a few days. differential scanning calorimetry experiments confirm that lysozyme is mainly unfolded above • c at ph . we monitored the growth of aggregate size by dynamic light scattering for some days at different temperatures. our results show that the fibrillogenesis is characterized by an initial apparent lag phase and a subsequent growth with quadratic dependence upon time of the scattering intensity. this behaviour recalls a simple kinetic model of nucleation and elongation, with nuclei in equilibrium with monomers. at the end of the incubation at high temperature, we collected atomic force microscopy images which show fibrils with a diameter of a few tens of nanometers and a length of a few microns, characterized by a periodicity along the elongation axis. interestingly, the fibrils morphology exhibits no branching or thickening. this is consistent with the non-exponential growth observed in light scattering experiments. in order to elicit the role of repulsive electrostatic interaction in protein unfolding and self-assembly we extended our study at different ph. in this work we used afm to follow the amyloidogenesis pathway of transthyretin (ttr) to form protofilaments. single-molecule force spectroscopy (smfs) of native ttr and protofilaments were also compared in order to evaluate dynamic and structural differences. we observed that this pathway proceeds through the formation of transient amorphous aggregates, followed by the occurrence of annular oligomers. although implicated in cytoxicity, the role of such oligomers within the amyloidogenesis pathway is poorly understood. we show that the annular species display a tendency to stack, forming tubular-like structures that precede the formation of protofilaments. the protofilament height and pitch resemble those of ttr amyloid reported in previous structural studies. upon solvent exchange, we also observed protofilament disassembly that revealed structures reminiscent of the initial ttr annular oligomers. smfs of protofilaments showed a time-dependent increase in the length of the manipulated structures, suggesting that associations between monomers stabilize with time. force spectra of native ttr and protofilaments contained transitions spaced by ∼ nm, indicative of sequential unfolding of individual β-strands. based on these results a model of ttr protofilament assembly is proposed. ability to undergo amyloid aggregation is affected by protein size c. parrini , h. ramshini , f. chiti , a. relini physics department, university of genoa, italy, biochemical sciences department, university of florence, italy short polypeptide chains, typically between and residues, are generally involved with the conversion from the native state into insoluble fibrillar aggregates. the low prevalence of large proteins is disproportionate with their high occurrence in the human proteome. in order to explore the propensity of large proteins to form amyloid-like fibrils, the -residue hexokinase-b from saccharomyces cerevisiae (yhkb) has been induced to aggregate under two separate conditions, at low ph in the presence of salts and at ph . in the presence of trifluoroethanol. such conditions are among the most promising to form amyloid-like fibrils by normally globular proteins. under both conditions yhkb aggregates very rapidly into species with significant β-sheet structure, as detected by circular dichroism, and a weak thioflavin t and congo red binding. atomic force microscopy revealed globular aggregates eventually clustering into large amorphous aggregates at low ph, while in the presence of trifluoroethanol ribbon-like structures with distinct morphology from typical amyloid fibrils were observed. they had irregular width, no twist, and were connected by thinner fibril segments perpendicular to them. in general, yhkb aggregates displayed an unusual softness, as they were very easily perturbed by the afm tip. these results suggest that inability to form amyloid fibrils may prevent large proteins from being associated with protein deposition diseases. the initial stage of proteins aggregation leading to amyloid fibrils: a saxs study m. g. ortore , f. spinozzi , f. carsughi , t. narayanan , g. irace , s. vilasi , p. mariani dip. saifet, univ. pol., ancona, italy, esrf, grenoble, france, dip. biochim. biofis., ii univ., napoli, italy under some conditions, a protein converts from its soluble form into highly ordered aggregates called amyloid fibrils, which are associated with many human diseases. in order to tackle the prevention and treatment of these diseases, we need to understand the mechanism of the pathological aggregation of proteins. in vitro amyloid fibril formation is preceded by the formation of metastable non fibrillar forms, which are responsible for cytotoxicity underlying neurodegeneration. the molecular mechanisms leading proteins into prefibrillar aggregates are still unclear. we present a saxs study performed at id beamline of esrf on the apomyoglobin mutant w fw f, which at physiological ph, firstly aggregates in prefibrillar forms that are cytotoxic and then forms amyloid fibrils. the first stages of w fw f oligomerization are induced by a ph jump. data show that big changes in w fw f in solution happen in less than ms. singular value decomposition (svd) of the data yields a set of functions, from which all the scattering curves can be reproduced. the major result of our study is the determination of the presence of different oligomers in each step of the process. hence, time-resolved saxs experiments together with the estimation of different oligomers via svd method, can be a new and useful approach to investigate the first stages of amyloidogenesis. -condensed colloidal phase in biology: from proteins crystals to amyloid fibrils - study of structure/toxicity relationship of amyloids by infrared spectroscopy h. p. ta amyloid diseases (alzheimer, parkinson, type ii diabetes, prion) correlate with protein aggregation. all the proteins associated with these pathologies aggregate into amyloid fibrils with a common ß-cross structure. according to many in vitro studies, the toxicity of various amyloids seems to be linked to some intermediates formed during the aggregation pathway and to their interaction with membranes. our aim is to understand the link between structure and toxicity of amyloids by studying their interaction with model membranes. for this, we use as an amyloid model, the prion-forming domain pfd - (wild type wt) of het-s, a prion protein of the fungal p. anserina. when expressed in yeast, wt is not toxic whereas one of these mutants, m is toxic. in vitro, m forms very unusual short amyloid fibers contrary to wt which polymerizes as long fibers. furthermore, atr spectra have shown that m is essentially assembled into mixed parallel and anti-parallel ß-sheets whereas wt displays a predominant parallel organization. we are currently studying the interaction between wt or m with lipid langmuir film by polarized modulation-infrared reflexion absorption spectroscopy (pm-irras). this technique allows determining the secondary structure of proteins and their orientation in the membrane. the study of interactions with different charged phospholipids is following. a. stirpe, m. pantusa, r. bartucci, l. sportelli, r. guzzi dipartimento di fisica, laboratorio di biofisica molecolare & udr cnism, università della calabria, rende (cs), italy an increasing number of experimental studies is demonstrating that the propensity of forming amyloid fibrils is not limited to proteins related to neurodegenerative diseases but it is a more general phenomenon. we present data on human serum albumin (hsa) aggregation induced by a combination of thermal and metal ions effects investigated by optical density, fluorescence and electron paramagnetic resonance (epr). the turbidity experiments as a function of temperature show that the hsa aggregation starts at about • c which is higher than the denaturation temperature (∼ • c). in the presence of copper and zinc metal ions the onset temperature for protein aggregation is markedly reduced as the metal:protein molar ratio is increased from : to : . moreover, the copper is more effective in inducing protein aggregation compared to zinc ion. the hsa aggregation analyzed by tht fluorescence at different incubation temperatures lacks a lag phase and the kinetic traces can be fitted by double exponential functions. the tht fluorescence increase evidences the formation of protein aggregates with fibrillar features. epr experiments for the cu(ii)-hsa complex show the binding of cu(ii) in the protein native state in a square planar coordination with equatorial n atoms, and is not influenced by the heat treatment of the protein. the overall results suggest that hsa aggregation is compatible with a downhill process that does not require the formation of an aggregation nucleus. protein condensation diseases -a colloid physicists viewpoint p. schurtenberger adolphe merkle institute, university of fribourg, ch- marly , switzerland a broad class of diseases, such as cataract, alzheimer's disease and sickle-cell disease, involve protein association phenomena as an essential aspect. the basic element common to all members of this class of molecular condensation diseases is the subtle interplay between protein interactions that produces condensation into dense, frequently insoluble mesoscopic phases. among this class of diseases, cataract is particularly important as the world's leading cause of blindness. this disease is most often the consequence of an uncontrolled aggregation (or phase separation) of the proteins in the eye lens that results in a loss of its transparency. the high concentration protein mixtures present in the eye lens are normally stable and produce a high refractive index that aids the eye in adaptive focusing of light. moreover, the proteins themselves exhibit a rich variety of repulsive interactions, attractive interactions, sizes, phase transitions and self-association. these mixtures also exhibit the pathological aggregation and opacification of the cataract disease that has inspired their study. in my presentation i will illustrate how we can use a combination of small-angle neutron and xray scattering experiments combined with molecular dynamics computer simulations to identify, measure and model the molecular interactions and emergent optical and viscoelastic properties and the phase behavior of the relevant, complex cytoplasmic mixtures. fluorescence microscopy studies of iapp fibrillation at model and cellular membranes d. c. radovan , n. opitz , r. winter department of physical chemistry i -biophysical chemistry, tu dortmund university, dortmund, germany, max-planck institute for molecular physiology, dortmund, germany type diabetes mellitus (t dm) is characterized by islet amyloid deposition and beta cell death, the main culprit being a small a.a. peptide hormone, islet amyloid polypeptide (iapp), which forms fibrils under pathological conditions. we studied the interaction of iapp with giant (guvs) and large (luvs) unilamellar vesicles as well as with ins- e cells. by using confocal / two-photon excitation fluorescence microscopy and guvs, we tested the influence of charge (dopc:dopg : ) and model raft systems, displaying liquid-ordered (l o ) / liquid-disordered (l d ) phase coexistence (such as dopc:dppc:cholesterol : : ), respectively, on the kinetics of iapp fibril formation. a preferential partitioning into the l d phase was observed and fibrils grew along with lipid uptake. fluorescence spectroscopy leakage experiments with carboxyfluorescein-filled luvs and the corresponding tht kinetics of iapp fibril formation were carried out as well. moreover, using the wst- reduction assay and fluorescence microscopy, we could show that the red wine compound resveratrol is a potent inhibitor of iapp fibrillation and its cellular toxicity on ins- e cells, these findings highlighting the potential role of resveratrol in future clinical applications, i.e., in the treatment of t dm. a remarkably high viscosity has been induced in aqueous solutions of lysozyme by the addition of certain structurally related organic solvents, such as tetramethylurea (tmu), dimethylsulfoxide (dmso), dimethylformamide (dmf), and hexamethylphosphortriamide. dmso-induced gelation is observed in samples fulfilling the two following requirements: ( .) lysozyme concentration in excess of mm, and ( .) volume fractions of dmso exceeding . . based on spectroscopic data, the whole process was characterized as consisting of two mutually independent stages. the first involves an extensive transition of the polypeptide backbone, from a predominantly helical to increased random coiled and beta-sheet structures, with the occurrence of non-orthodox protein secondary structures at regions above the solvent critical point. the second stage consists of short-lived interchain contacts leading to an entanglement of the macromolecular system as a whole. here we present a set of scattering and microrheology experiments that investigate both the structural and dynamic properties of the gels under various experimental conditions. studying alpha-synuclein aggregation by fluorescence polarization based kinetics l. tosatto, f. munari, i. tessari, g. de franceschi, m. pivato, p. polverino de laureto, m. bisaglia, l. bubacco università degli studi di padova, padova, italy alpha-synuclein (syn) is linked to parkinson's disease (pd) by two evidences: the accumulation of amyloid fibrils of the protein and the autosomal dominant forms of the disease (a t, a p and e k mutants). protein oligomers seem to be the most toxic species, causing dopaminergic neuronal death, probably disrupting cell membrane (volles & lansbury, ) . the aggregation of syn follows a nucleation dependent mechanism, i. e., aggregation is favoured only after the formation of an oligomer composed of a critical number of monomers (wood et al., ) . as the constitution of the nucleus is a rare event, early aggregation stages are difficult to study. to explore the early stages of the aggregation process a fluorescence polarization (fp) based method (luk et al., ) was applied to study syn oligomerization. fp depends on the size of the fluorescent molecule, therefore it is suitable for the detection of oligomers formation. we measured aggregation kinetic properties under several different conditions. a to mixture of syn and oregon green labelled syn was used to analyze the aggregation behaviour of wild type (wt) and pd mutants. the wt protein shows the fastest aggregation rate. this aggregation process is slowed down by the presence of the chaperone - - eta in wt syn samples. finally, dimers formed by a disulfide bond at the n-terminus or c-terminus of syn, when tested for their aggregation behaviour, show a different propensity to aggregate. a. pardini , r. bizzarri , a. diaspro , p. bianchini , c. usai , p. ramoino , g. checcucci , g. colombetti istituto di biofisica, cnr, italy, a) nest, sns, iit udr, pisa, italy; b) nest, cnr-infm, pisa, italy, univiversity of genova, genova, italy the colored ciliates blepharisma japonicum and fabrea salina show photomotile responses triggered by endogenous pigments of the hypericin family. b. japonicum exists in two forms: the wild one contains red blepharismin; the other one, generated by irradiating the cells with dim visible light, blue blepharismin. b. japonicum shows step-up photophobic response, whereas f. salina, that contains fabrein, shows step-down photophobic responses and positive phototaxis. we showed by confocal microscopy that the pigments are localized not only in pigment granules, but also in the cilia. this fact implies that the structure of the pigment in the cilia differs from that in the cell body, (pigment granules are too big to fit in the axoneme) and suggests that ciliary pigments might play a decisive role in photoreception. fluorescence lifetime imaging microscopy (flim) shows that there is a spatial distribution of lifetimes, which are shorter for the pigment in the cilia. this might indicate a functional role for these pigments. furthermore, lifetimes for f. salina are always longer than those of blepharisma. in order to further characterize the structure of ciliary pigments by means of spectroscopic methods, we have also performed spatially resolved static and time-resolved fluorescence anisotropy measurements. biosensing applications of micro/nano structured silicon several topics related to porous silicon (ps) biosensing properties were carefully considered in this study. ps allows the increasing of the immobilised biomolecule number on its surface and the creation of stable covalent bonds due to its controllable chemistry. beside this, ps is suitable for electrical (conductance, impedance), electrochemical and optical amplification of the detected signal. the experimental results on the fabrication of the ps microstructures such as: (i) protein immobilization and detection using microarray technique; (ii) dna biomolecule detection by impedance or by fluorescence spectroscopy; (iii) very sensitive sers biosensors (raman signal of -mercaptoundecanoic acid); (iv) sensitive element for neurons in nutmix culture are in detail presented. various characterisation techniques have been used, optical and scanning electron microscopy (sem), x-ray diffraction, raman, laser fluorescence and impedance spectroscopy for investigation molecule attachment on the au/ps structures. we have demonstrated that different morphologies of ps as-prepared or coated with gold nanoparticles have an important role in biomolecule/cell detection, due to its large internal surface combined with specific optical properties, being in the same time sensing element/support for immobilization of sensing biomolecules as well as transducer for biochemical interactions. heterotrimeric g-proteins interact with their g-protein coupled receptors (gpcrs) via key binding elements comprising the c-terminal segment of the α-subunit and the two lipid anchors at the α− and γ−subunit. direct information about diffusion and interaction of gpcrs and their g-proteins is mandatory, as these properties will affect the timing of events in the complex signal transduction cascade. in the case of the photoreceptor rhodopsin, receptor packing in the membrane and the related diffusion coefficients are discussed controversially ( , ). by using single particle tracking we show that the encounters of rhodopsin with the fluorescently labeled cterminus of the α-subunit as well as with the holo-g-protein transducin change upon rhodopsin light-activation. our results indicate confined areas of interaction for the c-terminal segment of the α-subunit with inactive rhodopsin disk membranes and less restricted diffusion of the receptor-bound cterminal segment after light-activation. this suggests dynamic short-range order in rhodopsin packing and specific structures for efficient interaction ( ) in vivo, skeletal muscle actively shortens and also resists lengthening, for example when landing after a jump. we study the energy during shortening and during lengthening by measuring the rate of pi release which results from atp hydrolysis. we show here the rate of pi releases in an in vitro protocol that involves muscle stretched followed by muscle shortening. it is hypothesised that the magnitude of deceleration of pi release in the stretch phase is less than the acceleration during the release due the energy input of the motor that is used to apply the length changes. therefore the end point of pi release is similar to isometric conditions. pi release responses to a ramped stretch ( %) followed ms later by a symmetrical release at low velocities ( . l /s − ) were measured in permeabilised fibre bundles of rabbit psoas at • c. laser diffraction and high speed video were also recorded to confirm length change. we show that the rate of pi release drops during the stretch phase, returns to isometric levels during the length hold phase, and finally accelerates during the ramped release. tracking of sarcomere length change using video analysis demonstrated that laser diffraction is unreliable at times during stretches due to lack of uniformity of the sarcomere spacing. microbial rhodopsins: receptors, channels, and pumps from a single design j. l. spudich, e. negri-spudich center for membrane biology, university of texas, houston, texas, u.s.a. the microbial rhodopsin family is comprised of ∼ homologous proteins containing transmembrane helices forming a pocket for the chromophore retinal. most are lightdriven ion pumps ("transport rhodopsins") and others are photosensory receptors ("sensory rhodopsins"). phylogenetic analysis indicates frequent lateral gene transfer of proton pumps among prokaryotic and unicellular eukaryotic species, followed by coupling of the pump's mechanism to the cell's existing signal transduction machinery to create photosensors. this evolutionary path is strongly supported by our studies of sensory rhodopsins in various organisms which demonstrate remarkably diverse signaling mechanisms with diverse transducer partners. the best studied are the phototaxis receptors in haloarchaeal prokaryotes (sri and srii) and in eukaryotic algae (channelrhodopsins). sri and srii transmit signals by protein-protein interaction to control a phosphorylation cascade that modulates motility. channelrhodopsins are light-gated cation channels that depolarize the membrane mediating calcium ion influx into the flagellar axoneme. crystallography and molecular biophysics have begun to clarify how modifications of the same architecture enable the rhodopsins to carry out their distinctly different molecular functions. interconversions of their functions by mutation reveal the elegant simplicity by which evolution uses existing genes to create proteins with novel functions. gold colloids-fluorophore complexes for protein detection assay l. sironi , s. freddi , l. d'alfonso , m. collini , m. caccia , g. tallarida , s. caprioli , g. chirico dipartimento di fisica, università di milano-bicocca, italy, laboratorio nazionale mdm, agrate brianza (mi), italy noble metal nanoparticles (np) are endowed with peculiar optical properties related to the surface plasmon resonances (spr). the interaction of surface plasmons of gold nanoparticles with fluorophores a few nanometers away from the surface modifies their brightness and excited-state lifetime, and this effect can be exploited to obtain nanodevices for proteinprotein recognition. we studied different types of constructs based on gold nps on which derivatives of fluorescein were bound. the interaction of this fluorophore with the gold surface plasmon resonances, mainly occurring through quenching, affects its excited-state lifetime, that is measured by fluorescence burst analysis in standard solutions. the binding of proteins to the gold nps through antigen-antibody recognition further modifies the dye excited-state lifetime. this change can therefore be used to measure the protein concentration. streptavidin-functionalized gold nps of size - nm are used to bind biotin-fluorescein and biotin-antibodies for specific proteins. we have first tested the constructs for bovine serum albumine (bsa) detection. the data reported here indicate that one can measure the concentration of bsa in solution with an apparent limit of detection of ± pm. we have then extended the study to the antitumor protein p -p antibody interaction in standard solution and directly in cellular extracts. calcium transport and phototransduction in isolated rod outer segments g. rispoli dip. biologia ed evoluzione, università di ferrara, italy ca + concentration in photoreceptor rod outer segment (os) strongly affects the generator potential kinetics and light adaptation. light stimuli may produce voltage changes exceeding mv: since the os ca + extrusion is entirely controlled by the na + :ca + ,k + exchanger (nckx), it is important to assess how the nckx ion transport is affected by voltage and intracellular factors. the nkcx regulation was investigated in whole-cell recorded os, using ionic conditions that activated maximally forward and reverse exchange. in all species examined of amphibia and reptilia, the forward (reverse) exchange current increased about linearly for negative (positive) voltages and exhibited outward (inward) rectification for positive (negative) voltages. since hyperpolarization increases ca + extrusion rate, the recovery of the dark level of ca + (and of the generator potential) after light stimuli results accelerated. mg-atp doubled the size of forward and reverse exchange current without modifying their voltage dependence, indicating that mg-atp regulates the number of active exchanger sites and/or the nckx turnover number. ca + jumps achieved via photolysis of caged-ca + produced current transients, possibly originating from electrogenic partial reactions. no monovalent cation substituted for na + at the nckx binding sites, but rb + substituted for k + , while sr + , ba + , mg + substituted for ca + with an apparent permeability ratio of . , . , < . : , respectively. a. pfeifer , k. zikihara , s. tokutomi , j. heberle , t. kottke bielefeld university, bielefeld, germany, osaka prefecture university, sakai, japan the blue light receptor phototropin regulates the growth of plants towards the light. it contains two light-, oxygen-, or voltage-sensitive (lov) domains and a kinase domain. lov domains bind noncovalently flavin mononucleotide (fmn) as chromophore. upon illumination, the triplet excited state of flavin reacts within few microseconds with a nearby cysteine under formation of a photoadduct, which represents the signaling state. in response to adduct formation, a jα helix adjacent to the lov domain dissociates and allows for autophosphorylation by the kinase domain. the mechanism of the photoreaction and the signaling pathway from fmn to the jα helix are still unclear. we have investigated the lov domain of arabidopsis phototropin by microsecond ft-infrared spectroscopy. the difference spectrum recorded at µs provides evidence that the flavin is unprotonated in the triplet excited state. therefore, a previously proposed ionic mechanism of bond formation is disfavored. changes in secondary structure were detected concomitant with adduct formation that relax with a time constant of µs. this early adduct intermediate has not been previously characterized. the final adduct state is formed in milliseconds by further alterations in secondary structure. these findings raise the question of whether the early or late adduct intermediate propagate the signal to the jα helix. -photosensory biophysics - the psychostimulant amphetamine increased no generation measured by epr as well as amino acid release in the rat brain nitric oxide (no) is a novel messenger that modulates many functions of the nervous system. the involvement of no in brain damage was shown mainly by indirect evidence and the data are controversial. the short half-life of no makes its direct detection difficult. we measured no generation using epr spectroscopy based on determination of the amount of paramagnetic mononitrosyl-iron complexes. the aim was to elucidate whether psychostimulant drug amphetamine (amph) modulates formation of no and lipid peroxidation (lpo) products as well as the neurotransmitter release in rat brain. the output of glutamate, aspartate, gaba and acetylcholine (ach) was monitored in striatum by microdialysis with hplc detection. amph produced fold elevation of no generation and lpo formation in brain areas. while amph increased the aspartate, gaba and ach release, the glutamate output was not affected. pretreatment with the neuronal nos inhibitor was highly effective in abating the rise of no and neurotransmitter levels but failed to influence the lpo intensity elicited by amph. the findings suggest that activation of no synthesis is a potent factor in the amph-induced neurotransmitter release. light scattering study of dna over a wide chain length range: comparison with wormlike model p. baeri, m. zimbone dipartimento di fisica e astronomia, università di catania, italy this work reports light scattering measurements on dna in aqueous solutions ( mm nacl , mm edta and mm tris-hcl buffer, ph . ) over a wide range of molecular weights ( - base pairs) and shows that, in the above standard solvent, shorter chains ( < base pairs) behave as a "wormlike chain" and their diffusion coefficient as obtained by dynamic light scattering measurements, confirm the prediction of standard wormlike model, whilst longer chains ( > base pairs) behave in a different manner. dynamic and static light scattering and sem analysis indicate that dna molecules base pairs long, condense into compact structures in our solvent condition. calculations done using a wormlike model are also presented and discussed in comparison both to our experimental data and to other data reported in the literature. a. asandei, t. luchian 'alexandru i. cuza'university, faculty of physics, laboratory of biophysics and medical physics, blvd. carol i no. , iasi, romania amphotericin b (amb) is an antifungal antibiotic which, despite the severe side effects, is still used for the treatment of systemic fungal infections. in this study we investigated the influence of ph upon the selectivity and the transport properties of amb channels inserted in reconstituted, ergosterolcontaining zwitterionic lipid membranes. our electrophysiology experiments carried out on single and multiple amb channels prove that at ph= . these channels are anion selective, whereas at neutral and alkaline ph's (ph= and ph= ) they become cation selective. we attribute this to the ph-dependent ionization state of the carboxyl and amino groups present at the mouth of amb molecules. surprisingly, our data reveal that the single-molecule ionic conductance of amb channels varies in a non-monotonic fashion with ph changes, which we attribute to the ph-dependent variation of the surface and dipole membrane potential. we demonstrate that when added only from one side of the membrane, in symmetrical salt solutions across the membrane and low ph values, amb channels display a strong rectifying behavior, and their insertion is strongly favored when positive potentials are present on the side of their addition. we report a detection method for the redox state of proteins which combines fret-based fluorescence/confocal microscopy on dye-labeled protein with cyclic voltammetry. by using this combined method, electron transfer properties can be revealed from protein to electrode or from redox enzyme to substrate. we applied the fluorescent detection to azurin, a blue copper protein from the bacterium ps. aeruginosa, fluorescently labeled on the n-terminus for monitoring the redox state of the protein. the dye fluorescence is quenched by energy transfer to the copper in oxidized, but not in reduced azurin. fluorescence results demonstrated that cy -labeled wtazurin switched in fluorescence intensity by up to % by varying the applied potential. labeled zinc-azurin was used as a control sample and did not show any fluorescence switching. for single molecule studies wt-azurin was labeled with atto dye and a mixed sam was used, with -hydroxy- octanethiol as a blocking agent to prevent non-specific binding of protein on the surface, and , decanedithiol for the specific covalent binding to the protein. preliminary data show that single-molecule fluorescence switching with the potential is indeed possible. -single molecule biophysics - is there a specific erythrocyte membrane receptor for fibrinogen? an atomic force microscopy approach f. a. carvalho , s. connell , r. a. ariëns , n. c. santos instituto de medicina molecular, univ. lisbon, portugal, university of leeds, u.k. fibrinogen (fg) contributes to erythrocyte (rbc) hyperaggregation by an increase in. rbc-fb protein binding considered to be non-specific. glycoprotein α iib β is a specific integrin receptor for fibrinogen on platelets. we showed that there is a single molecule interaction between fg and an unknown receptor on rbc membrane, with a lower affinity when compared with platelet binding. we evaluated if rbc-fg binding is through an integrin-like receptor or not. interactions between fg and platelet/rbc receptors were studied by force spectroscopy. force curves were performed between fg-functionalized atomic force microscope tips and rbc or platelets. to evaluate if the fg-rbc binding is calciumdependent, similar studies were performed in the presence of ca + or edta. we also carried out studies in the presence of a α iib β inhibitor and of methyl-ß-cyclodextrin (to disrupt lipid rafts by cholesterol depletion). rbc-fg single forces were of - pn and of - pn for platelet-fg binding in presence of calcium. a significant decrease of the platelets-fg force-rupture was obtained in the presence of edta or α iib β inhibitor. significant lower fg-rbc force value was obtained in the presence of mβcd, but not in the presence of edta. conclusion: fg-rbc binding seems not to be calcium-dependent but the existence of cholesterol on rbc membrane is important. nanoscopic and spectroscopic investigation of p -based complexes at single molecule level a. r. bizzarri biophysics and nanoscience centre, cnism, facoltà di scienze, università della tuscia, largo dell'università - viterbo, italy p is a transcription factor that plays a widely recognized role in preventing cancer development in response to dna damage. the tumor suppressor activity of p involves the formation of several complexes whose detection and study, at single molecule level, could be extremely relevant to understand, in detail, the mechanisms governing the cancer defense processes, as well as to develop ultrasensitive biosensors. p -based complexes, are investigated at molecular level, by combining atomic force microscopy (afm), atomic force spectroscopy (afs) and surface enhanced raman spectroscopy (sers) with the support of computational docking. our attention is mainly devoted to study complexes between p and the electron transfer azurin which has been demonstrated to interact with p , by promoting its stabilization. a possible competition between azurin and the cellular oncogene mdm is also investigated. unwinding the dna helix in force clamp condition p. bianco , l. bongini , m. dolfi , l. vincenzo physiolab, dbe, university of florence, italy, university of florence and centro interdipartimentale studio dinamiche complesse, firenze, italy we use a dual-laser optical-tweezers (dlot, smith et al., science, ) to define the highly cooperative conformational transition in the molecule of dna, where the natural b-dna has converted into a new overstretched conformation called s-dna (bensimon et al., phys. rev. lett., ; cluzel et al., science, ) . single molecules of double stranded λ-phage dna (in a solution with mm naci, mm tris-hcl, mm edta, ph . . and • c) are stretched either in length clamp or in force clamp mode. when the dna molecule is stretched in length clamp mode with a ramp lengthening, it shows the previously described highly cooperative overstretching transition at ∼ pn, attributed to unwinding from the b-form to the . times longer s-form. stretching the molecule in force clamp mode with a staircase of force steps at s intervals (step size - pn, rise time - ms) shows, for any given clamped force f in the region of the overstretching transition, different amounts of dna elongation (∆l) with exponential time courses. the analysis of the elongation rates allows to recover all the necessary parameters for an effective two-state model able to reproduce the out-of-equilibrium properties of the system. the results imply an unwinding cooperativity of bps. this value is significantly lower than that obtained assuming force independent rate constants. supported by miur, ente cassa di risparmio di firenze and itb-cnr (milano). c. m. becker , a. benedix , b. l. de groot , a. cafisch , r. a. böckmann saarland university, saarbrücken, germany, mpi for biophysical chemistry, göttingen, germany, university of zürich, zürich, switzerland modifying the stability or the binding behavior of the involved proteins by mutation can influence the activity of cellular processes. for an efficient identification of possible mutation-sites a fast calculation of the free energy of proteins is crucial. here we developed a fast and reliable method (cc/pbsa) [ ] for the prediction of the change in stability of proteins and binding affinity of protein-protein complexes upon mutation. the energy function of cc/pbsa is based on gas phase energies, solvation free energies and entropic contributions. the protein flexibility is taken into account by generating random conformations based on geometrical constraints only applying the concoord [ ] program. we applied cc/pbsa on the tem -blip complex, which is important in bacterial antibiotic resistance. the results of single-and double-point alanine scanning are used to detect hot spots, cooperative effects, and the corresponding energy distribution. cc/pbsa is freely accessible on our web-server: http://ccpbsa.bioinformatik.uni-saarland.de the human recombinase hrad is a key protein for the maintenance of genome integrity and for cancer development. this protein plays a central role is the dna strand exchange occurring during homologous recombination. here we report the polymerization and depolymerization of hrad on duplex dna observed with a new generation of magnetic tweezers, allowing the measurement of dna twist with a resolution of • in real time. at odds with earlier claims, we show that, after initial deposition of a multimeric nucleus, nucleoprotein filament growth occurs by addition of single proteins, involving dna twisting steps of ± • . simple numerical simulations support that this mechanism is an efficient way to minimize nucleoprotein filament defects. this behavior, consisting of different stoichiometry for nucleation and growth phases, may be instrumental in vivo. fast growth would permit efficient continuation of strand exchange by rad alone while the limited nucleation would require additional proteins such as rad , thus keeping this initiation step under the strict control of regulatory pathways. besides, our results combined with earlier structural information, suggest that dna is somewhat less extended ( . versus . Å per bp) and more untwisted ( . versus • per bp) by hrad than by reca, and confirm a stoichiometry of - bp per protein in the hrad -dsdna nucleoprotein filament. biofunctional micropatterned surfaces to study the spatio-temporal organisation of lfa- r. diez-ahedo , d. normanno , c. g. figdor , a. cambi , m. f. garcia-parajo bionanophotonics, ciber-bbn and ibec, barcelona, spain, tumor immunology, nijmegen center for molecular life sciences, the netherlands lymphocyte function associated antigen- (lfa- ) adhesion depends on receptor occupancy and lateral organization on the cell membrane. however, the signals and mechanisms which dynamically reorganize lfa- into high avidity clusters are still a subject of many studies. to obtain deeper insight on the mechanisms that control and regulate lfa- clustering, patterned surfaces of immobilized lfa- ligand areas were fabricated using microcontact printing. the diffusion of lfa- expressed by monocytes stretched over patterned surfaces was followed in time using single molecule tirf microscopy. single lfa- nanocluster trajectories on individual cells showed an increase of immobile lfa- fraction and a slow-down of diffusing lfa- on the ligand areas compared to the non-ligand areas. moreover, single-cluster intensity analysis indicated a reorganization of lfa- nanoclusters in microclusters upon ligand binding. finally, single particle motion analysis of lfa- trajectories in close neighborhood to the ligand areas showed no assisted diffusion of lfa- towards the adhesive regions, consistent with random ligand-encountering and binding. we are currently investigating the effect of cell membrane organizers to regulate the spatio-temporal organization of lfa- . r. diez-ahedo et al, small, in press. optical and electrophysiological detection of single phages across a lipid membrane n. chiaruttini , p. boulanger , m. de frutos , l. letellier , u. bockelmann , v. viasnoff nanobiophysique, espci paristech, cnrs, paris, france, ibbmc, université paris xi, cnrs, orsay, france, lps, université paris xi, cnrs, orsay, france we present an investigation study of the ejection of single t bacteriophages. in vivo studies of dna ejections from the bacteriophage capsid show that the t genome is introduced in the bacterial host in two steps. first % of the genome is ejected then after a pause of a few minutes the rest is internalized. bulk in vitro studies showed that various mutants of t eject their genome in solution following a single or a multistep process. by immobilizing single bacteriophages on a surface and following their ejection by fluorescence microscopy we showed that in all cases the ejection occurs in one step, but some mutants seem to have a subpopulation for which the triggering signal of the ejection is transmitted more slowly to the capsid entrance. we then reconstituted the phage receptor fhua into giant liposomes and followed the ejection of the dna into the liposome by fluorescence. finally we incorporated fhua in a suspended bilayer and followed the infection of the phages through the bilayer both by fluorescence labeling and electrophysiological measurements. we will discuss the influence of the cross membrane potential on the ejection speed of the dna. helixlike pili is a prerequisite of uropathogenic e. coli to adhere to host and withstand urine flow the gram-negative uropathogenic escherichia coli (upec) bacteria, invades the urinary tract region and cause in some cases severe infections, pyelonephritis, if they can withstand the rinsing action of urine and ascend to the kidney, via the bladder and ureters. to mediate adhesion, upec express quaternary surface organelles that are assembled from ∼ identical subunits into a helix-like coil, with a single adhesin located at the tip. it is believed that the single adhesin mediate attachment to host cells while the helix-like structures act as shock absorbers to dampen the irregularly shear forces induced by urine flow. to unravel the biomechanical properties of such quaternary structures, in particular in terms of their force-elongation and kinetic behavior, force-measuring optical tweezers (fmot) have been used. a plethora of different types of pili have been identified in the literature and we show, using fmot, that those dissimilarities might reflect the host environment. for example, we have found differences among pili expressed at diverse environment inside the urinary tract, which imply that pili presumably have evolved to resist specific forces under in vivo conditions. it is thus worth striving for understanding bacterial adhesion in order to figure out alternative to the over-abundance of antibiotics worldwide. single molecules studies have probed protein conformational fluctuations and monitored the oscillating activity of enzymes that remained masked in ensemble measurements. various statistical models have attempted to connect the conformational motions to the fluctuating activity of enzymes and suggested that they adopt inter-converting conformations each one of them exhibiting different catalytic activity. the main drawback of these studies is the non specific interactions that may bias the observed catalytic behavior. we have developed new innovating tools to study the behavior of single enzymes. as a first step we utilized liposomes as scaffold to confine enzymes while keeping them under physiological conditions. keeping them in their native conformation allowed us to monitor the inherent properties of their behavior. as a second step we developed a new model that accurately connects enzyme activity behavior to conformational motions. using this approach we accurately predicted the behavior of single lipases in a highly controlled environment. we modulated the enzyme's conformational mobility and activity by systematically varying its accessibility to liposomes. as we anticipated, that resulted in altering the time the enzyme spends on active conformations.our results provide new insights on interpreting the behavior of enzymes. simulations of single-molecule fret experiments on ribozymes m. hajdziona, a. molski adam mickiewicz university laboratory for dynamics of physicochemical processes, poznan, poland rnazymes are important biological molecules and that can catalyze the cleavage of their own nucleotide chains. this precess is called self splicing [ ] . there are several methods to to study kinetic properties of ribozymes at the singlemolecule level. in this work we focus on fret (förster resonance energy transfer) of single, immobilized molecules. single-molecule fluorescence spectroscopy gives an insight into the behavior of individual molecules rather than the average behavior of an ensemble of molecules, which makes it possible to observe the kinetic heterogeneity. we simulated fret trajectories for single immobilized ribozymes. in our computer simulations we consider two-and three-state kinetic models motivated by actual experiments, published in [ ] and [ ] . we fitted the histograms of on and off times to recover the kinetic parameters of the models. we investigated the bias and standard deviation of the recovered parameters when one or two thresholds are applied to fret trajectories between adjacent states. we found that two thresholds give better parameter estimates than one-threshold analysis. references frap probes the average dynamical properties of a population of fluorescently labeled molecules whereas spt obtains these properties from observations of the trajectories of individual molecules tagged with a colloidal particle (the "bead"). when results of frap are compared with those of spt, frap yields significantly higher diffusion constants than spt. to understand the origin of this difference, we have developed and tested a model system to evaluate the influence of the bead on the dynamics of a diffusing molecule. we use a dsdna tether to attach a bead to a supported lipid bilayer (slb). the dna tether is modified at one end by cholesterol for anchoring to and diffusion in the membrane, and at the other end by biotin for tagging with a bead. with this system, we can vary several parameters: distance between slb and bead, size and nature of the bead, lipid composition of the slb, external force. we can also model the brownian motion of the bead and the hydrodynamic flow around it. we will present results using φ = nm neutravidin-coated latex particles attached to an eggpc slb via a bp to bp dna tether. elucidation of the mechanism of the lambda bacteriophage epigenetic switch l. finzi physics department, emory university, dowman dr, atlanta, ga , usa the lambda bacteriophage epigenetic switch determines the growth lifestyle of the virus after infection of its host (e. coli ). it is now clear that the switch consists of a ∼ . kbplong dna loop mediated by the lambda repressor protein. using tethered particle microscopy (tpm), magnetic tweezers and afm, our laboratory has novel, direct evidence of loop formation and breakdown by the repressor, the first characterization of the thermodynamics and kinetics of the looping reaction and its dependence on repressor non-specific binding and dna supercoiling. these in vitro data provide insight into the different possible nucleoprotein complexes and into the lambda repressor-mediated looping mechanism which leads to predictions for that in vivo. the significance of this work consists not only of the new insight into a paradigmatic epigenetic switch that governs lysogeny vs. lysis, but also the detailed mechanics of regulatory dna loops mediated by proteins bound to multipartite operators and capable of different levels of oligomerization. mechanical force at the molecular level is involved in the action of many enzymes. for example, the phi dna polymerase mechanically unwinds the dna helix as it moves processively along the dna replicating one strand of the dna molecule. using optical tweezers we have developed a single molecule mechanical assay to elucidate the physical mechanism of dna unwinding by the phi dna polymerase as the protein replicates the dna. a single dna hairpin is hold between an optical trap and a mobile surface. as a single polymerase works on the dna hairpin, its replication and unwinding reactions can be measured in real time by measuring the change in extension in the dna polymer, revealing the fluctuations of its rate in response to the dna sequence. moreover, by gradually pulling on the opposite strands of the dna hairpin we can promote the controlled mechanical unwinding of the dna helix and determine the effect of increasing unwinding forces on the polymerization and unwinding rates. the effect of force and dna sequence on the activities of the wild type and an unwinding-deficient polymerase mutant will allow us to determine the inner workings of this molecular motor. single centrosome manipulation reveals its electric charge and associated dynamic structure we demonstrate laser manipulation of individual early drosophila embryo centrosomes in between two microelectrodes to reveal that it is a net negatively-charged organelle with a very low isoelectric region ( . ± . ). from this single-organelle electrophoresis, we infer an effective charge smaller or of the order of electrons, which corresponds to a surface-charge density significantly smaller than that of microtubules. by investigating the centrosome hydrodynamic behavior, we show that its charge has a remarkable influence over its own structure. specifically, we find that the electric field which drains to the centrosome expands its structure to a physiological size a % larger than previous electron microscopy determinations, a self-effect which modulates its structural behavior via environmental ph. this methodology further proves useful to study the action of different environmental conditions, such as the presence of ca + , over the thermally-induced dynamic structure of the centrosome. magnetic contrast neutron reflectivity delivers significant improvements in resolution for membrane structure analysis neutron reflection (nr), with its high resolution and use of contrast variation, is a unique tool to gain information on the orientation and structure of lipid bilayers in the z-axis perpendicular to the surface. to improve the use of nr we have used a highly oriented and stable layer of membrane proteins and lipids made possible by self-assembly upon a gold surface. this also provides a model of the bacterial outer membrane. as the dimensions of the layer can be predicted with accuracy, the system provides a molecular ruler for improvements in methodology and the complexity of the layer structure adds significantly to the modelling challenge. early improvements in resolution were obtained through sample preparation and gold smoothness. further improvement however required clearer discrimination between similar models. this was achieved using the new approach of magnetic contrast variation which uses a magnetic layer to provide two different scattering length densities for oppositely polarised neutrons. during this data collection the sample is unchanged. we show that this approach delivers significant improvements in data analysis and resolution of the protein, lipid and solvent structures. the method will provide a new level of understanding of membrane structure and dynamics. single molecule force spectroscopy and recognition imaging p. hinterdorfer institute for biophysics, johannes kepler university of linz, altenbergerstr. , a- linz, austria in single molecule force spectroscopy, interaction forces of ligand-containing tips with receptors on probe surfaces are quantified. here he attachment of human rhino virus (hrv ) to the cell surface, the first step in infection, was characterized. sequential binding of multiple receptors was evident from recordings of characteristic quantized force spectra. this suggests that multiple receptors bound to the virus in a timely manner. unbinding forces required to detach the virus from the cell membrane increased within a time frame of several ms. the number of receptors involved in virus binding was determined and estimates for on-rate, off-rate, and equilibrium binding constant were obtained. furthermore, we show that accurate free energy values of membrane protein unfolding can be obtained from single molecule force measurements. by applying a statistical theorem developed by jarzynski, we derived equilibrium unfolding free energies of from unfolding force data acquired at different force loading-rates and temperatures. finally, we present a method for the localization of specific binding sites and epitopes with nm positional accuracy. a magnetically driven afm tip containing a ligand covalently bound via a tether molecule is oscillated at a few nm amplitude, during scanning along the surface. in this way, topography and recognition images on membranes and cell surfaces were obtained simultaneously. mapping of the forces acting on biomolecules in cell membranes has spurred the development of effective labels, e.g. organic fluorophores and nanoparticles, to track trajectories of single biomolecules [ ] . standard methods use particular statistical observables, namely the mean square displacement (msd), to extract cues on the underlying dynamics. yet, msd is not an appropriate tool to access force fields and becomes easily a biased estimator in the presence of positioning noise. here, we introduce general inference methods [ ] to fully exploit information hidden in the experimental trajectories, providing sharp estimates for the forces and the diffusion coefficients within membrane microdomains. rapid and reliable convergence of the inference scheme is demonstrated on trajectories generated numerically. the inference method is then applied to infer forces and potentials acting on the receptor of the ε-toxin labelled by lanthanide-ion nanoparticles. results show a constant diffusivity inside a complex force field confining the receptor inside a specific domain. our scheme is applicable to any labelled biomolecule, and results presented here show its general relevance to the issue of membrane compartmentation and protein motion. large scale domain motions are structural rearrangements often adopted by enzymes to achieve their full functionality. understanding the mechanism responsible for such movements by means of computational approaches is of great interest especially in rational drug design, since induced fit or population shift effects are closely related aspects of the problem. unfortunately, the simulation time required to sample these events by conventional techniques such as plain molecular dynamics, is unfeasible. here, the domain motion required by adenosine kinase (ak) to achieve its (pre-)catalytic conformation was studied using well-tempered metadynamics [barducci, a. et al. phys rev lett. ( ) , : ] with path collective variables (pcvs) [branduardi, d. et al. j chem phys. ( ) , : ]. first, a low energy path for the apo form of the enzyme was obtained, and the potential of mean force along the pcvs was reconstructed. then, the large scale movement was simulated in the presence of two inhibitors known to bind to the enzyme in different conformational states. the adopted approach was proven to be successful both to understand the mechanistic features of the ak domain motion and to provide a picture of the population shift effects upon ligand binding. force measurement study of the interaction between s rrna and the ribosomal protein l single molecule force measurements enable to probe the complex structure and folding dynamics of rna molecules and furthermore to investigate the numerous interactions with proteins that affect rna folding in vivo. we use a double optical tweezers setup to study a region of the ribosomal rna s from e.coli and its interaction with the essential ribosomal protein l . the apparatus permits us to probe the dynamics of the rna structure with milisecond time-resolution. first, we pull the rna and show that it mechanically unfolds in several reproducible steps. we use these results in combination with structure prediction tools to evaluate the possible structures of the rna fragment in vitro. the most probable structure exhibits a unique binding site for l . then, force measurements are done on the rna in presence of l . we show that the protein specifically binds the rna and stabilizes it. the binding site is recognized with a resolution of a few bases. finally, two bases are mutated on the rna fragment. in presence of these mutations, in vivo and in vitro binding of l to s rna is abolished. the single molecule approach gives an explanation of this result: the force measurements show that the mutated rna folds differently from the natural one and that it does not bind l . control of the translocation of single dna molecules through alpha-hemolysin nanopores g. maglia, h. bayley department of chemistry, university of oxford, ox ta, oxford, uk the analysis of single nucleic acid molecules by electrophoretic threading through nanopores is under intense investigation as a rapid, low cost platform for dna sequencing. biological nanopores such as staphylococcal alpha-hemolysin (hl) have an added advantage over solid-state nanopores because they can be modified by genetic engineering with atomistic precision. although we showed that all four dna bases can be identified in an immobilized ssdna molecule ( ), the translocation of free dna is too quick to observe single bases. here we show that by a small increase of the net internal charge of the hl nanopore (e.g. by introducing a ring of arginine residues), we have augmented the frequency of dna translocation events through the pore and dramatically lowered the voltage threshold required for dna translocation ( ) . by further increasing the net positive charge of the transmembrane barrel region of the pore (e.g. by introducing extra positive charges) we have also reduced the speed at which dna translocate the pore by more than two orders of magnitude. these experiments provide a means of controlling dna translocation with protein nanopores, which might be translated to solid-state nanopores by using chemical surface modification. the atomic force microscope (afm) is a tool for imaging, measuring and manipulating matter at the nanoscale. single-molecule pulling experiments give information on the thermodynamics and kinetics of biomolecules. the purpose if this work was to develop software to simulate single-molecule pulling experiments and to analyze singlemolecule pulling data. the long term objective is to asses the accuracy and precision of the parameters recovered from single-molecule pulling data.we carried out simulations for two different models [ , , ] , using a wide range of loading rates. from the simulated force-extension curves we extracted the kinetic parameters and compared them with the values used for simulations. the kinetic parameters were the intrinsic rate coefficient (k kramers rate), the location of transition state (x ) and the free energy of activation (∆g). we have found that the loading rates have a small effect on the recovery of the free energy of activation, but have a significant effect on the recovery of the kramers rate. we report the tracking of single myosin v molecules in their natural environment, the cell. myosin v molecules, labeled with quantum dots, are introduced into the cytoplasm of living hela cells and their motion is recorded at the single molecule level with high spatial and temporal resolution. we perform intracellular measurements of key parameters of this molecular transporter: velocity, processivity, step size and dwell time. our experiments bridge the gap between in vitro single molecule assays and the indirect measurements of the motor features deduced from the tracking of organelles in live cells. a mathematical model of neurotransmission at the input stage of the cerebellum t. nieus , s. solinas , l. mapelli , e. d'angelo dept. neuroscience and brain technologies, iit, italian institute of technology, genova, italy, dept. of biomolecular sciences and biotechnology, milan, italy, dept. physiological and pharmacological sciences and cnism, university of pavia, italy the granule cell (gc) of the cerebellum has some peculiar properties compared to other cells of the vertebrate brain. the gc has a small soma (diam= microm) and just a few ( to ) excitatory and inhibitory inputs (e&i). the e&i gc synaptic inputs are formed inside the cerebellar glomerulus, which favors neurotransmitter diffusion between neighboring sites ( ) protracting postsynaptic receptor activation and ( ) causing cross-tall between e&i synapses through presynaptic receptors. neurotransmitter release probability (p) can be regulated by long-term plasticity (ltp and ltd at e synapses) and by gaba b and mglu presynaptic receptors (both at e&i synapses). the p change in turn modifies shortterm plasticity, affecting the first response in a train much more than the followings. to gain insight into the role that p changes might have in computations performed at the cerebellum input stage, we have built detailed biophysical models of the e&i synapses. the model has allowed to investigate glomerular processing of high frequency inputs reaching the cerebellum. optimal synaptic transmission through the mfs inputs resulted when gos inhibited synchronously the gcs. the role of feed-forward inhibition onto synaptic transmission is under investigation. unzipping dna with a nanopore j. muzard, n. chiaruttini, u. bockelmann, v. viasnoff cnrs/espci nanobiophysique, rue vauquelin, paris, france the use of proteinaceous or artificial nanometer size pores has become a promising approach for sensing biomolecules at the single molecule level. it was shown that alphahemolysin, a toxin from staphylococcus aureus, can be employed to sense the translocation of dna strands through a lipid bilayer. the pore dimension allows the electrophoretically driven passage of single stranded dna whereas double stranded structures need to open prior their translocation. we study the unzipping process of the double stranded part of dna both experimentally and theoretically. we show that in a first approximation the duplex opens progressively in a sequence dependent manner. the experimental results can be accounted for by modeling the unzipping process as a free diffusion of the unzipping fork in the energy landscape defined by the sequence of the basepaires. we then discuss the effect of the pore/dna interaction on the translocation process. we further characterize the effects of the pore geometry, showing that the pre-confinement of the dna in the pore vestibule is essential to the unzipping process. we eventually discuss the possibility to use this nanopore approach to sequentially probe rna secondary structures. we report on the interaction forces in the range -- pn determined between pairs of the lectin soybean agglutinin (sba) and a modified porcine submaxillary mucin (tn-psm) using a single-molecule approach. lectins are carbohydrate-binding proteins with biological activities related to i.e. cellular recognition, adhesion, growth and metastasis. here, dynamic force spectroscopy is used to investigate pairs of sba and tn-psm with the aim to understand the mechanisms of binding and cross-linking of multivalent lectins. the unbinding force increased from pn to pn with increasing force loading rate and the lifetime of the complex in the absence of applied force was . - . s. published kinetic parameters describing the rate of dissociation of other sugar lectin interactions are in the range . x − - . x − s. the long lifetime of the sba -tnpsm complex is compatible with a previously proposed "bind and jump" mechanism. this mechanism has also been suggested for lectins binding to multivalent carbohydrates and globular glycoprotein. the bind and jump mechanism is also similar to that observed for binding of proteins to dna, and suggest a common conserved binding mechanism of ligands to the two biopolymers and possibly between ligands and all biopolymers, as recently suggested. the rate of topoisomerase ii activity correlates with persistence length of dna q. shao , l. finzi , d. dunlap dept. of physics, emory university, atlanta, georgia, u.s.a., dept. of cell biology, emory univ. med. school, atlanta, georgia, u.s.a. type ii topoisomerases catalyze the transection of one double helical segment by another to modify the topological state of dna. reversible ' linkages to the phosphate-sugar backbones are established on each strand of the "gate" segment to create an opening through which the enzyme drives the "transfer" segment. a recent crystal structure shows that the "gate" segment bends approximately • upon binding to the enzyme. bending a stiff polymer like dna requires considerable energy and could represent the rate limiting step in the catalytic (topological) cycle. using modified deoxyribonucleotides in pcr reactions, more rigid dna fragments have been produced and used as substrates for topoisomerase ii-mediated relaxation of plectonemes introduced in single molecules using magnetic tweezers. before relaxation the persistence lengths were measured by fitting force extension data with the worm-like chain model. topoisomerase ii was found to release mechanically introduced supercoils more slowly in stiffer dna suggesting that dna bending might be a rate limiting step in topoisomerase ii activity. h. seidel , t. klose , h. lilie , c. g. hübner institute of physics, ratzeburger allee , lübeck, germany, institute of biochemistry and biotechnology, kurt-mothes-str. , halle, germany one essential element of a virus is its protein shell, the viral capsid, which encloses the viral genome. the murine polyomavirus is a non-enveloped dna tumor virus with an icosahedral t= d structure. besides the knowledge of the structure, it is of utter importance to understand the process of viral assembly [ ] . the assembly reaction of polyoma vp does not show the typical sigmoidal kinetics in light scattering experiments. the apparent kinetics is of fourth order, which appears rather unrealistic. in order to gain knowledge on capsid composition during assembly beyond ensemble average, we apply methods of single molecule fluorescence, namely fluorescence correlation spectroscopy (fcs), fluorescence-intensity-distribution-analysis (fida), and single-particle-imaging (spi). the molecular brightness and the diffusion time reported by fcs are in agreement with the results from light scattering. fida as well as spi, moreover, point to a polymerization of subunits to complete capsids along the assembly pathway without pronounced intermediates. mixing experiments show that already early in the course of the assembly reaction no exchange of pentamers between capsids occurs, and that the effect of breathing [ ] can be excluded for polyoma vp . genetic information coded in dna provides complete instructions to cells regarding metabolism and proper functioning. a number of endogenous and exogenous sources can damage the genomic dna. unrepaired dna damage can give rise to mutations and may cause cell death. cells have evolved different mechanisms to repair damaged dna and to protect genetic integrity. uracil in dna occurs as a result of incorporation of dump in the place of dtmp during replication and deamination of cytocine, resulting in u:a and u:g base pairs, respectively. uracil-dna glycosylase (ung) is a dna repair protein that searches and removes uracil from dna. ung removes mismatched base by flipping it out from the base stack into the active site. the exact mechanism by which ung searches dna for uracil is unknown. therefore, in our study we use atomic force microscopy (afm) to investigate the interaction between single ung molecules with dna carrying the u:a or u:g mismatches. furthermore, we study the complexation of dna and ung protein. afm images of ung bound to dna reveal the structural changes at the level of single complex, e.g. dna kinking that may occur upon binding of ung to dna. imaging of dnaprotein complexes can provide a a new level of understanding of ung-dna interaction. atomic force microscopy (afm) has been a useful device to visualize cellular and molecular structures at a single-molecule resolution. especially, afm imaging under the trec t m (topography and recognition) mode (recognition imaging) can map a specific protein of interest within an afm image. in this study, we employed an antibody-coupled afm cantilever in recognition imaging, and dissected the structural/functional domains of α actinin- , an actin binding protein that cross-bridges actin filaments and anchors it to integrin via tailin-vinculin-α actinin adaptor-interaction. a use of monoclonal anti-α actinin- antibody enabled us to map its epitope in the amino-terminal actin-binding domain within a single molecule afm image of α actinin- . counting fluorescent molecules by photonantibunching h. ta, m. schwering, d. p. herten bioquant, heidelberg university, germany information on the stoichiometry of labelled biomolecules is highly desirable. a method called photo-antibunching has successfully been used to determine the number of fluorescence emitters in multichromophoric systems. a statistical model to estimate the number of fluorescent molecules in a confocal observation volume is established based on photonantibunching in time-correlated single-photon counting (tcspc) scheme with avalanche photon diodes (apds) for detection of individual photons. numerical simulations based on realistic experimental conditions show that the model is able to estimate the number of molecules with moderate errors. experiments on immobilized double-strand dna oligonucleotides with photon stabilizing agents show promising results even when the number of molecules is ∼ . the proposed method allows us to monitor labelled single molecules and in the near future we plan to implement it in complex biological systems (cell extracts/live cells). single molecule afm force spectroscopy and steered molecular dynamics of contactin- protein j. w. strzelecki, k. mikulska, a. balter, w. nowak institute of physics, nicolaus copernicus university, grudziadzka , - torun, poland contactin- (cntn ) is an axonal cell adhesion protein that contains six igc and four fniii domains and is responsible for creating neural outgrowths. recent research shows that mutation of cntn gene may be responsible for autism, while its absence in transgenic mice results in lack of smell sense. we use a homemade afm single molecule puller and steered molecular dynamics simulation to test its elastic properties through force spectroscopy. stretching experiments show unfolding of fniii domains while igc domains stay coiled as they are stabilized by disulfide bonds. unfolding of contactin- molecule appears to play role of buffer that helps to protect neural contacts from damage when neural cells are subjected to shock or tumor. single molecule studies of the thermophilic bacillus ps f -atpase have revealed kinetic and structural information that cannot be discerned using other methods, including the presence of and degree physical substeps (yasuda et al. ) and the order and kinetics of chemical substeps. we are interested in using single molecule techniques to observe the effects of mgi mutations on enzyme kinetics and torque production in f from the yeast saccharomyces cerevisiae. using a high speed imaging camera, we have captured the rotation of wild-type and mutant forms of yeast f -atpase. rotation data for the wild-type and preliminary data for some mgi strains will be presented. we show for the first time that at saturating atp, wild-type yeast f rotates approximately four times faster than the thermophilic f . kinetic and substepping behaviour in yeast appears to be similar to that observed in bacterial f . -single molecule biophysics - biophysical characterization of a dna gquadruplex formed in the promoter of human mef d gene w. zhou , l. ying molecular medicine, national heart and lung institute, imperial college london, london, sw az, uk, chemical biology center, imperial college london, sw az, uk g-quadruplex is believed to be involved in many crucial biological processes, such as the gene regulation and the maintenance of human telomere. mef d, a member of mef (myocyte enhancer factor- ) family of transcription factors, regulates the response of heart to cardiac stress signals. we found that a g-rich sequence starting at - bp of mef d promoter can form a very stable intramolecular g-quadruplex. here we present a detailed biophysical characterization of this quadruplex. we also found that this quadruplex is more stable than its duplex form under physiological conditions by cd melting and single molecular fret. we observed subpopulations in smfret measurements possibly due to different conformations of the quadruplex. we characterized its unfolding process by monitoring the change in fret when it hybridizes to its c-rich complimentary strand. finally, we proposed several possible structures of this quadruplex based on the smfret and fluorescence dms footprinting results. this research is supported by national heart and lung institute foundation. by stretching a polymer in solution using single molecule techniques it is possible to infer about its physical properties. afm stretching experiments allow for a full characterization of the elasto-mechanical properties of the sample under study. in the presented work, single molecule afm force spectroscopy experiments were used to determine mechanical properties of a peptide obtained from exon (ex ) of the human elastin gene. elastin is a protein with important mechanical properties and, in particular, it shows quasi ideal elastic behavior associated to the presence of many hydrophobic unstructured domains (such as ex ) into the protein structure. ex coded polymer was used as a starting point to obtain bio-materials with specialized elastomechanical functions. in particular, a mutated polypeptide based on the ex sequence was synthesisized with the aim of obtaining a new polymer with the same mechanical and physical properties of the native molecule but with increased aggregation properties, induced by a cross-linking reaction. afm stretching experiments were used to verify the mechanical properties of the engineered proteins at a single molecule level. the obtained results allowed not only to address this question, but also to give some insight into the first aggregation steps of the polymer towards the formation of reticulated structures. luminescent lanthanide-ion doped nanoparticles (nps) are attractive single-biomolecule labels. they are synthesized directly in water, are highly photostable, and display narrow emission without intermittency. we coupled y . eu . vo nps to ε-toxins produced by clostridium perfringens, which bind to a specific receptor on mdck cells. single-molecule tracking experiments using these labels produce long ( min) uninterrupted trajectories with temporal resolution down to ms or localization precision down to nm. we found that the toxin receptor exhibits confined motion in cell membrane microdomains. to analyze the receptor trajectories, we used a novel approach based on an inference method [ ] . this method fully exploits the information of the ensemble of the trajectory, in contrast to the usual mean square displacement analysis. applying both techniques to recorded trajectories, we highlight the difference in extracted parameters. from the shape of the confining potential, obtained by mapping the forces inside the domain, we can deduce information about the mechanism of confinement. in combination with experiments on cholesterol depletion and cytoskeleton destruction, this technique will shed light into the nature of the membrane micropatterning. background: implantable cardioverter defibrillators (icds) are save-life device for patients at risk of sudden cardiac death, and help cardiac patients to avoid slow beat-rate by means of anti-bradichardia pacing (abp) feature. however, recent literature evidenced the presence of ventricular tachyarrhythmias (vts) immediately following abp, leading to the hypothesis that icd devices might be pro-arrhythmic. aim: study differences between pacing-associated tachyarrhythmias (pat) and other spontaneous vts. signals and methods: spontaneous vt episodes are retrieved from patients with icd. vts are examined and pat episodes, classified. characterization of vts is based on the analysis of seconds immediately preceding vt-onset quantified by: heart cycle (hc-prevt), prevalence of premature ventricular contraction (pvcprev) and prevalence of paced beats (pprev). significant differences are evaluated by student-t or chi-square tests with p< . . results: vt episodes from patients are pat episodes ( %). cardiac activity preceding pats vs. non-pats differs: pat episodes have higher hc-prevt (p< . ), greater pprev (p< . ), and greater pvcprev (p< . ). analysis also shows that pat episode often occur when the paced beat immediately follows a premature contraction. conclusion: the study indicates that new icd generations should avoid abp after premature ventricular contraction. towards mechanistical understanding of adsorption: combining technologies in situ and in real time p. h. bjöörn q-sense ab, västra frolunda, sweden a growing number of researchers in different surface related fields present evidence from more than one analytical technique when detailing their findings. thus a logic and useful development is to combine technologies for simultaneous measurements on a single sensor surface. to develop a biosensor platform such as an assay fast and accurately, mechanistical understanding of why the platform works is essential. quartz crystal microbalance-with dissipation (qcm-d) technology and instrumentation provides an open platform and enable easy and precise quantification of mass, thickness and viscoelastic properties of surface bound molecules. these parameters provide both a reference tool in assay development, but also detecting and identifying your target molecules as the combination of mass and material property info in many cases provide a unique response for a specific molecule. by combining qcm-d with other technologies, a range of useful info is put within easy reach of the researcher. recent advances will be presented where simultaneous real time and in situ measurements using qcm-d together with electrochemistry, ellipsometry and fluorescence microscopy enables manipulation of films as well as quantification of the variation of water content as a result of conformational changes in immobilized molecules. examples will include new data from the formation of protein films, polyelectrolyte multilayers and polymer brushes. intrinsic gravity versus metabolically inert infrastructure and basal metabolic rate in living mass i. r. bhattacharjee , r. kashyap , b. shaptadvipa assam agricultural university, jorhat- , india, international institute of intrinsic gravitation biology (i gb), jorhat- , india 'self gravitation bio' is an emerging concept in biophysics. intrinsic or 'self' gravitational force might exert when mass increases beyond critical level in biological particle pyramid. (http://en.wikipedia.org/wiki/biomechanics of intrinsic gravity) 'metabolically inert infrastructure' (mii) consists of total body mass (body water, dissolved substances, mineral and organic deposits) and serves as storage of nutrients, transport and distribution of these materials. to act independently as living body, mii is suggested also to provide structural support to the organism with density-gradient buoyant force against intrinsic and extrinsic gravitational attraction for the biological mass. it is shown that 'amniotic fluid', 'isotonic saline to ailing patient', 'growth factors', 'cultural medium' and other 'medium matrices' act as counter-gravity mechanism for micro to macro living organisms under center-of-biomass reference frame. controversy over relationship between bmr (basal metabolic rate), rmr (resting metabolic rate), pal (physical activity level), on one hand, and mass of the living organism, on the other, (as described in rubner's / law, kleiber's / law that continued to be contested by many) can be favorably resolved substituting the concept of self gravitation bio, considering 'mass' as synonym of gravitational force under center-of-biomass reference frame. bioenergetics would be an unequal but opposite entity of self-gravity reinforced by extrinsic gravity. platelet arrest on von willebrand factor (vwf) occurs transiently via the platelet receptor gpib. depending on the combination of shear stress and surface density of vwf binding sites the platelets either only adhere, pull tethers or generate microparticles. these processes are influenced by the properties of the platelet membrane and the cytoskeleton. under shear flow conditions these platelet characteristics were examined with reflection interference contrast microscopy and a viscosimeter. in addition we quantified platelet adhesion energy and tether pulling forces. disrupting platelet f-actin had several effects. the tethers were shorter, the membrane contact area was larger, the receptor αiibβ density of the microparticles was depleted and no platelet spreading occurred. breaking up the microtubular system had different implications. the number of severed tethers tripled, the contact area was smaller, microparticle formation was increased and the area of spread platelets was reduced. however changes in the membrane elasticity had no effect on the platelets. our results suggest that platelet attachment and adhesion is not only determined by the platelet adhesion receptors and their cytoplasmic linker proteins, but that cytoskeletal structures have also a crucial influence on how platelets interact with thrombogenic surfaces. gene expression is orchestrated by a host of regulatory proteins that coordinate the transcription of dna to rna. regulatory proteins function by locating specific binding sequences of dna and binding to these sequences to form the transcription initiation complex. in many instances, these regulatory proteins only have several hundred copies that must efficiently locate target sequences on the genome-length dna strand. the non-specific binding of regulatory proteins to random sequences of dna is believed to permit the protein to slide along the dna in a stochastic manner. periodically, a thermal kick or an interaction with another bound protein will disengage the regulatory protein from the dna surface, leading to three-dimensional diffusion. eventually, the protein will reattach to the dna at some new location that is dictated by both the diffusivity of the protein and the dna configuration. cycling through these random events constitutes a search strategy for the target site. we build a reaction-diffusion theory of this search process in order to predict the optimal strategy for target site localization. the statistical behavior of the dna strand acts as a necessary input into the theory, and we consider several governing behaviors for the dna strand. we explore the impact of dna configuration and protein occlusion on target site localization in order to predict how protein expression will vary under different experimental conditions. a. di garbo , s. alloisio , s. chillemi , m. nobile istituto di biofisica cnr, via g. moruzzi , pisa, italy, istituto di biofisica cnr, via de marini , genova, italy in the nervous system of vertebrates there are more glial cells than neurons: from to times, depending on the animal type. glial cells are not able to generate action potentials but, nevertheless they play an important role for the functioning of the different brain's area. the astrocytes are the more abundant cells of the macroglia and through their processes they modulate synaptic activity. in this contribution a biophysical neural network model consisting of a pyramidal neuron, an interneuron and the astrocyte is studied. the corresponding dynamical properties are mainly investigated by using numerical simulations. the results show that the presence of the atp and of the interneuron strongly impacts the neural activity. moreover, it is shown that the fluxes of calcium through the cellular membrane strongly affect the modulation of the neural activity arising from the astrocyte. microscopic origin of the very-low energy vibrational dynamics in proteins g. d'angelo , v. conti nibali , c. crupi , a. paciaroni , u. wanderlingh department of physics, faculty of science, messina university, italy, department of physics, faculty of science, perugia university, italy important functions of biological processes require large atomic rearrangements and conformational fluctuations. for proteins, atoms are mostly displaced along the soft directions identified by the delocalized, low frequency vibrations. the study of very low energy vibrational spectrum of proteins is therefore of particular interest. as a step toward understanding their functional role, we have investigated the low frequency vibrational motions ( . ÷ mev) in different proteins by performing inelastic neutron scattering and low temperature ( . ÷ k) specific heat measurements. for the first time for biological systems, the well-known boson peak found in neutron scattering spectra at ∼ mev is put in relationship with a clear anomaly of the measured specific heat located at around k. this departure from the debye-like behavior further expands the analogy of proteins with glassy systems. quite interestingly, in the very low sub-mev energy range and below ∼ k, we observe an additional anomalous behaviour, which could be ascribed to the existence of twolevel-systems states. increasing the hydration degree, the low energy vibrational region is drastically altered, revealing that the addition or removal of the hydrogen bond network around the protein deeply modifies the interatomic forces, affecting the character of the vibrational modes. a. cupane , m. levantino , m. cammarata , g. schirò department of physical and astronomical sciences, university of palermo, via archirafi , i palermo, italy, european synchrotron radiation facility, grenoble, france our efforts in recent years have been to study in great detail the way hemoglobin works in confined geometries [ ] [ ] [ ] . to this aim we have contributed to the development of a new experimental technique, time-resolved wide-angle x-ray scattering (tr-waxs), that enables one to watch proteins at work in solution [ , ] . a very recent and challenging application of this technique is the study of hemoglobin functioning inside intact red blood cells (rbc). our preliminary results show that, by using laser pulses of about ns, it is possible to photolyse hemoglobin inside rbcs obtaining about % - % photolysis. good structural signals from hemoglobin are obtained, with limited radiation damage to the cells: this opens the possibility of studying the conformational transitions of hemoglobin in its "natural" environment. preliminary results concerning the timing of the r->t quaternary transition inside the rbc and comparison with the behaviour in dilute solution will be discussed. physical review letters and physical review e invite submission of your best work in biological physics. submissions must present significant new results in physics; topics may range from the microscopic to the macroscopic. we will provide information on the different types of articles published in the journals, on authors and referees, and on the review process. for publication in physical review letters, the work should be important and of broad interest. for rapid communications in physical review e, the work should be important for the field. biological physics papers published in physical review are indexed in medline. in an effort to bring important research to the attention of a wider community, the physical review journals have recently begun highlighting important work with viewpoints in the online publication physics. all physical review journals may be accessed through the website http://publish.aps.org/. modeling of fibrin gels using confocal microscopy, light scattering and turbidimetry f. ferri , d. magatti , b. cardinali , a. profumo , m. rocco dipartimento di fisica e matematica, università dell'insubria, como, italy, istituto nazionale per la ricerca sul cancro (ist), genova, italy fibrin gels are biological networks playing a fundamental role in blood coagulation. confocal microscopy of fibrin gels shows a collection of straight fibers, not uniformly distributed in space, connected together at low-order ( ) ( ) branching points. although each fiber is quite straight (mass fractal dimension d m = ), for the overall system d m > . based on the confocal images, we generated threedimensional ( d) synthetic gels made of cylindrical sticks of diameter d, joined together at randomly distributed nodal points. the resulting d network is no more random but ordered on length scales of the order the average fiber length, and exhibits a fractal morphology with d m ∼ . - . . the gel structure is analyzed by means of its d correlation function g d (r)=<n(x) n(x+r)> x , where n(x) is the gel local density. since the gel is isotropic, g d depends on the modulus r=|r| and can be obtained by averaging d correlation functions evaluated at different heights of the gel volume. from this analysis we recover the fiber diameter d (fwhm of g d ), the fractal dimension d m (power-law decay of g d ) and the gel mesh-size ξ (minimum in g d ). furthermore, the d-fourier transform of g d (r) gives the gel power spectrum i(q), which compares quite well with elastic light and multi-wavelength turbidimetry data taken on real gels. a model coupling vibrational and rotational motion for the dna molecule e. drigo filho, j. r. ruggiero, r. a. d. s. silva unesp -sao paulo state university, brazil a largely used mechanical model for vibrational motion of dna has been proposed by peyrard and bishop (pb). in this model, dna is represented by a pair of harmonic chains coupled by a nonlinear potential. the most frequently used nonlinear potential for this purpose is the morse potential. some extensions of the original model are proposed considering, for example, the helicoidal structure of dna. an important objective for this kind of model is to understand the phenomenon of thermal denaturation and, through this, get some knowledge about other processes such as the genetic transcription and drug intercalation. it is possible to obtain from this type of model interesting properties, such as the average stretching between base pairs as a function of the temperature using the transfer integral operator. dynamical properties of this model were also explored and several phenomenological quantities are studied, such as energy localization. in this work, we extend the original pb model by introducing rotational motions for the nucleotides. in this way, both the vibrational and rotational motion for each nucleotide are considered. the stretch of the base pairs is given by the morse potential in the same way as in the original pb model. however, the coupling between the two kinds of motion, rotation and vibration, is obtained through a nonlinear combination of them in the morse potential. a. dobovišek , m. brumen , p.Županović , d. juretić university of maribor, faculty of natural sciences and mathematics, maribor, slovenia, jožef stefan institute, ljubljana, slovenia, faculty of science, university of split, split, croatia mepp is a physical principle, widely used for quantitative explanation of non equilibrium phenomena in physics, chemistry and biology [ ] [ ] [ ] . here, we applied mepp to study two and three state reversible michaelis-menten kinetic schemes of enzymatic reactions. by applying constraints as a diffusional limit for kinetic constants, constant free energy differences between enzymatic states and constant thermodynamic force, we calculated shannon information entropy and entropy production of the entire reaction system as a function of forward rate constants. we found maxima in the net steady-state metabolic flux, total entropy production and shannon entropy for equal values of forward rate constants. moreover, for these values an analytical expression derived gives a relation between substrate and product concentrations at which enzymes operate in the optimal way. in conclusion, we demonstrated that mepp is an appropriate selection principle for evolutionary optimization of enzymes. attractive interaction between like-charged lipid surfaces mediated by spherical nanoparticles with spatially distributed charge is theoretically described by using functional density theory and mc simulations. the spatial distribution of charge within a single nanoparticle is considered by two effective charges at a finite distance. the minimization of the free energy is performed to obtain the equilibrium configuration of the system. both, the rigorous solution of the variational problem and the mc simulations show that orientational ordering of nanoparticles subject to the gradient of the electric field gives rise to an attractive interaction between charged lipid surfaces for high enough charge densities of the interacting surfaces and large enough separations between charges within the nanoparticle. the attraction is explained by orientational ordering of dimeric charges in the electric field which lowers free energy. viral genomes encode for a series of membrane proteins which are embedded or attached to the lipid membrane of the host, or penetrate them during the very first step of viral invasion. we are focussing especially to those of the former which are known to assemble and from channels or pores for small ions or substrates. with these channel forming proteins the virus manipulates the host cell interior for the benefit of its replication. strategies are developed to answer the question about the assembly process of these proteins based on the 'two stage model' and the substrate flux. only few experimental data are available to answer these questions, consequently we stress computational methods to derive the adequate answers. the methods applied are ab inito molecular dynamics (md) simulations based on density functional theory (dft) and conventional md simulations. potential routes for assembly of the three transmembrane domains of a protein from sars-cov will be outlined and compared with computational data from other viral membrane proteins. with a novel pore lining motif suggested for a, the dynamics of ions at selected positions within the putative pore will be assessed. probing the molecular mechanism of antibiotics diffusion through the ompf channel e. hajjar , a. kumar , m. winterhalter , p. ruggerone , m. ceccarelli department of physics, university of cagliari, italy, jacobs university, bremen, germany in gram-negative bacteria, the outer membrane porin-f (ompf) is the preferred entry point of antibiotics. bacteria can resist to antibiotics by altering the expression and the structures of ompf. a key feature in the structure of ompf is the presence of a constriction zone, characterized by both spatial and electrostatics restrictions. to study the process of antibiotics translocation at a molecular scale, we performed molecular dynamic simulations accelerated with the metadynamics algorithm. we studied the diffusion of antibiotics with different structural and chemical properties through ompf wild-type and variants. the free energy surface suggests faster translocation for the cephalosporins compared to the penicillins antibiotics, and also for ompf mutants compared to the wild type. further, the conservation of favored orientation and affinity sites of antibiotics inside the ompf channel reveal which specific interactions govern translocation. the calculated energy barriers and rate determining interactions for translocations compared well with the electrophysiology measurements and liposome swelling assays from our collaborators. this study demonstrates how theory and experiments can be combined to reveal the structural determinants and mechanism of ompf permeation. this will benefit to the design of antibiotics with improved transport properties. m. g. gauthier, j. bechhoefer dept. of physics, simon fraser univ., burnaby, bc, canada dna replication requires two distinct processes: the initiation of pre-licensed replication origins and the propagation of replication forks away from the fired origins. experiments indicate that these origins are triggered over the whole genome at a rate i(t) (the number of initiations per unreplicated length per time) that increases throughout most of the synthesis (s) phase, before rapidly decreasing to zero at the end of the replication process. we propose a simple model for the control of dna replication in which the rate of initiation of replication origins is controlled by the interaction with a population of ratelimiting proteins. we find the time set by reaction-diffusion kinetics for such proteins to find, bind to, and trigger a potential origin. the replication itself is modeled using a formalism resembling that used to study the kinetics of first-order phase transitions. analyzing data from xenopus frog embryos, we find that the initiation rate is reaction limited until nearly the end of replication, when it becomes diffusion limited. initiation of origins and hence i(t) is suppressed when the diffusionlimited search time dominates. we find that, in order to fit the experimental data, the interaction between dna and the rate-limiting protein must be subdiffusive. we also find that using a constant nuclear import of the limiting proteins leads to a more accurate description of the experimental data. the microsoft research -university of trento centre for computational and sisytems biology, trento, italy we propose a new method for inferring the structure of a biochemical network from the time-series of the reactant species. it consists of two parts: the first is the quantification of the correlation between the time-series profiles. correlation in time series data can be used to reveal dependencies between variables and to infer the graph of connectivity among species. the second part consists in the elimination from the connectivity graph of the relationships that have a non-null correlation coefficient, but that are not biochemically plausible. the cutting of false correlations from the graph is performed through the estimation of the parameters ("calibration") of the network. to calibrate the network for detecting null dynamics correlations, we developed the software tool kinfer (knowledge inference). based on a new probabilistic model of the variations in reaction concentrations, kinfer infers the values of the kinetic rate constants, their confidence regions and the level of noise in the input data by maximizing the likelihood to obtain the observed variations given the network model. the a priori knowledge required as input is minimal, as it consists only in the time-series of reactant concentrations. the minimal a priori knowledge and the probabilistic formulation of the calibration method make the accuracy of the predictions strong against experimental, biological and stochastic noise, and allows to use it to cut the null-dynamics edges of the connectivity graph. a. kuzmanic, b. zagrovic mediterranean institute for life sciences, split, croatia root-mean-square deviation (rmsd) is a measure used to give information on the global structure of macromolecules. for example, pairwise rmsd (prmsd) is used to assess similarity of the lowest energy nmr structures or for clustering large ensembles of structures. on the other hand, to obtain information on the local structure of a macromolecule and its dynamics, root-mean-square fluctuation (rmsf) is often used. rmsf can be calculated from md simulations, but also from experimental x-ray b-factors. since prmsd and rmsf report on different features, it is interesting to ask what the relationship between them is. first, we provide a mathematical derivation showing that, given a set of conservative assumptions, the <prmsd> rms is directly related to the <rmsf> rms and, consequently, experimental b-factors. second, we demonstrate this on structures taken from distributed-computing md simulations of the native and unfolded state of villin headpiece. both our analytical and computational results suggest a strong correlation: <rmsf> rms = [(s- )/ s] / <prmsd> rms , where s is the number of compared structures. furthermore, if <prmsd> rms is defined as a generalized radius of gyration in the space of d structures and using rmsd as a measure of distance, the following identity holds: <rmsf> rms = <prmsd> rms. our results provide a basis for determining the level of structural diversity of molecular ensembles, as captured by <prmsd> rms , directly from experiment. charge migration along dna helices may be biologically important because extended electronic states could play a role in the processes of sensing of dna damage and/or dna repair via long-range charge transfer. we measured conductivity and other physical characteristics of several models of natural and diversely damaged molecules of dna. dna polymers were mimicked by various sequences of nucleotide-long double helices with fully watson-crick (wc) paired bases, with several central bases mismatched, and also with chemical modifications that included removal of bases from a few central nucleotides (abasic dna), and neutralization of phosphate charges by their derivatization. the model dnas were investigated by scanning tunneling microscopy, time-resolved thz spectroscopy, raman spectroscopy, circular dichroism, and modeled by molecular dynamics simulations. dna has the highest conductivity in its biologically most relevant double helical form with wc base pairs and negatively charged phosphates equilibrated with counterions. mismatches and all chemical modifications always lower the conductivity. the mechanism of charge transfer is consistent with electron or hole hopping between parallel stacked bases. these observations and data showing that the natural dna has also the most regular double helical form suggest that the continuous base stacking is critical for charge transfer. to control the passage across the bacterial cell wall nature created a large number of "nano"-channels which may act as selective gates for water soluble molecules. here we focus on porins from e. coli which control permeation through interactions with the channel surface. comparing single channel temperature dependent conductance measurements with all atom modeling allow conclusions on the mode of permeation. for example, surprisingly modeling ompf-conductance revealed not only a good agreement with the experiment over a broad range of temperature but also the selectivity for ions. the primary task of porins is to provide facilitated diffusion. we investigated facilitated diffusion of maltooligosaccharide or antibiotics. time resolved conductance measurements allows conclusion on the flux and molecular modeling identifies the limiting interactions with the surface, reveal potential barriers and pathways. exploiting the selectivity of natural or bioengineered channels has promising applications for detecting molecules, characterizing molecular interaction, sequencing dna, protein folding etc. traditionally, studies of diffusion-controlled reaction of biological macromolecules have been made in diluted solutions. however, the high concentration of macromolecules in intracellular environments results into non-specific interactions (macromolecular crowding), which have a great importance on the kinetics and thermodynamics of possible reactions that occur in these systems. in the literature there are studies concerning monte carlo (mc) simulations, giving results that are satisfactory agreement with experimental data, showing, for example, that the protein diffusion in cell cytoplasm is reduced considerably. in addition, there are mc studies about enzymatic reaction, which predict a temporal dependency of the velocity constant in macromolecular crowding. in this work, we try to compare the predicted behavior by mc simulation with the results obtained from the study of the diffusion and reaction of a model protein (alpha-chymotrypsin) using spectroscopic techniques in highly confined media in order to study experimentally the temporal dependence of its diffusion and reaction coefficients. a. paciaroni , a. orecchini , c. petrillo , a. de francesco , f. sacchetti university of perugia, italy, cnr-infm, genova, italy the single-particle and collective dynamical properties of protein hydration water have been studied by neutron scattering experiments in a wide temperature and hydration range. an unprecedented accuracy has been achieved thanks to the availability of a large amount of fully deuterated protein powder and the use of the high-flux spectrometers in and brisp. the protein under investigation was the maltose binding protein (mbp), which is a well-known and widely studied model of biosensor systems. we found that the low-temperature single particle dynamics of mbp hydration water shows clear features that can be traced back to amorphous systems. more in detail, its vibrational density of states is simply described as the superposition of the contributions of low-density and high-density amorphous ice. the quasielastic signal, which appears at the higher temperatures, can be excellently described with a fractional power law which may put in relationship with the peculiarities of fractal systems. quite strikingly, there is a strong similarity, on both the qualitative and quantitative point of view, with the behaviour of hydrated proteins. the collective dynamics of protein hydration water is characterised by the presence of two modes, whose dispersion curves are reminiscent of those of bulk water. however, the relevant damping factors suggest a strong similarity with glassy systems. m. malferrari , f. francia , s. sacquin-mora , g. venturoli università di bologna, bologna, italy, cnrs upr , paris, france the coupling between electron transfer and protein dynamics has been compared in reaction centers (rc) from the wild type (wt) and the carotenoid-less mutant r , by combining brownian dynamics simulations and the kinetic analysis of charge recombination. upon incorporation of the rc into a progressively dehydrated trehalose matrix the electron transfer between the primary photoreduced quinone and the photoxidized donor accelerates progressively and becomes broadly distributed. this behaviour reflects the hindrance of protein relaxation following charge separation and the inhibition of interconversion between conformational substates. in extensively dehydrated matrices the recombination kinetics is two-times faster and three-times more distributed in the wt rc, indicating a larger inhibition of the internal protein dynamics. in line with this findings brownian dynamics simulations reveal a larger rigidity of the carotenoid-containing structure, in which a cluster of residues close to the quinone acceptors is stiffened as compared to the r rc. the in silico and experimental results indicate that the introduction of an internal void in the rc structure has long-range effects on the protein dynamics and that the coupling between the glassy matrix and the rc interior depends markedly on the local mechanical properties of the protein. n. maghelli , v. krstic , n. pavin , f. julicher , i. tolic-norrelykke mpi-cbg, dresden, germany, mpi-pks, dresden, germany in the fission yeast schizosaccharomyces pombe, the nucleus is positioned at the cell center. since the nucleus determines the cell division site, keeping the nucleus at the center is crucial for ensuring symmetrical cell division ( ) . microtubules push against the cell ends and exert force on the nucleus ( ), but how the cell regulates these forces in order to center the nucleus remains unknown. here we tackle this problem by using a combination of live cell imaging, cell manipulations by optical tweezers, and a theoretical model. we show that microtubule pushing forces can center the nucleus because of a larger number of contacts between the microtubules and the proximal cell end than the distal one. moreover, kinesin- motors (klp / ) increase the rate of microtubule catastrophe (transition from growth to shrinkage) in a microtubule length-and contact-dependent manner. thus, the motor behavior results in a longer contact between a microtubule and the proximal than the distal cell end. taken together, our experimental and theoretical results provide a novel centering mechanism, where kinesin- motors increase the efficiency of nuclear centering. electropermeabilization is a commonly used physical method which can induce a transient permeabilization of the cell membrane allowing the entry of therapeutic molecules into the cell and is thus of great interest in the fields of cancer treatment and gene therapy [ ] . however, very little is known about the mechanisms occurring at molecular level. there is clearly some microscopic reorganization of the membrane which is responsible for this change in its transverse transport properties. rather than studying the change of these transport properties, we adopt a simple strategy based on the use of giant unilamellar vesicles and videomicroscopy, as described below. we apply a series of permeabilizing electric pulses to the liposomes, and we observe a size decrease down to a critical radius beyond which their size no longer changes. this decrease in size points to the fact that during the physical processes leading to electropermeabilization, lipids are lost from the vesicles. our results published in [ ] suggest different possible modes for lipid loss, which can be small vesicles, pores, or tubules formation. imaging of brain activation using core techniques as fmri, pet and synchrotron radiation in parallel c. poitry-yamate, g. margaritondo, r. gruetter ecole polytechnique fédérale de lausanne, switzerland functional magnetic resonance imaging (fmri), magnetic resonance spectroscopy (mrs), positron emission tomography (pet) and synchrotron x-ray emission imaging form a highly complementary set of imaging core technologies for studying brain function and energy metabolism. owing to differences in the information each conveys and the temporal and spatial scales on which they measure labeled substances, a single working hypothesis can be tested from different angles to provide a cross-validated, consistent and coherent explanation. the cibm is a unique research facility in europe for advancing our understanding of biomedical processes in health and disease. the housing of a -tesla human magnet, . and . -tesla animal magnets, an animal pet imaging facility and fully-equipped neurochemistry and rf laboratories has enabled us to develop and perform well-targeted experiments around one research theme. in parallel, a longterm collaborative project using synchrotron x-ray transmission and emission imaging at elettra enables us to combine these core technologies towards understanding brain function in vitro and in vivo, from tissue to cells. a brief presentation of these methods will be followed by their application in studying the visual system from man to mouse. p. picone , r. carrotta , d. giacomazza , m. di carlo dip. chimica e tecnologie farmaceutiche, università di palermo, italia, ibf -cnr, palermo, italia, ibim -cnr, palermo, italia diabetes and alzheimer's disease are connected in a way that still is not completely known. diabetes has been implicated as a risk factor for developing alzheimer's disease. some diabetes drugs appear to decrease the cognitive decline associated with alzheimer's disease. it has been recently demonstrated that extracellular injection of insulin is able to protect neurons against a-beta amyloid cell death. one of the proposed theories to explain such an effect is that the hematic glucose levels affect the metabolism of the hippocampus, a part of the brain (associated with memory, emotion and motor skills), which is strongly damaged in alzheimer's patients. the aim of this study is to investigate the effect of insulin on the a-beta induced degeneration and oxidative stress on the neuroblastoma lan cell line. in particular, the present study looks into the role of insulin in the inhibition of abeta specific degenerative apoptotic pathways. preliminary results indicate that insulin dissolved in culture medium in its hexameric form (as tested by absolute scale light scattering) is able to reduce neurodegeneration induced by a-beta amyloid in a dose dependent manner. the link between diabetes and alzheimer's disease may provide new targets for future alzheimer's treatments. moreover, due to the increased incidence of diabetes in western countries, a deeper understanding of such a link is relevant in order to control the escalation in the number of people dealing with dementia. a. pelizzola dipartimento di fisica, politecnico di torino, torino, italy many features of protein folding have been shown to be described by an ising-like model (one-dimensional, with longrange, multispin interactions) whose equilibrium thermodynamics is exactly solvable [ ] [ ] [ ] . we have generalized such a model to the problem of mechanical unfolding. the equilibrium thermodynamics is still exactly solvable, and the characteristic kinetic responses found in force ramp and force clamp experiments are well reproduced [ , ] . unfolding and refolding pathways and intermediates can also be studied, again with good agreement with experiments [ ]. applications to various proteins and rna fragments will be discussed. [ the key role of water in living systems has been widely studied in literature, along with its anomalies, consequence of the extensive three-dimensional hydrogen bonding of water molecules. moreover protein-water interactions take place at protein surface where cell water has been recognized to behave differently from bulky water. the two-states theory of water assumes that water is a mixture of microdomains of different structure and density, the low-density water (ldw) and the high-density water (hdw) domains, and ions partition selectively into ldw or hdw domains. the idea developed in this work was to explore the ordered water structure by measuring delayed luminescence (dl) from salt aqueous solutions in which water structuring is anticipated. it appeared that dl signal from salt solutions is significantly relevant when prevalence of ldw domains is foreseen, with a decay time probability distribution function characterized by a broad maximum in the microsecond range. the obtained results support the ability of dl to reveal the different properties of ldw and hdw domains induced by salt molecules. moreover, the results reveal the existence of clusters, whose characteristics strongly depend on the specific ion effects, of surprisingly long lifetimes not observed till now. this could give new insight into biological water properties. self assembly of patchy particles and dnafunctionalized dendrimers f. sciortino dipartimento di fisica e infm-cnr-soft università 'la sapienza', roma, italy i will report numerical results on the phase behavior of very simple models of patchy particles with the aim of understanding the interplay between phase separation and selfassembly and how the fraction of surface allowing for attractive interactions controls the collective behavior of the system. the case of janus particles, particles characterized by a surface divided evenly into two areas of different chemical composition, will be discussed. i will also discuss the self-assembly of a simple model for four single strands of dna tethered to a central core, and show that the model exhibits a rich phase diagram that includes at least four thermodynamically distinct amorphous phases (polyamorphism) in a one-component system. the dense phases consist of a hierarchy of interpenetrating networks, reminiscent of a woven cloth. peptide dimer motifs in the phospholipid environment -structure, interaction and molecular design p. e. schneggenburger , a. beerlink , t. salditt , u. diederichsen iobc, universität göttingen, germany., irp, universität göttingen, germany. based on recently reported homodimeric peptide pores with a d,l-alternating configuration a novel double helical hairpin-motif of a membrane active gramicidin a analog was designed. [ , ] the cd spectroscopic analyses of the peptide-lipid complexes revealed the structural preservation and elucidated the importance of a zwitterionic interaction of the peptide termini. [ ] the peptide design was enhanced regarding the versatile functionalization with analytical probes as well as molecular recognition moieties like peptide nucleic acids (pnas) to observe the effects of aggregation and specific organization within model lipid membranes even at high peptide-to-lipid ratios. [ ] x-ray reflectivity on lipid bilayer stacks in combination with heavy atom labeling and spectroscopic studies of vesicle systems provides information about the peptide structure and interaction in the native fluid state of the membrane system. [ , ] for this, the fmoc-diiodo-allylglycine building block was created to serve as a novel iodine label pinpointing at a certain position with respect to the membrane normal. we study thermal undulations of giant unilamellar vesicles (guvs) of lipids by flickering spectroscopy. getting values for the mechanical parameters of lipid bilayers requires the experimental fluctuation spectra to be scrutinized in view of the classical helfrich's theory. pure bending modes are revealed unable in predicting the large fluctuations systematically found at high wavevectors. hybrid curvature-dilational modes have been invoked as a more efficient mode of motion in producing high curvatures. a bimodal spectrum of the thermal undulations has been theoretically developed for the shell-like topology. from this new description, two important consequences emerge a priori, the dependence of the fluctuation dynamics on either vesicle size and on bending/compression parameters. for popc and dopc vesicles containing cholesterol the experimental fluctuation spectra are well described by the new spectrum. reconciliation between experiments and theory is achieved when this bimodal spectrum is considered. the new theory opens enormous possibilities for better exploring membrane mechanics in guv models. under normal conditions, platelets circulate in the vascular system having very low interaction with each other and with other cells. the platelets become activated when the biological system is disturbed, for instance by vascular damage in which blood gets in contact with collagen. upon activation, different types of receptors/molecules are exposed on the cell membrane to support adhesion, spreading and aggregation of the platelets onto the damaged vessel. the measurement of altered platelet function is particularly important in cardiovascular diseases such as thrombosis. we are investigating biosensor technologies for the detection of functional properties of platelets. an important study is the specific and non-specific stimulation of platelets in a biosensor cartridge. we will present experimental results on biosensor platelet activation using the platelet-specific membrane markers p-selectin and gp b. multi-joint analysis of locomotion in the first neonatal rats flown in space d. sulica, j. vinersan "carol davila" university of medicine and pharmacy, bucharest, romania the first mammalian neonatal animals in space were the rats flown on the space shuttle endeavor during a -day mission, sts- . the development of locomotion in weightlessness was evaluated using two litters of neonatal rats, launched at postnatal days and . age-and cage-matched animals were used as ground controls. free walking was videotaped from the landing day. although preliminary analysis of walking showed differences in both hindlimb and forelimb joint angles and a hyperextension of the hindlimbs was apparent, the numerical values reached the significance level only for the ankle angle measured at specific moments of the step cycle: foot contact, maximum loading with weight, foot lift and maximum flexion during swing. we report here on the behavior of all the joints during the whole step cycle, by computing the integral of these angles over the step cycle. the results were affected by the differences in the walking speed (the young animals walked faster than the controls), so we scaled the integrals by the step cycle duration. we found that, besides the ankle, the knee was also more extended throughout the whole step cycle in both groups of animals. moreover, all the joints (including the toe and the hip) were affected in the same way (hyperextended), since the differences were still significant when we added together these angles. the animals recovered slowly, with significant differences remaining after days of readaptation. the effect of dextran concentration on red blood cell deposit formation j. strzelecka, b. grzegorzewski department of biophysics, collegium medicum in bydgoszcz, nicolaus copernicus university - bydgoszcz, poland red blood cell (rbc) deposit formation was examined by means of an optical method. blood was obtained from healthy donors and measurements were performed at initial hematocrit %. the intensity of scattered light was measured during sedimentation of rbcs suspended in saline -dextran solutions at different polymer concentrations ( - g/dl). the changes in the intensity of the scattered light manifest rbc aggregate formation, their sedimentation and the process of deposit formation. the deposit formation curve was determined. it is shown that the concentration of dextran affects the deposit formation. an empirical model has been used to describe the experimental data. the parameters of the deposit formation curve as a function of dextran concentration are analyzed. water is essential to life and a major scientific interest lies in a detailed understanding of how it interacts with biological macromolecules in cells. we studied water dynamics in whole cells with neutron scattering [ , , ] . the cellular environment is extremely crowded with macromolecules and water molecules are permanently in close contact to biological interfaces. we measured water dynamics in e. coli and human red blood cells with neutron scattering [ , ] . the data revealed two populations of water in the cells: a major fraction which has dynamical properties similar to those of bulk water (relaxation times ∼ps) and a minor fraction in the order of ∼ % which is interpreted as bound hydration water with significantly slower dynamics (relaxation times > ps). in this contribution we report on investigation of model membrane dynamics by means of quasi elastic and inelastic incoherent neutron scattering and on the effect of membrane inserted pore forming peptide gramicidin. model membrane are realized by highly oriented, hydrated phospholipid bilayer stacks of dmpc ( , -dimyristoyl-snglycero- -phoshatidylcholine) hydrated with d o in excess of solvent condition. the bilayer were supported on mica substrates and prepared at different concentrations of gramicidin, a -residue oligopeptide showing antimicrobial activity by forming pores on the membrane surfaces which allow water and small ions to permeate across the membrane. incoherent qens and ins spectra, measured on in and in spectrometer at ill, allows to obtaining information on the mean dynamics of the hydrogen atoms in the system. moreover, by proper orientations of the membrane plane respect to the scattering wave vector q, we were able to derive information on in plane and out of plane motions of the phospholipids. the using of media products for the creating attracted bioenergetic brain rhythmus advertisement v. i. vlastopulo, v. g. nikolajev str. gen. petrova , app. , odessa, ukraine the technical efficiency of bioenergetical influence of advertisement is present with assistance of consciousness of memory at revision and hearing of advertisement on tv, radio stations, mobile phones, at supermarkets and other places. in a basis of useful model it is put a task to improve the method of creating the attracted advertisement, in which the creation of bioenergetical influence by the oscillation of not less electromagnetic fields or video-images is introduced with their creating as the base on the spatial or flat structure formative macro matrix or matrixes with repeatable structure with the brain α-rhythm frequency of extreme attention and δ-rhythm frequency of meditation. the point of the patent on the device is in the bioenergetical influence increases in addition to the information influence by advertisement of pictures and audio oscillations the bioenergetical influence increases at the consciousness contribution of human memory at the moment of watching or hearing of television, radio stations, working in the internet, music in the supermarkets, banks, clubs, metro and other places. cell adhesion and motility are processes involved in fundamental biological phenomena. they imply multimolecular scaffolds as anchorage points and actin cytoskeleton filaments to build internal stress and eventually crawl onto the substrate. these processes, very dynamic by nature are out of equilibrium. we study cell adhesion on micro-patterned substrates where the introduction of a finite distance between the possible anchorage points of the cell modifies drastically the organization of the cytoskeleton and the anchorage point's distribution. because statistical quantification shows that some shapes are more likely than other, we believe they represent particular organizations of the system which should minimize the energy dissipation. we checked this hypothesis by using the cellular potts model. shapes obtained by simulation are in excellent qualitative agreement with experimental shapes. they depend on phenomenological parameters such as interaction between cells and the extra cellular matrix, a line tension and an elastic modulus. the aim of this work is to link model parameters to physico-chemical properties of cells and to establish phenomenological relations between relevant biochemical regulators controlling the cytoskeleton organization. c. canale, d. ferrera, f. benfenati, l. gasparini the italian institute of technology, genova, italy alzheimer disease (ad) is characterized by cerebral extracellular deposits of β-amyloid (aβ) fibrils. aβ aggregation is a multi-step process involving the formation of various conformational species including soluble intermediate species (i.e. aβ oligomers), protofibrils and fibrils. such aggregates may have various effects on neuronal and glial function and differentially contribute to ad neurodegeneration. aim of this study was to investigate the structural properties of distinct aβ aggregated species and dissect out their effects on neuronal viability. recombinant aβ and aβ peptides were aggregated in vitro in conditions differing by ionic strength, temperature and ph and were analyzed by gel electrophoresis, thioflavin t binding assay and atomic force microscopy (afm). afm analysis was performed using both hydrophilic and hydrophobic substrates, to analyze the full spectrum of structural species. stable low molecular weight oligomers were obtained when aβ was incubated at • c for days in low salt concentration buffer. doughnut-shaped conformational species were detected by afm alongside globular aggregates ( . - . nm height range). acidic ph promoted aggregation of aβ into thioflavin-positive fibrils and protofibrils. protofibrils appeared as beaded chains having a mean height of . ± . nm. effects of aβ on viability of mouse hypppocampal neurons were assessed and correlated with their conformational features. a. bellova , e. bystrenova , m. koneracka , p. kopcansky , f. valle , j. bagelova , f. biscarini , z. gazova institute of experimental physics, slovak academy of sciences, kosice, slovakia, ismn cnr, bologna, italy peptide amyloid aggregation is a hallmark of amyloid diseases including azheimer's disease or type ii diabetes. recent works have addressed the potential of nanoparticles to affect amyloid aggregation. the experimental data are very controversial suggesting that particle characteristics markedly influence the final effect of nanoparticles on the amyloid aggregation (initiation, acceleration or inhibition of amyloid aggregation). we investigate the ability of electrostatically stabilized magnetic nanoparticles of fe o to affect the amyloid aggregation of lysozyme, as a prototype amyloidogenic protein. we have used a combination of spectroscopic (tht fluorescence) and local microscopic techniques (afm). we found, that the ability of magnetic nanoparticles to inhibit formation of amyloid aggregates or destroy pre-formed amyloids exhibit concentrationdependence. the values of inhibition ic and depolymerization dc were determined suggesting that nanoparticles interfere with lysozyme aggregation at stoichiometric concentrations. the observed features make magnetic nanoparticles of potential interest as a therapeutical agent against amyloid diseases. (this work was supported by project of esf and by slovak academy of sciences in frame of cex nanofluid, vega grants , and and eu-strp biodot.). a. bellova , l. balogova , b. chelli , e. bystrenova , f. valle , j. imrich , p. kristian , l. drajna , j. bagelova , f. biscarini , z. gazova institute of experimental physics, sas, kosice, slovakia, faculty of sciences, p. j. safarik university, kosice, slovakia, ismn cnr, bologna, italy numerous diseases have been linked to a common pathogenic process called amyloidosis, whereby proteins or peptide clump together to form amyloid aggregates in the body. an attractive strategy to develop therapies for these diseases seems to be reduction of polypeptide aggregation. we have tested several acridine derivatives characterized by various glycosyl groups for their potential to affect the lysozyme amyloid aggregation in vitro. the ability of glycosyl acridines to interfere with lysozyme aggregation was investigated by tht assay. we found that structure of acridine side chain is factor affecting their anti-aggregation activity significantly. for the most effective compounds the values of ic and dc were obtained. the reduction of protein aggregation was confirmed by afm. to investigate influence of the glycosyl acridines on the cell processes we examined effect of compounds on cell viability. we performed glycosyl acridines characterization by high anti-aggregation activity and low toxicity suggesting their possible application for therapeutical purpose. (this work was supported by project of esf and by slovak academy of sciences in frame of cex nanofluid and vega grants , and and eu-strp biodot.). a. j. beevers, a. m. dixon department of chemistry, university of warwick, uk due to the immense medical importance of proteins which span the membrane of cells, detailed molecular structural information of these systems is essential. practical difficulties in employing high-resolution structural elucidation techniques have resulted in a relative paucity of fully resolved membrane protein structures. therefore a variety of lower-resolution techniques are used to determine structural information of the transmembrane (tm) domains of proteins. one example of such a membrane protein is erbb- , a receptor tyrosine kinase responsible for triggering cell division and which is prone to a mutation in its transmembrane domain resulting in permanent activation and oncogenic effects. we have predicted an interface for the mutated tm domain dimer using site-specific infrared spectroscopy containing a repeating sequence of ile, val and leu . applying the in vivo toxcat assay to the tm domain sequence and to specific mutants of it, confirms this proposed interface whilst another proposed interface is discounted. current studies are focussing on the effect of the tm mutation to the activation of the erbb- receptor and to any possible change in this interface. bacteriophages are complex molecular assemblies which multiplication relies on bacteria infection. the process starts with the binding of the phage on its specific host receptor and the injection of its genome into the host cytoplasm. our work aims to determine the physical mechanisms and forces driving the dna transfer from the phage capsid. the in vitro dna ejection has been analyzed by using light scattering and gel electrophoresis measurements for three phages (t , spp and lambda) belonging to the same family (syphoviridae). our results reveal two forces contributing to drive the dna transfer: the first one is originated from the pressurization due to the strong confinement of dna into the capsid; the second one comes from a pulling mechanism originated by the presence of condensed dna outside the capsid. these two contributions were characterized in in vitro conditions but they likely play a role in the in vivo transfer. the ejection kinetics was also analysed and the characteristic time of the mechanism was studied as a function of the temperature. it appears to follow an arrhenius law, allowing the determination of the activation energy that governs the transfer. the energy values are close for the different phages, suggesting that the mechanism regulating the ejection is common for a given phage family. below these general features, our studies also reveal differences between the three phages. the effect of &beta-amyloid peptide on polymer cushioned membranes s. dante , r. steitz , t. hauss , c. canale , n. a. dencher istituto italiano di tecnologia, genova, italy, bensc, helmholtz center berlin, germany, tu-darmstadt germany beta-amyloid (aβ) is a peptide implicated in the neurodegenerative process characteristic of the alzheimer's disease (ad). to clarify its mechanism of action it is crucial to elucidate the interaction of aβ with the neural membrane. in previous work we demonstrated the capability of aβ to penetrate and perturb stacked lipid bilayers. in this study we considered polymer cushioned lipid bilayers as a model for neural membranes. the polymer cushion is aimed to preserve the membrane natural fluidity; it is obtained depositing charged polyelectrolites layer-by-layer; the lipid membrane is built on the top of the polymer film by fusion of unilamellar vesicles. the floating membranes were kept always in contact to the subphase. the kinetics of adsorption of the lipid double layer at the polymer/water interface was monitored by neutron reflectivity; different experimental conditions to obtain the best surface coverage were exploited. after administration of aβ to the subphase the lipid membrane still adhered to the polymer cushion, but its structure was modified by the interaction with aβ. neutron reflectivity showed a change of the scattering density profile in the direction perpendicular to the membrane plane, suggesting a penetration of aβ inside the double layer. a change in the surface morphology was detected by afm imaging; afm film-rupture experiments showed that aβ weakens the lipid packing. x. cheng , r. pacheco-gomez , a. rodger , h. matthew , d. i. roper university of warwick, u.k., university of birmingham, u.k. ftsz, the ancestral homolog of eukaryotic tubulins, is a gt-pase that assembles into a cytokinetic ring structure (z ring) essential for cell division in prokaryotic cells. the z ring also recruits other proteins (e.g. zapa, ygfe, zipa) to the division site, where they participate in formation of the septum that separates the two daughter cells. we have studied ftsz polymerization and its dynamic behaviour in real time by right angle light scattering. similar to tubulin, ftsz polymerizes into dynamic protofilaments in the presence of gtp; polymer assembly is accompanied by gtp hydrolysis. the kinetics of inorganic phosphate (p i ) released from the gtp hydrolysis have been studied as well, employing a fast and sensitive colourimetric assay. at ph . , approximate % of the p i was released into the media within minutes of gtp addition. the effects of gtp, ph, k + , and mg + were studied in both cases, and the results were used to build up a model for the mechanism of fibre assembly and disassembly. ygfe, a ftsz accessory protein, is identified as a functional zapa orthologue. finally, we have studied the ygfe bundling to ftsz polymers. it strongly promotes ftsz bundling and is an inhibitor of the gtpase activity. many genomes of viruses encode small membrane spanning proteins which are proposed to modify membrane permeability for ions and small molecules. these channel or pore forming proteins are getting into the focus for antiviral therapy since they are essential for some of the viruses. one of the general themes of the mechanism of function of the proteins is to self-assemble to form the functional form. we present a study on the open reading frame (orf) a membrane protein encoded in structural region of human severe acute respiratory syndrome coronavirus (sars-covs). the full length orf a protein is residues long and contains a single transmembrane (tm) domain. full length protein is synthesized using solid phase peptide synthesis and reconstituted into artificial lipid bilayers forms cation-selective ion channels. the bilayer recordings show cation selection channel activity with a major conductance level of around . ps also at elevated temperatures ( . • c). in silico studies with a amino acid tm domain are done to assess conformational space of the monomeric orf a helix. with this monomeric helix homooligomeric helical bundle models are built and embedded in a fully hydrated -palmitoyl- -oleoyl-sn-glycerol- phosphatidylcholine (popc) bilayer. results of both experimental channel recordings and computational modeling show sars orf a to act as a channel forming protein. -biomolecular self-assembly - microtubules are involved in many vital processes. their rigid structure can resist high forces while their intrinsic ability to switch stochastically between growth and shrinkage phases allows them dynamically to reorganise. in cells, a sizeable network of microtubule binding proteins control and regulate microtubule dynamics. alp and dis are members of the dis / xmap family that are major players in s.pombe. the deletion phenotypes of alp and dis are similar, but nonetheless distinct. both are involved in the formation of spindles but alp is also involved in the maintenance of cytosolic microtubules in interphase. the restrictive temperatures of alp -deletion and dis -deletion mutants are different. alp interacts with alp , a potential member of the tacc protein family. i am working to reconstitute alp /dis -dependent microtubule dynamics in vitro, using purified s. pombe tubulin. both alp and dis express well in insect cells and can be readily purified. preliminary data show that both proteins bind tubulin at low salt concentrations and that both influence the dynamics of pig brain microtubules. my goal is a complete functional analysis of alp and dis , individually and in combination, to test candidate molecular mechanisms for alp /dis -catalysis of s. pombe microtubule dynamics. the syphoviridae coliphage t is a well-suited model to study the assembly of large viral capsids. biochemical and biophysical approaches were used to reconstitute in vitro the assembly pathway of its capsid. the t structure was recently solved from cryo-em and image reconstruction. its icosahedral capsid (t = ) is built from the major head protein (pb , copies) forming both the pentons and hexons and from the portal protein (pb , copies) located at one vertex. its assembly proceeds by steps. pb and pb first assemble into a precursor structure called prohead i, which is converted to prohead ii by proteolysis of pb and pb by a head maturation protease. packaging of the kbp dsdna is then driven through the portal pore by a molecular motor, the terminase. this promotes expansion of prohead ii leading to the mature capsid. the different assembly steps and the conformational changes accompanying capsid maturation were characterized using proheads i either self-assembled from the overproduced and purified capsid proteins or isolated from a phage mutant. these precursor capsid structures were analysed by small angle x-ray scattering. the d structure of prohead ii and of the expanded capsid were solved from cryo-em. our data show that the assembly process of a large icosahedral capsid can be efficiently reconstituted in vitro. amyloid beta peptide fibril formation modulated by phospholipid membranes e. hellstrand , e. sparr , s. linse lund univ., department of biophysical chemistry, sweden, lund univ., department of physical chemistry, sweden disease-causing amyloid fibril formation can be modulated by many factors including interactions with biological lipid membranes. an increasing amount of evidence suggests that the process of fibril formation in vivo and the mechanism of toxicity both involve membrane interactions. alzheimer's is probably the most well-known amyloid disease and the associated amyloid beta peptide originates from the membrane incorporated amyloid precursor protein (app). we use recombinant abeta m - and abeta m - produced in escherichia coli, which allows us to perform large scale kinetics assays with good statistics where the amyloid formation process is followed in means of thioflavin t fluorescence. the lipid membranes are introduced in the system as large unilamellar vesicles composed of dopc, dppc and sphingomyelin, with and without incorporation of cholesterol. we find that the phase behanviour of the membrane in the vesicles has a large effect on the lag time of the amyloid formation process for both abeta m - and m - . all membranes increase the lagtime to some degree but dppc has the largest effect. by comparing different phases we can conclude that the translational diffusion in the membrane seems to be more important than the acyl chain ordering. furthermore, electrostatics, concentration dependence and membrane addition at different time points in the amyloid formation process have been investigated. equilibrium/non-equilibrium transitions in macromolecule interactions p. dumas , g. gibrat , s. bernacchi , e. ennifar ibmc-cnrs, strasbourg, france, llb (cea/cnrs), saclay, france usually, so called 'relaxation phenomena' occur on a fast time-scale and 'p-jump' or 't-jump' techniques are required to follow such events lasting (much) less than ms. we report that, during stability studies of proteins or nucleic acids, such relaxation events can be observed on astonishing long time-scales. we first performed 'melting studies' with nucleic-acid duplexes by using linear variations of temperature (t) with time (t). we observed that even very low rates dt/dt could lead to a frozen state for temperature values below a sharp temperature range, and relaxation to equilibrium beyond that range. this allows defining a 'relaxation temperature' t r separating the two regimes. numerical simulations very accurately described the related hysteresis phenomenon observed upon a heating-cooling cycle, which is the hallmark of departure from equilibrium. analogous observations were made with protein oligomers submitted to either a variable pressure, or variable concentration in denaturant. importantly, a single theoretical frame predicts that the critical relaxation value x r (x standing indifferently for temperature, pressure or denaturant concentration) depends on ln(dx/dt). one may ask whether some thermosensor rnas known for switching on or off genetic expression by 'feeling' a temperature variation, might also 'feel' dt/dt. if true, the exact switching temperature would depend on dt/dt and faster temperature changes would increase t r . -biomolecular self-assembly - the major component of amyloid plaques in the gerstmann-sträussler-scheinker disease is a prion peptide fragment from - to - residues. here, we present a structural study of prp - in form of oligomers and fibrils by fourier transform infrared spectroscopy (ftir) and atomic force microscopy (afm). after incubation at • c, the unfolded peptide was found to aggregate into oligomers characterized by intermolecular β-sheet infrared bands and by a wide distribution of oligomer volumes. after a lag phase, a conformational rearrangement of oligomers into fibrils, with a parallel orientation of the cross β-sheet structures, was observed. by afm, different morphologies were also detected for fibrils that displayed high heterogeneity in their twisting periodicity and a complex hierarchical assembly. in addition, we also studied thermal and random aggregation. the prp - peptide was found to undergo several aggregation pathways, whose end products display different structural properties and intermolecular interactions. these findings underline the high plasticity of the prion peptide, a peculiar feature of prion proteins to overcome species barriers (natalello et al. j.mol.biol. ; : - ). the role of proline isomerisation in the aggregation process and fibril formation of alpha-synuclein j. meuvis , m. gerard , v. baekelandt , y. engelborghs lab.of biomolecular dynamics, ku leuven, belgium, lab.of biochemistry, campus kortrijk, belgium, lab.for neurobiology & gene therapy, ku leuven, belgium alpha-synuclein (α-syn) plays a central role in parkinson's disease. the aggregation of this protein, which contains five proline residues (p ,p ,p ,p ,p ), is accelerated in vitro by fk binding proteins (fkbps), a family of enzymes with a peptidyl-prolyl cis-trans isomerase activity (ppiase). fkbps catalyze the cis-trans conformational change of proline, often a rate limiting step in protein folding. to elucidate the role of the proline residues in aggregation, we constructed a mutant p( , , , , )a α-syn . the kinetics of the aggregation of the mutant were studied with turbidity and thioflavin t fluorescence (tht). turbidity measurements show the formation of early, tht negative aggregates which is as fast for both wt and mutant. fibril formation however is faster for the proline-deficient mutant. we also studied the effect of fkbp on the aggregation of the mutant. although wt α-syn early aggregate formation is accelerated by the addition of µm fkbp , this effect disappears in the mutant. addition of ( pm- µm) fkbp accelerates the fiber formation of wt α-syn, which is abolished in the mutant. we can conclude that α-syn fiber formation is accelerated for the proline-deficient mutant, which suggests a role for the proline residues in fiber formation. furthermore all accelerating effects of fkbp are abolished in the mutant which suggests that the ppiase activity of fkbp is responsible for the accelerating effect on the aggregation of wt α-syn. materials used as gene delivery vehicles must be able to condense dna into small sizes to facilitate transport and crossing various barriers. one of the polycations investigated for dna compaction is chitosan, which has the advantage of being safe and biodegradable. as a step towards reducing the aggregation behaviour of dna-chitosan complexes, chitosans were modified by grafting peg-chains on the backbone. it is known that the transfection efficacy depends on the chitosan chain length. additionally, the degree of pegylation might influence the condensation process. here a systematic biophysical study of pegylated chitosans and how the interplay between chitosan chain length and degree of pegylation affect the compaction of dna in terms of particle size and structure, stability in pbs and when exposed to serum, and transfection efficacy is presented. three different chain lengths of chitosans are employed, and for each sample three pegylation degrees are investigated and the properties of the dna-pegchitosan complexes compared to complexes formed when employing the original, chitosan for dna compaction. it is found pegylation of chitosans can be used to increase both the stability of the dna-chitosan complexes when exposed to serum as well as increase their transfection efficacy in hek cells. max-planck institute for polymer research, mainz, germany model membranes mimic the essential function of a natural membrane. however, the complexity is reduced in order to allow the study of fundamental processes. tethered membranes consist in principle of a lipid bilayer that is covalently linked to a solid support through a spacer group. this architecture allows the characterization of the membrane itself as well as of incorporated membrane proteins using surface analytical techniques. we have established a versatile system of various anchor lipids, which allow membrane formation on different surfaces. the architectures have been characterized by surface plasmon techniques, neutron reflectivity and electrochemical methods. the membranes are electrically insulating and allow for the functional incorporation of ion channel proteins. polymerizable lipids allow to pattern the membrane and to study lateral diffusion processes. furthermore, the membranes can be used as a sensing platform, where embedded membrane proteins act as actual sensing units. a. perico, s. pietronave, l. arcesi, c. d'arrigo consiglio nazionale delle ricerche (cnr), institute for macromolecular studies (ismac) the electrostatic free energy (fe) of two parallel rigid likecharged polyelectrolytes (pes) is given as a function of the separation distance. for high linear charge density, z , the fe shows a minimum due to the increasing of the counterion condensation and condensation volume as the two pes approach. the interaction fe is governed by a critical linear charge density, z c , inversely proportional to the counterion valence. for highly charged pes (z > z c , like dna), the pes attract the stronger the smaller is the counterion valence, because the fe is dominated by the entropic term due to condensation of counterions in a volume displaying a maximum at short distances. for weakly charged pes (z < z c / ) the pes remain undercritical in the whole separation range and therefore repel. for moderately charged pes (z c / < z < z c ), the infinitely separated pes are undercritical but become supercritical as they approach a critical distance and charge condensation and condensation volume expansion start: in these circumstances the pes may attract if the counterion valence is sufficiently large. in the case of many dna rods, hexagonal clusters may be formed. upon interaction with hydrophobic surfaces, proteins show a tendency to expose regions that are normally buried in the hydrophobic core. unfolding is generally perceived as an undesired process in studies aimed to anchor functional proteins at surfaces. upon an upset of perspective the fine control of the unfolding/re-assembly process could be regarded as a strategy to build up molecular nanostructures for the development of organic-inorganic assemblies. we show that molecular layers patterned at the nanoscale, with longrange order properties extending over the microscopic scale, can be obtained upon adsorption of proteins onto the hydrophobic and ordered surface of pyrolytic graphite. upon adsorption, proteins lose their native folding and polypeptide chains re-assemble on the surface in a layered fashion, forming a molecular bilayer. the first layer, in contact with the substrate, and the second molecular layer show corduroy-like nanopatterns of different periodicity, with a relative orientation between the first and second layer patterns of • . surface-induced protein unfolding and polypeptide chain reassembly according to a layered ordered structure is a rather general phenomenon since it is observed for different proteins irrespectively of their specific structural properties. the possibility of using these ordered molecular structures as templates for the subsequent patterned deposition of supramolecular aggregates will be discussed. understanding protein-protein interactions and assemblies to control the hierarchical building of well-ordered supramolecular structures is highly relevant to new tailormade biomaterials. we previously evidenced that contrary to native calcium-loaded α-lactalbumin (holo α-la), calcium-depleted form (apo α-la) has the ability to selfassemble with lysozyme (lys) to form different supramolecular structures in temperature-dependent manner. in the present work, the events occurring at molecular scale were explored using fluorescence techniques. fluorescence anisotropy and fluorescence lifetime measurements provide a powerful and sensitive mean to measure intermolecular interactions. we showed that lys interacts with both apo α-la and holo α-la to form oligomers, assumed to be heterodimers, at • c and • c. the dissociation constants for dimerization, found to be in the µm range, were sensitive to the ionic strength. correlation time calculations suggest that formed heterodimers holo α-la/lys and apo α−la/lys differed in their shape and/or conformation. such conformation differences could explain why holo α-la/lys complexes are trapped as heterodimers while the apo α-la/lys complexes have the ability to further self-assemble into previously reported various supramolecular structures. polyglutamine aggregation and neurodegeneration g. nicastro, l. masino, a. pastore national institute for medical research, the ridgeway, nw aa london, u.k. polyglutamine (polyq) diseases are rare but dominant misfolding diseases linked to neurodegeneration. they are caused by the expansion of cag codon repeats, which encode polyq tracts in the corresponding gene products. aggregation of polyq proteins is thought to be triggered by polyq expansion but be strongly modulated by the protein context. in the attempt of understanding the molecular bases of polyq diseases, we are studying the structures, interactomes and aggregation properties of selected polyq proteins. here, we present recent work on ataxin- , taken as a representative example of the whole family. ataxin- is a ubiquitin specific cysteine protease, involved in the ubiquitinproteasome pathway and known to bind poly-ubiquitin chains of four or more subunits. the enzymatic site resides in the n-terminal josephin domain of ataxin- . we have characterized, using different biophysical techniques, the structure in solution and the aggregation properties of josephin both in isolation and in a ubiquitin complex. we demonstrate that interaction with ubiquitin strongly modulates the aggregation properties of ataxin- and suggest the importance of protein-protein interactions in preventing aggregation. our study also provides new insights into the molecular mechanisms which determine ataxin- specificity for poly-ubiquitin chains of the correct length and cross-linking. förster resonant energy transfer (fret) from an optically excited to a non-excited molecule has been widely used to probe molecular interactions in living cells. changes in the molecular makeup of a cellular region occurring during the acquisition of fluorescence images place tight constraints on the fret technology and data analysis, which could not be addressed satisfactorily until recently. we will describe a method for imaging protein complex distributions in living cells with sub-cellular spatial resolution, which relies on a spectrally resolved two-photon microscope (raicu et al, , nature photon. : - ) and a simple theory of fret in oligomeric complexes (v. raicu, , j. biol. phys. : - ) . then, we will overview recent results on the determination of the supra-molecular structure and distributions in living cells of oligomeric complexes of some g protein-coupled receptors. observing protein aggregates on surfaces m. rabe, d. verdes, s. seeger institute of physical chemistry, university of zurich, switzerland protein aggregation is an important topic of current protein research as it is associated with several human diseases including alzheimer's disease, parkinson's disease, and type ii diabetes. although protein aggregation mechanisms and conditions have been comprehensively investigated, studies on the formation and the fate of protein aggregates in contact with solid interfaces are scarce. we have comprehensively investigated the structure of protein assemblies that form spontaneously upon protein adsorption on solid interfaces using a surface sensitive fluorescence imaging technique based on super critical angle fluorescence (saf) detection. combining this technique with fret we not only succeeded to detect protein aggregates deposited on surfaces but also to characterize their behavior in real time, i.e., their emergence, growth, or spreading. the model protein bsa, for instance, was found to exhibit a certain tendency for aggregation in the buffer solution. these protein clusters can deposit onto solid surfaces and spread resulting in a large, flat structure after some time. a different possibility how protein aggregates emerge on surfaces consists of a direct deposition of protein monomers to pre existing aggregates. such a growth of protein aggregates on the surface has been observed in a model system using the protein α-synuclein, which is tightly associated with the parkinson's syndrome. we present the results of a spectroscopic ellipsometry (se) study of the adsorption process of yeast cytochrome c (ycc) on gold and graphite substrates, according to methods already applied to study the growth dynamics of organosulphur sams on gold [ ] . se investigation was carried out both in situ, at room temperature during protein deposition, and ex situ. on gold, se data demonstrate the formation of an about - nm thick layer, consistent with the formation of a dense monolayer of ycc molecules, confirmed by afm inspection. both in situ and ex situ measurements were characterized by well defined spectral features related to the soret band. analysis of the fine position of this feature allowed to obtain information on the oxidation state of the iron ion of the heme group. se data suggest that proteins have preserved their native structure. a completely different adsorption mechanism was observed on highly oriented pyrolytic graphite (hopg) [ ] . ex-situ se data on ycc/hopg, supported by afm observations, indicate the formation of an ultrathin molecular layer (∼ . nm) related to complete protein unfolding at the hydrophobic surface. the role of phospholipid anisotropy in the stability of inverted hexagonal phase was considered. the equilibrium configuration of the system was determined by the minimum of the free energy involving the contribution of the isotropic and deviatoric bending and the interstitial energy of phospholipid monolayers. the shape and local interactions of a single lipid molecule were taken into account. the minimization with respect to the configuration of the lipid layers was performed by the monte carlo simulated annealing method. at high enough temperature the lipid molecules attain a shape exhibiting higher intrinsic mean and deviatoric curvatures which fits better into the inverted hexagonal phase than into the lamellar phase. for the mathematical model the advanced geometry with non-spherical cross-section of inverted hexagonal phase was calculated, resulting in lower energy in non-spherical cross-section. theoretical results are in a good agreement with the small angle x-ray scattering experimental data. for a long while the conventional view has been that alzheimer disease is brought about by the beta-amyloid fibrils found in the senile plaques, but more recently it has been suggested that the main neurotoxic species would be the soluble oligomeric species, apparently prone to interact with cell structures and macromolecules potentially inducing neuronal dysfunction. peptide-peptide interactions resulting in self-assembly phenomena of beta-amyloid yielding fibrils can be modulated and influenced by small organic molecules that might also be effective therapeutic tools to ideally target both oligomeric and fibrillar species. in this perspective, polycyclic aromatic molecules are of special interest because they might disrupt the molecular architectures precursors of beta-amyloid fibrils by means of weak, non-covalent aromatic interactions, like stacking interactions. we have performed an in vitro spectroscopic study (light scattering, circular dichroism, ftir and fluorescence) of the effects on beta-amyloid fibrillogenesis of the natural pigment hypericin extracted from hypericum perforatum. our results show that, thanks to its structural characteristics and peculiar spectroscopic features, hypericin can be easily used to in vitro monitor the appearance of initial aggregation states of beta-amyloid peptides and, more importantly, that hypericin can interfere with the early stages of polymerization process, playing the role of an aggregation inhibitor. peptaibols are peculiar peptides produced by fungi associated to plants. they are composed by to amminoacidic residues and exhibit antibiotic and antifungal properties. due to their amphypatic nature, they can form ion channels in biological membranes. by making use of experimental models of biological membranes (biomimetic membranes) currently employed in the laboratory of bioelectrochemistry, and models of plant membranes (corn seed root), that are used in the international laboratory of plant neurobiology (linv), we characterized synthetic peptides such as trichogin gaiv and its shorter homologues ( and residues). we studied these peptaibols in a dioleoylphosphatidylcholine monolayer supported by hg using different electrochemical techniques (ac,vc,eis). the experimental technique employed in the linv (clark microelectrode coupled to mife system) allows to measure oxygen flux in the solution contacting plant cell membranes, after treatment with different peptide concentrations. preliminary results might indicate that short peptides can influence the whole metabolism of the plant and can therefore be used as "elicitors" in order to induce an acquired systemic resistance. supported biomimetic membranes (sbm) were developed for protein-membrane interactions studies. phospholipid vesicles were chemically linked onto amine grafted gold or glass surfaces; after an osmotic choc and liposomes fusion a continuous membrane bilayer was formed. the anchoring phospholipid molecule (dspe-peg-nhs) incorporated into the vesicles allowed the formation of a water-filled compartment between the surface and the bilayer. this first sbm model was used to monitor the membranes binding properties (dependent of calcium) of the adenylate cyclase toxin (cyaa) from bordetella pertussis. the sbm model was improved in order to study the translocation of the catalytic domain of cyaa across the bilayer. naturally, the cyaa catalytic domain, when it reaches the target cell cytosol, associates with intracellular calmodulin (cam) an activator of the adenylate cyclase activity of cyaa. to mimic this biological phenomenon, cam was first immobilized on the surface (gold or glass) and in a second step membrane construction was performed over the cam layer. the formation of the biomimetic membrane onto the cam layer was monitored by spr while membrane fluidity and continuity were analysed by fluorescence. our results demonstrated the potentialities of sbm for the study of protein insertion into and translocation across biological membranes. a multi-resolution approach to the structure and function of integrin αiibβ m. rocco , c. rosano , j. w. weisel , d. horita , r. r. hantgan istituto nazionale per la ricerca sul cancro (ist), genova, italy, university of pennsylvania, philadelphia, pa, usa, wake forest university, winston-salem, nc, usa. integrins are heterodimeric transmembrane receptors involved in mechanical anchoring and two-way signaling. each α and β subunit has a modular structure with a large extracellular portion, a single transmembrane region, and a cytoplasmic domain. integrins activation mechanism is regulated by controversial conformational changes: while crystallography revealed similar bent shapes for resting and primed extracellular region constructs, ligand binding-induced large structural rearrangements in smaller constructs suggested extension, "opening" and tails separation. in a multiresolution approach, we used experimental and computed hydrodynamics to discriminate among αiibβ integrin models built on x-ray, nmr, and em data. in contrast with xray data and d em maps, an extension is needed to match the hydrodynamics of full-length, solubilized αiibβ ; an electron tomography-based model fares better. consistent with that, and with our averaged d em images, a conformational change in the head region (β hybrid domain swingout) coupled to a simple transmembrane helices shift matches priming agents-induced frictional changes in full-length αiibβ . our multi-resolution study thus suggests that in integrins extension and immediate tail separation are uncoupled from head domain rearrangements following activation. -biomolecular self-assembly - stabilizing effects of α s -casein, a natively unfolded protein, on the aggregation of biomolecules a. trapani, r. carrotta, p. l. san biagio, d. bulone ibf-cnr palermo, italy α s -casein is one of the four types of caseins, a group of calcium phosphate-binding proteins that, in the form of micellar aggregates, makes up the largest protein component of bovine milk. the structure of α s -casein is that of a triblock polymer with a hydrophilic tract interposed between two hydrophobic blocks. due to the lack of a compact folded conformation, this protein can be classified as one of the intrinsically disordered (or natively unfolded) proteins. this class of proteins is known to exert a stabilizing activity on biomolecules through specific interaction with hydrophobic surfaces that partially unfolded molecules may expose to the solvent. here we present results on α s -casein effects on the thermally induced aggregation of gluthathione stransferase, a ligand-binding anzyme, and - β-amyloid peptide involved in alzheimer's disease. by means of light scattering and circular dichroism experiments, we attempt to reveal the molecular details of α s -biomolecules interaction. bacterial protein self-assembly on surfaces of well-defined chemistry s-layers are one of the most common cell envelope components of prokaryotic organisms and represent the simplest biological membrane developed during evolution. these (glyco)proteins, which can self-assemble into -d crystalline nanostructures on lipid films, liposomes, and polymers, play already an important role in nanobiotechnology. in this work, we present new findings concerning the recrystallization of bacterial proteins on substrates with defined chemistry. manipulation of the protein-sample interaction was carried out by changing the relative height of oh and ch terminated moieties in self-assembled monolayers (sams). we have found that differences in chain length lead to: i) protein bilayer-protein monolayer transition, ii) preferential protein side adsorption, and iii) increase of the crystal lattice parameters. further manipulation of the protein-sample interaction was achieved by using silane chemistry. we will show that sample hidrophobicity speeds up recrystallization kinetics and reduces the crystalline domain size (and layer compliance). the protein-adsorbed mass per unit area on these substrates is reported for the first time. self-assembly of phenylalanine oligopeptides: insights from experimental and computational studies p. tamamis , l. adler-abramovich , m. reches , k. marshall , p. sikorski , l. serpell , e. gazit , g. archontis dpt. of physics, univ. of cyprus, cyprus, dpt. of molecular microbiology and biotechnology, tel aviv univ., israel, dpt. of biochemistry, univ. of sussex, u.k. the diphenylalanine peptide (ff) forms well ordered nanotubes and its derivatives form nano-assemblies of various morphologies with promising material applications. we demonstrate for the first time by electron and laser microscopy and ftir spectroscopy that the related, triphenylalanine peptide (fff) assembles into rather planar nanostructures, rich in β-sheet. in addition, we conduct . -µs replica exchange m.d. simulations of aqueous ff and fff solutions in implicit solvent. the peptides coalesce into aggregates and participate frequently in open or ring-like linear networks, as well as elementary and network-containing structures with β-sheet characteristics. polar and nonpolar interactions, as well as the surrounding aggregate medium contribute to the network stabilities. within a network, consecutive peptides are linked by head-to-tail interactions; the aromatic sidechains of neighbor peptides assume approximately "t-shaped" orientations. these features are observed in ff crystals and could characterize early formations, or stabilize the mature nanostructures. the fff aggregates acquire higher stability and peptide-network propensity compared to the ff aggregates due to energetic contributions , . chlorophyll biosynthesis is light-dependent in angiosperms because the reduction of protochlorophyllide (pchlide) into chlorophyllide is driven by a photoenzyme, nadph:pchlide oxidoreductase (por). the unique properties of por are due to its ability to assemble into dimers and oligomers within the prolamellar body (plb) membranes of etioplasts studied mainly in leaves of dark-grown seedlings under laboratory conditions. we extended these studies to plant organs developed in the nature: cabbage heads, leaf primordia inside buds, pericarp-covered regions of sunflower cotyledons, potato tubers and seedlings germinating under the soil. in electron microscopic and fluorescence spectroscopic studies we found in many of these organs poorly developed plbs in which por was mainly in monomer state. as a consequence, the chlorophyll accumulation was slow and photo-oxidation processes occurred at illumination. in vitro we artificially induced the aggregation of por monomers into oligomers in glycerol and sucrose containing buffers. this resulted in the increase of the photoreduction rate at the expense of photo-oxidation. these results underline the importance of the self-assembly of por and the plbs in chloroplast development and chlorophyll synthesis in nature. v. vetri , g. ossato , v. militello , m. a. digman , m. leone , e. gratton dsfa, university of palermo, palermo, italy, lfd, university of california, irvine, ca, usa we report an experimental study on concanavalina (cona) aggregation in live cells. in vitro, close to physiological temperature, cona readily forms fibrils involving secondary structure changes leading to β-aggregate structures. the effect of cona on cell cultures and formation of protein aggregates were measured by confocal fluorescence microscopy. in particular, we monitored protein aggregation in live cells by means n&b analysis, cross-n&b and rics. n&b showed the aggregation kinetic and the progressive formation of cona oligomers at cell surface. this suggests that, at cell membrane where local concentration is higher, nucleation sites for aggregation are provided. in parallel, the morphology of the cells changes indicating the progressive cell compaction and death. aggregation and binding of small aggregates to the cell surface were assessed by rics: it is possible to distinguish regions where small aggregates are diffusing and regions where they are bound to the cell. oligomers formation may stimulate non-specific cellular responses due to the exposure of reactive regions of protein structure and of progressive formation of cross−β structures. moreover, aggregates stoichiometry was measured during the kinetic by cross-variance n&b. the two conductive pathways of p x purinergic receptor: different modulation and selectivity r. barbieri , s. alloisio , a. di garbo , m. nobile institute of biophysics, cnr, via de marini , genoa, italy, institute of biophysics, cnr, via g. moruzzi , pisa, italy the p x purinoceptor (p x r) is an atp-gated cation channel that is able to activate a cell permeabilizing pore. p x r cytosolic c-terminal tail is thought to modulate this function. this study was aimed to characterise the biophysical properties of p x r compared to those of the variant lacking the entire c-terminus tail (trp x r) by measuring whole-cell currents and intracellular ca + variations. a mathematical model is used to describe the experimental results. in p x r expressing hek- cells, the potent agonist '-o-( -benzoyl)benzoyl adenosine '-triphosphate (bzatp) -evoked ionic currents depending on concentration and frequency of agonist applications. the currents were strongly inhibited by extracellular mg + in a noncompetitive way. by contrast, in trp x r cells, only high bzatp concentrations elicited small currents not affected by mg + . interestingly, bzatp-induced ca + influx was present both in p x r and in trp x r cells, albeit in the latter the intracellular ca + elevation was smaller. importantly, in trp x r the intracellular ca + rise maintained a competitive mechanism of mg + inhibition similar to that observed in p x r. the experimental data and the modelling findings support the tenet of a functional structure of p x r possessing two distinct conductive pathways. the review of our data on the effects of physical and chemical weak signals on physicochemical properties of water, cell volume, activity and the number of membrane proteins (receptors, ionic channels and enzymes, na + /k + pump and na + /ca + exchanger), intracellular signal systems in norm and pathology (cancer and nerve disorders) would be presented. light microscopic, cell voltage-and patch-clamp, isotope, standard biochemical and genetic engineering methods were used. weak signal-induced effects on cell functional activity (intracellular enzymes activity, the number of functionally active membrane proteins) are realized by changing the physicochemical properties of extra-and intracellular aqua medium. the latter induces the modulation of na + /k + pump-induced cell hydration, which serves as a primary mechanism through which the autoregulation of pump and regulation of membrane excitability and chemosensitivity are realized. by genetic engineering method in oocytes it was shown that the correlation between na + /k + pump and na + /ca + exchanger, which is realized through intracellular messenger systems, plays a crucial role in weak signals transduction in cells and determination of aging-induced increase of cell pathology. listeriolysin pore forming ability in planar lipid membranes at different ph listeriolysin o (llo) is a cholesterol-dependent cytolysin secreted by the intracellular pathogen listeria monocytogenes. its main task is to enable escape of bacteria from the phagosomal vacuole into the cytoplasm. llo exhibits optimal cytolytic activity at low ph but it is still able to bind membranes at physiological or even slightly basic ph values in a cholesterol-dependent fashion. high cholesterol concentrations in the membrane restore the low activity of llo at high ph values. based on this broad ph activity we investigated the electrophysiological properties of pores formed by llo at room temperature and at different phs using planar lipid bilayer technique. llo is able to form pores both at ph . and . with a similar permeabilizing ability and similar heterogeneous conductances in the range of picosiemes to nanosiemens. cholesterol content directly correlates with llo activity but it does not change the pore characteristics. collectively, our results demonstrate that llo activity at physiological ph cannot be neglected and that its action at sites distal to cell entry may have important physiological consequences for listeria pathogenesis. s. aimon, g. toombes, p. bassereau institut curie, paris, france the physics of membrane/channel and channel/channel interactions is difficult to investigate in cells where it is nearly impossible to modify relevant parameters to deduce physics laws. to overcome these difficulties we built a model system in which voltage gated ion channels were reconstituted in giant unilamellar vesicles (guvs) for the first time. as a first step, we successfully expressed kvap (a voltage gated potassium channel) in e-coli. the channel was purified, fluorescently labelled and reconstituted in small liposomes. its functionality was checked with electrophysiology via fusion of these liposomes into black lipid membranes (blm). as a second step, guvs were formed from these small proteoliposomes using electroformation in a buffer containing mm kcl salt. the proper incorporation of proteins into guvs was controlled using confocal microscopy. functional proteins were detected using the patch clamp technique. with our protocol, we are thus able to prepare guvs containing functional voltage-gated ion channels. one goal is now to study the effect of channel activity on its spatial distribution in these guvs. ryr activation in cultured shr cardiomyocytes at the end of the prehypertensive period the rate of [ca + ] i elevation after the ryanodine receptor (ryr) activation by -chloro-m-cresol ( -cmc) and l-type ca + channels (dhpr) activation by bay k was studied in cultured ( days) cardiomyocytes of spontaneously hypertensive (shr) and normotensive rats (wky, wistar) during weeks of postnatal development. the differences in dh-prs and ryrs activities began to be evident after weeks age when cicr formation has finished and became more expressed at the end of prehypertensive period ( weeks). in response to -cmc ( mm), a drastic increase in the rate of [ca + ] i accumulation ( . ± . times) in shr myocytes was registered after days age versus a decrease in the rates of ca + efflux from the sarcoplasmic reticulum of wistar and wky rat cardiomyocytes. bayk ( µm) also induced more sharp [ca + ] i elevation in shr myocytes ( . ± . times) as compared with wistar ( . ± . times) and wky ( . ± . times) ones of the same age. our results argue that in shr and wky cardiomyocytes, as opposed to normotensive wistar rats, gradual growth of dhpr activity is observed, which follows in parallel with cicr formation in the excitation-contraction coupling during early postnatal ontogenesis, and drastic activation of ryr in shr myocytes after the process termination. cytochrome ba from thermus thermophilus belongs to the large family of structurally related heme-copper oxidases. it accepts electrons from cytochrome c at the p-side of the membrane and uses them to reduce oxygen to water. the energy released in this reaction is used for proton pumping across the membrane to form of an electrochemical proton gradient, used by the cell for formation of atp. in this work we followed the kinetics of single-electron injection into the oxidized nonrelaxed state (o h →e h ) of cytochrome ba by time-resolved optical spectroscopy. two main phases of electron transfer were resolved. the first (τ∼ µs) includes oxidation of cu a and simultaneous reduction of both low and high spin hemes. the second (τ∼ µs) reflects reoxidation of both hemes by cu b . this is in significant contrast to the o h →e h transition of aa -type oxidases, where the fastest phase is due to transient reduction of the low-spin heme a only. on the other hand, the single-electron reduction of the relaxed o state in ba oxidase consisted of only rapid electron transfer from cu a to heme b, which is similar to that in aa oxidase. this indicates a functional difference between the relaxed o and the pulsed o h states of cytochrome ba . as opposed to the phospholamban pentamer, sarcolipin forms anion-selective channels in biomembranes l. becucci , c. b. karim , d. d. thomas , g. veglia , r. guidelli chemistry department, florence university, florence,italy, chemistry department, university of minnesota, minneapolis, mn , department of biochemistry, molecular biology, and biophysics, university of minnesota, minneapolis, mn sarcolipin (sln) and the phospholamban pentamer (pln) are two membrane proteins that inhibit ca-atpase of the sarcoplasmic reticulum at low concentrations. in contrast to pln, sln stimulates maximal ca + uptake rates. sln and pln were incorporated in a bilayer lipid membrane (tblm) tethered to a mercury electrode through a hydrophilic spacer. electrochemical impedance spectroscopy measurements show that sln forms channels permeable to chloride ion, weakly permeable to phosphate ion and impermeable to inorganic cations such as na + and k + . a relationship between this property of sln and its regulatory function on ca-atpase of sarcoplasmic reticulum is proposed. atp increases the permeability of a tblm incorporating sln to phosphate ion by associating to sln with an association constant of . µm. an explanation for this behavior is provided. sln can be identified with the " p i transporter" described by a.g. lee et al. conversely, both electrochemical impedance spectroscopy measurements and molecular dynamics simulations provide strong evidence that the pore of the pentameric form of pln does not act as a chloride channel. "social" domain organization and dynamics of nicotinic acetylcholine receptor at the cell membrane f. j. barrantes unesco chair biophys. & mol. neurobiology. univ. nac. sur, b. blanca, argentina a combination of experimental techniques (patch-clamp, confocal frap and fcs, single-particle tracking, highresolution fluorescence microscopy) has been used to analyze the supramolecular organization of the acetylcholine receptor (achr), the dynamics of the receptor at the cell surface, and the kinetics of receptor internalization. changes in cholesterol (chol) content affected muscle and neuronal-type achr organization and dynamics at the cell surface. chol depletion produced gain-of-function of single-channel dwell time. submicron-sized (∼ nm) domains, stable over a period of hours at the cell membrane, could be resolved into achr "nano-clusters" with a peak size distribution of ∼ nm by sted microscopy. chol depletion reduced the number of nanoclusters, increasing their size, and changed their supramolecular "social" organization on larger scales ( . - . microns). frap, fcs and spt experiments provided information on the dynamics of achr nanoclusters, disclosing the dependence of their mobility on chol content and cortical cytoskeleton. chol content at the plasmalemma may thus modulate cell-surface organization and dynamics of receptor domains, and fine-tune receptor channel function to temporarily compensate for acute achr losses from the cell surface. m. czaplinska, k. gwozdzinski, a. koceva-chyla division of research of structure of biopolymers university of lodz, lodz, poland doxorubicin (dox) and paclitaxel (ptx) are anticancer drugs commonly used in chemotherapy of breast cancer therapy, however, the use of these drugs is limited by the risk of developing heart failure. generation of reactive oxygen species contributes to the cardiotoxicity of doxorubicin. nitroxides are low molecular weight, stable free radicals reacting with ros and they present antioxidant properties. the aim of this study was to analyze the effects of pirolid (pd) on the oxidative stress induced by dox and taxane in mcf- breast cancer cell line. results from mtt test revealed that ptx is more cytotoxic towards mcf- cells than dox. the ic was . µm and µm, respectively. pd alone does not influence cell viability. pd in combination with both drugs did not change viability of cells. both drugs increased the level of carbonyl groups in cells. the highest level of peroxide was observed in cells incubated with dox (approx. -fold). nitroxide alone did not influence the level of peroxide in the whole range of concentrations. combination of pd with dox and ptx reduced the level of carbonyls depending on its concentration. pd did not affected on the level of peroxide in cells suspension. dox and ptx increased ( -fold) the level of carbonyls. pd decreased the level of peroxides in cells treated with dox and ptx. the lowest concentration of peroxide was observed at µm of pd. these results show that pd protect mcf- cells against oxidative stress induced by drugs. open channel structure of mscl from fret microscopy and simulation mechanosensitive channels open in response to membrane bilayer deformations occurring in physiological processes such as touch, hearing and osmoregulation. here, we have determined the likely structure of the open state of the mechanosensitive channel of large conductance from e. coli (mscl) in a natural environment using a combination of patch-clamp studies, fret spectroscopy, epr data, molecular and brownian dynamics simulation. structural rearrangements of the protein are measured while controlling the state of the pore by modifying lipid bilayer morphology. fret efficiency changes can be related to distance changes using a monte carlo analysis program in conjunction with detailed orientational analysis. these measurements are used as restraints in all atom molecular simulations in order to determine the likely structure of the open state, whose probable conductance is derived from brownian dynamics simulations. transition to the open state occurs via large rearrangements throughout the protein that create a wide pore nearly a in diameter. both transmembrane helices are found to line part of the pore. the n terminal helix is found to lie along the face of the membrane where it can act to sense membrane tension and directly transfer this to the pore lining helices. the method of coupling spectroscopic data with simulations is likely to be of great value for studying conformational changes in a range of membrane proteins. putative potassium channels in synechocystis sp. pcc v. checchetto, m. zanetti, g. m. giacometti, i. szabò, e. bergantino department of biology, university of padova, italy we are interested in the identification and characterization of potassium channels in the cyanobacterium synechocystis sp. pcc , an organism which is considered the ancestor of plant chloroplasts. a bioinformatic screening of synechocystis proteome identified, among others, two proteins on which we focused our attention. the first one (syncak) displays sequence homology to mthk, a ca + -dependent potassium channel from m . thermoautotrophicum. the second one (synk) is predicted to contain six transmembrane regions and the typical selectivity filter of all potassium channels. our goal is to understand their roles in the physiology of cyanobacteria. we cloned their coding sequences in fusion with gfp and the hybrid proteins were expressed in chinese hamster ovary cells. we evaluated the presence of both proteins in plasma membrane by fluorescence microscopy and then we proceeded to their functional characterization using patch clamp technique.this analysis will allow us to gain information about channel activity, regulation and pharmacology. we also plan to evaluate the importance of syncak and synk channels in photosynthesis. to test the hypothesis that they could be involved in regulating this process, we will produce deletion and site-specific mutants in synechocystis. finally we would also identify the homologous of these channels in the higher plant a. thaliana and obtain some information about their localization and function. j. braunagel max-planck institute for polymer research, ackermannweg , mainz, germany the cyclododecadepsipeptide valinomycin is composed of two amino acids (l-valine and dvaline) and two hydroxyl acids (d-α-hydroxy-isovaleric acid and l-lactic acid). they form a membered ring of alternating amino and hydroxyl acids. in the cyclic structure, the polar groups are oriented towards the central cavity, whereas the rest of the molecule is relatively nonpolar. this enables the complexation of ions and valinomycin acts as a selective ion transporter (k + ) in lipid membranes. when one of the amino acids is exchanged, e.g one l-valine by an l-lysine, selected functionality can be engineered into the depsipeptide while maintaining its ion conducting properties. we induced several modifications into valinomycin, i.e. a biotin binding unit or a ferrocene group to induce an electrochemical active center. the ion conducting properties of the modified ion carriers have been probed in planar lipid bilayers as well as in solid supported membranes. the role of the membrane dipole potential (ϕ d ) is of a particular interest due to a powerful impact of this potential on the membrane permeability and lipid-protein interactions. channel forming activity of gramicidin a, alamethicin, syringomycin e, hpa peptide, and ompf porin are influenced by ϕ d . we have studied the effect of the membrane dipole modifier, phloretin, on the properties of single channels formed by a wild-type alpha-hemolysin in planar lipid bilayers. the single channel of a ∼ ps conductance exhibits transitions into a number of low-conductance states as the transmembrane voltage exceeds ∼ mv (regardless of the voltage polarity). the phloretin addition to the bathing solutions ( µm) (after the hemolysin channel was formed in the membrane) shifts dramatically the channel voltagedependence. transitions to the low-conductance states are observed at ∼ mv. the effect of the phloretin addition was not observed in the case when it was introduced into the bathing solution before alpha-toxin. since phloretin reduces ϕ d , the data may report on the influence of the electric potential profile on the energy of the low-conductance state of the alpha-hemolysin channel. the alternative explanation of this effect consists in a specific interaction between the phloretin and toxin channel. the work is supported in part by rfbr (# - - ), ss (# . . ) , and the program of the ras «molecular and cell biology». atypical mechanism of conduction in potassium channels c. domene , s. furini physical and theoretical chemistry laboratory, department of chemistry, university of oxford, oxford, u.k., department of electronics, computer science and systems, university of bologna, bologna, italy potassium channels can conduct k + ions with rates of up to ∼ ions per second at physiological conditions, and they are selective to these species by a factor of over na + ions. ion conduction has been proposed to involve transitions between two main states, with two or three k + ions occupying the selectivity filter separated by an intervening water molecule. the largest free energy barrier of such a process was reported to be of the order of - kcal mol − . here, we present an alternative mechanism for conduction of k + in k + channels where site vacancies are involved, and we propose that coexistence of several ion permeation mechanisms is energetically possible. conduction can be described as a more anarchic phenomenon than previously characterized by the concerted translocations of k + -water-k + . we exploited the au-deposited self-assembled monolayers of the type: [−s−(ch ) n −ch ] (where n = , and ) with hydrophobically adsorbed redox protein -azurin to verify intrinsic electron transfer mechanisms according to the charge-transfer theory. the enthalpies and volumes of activation were determined through the variation of temperature ( - o c) and pressure ( - mpa) and experimental values were compared with those expected on the theoretical grounds. for the case of n = the activation enthalpy definitely contains a large contribution originated from frictional-like dynamics of protein, and the activation volume has a small positive value. for n = the value of activation enthalpy directly matches / of that for the reorganization energy, and the activation volume attains substantially negative value. for n = we observed the intermediary performance. the whole kinetic pattern is consistent with the smooth changeover between adiabatic and nonadiabatic mechanisms of electron transfer. two gating modalities in the pore of the miniature k + channel kcv kcv is a viral protein that forms functional k + channel in heterologous systems. because of its miniature size ( amino acids) we use kcv as a model system to study and manipulate basic properties of the k + channel pore. by analysing single-channel recordings we highlighted two voltage-dependent modalities of gating in kcv: a slow and a fast gating. the presence of a slow gating is revealed by the very low (in the order of - %) mean open probability. slow gating is not related to the presence of a bundle crossing, as shown by accessibility of the cavity to mts reagents. channel opening might involve the transient formation of salt bridges between residues at the n and c termini of the channel, as suggested by mutational experiments inspired by molecular dynamics simulations of kcv. fast gating, analyzed by beta distributions, is responsible for the negative slope conductance in the single-channel i/v curve at extreme potentials and can be explained by depletion-aggravated instability of the filter region. aca is a type b caatpase with a regulatory n-terminus whose autoinhibitory action can be suppressed by binding of calmodulin (cam). aca n-terminus is able to bind a region of the small cytoplasmic loop connecting transmembrane domains and . to define the role of this interaction in autoinhibition we have analysed a number of single point mutants produced by mutagenesis of aca e -n sequence. mutation to ala of any of acidic residues (e , d , d , d , e , d ) originates an enzyme with normal activity in the presence of cam, but less camstimulated. these results highlight the relevance of a negative charge of the surface area of the small cytoplasmic loop in aca autoinhibition. the most deregulated mutant is d a aca , which is less activated also by controlled proteolysis or by acidic phospholipids; moreover, the phenotype of the d a mutant is stronger than that of d n aca suggesting a more direct involvement of this residue in autoinhibition. of the other mutants (i a, n a, p a, p a, v a, n a), only p a aca has a basal activity higher than that of the wt. these results provide the first evidence that the small cytoplasmic loop of a type b caatpase plays a role in the attainment of the autoinhibited state. complex i, the first member of the respiratory chain, serves as a proton pump catalyzing transfer of two electrons from nadh to ubiquinone coupled with the translocation of four protons across the membrane. so far the mechanism of energy transduction by complex i is unknown. the nadh-binding cavity of complex i has a very prominent feature -the presence of two invariant amino acid residues, glutamate and tyrosine, that are exposed to the solvent and located in the vicinity of the fmn, the primary electron acceptor in the enzyme. it was suggested that they might be involved in the binding of nadh through interaction with its nicotinamide moiety. in this work we assessed the function of corresponding glu from the nuof subunit of e. coli complex i by mutation for glutamine. we showed that the negative charge of glutamate in the catalytic site is needed for the electrostatic repulsion of negatively charged phosphates of nucleotides. this process facilitates release of the product nad + and, as a result, accelerates turnover of complex i. we also found that glutamate, as one of the four negatively charged amino acid residues surrounding the isoalloxazine ring of the fmn at a distance of - Å, has a share of mv of the overall mv depression of the midpoint potential of this redox cofactor. l. erokhova , p. kügler , p. pohl institute of biophysics, johannes kepler university, linz, austria, ricam, austrian academy of sciences, linz, austria the mechanism of water transport through epithelia is still under debate. in the present work we tested the hypothesis of isosmolal water transport using mdck cells stably expressing the human sodium-glucose cotransporter (hsglt ). tagging hsglt with egfp enabled determination of its abundance in the plasma membrane by fluorescence correlation spectroscopy (fcs). by monitoring tiny shifts in the concentration of water-soluble dyes in the vicinity of epithelia, fcs also allowed assessment of the water fluxes through confluent cell monolayers grown on permeable supports. fitting the set of differential equations for the osmotic drift and for the back diffusion to the experimentally determined dye distribution permitted calculation of water flow both in the presence and in the absence of an osmotic gradient. from the calorimetric measurements of glucose transported across the cell monolayer, the water:glucose stoichiometry was derived. dividing the increment in osmotic water flux due to hsglt expression by the number of hsglt copies in the plasma membrane resulted in a single transporter water permeability p f of . x − cm /sec. thus, p f is close to the single channel water permeability of aquaporin- . consequently, even small osmolyte concentration differences between the cytoplasm and the basolateral buffer solution are sufficient to drive a substantial water flux. m. emre, s. kavak cukurova university school of medicine, department of biophysics, balcali-adana, turkey experimental studies have shown that the at -receptor antagonists telmisartan (tel) has a ppar-activating property, but there does not appear to be a class effect. to test telmisartan's importance, we investigated its effect on electrical activities (ea) in diabetic (d) rats. the purpose of this study was to investigate the effects of the tel on ea of diabetic papillary muscle (dpm) with stz-induced. in this study, we used four groups: ( ) nondiabetic control (ndc) group (c), ( ) tel-treated ndc group (c+tel), ( ) diabetic group (d), and ( ) tel-treated d group (d+tel). diabetes was induced by a single i.v injection of stz. in the study, membrane potential (mp) and action potential (ap) recorded after the establishment of diabetes. ) mp was decreased significantly in both tel-treated c and d rats (from - , ± , to - , ± , mv and from - , ± , to - , ± , mv). ) ap unchanged in d group, whereas c+tel and d+tel groups showed increase in ap compared with c and d groups. ) repolarization time was prolonged in diabetic rats. ) in c+tel and d+tel groups depolarization rate values increased significantly. on the other hand, in d group repolarization rate values descreased increased significantly compared to baseline values in tel solution. as a result, our data suggest that the beneficial effects of tel-treatment on the ea of the dpm appear to be due to the diminished k + currents. cytochrome c oxidase is a terminal complex (cco, complex iv) of a respiratory chain that is located in an internal membrane of mitochondria or plasma membrane of bacteria. cco is an electron transfer enzyme that reduces o and uses the redox energy of the o reduction for the proton translocation across the membrane. the electron-and proton-transfer generate a transmembrane electrochemical gradient (∆µh + ) that is used for atp synthesis and all other kinds of work for the cell needs. the proton translocation mechanism of cco requires 'channels' for the h + uptake and expulsion within the enzyme. the proton transfer occurs on a time-scale of micro-to-milliseconds. in order to study the proton transfer in cco, a flow-flash approach based on a time-resolved ftir spectroscopy was developed and applied. our ftir flow-flash approach (the measurement of the reaction of cco with o ) allows to reach a time resolution up to tens milliseconds. with this approach and site-specific mutants of cco where the catalysis is slowed down, separate steps of the proton transfer were studied. the results showed that a unique cross-linked tyr- (in a combination with a time-resolved visible spectroscopy and electrometry) serves as a proton donor for the dioxygen bond cleavage during the o reduction by cco. furthermore, the protolytic transitions of glu- -a key amino acid in the proton transfer mechanism in cco -were shown for the first time. role of calcium ions in nickel potentiation of nmda currents p. gavazzo, i. zanardi, p. guida, c. marchetti biophysics institute ,cnr, genova, italy nmda receptors are glutamate-gated channels distributed throughout the brain in the excitatory synapses and are critical for the nervous system function. they are assembled from two types of subunits, the essential nr and at least one nr (a,b,c,d). nickel (ni) modulates the current flowing through nmda receptors in a different way depending on the nr subunit present. we have recently identified several domains of the channel involved in ni interaction, but many aspects of this modulation remain elusive. in this work we intended to determine the role of calcium (ca) ions in the potentiation induced by ni on the current through nr /nr b recombinant nmda receptors. when ni was applied in the presence of the physiological concentration of ca ( . mm) , a voltage-independent potentiation of the current was observed with a kp of . µm. this effect was progressively reduced by decreasing ca concentrations and it was no more detectable with . mm ca or in the presence of barium (ba, . mm). in this last case the effect of ni on nr /nr b receptors was mainly inhibitory (ki(- mv)= µm). therefore a physiological concentration of ca is necessary to induce ni amplification of the current. many data in the literature indicate a correlation between ca ion entrance through the channel, nmda current facilitation and cytoskeleton; however, in our experiments, the application of the actin perturbing agent cytochalasin-d did not produce major modifications in ni effect. heteromerization properties of voltage dependent potassium channels f. gambale , l. pedemonte , a. naso , i. testa , c. usai , a. diaspro , c. picco istituto di biofisica, cnr, genova, italy, lambs-microscobio, department of physics, genova, italy voltage-gated potassium channels are either homomeric or heteromeric tetramers composed of four α-subunits. in order to bring a contribution to the comprehension of channel heteromerization we have been investigating the properties of two plant voltage-gated k + -channels by using electrophysiological and fluorescence techniques. experiments were focussed on kdc and kdc , coexpressed in xenopus laevis oocytes. kdc , the first potassium channel cloned from daucus carota, belongs to the subfamily of α-modulatory silent channels as it doesn't form functional homomeric channels by itself. on the contrary kdc forms functional heteromeric channels when coexpressed with homologous subunits. kdc , the last k + channel cloned from d. carota, belongs to the kat family and shares an overall identity of % with kat . to correlate kdc functional properties with its localization in oocytes, kdc and/or kdc subunits were labelled with gfp and their properties investigated by confocal microscopy and voltage-clamp. we found that the kdc -egfp fusion protein is not targeted to the plasma membrane unless it is coexpressed with kdc . moreover electrophysiological experiments demonstrated that the heteromeric kdc -kdc channel has altered selectivity and activation properties with respect to homomeric kdc channel. circulating leukocyte sequestration in pulmonary capillaries is arguably the initiating event of lung injury in acute respiratory distress syndrome (ards) [ ] . we present a microfluidic investigation of the roles of actin organization and myosin ii activity during the different stages of leukocyte trafficking through narrow capillaries using specific drugs. the deformation rate during entry reveals that cell stiffness depends strongly on f-actin organization and hardly on myosin ii activity, supporting microfilament role in leukocyte sequestration. in the transit stage, cell friction is influenced by stiffness, demonstrating that the actin network is not completely broken after a forced entry into a capillary. conversely, membrane unfolding was independent of leukocyte stiffness. the surface area of sequestered leukocytes increased by up to % in absence of myosin ii activity, showing the major role of molecular motors on microvilli wrinkling and zipping. finally, cell shape relaxation was largely independent of both actin organization and myosin ii activity, whereas a deformed state was required for normal trafficking through capillary segments [ ] . [ ] g.s. worthen et al., science, , - ( ) excitation-contraction coupling in skeletal and cardiac muscle is tightly regulated by the calcium release channel of the sarcoplasmic reticulum, the ryanodine receptor (ryr). we could previously show that suramin is a potent activator of the ryr via the calmodulin binding site. calmodulin shows dualistic action, i.e. activation or inhibition of the ryanodine receptor, depending on the absence or presence of ca + . screening of suramin analogues identified nf as a use-dependent inhibitor of the skeletal muscle ryr (ryr ). here we show that nf inhibits high affinity [ h]ryanodine binding and single channel recordings of the purified ryr . nf induced a reduction of open probabilities in a concentration dependent manner, with no effect on current amplitude and unitary conductance. importantly, nf triggers flickering episodes of channel openings and closings before the ryr is frozen in a complete non-conducting state, which is fully reactivated by the ryr agonist atp. moreover, zwitterionic behaviour of nf facilitates plasma membrane permeation, which prevented caffeine induced ca + transients in skeletal muscle cells and cardiomyocytes. conversely, ip mediated ca + signals were not altered by nf . this work was supported by herzfelder'sche familienstiftung and fwf . beyond steady-state protein dynamics t. hauß , j. pieper , a. buchsteiner , r. e. lechner , n. a. dencher helmholtz-zentrum berlin für materialien und energie, berlin, germany, technische universität darmstadt, germany, technische universität berlin, germany to study protein dynamics beyond steady-state experiments we have developed a novel laser-pump:neutron-probe experiment which allows us to monitor temporal changes in protein dynamics during a working cycle of a protein. protein dynamics has been extensively studied, but so far, the correlation of internal protein dynamics with the function of proteins was investigated only indirectly in steady-state experiments by variation of external parameters by variation of external parameters like temperature or hydration. the method comprises of an in-situ optical activation of a protein and a time-dependent sampling of the dymamic response using quasi-elastic neutron scattering. with the membrane protein bacteriorhodopsin, a light driven proton pump, we can demonstrate for the first time temporary alterations in the protein dynamics after triggering the working cycle. this observation is a direct proof for the functional significance of protein structural flexibility, in connection with the largescale conformational changes in the protein structure occurring during the operation of a "molecular machine". the slow vacuolar (sv) channels are ubiquitous in all tissues of higher plants. the sv channel is a non-selective cation channel permeable to both monovalent and divalent cations. sv currents recorded in a typical patch-clamp experiment require unphysiologically high cytosolic and low vacuolar calcium concentrations for full activation. we aim at looking for endogenous plant substances which might be able to modify or shift the voltage activation threshold of this channel towards more physiological conditions. flavonoid naringenin [nar] is present in all plant species where it plays a central role in the flavonoid biosynthetic pathway. nar is stored in the vacuoles in glycosylated form called naringin. when nar was added to cytosolic bath solution, we recorded a dose-dependent reversible decrease in sv channel activity. when we investigated the effect of nar on the voltage dependence of the channel, we observed that the activation threshold of the sv channel is shifted towards more positive voltages. our group has evidences that approximately % of the total sv current at high (e.g.> mv) positive voltages is mediated by calcium. therefore, in order to verify whether nar affects both potassium and calcium conductance, we performed experiments by combining the patch clamp technique with fluorescence measurements using the fluorophore fura- : both sv currents and calcium signals were abolished by mm [nar]. determination of calcium currents in cation channels using a novel fluorescence/patch-clamp approach p. v. k. gutla, a. gradogna, a. carpaneto istituto di biofisica, consiglio nazionale delle ricerche, via de marini , genova, italy the patch-clamp technique combined with fura- fluorescence detection is suitable to investigate calcium fluxes. we used the excised patch configuration and focused the photomultiplier to the tip of the recording pipette where the fluorescent dye was present (fluoresence combined with excised patch = flep). this configuration has several advantages, i.e. absence of delay in loading the fluorophore, of interference by endogenous calcium buffers and of photobleaching. here we present an application for the determination of fractional calcium currents (pf) in a plant non-selective cation channel, showing that pf can be modulated by cytosolic calcium and potassium. flep is very efficient for measuring small calcium currents (< pa) of sufficiently long duration; fluorescence signals are amplified by integration in time, as the calcium/fura- complex accumulates at the tip of the recording pipette and diffuses slowly. we propose this technique not only for the study of calcium transport pathways, but also for other transporters of divalent cations as nickel and manganese known to quench fluorescence thus reducing both and nm components. moreover, using the appropriate fluorophore the technique may be extended to further ion species, e.g. bcecf for investigating proton transport pathways. ref: we introduced an original method for the monitoring of the changes in the electrostatic surface potential, using the quenching of the intrinsic tryptophan fluorescence by acrylamide or iodide. this approach opens new way to understanding the dynamic processes within the proteins. our experiments revealed that the conformation of the na + /k + -atpase large cytoplasmic loop (c ) in the presence of the atp (without magnesium) substantially differed from the conformation in the presence of mg + or mgatp or in the absence of any ligand not only in the sense of geometry but also in the sense of the electrostatic surface potential. moreover, our data indicate that the effect of the ligand binding is not restricted only to the close environment of the binding site and that the information is in fact transmitted also to the distal parts of the molecule. this property could be important for the communication between the cytoplasmic headpiece and the cation binding sites located within the transmembrane domain. influx of antibiotics into the periplasm of gram-negative bacteria is facilitated by porins that form channels in the outer membrane. we propose that certain natural antibiotics have been optimized by co-evolution to take advantage of the charge distribution in non-specific porins to achieve binding and thereby facilitating their uptake in bacteria. we investigate the permeation pathways of antibiotics into bacteria by reconstitution of a single porin into an artificial lipid bilayer and measuring the binding of antibiotic molecules through the time-resolved modulation of a small ion current. we have been able to characterize facilitated translocation of several antibiotics through escherichia coli and enterobacter aerogenes porins. noise analysis of ion currents through a porin in the presence of effective antibiotics revealed binding kinetics at a single molecule level. we report for the first time temperature dependent antibiotic translocation that revealed complete energy profile. combining these results with microbiological assays and molecular dynamics simulations, we conclude the molecular mechanism of antibiotic permeation. our approach may contribute to the rational design of new antibiotics against clinical bacterial strains for the most efficient delivery to target sites. mitochondria regulate ca + influx and determine patterns of er ca + refilling in acinar cel o. kopach, i. kruglikov, t. pivneva, n. voitenko, n. fedirko bogomoletz institute of physiology, kiev, ukraine store-operated ca + entry (soce) is mediated by activation of soc-channels of plasma membrane following the emptying of endoplasmic reticulum (er) ca + stores. the soce is required for calcium signaling, secretion of neurotransmitters and proteins, but the mechanisms of natural soce regulation are not well understood. we utilized several imaging methods to measure ca + signals in cytoplasm ( ] mit transients, observed both in egta-and bapta-buffering inside solutions. these data suggest that ca + sequestration by mit is associated with the formation of microdomains and prevents ca + -dependent socc inactivation. we also found that inhibition of mit under prolonged cell stimulation resulted in complete inhibition of soce as well as decrease and deceleration of er refilling. thus, mit regulate calcium recycling and maintain the soce controlling dynamic interplay between soce and sustained er refilling under prolonged stimulation. bioelectrochemical devices composed of au electrodes coated by self-assembled monolayers (sams) of different composition and thickness are ideal systems to probe et patterns and mechanisms for redox proteins. representative proteins, cytochrome c and azurin were studied by using the combinations of four different strategies including the variation of sam thickness ([−s−(ch ) n −ω], with n running over the range to , throughout), solution viscosity (varied by adding of the viscose additive -glucose), temperature ( to o c) and hydrostatic pressure (up to mpa), aiming the identification of different intrinsic et patterns and interplay between them in the framework of generalized charge-transfer theory. we demonstrated the full adiabatic (frictional) control for the case of thinner sams, the intermediate (mixed) regime, and the complete changeover to the nonadiabatic mechanism (long-range tunneling) for the case of thick sams owing to the variation of electronic coupling, in a nice agreement with theoretical predictions. h. nury , f. manon , b. arnou , m. le maire , e. pebay-peyroula , c. ebel institut de biologie structurale (ibs) cea cnrs ujf grenoble france, cea ibitec cnrs ura univ. paris-sud gif-sur-yvette france adp/atp carriers (aacs) are major and essential constituents of the inner mitochondrial membrane. they drive the import of adp and the export of newly synthesized atp. they were described as functional dimers from the s until the structures of the aac shed doubt on this consensus. we aimed to ascertain the published biophysical data claiming that aacs are dimers and to characterize the oligomeric state of the protein before crystallization. analytical ultracentrifugation sedimentation velocity experiments clearly show that the bovine aac is a monomer in -laurylamido-n,n dimethylpropylaminoxide (lapao), whereas in triton x- and reduced triton x- , higher molecular mass species can also be identified. neutron scattering data for monomeric bovine aac in lapao does not give definite conclusions on the association state, because the large amount of detergent and lipids is imperfectly matched by contrast methods. we discuss a possible way to integrate previously published biochemical evidence in favor of assemblies, the lack of well-defined multimers that we observe, and the information from the high-resolution structures, considering supramolecular organizations of aacs within the mitochondrial membrane. local anaesthetic binding to shaker channels: role of aromatic residues j. nilsson, h. ullman, k. sahlholm, p. arhem the nobel institute for neurophysiology, department of neuroscience, karolinska institutet, se- stockholm, sweden local anaesthetic, antiepileptic and antiarrhythmic drugs acting on nav and herg channels have been assumed to bind to aromatic residues in the internal vestibule; to f and y in nav (ragsdale et al., and to y and f in herg (mitcheson et al., ) . despite a lack of such residues in kv channels, local anaesthetics, antiepileptic and antiarrhythmics bind to kv channels with a considerable affinity. to explore the role of aromatic residues for the binding we investigated the effect of bupivacaine, benzocaine, phenytoin and quinidine on shaker channels mutated to residues corresponding to the most c-terminal of the two aromatic residues in the s segment of the nav and the herg channels, (v y and p f respectively). the channels were expressed in xenopus oocytes and the currents measured with the two-electrode voltage-clamp technique. the results suggest that aromatic residues do not increase the binding affinity of the studied compounds to kv channels. rather, the affinity decreases (as reflected in typical k d values for bupivacaine on v f, p f and wildtype channels, being , and µm, respectively). thus, aromatic residues seem not to be necessary for high-affinity binding of the studied compounds to kv channels. how this relates to their suggested roles in the nav and herg channels, remains to be evaluated. (molina et al, ) . the occurrence of a similar behavior in other channels points out to clustering and coupled gating as a potentially important drug target to modulate channel activity. we have identified molecular determinants involved in single and coupled channel gating in kcsa. first, we detected that clustering and coupled gating of kcsa is modulated by anionic lipid. also, a model for the interaction between two kcsa open channels was built. the docking predicts intermolecular sites which includes the non-annular lipid binding site. this explains how an excess of anionic lipid disrupts interactions between channels, destabilizing clustering and coupled gating in kcsa. in addition, the docking model reveals molecular determinants involved in single and coupled channel gating. this interaction involves w , which affects the neighbouring channel through specific interactions in the extracellular mouth stabilizing the selectivity filter in an open conformation. the coupled gating is also explained since this mechanism affects the opposite channel in a mutual manner. finally, mutants kcsa e a and kcsa w a disrupt the coupled gating of kcsa, thus, supporting the model. we think that this coupled gating phenomenon could correspond to the second gate previously detected by fluorescence methods (blunck, et al, ) . supported by grants from the spanish bfu - /bmc and csd - . a. kumar, e. hajjar, p. ruggerone, m. ceccarelli university of cagliari, monserrato, italy the striking presence of outer membrane (om) in gramnegative bacteria of e.coli represents a strong barrier for any molecule to penetrate inside bacteria. in particular for β-lactam antibiotics, which have their target located inside the bacteria; the first step towards reaching inner part of bacterium is the cellular uptake. ubiquitous presence of porins (such as for instance ompf, ompc) in the om, function as a channel facilitating the transport of molecules (such as, for instance antibiotics) across the om. bacteria can exhibit resistant towards antibiotics by: (i) decreasing their uptake by under-expressing the porins or/and (ii) production of inactivating enzymes such as ß-lactamases. to combat the latter mechanism ß-lactamase inhibitors (such as, sulbactam for instance) are prescribed together with the antibiotics. like the antibiotics, the inhibitors must penetrate the om, the main path being through porins. it is thus evident the biological relevance of investigating the mechanisms by which porins can regulate entry/exit across the om. to achieve this goal, molecular dynamics simulations were performed to explore the structure and dynamics of pores formed by ompf and ompc porin. from the analysis of data obtained from our simulations, we identified the key residues buried behind the l loop, which may play be crucial for porins to exert their biological role. as a case study, we report results about the diffusion of sulbactam through the two porins. the pentadecapeptide gramicidin forms a cation-specific ion channel in membrane environment. the two main conformations are the head-to-head helical dimer (hd) known as the channel conformation and the intertwined double helical form (dh) often referred to as non-channel conformation. in this comparative study [ ] , the energetics of single potassium ion permeation by means of the potential of mean force (pmf) for both gramicidin conformations embedded in a dmpc bilayer has been addressed by molecular dynamics simulations. a significantly decreased free energy barrier by ∼ kj/mol for potassium ion passage through dh as compared to hd is reported. favorable electrostatic side chain-cation interactions in hd are overcompensated by phospholipid-cation interactions in dh. the latter are coupled to an increased accessibility of the channel entrance in dh due to distributed tryptophans along the channel axis. this result underscores the importance of the lipid environment of this channel not only for the equilibrium between the different conformations but also for their function as cation channels. y. sawada , m. murase , m. sokabe dept. physiol. nagoya univ. grad. sch. med., nagoya, japan, icorp/sorst cell mechanosensing, jst, nagoya, japan the bacterial mechanosensitive channel of large conductance mscl is constituted of homopentamer of a subunit with two transmembrane inner and outer α-helices, and its d structure of the closed state has been resolved. the major issue of mscl is to understand the gating mechanism driven by tension in the membrane. although several models for the opening process have been proposed with molecular dynamics (md) simulations, as they do not include mscllipid interactions, it remains unclear which amino acids sense membrane tension and how the sensed force induces channel opening. we performed md simulations for the mechano-gating of mscl embedded in the lipid bilayer. upon tension in the bilayer, phe in the outer helix was dragged by lipids, leading to a tilting of the helices. among amino acids in the outer helix facing the bilayer, phe at the water-lipid interface showed the strongest interaction with lipids, thus may work as a major tension sensor. neighboring inner helices cross each other in the inner leaflet, forming the most constricted part of the pore. as tension increases, the crossings move toward the cytoplasm associated with an expansion of the constricted part. during the movement, a hydrophobic water block environment around the constricted part was broken followed by water penetration and permeation. the k + channel kcsa is an integral membrane protein from s.lividans, used as a model system for studies on ion channels and oligomeric membrane proteins. its atomic structure has been solved by x-ray diffraction, which shows an assembly of four identical subunits around a central aqueous pore, including the so-called selectivity filter. this channel is able to permeate k + at high flux rate and it is blocked by na + (physiological blocker). fluorescence, circular dichroism and fourier transform infrared experiments carried out in our laboratory demonstrated that k + and na + are able to bind to kcsa in a competitive manner. this binding is indeed associated with channel conformational changes, which seem to be related to the permeation and blockade processes. to further investigate this phenomenon we carried out a detailed study of chemical and thermal denaturation of wild-type and mutant kcsa channels. these two types of experiments were in agreement and indicate that both cations are able to stabilize the channel through conformational changes, being k + the more efficient one, even more when lipids are present. particularly, mutant channels with a structurally altered selectivity filter show that ion and lipid-induced global conformational changes are intimately associated to the conformation of this selectivity filter (conductive and non-conductive forms). modulation of the voltage-gated sodium channel nav . by rcssii, a toxin from the scorpion centruroides suffusus c. picco , g. corzo , l. d. possani , g. prestipino institute of biophysics, cnr, genova, italy, instituto de biotecnologia, unam, cuernavaca, mexico the main cardiac voltage-gating sodium channel, na v . , generates the fast depolarization of the cardiac action potential and plays a key role in cardiac conduction. its importance for normal cardiac function has been exemplified by the description of numerous naturally occurring genetic variants of the gene scn a, which encodes na v . , that are linked to various cardiac deseases. subsequently, studies of this channel localization have led to its identification in immature and denervated skeletal muscle and in the brain neurons. in our effort to identify high affinity ligands for this channel, we have investigated the effects of the recombinant cssii (rc-ssii), a four disulfide-bridged scorpion toxin isolated from the venom of the scorpion centruroides suffusus. human cardiac sodium channel α subunit scn a was expressed in cho cells and macroscopic na + currents were recorded with patchclamp technique in whole cell configuration. the electrophysiological experiments have highlighted a strong affinity for the channel at low nanomolar concentration. compared with control conditions, rcssii toxin affects in reversible way the kinetics of activation and inactivation and marked decrease the peak na + influx. the extrusion mechanism of substrates in rnd family efflux pumps: a molecular dynamics study a. v the rnd transporters of the acrab-tolc (e.coli) and mexab-oprm (p.aeruginosa) systems are able to export structurally and chemically different substrates outside bacteria through the membrane, being responsible of multidrug resistance. on the basis of crystallographic information, an extrusion process conceived as a three-cyclic peristaltic pumping has been proposed, but further microscopically well-funded investigations are needed to understand the mechanism. using different computational methods like adaptive bias force (abf) and targeted molecular dynamics (tmd), we have investigated the mechanism of substrate uptake and pumping at a molecular level. with the first method we have investigated the passage of antibiotics from the periplasm into the internal pore of the pump, while tmd has been used to assess the effect of conformational changes on the extrusion of drugs (which have been located into one of the proposed binding pockets). comparison between the active pumps acrb and mexb (which show different resistance patterns despite their homology) provide insights into the microscopic details of their functioning. in arabidopsis thaliana there are twenty genes, grouped into three subfamilies, encoding for homologues of animal ionotropic glutamate receptors (iglrs). each protein displays a pore-forming loop, flanked by two conserved helices (plus a third c-terminal helix), a glutamate-binding domain and an n-terminal region. through pharmacological and/or genetic approaches, many physiological functions have been attributed to plant glurs, such as the regulation of cytosolic calcium, photomorphogenesis, water balance and carbon/nitrogen sensing and assimilation. according to the endosymbiotic theory, the cyanobacteria are considered to represent the precursors of the present chloroplasts. the first prokaryotic glutamate receptor (glur ) was identified in the cyanobacterium synechocystis. the putative products of the atglr . and atglr . genes display a possible targeting sequence for chloroplast location and show a high degree of homology with glur . using specific antibodies and confocal microscopy, we have localized the two members of the atglr subgroup , glr . and glr . (splicing variant) , to the chloroplast in arabidopsis and to the inner envelope membrane in spinach. electrophysiological experiments indicate the presence of an activity which is compatible with that of glutamate receptors. furthermore, oxygen evolution measurements suggest that chloroplast-located glutamate receptors may play a role in the regulation of photosynthesis. under extreme conditions many cells control their volume responding to osmotic challenges by unloading or loading solutes to recover their original volume. a faster volume regulatory role triggered by membrane tension has been disclosed for aquaporins in kidney proximal tubule cells, where aqp is the main water channel, using isolated brush border membranes. in conventional osmotic studies in animal cells it is common to disregard internal hydrostatic pressures because they are insignificant compared to osmotic forces. however, by using low osmolarity buffers in small radii vesicles, we detected a rise on the internal pressure that creates surface tension and causes membrane stress, with a negative outcome on aquaporin water permeability. these findings suggested a mechanism for volume regulation in kidney proximal tubule epithelia where massive solute and fluid transport occurs. to further explore aquaporin regulation by membrane tension, yeast cells were used as a model that could bare surface tension some orders of magnitude higher than animal cells due to the existence of a cell wall. the effect of increasing levels of membrane tension on yeast water channel activity was evaluated. an impairment of aquaporin activity correlated with the increase of membrane tension corroborates the volume regulatory role of aquaporin in different cells. deuterium isotope effects on fast gating of the chloride channel clc- g. zifarelli, a. r. murgia, p. soliani, p. michael istituto di biofisica, cnr, via de marini, , i- genova, italy gating of the torpedo cl − channel clc- is modulated by intracellular and extracellular ph, but the mechanism responsible for this regulation has remained so far elusive. using inside-out patch clamp measurements we studied the dependence of the fast gate on ph int and [cl − ] int . only the closing rate, but not the opening rate showed a strong dependence on these intracellular factors. using mutagenesis we excluded several candidate residues as mediators of the ph int dependence. we propose a model in which a proton generated by the dissociation of an intrapore water molecule protonates e leading to channel opening. deuterium isotope effects confirm that proton transfer is rate limiting for gate opening and that channel closure depends mostly on [oh − ]. the model is in natural agreement with the finding that only the closing rate constant, but not the opening rate constant, depends on ph int and [cl − ] int . deletion of the c-terminus destabilizes phosphorylated na/k pump state containing na ions n. vedovato, d. c. gadsby the rockefeller university, new york, u.s.a. the na/k pump's extended c-terminus (compared to the serca ca pump's) links transmembrane helices, and its truncation lowers cytoplasmic na affinity for forming the occluded e p(na ) state. here we test the effects of c-terminal truncations on interactions with external na. we deleted the last (yy) or (kesyy) residues in xenopus α β pumps made ouabain resistant by mutations q r-n d (rd) or c y (c-y), and then used two-microelectrode voltageclamp recording in xenopus oocytes to measure pump currents as mm ouabain-sensitive currents while endogenous na/k pumps were silenced with µm ouabain. inhibition by external na of steady outward pump current ([k] o = mm) at large negative voltages was somewhat weaker in both rd and c-y pumps than in wt pumps, but was severely impaired in all c-terminal truncated pumps. consistent with this, the voltage dependence of transient charge movements under na/na exchange conditions ([k] o = mm) was strongly shifted to more negative potentials in the truncated pumps relative to the parent rd or c-y pumps, shifts comparable to those seen in wt pumps on decreasing [na] o several-fold. together, the results suggest that these c-terminal deletions lower the apparent affinity for external na ions to bind and become occluded in the na/k pump. the c-terminus therefore provides contacts important for stabilizing the occluded e p(na ) conformation, regardless of the route of na ion entry into the binding pocket. muscle contraction is driven by molecular motors that adapt their energy utilization according to the demands made on them. we test the hypothesis that rate constants controlling the biochemical steps involved in atp hydrolysis by myosin atpase are affected by the force of the muscle. here we use fluorescence lifetime imaging microscopy (flim) of a fluorescently labelled atp analogue to investigate changes in the environment of the myosin atpase, caused by different loads applied to skeletal muscle. single muscle fibres were subjected to cycles of stretches and releases in the presence of rigor solution and µm of coumarin-labelled atp. flim acquisition was synchronised with stretch/release cycles and force measurements, which allow us to investigate the effect of strain on the lifetime of the labelled atp bound to the actomyosin complex. characterization of the fluorescence decay by a bi-exponential function resolved the time constant of two populations, namely, free fluorophore (τ = . ± . ns; mean ± s.d.) and fluorescent nucleotide bound to the actomyosin complex (τ = . ± . ns at low strain). these experiments showed that while the time constant of the free fluorophore did not change with force, the time constant of the fluorescent nucleotide bound to actomyosin showed a linear dependence with the force applied to the muscle of . ± . ps/kpa. neck linker docking coordinates the kinetics of kinesin´s heads i. derenyi, a. czovek, g. j. szollosi department of biological physics, eotvos university, pazmany p. stny. a, h- budapest, hungary conventional kinesin is a two-headed motor protein, which is able to walk along microtubules processively by hydrolyzing atp. its neck linkers, which connect the two motor domains and can undergo a docking/undocking transition, are widely believed to play the key role in the coordination of the chemical cycles of the two motor domains and, consequently, in force production and directional stepping. although many experiments, often complemented with partial kinetic modeling of specific pathways, support this idea, the ultimate test of the viability of this hypothesis requires the construction of a complete kinetic model. considering the two neck linkers as entropic springs that are allowed to dock to their head domains and incorporating only the few most relevant kinetic and structural properties of the individual heads, we have developed the first detailed, thermodynamically consistent model of kinesin that can (i) explain the cooperation of the heads during walking and (ii) reproduce much of the available experimental data (speed, dwell time distribution, randomness, processivity, hydrolysis rate, etc.) under a wide range of conditions (nucleotide concentrations, loading force, neck linker length and composition, etc.). besides revealing the mechanism by which kinesin operates, our model also makes it possible to look into the experimentally inaccessible details of the mechanochemical cycle and predict how certain changes in the protein affect its motion. the positive role of noise on the transport efficiency of na, k atpase c.-h. chang , t. y. tsong institute of physics, national chiao tung university & physics division, national center for theoretical sciences, hsinchu, taiwan, institute of physics, academy of sciences, taipei , taiwan na, k atpase is a molecular motor which is able to transport ions through cell membranes, even against the transmembrane ion concentration gradient. while in vivo this nanoscale soft machine consumes atp, it may be driven by external fluctuating electric fields, no matter they are periodic or random. theoretically, the motor conformations can be described by a conformation vector v(t) governed by a multi-dimensional kinetic equation. given an oscillating electric field with a slight fluctuation, the boltzmann distributions of these conformations will change with time. the instantaneous transported ion flux is a functional of the quasi-cyclic trajectory v(t) of this non-autonomous dynamical system. various interesting dynamical properties of this ion pump, including stochastic resonance, can be studied theoretically, some of which have good agreement with recent experimental findings. in situ measurements of the molecular motor of muscle with nanometer-microsecond resolution in a contracting muscle, arrays of the dimeric motor protein myosin ii pull the actin filament towards the centre of the sarcomere during cyclical atp driven interactions. when the external load is smaller than the array force, the sarcomere works as a motor, converting metabolic energy into mechanical work; when the external load is larger than the array force, the sarcomere acts as a brake resisting the load with reduced metabolic cost. to investigate the molecular basis of the work production and the braking action of muscle, we use sarcomere-level mechanics and x-ray interferometry in intact single cells from frog skeletal muscle. during isometric contraction, each motor bears a force of about pn. during shortening against high and moderate loads, the number of myosin motors attached to actin reduces in proportion to the external load while the force per attached motor is maintained similar to the isometric value (piazzesi et al., cell , - , ) . rapid stretches of - nm between each overlapping set of myosin and actin filaments in a muscle sarcomere cause the stiffness of the array of myosin motors to increase up to twice the isometric value within ms (brunello et al., pnas usa , - , ) , indicating that the high resistance of active muscle to stretch is due to recruitment of the second motor domain of the myosin molecules with the first domain already attached to actin. supported by miur and ente crf (italy), nih (usa), mrc (uk), embl, esrf. kinesin- is a molecular motor that moves cellular cargo along microtubules. its functional mechanism is well understood for individual motors. however, the way that many kinesin- motor proteins bound to the same cargo move together is not. we addressed the structural basis for this phenomenon using video microscopy of single microtubulebound full-length motors and various spectroscopy methods were employed to study synthetic peptides derived from hinge- region. these peptides show an unexpected profile of secondary structure forming propensities. video microscopy of single microtubule-bound full-length motors reveal the sporadic occurrence of high compliance states alternating with longer-lived, low compliance states. the deletion of hinge- abolishes transitions to the high compliance state. from the results we hypothesize that strain accumulated during multiple kinesin motility populates the high compliance state by unfolding helical secondary structure in the central hinge domain flanked by unordered regions, thereby preventing the motors from interfering with each other in multiple motor situations. titin is a giant protein of vertebrate skeletal and cardiac muscles. cardiac titin is expressed in two main isoforms: short n b (∼ kda) and long n ba (∼ kda). we have studied changes of titin isoform composition in myocardium of hibernating ground squirrels and spontaneously hypertensive rats (shr). using electrophoresis we have revealed considerable decrease (by - times) in the content of titin relative to myosin heavy chains in shr heart as compared with that for normotensive rats. surprisingly that the data of qrt-pcr showed the increase in mrna content of n ba and n b-isoforms in hypertrophic heart more than times in comparison with norm. we suppose that such a result is an effect of depressed translation of mrnatitin in pathology. we have observed the decrease (by , times) of total titin amount in heart of hibernating animals in comparison with that for summer active animals. however n ba/n b ratio in the heart upon hibernation was increased by times. similar trend was not revealed for the mrna level of corresponding isoforms, although we have showed the decrease of mrna of both titin isoforms in heart of hibernating ground squirrels as compared to their content of summer animals. the decrease in total mrna level may be explained by repressed transcription or mrna degradation in the cell during hibernation. these discrepancies in protein and mrna levels may be considered as the posttranscriptional regulation of titin isoforms expression. actomyosin cross-bridges formed when the globular heads of myosins bind to actin filaments are the molecular engines that drive muscle contraction, fuelled by atp hydrolysis. critical to this process is the change in shape of the cross-bridge and the change in the interactions with actin, in response to force applied to the muscle, and to the status of the nucleotide in the binding pocket. although molecular detail is known from x-ray crystallography and biochemistry, understanding of the interplay between cross-bridge shape and chemical state requires studies in muscle fibres generating force. we use fluorescence life time imaging microscopy (flim) as a probe of the cross-bridge environment.with a fluorescent analogue of atp , fluorescence life-time (flt) changes when the crossbridge binds to actin. now, we show preliminary experiments on the effect of force on flt. the essential light chain of myosin (elc) is a ∼ kda peptide that wraps around a nmlong α-helix of the myosin cross-bridge known as the lever arm which tilts during force generation. using a recombinant elc, labelled with a fluorophore at a strategic cys, we replace the native elc and introduce the fluorescent elc in muscle fibres. preliminary experiments demonstrate that the elc fluorophore also is sensitive to force applied to the muscle fibre. in addition, förster resonance energy transfer occurs between the nucleotide and elc fluorophores, opening the way for studying structural changes in cross-bridges during force generation by fret. muscle contraction: pitfalls in the determination of the contractile response e. grazi dipartimento di biochimica e biologia molecolare, università di ferrara, ferrara, italy the contractile response of an active muscle depends on the load. the load is a force /cross-section. there are three fundamental dimensions: the mass, m; the space, l; and the time, t. from these three dimensions are built up all the physical dimensions. as an example the acceleration, a, is given by, a=l.t − . once the direction and versus are settled the modulus fully defines the physical effect of the acceleration. what about the force? the force, f, is given by, f=m.a. at variance with the acceleration, once the direction and versus are settled, the modulus does not define the physical and the biological effects of the force: the same force is generated by an infinite number of mass-acceleration couples that display different physical and biological effects. the same occurs with the load. thus defining the load that opposes the contractile force does not define the contractile system. in the studies on muscle contraction the acceleration of the load is not considered nor it is provided a way to extract this information. thus these systems are poorly defined from the physical as well as from the biological point of view. models of muscle contraction that consider explicitly both the mass and the acceleration of the load show that, at the same load, the decrease of the acceleration of the load significantly delays the pre-steady state of the contraction and decreases the stiffness of the active fibre. r. shahapure , f. difato , a. laio , d. cojoc , e. ferrari , j. laishram , g. bisson , v. torre int. school for advanced studies, trieste, italy, iit-sissa unit, trieste, italy, lab. nazionale tasc, trieste, italy polymerization of actin filaments is the main source of motility in lamellipodia and is controlled by many regulatory proteins. the underlying molecular mechanisms are only partially understood and now a determination of the dynamical properties of force generation is needed. using optical tweezers we measured with millisecond temporal resolution and pn sensitivity the force-velocity (fv) relationship and the power dissipated by lamellipodia of dorsal root ganglia neurons. when force and velocity are averaged over - s, fv relationships can be flat. on a finer time scale, random occurrence of fast growths and sub-second retractions become predominant. maximal power dissipated by lamellipodia over a silica bead with a diameter of µm is − w. due to the presence of adhesion forces, beads in close contact with a lamellipodium can seal on its membrane reducing the amplitude of brownian fluctuations often by more than times. under these conditions, when lamellipodia grow and push the beads, discrete jumps varying from about to nm are detected. when lamellipodia retract, pulling the beads, no discrete events are observed. our results on the dynamical properties of force generation are: a) force generation is a probabilistic process; b) underlying biological events have a bandwidth up to at least hz; c) fast growths of lamellipodia leading edge alternate with local retractions; d) force generation is produced in discrete steps with varying amplitude up to . pn. pushing on microtubules: dominant spindle centering mechanism in c. elegans embryo? j. pecreaux, s. redemann, a. a. hyman, j. howard mpi-cbg, pfotenhauerstr , dresden, germany asymmetric cell division, where the content of the two daughter cells -as well as their sizes -differ, is found in many organisms. strikingly, the spindle, first centered, starts to be displaced out of the center only in late metaphase. in c elegans embryo, the spindle rocks and is posteriorly displaced during anaphase by force generators asymmetrically localized on cell cortex. prior to anaphase onset, the spindle is usually assumed to be centered by the same pulling force. it thus requires the force generators to be carefully repressed to distribute forces symmetrically. on live embryos, we measured positional fluctuations of centrosomes during metaphase with nm accuracy. fourier analysis shows an extremely accurate centering respect to the number of force generators and microtubules. furthermore, spectrum is close to a lorentzian, modeled by a spring and a dashpot, suggesting a spindle centering more likely by pushing on microtubules than pulling. deviation at high frequencies indicates a subdominant pulling force. rnai of gpr- / , known to control force generation, increases slightly centering accuracy; this result supports the hypothesis of an independent centering mechanism. conversely, zyg- (rnai), a microtubule growing factor, decreases centering accuracy, modeled spring stiffness and damping modulus. conclusion: first, the spindle centering mechanism is independent of cortical pulling force generator. second, microtubules pushing is likely to center the spindle. mechanical forces are important in the regulation of cellular adhesion and migration. the focal adhesion kinase (fak) has been suggested to transduce cellular forces and govern cell migration. to obtain more insight in the functioning of fak, a fret-based optical biosensor for fak was designed to relate integrin-mediated conformational changes in its ferm domain to focal adhesion behavior during cell spreading and migration in living cells. imaging of the kinetics of ferm-based fak conformational changes in spreading cells revealed two consecutive stages of focal adhesion activation. heterogeneous ferm conformational responses were observed in individual focal adhesions of adherent motile cells, with the active ferm conformation being enriched in growing and sliding fas, but not in stable and shrinking focal adhesions. inhibition of the cellular actomyosin system revealed the involvement of rho-rock rather than mlckinduced tension signaling in the modulation of the ferm response. our results place the ferm conformational change of fak at the interface between integrin and force sensing. the time course of inorganic phosphate release in permeabilized cardiac trabeculae of the rat c. mansfield, t. west, m. a. ferenczi imperial college london, u.k. the rate of p i release was determined in permeabilized rat trabeculae. contraction was elicited at • c by laser-flash photolysis of npe-caged atp, and time-resolved p i release was monitored using mdcc-pbp, a coumarin-labelled phosphate binding protein, which increases its fluorescence intensity five-fold upon p i binding. the atpase rate during the first turnover of the total crossbridges (assuming µm myosin heads) was s − . the rate decreased to a steady state of s − after the eighth turnover ( . - . s after activation). this steady state rate is comparable to published values of - s − , made ∼ s after activation using an nadh-linked enzyme assay of adp release. the advantage of using mdcc-pbp is that the control of mechanochemical coupling can be examined from the onset of force production and as it progresses toward the steady state. force production and p i release were simulated using a seven step scheme. force was attributed to the states in the sequence a.m.adp.p i ↔ a.m.adp ↔ a.m.adp, with strain sensitivity incorporated into the isomerisation of a.m.adp. the a.m.adp.p i and a.m.adp states populated rapidly as force was increasing. in contrast, the a.m.adp state accumulated slowly after the force plateau was reached and became the dominant force bearing state at the time of the eighth crossbridge turnover. experiments are on-going to examine how the distribution of a.m states changes in response to rapid length-changes. pulling as a factor in forming the heterophasic structure of immunoglobulin proteins structure of proteins of immunoglobulin superfamily: human igg kuc and muscle protein titin, has been investigated by methods of electron microscopy and diffraction with the use synchrotron radiation. super elasticity of titin, the protein of immunoglobulin superfamily, is a key parameter that determines the mechanical properties of muscle. however, the structural-physical mechanism of titin elasticity under tension remains poorly understood. here both tension transduction and high elasticity of titin are explained in terms of crystalline polymer physics. x-ray data suggest a model of titin as a nanoscale, morphological, aperiodical array of rigid ig-and fn -type domains covalently-connected by conformationally variable short loops. the line group symmetry of the model can be defined as s m with axial translation τ ∞ . homologous domains would have similar stability, but the structure of different domains on stretching is subject to different forces because they have different orientations relative to the axis of the molecule. under the force influence the structure of any domain can become either rigid or flexible depending on its orientation in the titin strand. pulling geometry forms an active axial structure from latent isotropic random coil structure of titin strand. we are suddenly faced with nanophase-separated morphology of igg kuc. study was supported by rfbr grant - - . strain response of myosin essential light chain in permeabilized skeletal muscle fibres d. s. ushakov, d. ibanez-garcia, t. g. west, p. m. w. french, m. a. ferenczi imperial college london, uk we applied fluorescence lifetime imaging microscopy (flim) to investigate the relation between conformation of myosin head and mechanical force in skeletal muscle fibres. recombinant myosin essential light chain (elc) was expressed in e.coli and labelled at cys- with coumarin. the labelled elc was exchanged with native elc in single permeabilized rabbit m.psoas fibres. fluorescence lifetime was measured using leica sp upright confocal microscope equipped with becker & hickl time-correlated single photon counting module and x . na leica planapo dipping objective, with the two-photon fluorescence excitation at nm by sapphire pulsed laser. after acquiring flim images of muscle fibres in relaxed state, the solution was changed to ca-free rigor. further images were acquired in rigor with or without . - % stretch applied by a motor. both single and double exponential fluorescence decay analysis showed that the lifetime in rigor was lower compared to relaxed (about ps difference for single exponential fit) and to rigor fibres under strain (about ps). these data suggest a change in the microenvironment of coumarin induced by nucleotide binding and strain. this change is likely to be due to interaction between c-terminal domain of elc and the n-terminal domain of myosin heavy chain related to the lever arm re-orientation process. supported by bbsrc. alpha-synuclein and its a p mutant affects the actin cytoskeleton structure and dynamics v. sousa , s. bellani , g. ronzitti , f. valtorta , j. meldolesi , e. chieregatti department of neuroscience, hsr, milano, italy, department of neuroscience, iit, genova, italy alpha-synuclein (syn) is a soluble protein abundant in the brain, primarily enriched at pre-synapses. syn overexpression and the expression of its a p mutant participate in the pathogenesis of parkinson's disease. many roles have been proposed for syn, including the regulation of synaptic vesicle pools and of neurotransmitter release. the actin cytoskeleton regulates many aspects of synaptic function and its dysregulation may be a cause of neurodegeneration. working both in cell-free and in vivo conditions we demonstrate that syn and the a p mutant have different effects on the actin cytoskeleton dynamics. our results show that syn binds actin, and decreases actin polymerization rate probably by monomer sequestration. on the contrary, a p accelerates actin polymerization in vitro and disrupts the cytoskeleton of intact cells. in particular, during dynamic cytoskeleton remodeling, a p induces the assembly of discrete actin-rich foci. actin trapping and the impairment of filaments reassembly lead to inhibition of cell movement and of the re-establishment of cell-cell contacts. in a p expressing cells cytoskeleton-based processes, such as cell migration and the exo/endocytic traffic are inhibited. elucidating the dynamics of syn interaction with actin may contribute to the understanding of its role in neuronal physiology as well as in neurodegeneration. on the physics of muscle contraction m. l. shur ural state university, yekaterinburg, russian federation whichever energy source is chosen as an engine, its force will decrease with increasing velocity. this is connected with a limited power of any engine. thus, we state that hill's formula is a mere sequence of the law of energy conservation. to derive a mathematical dependence "force-velocity", all the means of consumption of fuel energy should be determined -in our case, the energy of the atp hydrolyze. moreover, the conformation energy of the crossbridges attached serves as the force source as well. we state that part of the energy release transforms into the energy of oscillations of myosin proteins; the other part goes into thermal energy of the sarcoplasmic solution. interaction of the oscillating myosin system with the sarcoplasmic solution controls the process of force generation by a muscle. it is just this interaction that leads to the temperature dependence of force. the presentation is devoted to constructing a theory based on these simple considerations. a discussion and constructive critic is especially wanted. -biological motility and molecular motors - meiotic nuclear oscillations in the fission yeast schizosaccharomyces pombe are crucial for proper chromosome pairing and recombination. we report a mechanism of these oscillations based on collective behavior of dynein motors linking the cell cortex and dynamic microtubules that extend from the spindle pole body in opposite directions. by combining quantitative live cell imaging and laser ablation with a theoretical description, we show that dynein dynamically redistributes in the cell in response to load forces, resulting in more dynein attached to the leading than to the trailing microtubules. the redistribution of motors introduces an asymmetry of motor forces pulling in opposite directions, leading to the generation of oscillations. our work provides the first direct in vivo observation of self-organized dynamic dynein distributions, which, due to the intrinsic motor properties, generate regular large-scale movements in the cell ( ) m. versaevel, s. gabriele, p. damman university mons-hainaut, mons, belgium the remodeling of blood vessels in response to changes in blood flow is mainly realized by endothelial cells (ecs) that convert mechanical stimuli from flowing blood into changes in cell signaling through a process called mechanotransduction. many of the biological responses to external forces originate at two types of microscale structures: focal adhesions linking cells to their extracellular matrix and adherens junctions that link adjacent cells. this study aims to elucidate the role of the cytoskeleton, cell-matrix and cell-cell junctions in transducing fluid shear stress into intracellular signals in ecs. by using microcontact printing of proteins, we design substrates with defined adhesive islands in order to control shapes of living cells. this confinement of ecs allows to study the organization and the contractile activity of the cytoskeleton in order to redistribute their intracellular forces in response to externally applied forces. we design microfluidic channels with sizes and geometries close to small blood vessels to apply a physiological range of shear stresses on ecs. our results indicate that cells deposited on a precisely defined adhesive area inside microchannels and subjected to shear stress reorganize their cytoskeleton, their focal adhesions and adherens junctions in response to blood flow. drugs interfering with the cytoskeleton are used to underline the role of its different components in the cellular adaptation to the mechanical environment. s. mahdavi , b. ranjbar , s. gharibzadeh , m. toosi , m. javan tarbiat modares university, tehran, iran, amirkabir university of technology, tehran, iran multiple sclerosis (ms) is the main known pathology of myelinating cells. an autoimmune reaction occurs against myelin sheets of neurons, so action potential (ap) propagation along the affected nerve fibers has been destroyed and it causes various disorders. here, we propose a novel strategy for ms symptoms treatment. we modeled neuron by orcad software and simulated the action potential propagation along the axon in normal condition. our model simulated normal neuronal behavior. then we destroyed the myelin sheet as it occurs in ms and observed destroyed ap propagation as it was reported in ms disease. we investigated the effect of changes in the voltage-gated sodium channel (vgsc) threshold on the efficiency of ap propagation. the results demonstrated that reduction of vgsc threshold improves the propagation of ap by increasing the amount of sodium flux during ap propagation. although, some researches have proposed vgsc blocker as ms symptom treatment, our result suggests that the increase of sodium current produced by reduction of vgsc threshold, improves ap propagation and probably cure some ms symptoms. so, we suggest that vgsc gating modifiers can be considered as novel strategy for ms treatment. surely, this results needs to be confirmed by experimental studies. a. gradogna, e. babini, a. picollo, m. pusch istituto di biofisica, consiglio nazionale delle ricerche, via de marini , genova, italy clc-ka and clc-kb are highly homologous cl − channels expressed in the kidney and the inner ear where they mediate transepithelial chloride transport. both channels heteromerize with the beta subunit barttin. mutations in clc-kb and barttin genes lead to bartter's syndrome. we analyzed the modulatory effect of extracellular ca + and h + on clc-k channels using the xenopus oocyte expression system. clc-ka currents increased with increasing [ca + ] ext without full saturation for [ca + ] ext up to mm. however, in the virtual absence of ca + , clc-ka currents are about % of currents measured in mm [ca + ] ext , demonstrating that ca + is not strictly essential for opening. vice versa, clc-ka was blocked by increasing the [h] + ext with an almost complete block at ph . among various reaction models tested, the model that best fitted all state-steady data predicts an allosteric regulation of channel opening by separate binding sites for ca + and h + . moreover, the best fit suggests that one ca + and two h + bind to the channel. kinetic analysis of current responses upon [ca + ] ext and ph jumps confirmed the allosteric character of modulation. in support of the presence of two separate binding sites we identified several mutations that selectively altered ca + or h + sensitivity. our data represent a first step towards a molecular picture of ca + and proton regulation of clc-k channels and suggest that it is of physiological relevance. the extracellular matrix molecule hyaluronic acid modulates l-type voltage-dependent ca + channels e. dvoretskova , g. kochlamazashvili , o. bukalo , c. henneberger , d. rusakov , m. schachner , a. dityatev italian institute of technology, genova, italy, centre for molecular neurobiology, hamburg, germany, institute of neurology, university college london, london, uk we studied the effects of hyaluronic acid (ha), a major extracellular matrix molecule, on activity of l-vdccs in a heterologous expression system and in hippocampal slices. we recorded currents mediated by a major neuronal subtype of l-vdccs (ca v . c, β b, and α δ ) expressed in cho cells. a five-minute application of . mg/ml ha potentiated l-vdcc currents at - , - , and + mv by approximately %. analysis of boltzmann curves showed that ha increased maximal conductance rather than other parameters (v . or k ). treatment with hyaluronidase removed endogenous ha in murine hippocampal slices and specifically impaired long-term potentiation (ltp) induced at ca -ca synapses by repetitive theta-burst stimulation. blockade of l-vdccs reduced ltp in control slices to the levels seen after hyaluronidase treatment. a potentiation of l-vdccs with bay k fully restored ltp after hyaluronidase treatment. removal of ha reduced ca + transients elicited by backpropagating action potentials in individual dendritic shafts and spines of ca pyramidal cells, whereas pretreatment with nifedipine fully occluded this effect. thus, ha potentiates postsynaptic l-vdccs and by this way influences use-dependent synaptic plasticity. whole-cell patch clamp recordings from a variety of human cancer cells showed that functional voltage-gated sodium channel (vgsc) expression occurred specifically with strongly metastatic cells. in addition, where studied, this was accompanied by down-regulation of outward (mainly potassium) currents. this has led to the celex ("cellular excitability") hypothesis of cancer according to which metastatic cell membranes are excitable and this promotes their hyperactivity. importantly, the vgsc genes expressed are embryonic splice variants, which are normally developmentally regulated, hence the phenomenon is 'oncofetal'. in breast cancer, where the predominant vgsc is nav . the neonatal and adult forms are significantly different and this is reflected in channel activity whereby the neonatal vgsc has much slower inactivation kinetics. the double-charge change at position is critical for this difference. the slow kinetics results in much greater influx of na + into cells and one consequence of this is activation of protein kinase a. this is a tonic effect and, under steady-state resting conditions, it results in a positive feedback effect promoting post-translational trafficking of vgsc protein to plasma membrane. the unique amino acid sequence of the spliced region has enabled the production of a polyclonal blocking antibody specific to neonatal nav . . it is concluded that vgscs represent novel biophysical targets for clinical management of metastatic disease. m. pusch cnr, istituto di biofisica, genoa, italy clc proteins form an evolutionary conserved gene-family that comprises members in mammals. four of the human clcs are passive plasma membrane cl − ion channels. the other clcs are expressed in intracellular organelles. clc- and clc- , mutations of which lead to dent's disease, are secondary active cl − /h + antiporters, similar to the bacterial clc-ec , and with identical cl − : h + stoichiometry. cl − and h + transport activity of the exchanger clcs depends on two glut residues. mutating a 'gating glutamate' (e in clc- ) converts the exchanger into anion conductances. neutralizing the 'proton glutamate' (e ), but not its replacement by some other titratable groups, abolishes cl − and h + transport. noise analysis indicated that clc- switches between silent and transporting states with an apparent unitary conductance of . ps, indicating a very large transport turnover. no − uncouples h + transport but mutating the highly conserved s to p, as found in the plant no − / h + antiporter atclca, led to coupled no − : h + exchange. clc proteins are a fascinating example of how a very similar protein architecture can be used to provide either a passive electrodiffusive permeation pathway or a strictly coupled secondary active ion transporter. (supported by telethon italy -grant ggp ). the role of ion dynamics in zebrafish fin regeneration the specific and directional ion transport across cell membranes or tissue layers results in differential accumulation of ions and endogenous electric currents. these phenomena have been shown to be important for vertebrate organs regeneration. however, the specific ion nature of such electric currents remains unknown, as well as the role of cellular ion dynamics during regeneration and the molecular signalling pathway that transduces electric cues into cellular responses. we use zebrafish caudal fin as an adult regeneration model to unveil the specific ion composition of the currents associated with wound healing and regeneration, using a non-invasive ion-specific scanning microprobe setup. our data suggests a role for potassium (k + ), calcium (ca + ) and protons (h + ) at different stages of the regeneration process. k + and ca + extracellular effluxes have both been detected during the wound healing stage. h + efflux is triggered during wound healing and is maintained throughout regeneration. we are validating these data with genetic and pharmacological approaches, as well as advanced ion imaging. overall, our results suggest ion-driven mechanisms underlie adult tissue regeneration and its comprehension may open way for new therapeutic strategies, both in regenerative and developmental medicine and in cancer therapy. cardiac effects of anabolic steroids: an electrophysiological approach anabolic androgenic steroids (aas) have been used by athletes and non athletes for almost five decades in order to improve performance. however, the illicit abuse of high-doses of aas has been attributed as a main cause of several cardiovascular disorders such as arterial hypertension, lipid profile abnormalities, heart failure, hypertrophic cardiomyopathy, arrhythmia and sudden death. the aim of this study was to investigate qt interval and transient outward potassium current (i to ) changes in rats treated with nandrolone decanoate (deca). male wistar rats received weekly mg/kg of deca (n= ) or vehicle (control, n= ). electrocardiogram was recorded weekly, and qt interval was measured. after weeks hearts were excised and single myocytes were isolated from the ventricles of animals. i to was recorded by means of the whole cell patch clamp technique. qt interval was larger in deca group from th to th week (p < . ). analysis of i to showed a decreased current density (p < . ) in ventricular cardiomyocytes of deca group, compared to control group. in conclusion, our results show that alterations on ventricular repolarizaton may constitute an early consequence of the chronic administration of high doses of anabolic steroids in rats, and demonstrated qt prolongation and i to density reduction, which may constitute an important marker of arrhythmia vulnerability and sudden death. -ion channels in channelopathies and cancer - denitrifying bacteria control no and no cytosolic levels by regulating the expression of denitrification gene clusters via redox signalling of specific transcriptional factors that may act as no sensors in vivo. a protein belonging to the subclass dnr (dissimilative nitrate respiration regulator) from pseudomonas aeruginosa has been recently suggested to be a heme containing protein. very recently the three dimensional structure of the apo-form of dnr (in the absence of heme) has been determined by x-ray crystallography, whereas the holo-form (in the presence of heme) has not yet been crystallized. we have investigated the heme local structure in solution of ferric, ferrous, co bound and no bound holo-dnr by x-ray aborption spectroscopy (xas) and we added a kinetic study of the co bound form by means of a flash photolysis setup using uv-visible absorption as a spectroscopic probe. the combination of fe k-edge xanes fingerprints and kinetic study reveal a heme pocket able to bind exogenous ligands like no and co with increased plasticity, thus supporting its role as the cofactor involved in no sensing activity. molecular examination of motifs that lead to the formation of s-nitrosylated proteins i. alicea, e. r. schreiter university of puerto rico rio piedras, san juan, puerto rico physiologically, a wide range of proteins experience structural and functional modifications after the addition of a nitric oxide (no) moiety to cysteine thiol. this posttranslational modification, known as s-nitrosylation, regulates a large number of cellular processes like vasodilatation, cell signaling and others, and the products of s-nitrosylation can be involved during the development of different human diseases. however, little is known about the mechanism by which different proteins specifically bind the no moiety to their cysteine. here we show a bio-statistical analysis of some properties of cysteine that are s-nitrosylated in different proteins, including the pk, electrostatic environment, solvent accessibility of the target cysteine and identity of surrounding amino acids. we also chose model proteins (human thioredoxin and s protein) to make specific targeted amino acid substitutions around selected cysteines to alter the properties described above. the reactivity and stability of these mutant proteins towards s-nitrosylation will be examined. sampling the flexibility of ppar-γ s. aci-sèche , n. garnier , d. genest , s. bourg , c. marot , l. morin-allory , m. genest upr cnrs , orléans, france, fr pcv cnrs , orléans, france, umr cnrs , université d´orléans, france a promising approach to consider the flexibility of proteins in docking studies consists in performing multiple rigid docking on a representative set of the receptor conformations. molecular dynamic (md) simulation is one of the best adapted methods for structural sampling, but exploring the conformational diversity of a protein is computationally expensive. we present a protocol for generating a wide range of conformational states of a receptor using restrained md and a partitioning protocol to select a few representative conformations of the binding site from this md. a way to speed up efficiently md calculation is using an implicit model to represent the solute-solvent interactions. we explore a protocol using a distance-dependant permittivity function to represent solvent effect and an ensemble of controlled restraints applied on a subset of specific atoms in order to prevent artefactual structural distortions, but preserving receptor's flexibility. ten ns simulations have been performed using different sets of parameters and compared to a reference ns simulation with explicit solvent. to select a representative set of conformations, partitioning (k -means algorithm) was applied on the ensemble of simulated conformations. this methodology was applied to the ligand binding domain of peroxysome proliferator-activated receptor-γ. department of biomedical sciences, university of antwerp, antwerp, belgium the complex system of cavities identified in human neuroglobin (ngb) has been postulated to be of functional significance to the putative no dioxygenase activity of the protein. the interconnected hydrophobic cavities may support this catalytic activity by acting as reservoir for reactants and providing preferential pathways assisting product removal from the active site. we thus decided to investigate co rebinding kinetics to ngb embedded in silica gels to expose ligand migration processes in the geminate phase. encapsulation of the co complexes of reduced neuroglobin, leads to a slight increase in geminate recombination after nanosecond laser photolysis. increasing the viscosity of the medium, by soaking the gels in glycerol, completely inhibits escape of the photodissociated ligand to the solvent, and highlights a complex, multiphasic kinetic pattern. this finding can be rationalized by assuming the existence of a discrete set of temporary docking sites, capable of trapping the photodissociated ligand for very long times, up to a few ms after photolysis. g. bartolommei, f. tadini-buoninsegni, m. r. moncelli bioelectrolab -department of chemistry, university of florence, italy ion pumps are integral membrane proteins devoted to ion transport through a lipid membrane phase. the ion pumps ca-atpase and na,k-atpase are prominent members of the p-type atpases family. due to fundamental physiological roles of these proteins, they are very promising drug targets. bioelectrolab has a wide expertise in the study of ion transport by these proteins [ ] . our attention has been recently focused on the interaction of these enzymes with molecules of potential pharmacological interest. frequently, drugs exert an inhibitory action on the transport activity of an ion pump, usually confining it in an inactive conformation. molecules like thapsigargin and cyclopiazonic acid belong to high (nanom) affinity inhibitors of the ca-atpase, whereas clotrimazole and curcumin are medium (microm) affinity inhibitors of both ca-atpase and na,k-atpase. for each of these compounds a mechanism of action is proposed. moreover, recent results concerning ca-atpase inhibition by clotrimazole analogues will be shown: the relevance of this type of molecules is due to their potential employment as an alternative to traditional drugs against malaria parasite. financial support of ente cassa di risparmio di firenze and of miur (prin project) is gratefully acknowledged. [ ] tadini-buoninsegni f., bartolommei g., moncelli m.r., fendler k. . arch. biochem. biophys. : - (review). s. asthana , s. shukla , g. giliberti , f. luliano , m. ceccarelli , r. loddu , p. ruggerone , p. la colla department of biomedical science and technology, università di cagliari, cagliari, italy, department of physics, università di cagliari, cagliari, italy studies of protein-inhibitors interactions are helpful to elucidate the mode of action of ligands and thereby providing clues for rational drug design. bvdv, is an important target of drug discovery activities largely because it is essential for viral replication. in bvdv rdrp no specific nni binding site has been reported till now. experimental results have shown that different class of inhibitors (benzimidazole, imidazoquinolines and pyridoxyquinolines), have resistant mutations located in the finger domain of the rdrp. all the reported mutations are spatially very close to each other. thereby, indicating that binding sites of nni's may lie in the finger domain for these different class of inhibitors.herein, we have utilized docking procedure to investigate binding sites, binding modes as well as binding affinity of different class of inhibitors.we then used all atom molecular dynamics (md) simulations to investigate the stabilizing interaction between inhibitor-receptor pairs. our md results are in good agreement with experimental data and provide deep insights into the dynamical features of the high affinity inhibitorreceptor binding. thus identifying the binding modes of our inhibitors and mechanism leading to inactivity of the enzyme can help us to build a microscopically well-funded picture of the functioning of these enzymes. we present an investigation of the molecular basis of ligand binding and reactivity of heme proteins using computer simulation. a combination of classical molecular dynamics and hybrid quantum-classical (qm-mm) calculations are applied to explore distal and proximal effects on diatomic ligand binding to the heme. trends in binding energies and in the kinetic constants are illustrated through a number of selected examples. an investigation of the interplay between ligand migration and protein dynamics obtained through classical molecular dynamics techniques in combination with advanced sampling tools is also presented to yield information about free energy profiles and possible secondary docking sites. results for truncated n hemoglobin of mycobacterium tuberculosis, presented as an illustrative example, suggest that the truncated hemoglobin n has evolved a dual-path mechanism for selective/distinct migration of o and no to the heme, to achieve efficient no detoxification. finally, we present also an analysis of the molecular basis of hexacoordination in human neuroglobin, which suggest that the flexibility of the cd plays a key role in determining the ligand binding properties. thermodynamic bases of nucleoplasmin-histone complexes recognition by the nuclear transport machinery i. arregi, j. falces, s. bañuelos, m. a. urbaneja, s. g. taneva unidad de biofísica (csic/upv-ehu), departamento de bioquímica y biología molecular, universidad del país vasco, spain the nuclear transport of the chromatin remodeling (nucleoplasmin) and chromatin building (histones) proteins is mediated by importins. nucleoplasmin (np) contains a classical bipartite nuclear localization signal (nls) that is recognized by importin α, while histones present multiple sequence elements (nls-like motifs) for nuclear targeting. besides, ternary importin/np/histone complexes might represent a putative coimport pathway for nuclear import of linker (h ), nucleosomal core (h ah b) histones and their chaperone protein np, enhancing the histone import efficiency. to better understand np and histone recognition by the transport machinery we studied the thermodynamics of complex formation of importin α (a truncated form) and importin β with histones and np, and with np/histone binary complexes by means of isothermal titration calorimetry. data show that importins interact with the two histone types and np, and that importin and histones can simultaneously bind to np. analysis of the binding energetics reveals an enthalpy driven formation of high affinity binary and ternary complexes. we demonstrate that different amount of importin molecules can be loaded on np/binary complexes dependent on the histone type, linker or core, and the amount of the bound histones. g. breuzard, i. di maïo, p. barbier, d. allegro, c. brault, v. peyrot cro inserm u , ufr de pharmacie, marseille, france since a decade, our laboratory has already studied the interaction of tau variant with tubulin (tub) or microtubules (mts) and phosphorylation process on this interaction. indeed, fret assay was a powerful tool to achieve binding parameters between tau and tub in vitro: / with mts stabilized by fluorescein-coupled taxol (flutax- ) as donor and rhodamine-labelled tau (rho-tau) as acceptor, and / in living cells by confocal laser scanning microscopy (clsm) with tau/α-tub fused to egfp/mcherry, respectively. results revealed ± % energy transfer efficiency from flutax- to rho-tau and a donor-to-acceptor distance of ± Å. by titration, the dissociation constant of tau was determined to . ± . µm. a cleavage procedure of αβ-tub was performed to determine the influence of the c-term tails of αβ-tub on the tau-mt interaction. no difference in distances and binding parameters was observed. clsm images displayed a heightened concentration of fluorescent tau in patches along mts. fret experiments revealed in particular higher efficiencies between gfp-tau to mcherry-α tub proteins in these locations. overall, our results suggested no involvement of the hypervariable and highly acidic c-term tails of tub in mt/tau binding. a molecular model is proposed in which flutax- is directly accessible to tau molecules. besides, the modified distribution of fluorescent tau could be implicated in local change of mechanical properties of mts. a. boreham , k. winkler , c. gebhard , k. rueck-braun , p. henklein , e. michalsky , r. preissner , r. misselwitz , a. ziegler , u. alexiev freie universität berlin, berlin, germany, technische universität berlin, berlin, germany, charité-universitätsmedizin berlin, berlin, germany peptide presentation by major histocompatibility complex (mhc) molecules is crucial for immune responses. photocontrol of peptide dynamics by means of photo-switchable peptide analogs will provide insights in peptide dynamics and its dependence on mhc polymorphism ( ) ( ) ( ) . we have designed a hemithioindigo (mhti) photo-switch bearing peptide using the viral epitope rrrwrrltv (plmp ) as a template. incorporation of mhti in the peptide backbone should result only in a minor change of the overall peptide structure given the relaxed conformation of the zisomer. indeed, the human mhc molecule hla-b* can tolerate nonpeptidic elements in plmp , as evidenced by normal peptide binding. using the autofluorescent properties of mhti we determined the stability of the hla-b* /mhti-plmp complex. photoswitching from z →e results in a decrease of hla-complex stability. based on computer modeling this decrease is due to a reduced interaction of the peptide c-terminus with hla-b* . truncated hemoglobins (trhbs) are heme proteins present in bacteria, unicellular eukaryotes, and higher plants. three phylogenetic groups (n, o, and p) have been identified in trhbs. the crystal structure of truncated hemoglobin o of b.subtilis, does not show an evident tunnel/cavity system connecting the protein active site with the solvent, a fact that cannot be easily rationalized considering the very high oxygen association rate. moreover, resonant raman results of the co bound protein, showed that a complex hydrogen bond network exists in the distal cavity, making it difficult to assign unambiguously the residues involved in the stabilization of the bound ligand. for these reasons we performed classical molecular dynamics simulations of the oxy, carboxy and deoxy protein, and computed the free energy profiles associated with ligand migration to the active site. our results suggest that there is a key residue, glne , that may present an alternate conformation in which a wide ligand migration tunnel is formed, consistently with the kinetic data. the results for the co and o bound protein show also that glne is directly involved in the stabilization of the coordinated ligand, playing a similar role as tyrb , and trpg in other trhbs. our results not only reconcile the structural data with the kinetic information, but also provide additional insight about the general behaviour of trhbs. patterned functionalization of surfaces for guided transport on molecular motors tracks m. bhagawati , s. ghosh , t. surrey , j. piehler department of biophysics, university of osnabrueck, osnabrueck, germany, european molecular biology laboratory, cell biology and biophysics unit, heidelberg, germany chelator head groups with multiple nitrilotriacetic acid (nta) moieties have been very well characterized and successfully utilized as high affinity adapters for functional immobilization of oligohistidine tagged proteins to surfaces. we have recently established a generic method for patterning nta functionalized surfaces by selective photodestruction via a light induced fenton reaction. efficiency of different transition metal ions for catalyzing this reaction was tested. functionality of the patterned protein was confirmed using the interaction between interferonα and its receptor. implementation of this technique in a confocal laser scanning microscope allowed us to control surface density of binding sites, providing the possibility to vary the surface concentration of immobilized proteins in a spatially resolved manner. we also applied this approach for exploring guided transport of microtubules by kinesin selectively immobilized onto trisnta patterns. rheumatoid arthritis (ra) is an autoimmune disorder, leading to pathological damage at the level of joints, associated with the hla class ii allele hla-dr . although etiology of ra is unknown, type ii collagen (cii) is a potential antigen candidate and it is believed that t cell responses in collagendependent ra are directed towards the immunodominant pathogenic epitope cii( - ). despite recent advances in characterization of class ii major histocompatibility complex (mhc) and t-cell receptor (tcr) contacts in this epitope, the atomic details of tcr-cii( - )-mhc complex are not known. here, homology modeling and molecular docking studies have been used to derive a three-dimensional model of tcr β chains, obtained from a dr + subject, in complex with cii( - )/hla-dr . the best complex from docking was further refined using molecular dynamics simulations for ns. the proposed model represents a reasonable structural basis for understanding cii( - )-mhcii complex recognition by tcr and for rational design of inhibitors targeting tcr-pmhc interface. probing bio-molecular bonds with magnetic force for biosensor applications a. m. de jong , a. jacob , x. j. janssen , j. m. van noorloos , l. j. van ijzendoorn , m. w. prins eindhoven university of technology, eindhoven, the netherlands, philips research laboratories, eindhoven, the netherlands we investigate new technologies to be applied in next generation biosensors, which not only measure biomarker concentrations but also probe bio-molecular interactions. the concept is based on the response of ligand-receptor pairs to an applied force or torque [ , ] . we use functionalized magnetic beads and magnetic fields to apply translational and rotational forces on the molecular bonds. in a model experiment, polystyrene surfaces were coated with anti-biotin and beads were coated with biotin. after incubation, a constant magnetic force was applied to the beads and the number of bound beads was measured as a function of time. this was repeated for a range of forces. the dissociation rate (k o f f ) is determined for each force and k o f f at zero force is extracted from these data. rotational forces were exerted on protein-g coated beads bound to igg antibodies immobilized on a surface. rotating fields revealed an oscillating behavior, which can be understood from the balance between the applied magnetic torque and the torque due to the deformation of the biological bond. studying interactions between cop regulatory protein and hy transcription factor d. s. dalafave the college of new jersey, ewing, nj , usa this work addresses important questions of protein-ligand interactions and selective protein recognition. proteinligand bindings are crucial for many cellular processes. dependable methods for predicting binding sites would lead to a better understanding of proteins' selective recognition and would, in turn, help research on fighting diseases. presented here is a study of interactions between the wd domain found in a regulatory protein cop and the motif v-p-e/d-Φ-g (Φ=hydrophobic residue) found in a transcription factor hy . cop and hy proteins have opposing roles in developmental regulation. cop can repress hy by directly binding with it. the repression involves specific interactions between the wd domain and the motif v-p-e/d-Φ-g. previous experimental research showed that mutations in the motif's v-p pair resulted in a large decrease in hy repression. to study effects of similar mutations, residues in the motif v-p-e/d-Φ-g were systematically substituted with other residues. interactions between the mutated motif and the wd domain were studied. distributions of binding sites, bond lengths, local shape complementarity, and interaction potentials were modeled for each residue substitution. the study identified binding sites critical for the cop -hy binding. some hy residues in the vicinity of the motif were also found to be important in the binding. the significance of the results for understanding selective protein recognition is discussed. determination of protein-ligand binding thermodynamics by thermal shift assay p. cimmperman, a. zubriene, l. baranauskiene, e. kazlauskas, j. matuliene, d. matulis laboratory of biothermodynamics and drug design, institute of biotechnology, vilnius, lithuania thermal shift assay determines the effect of ligand binding on the protein thermal denaturation equilibrium. several models have been derived to describe the general cases of protein-ligand binding. first, the model describing protein stabilization and destabilization by ligand binding to the native and unfolded states. second, the split of protein melting transitions by tightly binding ligands is presented when the transition of free protein and ligand-bound protein occur separately. mathematical equations describing the protein unfolding and ligand binding thermodynamic parameters at various temperatures are presented. the protein melting temperature shift can be determined by various techniques such as differential scanning calorimetry, circular dichroism, and intrinsic or extrinsic fluorescence. the advantages of fluorescent techniques are presented. the melting temperature shift caused by ligand binding is dependent on the thermodynamic parameters of protein unfolding and ligand binding, including enthalpy, entropy, and heat capacity, thus allowing determination of binding thermodynamics. application of the models in the design of hsp chaperone and carbonic anhydrase inhibitors is discussed. comparison with isothermal titration calorimetry data is presented. recent reports have shown that the bacterial redox protein azurin can enter into cancer cells and induce apoptosis by stabilizing p . the formation of a complex between the two proteins has been demonstrated, but little is known about binding features. for the first time, we show here that azurin binds to the n-terminal region of p with a dissociation constant in the - µm range. trp phosphorescence lifetime measurements revealed conformational changes of azurin induced by the interaction with p ( - ). acrylamide quenching of trp phosphorescence also indicated a significant increase of the overall flexibility of azurin upon binding to p . no change of the fluorescence emission of p ( - ) was detected in the presence of azurin. the latter finding suggests that w of p is not directly involved in domain binding to azurin, indicating that the binding site is distinct from that of mdm . the present results may assist the design of novel cancer treatments based on p stabilization by azurin. a. eleta , r. georgieva , h. bäumler , j. l. toca-herrera cic biomagune, donostia-san sebastián, spain, charité-universitätsmedizin berlin, berlin, germany human serum albumin (hsa) is the most abundant nonglycosilated plasma protein in the human body. this multifunctional protein has ligand-binding and transport properties, antioxidant functions and enzymatic activity [ ] . bilirubin interacts with albumin in order to be transported from the blood to the liver where it is secreted. the interaction occurs specifically in hsa i-domain of its three domains [ ] . in our work, we investigate the interaction between hsa and bilirubin by quartz crystal microbalance with dissipation (qcm-d) and atomic force microscopy (afm) [ , ] . albumin was adsorbed on negatively charged silicon oxide. however, hsa was removed after rinsing with pbs. hsa adsorbed on positively charged polyelectrolyte multilayers leads to a stable layer of surface mass density of ng/cm . cross linked albumin with glutaraldehyde after its adsorption on nh -terminated thiols is also stable reaching surface mass density of ng/cm . a preliminary bilirubin adsorption results on cross linked hsa substrate show that ng/cm is immobilized. taking into account that hsa-bilirubin stoichiometry is : , the outcome demonstrates that the % of hsa i-domains remain active. antimicrobial peptides (amps) are short positively charged polypeptides. they are important due to their potential to provide an alternative to conventional therapy against bacterial infections. rbpi is a kda peptide based on the nterminal region of the neutrophil bactericidal/permeabilityincreasing protein (bpi). it was shown that this amp possesses bactericidal effects on gram-negative bacteria and higher affinity for lipopolysaccharide (lps), neutralizing its effect. the peptide use against meningitis, is in phase iii clinical trials. here, we demonstrate that rbpi promotes aggregation of negatively charged large unilamellar vesicles (luv) and lps aggregates, by dynamic light scattering, while for zwitterionic phosphatidylcholine (popc) luv the size remains unchanged. the aggregation increases with peptide concentration until peptide promotes massive aggregation followed by sample flocculation/precipitation. with the rbpi -lipid interaction there is a progressive change in the zeta-potential of the luv systems and lps aggregates. luv systems composed of phosphatidylglycerol (popg) and popc:popg mixtures have higher zeta-potential variations than popc luv. for lps aggregates, rbpi neutralizes the surface charge and at higher peptide concentrations overcompensates it. results demonstrate that the interaction of the peptide rbpi with lps aggregates and luv systems has electrostatic and hydrophobic contributions. serratia marcescens heme acquisition system: heme transport and protein:protein interactions m. delepierre unité de rmn des biomolécules cnrs ura , institut pasteur, paris, france heme transport systems in bacteria are required and might be potential target for antibacterial drugs. the heme acquisition system, has, exists in pathogenic as well as in opportunistic bacteria but only for the latter one extensive studies have been conducted, constituting as such a model system. the outer membrane receptor hasr, the central component of this system, functions in synergy with a secreted high affinity heme binding protein, the hemophore hasa. hasa extracts heme from host hemoproteins and returns it to hasr. then, the energy given by a protein complex of the inner membrane is used to allow heme entrance across the bacterial membrane and to eject the empty hemophore from the receptor. reconstitution of this heme acquisition system in e coli, overexpression and purification of its various components have allowed us to obtain sufficient amount of protein to perform nmr and biophysical studies to analyse at the molecular level the different steps of heme acquisition by hasr. protein-protein interactions (ppi) are the central pillar supporting most of biological functional activity on the molecular level. a binding event between two proteins typically consists of two stages: ) diffusional search of the binding partners for each other, and ) specific recognition of the compatible binding surfaces followed by the formation of the complex. we focus here on the non-specific component of ppi, which refers to all physico-chemical properties of the binding partners (such as size, charge, isoelectric point, hydrophilicity etc.) that are independent of the exact details of their binding sites, but which could in turn affect their localization or diffusional search for one another. it is known that proteins co-localize due to segregation into different cellular compartments, sequestration via anchor and scaffold proteins or even chemical modifications. we suggest that the non-specific component of ppi determines in part the co-localization and clustering of the binding partners, which then directly in a non-specific fashion influences their interactions. we examine the possibility that such signature might be encoded within the experimental d structures of a large set of known mutually interacting proteins. we provide preliminary evidence that this indeed may be the case, and corroborate our findings by using different statistical tests to compare those features of the known interacting partners, and ascertain correlations and commonalities between them. a link between hinge-bending domain motions and the temperature dependence of catalysis in ipmdh i. hajdú, a. szilágyi, j. kardos, p. závodszky institute of enzymology, hung acad sci, budapest, hungary enzyme function depends on specific conformational motions. since conformational flexibility strongly depends on temperature, temperature dependent enzyme kinetic studies with measurements related to dynamics can give us some insight at atomic level into these functionally relevant motions. the catalytic efficiency (k cat /k m ) of -isopropylmalate dehydrogenase for its substrate (ipm) has unusual temperature dependence, showing a local minimum at • c. in search of an explanation, we measured the individual constants k cat and k m,ipm as a function of temperature, and found that the van't hoff plot of k m,ipm shows a sigmoid-like transition in the - • c temperature range. by means of various measurements including h-d exchange and fret, we showed that the conformational fluctuations, including hinge-bending domain motions increase more steeply with temperature above • c. the thermodynamic parameters of ligand binding determined by itc as a function of temperature were found to be strongly correlated to the conformational fluctuations of the enzyme. because the binding of ipm is associated with a hinge-bending domain closure, the more intense hinge-bending fluctuations at higher temperatures increasingly interfere with ipm binding, thereby abruptly increasing its dissociation constant and leading to the observed unusual temperature dependence of the catalytic efficiency. a simulation approach to multiple sclerosis: study of a peptide with a pharmaceutical potential c. guardiani , s. marsili , p. procacci , r. livi centro dinamiche complesse, università di firenze, italy, dipartimento di chimica, università di firenze, italy, dipartimento di fisica, università di firenze, italy multiple sclerosis (ms) is an autoimmune disease of the central nervous system, leading to premature death. one of the potential targets of the autoimmune reaction is the myelin protein mog that has been crystallized in complex with the - c ms-autoantibody. the analysis of contacts and buried surface area combined with an alanine scanning computation reveals the key role of mog fragment - for the interaction with - c . a docking simulation shows that the - fragment, excised from mog, and kept in crystal-like conformation, is still capable of fitting into the binding pocket of the antibody. we then studied, through replica exchange molecular dynamics simulations, the structural equilibrium distribution of the free peptide and of a number of analogs stabilized by a disulfide bond. we found that the free peptide yields a significant fraction of crystal-like conformations and the proportion of native-like structures is further increased by the disulfide bridge. when we tried to dock the centroids of the most populated clusters to - c , we discovered the existence of a docking funnel whose bottom is populated by stable complexes where the peptide occupies the same spatial region as in the crystal. we therefore conclude that the mog - fragment may be used to develop a diagnostic assay or a drug for ms. the escherichia coli membrane insertase yidc reversibly binds its substrate pf coat protein u. gerken, s. winterfeld, a. kuhn institute of microbiology and molecular biology, university of hohenheim, germany the membrane insertase yidc of e. coli belongs to the oxa family of mitochondria and plays an essential role in facilitating the insertion and assembly of membrane proteins. we have previously shown with detergent-solubilized (c pc) yidc, labelled with ans, and pf coat that the initial step of the membrane insertion process, the binding of the substrate pf coat to yidc, is reversible [biochemistry , - ( ) ]. the dissociation constant k d for that particular system is about µm. in order to obtain data for the native system we used in this study membrane-reconstituted (dopc and dope/dopg) yidc. the effect of the initial binding was examined in vitro by fluorescence quenching of the tryptophan (trp) residues of yidc which are highly sensitive fluorescent probes for changes of the tertiary structure. quenching of the trp fluorescence after titration with a trp-free pf mutant indicates a change in the yidcs tertiary structure upon binding to its substrate. the binding data show a k d value in the range of . - . µm. the influence of different environments (lipid membranes, ddm micelles) on the secondary structure of yidc as well as on the yidc large periplasmic domain p was investigated by circular dichroism (cd). the cd data show that the secondary structure of yidc changes upon reconstitution into a membranes when compared to the detergent solubilized state. particularly, the p domain is considerably affected by the detergent c pc. b. karasulu, b. erman, o. keskin koc university, istanbul, turkey histone proteins are fundamental to the cells since they are involved in cell regulatory processes, such as chromatin regulation, gene silencing and transcription, cell cycle control, and epigenetics, which are controlled via post-transcriptional modifications of the histone protein tails. these modifications are categorized under four main groups: methylation, acetylation, ubiquitination, and phosphorylation. among them methylation has been very recently proven to be reversible with the discovery of histone demethylase proteins and this modification type has been extensively studied, because the abnormal methylation rates cause the excess proliferation of the cell, which, in turn, triggers the cancer. therefore, understanding of the details of reaction mechanisms of histone methylation/demethylation dynamics provide the required knowledge basis for preventing the abnormal methylation rates by designing proper inhibitor (drug) molecules. in this study, we display possible reaction mechanisms (such as amine oxidation via lsd ) for the demethylation of specific histone tail proteins. we try to explain the role/importance of residues that take place in or near to the reaction pocket for the demethylation reaction. we also carry out md simulations and reaction path (free energy profile) analysis using free energy perturbation (fep) method for qm/mm hybrid systems in order to compare different possible reaction pathways. the sonic hedgehog (shh) signalling pathway plays an important role both in embryonic development and in adult stem cell function. inappropriate regulation of this pathway is often due to dysfunction between two membrane receptors patched (ptc) and smoothened (smo) , which lead to birth defects, cancer or neurodegenerative diseases. however, little is known about ptc, the receptor of the shh protein, and the way ptc regulates smo, the receptor responsible for the transduction of the signal. to develop structure-function studies of these receptors, we expressed human ptc (hptc) in the yeast saccharomyces cerevisiae. we demonstrated that hptc expressed in a yeast membrane fraction is able to interact with its purified ligand shh, indicating that hptc is produced in yeast in its native conformational state. using surface plasmon resonance technology, we showed that fluorinated surfactants preserve the ability of hptc to interact with its ligand after purification. this is the first report on the heterologous expression and the purification of a native and stable conformation of the human receptor ptc. this work will allow the scale-up of hptc production enabling its biochemical characterization, allowing the development of new therapeutic approaches against diseases induced by shh signalling dysfunction. dissecting the colicin translocon c. l. johnson , a. solovyova , p. callow , s. a. holt , l. a. clifton , k. weiss , j. h. lakey inst. for cell and molecular biosciences, newcastle univ., uk, inst. laue langevin, grenoble, france, isis, rutherford appleton lab., didcot, uk, oak ridge national lab., centre for structural molecular biology, oak ridge, usa pore-forming colicin n hijacks e. coli outer membrane protein ompf and exploits it as both a receptor and translocator to cross the outer membrane [ ] . it is currently a matter of debate if the translocation route is through the ompf lumen or the interface between ompf and the lipid bilayer. recent electron microscopy data from our laboratory suggests the latter route for translocation [ ] . the colicin n/ompf complex in detergent has been studied by sans to examine the translocation pathway undertaken by colicin n. by using a combination of deuterated ompf and hydrogenated colicin we have been able to derive a low resolution structure of individual proteins in the binary complex. low resolution structural studies supplemented by targeted mutagenesis and screening techniques including itc, potassium efflux assays and auc have allowed us further our understanding of colicin n translocation. dual-color fluorescence cross correlation spectroscopy (fccs) has been used to explore the molecular dynamics at immune cell surfaces, with a particular focus towards the regulation mechanisms of natural killer (nk) lymphocytes. nk cells are critical mediators of anti-viral immunity and protectors against cancer spread. their activity is governed by a fine-tuned balance between inhibitory and activating receptors, where ly a and kir receptors represents the inhibitory ones. their ligands are mhc class i receptors. fcs is a technique based on the analysis of intensity fluctuations of fluorescent molecules excited by a focused laser beam. the technique offers information about molecular dynamics at the single molecular level, in the nanosecond to millisecond range. dual color fccs expands fcs by correlating the intensity from two different colors. by labeling two potential interaction partners with dyes emitting at different wavelengths, the amount of interaction can be determined. here, we will report on recent fccs data exploring the interaction between the inhibitory receptors and their ligands, as well as different labeling strategies used to enable these measurements. effect of osmolytes on the dhfr activity, structure and dynamics b. legrand , s. renaud , m. collen , c. tascon , s. bonnassie , e. gautier , j. mellet , c. blanco , e. le rumeur , j.-f. hubert , a. bondon rmn-ilp, duals, cbp, umr cnrs , univ. de rennes , france osmolytes are small molecules accumulated by a wide variety of organisms in response to hyperosmotic stress. they contribute to save the cellular integrity and to stabilize the macromolecules from environmental stress. dihydrofolate reductase activity is inhibited by several osmolytes. we studied the impact of osmolytes on the dhfr structure and dynamics by various techniques. we observe that substrate (dhf) and cofactor (nadph) diffusions are quite different in glycerol and betaine despite similar viscosities. we demonstrate that the overall structure is maintained at high osmolyte concentrations while no direct interactions can be detected with the enzyme. the k o f f of substrate analogues decreases with increasing the osmolyte concentrations. the enzyme dynamics, in various media, has been compared with the dhfr behaviour in water described in the literature. the osmolyte impact appears only partly conditioned by its viscogenic properties which reduce the molecules diffusion and the k o f f of the product controlled by the m loop of the dhfr. we suggest that the osmolytes decrease m loop mobility. comparing the results obtained with different osmolytes, we offer a better understanding of the osmolyte nature dependence of the dhfr inhibition. estrogen receptor (er) is a well characterized member of the nuclear receptor superfamily that modulates the expression of estrogen-responsive target genes in response to estradiol and other natural and synthetic chemicals mimicking the estradiol structure. in human, two ers, erα and erβ, lied on two distinct chromosomes, are known. er exhibits several functional domains: two conserved domains, a short domain c involved in dna-binding and a large domain e/f responsible for ligand-binding and hormone-dependent transcription activation, are linked by a hinge domain d; a poorly conserved a/b domain, at the n terminus, mediating interactions with the general transcription machinery, is involved in hormone-independent transcription activation. upon estrogen binding, ers can specifically bind to a dna fragment, called estrogen response element ere, and activate the transcription. the optimal ere sequence consists of two six base-pair half-sites, aggtca, organized as inverted repeats with a three base-pair spacing. in this study, we have investigated, by fluorescence methods, the effect of kcl concentrations, on the protein conformational flexibility and the thermodynamic stability of hers -eres complexes. we show, here, that electrostatic interactions, inside hers, contribute to its conformational flexibility and its thermal stability. moreover, the specific interaction between hers and eres is poorly sensitive to changes in ionic strength, in opposite to unspecific complexes. kinetic and structural explanation for the low enantioselectivity of human -phosphoglycerate kinase p. lallemand , j. rouhana , l. chaloin , b. roy , s. arold , t. barman , c. lionne cpbs umr , bd henri iv cs , montpellier cedex , france, ibmm umr , cbs umr l-nucleosides comprise a new class of antiviral and anticancer agents that are converted to pharmacologically active nucleoside triphosphates in vivo. the last step of the cascade may be catalyzed by -phosphoglycerate kinase (pgk), an enzyme that has low specificity for nucleoside diphosphate: ndp + , -bisphosphoglycerate ↔ ntp + -phosphoglycerate. here we compare the kinetics of formation of the complexes of human pgk with different d-and their mirror images, l-nucleoside diphosphates, and the effect of -phosphoglycerate thereon. two types of experiment were carried out: equilibrium experiments allow the estimation of dissociation constants, and stopped-flow experiments the transient kinetics of the interactions. in addition, by the rapid-quench-flow technique, we compare the kinetics of the phospho-transfer and product release steps with each d-or l-nucleotide. crystallographic and molecular modelling studies allow defining the structural reasons for the low enantioselectivity of pgk. the aim of this basic work on the mechanism of action of human pgk with non-natural nucleotides is to obtain information for the optimization of therapeutic nucleoside analogues that are poorly phosphorylated by pgk. anrs is gratefully acknowledged for financial support. a new itc assay for measuring high-and lowaffinity protein-ligand interactions g. krainer , j. fanghänel , s. keller leibniz institute of molecular pharmacology (fmp), berlin, germany, bayer schering pharma ag, berlin, germany isothermal titration calorimetry (itc) is the gold standard for the quantitative characterisation of protein-ligand and protein-protein interactions [ ] . however, reliable determination of the dissociation constant (kd) is typically limited to the range µm > kd > nm. nevertheless, interactions characterised by a higher or lower kd can be assessed indirectly, provided that a suitable competitive ligand is available whose kd falls within the directly accessible window [ ] . unfortunately, the established competitive itc assay requires that the high-affinity ligand be soluble at high concentrations in aqueous buffer containing only minimal amounts of organic solvent. this poses serious problems when studying protein binding of small-molecule ligands taken from compound libraries dissolved in organic solvents, as is usually the case during screening or drug development. here we introduce a new itc competition assay that overcomes this limitation, thus allowing for a precise thermodynamic description of high-and low-affinity protein-ligand interactions involving poorly water-soluble compounds. we discuss the theoretical background of the approach and demonstrate some practical applications using examples of both high-affinity (kd < nm) and low-affinity (kd > µm) protein-ligand interactions. a molecular dynamics study of the cftr nucleotide binding domains interaction v. martorana , r. noto , o. moran cnr-istituto di biofisica, palermo, italy, cnr-istituto di biofisica, genova, italy the cystic fibrosis transmembrane conductance regulator (cftr), the dysfunctional protein in cystic fibrosis, contains two transmembrane domains, two nucleotide-binding domains (nbds), and a regulatory domain. opening of the pore have been linked to the atp-driven tight dimerisation of nbd. we have studied the nbd -nbd interactions on wild type and cystic fibrosis-related mutations by steered molecular dynamics simulations (smd). a fully solvated dimer, including the two bound atps, was separated by pulling one monomer with an external, increasing force. interestingly, the force needed to break the mutated (g d) dimer is significantly smaller than in the wild type case. the effect of a cftr potentiator, the genistein, has also been tested by repeating the smd simulations with the small molecule docked at the interface between the two nbd domains. to test the validity of our results we have repeated the separation process for different simulation lengths and force strengths. the amount of distortion on the pulled nbd domain has also been studied. this work is partially supported by the italian cystic fibrosis foundation (prog ffc # / ), with the contribution of "mille bambini a via margutta"onlus"blunotte", "lega italiana fc toscana" stability and aggregation studies of human septin (sept ) j. n. a. macêdo, r. c. garratt, a. p. u. araújo instituto de física de são carlos, usp, são carlos, brazil the septins are a conserved family of nucleotide binding proteins firstly identified in saccharomyces cerevisae as proteins required for the completion of the cell cycle. septins are implicated in several cellular functions such as cytokinesis, vesicle trafficking, exocytosis, cytoeskeletal dynamics, cell polarity and sperm motility. furthermore, they are associated with alzheimer's and parkinson's disease. sept was identified in rat brain being highly enriched in presynaptic nerve terminals. it colocalizes with synaptophysin and dynamin i and is associated with synaptic vesicle. in this work, human sept without its n-terminal domain (sept gc) was expressed in e. coli and purified by affinity and size exclusion cromatographies. structural stability studies were performed with recombinant sept gc using circular dichroism spectroscopy (cd), right-angle light scattering, and fluorescence spectroscopy. the sept gc cd spectrum showed a conformational transition from a predominantly α-helical starting structure to one dominated by β-sheet, just above physiological temperatures. the formation of irreversible aggregates, detected by light scattering, and their ability to bind thioflavin-t suggested that sept forms amyloidlike structures, as has been previously observed for human septins and . our data suggest that amyloid formation by isolated septins in vitro may be a general phenomenon whose physiological relevance needs to be further investigated. pln is an antimicrobial peptide produced from lactobacillus plantarum nric . analog pln (pln a) and a ser-derived (pln s, tyr to ser replacement) were synthesized on solid phase and their interactions with biomembrane model systems and inhibitory property on s.aureus and p.aeruginosa were investigated by cd, leakage assays, fluorescence spectroscopy, and differential scanning calorimetry. both peptides share the same unordered structure-like cd spectrum in aqueous solution, but a helicoidal induction in the presence of negative vesicles were observed, however pln s showed lower helical content than pln a. the ser-derived peptide presented % decrease of its leakage activity in different liposome compositions, a threefold increase in the dissociation constant from the liposomes than pln a, and close to % reduction for the inhibitory activity against p. aeruginosa growth. we can conclude that besides the electrostatic contacts between the amphipathic &alpha-helix, formed by the cationic residues from pln and negatively charged phospholipids, the pln -membrane binding is a process driven for the hydrophobic interactions from non-polar residues. in this case, there was a significant contribution of the tyr residue, which must be allocated in a lipid interface, described as the preferential position to this residue in membrane proteins. supported: fapesp label free detection using deep-uv laser-based fluorescence lifetime imaging microscopy q. li, s. seeger physikalisch-chemisches institut, universität zürich, winterthurerstrasse , ch- zürich, switzerland label free detection based on native fluorescence excited at uv region shows great potential for the life sciences. it offers simple, low-cost and fast method for sensitive detection of important biological analytes without modification. in this contribution we present a deep uv fluorescence lifetime imaging microscopy system (duv-flim) based on a picosecond deep uv laser using time-correlated single-photon counting method. the described setup is well-suited for biological applications for ultrasensitive detection. we have showed single uv dye (bmq) and single protein (ecβ gal ) molecules detection using duv-flim. further, the label free detection of protein interaction between ecβ gal and monoclonal anti-ecβ gal has been demonstrated by means of steady-state and time-resolved fluorescence spectroscopy. we achieved detection sensitivity for the ecβ gal/ anti-ecβ gal pair down to the nanomolar concentration range. we also extended this method to study the interaction of therapeutic drug porphyrin with bsa protein. fluorescence resonance energy transfer between protein and alexa fluor has been investigated using duv-flim. the intrinsic fluorescence and fluorescence lifetime changes of donor biotin β-galactosidase have been measured. energy transfer efficiency and donor acceptor distance have been obtained. fluorescence images of acceptor af due to fret have been observed when excited at nm. the monomeric heme-containing indoleamine , dioxygenase (ido) catalyses the oxidative cleavage of the indole moiety of l-tryptophan (l-trp). enhanced l-trp degradation contributes to various physiological disorders including depression or failure of the immuno-regulating system. the regulation of ido by inhibitors is extensively studied. however, the catalytic mechanism of ido on the molecular level is still unknown. in addition to l-trp, a wide range of substrates are degraded including d-tryptophan, melatonine or tryptamine. in contrast, indole or histidine do not function as substrates for ido. to understand the determinants for substrate specificity, we investigate the interaction of the heme iron, the heme-bound ligand and the substrate. we use (time-resolved) uv/visible and fourier transform infrared (ftir) spectroscopy over a wide temperature range ( - k) to monitor the binding of diatomic ligands to the heme iron and the binding of different substrates to coligated ido. changes in the spectra upon addition of l-trp are analyzed and compared to those induced by other substrates or inhibitors. archaeal protoglobin d-structure: novel ligand diffusion paths and heme-reactivity modulation protoglobin (pgb) from methanosarcina acetivorans c a, a strictly anaerobic methanogenic archaea, is the latest entry in the hemoglobin superfamily. our previous crystallographic studies on pgb have shown that protoglobinspecific loops and a n-terminal extension completely bury the heme within the protein matrix ( ) . access of o , co, and no to the heme is granted by protoglobin-specific apolar tunnels reaching the heme distal site from locations at the b/g and b/e helix interfaces. here we report structural and kinetic data on pgb mutants engineered to probe the protein structural and kinetic properties. six crystal structures (pgb mutants: ∆ (missing nter residues), y(b ) →a, y(b ) →w, f(b ) →w, f(g ) →w, i(g ) →f) show that the mutations engineered essentially restrict access to ligand tunnel . an accurate molecular level description of the signaling mechanism in ca + transducers necessitates the knowledge of the kinetics and energetics of conformational changes associated with ca + binding to various calcium binding proteins. with this in mind, we have developed an approach that combines the laser-induced photolysis of photolabile "caged" ca + compound, dm-nitrophen, with the photothermal beam deflection (pbd) technique to determine thermodynamic profiles associated with the ligand binding to calcium chelator, edta, and ca + sensor, calmodulin. this approach allows us to monitor time profiles of volume and enthalpy changes on the microsecond to millisecond timescale. the initial pbd study of ca + photo-relase from ca + loaded dm-nitrophen reveals the presence of two phases. the first step takes place within first µs upon and is associated with a volume decrease of - ml mol − and enthalpy change of kcal mol − . on the longer timescale (τ = µs), the second event with a positive volume change of ml mol − and enthalpy change of kcal mol − was detected. on the other hand, ca + photorelease in the presence of calmodulin is accompanied with an additional phase with a distinct lifetime and volume and enthalpy changes that reflects the metal binding to calmodulin and concomitant structural changes. antimicrobial peptides: linking partition, activity and high membrane-bound concentrations antimicrobial peptides (amps) have been intensively studied at micro and macroscopic levels for over twenty years. knowledge from these two domains has, however, contributed little to a comprehensive understanding of amp action; rather, in vivo amp performance has been only remotely correlated to biophysical properties. we focus on the assessment of peptide accumulation on bacterial membranes as an example of this separation: amp-bilayer interactions have been subject to extensive biophysical characterization, but conversion of that information into educated estimates of in vivo membrane-bound amp concentrations is lacking. this has led to the overlooking of important factors for activity. using simple partition models we were able to analyze available information on amp activity and interaction with membranes to show that unexpectedly high membranebound peptide concentrations are likely in vivo and may, in some cases, be required for triggering bacterial death. e. e. schäfer , s. schuy , a. janshoff georg august-university göttingen, germany, johannes-gutenberg-university mainz, germany retrovirus entry into cells occurs through fusion of the lipid bilayers that surround the virus and the lipid bilayer of the host cell. fusion proteins, present on the surface of the virus membrane play an essential role in the early stage of virus entry. understanding of the molecular mechanism is important for the design and function of modern fusion inhibitors. in this project we analyze the fusion of active conformation of the envelope gp from the human and simian immunodeficiency viruses (hiv and siv). during the infection process gp undergoes a sequence of conformational changes where the n -terminal nhr develop a trimer pre-hairpin intermediate. afterwards the three chr fold back towards the central nhr and a six helix bundle is formed. this rearrangement forces the viral and the host membrane into close contact and fusion pores may induce membrane fusion. this decisive molecular step in retroviral fusion has been modeled by rational design of lipopeptide assemblies that mimic a coiled-coil structure serving as a receptor for potential antagonists. for this purpose, sslbs were functionalized in an in situ coupling reaction with peptides originating from the nhr (n-peptides) of siv and hiv to monitor the interactions with the specific chr peptides (c and t ). binding of antagonists to surface confined coiled-coil structures has been quantified by ellipsometry, quartz crystal microbalance and was visualized by atomic force microscopy. a. rupprecht , e. sokolenko , e. e. pohl institute of cell biology and neurobiology, charité -universitätsmedizin, berlin, germany, frumkin institute of physical chemistry and electrochemistry russian academy of sciences, moscow, russia the production of reactive oxygen species (ros) in mitochondria is very sensitive to the proton motive force and can be decreased by mild uncoupling, mediated e.g. by uncoupling proteins (ucp) . in contrast, the activation of uncoupling proteins by ros as a negative feedback loop is a highly controversial hypothesis. ucp activation in mitochondria by -hydroxy- -nonenal (hne, aldehydic product of lipid peroxidation) was first demonstrated by echtay et al. . here we investigate the ability of hne to activate and/or to regulate the expression of ucp in two different systems: (i) in lipid membranes reconstituted with recombinant ucp and (ii) in primary neuronal cells. total bilayer conductance was enhanced in the presence of hne, but this effect was independent on ucp and ucp . the results concerning the hne-mediated ucp expression after induction of ros-production and/or after exogenous addition of hne are discussed for three brain-associated proteins (ucp , ucp , ucp ) in view of their possible functions. . beck, v et al. . faseb j. : - . . echtay, k. s. et al. . t. rudack, j. schlitter, k. gerwert ruhr university, department of biophysics, nd north, bochum, germany the gtpase ras p which is linked to the membrane via a lipid anchor, is a crucial switch in the cellular signal transduction processes that control cell growth and proliferation. docking simulations can be seriously hampered by the great difficulty to accurately estimate the ligand-protein binding affinity constant k, which is usually derived via the computation of the binding free energy ∆g. unfortunately, due to the involved logarithmic relationship, errors of less than . kcal/mol in the computation of ∆g result in about one order of magnitude inaccuracy on k. this can hinder computational methods from discriminating micromolar from nanomolar compounds. to improve the reliability of docking prediction, we make use of enhanced sampling methods, ranging from steered molecular dynamics to metadynamics . we also test several descriptors, such as the recently developed path collective variables , to identify the most suitable reaction coordinates accounting for binding and unbinding processes. using these approaches we investigate ligand docking and undocking and we attempt to describe at an atomistic level the kinetics of binding, which we intend to exploit for drug design purposes. here, an example of application in drug design is reported. . isralewitz, b. et al.; j. mol. graph. model. , , - . . laio, a. and parrinello, m.; pnas , , - . . branduardi, d.et al.; j. chem. phys. , , . prediction of protein-protein complex structures using wang-landau simulations a. solernou, barcelona supercomputing center, jordi girona , barcelona, spain protein-protein interactions are essential in the majority of life processes, so they have keen interest in several knowledge areas. however, experimental data on protein complexes is being produced at a quite low pace in comparison to that of the individual components. thus, in recent years attention has focused into computational approaches to the proteinprotein docking problem. a variety of docking protocols have been recently reported, sharing usually the following strategy: fast rigid-body search of the interacting subunits, followed by scoring and refinement of the interfaces. although this kind of strategy has proven some good results in the capri blind test it has two main limitations. on one hand one should include full flexibility on the protein structures. on the other, the evaluation should be made with free energy calculations instead of using a scoring function. in this work we propose to search the native complex structure in the minimum of the free energy landscape. we use the coarse grained potential unres to get the potential energy of the possible conformations. they are generated changing the dihedral angles, the side chain rotamers, and lastly the mutual orientation using a new sampling protocol we have developed (rotation-based uniform sampling; rotbus). finally, the free energy calculations are performed using an omp parallelization of the wang-landau algorithm. s. shukla , s. asthana , g. giliberti , f. luliano , m. ceccarelli , r. loddu , p. ruggerone , p. la colla department of biomedical science and technology, università di cagliari, cagliari, italy, department of physics, università di cagliari, cagliari, italy the virus encoded rdrp has emerged as a prime target in the search for specific hcv and other flaviviridae antivirals. recently, the determination of the hcv rdrp structure in complex with certain benzimidazoles has been reported, these nni's bind to the surface of the thumb domain, thereby disrupting its interaction with the finger domain, which is necessary for catalytic activity. on the other hand, we have found that, in bvdv, the mutations conferring resistance to same class of inhibitors lie in the finger domain of rdrp, indicating that the mode of inhibition of benzimidazole class of compounds is different in both hcv and bvdv rdrp. herein, we have applied docking approaches (binding orientation), molecular dynamics (md) simulations combined with metadynamics to elucidate the microscopic mechanism of the interactions between the ligand and the receptor in order to identify features barely seen in experiment. the recently designed algorithm overcomes the time scale problem by accelerating properly defined reaction coordinates. it mimics the real dynamics of a ligand staying or leaving the receptor and in doing so reconstructs the free energy surface, which in turn gives an idea of the residence time of the inhibitor in the cavity.finally we identified the binding modes and different mechanism of inhibition of benzimidazole class of compounds in rdrp of two closely related rna viruses. . c. seifert , w. stacklies , f. graeter protein mechanics and evolution, bioquant, inf bq , heidelberg, germany, ag graeter, picb, yueyang lu, shanghai , china we use a new method that detects force distribution in proteins. based on molecular dynamic simulations, changes in inter-atomic forces are calculated, here caused by different ligands. these changes will then be analyzed to detect a signal transfer through a protein initiated by the binding of a ligand to the protein. chaperones are ubiquitous proteins, which help other proteins to fold into a native conformation. they are able to refold misfolded proteins with a great variety of mechanisms. in this project, our group focuses on molecular dynamic simulations of htpg, an e. coli homolog of the human hsp (heat shock protein kda). the full structure of htpg was published by shiau et al. in , but the mechanism of how htpg performs its function is still not understood. the folding mechanism is an atp driven reaction cycle, in which the functional entity is a homodimer of htpg. we separate the cycle in three main states: apo, adp-and atpbound state. conventional molecular dynamics simulations are used to build a stable simulation system and provide structure trajectories and primary information about the behavior of htpg in its different states of the folding process. experiments indicate that the molecular movement is atp driven. we use force distribution analysis to elucidate how atp effects htpg conformation and dynamics. the small size of myoglobin makes it the preferred candidate to investigate the structure-function paradigm. in its interior five docking sites have been identified and for long time these xenon cavities have been classified as packing defects. recently, it was shown that they might be involved in ligands migration path. however, some questions regarding its role as oxygen carrier no scavenger remain yet open as well as the microscopic mechanism regulating these biological functions. in this work we made use of standard md simulations of solvated myoglobin to characterize internal cavities. our principal results is that we have found several secondary cavities showing volume and occurrence at least comparable to that of xenon cavities. in order to analyze and rationalize internal cavities we applied special cluster-analysis: we classified all cavities with respect to the position, size and occurrence ascribing them to different clusters. this analysis highlights possible intrinsic migration paths for small ligands within the protein matrix controlled by spontaneous fluctuations of the protein itself. moreover, we identified some key residues playing a fundamental role in controlling internal pathways. our suggestion that the secondary cavities constitute the preferred path for ligand escape is also supported by explicit metadynamics simulations of ligand escape. a. varga , p. lallemand , j. szabó , p. závodszky , m. vas , t. barman , l. chaloin , c. lionne institute of enzymology, brc, hungarian academy of sciences, budapest, hungary, cnrs-université montpellier -université montpellier , institut de biologie, umr , montpellier, france -phosphoglycerate kinase (pgk) is a promising candidate for the activation of nucleotide analogues used in antiviral and anticancer therapies. pgk is a key enzyme in glycolysis; it catalyses the reversible reaction , -bisphosphoglycerate + adp ↔ -phosphoglycerate + atp. here we explored the catalytic role in human pgk of the highly conserved lys that has been proposed to be essential for pgk function, by a transient and equilibrium kinetic study with the active site mutant k a. by the stopped-flow method we show that the kinetics of substrate binding and the associated protein isomerization steps are fast and identical for the wild-type pgk and mutant k a. by the use of a chemical sampling method (rapid-quench-flow) under multi and single turnover conditions and in both directions of the reaction, we show that the rate-limiting step with wild type pgk follows product formation, whereas with the mutant it is the phospho-transfer step itself that is rate limiting. these data are supported by saxs measurements which showed no direct role of lys in the domain closure of the enzyme i.e. the isomerisation step of the reaction. the results are explained by the structural data of the enzyme. characterization of different recombinant nrp proteins and interactions with heparin k. a. uniewicz, y. ahmed, d. g. fernig school of biological sciences and centre for glycobiology, biosciences building university of liverpool, l zb liverpool, u.k. neuropilin- (nrp ) is a mammalian membrane glycoprotein involved in tip sprouting processes like angiogenesis and neurogenesis. it has been shown that the interaction of nrp with heparin/heparan sulfate is implicated in its enhancement of growth factor signalling, however the mechanism is not yet known. commercially available extracellular domain of nrp is available either as a truncated his-tagged human protein (hnrp ) or as the rat protein fused to histagged human fc and expressed as a dimer (fcrnrp ). biochemical properties such as kinetics of heparin binding and structural requirements for sugar binding together with biochemical tests of protein properties were performed in order to characterise these commercial proteins. fc rnrp shown high affinity to heparin (kd= . nm) and required a minimum of dp to effectively compete with fc rnrp binding to immobilised heparin. competition experiments with various modified sugars show that interaction of fc rnrp with heparin is highly ionic and dependent on the position of sulfate groups along the heparin chain. the hnrp did not bind to heparin immobilised via nhs-biotin, though it did bind to some heparin affinity chromatography matrices. organic compounds of tin are among wide spread environmental pollutants. due to physical and/or chemical actions, poly-substituted alkyltins speciate into less substituted, more toxic species. tetra-or tri-alkyltins show a marked delay in their toxic action with respect to the corresponding di-and mono-alkylated analogs. it has been hypothesized that the delayed toxicity may results from the progressive dealkylation of alkyltins in more toxic di-and mono-derivatives, which bind and inhibit essential enzymes. it has been proposed that alkyltins preferentially target enzyme sulphydryl groups. previously, we showed that a nine amino acid linear peptide (i lgcwcylr ) containing a cxc motif is able to bind and dealkylate tri-substituted alkyltin compounds into the corresponding dialkyl derivatives. here, we investigated the time dependence of the degradation of the most common alkyltin derivatives by this peptide. we monitored the reaction kinetics using the intrinsic fluorescence of the tryptophan residue in position of the peptide. we found that all of the alkyltins analyzed are progressively degraded to dialkyl derivatives, following a pseudo-enzymatic reaction mechanism. the end-point of the reactions was the formation of a covalent complex between the disubstituted alkyltin and the peptide. these data agree with the speciation profiles proposed for polysubstituted alkyltins in the environment and reveal a possible biotic degradation pathway for these toxic compounds. parkinson's disease (pd) is a multifactorial neurodegenerative condition characterized by the progressive loss of dopaminergic neurons in the substantia nigra and by the presence of intracellular inclusions, composed predominantly of fibrillar alpha-synuclein (as). post-mortem studies indicate the presence of oxidative damage in the nigral neurons. dopamine oxidation, which leads to the formation of highly reactive quinones (daq), may account for the specificity neurodegeneration observed in pd. daq have many potential protein targets for chemical modifications. among them, we focused our attention on dj- , of which mutated forms have been found in familial cases of pd. a possible function of dj- is its redox-dependent chaperone activity that could prevent as aggregation and fibril formation. in the present work, we analyzed the structural and functional modification induced on dj- by daq. n-hsqc spectra of dj- were recorded in the presence of different amounts of daq and chemical shift perturbations were used to identify the dj- residues target of daq and their relative reactivity toward daq. aggregation assays were also performed to evaluate the functional effects of the daq modifications on the chaperone activity of dj- . rabies virus (rabv) infects neurons exclusively and causes lethal encephalitis. pathogenic rabv strains favor neuronal survival, whereas non-pathogen strains lead to neuronal apoptosis. the use of recombinant rabv showed: / the g protein determined the induction of the survival or death phenotypes; / the last cooh amino acids of the g protein cytoplasmic domain (cytog) are critical. these residues form a binding site for pdz domain (pdz-bs). one of the amino acid differences between survival and death gproteins are located in this pdz-bs. results of two-hybrid experiments showed that cytog survival interacted pdz domain of ser/thr kinases (mast), while cytog death interacted also with additional pdz containing host proteins. to understand the fine structural basis for the specificity of the pdz-cytog complexes, we determined the d structure and the dynamics of the mast-cytog complexes by nmr. the structures, as well as the affinities and constant kinetics, of the mast pdz complexes with both cytog are similar. we conclude that difference by one aa in the pdz-bs of the two strains cannot drastically modify the interaction with mast -pdz, in agreement with the two-hybrid data. preliminary results suggest that the interaction of cytog death with one additional cellular partner blurs the pro-survival signals engaging the infected cells through apoptotic trails. functional protein immobilization on glass-type surfaces s. waichman, m. bhagawati, y. podoplelova, j. piehler institute of biology, department of biophysics,university of osnabrück , barbara st. osnabrück, germany the immobilization of membrane proteins onto solid supports enables protein interactions and conformational changes to be probed by spectroscopic techniques under highly defined conditions. here, we present a novel method for covalent protein immobilization on glass-type surfaces using a bottom-up approach. in this approach the 'phosphopantetheinyl group of coenzyme a (coa) was transferred to the acyl carrier protein-derived ybbr tag of the target protein by means of the phosphopantetheinyl transferase sfp. the glass-support was rendered biocompatible by coating it with an ultrathin layer of peg (polyethylene glycol), followed by functionalization with coa through maleimide chemistry. immobilization of ybbr-tagged proteins in presence of sfp was followed in real time by label-free detection using reflectance interference spectroscopy. the immobilization procedure was thus systematically optimized by means of binding specificity, enzyme activity and functionality of the immobilized protein. this approach was employed for immobilizing the type i interferon ifnα in order to probe ligand recognition by ifnar and ifnar and ligand-induced ternary complex formation. the versatility of this technique was further enhanced by its combination with photopatterning methods. this immobilization technique can provide a beneficial tool for bioanalytical and biophysical applications at the single molecule level. r. vijayan, p. c. biggin department of biochemistry, university of oxford, south parks road, oxford, ox qu, uk ionotropic glutamate receptors mediate excitatory synaptic transmission in the brain and are heavily implicated in memory and learning as well as in numerous neuropathological conditions. one family of iglurs, the kainate receptors, show unusual sensitivity to changes in external ion species resulting in an apparent requirement for both sodium and chloride ions for activation. our recent work revealed the location and selectivity of the cation binding sites. despite this progress, it is still unclear how the cation binding sites confer sodium selectivity and how apparent affinity for chloride is influenced by the presence of cations. we have attempted to address these questions by performing extensive free energy calculations using all-atom molecular dynamics simulations. the rank order of cation binding obtained from relative binding free energy calculations is in agreement with experimental measurements of apparent affinity. these calculations also reveal that the pair of cation binding sites in the dimer interface act independently. binding free energy calculations performed using a reduced model of the binding site show that cation selectivity can been attributed to both the rigidity and high charge density of the binding sites. finally, a potential of mean force derived from umbrella sampling simulations indicate that the presence of cations stabilize the anion binding site considerably. the effect of toxins on the inorganic phosphate release during the actin filament formation a. vig, t. kupi, g. hild, m. nyitrai university of pécs, faculty of medicine, department of biophysics, szigeti str. , pécs, hungary actin can be found in monomeric (g-actin) and filamentous (f-actin) form in eukaryote cells under physiological circumstances. the first step of actin polymerisation is the formation of actin nuclei by atp-binding actin monomers. the next step in is the elongation when monomers are associated to the previously formed nuclei or to the ends of the growing filaments. after the association of monomers the bound atp is hydrolysed to adp.p i , and with first order kinetics the release of inorganic phosphate occures. the rate constant of the release is , s − , which is a slower process than the hydrolysis itself ( , s − ).phalloidin, a cyclic peptide from amanita phalloides can bind to the filaments and stabilizes their structure. jasplakinolide is another cyclic peptide from marine sponge (jaspis johnstoni) which binds actin filaments. the aim of this study was to investigate whether the binding of toxins to the newly created filaments has an effect on the kinetics of the inorganic phosphate release or not. we used absorption photometry measurements to measure the rate of phosphate release. phalloidin decreased the rate of the release substantially. although the effect of jasplakinolide was weaker, the results showed that the binding of these toxins to the actin can modify the rate of the release of inorganic phosphate from the filaments. these observations are in agreement with the molecular mechanisms by which these toxins stabilise the actin filamens. fluorescent proteins (fps) are invaluable fluorescent markers in cell biology. however, their use is often limited by photobleaching of the chromophore, notably in single-molecule, time-resolved or super-resolution imaging studies. we will present the crystallographic studies at near atomic resolution of a photo-activatable fluorescent protein irisfp that has been observed in a transient radical state en route to photobleaching. we took advantage of x-rays to populate the radical, which, under illumination with visible light, presumably forms with low probability from the triplet state. the combined x-ray diffraction and in crystallo spectroscopic data (from uv-vis and raman spectroscopies) reveal that radical formation in irisfp involves strong but reversible distortion of the chromophore, suggesting a transient loss of pi-conjugation. these results will help unravel the mechanisms of blinking and photobleaching in fps, which is of importance to rationally design variants of higher photostability. optimizing photoactivatable fluorescent proteins for live-cell imaging recently, novel fluorescent proteins (fps) have been reported which perform spectral changes in response to irradiation with light of a particular wavelength. reversibly photoactivatable proteins switch between a bright and a dim fluorescent state. this process is accompanied by photoisomerization of the protein chromophore. irreversible photoactivation results from photochemical processes within the protein, e.g., photolysis of an amino acid side chain, or an extension of the alternating π-electron system of the chromophore by a β-elimination reaction. fps have became valuable tools in live-cell imaging because they allow intracellular protein labeling by using them as fusion tag, and photoactivatable fps are powerful tools for application in novel subdiffraction imaging techniques. however, the available fps still offer potential for improvement in various ways. they frequently show a tendency to aggregate or oligomerize, incomplete chromophore maturation, fluctuating emission and low photostability. here, we will present our recent progress towards engineering the 'perfect' fp. simultaneous intracellular chloride and ph measurements using gfp-based sensor in ldcv d. arosio , f. ricci , l. marchetti , l. albertazzi , f. beltram italian institute of technology, udr pisa, italy, nest, scuola normale superiore, pisa, italy, nest, infm cnr, pisa, italy chloride ion participates in many physiological functions including control of neuronal resting potential, charge balance during endosome acidification, and regulation of cell volume. as a consequence dysfunctions in regulating membrane chloride permeability lead to severe diseases including motor disorders, cystic fibrosis and epilepsy. at present processes regulating intracellular chloride ion concentrations are still widely unexplored mainly as a consequence of limiting methods to quantify chloride fluxes in living cells. in the present work a highly specific, genetically encoded sensor is developed for detecting simultaneously intracellular ph and chloride concentration. the sensor is obtained by fusion of a red fluorescent protein (dsred-monomer), insensitive to chloride and ph, to a gfp variant containing a specific chloride-binding site (gfp-chl). dsred-gfp-chl binds the chloride ion following a fluorescence static quenching mechanism, which allows measurements of intracellular ph in a chlorideindependent manner. the sensor has been successfully tested in different living cells, in a ph range - and chloride concentration up to mm. for the first time, to the best of our knowledge, it allowed to measure the chloride concentration of dense core vesicles in the secretory pathway. applicability to high-throughput screening, range of validity and accuracy of time-lapse maps will be discussed. we aim to understand the basis of the photophysical changes in fluorescent proteins (fp) induced by reactive oxygen species (ros). indeed, ros might be involved in photochemistry of fp, leading to their photobleaching or photoconversion. in addition, fp may be used to investigate cellular events like phagocytosis or mitochondrial activity, where ros are naturally produced. in the latter cases, an accurate analysis of fp's fluorescence signals requires the full knowledge of reactions between ros and fp. in the future, this work may help in developing either photoresistant or photoswitchable fp and improving their use for imaging under oxidative stress conditions. using γ-radiolysis as a quantitative source of ros, we investigated the reactions of oh and o − radicals on the cyan fluorescent protein (cfp) and the modifications of the cfp's photophysical properties by oh radicals were explored in detail (submitted). in order to address the corresponding chemical changes in the protein, we devised a mild proteolysis protocol that for the first time offers a peptide mass fingerprint almost covering the cfp sequence (alvarez et al. biochemistry ). then, we achieved the meticulous characterization of the cfp oxidation products by mass spectrometry and proposed a mechanism to account for their formation by pulse radiolysis. since the cloning of the green fluorescent protein from aequoria victoria, numerous screens have been performed to improve the brightness of this protein, its spectral variants and fluorescent proteins from other species. the improvement is evaluated by comparing fluorescence intensity of individual bacteria or colonies. in this way also expression level, folding and maturation efficiency, and thickness of the bacterial colony contribute. here we report a screening method that, in addition to fluorescence intensity, quantifies the excited state lifetime of a fluorescent protein, which is independent of intensity or expression level, and provides a direct measure for the quantum efficiency of the fluorescent protein. the novel approach was used to screen a library of cyan fluorescent protein (cfp) variants randomly mutated at position , and , yielding an improved bright cyan fluorescent protein named, mposeidon, with a markedly increased fluorescence lifetime and quantum yield, increased photostability and improved fluorescence intensity in vivo. it is shown that mposeidon is the brightest cyan fluorescent protein in mammalian cells. in addition, several lifetime variants were identified that can be used for lifetime unmixing. it is demonstrated that three cfp variants can be separated and their distribution quantified in a single detection channel. a. r. faro , p. carpentier , d. bourgeois , e. rosny irtsv, cea, ufj, grenoble, france, ibs, cea, cnrs, ufj, grenoble, france, ibs, cea, cnrs, ufj, grenoble, france, ibs, cnrs, ufj, grenoble, france enhanced yellow fluorescent protein (eyfp) is extensively used as a fluorescent marker. like other photo activatable fluorescence proteins (pafp), it exhibits photo-switching properties. however, the mechanism by which fluorescence can be swiched on or off upon light irradiation is not fully understood at the molecular level. the bright to dark conversion involves a protonation step and structural rearrangements of the chromophore, but it is not clear which of these two steps is the triggering event. to answer this question, we carried out photo-switching experiments at cryotemperatures. our data suggest that a photo-induced protonation step (probably in the triplet state) is the primary event in the bright to dark conversion. our results may bear relevance to other pafps, such as iris, eos, dronpa, padron or kaede. how misfolding and aggregation of proteins constitutes a toxic insult to neurons remains largely unknown. in order to obtain insight into the molecular biology of neurodegenerative disease, we have developed a number of gfp-based biosensors for the detection and quantification of cellular clearing mechanisms for aggregated proteins. the high load on these protein quality control mechanisms, and their failure to meet normal physiological demand ultimately results in a "de-compensation" condition from which the nerve cell cannot recover. our fret/flim-based bioassays visualize protein ubiquitination and degradation; proteasomal activity; foldase activity using a folding-impaired gfp mutant which gains fluorescence conditional on the upregulation of chaperone activity; chaperone binding to unfolded proteins; and autophagosome formation/lysosomal integrity via the targeted and sensitive fret-based measurement of ph changes. these sensors are employed in cellular model systems for parkinson's and alzheimer's disease, and amyotrophic lateral sclerosis (als) to delineate the molecular pathway of cellular demise, and to gain a mechanistic understanding of the toxicity of protein aggregates and the basis for the vulnerability of neurons. millisecond photo-switching dynamics of e q gfp mutants for sensor and imaging applications m. collini , v. quercioli , l. d'alfonso , g. baldini , b. campanini , s. bettati , g. chirico dipartimento di fisica, università di milano bicocca, italy, dipartimento di biochimica e biologia molecolare, università di parma, parma, italy e q mutants of the green fluorescent protein are known to possess photo-chromic properties: the anionic emission, primed by a pump nm laser beam, can be switched between two levels of different brightness by irradiation with a blue, ∼ = nm, probe laser light. we have studied here the amplitude and the dynamics of the brightness enhancement of the e q mutant of gfpmut . the fluorescence emission increases almost threefold, under saturating probe laser excitation, for pump excitation intensity in the linear regime. two characteristic activation times, estimated by means of modulated two colour fluorescence correlation spectroscopy, are detected in the - ms range, independent of solution temperature and viscosity. the brightness enhancement factor and the characteristic activation times depend markedly on the solution ph. these results indicate that this mutant can be used as a high sensitive intracellular marker for local proton concentration and for modulated excitation imaging. the advent of fluorescent proteins (fp) gave researchers the opportunity to study proteins in situ. fluorescence resonance energy transfer (fret) benefited from this. cell fixation is a commonly used approach when working with microscopy. however, we have found that fret efficiency (e) in cells transfected with cerulean and venus chimeras could not be reproducibly measured after fixation. to evaluate this problem in detail, we measured e of cerulean-venus standard constructs by acceptor photobleaching fret, intensity-based ratiometric fret and flim-fret. the constructs were produced as standards (biophys j, , , ) with , and % e values, comprising donor-acceptor separations of , and amino acids, respectively. transient transfection of the fusion plasmids was performed into hela cells and e was measured in live and pfa or methanol fixed cells. literature e values were reproduced when measuring live cells. conversely, cell fixation caused a deviation of e values. methanol fixed cells showed e between - % for all the constructs. the effect of pfa fixation on both fluorescence intensity and fret varied vastly among independent experiments regardless of the measurement modality. thus, fixation should be avoided due to the effects it has on fp's fluorescence and consequently on fret efficiency. to explain the improvement in the fluorescence properties of cerulean when compared to ecfp, we have determined the x-ray crystallographic structures of these two proteins at physiological ph, and performed molecular dynamics simulations. both proteins exhibit a structural heterogeneity in the nterminal half of their seventh strand, which forms a specific set of van der waals interactions with the chromophore. the critical h d mutation present in cerulean induces a modification of these interactions, and allows the chromophore to be more planar and better packed, albeit only intermittently. as a consequence, the probability of non-radiative decay is significantly decreased. our results highlight the considerable dynamical flexibility that exists in the vicinity of the tryptophan-based chromophore of these engineered fluorescent proteins, and provide insights which should allow the design of mutants with enhanced optical properties. the fluorescence lifetime of green fluorescent protein g. jung biophysical chemistry, saarland university, saarbruecken, germany biotechnological design of the green fluorescent protein (gfp) and the discovery of other proteins boosted the development of the life sciences in the past decade. tracking protein movements and high resolution microscopy are only a few recent applications which were realized by fluorescent protein technology. among these examples, the switching between two chromophore state is exploited. our aim is to establish fluorescent proteins for bioanalytical fluorescence lifetime measurements. despite the progress in other fields, quantification with gfp still imposes practical problems [ ] . in the past, we observed that the uv-light driven decarboxylation of the nearby aminoacids glu distorts the fluorescence lifetime of gfp [ ] . we found out that this chemical reaction also occurs under excitation of the anionic chromophore state with a high quantum yield [ ] . recently, we could show by time-resolved spectroscopy that, indeed, the more susceptible state for this kind of photoconversion is the anionic chromophore state [ ] . suppression of this reaction therefore enables the design of autofluorescent proteins which can be used e.g. for the quantification of ions and which are beneficial as donors in fret applications. the fluorescent properties of tryptophan residues (w) in proteins are highly dependent on their immediate protein environment. however, the multi exponential decay of single w proteins is not completely understood. the most cited hypothesis contributes a multi exponential decay to the existence of several micro conformations (rotamers) of the w residue within the protein matrix. to determine rotamers we apply a method based on dead-end elimination (dee) and molecular dynamics simulations (md). low energy rotamers are calculated by dee while dynamics and further refinement is accomplished using md. the method was applied on several test cases including the protein mutant bc-csp l e from bacillus caldolyticus, which contains a solvent exposed w residue. as resolved by x-ray crystallography, this w residue occupies two conformations. using dee and md we were able to retrieve the w conformations found in the x-ray structure. the results demonstrate the ability of the method to obtain valuable w rotamers, both for solvent shielded as exposed residues. the determined conformations were compatible with the findings based on a method using replica exchange simulations. k. nienhaus , h. nar , r. heilker , j. wiedenmann , g. u. nienhaus inst. of biophysics, universität ulm, ulm, germany, dept. of lead discovery, boehringer ingelheim, biberach/riss, germany, national oceanography center, university of southampton, southampton, uk, inst. of applied physics, universität karlsruhe (th), karlsruhe, germany eqfp is a red fluorescent protein (rfp) with the chromophore in a co-planar trans orientation, whereas the cis isomer is preferred by most other rfps. by using x-ray crystallography, we determined the structures of the dimeric variants d eqfp and d eqfp at high resolution (up to . Å). for d eqfp , we had previously seen a redshifted species upon irradiation with -nm light. concomitant changes in the raman spectrum were interpreted as evidence of a trans-cis isomerization of the chromophore. here we have combined x-ray crystallography and site-directed mutagenesis to assess whether we can create a stable fluorescent, red-shifted eqfp variant with a cis chromophore. in a first step, we introduced the n s substitution. this variant, d rfp , is highly fluorescent, with the absorption (emission) maximum red-shifted by ( ) nm. with an additional s c mutation, the chromophore is found completely in the cis form. the variant, rfp , is highly fluorescent, with excitation and emission maxima at and nm. still further red shifts appear to be in reach. k. nienhaus , v. adam , d. bourgeois , g. u. nienhaus of biophysics, universität ulm, ulm, germany, esrf, grenoble, france, institute of applied physics, universität karlsruhe (th), karlsruhe, germany dendra is an engineered, monomeric gfp-like protein that belongs to a sub-class of fluorescent proteins undergoing irreversible photoconversion from a green-to a red-emitting state upon exposure to purple-blue light. this process occurs in the neutral state of the chromophore and is known to result from backbone cleavage accompanied by an extension of the delocalized π-electron system. we have measured the x-ray structure of green dendra and performed a comprehensive characterization of the optical absorption and fluorescence properties of the protein in both its green and red forms. the structure, which is very similar to those reported for the closely related proteins eosfp and kaede, revealed a local structural change next to the chromophore, involving mainly arg and a water molecule. we propose that this structural change explains the blue shift of the absorption and emission bands, as well as the markedly higher pks of the hydroxyphenyl moiety of the chromophore. the -fold enhancement of the neutral species in dendra at physiological ph accounts for the observed higher photoconversion yield of this protein in comparison to eosfp. photochromic green fluorescent protein mutants: chromophore states unveiled by raman spectroscopy s. luin, v. voliani, g. lanza, r. bizzarri, r. nifosì, p. amat, v. tozzini, m. serresi, f. beltram nest, scuola normale superiore, cnr-infm and italian institute of technology, pisa, italy the most widespread genetically-encodable fluorescent markers used for studies in living cells and tissues belong to the green fluorescent protein (gfp) family. reversibly switchable fluorescent proteins (rsfps) were developed that can undergo repeated transitions between different states, e.g. bright and dark forms. this property makes rsfps particularly attractive as active labels in biological systems for selective photolabeling applications or subdiffraction imaging. we shall present pre-resonant raman results unveiling the photophysical mechanism underlying the observed photochromic behavior. the variation of spectral properties before and after photoconversion of chemically-synthesized isolated chromophores under different protonation and/or isomerization have been analyzed, and compared to results obtained for the case of complete folded proteins comprising the same chromophores. experimental results have been analyzed within a time-dependent density functional theory, allowing us to assign all relevant vibrational modes. these results make it possible to discriminate between the effect of cis-trans isomerization and of diverse protonation states of the chromophore in the photoproducts of these proteins. s. luin et al, j. am. chem. soc. , - ( ). r. bizzarri et al., anal. bioanal. chem. , - . a combined study of the interaction of outer membrane proteins with cephalosporin antibiotics m. lovelle, i. barroso, m. j. feio, p. gameiro requimte , fac. ciências, universidade do porto, portugal gram-negative bacteria characteristically are protected by an outer membrane that serves as a selective permeation barrier. most of the β-lactam antibiotics appear to penetrate the outer membrane through these non-specific channels, and it becomes important to understand the possible interactions between β-lactams and the porin. fluorescence techniques have been largely used to characterize both the conformation and the dynamic behavior of large biological structures such as membranes and proteins. the fluorescence of the tryptophan residues is quenched in the presence of the different cephalosporin antibiotics. this reaction between the excited state of the fluorophores and the drug can be described as a formation of a non-fluorescent complex. the dependence of the fluorescence intensity upon quencher concentration for static quenching is proportional to the binding constant for complex formation. since β-lactam susceptibility is closely related to the presence of these non-specific porins, minimum inhibitory concentration (mic) by micro-broth dilution in microplate were used to assess the bactericidal activity of cephalosporin antibiotics upon on escherichia coli strain bl (de ) and a series of bl (de ) mutated in different outer membrane proteins. this combined study of the interactions at single molecular level and at in vivo level provides new insights for a better understanding of the antibiotic translocation. when used in combination with e.g. a cyan fluorescent protein, keima offers the unique opportunity to perform dual color fluorescence cross-correlation spectroscopy using a single laser line to excite both fluorophores. the molecular determinants of the large stokes shift of mkeima have been characterized structurally by combining x-ray crystallography with in crystallo uv-visible absorption, fluorescence and raman spectroscopy. our results reveal a ph-dependant "reverse chromophore protonation" of mkeima, driven by the key residue asp . moreover, the chromophore protonation state is shown to be coupled with different chromophore conformations, cis conformation at ph . , and mostly trans conformation at ph . . these results will help unravel the mechanisms of fluorescence in fps, which is of importance to rationally design and develop new fluorescent markers. recently reversibly switchable fluorescent proteins (rsfps) have become a new branch of the green fluorescent protein (gfp) like family. the rsfps may be reversibly switched between a fluorescent and a non-fluorescent state by irradiation with light of distinct wavelengths. the key process of this switching behaviour is a light induced change of the chromophore between a cis-and a trans-isomeric state. the proteins are becoming increasingly important for diverse applications like protein tracking, sub-diffraction resolution microscopy and data storage. based on the switching mechanism, we created novel rsfps with unique and improved characteristics. padron and bs-dronpa are two of these new switchable proteins. padron features a reversed switching mechanism as compared to all other green rsfps known to date while bsdronpa exhibits a very broad absorption spectrum extending into the uv. utilizing the characteristics of both proteins, we performed multi label single detection colour microscopy as well as dual colour sub-diffraction microscopy, the latter with a resolution of nm. further, we recently introduced the first monomeric rsfp exhibiting red fluorescence: using a semi rational mutagenesis approach on the non-switchable mcherry, we generated the switchable monomeric protein rscherryrev. the use of this protein in single molecule switching microscopy allowed us to record time-lapse live-cell images of the endoplasmic reticulum with sub-diffraction resolution. a spectroscopic approach to the study of chromophoric dissolved organic matter (cdom) in the sea c. santinelli, r. lavezza, l. nannicini, a. seritti cnr-ibf, via moruzzi , pisa, italy dissolved organic matter (dom) in the ocean represents the largest reservoir of reactive carbon on the earth. it is produce at each level of the marine food web and it represents the food for heterotrophic bacteria, which can use the different pools of dom (labile, semi-labile, refractory) with a large range of turn-over times (from minutes to millennia). dom plays a central role in the global carbon cycle and it has a high ecological significance. chromophoric dom (cdom) is the fraction of dom capable of adsorbing light at uv and visible spectral regions. it determines the underwater light availability in open and coastal regions with important implication on primary production and biological activity. it is photodegraded to co , co, with a significant impact on the role of the ocean as source and/or sink of atmospheric co and to highly reactive compounds, dangerous for marine organisms. in its pool "humic-like" and "protein-like" fluorophores have been individuated. cdom optical properties (absorption and fluorescence) together with doc data collected in some key regions of the mediterranean sea will be presented and discussed, in order to (i) investigate the powerful of cdom optical properties to get information on cdom "quality" (i) asses the role of dom in carbon export at depth, (iii) underline the main unresolved question on dom and cdom dynamics in the ocean, with particular emphasis to their biological lability. mechanism and applications of photo-and redox-switchable fluorescent proteins s. j. remington, j. n. henderson, x. shu, j. lohman institute of molecular biology, university of oregon, u.s.a. photoswitchable fluorescent proteins have significant advantages over conventional fluorescent labels, and in a revolutionary application, now allow cell biologists to exceed the diffraction limit in light microscopy by a factor of ten. atomic resolution crystal structures and time resolved spectroscopy of both reversible (mtfp . ) and irreversible (pa-gfp) photoswitchable fluorescent proteins in the light and dark states explain the long term stability of either state, as well as how illumination at the appropriate wavelength causes the molecules to switch between states. the photoswitching mechanisms will be discussed in terms of photochemistry, light induced chromophore isomerization and excited state proton transfer (espt). mutagenesis of espt pathways provide insight into the nature of the ratedetermining steps in proton transfer and lead to practical applications, such as redox-sensitive gfp biosensors. k. i. willig, b. hein, s. w. hell max-planck-institute for biophysical chemistry, goettingen, germany stimulated emission depletion (sted) nanoscopy is a light microscopic technique offering a resolution far beyond the diffraction limit: the excitation beam is overlapped with a doughnut-shaped, red-shifted sted beam, which switches off the ability of the molecules to fluoresce in the outer region of the excitation spot. scanning the nanosized focal spot through the sample renders sub-diffraction images with a sub-second frame rate. we used the yellow fluorescent protein citrine to image individual structural elements of living mammalian cells: vimentin and the tubular network, structures of the cytoskeleton, were recorded with a lateral (x,y) resolution < nm inside the living cell, corresponding to a -fold improvement over that of a confocal microscope. also, time lapse sted imaging of dendritic spines of yfppositive hippocampal neurons in organotypic slices outperforms confocal microscopy in revealing important structural details. as an alternative to fluorescent proteins we used a genetically encoded protein tag which can be labelled with modified organic dyes within living samples. thus nanoscale imaging of structures in the interior of living cells greatly expands the scope of light microscopy in cell biology. pulling membrane tubes from solid-supported lipid bilayers with atomic force microscopy j. w. armond , j. v. macpherson , m. s. turner department of physics, university of warwick, u.k., department of chemistry, university of warwick, u.k. information on the elastic and dynamic properties of membranes is essential for a thorough understanding of biological processes such as exocytosis, endocytosis, cell division, and pore formation. we use an atomic force microscope to pull on solid-supported lipid bilayers and observe that an energy barrier must be overcome prior to the formation of membrane tubes. we have modified elastic models of lipid bilayer vesicles to describe the free energy of a planar lipid bilayer in adhesive contact with a surface. from this model we are able to extract force-distance curves for the formation of a membrane tether, including the associated energy barrier. the experimental data can thus be understood in a quantitative fashion. this work enables the atomic force microscope as a quantitative instrument for measuring membrane pulling mechanics, and in future work will allow two-dimensional mapping of elastic properties under tension. from pores to micelles -a peptide-membrane interaction study t. l. andresen, j. r. henriksen technical university of denmark, dtu nanotech, frederiksborgvej , b , roskilde, denmark. email: thomas.andresen@nanotech.dtu.dk. research in the partitioning of peptides into lipid membranes has been intense for several years. along with scattering techniques and nmr, isothermal titration calorimetry (itc) has proven to be a powerful tool for thermodynamic characterization of peptide-membrane interactions. we have studied two antimicrobial peptides, mastoparan-x and melittin, and found that these peptides induce a range of structural transitions of popc and popc/popg membrane systems at different peptide-lipid ratios. we have found that itc can be used to elucidate the threshold where transitions occur, including the threshold for pore formation and micellation. this has been achieved using a thermodynamic model based on gouy-chapman theory, which provides the partitioning constant of the peptide-membrane interaction and thereby the concentration of peptide on the membrane surface. the structural changes have furthermore been visualized by cryo-tem. we have further investigated the pore formation in detail and found that the thermodynamic parameters of pore formation can be fully characterized using a system specific temperature where the enthalpy of peptide partitioning becomes zero. this allows for an exclusive study of the pore formation process. lactoferrin is a glycoprotein with two globular lobes, with of two domains each. since its discovery, the research on antimicrobial properties has been extended to peptides derived from this protein. the largest family studied so far is known as lactoferricin b, obtained from the protein by digestion with pepsin. more recently, a new family of antimicrobial peptides derived from lactoferrin was discovered by bolcher et al and named lactoferrampin (lfampin). the original sequence of lfampin contained residues - from the n domain of lactoferrin. we studied peptides derived from lfampin obtained by extension and/or truncation at the c-or n-terminal sides, keeping the essential characteristics, in order to unravel the main structural features responsible for antimicrobial action. the peptides were tested against model membranes. the ability to adopt helical conformation was followed by cd, the perturbation of the membrane phase transition by dsc, the energetics of interaction by isothermal titration calorimetry (itc), the partition of the peptide to the membrane by trfs and the importance of charge effects assessed by zeta potential measurements. the results are discussed and compared to the antimicrobial and hemolytic activities obtained by microbiology techniques. defensins are small cysteine-rich cationic proteins or peptides found in both vertebrates and invertebrates. they can be active against bacteria, fungi and many enveloped and non-enveloped viruses; thus, being also classified as antimicrobial peptides (amps). in the present work a comparative study between psd (a plant defensin with antifungic properties obtained from the garden pea pisum sativum) and hnp (a human neutrophils defensin) was conducted, in order to shed some light on the mechanism of action at the molecular level of these defensins. large unillamelar vesicles with different lipid compositions were used for this purpose as biomembranes model systems; namely, popc/cholesterol (characteristic of the outer leaflet of vertebrates cell membranes) and popc/ergosterol (fungal) mixtures. changes on the intrinsic fluorescence of the tryptophan residues present in these peptides upon membrane binding/insertion were followed by fluorescence spectroscopy. experimental results show the affinity of both defensins for mammalian and fungal sterols. the partitioning behavior of psd showed a high selectivity for ergosterol rich membranes, while hnp has preference for cholesterol rich membranes. preliminary characterization of atomistic computer models of galactolipid bilayers k. baczynski, m. pasenkiewicz-gierula faculty of biochemistry, biophysics and biotechnology, jagiellonian university, krakow, poland the main lipid component of thylakoid membranes are galactolipids, which constitute more than % of its lipid composition. the most common types of galactolipids found in thylakoid are monogalactosyldiacylglycerol(mgdg), whose headgroup consists of a single molecule of beta-d-galactose and digalactosyldiacylglycerol(dgdg), whose headgroup consists of two galactose molecules: alpha-d-galactose and beta-d-galactose linked by o-glycosidic bond majorty of thylakoid galactolipid have gamma-linolenic acid both in the sn- and sn- position. atomistic computer models of mgdg and dgdg molecules have been constructed and parametrized in the opls-aa force field. the molecules were used to construct three bilayer systems for molecular dynamics (md) simulations:( ) composed entirely of dgdg molecules,( ) composed entirely of mgdg molecules,( ) composed of a mixture of dgdg and mgdg molecules the ratio : . the systems have been md simulated using the gromacs . . package. the generated trajectories were analysed to determine a number of structural parameters among them: membrane thickness, average area per lipid, electron density profile accross the bilayer, order parameters, organisation of the bilayer/water interface as well as several dynamic parametrers like diffusion coefficients, lifetimes of conformational states, lifetimes od lipid lipid interactions. single mixed-lipid guv method reveals interaction of viper venom with lipid membranes n. m. ayvazyan, n. a. zaqaryan, n. a. ghazaryan dpt. biophysics, yerevan state university, armenia studies on the interaction of snake venom and organized lipid interfaces have been conducted using a variety of systems, including bilayer lipid membranes (blms), small and large unilamellar vesicles. giant unilamellar vesicles (guvs) with a mean diameter of µm have a minimum curvature and mimic cell membranes in this respect. guvs are ideal for studying lipid/lipid and lipid/protein interactions using microscopy techniques with membrane fluorescence probes. guvs were formed from the total lipid fraction from bovine brain by the electroformation method, developed by angelova and dimitrov ( ) . vipera lebetina obtusa venom was added to the sample chamber before the vesicles were formed. the membrane fluorescence probes, ans and pyrene, were used to assess the state of the membrane and specifically mark the phospholipid domains. ans and pyrene allows us to quantify the fluidity changes in the membrane by measuring of the fluorescence intensity. the presence of viper venom in guvs media reveals a noticable decreasing of membrane fluidity compare the control, while the binding of fluorophores with guvs modified by venom lead to appearance of channel activity. it was recognized early that the vipers' venom components preferred an organized lipid substrate near the lipid's phase transition and were particularly active against micellar lipids. these studies also emphasize the importance of a membrane surface curvature for its interaction with enzymatic components of venom. the attenuated total reflection fourier transform infrared (atr-ftir) spectroscopy is ideal for highly absorbing samples such as water suspensions or even bulk water due to the small light penetration depth. it is also suited for experiments on lipid membranes in excess water. for example, we have studied the hydrogen bonding (h-bonding) between cholesterol (ch) and phosphatidylcholine (pc) or sphingomyelin (sm), which could be important for the stabilization of the cholesterol-rich lipid domains. the atr-ftir method enabled comparison of the carbonyl band shape for pc/ch samples in excess h o or d o, and has confirmed similar behavior for both [ ] . secondly, we were able to analyze the amide ii band for sm/ch samples in excess h o. the observations confirm the presence of h-bonds between ch and n-h group in sm [ ] . another example is the study of the interlamellar water structure, which could influence the water-mediated phenomena in membranes. h-bonds in interlamellar water in partially hydrated lipid multibilayers are stronger with respect to bulk water. in contrast, we show by atr-ftir spectroscopy that the h-bonds are weaker in multibilayers in excess water [ ] . the bactericidal activity of antimicrobial peptides is linked to their ability to perturb the permeability of bacterial cells. they often show α-helical conformation, and many peptides have a kink in the middle of this structure, caused by pro or gly. in order to understand the role of the kink we designed various analogues of p , in which the central pro residue was moved from its central position, or removed altogether (in analogue p f). displacement of the pro residue caused a decrease of the antimicrobial activity, and an increase in the toxicity against erythrocytes, with the less active and more toxic peptide being p f. fluorescence studies suggest that both p and p f bind on the membrane surface. fluorescence experiments show that the activities of the two analogues correlate with their affinity for different kinds of lipid bilayers: the kinked p peptide has a dramatically higher affinity for negatively charged vesicles (mimicking the composition of bacterial membranes) than for neutral liposomes (similar to mammalian cells), while analogue p f exhibits comparable affinities for both membranes. therefore, our data suggest that the central pro-induced kink is involved in selectivity, inhibiting peptide binding to the membranes of eukaryotic cells. d. behn , h. hoffmeister , r. witzgall , c. steinem institute for organic and biomolecular chemistry, university of göttingen, germany, institute for molecular and cellular anatomy, university of regensburg, germany polycystin- , encoded by pkd , is an integral membrane protein with a size of kda and amino acids. the protein, which is known to be a ca + permeable, non selective cation channel, interacts with several proteins such as polycystin- , pigea or pacs- /- etc. the interaction takes place through a proposed coiled-coil domain of polycystin- located at the c-terminus of the protein.if mutation of either pkd or pkd (gene product of polycystin- ) occurs, the interaction between the proteins is disturbed leading to increased formation of renal cysts. this autosomal polycystic kidney disease (adpkd) is one of the most common genetic diseases causing renal failure due to the enormous cyst formation. in this study, the interaction of the c-terminus of polycystin- with its postulated specific interaction partners has been investigated in terms of its biological relevance. binding affinities as well as kinetic parameters of the interaction were determined. in particular, the interaction of c-polycystin- and pigea immobilized on solid supported membranes with c-polycystin- has been quantified by means of the quartz crystal microbalance (qcm) method. a dissociation constant of about nm was obtained. the results are compared with those obtained by surface plasmon resonance (spr) technique using a different immobilization strategy. correlation of the lateral structure of lipid bilayers and monolayers using two photon excitation fluorescence microscopy l. a. bagatolli membrane biophysics and biophotonics group/memphys -center for biomembrane physics, bmb, university of southern denmark most of the reported fluorescence microscopy applications on langmuir lipid films are focused in obtaining fluorescence intensity images using particular fluorescence probes. in this type of experiments the probes are generally utilized to obtain "contrast" between membrane regions (lipid domains) displaying dissimilar physical properties. the obtained information largely depends on the partition of the fluorescent probes for the lipid domains and the obtained fluorescence intensity pictures are not providing any information about the local physical properties of the lipid film. local physical properties of these distinct regions can be determined by exploring fluorescent probe related parameters such lifetime, emission shift, polarization. however these fluorescent probe's properties are almost unexplored in this type of experiments. this presentation will focus in describing a new experimental setup that includes a specially designed film balance on top of a custom built multiphoton excitation fluorescence microscope. this setup allows obtaining laur-dan gp images ( ) many studies on phase separations of lipids in bilayers and their leaflet asymmetry make use of fluorescence techniques. we used a dithionite assay, steady-state fluorescence anisotropy and fluorescence resonance energy transfer (fret) for characterization. dithionite assay was performed to measure the fluorophore distribution in the inner and outer leaflets of symmetric membranes. for low concentrations, the fluorophore is distributed almost homogeneously, whereas for concentrations > . mol % it accumulates in the outer leaflet. we discuss these effects taking into account the presence of multilamellar liposomes and dynamic effects produced by higher local bending elasticities. the results point out possible artifacts in the use of fluorophores to characterize bilayers under the assumption of their homogeneous distribution. dithionite relative bilayer permeability is discussed. the results won by fret and steady-state fluorescence anisotropy regarding lipid phase transitions are in good coincidence to each other and to results reported in the literature. the photosensitizing properties of three chlorins are compared in solution and when incorporated in dioleoylphosphatidylcholine vesicules. in solution, they possess a similar efficacy to generate singlet oxygen and a similar ability to induce the peroxidation. however, the role of the photosensitizer localization within the lipidic bilayer is strongly highlighted, when chlorins are incorporated in liposomes, both by the changes in order of peroxidation efficacy but by the measurements of the photoinduced permeation of the liposomes. the results are discussed in relation with the technology of photochemical internalization, pci. then, using giant unilamellar vesicles, we asymmetrically oxidize the membranes. we observed different shape transitions, such as budding, typical of membrane curvature modifications. the asymmetry of the shape transitions are in accordance to a lowered effective spontaneous curvature of the leaflet targeted. this effect is interpreted as a decreased preferred area of the targeted leaflet compared to the other, due to the secondary products of oxidation. permeabilization of guv by photooxidation is interpret as the opening of a pore above a critical membrane tension due to the budding of vesicles. the effective spontaneous curvature of photosensitized vesicle at lysis was estimated. additionally photooxidation was shown to be fusogenic. influence of polyphenol extracts from fruits on biological and model membranes d. bonarska-kujawa, h. pruchnik, j. sarapuk, j. oszmiański, h. kleszczyńska univ. of environmental and life sciences, wroc law, poland biological activity of polyphenol extracts from apple, strawberry and chokeberry was studied. polyphenols were shown to be good antioxidants and to act as antyhemolytic agents. both the activities are the result of polyphenols incorporation into biological membranes. to check potential changes they induce in membranes some experiments were performed with the use of erythrocytes, and lipid membranes. it was found that the extracts studied induced shape transitions of erythrocytes. they were classified according to the bessis-brecher scale. strawberry extract induced mainly discocytes and discoechinocytes. populations of discocytes, echinocytes and some discoechinocytes were found on applying apple extract, while chokeberry induced mainly the formation of echinocytes and spheroechinocytes. the results evidence that the polyphenols incorporate into the external monolayer of lipid bilayer of the erythrocyte membrane. the results of the fluorimetric experiments showed that all the extracts changed fluidity in the hydrophilic part of rbc membrane. the changes observed were biggest for chokeberry extract and smallest for strawberry one. incorporation was also followed by changes in electrical resistance of black lipid membranes and in shifting the temperatures of main transition (t m ) and pretransition (t p ) in liposomes. this work was sponsored by ministry of science and education, scientific project no. n n . a. boll , n. czudnochowski , m. geyer , c. steinem institue of organic and biomolecular chemistry, university of göttingen, germany, mpi for molecular physiology, dortmund, germany hiv- tat belongs to the accessory proteins of hiv and has regulatory functions. tat is concentrated in the nucleus and nucleolus of infected cells. the protein is composed of amino acids with a molecular weight of kda. tat is a transcriptional activator protein, which stimulates rna polymerase ii-mediated transcription elongation. therefore, tat interacts with cyclin t and binds to the tar rna element. tat has different domains. with respect to the interaction with lipid membranes, the most important structural motif is its basic region, including arginine and lysine residues. the peptide derived from this basic region belongs to the cell-penetrating peptides and is able to translocate through lipid membranes. the major aim of this study is to investigate the influence of full length hiv- tat on artificial lipid membranes. as a starting point solid-supported membranes composed of -palmitoyl- -oleoyl-sn-glycero- phosphocholine (popc) immobilized on silicon dioxide were used. the influence of the lipid head groups on the interaction with tat was analysed by using membranes composed of the negatively charged lipid -palmitoyl- -oleoyl-sn-glycero- -[phospho-l-serine] (pops) in a mixture with popc. fluorescently labelled tat was used to localise the protein in the membrane. the impact of tat on lipid membranes was investigated by fluorescence and atomic force microscopy, showing that it is capable of perturbing lipid membranes. g. bocchinfuso , a. palleschi , b. orioni , g. grande , f. formaggio , c. toniolo , y. park , k. s. hahm , l. stella univ. of rome tor vergata, dept. of chemistry, italy, dept. of chemistry, univ. of padova and cnr, italy, chosun univ., rcpm, south korea. most antimicrobial peptides exert their activity by perturbing the permeability of bacterial membranes, but the molecular details of this process are still debated. here, we compare fluorescence experiments and molecular dynamics simulations regarding the interaction of two antimicrobial peptides (pmap- and trichogin ga iv) with lipid membranes, showing that their mechanisms of bilayer perturbation are significantly different. pmap- , a cationic peptide member of the cathelicidin family, associates to the membrane close to its surface and parallel to it, and in this arrangement it causes a severe perturbation to the bilayer, both regarding its surface tension and lipid order. on the other hand, trichogin ga iv, a neutral peptide member of the peptaibol family, undergoes a transition from a surface-bound state to an inserted orientation. in the first arrangement it does not cause any strong membrane perturbation, while in the second orientation it may span the bilayer from one side to the other, despite its relatively short length, by causing a significant thinning of the membrane. lipopolysaccharide (lps) is the main component of the outer membrane of gram negative bacteria. lps is also known as endotoxin because of its potency to induce sepsis, a serious source of mortality in many clinical cases. among lps-neutralizing agents, polymyxin b (pxb) is considered as the "gold standard" due to its high efficiency of binding and detoxification of endotoxin. in this work, we have further explored the interaction of pxb to lps from the rough mutant of salmonella minnesota (re-lps) both in the gel and in the liquid crystalline phase, using isothermal titration calorimetry (itc) and fluorescence based techniques. the effect of pxb binding on lps-membrane integrity was determined with a fluorescence quenching assay treating vesicles of lps labeled with nbd-pe with dithionite. thermodynamic parameters associated with the binding process as well as binding stoichiometry were obtained from itc experiments. finally, itc was conducted with enterococcus faecalis and salmonella typhimurium, as representative examples of a gram negative and a gram positive bacterium respectively. pressure jumps -an accessible trigger for biomolecular transformations high pressure can be used to induce many structural changes in biological systems: from triggering phase changes in model membranes to causing protein unfolding, in fact any change involving a volume reduction is promoted by pressure. as well as being broadly applicable, pressure changes can be applied very quickly both up and down in contrast to other structure change triggers such as temperature jumps. fast pressure jumps allow the trigger to be decoupled from structural changes, so with fast structure probe techniques such as time resolved x-ray diffraction, the out-of-equilibrium evolution of these systems can be monitored. despite great advantages, high pressure remains under-utilised primarily due to its technical difficulty. in response to this technology vacuum a high pressure user facility based around a pressure jump cell for small and wide angle x-ray diffraction has been commissioned at beamline i , diamond light source, uk and will be freely available to the user community. the cell is highly robust requiring virtually no user intervention during an experiment and the pressure system is computer controlled with a graphical user interface and is integrated with the beamline. pressures between and . gpa are accessible and jumps can be carried out in approximately ms at temperatures from - to • c. sample changing has been made simple and fast with a dedicated sample loading port and modular sample holders allow optimisation for a broad range of samples. the transformation of vesicle and lateral distribution of mobile membrane inclusions b. bozic institute of biophysics, faculty of medicine, university of ljubljana, lipičeva , si- ljubljana, slovenia membrane inclusions such as membrane embedded peptides or proteins exhibit curvature dependent interaction with the surrounding lipid matrix due to the mismatch between their intrinsic curvature and the local membrane curvature. this interaction causes an inhomogeneous lateral distribution of the inclusions and a corresponding adjustment of the vesicle shape. by taking into consideration that the membrane free energy includes elastic energy of the lipid bilayer and a contribution due to an inclusion-membrane interaction, the configurations of lipid vesicles with mobile inclusions have been studied theoretically. the variational problem to calculate equilibrium vesicle shapes is solved by applying a ritz method based on an expansion in spherical harmonics. in general, vesicle shapes adjust to the presence of inclusions by increasing regions with favorable curvature and decreasing regions of unfavorable curvature in such a way that the lateral distribution of inclusions becomes inhomogeneous. if the number of inclusions or the inclusion-membrane interaction exceeds a certain value, the prolate shapes become globally stable. investigating the structure of pores formed by antimicrobial peptides using epr spectroscopy m. bortolus , k.-s. hahm , a. l. maniero universita' di padova, padova, italy, chosun university, kwangju, south korea spin label electron paramagnetic resonance (epr) is a spectroscopical technique effective to study the molecular mobility of membrane components and the membranepeptide interactions, as the timescale of epr is optimally matched to the rotational motions of lipids in membranes. we applied epr to study the pore-forming mechanism of two antimicrobial peptides (amp) that create pores of different dimensions when interacting with liposomes. hp ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) is derived from the n-terminus of helicobacter pylori ribosomal protein l , and hpa is an hp ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) analogue where gln and asp at positions and were substituted by trp. we studied the interaction of the two amp with zwitterionic and negatively charged vesicles, doped with phospholipids spin labelled in the lipid head or at the c or c positions; the different phospholipids allow us to study the peptide-membrane interaction at different depths relative to the membrane surface. we studied the interaction of the amp with vesicles following the influence of amp on label mobility as a function of temperature and membrane depth. we also prepared spin-labelled dmpc/dhpc bicelles, doped with lanthanide ions (dy + /tm + ) that allow us to macroscopically orient the system using the magnetic field of the epr spectrometer. we studied the interaction of amp with the oriented bicellar system monitoring the effect of amp on the order parameter of the phase. a. chattopadhyay centre for cellular and molecular biology, hyderabad, india we addressed the organization and dynamics of the human serotonin a receptor fused to enhanced yellow fluorescent protein (serotonin a r-eyfp) expressed in cho cells. serotonin a receptors are prototypical members of the gprotein coupled receptor superfamily and represent a prime target for therapeutic actions of several anxiolytic and antidepressant drugs. interestingly, we observed significant retention in fluorescence of serotonin a receptors upon triton x- treatment of intact cells at low temperature demonstrating their detergent insolubility. we analyzed the role of cholesterol in the plasma membrane organization of the serotonin a receptor by fluorescence recovery after photobleaching (frap) measurements with varying bleach spot sizes. our results show that lateral diffusion parameters of serotonin a receptors are altered in cholesterol-depleted cells in a manner that is consistent with dynamic confinement of serotonin a receptors in the plasma membrane. interestingly, results from frap measurements performed under conditions of mild cytoskeletal destabilization suggest that receptor signaling is correlated with receptor mobility, in agreement with the 'mobile receptor hypothesis'. our current work is focused on exploring the oligomerization of the receptor using photobleaching anisotropy measurements and indicates the presence of constitutive oligomers of the serotonin a receptor in live cells. expression and reconstitution of connexin in pore-suspending membranes c. carnarius , s. kaufmann , m. tanaka , c. steinem institute of organic and biomolecular chemistry, university of göttingen, tammannstrasse , göttingen, germany, institute of physical chemistry, university of heidelberg, im neuenheimer feld , heidelberg, germany the intercellular communication and electronic coupling between adjacent cells of vertebrates are mediated by gap junctions. these proteins are composed of two connexon hemichannels, whereas each connexon consists of six connexin subunits. each subunit is characterized by two conserved motives: two extracellular loops and four transmembrane α-helices. in this study, we focused on cx . to obtain large amounts of this protein, we expressed cx and cx +gfp in a rather new expression system: dictyostelium discoideum. in contrast to human tissue cultures, the system allows for high cell densities up to million cells per ml and the cells can be cultivated by fermentation. cx +gfp was successfully visualized in d. discoideum by confocal laser scanning microscopy, where it was preferentially found in the plasma membrane.after the cells were harvested, plasma membranes were prepared and both proteins (cx and cx +gfp) were verified by western blot analysis. the proteins were solubilized by addition of % n-octyl-β-d-glucopyranoside and purified by ion metal chelate affinity chromatography. the activities of both proteins were confirmed by a cytochrome c assay. after the purification of both proteins, they were reconstituted in µm-sized pore-suspending membranes. in the near future, we plan to determine the mobility of cx and cx +gfp in these membranes by fluorescence recovery after photobleaching. o. cañadas, c. casals dpt. biochemistry and molecular biology i, and ciber enfermedades respiratorias, complutense university of madrid, madrid, spain inhaled bacterial lipopolysaccharide (lps) may incorporate into the lung surfactant monolayer. in this study, the effect of smooth lps (s-lps) on the surface activity of lung surfactant was evaluated. to that end we investigated the behavior of dppc films containing s-lps, with and without surfactant protein a (sp-a) in the subphase, using epifluorescence microscopy combined with a surface balance. our data show that s-lps injected into the subphase incorporated into dppc films forming mixed dppc/s-lps monolayers. cospread s-lps fluidized the dppc monolayer as demonstrated by epifluorescence images and changes in the compressibility modulus of the monolayer as a function of s-lps molar fraction (x s−lps ). the presence of low amounts of s-lps in the monolayer promoted early collapse, preventing high surface pressures to be reached. moreover, s-lps hampered the re-spreading of dppc molecules during dynamic compression at s-lps concentrations as small as x s−lps = . . such inhibitory effects could not be relieved by repeated compression-expansion cycles or by adding surfactant protein a. however, sp-a facilitated the squeeze-out of s-lps from dppc/s-lps mixed monolayers, suggesting that sp-a is an s-lps scavenger. cholesterol displaces ceramide from its tight packing with sphingomyelin in the absence of ld phase j. v. busto, j. sot, j. requejo-isidro, f. m. goñi, a. alonso unidad de biofísica (csic-upv/ehu) and departamento de bioquímica, u. país vasco (upv/ehu), spain a set of biophysical approaches have been applied to study the phase behaviour of palmitoylsphingomyelin (psm)/cholesterol (chol) model membranes upon palmitoylceramide (pcer) addition. fluorescence spectroscopy of di- -aneppdhq-stained psm/chol vesicles reveals no segregation of large liquid-ordered (l o ) microdomains. in contrast, the formation of disperse, compositionally homogeneous l o psm/chol ( : ) nanodomains over a psm gel (l β ) phase is proposed. dsc measurements show that pcer addition to vesicles with coexisting l o and l β phases (low [chol] ) induces the formation of large gel-like psm/pcer microdomains, coexisting with a l o psm/chol phase. the ∆h for the psm/pcer phase at high psm/(chol+pcer) ratio is close to that of the binary mixture in the absence of chol, supporting immiscibility, but no displacement, between chol and pcer-rich phases. on the contrary, both confocal microscopy of guvs and the dsc data coincide in showing that a rise in pcer increases the gel-like phase to a lower extent than in the pure psm/pcer mixture, revealing some cholinduced restriction. in the presence of a pure l o psm/chol phase (high [chol] ), pcer addition is unable to induce the formation of large psm/pcer microdomains. the present data support the role of chol as the key determinant in controlling its own displacement from l o domains by ceramide. hydroquinones modifying lipid membrane morphology c. di vitta , l. marzorati , v. rebbin , s. s. funari iqusp, university of são paulo, são paulo, brasil, hasylab, hamburg, germany quinones are important molecules in cells. flavina, ubiquinone and coenzyme q, coq, are associated with electron transfer. coq, having a hydrophobic tail, is soluble in the internal lipid membrane of mitochondria. their reduction leads to the equivalent hydroquinones. we synthesized novel alkylthioquinones aths, to investigate their interaction with phospholipids. one or more long hydrophobic chains attached to the quinone ring alter their hydrophobicity and electron availability. we aimed at their influence on the structure of membranes. different phs highlights the charge/polarity effect on the interaction and on the morphology of the bilayer. for that, pope and popc, lipids with different charges, but identical chains were selected. x-rays diffraction on pope/ , bath, / at different temperatures and ph, showed cubic phases coexisting with the usual phases formed by simply hydrated pope, at different phs. for investigation of the charge/polarity, we turned to the popc/ , bath system (smaller charge/polarity). despite decrease in temperature of phase transition and dimensions of the lattice, the structures were the same as in hydrated lipid, illustrating a less significant effect than on the similar lipid pope. another additive, , ath, mixed with pope showed the effect of multiple thioalky chains. no morphology change was seen, compared to pure lipid. interestingly, despite both additives differ by one thioalkyl chain only, their influence on the pope matrix is so different. cross-linking of phospholipid membranes by calcium-sensitive synaptotagmins synaptotagmins are vesicular proteins implicated in many membrane trafficking events. they are highly conserved in evolution and the mammalian family contains isoforms. we now show that the tandem c domains of several calcium-sensitive synaptotagmin isoforms tested, including drosophila synaptotagmin, rapidly cross-link phospholipid membranes. in contrast to the tandem structure, individual c domains failed to trigger membrane cross-linking in several novel assays. large-scale liposomal aggregation driven by tandem c domains in response to calcium was confirmed by the following techniques: turbidity assay, dynamic lightscattering and both confocal and negative stain electron microscopy. high-resolution cryo-electron microscopy revealed that membrane cross-linking by tandem c domains results in a constant distance of approximately nm between the apposed membranes. our findings show the conserved nature of this important property of synaptotagmin, demonstrate the significance of the tandem c domain structure and provide a plausible explanation for the accelerating effect of synaptotagmins on membrane fusion. membrane proteins can be challenging samples to work with and as such often require the use of multiple techniques. here we present an insight into the structure of the antimicrobial peptide melittin in lipid membranes using various techniques. we explore the conformational changes of membrane-bound melittin and its interaction with model membrane lipid systems with a series of spectroscopic methods that can be used in parallel. the techniques used include linear dichroism and ft-ir for orientation information and circular dichroism for conformation information. we use dynamic light scattering for molecular sizing, fluorescence emission for information on peptide environment, thin-layer chromatography for lipid identification, analytical ultracentrifugation to identify oligomerisation state and calorimetry to investigate the thermodynamics. we observe how the physical properties of both the peptide and the membranes affect the insertion kinetics of the peptide in the membrane. phospholipid membranes dynamics: molecular dynamics vs neutron scattering v. conti nibali , m. tarek , u. wanderlingh , g. d'angelo department of physics, faculty of science, university of messina, italy, unité mixte de recherche cnrs/uhp , université henri poincaré, nancy, france collective dynamics and single-particle dynamics of hydrated multilamellar phospholipid bilayers ( , -dimyristoylsn-glycero- -phosphatidylcholine, dmpc) have been studied by means of all-atom molecular dynamics simulations. here we report results of a gel phase bilayer at k and of a liquid crystal phase bilayer at k. coherent and incoherent dynamic structure factors and meansquare displacements have been calculated from the trajectories for both the inplane and out-of-plane lipid dynamics. moreover, the results have been compared to recent quasi-elastic and inelastic neutron scattering data. optical tweezers allow trapping of particles of different types in a wide range of sizes [ ] . among these, unilamellar vesicles are of interest as they are known to be effective vectors in drug delivery and they are also studied as models for cell membranes. this work focuses on the interactions between optically confined unilamellar vesicles and their cross-linking by proteins [ ] . synaptotagmins are vesicular proteins implicated in many membrane trafficking events having an accelerating effect on the membrane fusion. calcium-sensitive synaptotagmins are thought to confer calcium sensitivity to the fusion of secretory vesicles with target membranes [ ] . the novel optical assay reported here allowed us to visualize the cross-linking of nm liposomes mediated by synaptotagmin and calcium. the use of the optical tweezers approach to investigate the function of other fusogenic proteins related to exocytosis (i.e., snare proteins) is discussed as well. antimicrobial peptides (amps) have received increasing interest as the search for new potential antibiotics has become imperative due to increasing bacterial multiresistance. acylating the natural occurring polymyxin b (pmb) significantly enhances antibacterial activity towards two representative gram-negative bacteria e. coli and k. pneumonia compared to the nonacylated pmb. the aim of the presented work was to study various biophysical parameters, such as partitioning coefficients, zeta potentials and effective charges of a selected amp, mastoparan-x (mpx) and a propanoylated (pampx) and octanoylated (oampx) analogue, respectively. fluorescence spectroscopy, isothermal titration calorimetry and ζ-potential measurements were the main techniques used for the measurements. we employed different luv model systems; a partially charged (popg/popc : ) and a neutral system (popc). for the neutral luvs there was an increase in partitioning with increasing length of the acyl chain, whereas partioning into the partially charged luvs was governed by a balance of hydrophobic and electrostatic contributions. the selectivity for the partially charged luvs over the neutral luvs was in the order pampx, mpx and oampx. the modeled effective charge for the peptide followed the same trend as the partitioning coefficient for the three peptides for the respective luv systems. does the lysosomal membrane need triglycerides? a spectroscopic study of a simple model membrane l. duelund , k. pakkanen , m. vuento , j. h. ipsen memphys -center for biomembrane physics, university of southern denmark, odense, denmark, nanoscience center, university of jyväskylä, jyväskylä, finland lysosomes are intracellular organelles in which proteins and other macromolecules are degraded. morphological and functional changes in different compartments of the endocytic pathway are connected to several diseases. a crucial step in understanding biogenesis of lysosomes and their role in disease conditions, is to characterise the properties of the lysosomal membrane. by the use of tlc we have found that lysosomes contain non-neglible amounts of triglycerides (tg). to investigate how the presence of tgs could influence the lysosomal membrane, we have investigated the properties of a mixture of popc and triolein, as a simple model for the lysosomal membrane. we found the system to form two types of popc-rich membranes. these were determined as co-existing phases based on their spontaneous and stable separation and named heavy and light phase according to their sedimentation behaviour. by using epr and fluorescence spectroscopy the physical properties, including order, fluidity and water penetration, of these phases were found to differ markedly despite of their almost identical composition. the results suggest that presence of tgs on lysosomal membranes could have a crucial effect in the barrier functions and thus, the integrity of the organelle. model membranes with the capacity to align in magnetic fields such as bicelles and magnetically sensitive vesicles are of high interest, since their macroscopically alignment allows for the determination of structure, dynamics and topology of molecules within the membrane [ ] . we performed p solid state nmr on a magnetically sensitive lipid possessing a large positive magnetic anisotropy introduced in form of a biphenyl unit during lipid synthesis in one of its acyl chains ( -tetradecanoyl- -( -( biphenyl) butanoyl)-snglycero- -phosphocholine (tbbpc)). the phosphorus lineshapes of tbbpc mlvs studied at various magnetic fields ( . t, . t, . t, . t), revealed a drastic change in shape upon exposure to fields > . tesla: resonances resulting from phospholipid molecules oriented perpendicular to the magnetic field decrease whereas resonances resulting from parallel oriented molecules increase. this is a sign for magnetically induced vesicle deformation from vesicle to oblate ellipsoid shape. factors influencing this drastic deformation, such as magnetic anisotropy, membrane elasticity, lipid chain length and field dependency are discussed based on existing theories [ ] . hepatitis g virus (gbv-c/hgv) is an enveloped viruses belonging to the flaviviridae family. clinical findings have suggested that in people co-infected with gbv-c/hgv and human immunodeficiency virus (hiv), delayed progression of aids has been observed ( ). the mechanism by which this virus may inhibit the progression of aids remain to be elucidated. enveloped viruses acquire their lipid membranes by budding through host cellular membranes ( ) , in this process; fusion peptides play an important role. study of the interaction of hgv-c peptides with lipid membranes could lead us useful information about the mechanism that takes place. in this work we present a study on the effects of e peptides on the fluidity of and polarizability of model membranes (dmpc and dppc liposomes) by dsc and fluorescence polarization. both techniques showed that the presence of the studied sequences in phospholipid mixtures already affected the thermotropic properties of the gel to liquid-crystalline phase transition. systems were thermodynamically charac- small angle neutron scattering (sans) studies have been performed to study the structural changes induced in membranes of vesicles prepared from phospholipid and mixed phospholipid-sterol mixtures, in the presence of different concentrations of the anti-fungal drug, amphotericin b (amb).the vesicles, sonicated to a mean size∼ nm, were prepared using dimyristoylphosphatidylcholine(dmpc) or dmpc-cholesterol or dmpc-ergosterol mixtures -with both of the mixed systems involving mol% sterol. analysis of the sans data show that when the concentration of amb added is just above the drug's cmc (∼ µm) there is an increase in the membrane thickness of both the dmpc-chol and dmpc-erg vesicles (both cases + Å), but the thickness of the pure dmpc vesicle membranes remains the same as in the absence of amb. when amb is added at a concentration in excess of its cmc (∼ µm), the mixed-sterol vesicles show the same changes in membrane thickness as observed with the lower amb concentration, and the pure dmpc vesicles again remain unaffected. on the basis of these studies, therefore, there appears to be no difference in the structural changes induced by the insertion of amb into the model fungal cell membranes (mimicked by dmpc-erg vesicles and those resulting from its insertion into the model mammalian cell membranes (mimicked by dmpc-chol vesicles). the third transmembrane helix of bacteriorhodopsin, also known as phlip, is a unique model system for studying the interactions of a natural transmembrane domain with lipid membranes: depending on ph, the water-soluble peptide either adsorbs superficially or inserts as a transmembrane helix on addition of lipid vesicles [ ] . published values for the free energies of these processes were based on a stoichiometric model invoking two distinct sets of binding sites [ ] . however, discrepancies between data obtained from different experimental techniques and inconsistencies between experimental and expected temperature dependencies suggest that these values should be taken with caution. we therefore reassessed membrane interactions of phlip using titration calorimetry and fluorescence spectroscopy. if electrostatic effects at the membrane surface are taken into account, the data can be described quantitatively by a partition equilibrium, but not by a stoichiometric binding model. the thermodynamics of membrane partitioning differ substantially from those determined previously [ ] and draw a different picture of peptide-lipid interactions. beyond deepening our insights into the first step of the two-stage model of membrane protein folding, this also sheds light on the ability of phlip to drag cargo molecules across lipid membranes. adsorption of monolysine and polylysines at the surface of lipid membranes of varied composition was studied by two methods. both methods -the electrokinetic one, measured the surface potential of liposomes, and the intramembranouse field compensation sensitive to boundary potential of planar bilayer lipid membranes -detected the positive changes of the potentials for membranes with negatively charged component (cardiolipin, cl). neither monolysine, nor polylysines adsorbed at neutral membranes (phosphatidylcholine, pc). pentalysine show the difference between these potentials for the membranes composed from cl/pc mixtures. this difference is attributed to dipole part of boundary potential and indicates the changes in lipid packing. polylysines show high affinity to the membrane and saturation with plateau. these saturation levels correspond to surface charge densities . and . c/m for oligomers with and units, and . c/m for polymers with and units. these values do not depend on the ionic strength of background electrolyte but proportional to the content of negatively charged components in the lipid bilayer. the polylysine layer at the mica surface was studied by atomic-force microscope (afm) technique. it was shown that pentalysine molecules cover the surface by the layer of . nm thickness and polylysines of high molecular weight by the layer up to nm. effects of lysolipids on the mechanical stability of lipid membranes j. r. henriksen , l. feldborg , j. h. ipsen , t. l. andresen technical university of denmark, dtu nanotech, frederiksborgvej , b , roskilde, denmark., university of southern denmark, department of physics and chemistry, memphys center, campusvej , odense m, denmark lysolipids (lpcs) and fatty acids (fas) are the natural products formed by the hydrolysis of phospholipids. lpcs and fas form micelles in solution and thus act as detergents in the presence of lipid membranes. in the present study we investigate the detergent strength of a homologous series of lysolipids (lpcs) on popc lipid membranes by use of isothermal titration calorimetry (itc) and vesicle fluctuation analysis (vfa). the membrane partition coefficient (k) and critical micelle concentration (cmc) are determined and found to obey an inverse proportionality relation (cmc x k ∼ constant). the partition coefficient and critical micelle concentration are used for the analysis of lpc's effect on the membrane bending rigidity. the dependence of the bending rigidity on the lpc membrane coverage has been analyzed in terms a phenomenological model based on continuum elastic theory which yield information about the curvature inducing properties of the lpc molecule. the results reveal: (i) an increase in the partition coefficient with lpc acyl-chain length and (ii) the degree in acyl chain mismatch between lpc and popc determines the magnitude of the membrane mechanical perturbation per lpc molecule on the membrane. patch clamp electrophysiology remains the gold-standard for ion channel research because of the information richness of the data produced. automated planar patch clamp devices, with their higher throughput and high data information content, have made the technique accessible to a wider audience. nanion technologies offers two planar patch clamp workstations combining higher throughput with high data quality. the port-a-patch records from a single cell at a time and the patchliner from up to cells simultaneously with high success rates (typically - %). both, the port-a-patch and the patchliner are bench top patch clamp rigs which uses a planar borosilicate glass chip for obtaining a giga-seal for electrophysiological recordings. suction applied from the underside of the chip is used to attract a single cell to the recording site without the need for optical visualization. both workstations have been successfully used for whole cell, perforated patch, and cell attached recordings as well as for guv-bilayer recordings. due to the versatility of nanion`s products it is possible to study a wide variety of ion channels including nav, kv, cav, ligandgated, herg or reconstituted proteins such as ksca, cx , ompc. special features unique to nanion products, including but not limited to internal solution exchange and temperature control, expand the experimental possibilities. waves on lipid monolayers j. griesbauer, s. boessinger, a. wixforth, s. f. matthias univiversity of augsburg, experimental physics i, biological physics group, augsburg, germany " for the sake of illustration we shall try to provide a physical basis for the equations, but must emphasize that the interpretation given is unlikely to provide a correct picture of the membrane." [ hodgkin&huxley, ] to explain the occurrence of reversible heat production during nerve pulse propagation it has been suggested by multiple authors [wilke,kaufmann,heimburg] that travelling sound waves and not ion channels may offer a better explanation than the hodgkin&huxley theory. therefore, if sound waves are an essential feature of the nerve membrane they should also appear on lipid monolayers where ion conductivity is evidently absent. here we demonstrate that sound waves can be excited on a lipid monolayer by using a set of planar electrodes incorporated into the monolayer and driven by an alternating voltage. not only do our results indicate propagating waves on lipid monolayers in accordance with their thermodynamic predictions, but importantly, no significant attenuation is detected proposing an adiabatic phenomenon. in order to provide evidence that our physical explanation provides a correct picture of the membrane, direct detection of the waves was done, whereas a clear transduction of signals was shown. finally, the impact of toxins, neuropharmaca and anaesthetics needs to be integrated in our physical picture of the nerve membranes, what can easily be done now and could deliver a new approach for understanding their physical mechanism. our earlier studies showed that organometallic compounds (otc) in the presence of uvc radiation enhanced the degree of phosphatidylcholine (pc) liposome oxidation, whereas quercetin effectively protected the membrane. the present investigation is concerned with the effect of uvb and otc (chlorides of diphenyl-and dibutyltin, triphenyl-and tributyltin), both in combined action and separately, on oxidation of erythrocyte proteins, pc liposome and albumin. the degree of oxidation of proteins and liposomes induced by otc and radiation, also in the presence of selected antioxidants (trolox, quercetin) was determined on the basis of changes in the number of sulfhydryl groups, carbonyl groups and malone dialdehyde, respectively. the studies indicate that uvb induces pc liposome and erythrocyte oxidation, whereas in the case of albumin it causes both an increase in the number of c=o groups and free sh groups (most probably, braking sulphonic bridges). otc compounds interact with membrane biomolecules both as weak pro-and antioxidants, also in combined action (uvb plus otc). the prooxidative effects are markedly diminished by application of antioxidants. the action of quercetin results from its ability to incorporate into membranes and formation of complexes with otc (liposome>erythrocyte>albumine). this work was supported by grant n n . a phospholipase c/ sphingomyelinase from pseudomonas aeruginosa has been assayed on giant unilamellar vesicles (guv) consisting of phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and cholesterol, at equimolar ratios. the enzyme activity modifies the chemical composition, thus the physical properties of the bilayer, and conversely the latter influence the enzyme activity. biochemical assays of enzyme activity, together with confocal fluorescence microscopy examination of guv provide novel information about the system. the original lipid composition in the absence of enzyme gives rise to lateral phase separation of liquid-ordered and liquid-disordered domains in the guv. the two enzyme end-products, diacylglycerol and ceramide, have opposite effects on the bilayer physical properties, the former abolishes lateral phase separation, while the latter generates a new gel phase. morphological examination of individual guv shows that the enzyme binds preferentially the more fluid (or more disordered) domains, and that, in most cases, it causes the fluidification (disordering) of the other domains.. cholesterol incorporation into lipid bilayers, in the form of multilamellar vesicles or extruded large unilamellar vesicles, has been quantitated. to this aim, the cholesterol contents of bilayers prepared from phospholipid:cholesterol mixtures ( - mol% cholesterol) have been measured and compared with the original mixture before lipid hydration. there is a great diversity of cases, but under most conditions the actual cholesterol proportion present in the bilayers is much lower than expected. the maximum solubility of chol in bilayers containing saturated pc or pc with less than four unsaturations is - mol%, while in polyunsaturated pc, e. g. dic : , and in sphingomyelin the maximum chol contents is mol%. a quantitative analysis of the vesicles is thus required before any experimental study is undertaken. membrane fusion assay based on poresuspending lipid bilayers i. höfer, c. steinem institute of organic and biomolecular chemistry, university of göttingen, germany membrane fusion has attracted significant interest due to its biological relevance. thus in recent years a great variety of fusion assays has been developed. since the process of membrane fusion is not yet fully understood, our aim is to develop a new fusion assay based on pore-spanning membranes, which have been proven mechanically stable, highly insulating and rather tension free. the application of these so-called micro-blms should allow simultaneous monitoring of lipid mixing, content release as well as electrical readouts. furthermore both membrane sides can be addressed individually to apply transmembrane potential or fusion modulating compounds. first results show that the micro-blms provide the opportunity to investigate lipid bilayer fusion by means of fluorescence microscopy. the formation of porespanning membranes is achieved by the painting technique of dphpc doped with oregon green dhpe dissolved in ndecane. the addition of large unilamellar vesicles doped with texas red dhpe allows direct observation of single fusion events. lipid mixing during fusion leads to a decrease of the donor fluorescence (oregon green) and an increase in acceptor fluorescence intensity (texas red) in the plane of the planar bilayer due to fret. in future work simultaneous monitoring of lipid-mixing and content release combined with electrical measurements are planned to gain further insight into different intermediate steps of membrane fusion. the kinetics of membrane-peptide folding and orientation m. r. hicks department of chemistry, university of warwick, uk. understanding interactions of peptides with lipid membranes is essential if we are to be able to design new and better antibiotic peptides. there are different steps in these interactions and following the kinetics of these processes requires that we combine the information from different biophysical techniques. here we present data on the kinetics of peptidemembrane interactions using circular dichroism to report on conformation and linear dichroism to report on orientation of the peptides in/on membranes. these are combined with other techniques such as dynamic light scattering and fluorescence spectroscopy to elucidate the mechanisms of action of the peptides. one of the most important aspects of membrane-active peptide design is that of specificity. we investigate specificity for different types of membranes by using libraries of lipids with different properties of charge, chain saturation and curvature stress. in this way one can test for effects of e.g. negatively charged head groups found in bacterial membranes or cholesterol found in animal and human membranes. using these approaches it is proposed that we will be able to modify peptide sequences, test what part of the kinetic processes are affected and subsequently use this information to design new and better antibiotics. k. kubica, h. misiak wroclaw university of technology, wroclaw, poland reversible membrane electroporation, that can be observed as a effect of electric field influence on biological membranes, finds application in cell delivery of biologically active compounds. the membrane susceptibility for creation of stable pores, depends on electric field parameters applied to the membrane as well as on the membrane environment (ionic strength, ph, temperature) and membrane molecular composition. we have already shown that the changes of van der waals lipid chain interaction effect the process of pore formation. there are known factors which increase or decrease the membrane respond to electric field. because pores appear in lipid membrane matrix it make sense to study this phenomenon using model planar lipid membranes. as lipids are very receptive on oxidizing factors we decided to study the relation between lipid oxidation and membrane reaction on electric field. with the use of four electrode galvanostat we are examining the influence of factors initiating lipid oxidation (uv, fe + ) and the efficiency of some antioxidant compounds of lipid protection on electric properties of lipid membrane. the product of lipid oxidation is determined by characteristic reaction with tiobarbituric acid. we want to determine the role of polyunsaturated fatty acids on membrane properties also. results of our work will be useful in optimization of medicine distribution, aided by electric field, into cells. budding of giant phospholipid vesicles induced by β -glycoprotein i j. kovacic, b. bozic, s. svetina institute of biophysics, faculty of medicine, university of ljubljana, ljubljana, slovenia β -glycoprotein i (β gpi) is classified among amphitropic proteins: in its inactive form it is dissolved in plasma and in its active form it is bound to membrane. in comparison to other amphitropic proteins it exhibits a distinct membrane interaction behavior. a patch of positively charged amino acid residues contacts anionic phospholipids via electrostatic interactions, and a hydrophobic loop anchores the protein into the outer leaflet of the membrane via hydrophobic interactions. the binding constant depends on physical properties of the membrane such as membrane lipid composition, surface potential, lipid packing density and curvature. the binding of an amphitropic protein alters the membrane's spontaneous curvature as well as the difference between the equilibrium areas of membrane leaflets. theoretical model has been developed to predict vesicles shape transformations realized by the formation of buds. it was shown that the effects are stronger for flaccid vesicles which were therefore chosen for our experimental investigations. vesicles were composed of % pops and % popc, and flaccidity was achieved by an osmotic adjustment. the solution containing β gpi was subsequently injected to a chamber with flaccid vesicles by a syringe pump. observation took place under a phase-contrast microscope. experiments showed a concentration dependent occurrence of buds. their number and size were related to the degree of vesicle flaccidness. the interplay between detergent cohesion and peptide adsorption on the structure and dynamics of a glycophorin a tm-detergent complex j. khao, j.-p. duneau, j. n. sturgis lism, cnrs -aix marseille university, marseille, france in spite of the major interest of membrane proteins at functional, genomic and therapeutic scales, their biochemical and structural study remain challenging as they require, solubilization in detergent micelles. the complexity of this task arises from the structural dependence of membrane proteins on their anisotropic environment and in particular by a delicate balance between different physico-chemical properties. in order to study these properties in a small protein detergent complex, we have used molecular dynamics simulations on the transmembrane part of glycophorin a (gpatm) solubilized in micelles of the detergent di-hexanoyl-phosphatidylcholine(d pc). we show that the molecular aggregates organizes to give distinct populations of detergent molecules. those molecules which loosely interact with the peptide are preferentially involved in highly cohesive inter-detergent interactions that impose a global bilayer structure to the micelle. interaction profiles of other detergents with gpatm depend upon the nature of residues along the surface of the peptide. this topology dependence leads to different modes and strength of interactions that ultimately constrain the orientation of the micelles around the peptide. this simple model illustrates how differential detergent selectivity for faces and strong constraints coming from purely environmental features could influence transmembrane helix packing, membrane protein structure and assembly. in this paper we present a study of a new family of bolaamphiphiles. these amphiphiles are unsymmetrical bolalipids having one sugar polar head and the second glycine betaine polar head. they are potentially useful for pharmaceutical or cosmetics applications as vectors for drugs. therefore it is important to investigate their self-assembled properties. the chemical variations that we introduced in this new family concern the length of the main chain that connects two polar heads as well as the length of the side chain placed on the position of the sugar moiety. another variation concerns the introduction of a diacetylenic unit into the main chain in order to rigidify it. we have performed the saxs (small angle x-ray scattering) measurements on the dehydrated compounds as a function of temperature and observed the lamellar liquid crystalline structures. we also measured the saxs spectra of aqueous solutions of these compounds that have shown lamellar l α structure in all cases. these measurements are compared with polarised optical microscopy measurements that confirmed our interpretation. formation of membrane tubular structures induced by phase separation in giant vesicles y. li, r. lipowsky, r. dimova max planck institute of colloids and interfaces, science park golm, potsdam, germany tubular membrane structures (also known as tethers) exist widely in eukaryotes. abundant work has been done on tube extrusion from cells and model membranes under the application of external forces. we present a novel system allowing tube formation in the absence of external forces. aqueous solutions of two chemically dissimilar polymers, polyethylene glycol (peg) and dextran are encapsulated in giant vesicles, a cell-size model system. the exposure of vesicles to hypertonic solutions induces phase separation of the internal aqueous polymer solution. the excess membrane area created by this vesicle deflation, engages in the formation of tubular structures. membrane tube formation and phase separation are coupled processes. hydrodynamic flows and changes in the membrane spontaneous curvature during phase separation might be the driving forces for tube formation. the tubes are rather stable: without external perturbation, they can exist for several days. they prefer to be located in the peg-rich phase at low polymer concentration. at high concentration, they are absorbed at the interface of the liquid phases to lower the surface energy of the system by decreasing the contact area between the liquid phases. the membrane tubes can be retracted back to the vesicle surface by increasing the membrane tension via vesicle aspiration. membrane tubes, which can form and be retracted easily, might be relevant to lipid storage in cells. insight into the antimicrobial mechanism of a de novo auto-assembling peptide b. legrand , m. laurencin , e. duval , c. zatylny , j. henry , m. baudy-floc´h , a. bondon rmn-ilp, umr cnrs -univ. de rennes , france, icmv, umr cnrs -univ. de rennes , france, pemm, umr ifremer -univ. de caen, france a short ( residues) de novo antimicrobial peptide (k ) composed of a cationic polar head and a hydrophobic tail was studied. it exhibits a broad spectrum of antimicrobial activity on bacteria. no haemolytic activity or cytotoxicity on eukaryotic cells are observed at mic. when bacteria are lysed by the k , spherical objects are observed on the sem micrograph. k was structurally studied using various membrane mimetic media such as different micelles and small unilamellar vesicles (suv) of different composition. cd revealed that k adopt various structures (random, β-turn, α-helix) in the presence or the absence of detergents or phospholipids. nmr structures confirmed the α-helical structure of k hydrophobic tail in presence of sds. k self-assembles at high concentration as observed by sem and dls. we suggest that k may act as a surfactant building mixed microsomes composed of peptide and lipids. this destabilization mechanism of the bacterial membrane support the "detergent like model" previously described in the literature. oxidative stress and the membrane dipole potential; modulation with tocopherol s. le-nen-davey , b. m. davis , j. l. richens , k. a. vere , m. w. tilley , p. g. petrov , c. p. winlove , p. o'shea biomedical physics group, university of exeter, uk, cell biophysics group, university of nottingham, uk tocopherol, a component of vitamin e well know for its antioxidant properties, has recently been thought also to influence the structure of cellular membranes. tocopherol treatment reduces hyperglycaemia induced oxidative stress and the associated endothelial dysfunction which is a precursor to the vascular complications of diabetes. tocopherol and insulin interactions are also modified by hyperglycaemia. it is unclear whether these clinically important effects arise from the anti-oxidant and/or structural properties of tocopherol. we have therefore measured the dipole and surface potentials of phosphatidylcholine vesicles containing different amounts of cholesterol, ketocholesterol and tocopherol. we have also investigated the effects of hyperglycaemia, ketocholesterol and tocopherol, alone and in combination, on microdomain formation and interactions of insulin within the membranes of cultured endothelial cells. both sets of experiments indicate that tocopherol causes significant modifications of the membrane dipole and surface potentials. the physiological significance of these changes will be discussed. t. d. lazzara , a. janshoff , c. steinem georg-august university göttingen, institute for organic and biomolecular chemistry, tammannstr. , , germany, georg-august university göttingen, institute for physical chemistry, tammannstr. , , germany cell penetrating peptides (cpp) have been shown to penetrate cellular membranes. they have been of interest for their ability to translocate not only themselves through cellular membranes, but also carry along with them, cargo as large as iron nanoparticles. the exact entry mechanism remains unclear, but has been shown to vary with peptide sequence. their translocation properties have been demonstrated through different experiments involving vesicles, cells and living animals. we plan on using nano-black lipid membranes (nano-blm), which span the pores of nanoporous materials as a model system to study the interaction between cpp and lipid membranes. the nanopores can be used as cellular containers whose interior surface can be functionalized with receptors for biotin-streptavidin recognition. we hope that this system will provide greater control over experimental variables, such as the type of cpp and lipid used, as well as provide kinetic data that can be used to evaluate cpp activity and the kinetics of cargo transport. ethanol induces phospholipid acyl chain interdigitation. while much is known, important issues remain unclear, such as the role of lipid domains. the main purpose of this study was to follow, in real time, the changes induced by ethanol on supported lipid bilayers with nano to microdomains by in situ atomic force microscopy. to this goal, a pure lipid in the fluid phase (dopc), a pure lipid in the gel phase (dppc), binary phospholipid mixtures with gel/fluid phase coexistence, and cholesterol-containing, raft -forming mixtures (dopc/dppc/cholesterol and dopc/sphingomyelin/cholesterol) were investigated. from the height differences observed upon ethanol addition to pure lipids (dppc and dopc), and to dopc/dppc mixtures, it is shown that in the binary system the interdigitation of the fluid phase occurs prior to the gel phase. however, for the lipid rafts mixtures the simultaneous interdigitation of both raft and non raft portions of the membrane is observed both in mica and silicon substrates. for all compositions studied, domain formation or rearrangement accompanied by lipid bilayer expansion occurs as a consequence of interdigitation. these results show the ability of ethanol to influence the bilayer properties in different ways according to membrane composition. ethanol may exert its biological effects by reducing bilayer thickness, and also by changing membrane proteins conformation and lateral distribution, as a consequence of the altered properties of the lipid bilayer. interactions between non-steroidal anti-inflammatory drugs and a pc/cholesterol bilayer m. markiewicz , t. librowski , p. serda , m. pasenkiewicz-gierula faculty of biochemistry, biophysics and biotechnology, jagiellonian university " department of pharmacodynamics, medical college, jagiellonian university " regional laboratory, jagiellonian university, krakow, poland. the non-steroidal anti-inflammatory drugs (nsaids) are among the most frequently prescribed and used drugs [ ] . there are several side effects connected with frequent use of nsaid, mainly gastrointestinal ulcers and bleedings. a plausible molecular mechanism of these effects are direct interactions of nsaids with gastric phospholipids [ , ] . the influence of three well-known non-steroidal antiinflammatory drugs with a diverse gastric toxicity (aspirin, ketoprofen and piroxicam) and three newly-synthesized xanthone derivatives (belonging to the nsaids) on the structure and dynamics of lipid bilayers was studied using small angle x-ray scattering and molecular dynamics simulations. the results showed some correlation between nsaid toxicity and its binding to the lipid polar groups. this binding increases membrane fluidity by reducing its density due to an increased membrane surface area. a reduced lipid packing in the membrane most likely increases gastric mucosa permeability, which can result in a decreased resistance of the gastric mucosa to luminal acid. we studied the partition of the anionic amphiphile pyrenesulfonate (psa) into mlv and luv (produced by extrusion), composed by zwitterionic popc and zwitterionic/anionic mixtures with pops (until mol %). psa is an anionic amphiphile that mimics several xenobiotics (e.g. pharmaceuticals, pesticides) and endogenous substrates that interact with biological membranes. we found increasing b. lorenz, s. schuy, a. janshoff georg august university, göttingen, germany cell-cell and cell-virus interactions are ubiquitous in living organisms. the analysis of the forces acting between two cells or a cell and a virus gives insight into details of these interaction processes such as their stochastics, cooperativity, reversibility, and energy landscape. using colloidal probe microscopy in conjunction with solid-supported lipid bilayer techniques, we mimic the contact between two membranous compounds displaying sponge glycolipids or viral peptides on their surfaces. after spreading functionalized lipid bilayers on both -a colloidal probe and a silicon wafer surface -the molecular interactions are quantified by means of force-distance curves. by probing the dynamic interaction strength between the viral peptides n and c we aim for a deeper understanding of the role of these peptides in the complex process of the formation of the prehairpin intermediate as the key step in retroviral fusion. as far as the cellular interaction of marine sponge cells is concerned, we intend to investigate the strength, specificity, and the ca + dependency of the self-recognition between sponge glycans. the systems advantages are the flexibility of the membrane composition and the control over the distribution of receptor molecules in the membranes. since adhesion in biological systems relies heavily on cluster formation within the biomembrane, we plan to mimic the clustering by "printing" interaction domains and comparing the results to homogeneous samples. development of video-rate imaging microscope using laurdan and its applications to lipid raft t. ohba, k.-i. muto, t. kiuchi, k. ohki department of physics, graduate school of science, aobaku, sendai, japan - , ohki@bio.phys.tohoku.ac.jp various biological functions are located on cell membranes, and the biomembranes maintain heterogeneity as microdomain even in its dynamic structure. existence of such domain was reported as phase-separated 'rafts' in a cell. and the bio-functions of membrane protein are affected by physical property of its surrounding lipid bilayers. laurdan is a useful fluorescence dye to monitor membrane fluidity, and we have developed an instrument to image spatial and temporal change in membrane fluidity by use of laurdan. in order to examine role of 'micro-domain' in biomembrane, we applied this imaging instrument to a giant liposome, cho cells and pc d cells. fluorescence microscope was equipped with a home-build dual-view optical unit. microscopic image of membranes stained with laurdan is separated into an image at nm and an image at nm by the combination of monochromatic filters and dichroic mirrors. the each image is focused on an image plane of a ccd camera side by side. and generalized polarization (g.p.) image is calculated from those images by personal computer according to the definition of g.p. the g.p. imaging at video rate was applied to a giant liposome of composed of dmpc and dmpe in order to observe phase separation. it was also examined that specific interaction of sphingomyelin and cholesterol in living cho cells and neuritis protrusion from raft region of pc d cells stimulated by neuron growth factor. current fluctuations in biological lipid membranes from human cell lines s. nuschele, a. wixforth, m. f. schneider university of augsburg, experimental physics , univer-sitätsstrasse , d - augsburg, germany lipid membranes can undergo phase transitions at physiological temperatures. not only do single lipid component membranes show melting behaviour but also biological lipid membranes from eukaryotes as well as from prokaryotes. performing calorimetric and monolayer studies we are able to detect phase transitions in extracted membranes from different human cell lines (e.g. keratinocytes and melanoma cells). close to and in the melting transition regime we measured distinct channel like current fluctuations. the opening times of these current fluctuations can be predicted based on the lateral compressibility measured from the monolayer isotherm and agree well with the experimental data [*]. the applied method of extraction excludes the presence of functional proteins in the membrane rendering the lipid bilayer as the source of the observed current fluctuations. the molecules are composed of single hydrophobic tail and two hydrophilic aldonamide-type groupings (gluconyl c -dga or lactobionyl c -dla) linked by the propylene chain at the nitrogen atom. the micellization processes of c -dga and c -dla were studied by means of itc. the critical micelle concentrations, the enthalpies (∆h m ) and the entropies (∆s m ) of micellization as well as the contributions of the headgroups to the gibbs free energies (∆g m (hy)) were calculated. qspr analysis was also used to predict cmc of studied compounds. the interactions of c -dga and c -dla with model membranes (dppc and dppc/chol. bilayers) were studied by means of dsc. using quantum computations some basic molecular properties were calculated. the conformational space was explored using molecular mechanics. obtained results were compared with those for analogical compounds with single head groups, e.g. with also synthesised by us n-alkanoyl-nmethyllactitolamines (c n mela) and common sugar-based surfactants c gluc and mega- . enfuvirtide (t- ) was the first hiv- fusion inhibitor peptide approved for clinical use. t- is an inhibitor still under evaluation. previous studies, based on tryptophan intrinsic fluorescence, showed that the peptides interact differentially with membrane model systems (luv) with different lipid compositions. studies with human blood cells were necessary to further establish the role of membranes on these peptides mode of action. an experimental strategy was applied based on the membrane dipole potential, as measured by the fluorescent probe di- -anepps. human erythrocytes and peripheral blood mononuclear cells (pbmc) were successfully labeled. for both systems, a fusion inhibitor concentration-dependent decrease on di- -anepps fluorescence excitation ratio (a measure of the spectral shift and dipole potential) was observed. the quantitative analysis of these variations indicated that t- has an approximately ten-fold higher affinity towards erythrocyte and pbmc, when compared with enfuvirtide. this is in agreement with the previously known adsorption of t- on cholesterol-rich membrane domains and with its higher partition constants. hiv associates with erythrocytes in vivo, which can constitute a route to deliver peptide to the viral membranes (also rich in cholesterol). lymphocyte membranes can concentrate and accelerate the drug interactions with its molecular target, gp in its exposed conformation. oxidation of low density lipoprotein (ldl) is known to be a key step in atherogenesis, leading to inflammation, proliferation and apoptosis of cells of the arterial wall. these effects are largely exerted by oxidatively fragmented phospholipids, which are highly exchangeable between cells, tissues and lipoproteins. in particular, pgpc has been identified in minimally modified ldl and has been reported to elicit a wide range of pathophysiological responses in vascular cells, e.g. the activation of apoptotic signaling pathways. we investigated here the behavior of the fluorescent oxidized pgpe-alexa compared to dhpe-bodipy in artificial supported lipid bilayers with different cholesterol contents. the two labeled lipids differ in the type of membrane insertion: while dhpe-bodipy is anchored to the membrane via two fatty acids, pgpe is incorporated with only one fatty acid. the second chain is an acyl fragment in sn- position, which represents the oxidation product of an unsaturated acyl chain. with increasing cholesterol content we observed a decrease in the diffusion coefficient for both lipids. interestingly, the diffusion of the oxidized lipid was reduced in a higher degree compared to that of the non-oxidized lipid. the calculated ratios of the diffusion constants of pgpe-alexa and dhpe-bodipy suggest a different type of interaction with cholesterol. membrane proteins account for a third of all proteins encoded for by the human genome, and play a vital role in a number of cellular processes. few membrane protein structures have been determined to date in comparison to soluble proteins. this discrepancy is due to experimental difficulties in preparing membrane protein samples for structural analysis. traditional techniques for studying membrane proteins by x-ray crystallography or solution nmr use detergent solubilised proteins which can differ from their native confirmations. solid-state nmr allows the study of transmembrane proteins in lipid bilayers, representing a more native like environment in which to obtain biologically relevant structural information. we have been working on the development of reliable methods for reconstitution of transmembrane peptide into liposomes using glycophorin a as a model tm protein. using reconstitution methods based on the removal of detergent by bio-beads, we have used electron microscopy to screen the ideal conditions for insertion of gpa into liposomes in preparation for mas nmr experiments. em has allowed us to identify conditions favourable for insertion of peptide into lipid vesicles and those that result in aggregation. in order to confirm the secondary structure and insertion of gpa into lipid vesicles, techniques such as cd, ocd, ftir and dls have been used to provide quantitative information in addition to the visual results from em. nonesterified fatty acids regulate a broad spectrum of metabolic activities and are involved in many physiological, pathological and/or pharmacological processes in living cells. they spontaneously transfer between donors and acceptors such as fatty acid binding proteins and lipid membranes. we focus on protein-lipid dispersions of human serum albumin (hsa) and sterically stabilized liposomes (ssl) composed of dppc and appropriate amount of peg: -dppe in which stearic acids (sa) are inserted either in the protein or in the ssl. exploiting the fact that hsa has a single tryptophan residue and that the intrinsic trp-fluorescence emission signal is quenced by the presence of sa, the kinetics of sa transport between hsa and peg: -grafted dppc membranes is studied by means of fluorescence. it is found that the transfer of sa between hsa and ssl is a first-order process and the kinetics of transfer depends on the type of donor and acceptor matrix, on the temperature (i.e., on the physical state of the lipid bilayers), and on the grafting density of the peg-lipids at the protein/lipid interface. indeed, in the absence of polymer-lipids, the rate of transfer increases with temperature in both directions of transfer and it is faster for the passage from dppc bilayers to hsa. the presence of polymer-lipids reduces the rate of transfer both in the mushroom and in the brush regime of the polymer-chains, especially for lipid membranes in the fluid phase. a. orth , w. römer , l. johannes , c. steinem institute for organic and biomolecular chemistry, university of goettingen, germany, laboratoire trafic et signalisation, institut curie, paris, france shiga toxin (stx) from shigella dysenteriae is an ab -class bacterial toxin. infections with stx lead to the haemolyticuraemic syndrome, which is known to be a major cause for renal failure at an early age. the interaction of the homopentameric b-subunit (stxb), which is responsible for binding and intracellular transport of the holotoxin, with its cellular receptor, the glycosphingolipid gb , is the first step for endocytosis of the toxin. one b-subunit can bind up to gb -molecules to form stxb-gb -clusters causing negative curvature of a membrane. the binding of stxb to giant unilamellar vesicles, composed of dopc, cholesterol and gb induces tubular membrane invaginations, which were also found in experiments with energy-depleted hela-cells. in recent studies, protein and lipid reorganization processes after attaching stxb to solid supported membranes, composed of dopc/sphingomyelin/cholesterol/gb have been investigated. the compaction of gb -molecules led to an additional stxb-gb -enriched phase, which was also observed in lipid monolayers at the air/water interface. in this study, the influence of stxb on model membranes will be investigated combining the advantages of free-standing lipid membranes with those of ssms. the impact of stxb on pore-suspending membranes will be followed by confocal laser scanning mi croscopy and atomic force microscopy. a. robaszkiewicz , c. spickett , p. sicinska , g. bartosz , m. soszynski department of molecular biophysics, university of lodz, lodz, poland, strathclyde institute of pharmacy and biomedical sciences, glasgow, u.k. hypochlorite generated in vivo under pathological conditions is a known oxidant, able to initiate lipid peroxidation process, which affects the stability of biological membranes. the aim of this study was the analysis of the products formed during the reaction of hypochlorite with phosphatidylcholines containing unsuturated fatty acid residues ( -stearoyl- -oleoyl-, -stearoyl- -linoleoyl-, stearoyl- -arachidonylphosphatidylcholine) and their effects on the human erythrocytes. using electrospray mass spectrometry we observed complete conversion of the lipids into chlorohydrins, which resulted in the decrease of the rotational correlation time and rotational motion freedom of liposomes estimated by epr using spin probes ( -and doxylstearic acid). unilamellar chlorohydrin liposomes had lower diffusion coefficient for calcein than liposomes made of parent lipids. flow cytometry demonstrated fast incorporation of uni-and multilamellar chlorohydrin liposomes labeled with nbd-pe into erythrocytes. this effect was accompanied by the formation of the erythrocyte subpopulations of higher volume, decrease of the rate of fluorescein diacetate hydrolysis, estimated by flow cytometry, and increase of affinity and maximal velocity of the membrane enzyme acetylcholinesterase. extensive bilayer perforation coupled with the phase transition region of an anionic phospholipid k. a. riske , l. q. amaral , m. t. lamy depto. biofisica, universidade federal de sao paulo, sao paulo, brazil, instituto de fisica, universidade de sao paulo, sao paulo, brazil at low ionic strength dimyristoylphosphatidylglycerol (dmpg) exhibits a broad phase transition region characterized by several superimposed calorimetric peaks. peculiar properties, such as sample transparency, are observed only in the transition region. we use differential scanning calorimetry, turbidity and optical microscopy to study the narrowing of the transition region with the increase of ionic strength. upon addition of salt, the temperature extension of the transition region is reduced and the number of calorimetric peaks decreases until a single cooperative event is observed in the presence of mm nacl. the transition region is always coupled with a decrease in turbidity, but a transparent region is detected within the melting process only in the presence of up to mm nacl. optical microscopy of giant vesicles shows that bilayers first rupture when the transition region is reached and subsequently lose optical contrast. fluorescence microscopy reveals a blurry image in the transparent region, suggesting a different lipid self-assembly. overall sample turbidity can be related to the bilayer optical contrast. our observations are discussed in terms of the bilayer being perforated along the transition region. in the transparent region the perforation is extensive and the bilayer completely loses the optical contrast. financial support: fapesp. label-free imaging of biological membranes using surface imaging techniques j. l. richens, k.-a. vere, p. o'shea cell biophysics group, university of nottingham, nottingham, uk surface plasmon resonance (spr) is a detection technique traditionally used for specific protein detection, which is now exploited routinely as a generic label-free sensor. spr responds to changes on a metal surface conditioned to sense the binding of analytes. thus, the composition of the external medium and metal surface will be fundamental to the signal output. here we use a model phospholipid membrane system to investigate the effect of altered buffer and surface compositions on spr signals. comparisons are undertaken between continuous gold surfaces and gold nanoparticles. we demonstrate that surface electrostatics and the salt composition and molarity of a buffer all have significant impacts on spr output. the association of respiratory syncytial virus matrix protein with membrane microdomains the association of the matrix protein from respiratory syncytical virus with membranes has been characterised by tensiometry, brewster angle microscopy, and atomic force microscopy following deposition of langmuir monolayers onto modified silica substrates. association of the protein with monolayers containing phosphocholines and cholesterol leads to the formation of materials with new properties that differ from those of either of the pure components. the behaviour of the protein in monolayers rich in cholesterol and sphingomyelin exhibits a significant concentration dependence. at low concentrations, the protein exhibits a simple partioning behaviour. at higher concentrations, lipids are extruded from the monolayer below a critical surface area. these findings are discussed in relation to the recently published structure of the protein (pnas, , , - ) , the documented formation of viral filaments during key stages of the infection cycle and the isolation of the protein from detergent-resistant membrane fractions. structure and dynamics of closed melting membranes v. m. rondelli , s. santorsola , p. brocca , e. del favero , l. cantù , m. zimbone dep. of chemistry, biochemistry and biotechnologies for medicine, university of milan, segrate, italy., dep. of physics, university of catania, catania, italy. we studied the dynamical and structural properties of large unilamellar vesicles (≈ nm luvs) of phospholipids (dmpc, dc pc and dmpc : dc pc = : molar mixture) in the temperature range around the chain-melting transition, ≈ • c wide, with . • c resolution and . • c accuracy. small-angle (saxs) and wide-angle x-ray scattering (waxs) measurements show that across the transition the vesicle behaves as an 'evolving membrane', passing through several different states, each of them being characterized by different proportions of coexisting fluid-and gelchains molecules. noteworthy, no kinetics has been detected. on the same samples, a unique and very sensitive laser light scattering technique allows to determine the characteristic times of thermally induced shape fluctuations, connected to the elastic properties of the membranes. results indicate a clear softening of the membranes in correspondence to the chain-melting transition, as indicated by a manifold increase of the corresponding fluctuation characteristic time. meanwhile the overall size of the vesicle is not sensibly changed. this softening is likely to be due to the presence of structural defects, eventually driving to local morphological modifications. thermodynamics characterization of isolated lung surfactant assembled as lamellar bodies e. x. rodriguez , r. alvarez , r. barrio , c. irles , a. ortega biochemistry depto., school of medicine, inst. of physics unam, nat. inst. of perinatology, mexico city, mexico lungs are a large extension of ∼ m of one layer cells, which is structured as compacted sacs called alveoli, where lung function takes place: the gas exchange. alveolar endotelium is formed by two kinds of cells: neumocyte type i (nti), which covers ∼ % of the alveolar surface where gas exchange occurs and, ntii where lung surfactant (ls) is produced. ls are a mixture that lay over a water film in the luminal surface of the alveoli and it is thought to decrease the surface tension to avoid alveolar collapse during expiration. ls are made of phospholipids and proteins, which are determinant for the structure of ls under particular conditions of pressure and temperature. ls inside the cell is packed in organelles called lamellar bodies (lb), and is released to the water/air interphase in other conformation. in the present study we isolate lb from pig's lung and study lb thermodynamic characteristics by microcalorimetry in the presence and in the absence of structural proteins. phase transition profile of lb with and without proteins is basically the same; while ls in the alveolar lumen have a higher transition temperature (tm). mayor changes in tm are observed between lb and ls from alveolar lumen. although ls lipid composition in and outside the cell is assumed to be the same, the ground for the differences in tm under these two conditions is unknown. m. rodrigues , g. rádis-baptista , b. g. de la torre , m. castanho , d. andreu , n. c. santos instituto de medicina molecular, portugal, pompeu fabra university, spain, federal university of cearà, brasil nucleolar-targeting peptides (nrtps) have been recently designed by structural dissection of crotamine, a toxin from the venom of a south america rattlesnake (radis-baptista et al. . j med chem : ) . at µm concentration, nrtps penetrate different cell types and exhibit exquisite nucleolar localization. the aim of this work is to decipher the molecular mechanism for the translocation of nrtps into cells. quantification of partition into membranes was carried out, based on intrinsic tyrosine fluorescence. the role of the bilayer phase, anionic lipids, reducing agents and peptide concentration on the extent and kinetics of partition were studied. both nrtp and nrtp exhibited high partition to popc (neutral) lipid vesicles (k p ≈ × ), which was enhanced by the anionic lipid popg for nrtp , but not nrtp . the peptides showed a decrease in partition for popc:cholesterol (liquid ordered state) or dppc (gel) membranes. depending on the lipid composition, the peptides either increased or decreased their quantum yield upon membrane insertion. quenching experiments with acrylamide showed no peptide aggregation in solution. once the translocation mechanism is fully understood we will test nrtps as carriers of relevant cellular cargos, evaluating their potential clinical application in drug delivery or gene therapy among other applications. the ingestion of trans fatty acids (tfa) formed during the partial hydrogenation of vegetable oils has been linked to a detrimental impact on health by an, as yet, unknown mechanism. we synthesized deuterated analogs of -elaidoyl- -stearoylphosphatidylcholine (t : - : pc) that contains a single "unnatural" trans double bond and -oleoyl- -stearoylphosphatidylcholine (c : - : pc) that contains a single "natural" cis double bond. solid state h nmr, complemented by molecular dynamics (md) simulations, was then employed to compare molecular organization in model membranes prepared from these isomeric molecules. analysis of spectra recorded as a function of temperature reveals a higher chain melting temperature for the trans isomer, indicating tighter molecular packing in the gel state. in the liquid crystalline, however, the difference between the trans and cis isomers is subtle. order as probed by the perdeuterated [ h ] : sn- chain, and corroborated by computer simulation, coincides within < %. only in the conformation of the double bond is an appreciable difference implied. thus, our results contradict the conventional view that tfa resemble saturated fatty acids, which is > % more ordered. (supported by acs, prf -ac .) photooxidation and lateral membrane diffusion of dipole molecules v. s. sokolov , e. a. sokolenko , a. a. lents , p. pohl a.n frumkin institute of physical chemistry and electrochemistry, russian academy of science, moscow, russia, institut fuer biophysik, johannes kepler universitaet linz, austria the photodynamic oxidation of phloridzin, of the styryl dye di- -anepps and of unsaturated lipids was monitored "online" by measuring the collapse of the dipole potential which has been introduced to the membrane by these molecules. their photodamage occurred at different rates when the target molecules and the singlet oxygen generating photosensitizer (phthalocyanin) were adsorbed to the same or to opposite sides of the planar lipid bilayer. the difference in the oxidation rates were attribute to singlet oxygen transport through lipid bilayer and therefore we were able to estimate the permeability of lipid bilayers to singlet oxygen. however, the apparent permeabilities derived from experiments with different targets were differ from each other. therefore, we tested the hypothesis that the lateral membrane diffusion of target molecules and oxidation product may have biased our analysis. in line with this anticipation we found that the apparent permeability is dependent on the size of the planar bilayer. the development of a new mathematical model, which takes the mobility of all reacting species in the aqueous and lipid environments into account, allowed estimation of the real membrane permeability to singlet oxygen. it appeared to be very close to that of oxygen in the ground state. a number of complex three-dimensional lyotropic liquid crystal phases are already known, such as the bicontinuous cubic phases, but so far only a single example has been found -a cubic phase of spacegroup fd m -of a structure based upon a complex close packing of inverse micelles ( ) . we now report the discovery ( ) of a novel lyotropic liquid crystal phase, of space group, p /mmc, whose structure is based upon a hexagonal close packing of identical quasi-spherical inverse micelles. the model membrane system consists of a hydrated mixture of dioleoylphosphatidylcholine, dioleoylglycerol, and cholesterol. this novel phase has a number of unique features which may render it useful for a range of applications. firstly, it is the only known self-assembled lyotropic phase whose structure consists of a periodic close packing of identical inverse micelles. secondly, it is stable in excess aqueous solution, which is very important for potential biological or biomedical applications. references ( ) v. luzzati, v., r. vargas, a. gulik, p. mariani, j.m. seddon, and e. rivas, biochemistry , - ( ) . dystrophin is a rod-shaped muscular subsarcolemal protein. its deficiency is one of the root causes of duchenne muscular dystrophy. dystrophin rod domain contains homologous repeats, where the sub-domain constituted by the repeats to (r - ) was reported to bind actin and membrane lipids. we analyzed the interaction of r - with lipid monolayers. to better understand the assembly mechanism of this protein with lipids, we studied its adsorption behavior at the air-liquid and lipid/liquid interface using ellipsometry, surface pressure, and atomic force microscopy (afm). using two different mixtures of phospholipids, ellipsometry and pressure surface data show that r - interacts with the lipid monolayers, but is inserted into the monolayer formed by dopc-dops while it lies below the monolayer of dopc-dope. this indicates that r - interacts more strongly with dopc-dops than with dopc-dope. afm images show that the pressure of lipid monolayer influences the organization of r - . when the lipid surface pressure is mn/m, r - forms a striking network, indicating a protein-protein interaction in addition to the protein-lipid interaction. this unique behavior of one part of the central domain of dystrophin may explain its key role in muscle cell. studies on polyphenol extracts from helichrysum l., fagopyrum mill., crataegus l. and hypericum l. were performed in order to find if they may be applied as a natural free radical scavengers protecting biological membranes against peroxidation. ghosts erythrocytes were used in the experiments. they were suspended in tris-edta solution, ph , , then.irradiated with a bactericidal lamp without (control) or with a proper amount of the extracts studied. the product of lipid peroxidation was malonic dialdehyde (ma). the colour reaction of ma with thiobarbituric acid (tba) enables to determine the concentration of ma spectrophotometrically. it was found that peroxidation increased with the irradiation time. however, it significantly decreased when concentrations of poliphenols increased. the best antioxidtive property was found for hypericum l. the antioxidative efficiency sequence of the plant extracts studied was the following: hypericum l. > crataegus l. > fagopyrum mill. > helichrysum l. the results obtained indicate that polyphenol extracts exhibit excelent antioxidative properties that make them good free radical scavengers for efficient protection of biological membranes. this work was sponsored by ministry of science and education, scientific project no. n n . interaction of cationic porphyrins with neutral and negatively charged liposomes i. voszka , g. corradi , p. maillard , h.-j. steinhoff , g. csík institute of biophysics and radiation biology, semmelweis university budapest, hungary, research institute for solid state physics and optics, hungarian academy of sciences, budapest, hungary, institute curie, section de biologie, orsay, france, fachbereich physik, universitat osnabrück, germany porphyrin derivatives are used in photodynamic therapy of tumors. the knowledge of the photosensitizer location within the cell is important. the effect of porphyrin derivatives containing cationic side groups was examined on neutral and negatively charged liposomes by esr. the esr signal was first examined as a function of temperature and porphyrin concentration in the dark. a significant change related to the appearance of quasi free spin labels was obtained for spin probes at the th carbon atom and was more expressed for the asymmetrical derivative. illuminating the samples the esr amplitude decreased for all positions of the spin probe but to different extent. the effect was more expressed in case of the symmetrical derivative, especially for label positions and . for the asymmetrical derivative the effect changed from moderate to weak from the th to the th position. this indicates that the asymmetrical derivative is incorporated nearer to the lipid head groups while the symmetrical one may be located deeper in the membrane. under oxygen-free conditions both derivatives showed weaker but still pronounced effects.. single channel recording of α-hemolysin in nanopore-spanning tethered bilayer lipid membranes n. t. thet , i. pfeiffer , w. knoll , i. köper max planck institute for polymer research (mpi-p), ackermannweg , mainz, austrian research centers gmbh -arc, tech gate vienna, vienna, austria artificial lipid bilayer membranes mimic biological cell membranes in many aspects and can be used to study functional processes such as ion channeling, signal transducing, transport of nutrients etc. as most of the functions of a cell are accomplished by membrane proteins, research has been ongoing in studying and characterization of membrane proteins embedded in model lipid bilayer membranes. black lipid membranes (blms) were studied for decades but their potential for practical applications is hindered, mainly due to their lack of stability and limited lifespan. recently, tethered bilayer lipid membranes (tblms) proved to have long life span of up to several months without significant changes in membrane resistance and capacitance. in order to combine advantages of blms and tblms, we have designed a membrane system which is freely spanned across a single nanopore in a silicon nitride membrane. this system not only mimics cell membranes, but also it allows control over the chemical composition of buffer with unlimited ionic reservoirs on both sides of the membrane. this freestanding tblm maintains structural stability and lifetime of up to hours without significant decrease in its structural integrity and electrical sealing. for the validation of the tblm formation, we have inserted well known α-hl pores. we are able to control the amount of α-hl pores insertion and measure single channel ion transport across the tblm by α-hl pores using a capacitor feedback amplifier. the language of shape: biological reactions are dramatically affected by the shape of lipid membrane d. stamou university of copenhagen, copenhagen, denmark a plethora of biological process are taking place on the surface of lipid membranes. as a rule membranes in vivo are curved in a variety of complex geometries. here i will present a quantitave study on the influence of membrane curvature on protein-membrane and membrane-membrane interactions. to gain systematic access to a continuum of membrane curvatures we immobilized liposomes on a surface at dilute densities. using confocal fluorescence microscopy we imaged single liposomes of different size, and therefore different curvature, and monitored their interaction with a binding partner (proteins or other liposomes). i will discuss unpublished data on two important classes of biomolecular interactions that exhibited dramatic curvature dependence: a) snare-mediated docking and fusion b) anchoring of peripheral proteins. the following references provide partial information on the single-liposome assay: a. zettergren, c. gudmundsson, t. nylander, e. sparr physical chemistry , lund university, lund, sweden there is accumulating evidence of substantial amounts of phospholipids in the cell nuclei , although the function of these lipids is still not fully understood. it has been shown that the chromatin complex composed of dna, rna and proteins also includes phospholipids, and that rna colocalize with these . although the rna-phospholipid interactions may have important implications to biological function, in gene therapy and in medicine, very little work has been dedicated to the characterization of rna interaction with phospholipids. the objective of this work is to investigate the adsorption behavior of short single stranded bases long rna (ssrna ) molecules (similar to mirna) to lipid monolayers at the air-water interface as well as to study how the presence of rna affect the domain formation in the monolayers using fluorescence microscopy. monolayer studies have shown adsorption of ssrna to monolayers consisting of zwitterionic dppc as well as to monolayers consisting of cationic dodab. the adsorption behavior of these very short nucleic acids differ significantly from the adsorption process for longer nucleic acids as for example a base pairs long ds dna (dsdna ) which has been used as a reference system . the presence of ssrna significantly changes the compression isotherm of both dppc and dodab monolayers. plant defensins are cysteine-rich antimicrobial peptides found in various plant species. they share a common threedimensional structure, stabilized by eight disulphide-linked cysteines consisting of three antiparallel β-strands and one α-helix. most plant defensins show antifungal activity with no effect on mammalian and plant cells. expression of these peptides in plant tissue is induced by pathogen infection. the mechanism of defensin action is based on membrane permeabilization. this occurs through an interaction with high affinity binding sites on fungal membranes, resulting in alteration of membrane potential. the genes encoding for a peach ppdfn and a grape vvamp defensins were expressed in e. coli and purified to homogeneity. they were tested for antimicrobial activity against some fungi and showed to have an inhibitory effect on the spore germination. biophysical analysis showed that defensins were able to interact with artificial membranes. binding of defensins to membranes was dependent on lipid composition, increasing with the sphingolipids content. interaction between peptides and sphingolipids could lead to insertion of the defensins into the membrane resulting in its destabilization. extracts of the plant perilla frutescens have many uses in the asian kitchen, e.g. as a popular garnish used in sushi. the plant is also employed in eastern traditional medicine to treat a variety of ailments including colds, food allergy and depression. two of the main constituents of the plant are limonene and perilla aldehyde. by bio-oxidation, these molecules can be converted to perillic alcohol and perillic acid. these cyclic terpenes possess antibacterial and anticarcinogenic properties. the modes of action for these compounds are at present not understood, but their remarkably diverse pharmacological properties suggest that they might target the phospholipid matrix of the cellular lipid membrane. indeed, the cyclic terpenes can bind to, and alter the physiochemical properties of the lipid bilayer of the membrane, the effects of which can cascade down to several essential cellular processes. here, we use molecular dynamics (md) simulations to investigate the effect of limonene and its derivatives on the properties of lipid bilayers including the changes in acyl chain order parameters, bilayer thickness and the area per lipid. md can afford molecular-scale dynamic information, often not easily accessible from experimental measurements. this information can be used to interpret existing experimental data obtained by e.g. isothermal titration calorimetry, electron paramagnetic spectroscopy and differential scanning calorimetry. catalysis in the membrane: interfacial mechanism of phospholipase a h. p. wacklin , r. k. thomas institut laue langevin, grenoble, france, physical and theoretical chemistry laboratory, oxford university, u.k. phospholipase a cleaves the sn- acyl chains of membrane phospholipids and performs a range of physiological functions. one of the least well understood aspects of the its mechanism is how its activity is regulated by the interaction with the substrate membrane. we have used neutron reflection to monitor changes in membrane structure during lipid hydrolysis [ , ] . the penetration depth of pla depends on lipid packing and increases during the lag phase of porcine pancreatic pla . by using a selectively deuterated lipid substrate, d -popc, we determined the relative membrane-water partitioning of the lipid products in-situ. the lyso-lipid product partitions into the solution phase, while fatty acid accumulates in the membrane and increases the affinity of pla [ ] . pla is inhibited at ph , which is consistent with protonation of the catalytic histidine. however, irrespective of ph, pla is fully activated by me-betacyclodextrin, which facilitates the release of the lyso-lipid from the enzyme-substrate complex. me-beta-cyclodextrin does not interact directly with the membrane surface or the substrate lipids, indicating that product release occurs outside the immediate membrane-water interface. diffraction limits resolution in optical microscopy, but many interesting biological problems occur on shorter (molecular) length scales. recently, methods to circumvent the diffraction limit have been presented. fluorescence photoactivation localization microscopy (fpalm) uses activation of many small subsets of photoactivatable or photoswitchable fluorescent probes (pafps) to generate images with effective resolution in the tens of nanometers. pafp molecules are photoactivated, imaged, localized, and photobleached in small numbers. the process is repeated for many subsets to build up data on thousands to many hundreds of thousands of molecules. the positions of all localized molecules are used to construct an image of the sample with resolution limited not by diffraction, but by the localization precision and molecular density. results will be shown from a variety of biological systems, including live and fixed cells expressing a variety of pafp-tagged proteins. bi-plane fpalm can image in three dimensions with demonstrated resolution of nm x nm x nm. polarization fpalm can image both molecular positions and anisotropies simultaneously with lateral resolution of ∼ - nm. using these powerful capabilities, many potentially interesting biological problems can be addressed. binding of the hiv- ncp on oligonucleotides at the single-molecule level j. godet, p. didier, a. jouonang, y. arntz, y. mély laboratory of biophotonics and pharmacology, umr cnrs , university of strasbourg, france the nucleocapsid protein (ncp ) of the hiv- is a small basic protein which plays key functions in the viral life cycle. the activity of ncp mainly rely on its potent rna-and dna-chaperone activities that direct the rearrangement of numerous nucleic acid molecules into their most stable conformation. two main features of nc's chaperone activity are its abilities to aggregate and destabilize nucleic acids. in addition, the rapid kinetics of ncp interaction with nucleic acids was recently proposed as another major component of nc's chaperone function based on the apparent correlation between an indirect measurement of the nucleic acid dissociation kinetics of ncp and its overall chaperone activity. but so far, no direct measurement of the on/off rates of ncp binding to oligonucleotides was performed. in the present work, we realized single molecule fluorescence resonance energy transfer (smfret) measurements to probe the transient interactions of a tmr-labelled ncp with a short cy -labelled dna oligonucleotide confined into nanovesicles. after confirming the efficiency of the ncp /odn complex encapsulation into the nanovesicles by fcs, the vesicles were tethered to the surface for immobilization. integrity of the entrapping vesicles attached on the surface was confirmed by afm. finally, the association and dissociation constants retrieved from these smfret measurements were discussed in the context of the dna-chaperoning activity of the protein. high-resolution spatiotemporal organization of the integrin lfa- m. f. garcia-parajo , t. s. van zanten , g.-j. bakker , r. diez-ahedo , a. cambi , c. g. figdor bionanophotonics group; ibec, barcelona, spain, tumour immunology dept; ncmls, nijmegen, the netherlands lfa- is a leukocyte-specific integrin involved in different steps of the immune response. on monocytes, lfa- plays a key role in the regulation of monocyte-endothelial interaction during rolling, arrest and extravasation into the underlying tissue. i n-vivo experiments showed that blood borne lymphocytes can 'switch' within seconds from rolling to arrest. furthermore, tem observations of pro-active, ligandindependent nanoclusters confirmed that affinity and clustering are complementary processes required in adhesion. yet, the mechanisms leading to fast-switching remain obscure. in our group, we used a combination of single molecule fluorescence techniques to study the spatiotemporal organization of lfa- on monocytes. we performed optical nanoimaging of lfa- nanoclusters in relation to membrane rafts with a resolution of nm and accuracy of ∼ nm. in quiescent cells, lfa- nanoclusters do not associate with membrane rafts and diffuse freely on the membrane. binding of the integrin to its ligand icam- induces the formation of microclusters that further associate with rafts and exhibit reduced mobility, consistent with cytoskeleton interactions. our work highlights the markedly different spatiotemporal organization of lfa- that might explain its concerted action to form larger and stable platforms on the cell surface required for rapid and effective cell adhesion. c. ciobanasu, u. kubitscheck rheinische friedrich-wilhelms-universität bonn, germany cell penetrating peptides like the hiv tat peptide have the property to rapidly translocate the cell membranes and the capability to deliver a wide range of cargoes. the mechanism of the membrane translocation is still under investigation and object of considerable controversy. we applied and single-molecule and confocal laser scanning microscopy (lsm) to study peptide-membrane interactions. electron-multiplying ccd cameras yield images of single fluorescent molecules with a time resolution in the range of a few milliseconds only, which allows the tracking of fluorescently labelled peptides and lipids at bio-interfaces in realtime with a localization precision of a few nanometers. we formed giant unilamellar vesicles (guvs) from different lipid mixtures and examined their interaction with fluorescently labeled tat peptides. we found that the passive peptide internalization process depends on lipid composition, charge of the lipid bilayer, and the ionic properties of the medium. a translocation of cationic tat peptides was observed in membranes containing at least mol% of lipids with a phosphatidyl ethanolamine or a high mol fraction of the phosphatidyl serine head group. in salt-free solution tat efficiently bound to guvs, however, in a physiological nacl solution tat binding was completely abrogated, but the peptides efficiently equilibrated across the guv membrane. new approaches to measure interactions in the live cell plasma membrane g. j. schütz biophysics institute, johannes kepler university linz, austria in my lecture, i will show examples how to obtain insights into the organization of the cellular nanocosm by single molecule experiments. our primary goal is an understanding of the role of such structures for immune recognition. brightness and single molecule colocalization analysis allows us to study stable or transient molecular associations in vivo. in particular, i will present results on the interaction between antigen-loaded mhc and the t cell receptor directly in the interface region of a t cell with a surrogate antigenpresenting cell. in addition, we developed a method for in vivo micropatterning of plasma membrane proteins to measure molecular interactions. the method allows identifying and characterizing interactions between an arbitrary fluorescence labeled protein ("prey") and a membrane protein ("bait") directly in living cells. cells transfected with a fluorescent fusion protein of the prey are plated on micropatterned surfaces functionalized with specific antibodies to the extracellular domain of the bait; the fluorescence copatterning is used as readout for the interaction. we applied this tool for the study of the interaction between cd -the major coreceptor for t cell activation -and lck, an important tyrosine kinase in early t cell signaling. in addition to the well-known zinc-clasp structure, we found strong contributions of lck membrane anchorage for the binding of the two proteins. developing a fluorescent redox sensor for monitoring metal-ion mediated catalysis in biosystems a. rybina , a. kiel , b. thaler , a. sprödefeld , r. krämer , d. p. herten bioquant and, department of inorganic chemistry, heidelberg university, germany a fluorescent redox sensor is an electron photo-switching device that can be used for the characterization of the redox state of a given environment. it combines a fluorescent fragment with a redox-active unit that senses the media by a redox reaction and controls the light emitting properties of the fluorophore. such reversible sensor can help to examine the electrochemical state and changes in biological systems during biochemical processes in real time. recently a new fluorescent molecular sensor with a redox-active hydroquinoneunit covalently linked to fluorophore rhodamine b was developed. (kierat r.m. et al., bioorg. med. chem. lett., -accepted) . the reduced hydroquinone-form of the sensor is fluorescent while its oxidation to benzoquinone-derivative leads to a significant decrease of the fluorescence. although the above method shows great promise for applications in biological systems, the exact mechanism of this process is not fully understood yet. we use fluorescence spectroscopy to investigate oxidation reactions on the ensemble and single-molecule level and study kinetic rates. the proposed strategy is to use cu (ii) complex as oxidation mediator immobilized on surface via dna linker to examine the oxidation mechanism. fluorescently labeled atp as a probe of the outer mitochondrial membrane barrier: role of vdac fluorescence correlation spectroscopy (fcs) was applied for studying the distribution of fluorescently labeled atp (bodipy-atp) in isolated mitochondria. the setup and peak intensity analysis (pia) was described in our recent paper (perevoshchikova et al. biochim.biophys.acta : . the binding of bodipy-atp to mitochondria was maximal in the non-energized state, whereas the addition of succinate (respiratoty substrate) or atractyloside (adenine nucleotide translocase inhibitor) led to a decrease in the binding. nadh reduced the fcs signal from bodipy-atp added to non-energized mitochondria more than nad+ did under the same conditions suggesting the control of nucleotide transport through voltage-dependent anion channel (vdac) residing in the outer membrane. konig's polyanion also decreased the bodipy-atp binding to mitochondria with the effect being reduced by alamethicin or digitonin. control experiments showed that bodipy-atp did not bind to liposomes showing minor role of unspecific binding. it was suggested that bodipy-atp in combination with fcs can be used to monitor the functional state of mitochondrial vdac which is considered to be a principal regulator of mitochondrial function. fluorescence correlation spectroscopy studies of lysozyme partition to phospholipid vesicles a. m. melo , a. coutinho , m. prieto cqfm and in, ist, - lisboa, portugal, dqb, fcul, - , lisboa, portugal binding to membrane lipids has been increasingly recognized as an important step in the aggregation and cytotoxicity of several amyloidogenic proteins [ ] . in addition, it has been recently reported that membranes containing negatively-charged phospholipids can also trigger rapid amyloid-like fiber formation by a variety of several nonamyloidogenic proteins, such as cytochrome c and lysozyme [ ] . our study aims to elucidate the factors that govern the formation of these lipid-protein complexes. given the importance of electrostatic interactions between the proteins and the acidic phospholipids in the putative membrane-induced protein misfolding step, it is essential to first characterize quantitatively the protein partition behavior towards liposomes prepared with variable anionic lipid content. in this study, lysozyme was chosen as a model protein and fluorescence correlation spectroscopy (fcs) was used to monitor its binding to liposomes after its conjugation to alexa fluor . most organic dyes labelling techniques produce a mixture of populations of molecules labelled with a different number of fluorophores. the influence of this poli-dispersity of labelled molecules on the protein partition behaviour will be explored, namely the ability of the fcs technique to detect the production of non-competent membrane-binding species. t. wohland , p. liu , x. shi , y. h. foo , t. sudhaharan , s.-w. chong , v. korzh , s. ahmed chemistry dept., singapore nat. univ., medical biology inst., singapore, molecular & cell biology inst., singapore biomolecular interactions have been measured mostly under in vitro conditions because of higher accuracy and ease of measurement. however, it has become clear that the cellular environment has an important influence on these interactions. it was therefore necessary to develop new tools to allow the measurement of interactions in cells and organisms. recently, we have developed a modality of fluorescence cross-correlation spectroscopy (fccs) called single wavelength fccs (sw-fccs), which uses one-photon excitation to excite two fluorophores with overlapping absorption but separable emission spectra. sw-fccs has been used to determine e.g. dimer fractions of proteins in live cells. here we aim to extend the use of sw-fccs to cells and organisms. in the first part we determine the dissociation constants of a small rho-gtpase (cdc ) with an effector domain (crib) and two effectors (n-wasp or irsp ). in the second part we measure the binding between cdc and a scaffolding protein (iqgap ) in dependence of their expression levels in cho cells and in live zebrafish embryos. by using gfp/mrfp fusion proteins we can excite both fluorophores at nm and detect them separately in two different wavelength channels. we quantitatively determine the dissociation constants and compare their differences in vitro, in cells, and in embryos. these experiments demonstrate that sw-fccs is a powerful biophysical tool for the quantitation of biomolecular interactions in cells and organisms. addressing plasma membrane structure at the nanoscopic length-scale s. wieser, m. axmann, g. j. schütz biophysics institute, johannes kepler university linz, altenbergerstr. , a- linz, austria there is increasing interest in a detailed understanding of the structure and dynamics of the cellular plasma membrane, primarily based on recognizing its essential role for controlling cellular signaling processes. we employed single molecule fluorescence microscopy to study diffusion of cd , a gpi-anchored protein, in the plasma membrane of living t cells at sub-wavelength resolution, both on the cell body and on tunneling nanotubules connecting cells. the lateral motion of this single fluorescence labeled molecule was imaged on a millisecond time scale. within the experimental errors, no indications for confined diffusion for cd on the cell body in t cells have been found. furthermore by separating longitudinal and transversal mobility, we found isotropic diffusion behavior on the surface of tunneling nanotubules. in both studies we analyzed the mean square displacement as a function of the time-lag and the distribution of displacement steps. however, a closed analytical theory for these analysis is only available for the simplest models. to address a suspected diffusion process we reasoned that a full analytical description may not be required; it may well be sufficient to compare the experimental data with monte carlo simulations of the process. we demonstrated the working principle for this simulation based analysis for free diffusion, hop diffusion and transient binding of the tracer molecule to slowly moving receptors. n. chakroun , f. fraternali , m. malfois , h. rezaei , c. a. dreiss king's college london, u.k., diamond light source, didcot, oxfordshire, u.k., inra, jouy-en-josas, france prion(prp) diseases are fatal neurodegenerative diseases affecting mammals including human and sheep.they are characterized by the accumulation of extracellular βrich fibrillar deposits of a structurally modified form (prp sc ) of the cellular prp c .despite the increasing interest for prp diseases,the mechanism of prp c /prp sc conversion is still unknown.studies on prp diseases suggest that neurotoxicity arises from small pre-fibrillar oligomers.we have used a range of biophysical techniques combined with molecular dynamics simulations (md) to resolve the oligomerization pathways of sheep prp (sprp).we have shown that under well established conditions, sprp oligomerizes into three oligomers, which form in parallel.in addition, we have now identified the minimal region of sprp leading to the same oligomerization profile of the entire sprp,namely h h .low resolution shapes of sprp, h h and resulting oligomers have been determined by small-angle x-ray scattering.time-resolved studies have been used to follow the oligomerization of sprp and h h monomers into the oligomers.the conversion of sprp sc at the molecular scale was studied by md.simulations of the h h region recreating experimental conditions revealed a complete unfolding of h helix followed by h helix.these crucial steps are followed by the formation of β-sheet structures leading to a stable βrich double hairpin structure. single-molecule force spectroscopy investigation of the conformational equilibria of alphasynuclein m. brucale , a. rampioni , m. sandal , i. tessari , l. tosatto , l. bubacco , b. samorì istituto di biochimica g.moruzzi, università di bologna (italy), dipartimento di biologia, università di padova (italy) alpha-synuclein (asyn) is an abundant intrinsically disordered protein (idp) primarily located at presynaptic terminals. mutations in the gene encoding asyn have been linked to early-onset parkinson's disease (pd). by means of single molecule force spectroscopy (smfs) experiment, we show how the conformational equilibrium of monomeric wild type (wt) asyn shifts toward more compact structures in several unrelated conditions linked to pd pathogenicity [ ] . the conformational heterogeneity of pathological alpha-syn mutants a p, a t and e k has also been characterized, revealing marked differences in the conformational behaviors of the mutants with respect to wt asyn [ ] . all the mutants show a distinctively higher propensity, with respect to wt, to acquire a monomeric compact conformation that is compatible with the acquiring of beta structure. the same smfs experimental methodology is then used to characterize the conformational behavior of wt and mutant asyn in a variety of conditions, in an attempt to gain insight about the multiple and contrasting parameters controlling the equilibrium. in vitro protein folding studies using chemical denaturants have contributed tremendously to our understanding of the folding thermodynamics and kinetics of water-soluble proteins. this is not the case for integral membrane proteins, which constitute about one third of all eukaryotic proteins and more than half of all validated and potential drug targets. fully reversible denaturant-induced unfolding remains limited to a few β-barrel porins, whereas the much larger and more relevant class of α-helical membrane proteins has thus far evaded this approach. we report here the first example of an α-helical membrane protein that can be unfolded completely and reversibly by a chemical denaturant: mistic, a -residue protein from bacillus subtilis [ ] , dissociates from detergent micelles or lipid vesicles and assumes an unfolded monomeric state on titration with urea. using spectroscopic and microcalorimetric techniques, we exploited this unique property to provide (i) a quantitative comparison of membrane protein stability in different membrane-mimetic systems; (ii) an experimental test of controversial predictions [ ] regarding the folding core of mistic; and (iii) a convenient setup to study the spontaneous, translocon-independent membrane insertion of this unusual membrane protein. the mechanical functioning of biological tissues is important from many viewpoints such as diseases, clothing and even food. the protein collagen makes up the greater part of these tissues, and is remarkable for its many uses in the body, however there are at least two other major components. one of the most interesting properties of these tissues is their non-linear behavior under stress. this behavior is essential to prevent a catastrophic failure such as an aneurysm. at least three major theories have been proposed within the past few years to explain this behavior, but have been impossible to verify. in order to determine a correct description of the mechanical structure of the tissue we have been using cutting edge technological solutions to address the single molecules within the extra cellular matrix. this technique combines optical methods with single molecule force spectroscopy, allowing stiffness measurements over the nanoscale as well as determining the individual protein tensions within the extra cellular matrix. the results show that this method can be used to determine the network properties even in the complicated aortic wall enabling better understanding of disease states, which in this case include marfan's syndrome and ascending aortic aneurysms. beta amyloid peptide abetapy - shows a faster aggregation kinetics than abeta - c. d'arrigo , m. tabaton , a. perico institute for macromolecular studies, national research council, genova, italy, department of neurosciences, university of genova, genova, italy we test directly the differences in the aggregation kinetics of three important beta amyloid peptides, the full-length abeta - and the two n-terminal truncated and pyroglutamil modified abetapy - and abetapy - , found in different relative concentrations in the brains in normal aging and in alzheimer disease. we find by following the cd signal and the tht fluorescence of the solution in phosphate buffer, a substantial faster aggregation kinetics for abetapy - . this behavior is due to the particular sequence of this peptide which is also responsible of the specific oligomeric aggregation states, found by tem, during the fibrillization process which are very different from those of abeta - , more prone to fibril formation. in addition abetapy - is found here to have an inhibitory effect on abeta - fibrillogenesis, coherently with its known greater infective power. this is an indication of the important role of this peptide in the aggregation process of beta-peptides in alzheimer disease. the puzzle of the anomalously long denaturation kinetics of green fluorescent protein (gfp) mutants still is largely unveiled. in this study we have followed the effect of mutation h g on the stability of gfpmut (mut g) in the presence of guanidinium hydrochloride (gdnhcl). different techniques of fluorescence spectroscopy have been employed in order to obtain information concerning the unfolding event: time resolved fluorescence, fluorescence correlation spectroscopy (fcs), and fluorescence anisotropy. the substitution of the histidine with glycine affects protein stability versus ph: in particular mut g kinetics is not ph dependent and at basic ph values the protein is less stable. the fluorescence properties (quantum yield, lifetime) and the rotational correlation time are unchanged during the unfolding dynamics, while the number of fluorescent proteins decreases exponentially. an extrinsic probe, bound to cysteine , has been employed in order to gain more insights on the unfolding process, monitoring the stability of a different region of the protein. in particular, it has been found that a softer region is present around cysteine in both gfp variants, showing that the unfolding process does not follow a simple two step mechanism. recently, negatively-charged membranes were reported to catalyze amyloid fiber formation by amyloidogenic peptides/proteins and also to induce formation of "amyloidlike" fibrils by nonamyloidogenic proteins. here, we used an approach which combines steady-state and time-resolved fluorescence measurements to obtain structural information about these supramolecular assemblies and to gain insights about the factors that control their formation. by exploring a wide range of lipid concentrations, the interaction of alexa -lysozyme with phosphatidylserine-containing membranes was found to be a complex multi-step process, critically dependent upon the protein-to-lipid molar ratio (p/l) used. upon increasing the total lipid concentration in solution, there was a balance between an increased overall protein binding to the lipid vesicles and a progressive protein dilution on the membrane surface. as the surface potential of the vesicles decreased upon increasing the protein interfacial coverage of the liposomes, the protein binding mode was found to switch from a peripheral binding of lysozyme to the anionic headgroups (at low to intermediate p/l) to a partial insertion of the basic protein into the hydrophobic core of the membrane (at a high p/l). it is hypothesized that disruption of the protein tertiary structure might be a stepwise process beginning with loosening of the structure caused by its deeper insertion in the membrane bilayer. unexpected scaling laws in the mechanical unfolding of single protein molecules m. clusel, e. i. corwin, h. lannon, j. brujic center for soft matter research, physics department, new york university, new york, ny, usa it is a question of fundamental importance to understand the response of proteins to a stretching force, particularly in the case of mechanically active proteins, such as those in muscle fibers. we aim to understand how the structure and topology of a protein affect its resilience to external forces and presumably its function. owing to recent advances in single molecule force-clamp spectroscopy using the atomic force microscope (afm), we are now able to probe the structure and dynamics of single proteins under a constant stretching force by measuring their end-to-end length over time. the probability distribution of unfolding times at a given force allows us to estimate the strength of the protein in terms of a characteristic energy barrier, while the shape of the distribution provides a window into the microscopic mechanism by which the protein breaks apart. here we show a novel scaling of the unfolding kinetics with the stretching force, which deviates significantly from the currently accepted bell model. instead, we propose a physical picture for forced unfolding that is analogous to the mechanics of fracture. v. garcía-gonzález, j. mas-oliva instituto de fisiología celular. universidad nacional autónoma de méxico. méxico, d.f. méxico. studies focused on the thermodynamic and kinetic analysis have demonstrated that transfer of neutral lipids such as cholesterol esters through an aqueous phase is a highly costly biophysical event. therefore, nature has developed a series of lipid transfer proteins such as the cholesterylester transfer protein (cetp) designed to efficiently lower the energy barrier for transfer of cholesterol-esters between lipoproteins through an aqueous environment. employing circular dichroism we evaluated the secondary structure stability of a small peptide derived from the carboxy-end of cetp (helix y ) in a wide range of ph's. the percentage of α-helix is diminished only at extreme temperatures and acidic ph's in a reversible way. we report that while a mixture of phosphatidylcholine/cholesteryl-ester forms large aggregated particles independently of ph, inclusion of helix y to the mixtures close to neutral ph's allows the formation of small micellar-like structures confirmed by dynamic light scattering and electron microscopy. these results suggest that helix y when close to physiological ph values presents the ability to organize a micellar structure around itself. this type of organization allows the process to dramatically lower the energetic barrier for lipid transfer through aqueous media, phenomenon directly related with the facilitation of lipid transfer between lipoproteins. mimicking metastasis by a novel microfluidic approach there is increasing evidence that cancer metastasis shares commonalities with thrombosis. the von-willebrand-factor (vwf), a mechanical active blood clotting protein appears to be a particular potent candidate to bridge the gap between clotting and cancer extravasation. modeling the crucial physiological conditions of the blood circulatory system, for an in situ study of blood clotting-metastasis connections is not only, absolutely necessary, but also a challenging task. here, we present acoustically driven flow as a novel microfluidics method for mimicking the blood flow. this method enjoys very beneficial advantages of possibility of handling very little volumes of fluids, together with freedom to model most of the geometries present in our microcirculatory system. one technologically challenging, yet physiologically important factor, is the hydrodynamic condition in a bifurcated vessel, where complex shear profiles arise. we present a model to mimic these conditions and discuss the impact of hydrodynamics on vwf mediated cancer cell adhesion in bifurcated vessels of our microcirculatory system. protein structural changes occurring in flows stresses inherent to viscous fluid flow have previously been associated with protein unfolding, although structural changes have not been well documented as a function of relevant hydrodynamic parameters. we have used raman spectroscopy to monitor the structure of various protein solutions in situ for multiple flow scenarios within a concentric cylinder fluidic device ( ) . the flows, which ranged from circular couette to wavy taylor-couette flow, were characterised experimentally using particle image velocimetry. several proteins were observed to change conformation when exposed to these flows, although the nature of these changes was protein specific. shearing hen egg white lysozyme in water altered the protein backbone structure, while similar shear rates in a % glycerol, % water solution affected the solvent exposure of the side chain residues near the exterior of the α-domain. the solventdependent response may be due to the flow topology, viscous stress, or the surface hydration properties. comparison of spectra acquired at different time points, including before and after flow, confirmed that the observed changes are reversible and independent of fluid stress exposure time. the scripps research institute, la jolla, ca, usa. intrinsically disordered proteins are increasingly found to play major roles in cell biology and disease. we are utilizing single-molecule fluorescence methods to probe these complex and highly dynamic molecules, allowing more direct measurements of structural distributions and dynamics, while avoiding loss of information due to ensemble averaging. in one example, we investigated the structural dynamics of sup -nm, whose regulatable amyloid formation is believed to have functional significance in yeast. using a combination of single-molecule fret as a molecular ruler, coincidence to interrogate intermolecular interactions, and correlation analysis to probe conformational dynamics, we showed that the monomeric protein populates an ensemble of compact and rapidly interconverting conformations. a particularly interesting feature of intrinsically disordered proteins is that they are relatively unstructured in isolation, but can gain stable structure by interaction with binding partners. in this context, we used single-molecule fluorescence to characterize the complex folding pathway for the parkinson's-related idp alpha-synuclein induced by binding to a lipid-mimic. this combined single-molecule fluorescence methodology provides a powerful approach for detailed studies of the coupling of folding and dynamics with interactions and biology of this important class of proteins. m. ito , j. johansson , r. stromberg , l. nilsson department of biosciences and nutrirtion, karolinska institutet, huddinge, sweden, department of anatomy, physiology and biochemistry, swedish university of agricultural sciences, the biomedical centre, uppsala, sweden amyloid β-peptide (aβ) is a - amino acid polypeptide and known to aggregate and form insoluble amyloid fibril, which is regarded as a primary cause of alzheimer's disease (ad). the discovery of practical and effective treatments and drugs for ad has been waited eagerly. in a recent experimental study, it was suggested that stabilization of the helical conformation of the aβ middle region, which strongly favors collapsed coil formation in the extracellular environment, would reduce the aβ fibril formation. based on the experimental evidence, inhibition of the unfolding of the aβ α-helix can be a forceful strategy to repress the aβ fibril formation resulting in prevention of ad. however, the detailed mechanism of the unfolding of the aβ α-helix has remained unclear, because the x-ray or the nmr structure of the aβ α-helix in aqueous solution has not been reported due to its instability. the aim of this study is to find effective ways to inhibit the unfolding of the aβ middle region, which is a prerequisite for the aβ fibril formation. in this study, we attempted to elucidate the molecular mechanism of the aβ unfolding by molecular modeling and molecular dynamics (md) simulations. the md simulations were performed for α-helical structures of a wild-type aβ( - ) model and a mutant aβ( - ) model. linker average hydrophilicity as a tool to discriminate between extended and non-extended calcium binding proteins a. isvoran , e. quiniou , c. craescu , l. mouawad west university of timisoara, department of chemistry, pestalozzi , timisoara, romania, inserm u , centre universitaire paris-sud, bâtiment , orsay, france the ef-hand calcium binding proteins (cabps) may exist either in an extended or a compact conformation, sometimes correlated with their functions. for the cabps with know structure and function, calcium sensors are usually extended and calcium buffers compact, hence the interest to predict the form of the protein starting from its sequence. in this study we used two different procedures, the sosuidumbbell algorithm and a novel procedure that is based on the linker average hydrophilicity (lah). the two procedures were tested on known-structure cabps and then applied to unknown-structure centrins. the so-suidumbbell algorithm yielded the right conformations for of the known-structure proteins and predicted that all centrins should are compact. the lah procedure discriminated well between the extended and non-extended forms of all the known-structure cabps and it reflected well the phylogenetic classification of centrins being a simple and powerful means to discriminate between extended and nonextended forms of cabps. only few residues that constitute the linker are responsible for the form of the cabp, showing that this form is mainly governed by short-range interactions. (http://u .curie.u-psud.fr/modelisation/lah) lipid and protein organization of hepatitis b antigen characterized by fluorescence spectroscopy v. greiner , c. egelé , s. oncul , f. ronzon , c. manin , a. klymchenko , y. mély laboratoire de biophotonique et pharmacologie, umr cnrs, faculté de pharmacie, université de strasbourg, france, sanofi pasteur, av. marcel mérieux, marcy l'étoile, france. hepatitis b surface antigen (hbsag) particles are nm lipoprotein particles, mainly composed of the major s surface viral protein containing trp residues and yeast-derived lipids. since the structure of these particles is still missing, we further characterized them by fluorescence techniques. fcs indicated that the particles diffuse mainly as monomers and contain about proteins per particle. fluorescence quenching and time-resolved fluorescence experiments showed that the fluorescence signal is largely dominated by the trp residues at the protein surface. moreover, time-resolved anisotropy measurements indicate that the protein motion is restricted and that the surface trp residues exhibit both local and segmental motions. the lipid part of the particles has been studied by environment sensitive -hydroxyflavone probes and viscosity-sensitive dphbased probes, and compared to lipid bilayers and low density lipoproteins (ldls), taken as models. the results suggest that the lipid part of hbsag is closer to ldls than to model lipid bilayers. we present an extensive calorimetric study of bovine alphalactalbumin for various ca++ content. equilibrium dsc raw data are analyzed and the melting temperature tm, the specific heat jump deltacp, the heat of unfolding deltahm are directly extracted. the binding of calcium on the native (n) state greatly stabilizes the protein, essentially by the enthalpic difference between the unfolded (u) and n states. we show that subsequent addition of calcium in the mm range stabilizes further the n state. the equilibrium calorimetric measurements are completed with out of equilibrium stopped flow refolding kinetics by cd spectroscopy performed at different temperature and ca++ concentrations. we discuss the possible stabilization mechanisms compatible with our measurements. protein unfolding/refolding in cellular compartments: application of luciferase constructs our studies show that a reporter enzyme, firefly luciferase, can be used for evaluation of the stress-induced proteotoxicity within different cellular compartments such as the nucleus, cytoplasm or mitochondria. in transfected mammalian cells, firefly luciferase is localized in microsomes. we engineered plasmid constructs encoding luciferase with inserted specific sequences that ensure its cytoplasmic or intranuclear, or intramitochondrial localization. in addition, we fused luciferase to the green fluorescent protein (gfp) that enables to visualize patterns of the compartment-targeted product. using such gfp-labeled constructs we had a possibility to monitor protein unfolding, aggregation and refolding in the cytoplasm, nucleus and mitochondria of transfectants exposed to either stressful conditions. gfp-luciferase expressed in mammalian cells behaves as a relatively labile protein which can undergo reversible unfolding and aggregation in response to heat shock, atp depletion or action of toxic agents. in the case of cell recovery, refolding of denatured luciferase is carried out at the chaperone machine. we explored unfolding/refolding of gfp-luciferase in the cytoplasm, nucleus and mitochondria of ischemia-stressed rat cardiac cells and in several cancer cell lines treated with hyperthermia or some chemotherapeutic drugs. the results obtained have revealed intriguing correlations between the proteotoxic impact within either compartment and the viability of treated cells. amyloidogenic and conformational properties of proiapp and iapp in the presence of lipid bilayers s. jha , d. sellin , r. seidel , r. winter biophysical chemistry, department of chemistry, tu dortmund university, otto-hahn str. , d- , dortmund, max-planck-institute for molecular physiology, otto-hahn str. , d- , dortmund, germany the islet amyloid polypeptide (iapp), which is considered as the primary culprit for β-cell loss in type diabetes mellitus patients, is synthesized in the β-cells of the pancreas from its precursor, the pro-islet amyloid polypeptide (proiapp). proiapp is co-processed in the secretory granules and co-secreted to the extracellular matrix together with insulin as iapp. here, we compare the amyloidogenic and conformational properties of proiapp and iapp in the presence of lipid membranes, which have been discussed as loci of initiation of the fibrillation reaction. the two peptides show an enhanced amyloidogenic propensity in the presence of negatively charged membranes and similar secondary structural properties. however, proiapp shows a much less amyloidogenic propensity, probably due to the increased net charge on proiapp, compared to iapp. unlike iapp, proiapp forms small oligomeric structures at the lipid interface, having heights of ∼ . nm. this morphological distinction can be attributed to the presence of the pro-region, flanking the amyloidogenic iapp. the addition of proiapp to iapp marginally delays iapp fibrillation, probably by interfering with the interaction between amyloidogenic iapp cores of distinct iapp molecules. thus, it appears reasonable to speculate that the pro-region of the proiapp could serve to delay the fibrillogenesis of iapp at negatively charged lipid bilayers. the role of transmembrane domain interactions in the kinetics and folding of cpt z. a. jenei , k. borthwick , v. a. zammit , a. m. dixon chemistry dept., univ. of warwick, coventry, uk, clinicalȃsciencesȃres.ȃinst., warwick univ., coventry, uk carnitine palmitoyltransferase i (cpt ) enzymes are polytopic integral membrane proteins in the outer membrane of mitochondria (omm). cpt controls the rate of entry of long-chain fatty acids into the mitochondrial matrix for βoxidation. the two catalytically active isoforms, cpt a and cpt b, are different in their inhibitor binding kinetics and structure (interaction between n-and c-segments, interactions of transmembrane domains (tmd)). it has been suggested that inter-and intramolecular tmds interactions are important for cpt a, but not for cpt b function. cpt a has also been implicated in formation of oligomeric complexes through its tm segments. the study of tm helix-helix interactions in cpt isoforms could lead to a better understanding of their function and inhibitor binding kinetics, and will contribute towards the design of pharmacological strategies aimed at modulating the activities of cpt enzymes in conditions such as diabetes. to investigate the ability of the tmd in cpt to self-associate and the order of any oligomers formed, several biochemical and biophysical techniques have been used. we found the self-association of rcpt a tmds (tm , tm ) to be different as measured using the in vivo tox-cat assay. chemical cross-linking and analytical ultracentrifugation studies demonstrated formation of both trimers and hexamers by the rcpt a tm peptide. these results provide further evidence that tm plays role in formation of a channel in the omm. self-assembly of transmembrane domains in cpt : role of gxxxg(a) motif in possible channel formation z. a. jenei , k. borthwick , v. a. zammit , a. m. dixon department of chemistry and, warwick medical school, university of warwick, coventry, uk carnitine palmitoyltransferase a (cpt a), a membrane protein that controls the rate of oxidation of long-chain fatty acids, is of key importance in diabetes and has recently been reported to exist as an oligomer in vivo. we have investigated full-length cpt a and find that the protein exists as a hexamer in liver mitochondria. using mutants of cpt a expressed in yeast mitochondria, we have localised key protein interactions in the hexamer to the transmembrane (tm) domains of the protein. detailed study of the tm domains in isolation, in both e.coli membranes and detergent micelles, demonstrated that while tm shows little self-assembly, tm displayed significant self-association. biophysical analyses of a tm -derived synthetic peptide revealed oligomerization behaviour identical to native cpt a in mitochondria, providing a strong link between tm helixhelix interactions and cpt a hexamer formation. this is significant in light of a recent suggestion that cpt a oligomerization may lead to formation of a channel in the mitochondrial outer membrane through which acylcarnitine gains access to the inter-membrane space. our data supports this new theory, and we go on to demonstrate experimentally the structural determinants of hexamer (channel) formation, specifically gxxxg(a) motifs in the tm domain which pack favourably in the hexamer and stabilize the oligomer. investigation of flexible loop role in structure and thermodynamic stability of firefly luciferase p. maghami, b. ranjbar, s. hosseinkhani department of biochemistry and biophysics, faculty of basic sciences, tarbiat modares university, tehran, iran protein folding, like any chemical process, consists of two fundamental components, thermodynamics and kinetics, which determine the stability of the folded state and the pathway of folding, respectively. these processes are currently too difficult to be solved de novo by purely computational methods. experimental evidence is required to simplify the problem via protein engineering .in this research, the wild type firefly luciferase (photinus pyralis) and some of its mutants were over expressed and purified. then their unfolding thermodynamics were examined, using circular dichroism and conventional fluorescence measurements. the unfolding equilibrium constant were measured over a complete rang of denaturant conditions. the measurements were shown structural and physico-chemical changes between wild-type and mutant proteins. exploring intrinsic disorder of unstructured membrane proteins by surface polymer physics intrinsically unstructured proteins (iups) are considered as a separate class within the protein world because they lack a well-defined folded structure. because iup's function is indeed directly linked to structural disorder, they are assumed to be natively unfolded. several physicochemical techniques are available to discriminate the degree of disorder. however a clear structural classification is still lacking. in this context, polymer physics emerges as a powerful tool for getting inside on the conformational abilities directly related to structural disordered of iups. in the present contribution, surface pressure and ellipsometry experiments in conjunction with polymer physics have been used to infer structural data in terms of molecular conformation and flexibility of a membrane protein essential for bacterial division, zipa. this protein has been pointed to posses a high molecular flexibility and to adopt a random coil conformation. folding dynamics of peptides studied by timeresolved infrared spectroscopy c. krejtschi , o. ridderbusch , r. huang , l. wu , t. a. keiderling , k. hauser institute of biophysics, university of frankfurt, germany, department of chemistry, university of illinois at chicago, usa peptides are ideal model systems to study protein folding mechanisms. ir techniques provide both the necessary time resolution as well as the structural sensitivity. we initiate rapid heating by laser-excited ns temperature jumps (∼ • c) and study fast ns-to-µs relaxation dynamics [ ] . the dynamics of the α-helix to random coil transition of polyglutamic acid was analyzed under reversible folding/refolding ph-conditions. the observed relaxation kinetics allowed separation of the folding and unfolding process with additional use of ftir measurements in thermal equilibrium. sitespecific dynamics were monitored by use of isotopic labeling for a β-hairpin peptide whose conformation is stabilized by a hydrophobic core. various single and cross-strand isotopically labeled variants were analyzed. the isotope-edited kinetics show variations in local structural stability of the hairpin backbone. our data support a multistate dynamic behavior, and the site-specific kinetics are consistent with a hydrophobic collapse hypothesis for hairpin folding [ ] . small heat shock protein hsp was predicted to belong to the family of intrinsically disordered proteins. one of the features of these proteins is that they do not demonstrate cooperative thermal transitions on heating. we applied different methods (dsc, ftir and intrinsic tryptophan fluorescence) to investigate the thermal unfolding of hsp . dsc results have shown that thermal denaturation of hsp begins from o c and occurs, with very low cooperativity, within a broad temperature region (up to o c and above). the thermal unfolding of hsp is fully reversible. the ftir data show that heating of hsp from to o c results in complete disappearance of α-helices (from - % to ) and the decrease in β-sheets content from to %. studies on the temperature dependences of tryptophan fluorescence have shown a significant red shift of the spectrum. these changes occurred within temperature region from to o c with midpoint at ∼ o c. probably, this transition can be explained by destruction of β-sheets around trp , the only trp residue of the α-crystallin domain of hsp (other trp residues of hsp are localized in the n-terminal domain). the data obtained confirm the suggestion that hsp is a protein, whose significant part is intrinsically disordered. we propose that, on heating, the α-crystallin domain containing β-sheets melts at higher temperature than the n-terminal domain containing the most of α-helices. t. rosenkranz , a. katranidis , d. atta , j. enderlein , i. gregor , m. grzelakowski , p. rigler , w. meier , j. fitter isb- : molecular biophysics, research centre jülich, germany, institute of physics, biophysics/complex systems, georg august university, göttingen, germany, institut für physikalische chemie; universität basel, basel, swizerland the protein folding mechanism is the missing link in the biological flow of information from the dna to its specific function. since most of proteins within a cell consist of more than one domain studies on this protein class are of major importance. it is a common feature of multidomain proteins to aggregate under refolding conditions, which hinders a refolding. molecular encapsulation of single molecules prevents aggregation. by immobilizing the nanocapsule the observation period in a wide field microscope will be extended, so that slow or rare folding events can be detected. a major goal of this study is to investigate polymeric vesicles with respect to their suitability for protein folding studies [ ] . polymer vesicles maintain their structural integrity even under harsh unfolding conditions. furthermore the nanocontainer proved to be permeable to guanidinium hydrochloride. using encapsulated phoshoglycerate kinase, labeled with atto- , a dye which experiences fluorescence quenching by photo-induced electron transfer (pet) with tryptophans, we demonstrate the remarkable properties of polymeric nanocontainers. applying pet as a folding probe we detected multiple unfolding/refolding transitions of single proteins. proteins frequently become irreversibly modified by carbonylation, a process of introducing the carbonyl group (carbon monoxide) in a reaction with reactive oxygen species (ros) such as superoxide, peroxide or ozone. the main targets for carbonylation in proteins are amino-acid side chains of lysine, arginine and proline. products of carbonylation are aminoadipic semialdehyde from lysine (asa) and glutamic semialdehyde (gsa) from arginine and proline. importantly, carbonylated proteins are marked for proteolysis by the proteasome, but can escape degradation and form aggregates that can be cytotoxic. carbonylation increases with the age of cells and it is associated with ageing and age related disorders such as alzheimer's disease, parkinson's disease and cancer. we have used the molecular dynamics method to study the stability of carbonylated proteins villin headpiece and ubiquitin. simulations were run after mutations of arginine, proline and lysine into gsa and asa had been performed. in addition, we have used thermodynamic integration on lysine, arginine, proline, asa and gsa residues in order to estimate their solvation free energy (related to relative hydrophobicity and hydrophilicity). our results suggest that carbonylation markedly decreases the overall stability of proteins, and that one potential reason for that may be a disruption of the balance between hydrophilic and hydrophobic regions in the protein. a. martino, d. crane, i. m. feavers, b. bolgiano division of bacteriology, national institute for biological standards and control, potters bar, uk the sensitivity to protein's secondary structure and progress in computational calculations have made circular dichroism (cd) an attractive technique to explore the optical properties of three promising vaccine candidates to neisseria meningitidis. clinical batches of a c-term deleted form of nada (genome-derived neisseria antigen -gna ) and the fusion proteins gna - (fp- ) and gna - (fp- ) were therefore studied. increases in temperature and denaturant concentration on secondary structures and folding/unfolding profile were monitored by cd in the far and near uv regions and complemented by trp fluorescence spectroscopy data. furthermore, epitope conformational changes on binding activities to immune-sera were investigated. the calculated secondary structure content was in broad agreement with the available predicted or solved protein structures. upon temperature incubation, a structural transition from a highly α-helical nada to a more unordered conformation, with a mid point at ∼ • c, was observed. fp- and fp- maintained their conformation up to • c or m guhcl in the case of fp- . unfolding was not always reversible. reductions in binding to monoclonal ab titrated along with increasing unfolding. folding/unfolding studies have proven useful in better understanding the solution behaviour and extent of folding of proteins. cold denaturation of yfh offers the clue to understand the effect of alcohols on protein stability s. r. martin , v. esposito , p. de los rios , a. pastore , p. a. temussi national institute for medical research, the ridgeway, nw aa london, u.k., laboratoire de biophysique statistique, sb/itp, ecole polytechnique fédérale de lausanne (epfl), ch- , lausanne, switzerland, dipartimento di chimica, università di napoli federico ii, via cinthia, i- napoli, italy although alcohols are well known to be protein denaturants when present at high concentrations, their effect on proteins at low concentrations is much less well characterized. here we present a study of the effects of alcohols on protein stability using yfh . exploiting the unusual property of this protein of undergoing cold denaturation around • c without any ad hoc destabilization, we determined the stability curve on the basis of both high and low temperature unfolding in the presence of three commonly used alcohols: trifluoroethanol,ethanol methanol. in all cases, we observed an extended temperature range of protein stability as determined by a modest increase of the high temperature of unfolding but an appreciable decrease in the low temperature of unfolding. we suggest that alcohols, at low concentration and physiological ph, stabilize proteins by greatly widening the range of temperatures over which the protein is stable. our results also clarify the molecular mechanism of the interaction and validate the current theoretical interpretation of the mechanism of cold denaturation. biomolecular sciences and biotechnology tor vergata moro , rome, italy cholesterol plays an important role in regulating the structural properties of phospholipid and non-phospholipid membranes. in this study we have applied in situ energy dispersive x-ray diffraction (edxd) to investigate the effect of cholesterol on the structure of different phospholipid and non-phospholipid oriented membranes. in detail, phosphatidylcholine (pc) bilayers and niosomal membranes, made of a non-ionic surfactant centre for bioactive chemistry, department of chemistry department of chemistry this process is initiated at nuclear envelope remnants (ners) in the presence of atp and gtp. the mvs can be divided in two main populations: mv and mv . mv has a classical lipid composition while mv is enriched in phosphoinositides (pips: pi, pip, pip and pip ). ners have an unusual lipid composition, enriched both in cholesterol and pips. physicochemical properties of the pips were investigated as a function of ph and temperature (t) using nmr, saxs and dls to map out their phase state. pips-water dispersions are observed in lamellar, hexagonal or isotropic phases depending on t and ph. in parallel, model membranes mimicking mv and ners lipid composition were studied by h and p nmr. mv modelling shows a complex behaviour of pips on pc membranes: they order or disorder membranes, whereas the order of pc/pi/pip/pip membrane is lower than that of pc or pc/pi membranes c. manzo, t. s. van zanten, m. f. garcia-parajo bionanophotonics group, ibec-institut de bioenginyeria de catalunya, barcelona, spain membrane proteins play a fundamental role in intra-and inter-cellular functions. in particular, the proteins lateral mobility in the fluid membrane environment is crucial for the regulation of several mechanisms, as receptor-mediated signal transduction and establishment of immunological synapses. these mechanisms are controlled through protein crowding and reduced lateral diffusion, which induce macromolecular associations and limit the application of conventional single molecule fluorescence techniques. to measure proteins mobility on living cells membrane, we developed a fluorescent correlation spectroscopy (fcs) setup in which the sample illumination is obtained through near-field scanning optical microscopy (nsom) probes. the use of nsom probes is particularly suited for the observation of dynamics on the cell membrane and overcomes the drawbacks of other techniques. in fact, through a shear-force-based position control, the probe is kept at a fixed distance from the membrane and its sub-wavelength aperture (∼ nm) reduces the illumination area, allowing the observation of highly crowded regions of the membrane. on the basis of preliminary results, the nsom-fcs is expected to provide an additional insight on the proteins trafficking at the membrane level. the technique also presents several potential developments, as the further reduction of the illumination area and two-colors correlation. single molecule fluorescence microscopy of the store-operated calcium channel subunit orai j. madl, j. weghuber, d. bergmair, m. fahrner, m. muik, c. romanin, g. j. schütz johannes kepler university, institute for biophysics, linz, austria store-operated calcium entry (soce) is essential for many cellular signalling processes. the essential pore forming subunit of soce channels in the plasma membrane is orai . here we present single molecule fluorescence microscopy of orai which was performed in order to directly visualize the stoichiometry of mobile orai pores. the protein was fluorescently labeled with monomeric gfp. a novel single molecule fluorescence approach, toccsl (thinning out clusters while conserving the stoichiometry of labeling), was used for the determination of the stoichiometry. this technique allows reducing the density of fluorescently labeled molecules without affecting the stoichiometry of labeling. density reduction is achieved by completely photobleaching a defined area within the plasma membrane; nonbleached gfp-orai aggregates enter the bleached region subsequently by diffusion. our data indicate that there are different populations of orai present in the cell: most of orai is located in the plasma membrane. a second population of orai -mgfp was found to be localized in intracellular vesicles. a significant fraction of the plasma membrane orai exhibits a diffusive movement. we found by analyzing the bleaching characteristics of single orai -mgfp aggregates that in resting cells mobile orai is predominantly dimeric. a. kobitski , a. nierth , m. helm , a. jäschke , g. u. nienhaus university of ulm, germany, university of heidelberg, germany, university of karlsruhe, germany rna molecules have attracted enormous attention in recent years, and various novel roles were revealed for rna in biological processes. ribozymes are a class of rna molecules capable of catalyzing chemical reactions. we have studied a diels-alderase (dase) ribozyme, a small artificial -mer ribozyme, which is capable of catalyzing carbon-carbon bond formation between an anthracene diene and a maleimide dienophile in multiple turnovers. single-molecule fluorescence resonance energy transfer was employed to investigate the intramolecular dynamics of this rna molecule as a function of mg + ion concentration. folding into a functional state occurs via an intermediate state, and continuous fluctuations between these two states were observed on the ms time-scale at the midpoint concentration of mg + ions. an effect of substrates binding on the folding and catalytic reaction of the dase ribozyme is in the focus of our recent research with the ultimate goal to obtain a detailed structural view of the single-molecule conformational changes that accompany the catalytic reaction. a. katranidis , r. schlesinger , k. nierhaus , i. gregor , m. gerrits , g. bueldt , j. numerous studies showed that protein folding and maturation can differ substantially between de novo synthesized proteins and in vitro refolded proteins. here we present an approach employing a two color single molecule sensitive fluorescence wide-field microscope in order to visualize surface tethered fluorescently labeled ribosomes and de novo synthesized gfp molecules in real time [ ] . fluorescence of co-translational folded proteins was observed from mature fluorescent gfp molecules which carry additional amino acids at the c terminus remaining linked to the ribosome. thus it was possible to co-localize fluorescence from labeled ribosomes and from gfp molecules. we demonstrate that the green fluorescence protein mutant gfp emerald is produced with a characteristic time of five minutes. the fastest gfp molecules appeared already within one minute. processes precedent to chromophore formation, such as polypeptide synthesis and protein folding, are fast and last not longer than one minute. in fluorescence spectroscopy, photobleaching is a process which leads to irreversible loss of fluorescent properties of a dye molecule, usually due to photochemical reactions. it is especially important for fcs experiments on slow-diffusion systems since for high excitation intensities it can have a strong impact on fluorescence intensity correlation function. usually it is observed as apparent shortening of the mean diffusion time of the dye molecules. the behavior of tmr-labeled fd-virus rods in water solution under various excitation conditions was investigated. the experiments were conducted for low ( : ) and high ( : ) tmr:virus ratios and for increasing laser intensities. the correlation function was measured in the experiments. the results were fitted using origin software to estimate the influence of photobleaching, and compared with computer simulations. a strong effect of photobleaching was visible for rods labeled with a single dye molecule, while rods labeled with tmr molecules showed little to no bleaching. a prolongation of characteristic diffusion times for highly labeled virus rods in comparison to low-labeled ones was also observed. k. toth , a. gansen , a. valeri , v. böhm , c. a. seidel , j. langowski abt. biophysik der makromoleküle, deutsches krebsforschungszentrum, heidelberg, germany, lehrstuhl für molekulare physikalische chemie, heinrich heine universität, düsseldorf, germanythe nucleosome has a central role in the compaction of genomic dna and the control of dna accessibility for transcription and replication. we studied the effect of dna sequence and selective histone acetylation on the structure, stability and disassembly of the mononucleosomes. quantitative single molecule fret measurements between dyes attached to different parts of the nucleosome permitted us to detect the equilibrium between several subpopulations of reconstituted nucleosomes in solution. we obtained that the heterogeneity and stability of the samples are correlated with each other and influenced both by the dna sequence and the histone acetylation. the path of the linker dna is more sensitive to all studied effects than the dna on the core. intermediates of the disassembly pathway were identified and characterized. j. strömqvist , s. johansson , y. ohsugi , k. andersson , l. xu , m. kinjo , p. höglund , j. widengren experimental biomolecular physics, kth, stockholm, sweden, department of microbiology and cell biology, karolinska institutet, stockholm, sweden, laboratory of molecular cell dynamics, hokkaido university, sapporo, japan dual-color fluorescence cross correlation spectroscopy (fccs) has been used to explore the molecular dynamics at immune cell surfaces, with a particular focus towards the regulation mechanisms of natural killer (nk) lymphocytes. nk cells are critical mediators of anti-viral immunity and protectors against cancer spread. their activity is governed by a fine-tuned balance between inhibitory and activating receptors, where ly a and kir receptors represents the inhibitory ones. their ligands are mhc class i receptors. fcs is a technique based on the analysis of intensity fluctuations of fluorescent molecules excited by a focused laser beam. the technique offers information about molecular dynamics at the single molecular level, in the nanosecond to millisecond range. dual color fccs expands fcs by correlating the intensity from two different colors. by labeling two potential interaction partners with dyes emitting at different wavelengths, the amount of interaction can be determined.here, we will report on recent fccs data exploring the interaction between the inhibitory receptors and their ligands, as well as different labeling strategies used to enable these measurements. dynamic multiple-target tracing probes spatiotemporal cartography of cell membranes in order to decipher the non random and non homogeneity of the plasma membrane organization, we had performed fluorescence correlation spectroscopy measurements on live cells. this allowed us to establish the presence of nanoscale confining structures and to demonstrate their implication in signaling process . complementing these studies, we present here a new analytical method, namely multiple-target tracing (mtt) which takes advantage of the high resolution provided by singlemolecule sensitivity to generate dynamic maps at high densities of tracked particles. introducing deflation by subtracting detected peaks allows detecting peaks of lower intensity. we achieved an exhaustive detection of particles with performances reaching theoretical limits, and a reconnection of trajectories integrating the statistical information from past trajectories. we demonstrate the potential of this new method of analysis by applying it to the epidermal growth factor receptor labeled with quantum dots, in the plasma membrane of live cells. this has allowed us to build up a global representation of molecular dynamics in cell membranes. dual polarisation interferometry (dpi) is a surface analytical technique capable of dynamically measuring biophysical parameters of conformational change in biomolecular interactions. the technique measures three key parameters, namely layer thickness, layer density (ri) and mass, thereby enabling the resolution of conformational changes involved during binding. a number of different applications are presented. protein-protein interactions: understanding the biophysical nature of protein interactions can deliver insights into the mechanisms by which proteins interact, thereby elucidating protein function. dpi enables correlation between binding affinity and conformational change, greatly enhancing the study of structure-function relationships. lipid layers: the birefringence mode of dpi can be used to study the formation of lipid bilayers and biomolecular self-assembly. it is possible to use a combination of bilayer refringence and mass to study phase transitions associated with protein or peptide binding to the lipid bilayer. the individual stages of adsorption, absorption and micellisation can be distinguished. carbohydrate interactions: dpi uses a planar glass surface and a wide range of coupling chemistries. a carbohydratespecific surface can be used to study a wide range of biomolecular interactions, such as lectins, acidic proteins, extracellular matrix signaling and interactions with complex membranes. the experimental characterization of the elementary conformational steps constituting the protein folding pathway remains a big challenge. quenching of the triplet state of tryptophan by close contact with cysteine has been shown to provide a new tool for measuring the rate of intramolecular contact formation -one of the most elementary events in the folding process -in peptides and proteins using only natural probes. here we show an extensive study on a stabilized mutant of the second beta-hairpin of gb domain. steady state fluorescence and kinetics of contact formation between a natural tryptophan and a cysteine added to the c-terminal are measured for different temperatures and solvent conditions. we separately address the contributions of different structural elements to the overall stability of the hairpin. we extract kinetics parameter for contact formations in the unfolded state, formation of the hydrophobic core and tails pairing in the folded state. by means of a fragment peptide terminated with a tryptophan and a cysteine, we also estimate the structural propensity of the turn region. the data coherently combine with a simple model previously developed to describe the dynamics of unstructured chain [biophys. j. , ( )], here modified with the addition of attractive interactions between specific residues. catalytic power of partially denatured enzymes: implementation of molten-globule-like states m. shushanyan , d. e. khoshtariya , m. makharadze , t. tretyakova , r. van eldik institute of molecular biology and biophysics, gotua , tbilisi, georgia, department of physics, i. javakhishvili tbilisi state university, tbilisi, georgia, department of chemistry and pharmacy, university of erlangen-nürnberg, germany impact of nonspecific moderate denaturants, urea and dmso, on the kinetic (functional) and thermodynamic (stability) patterns of a hydrolytic enzyme, α-chymotrypsin (α-ct) has been investigated. furthermore, an impact of urea and guhcl in combination with of temperature on the kinetic pattern of carboxypeptidase a has been examined. for the case of α-ct, in particular, we have observed about tenfold increase of the apparent mickaelis constant when going from to m urea ( o c), whereas the value of catalytic constant remained almost unchanged, indicating that the protein is not unfolded even under those severe conditions. the matching microcalorimetric experiments revealed that both the temperature-induced melting temperature and transition enthalpy decrease gradually with the increase of the additive concentration. with m urea the peak-shaped calorimetric feature disappears totally. however, catalytic power of α-ct was preserved owing to its catalytic constant. for other studied enzyme/substrate/denaturant arrays diverse kinetic peculiarities due to mgls have been observed. rubredoxins are a class of iron-containing proteins whose biological role on electron transfer processes and metal binding is still unclear. the unfolding dynamics of the rubredoxin mutant rda c from the mesophile desulfovibrio vulgaris (dv) was studied on the temperature range from • c to • c and along time at • c. resonance energy transfer (ret) was used to determine the donor (d; trp ) to acceptor (a; , -iaedans) distance. from • c to • c the d-a distance increased Å. however the random coil expected d-a distance was only achieved after heating the protein solution during . hours at • c. from uv-vis absorption data it's clear that this protein is capable of maintaining its iron-sulfur center at • c. by melting the protein at the same temperature all iron-centers disintegrate and the protein unfold after . hour. the trp fluorescence also shifts nm to the red reflecting the partial exposition of the indole ring to the solvent. from fluorescence and anisotropy decay curves a breathing type movement of the protein structure was observed between • c to • c without lost of significant secondary structure. this structure flexibility should play an important role on the thermal stability of the dvrd the antimicrobial peptide novicidin (nc) -modified from the sheep self defense peptide smap- , for reduced mammalian cytotoxicity -is a cationic peptide (net charge +∼ . ) that adopts random coil structure in solution, but an α-helix in the presence of lipid vesicles. the conformation of nc in presence of the lipids dlpc, dlpg, dmpc, dmpg, dopc, dopg, dope, and dops in different combinations reveal the lipid selectivity, affinity, and phase dependent changes with varying l/p ratios and temperatures, observed from cd spectroscopy. it is understood that the conformational change is dependent on chain length and head group of the lipid. apart from the in vivo results on the nc activity, studies using qcm-d, dual polarisation interferometry, and calcein release assay reveal the kinetics and concentration dependent activity of nc in lipid bilayers and vesicles. preliminary studies on orientation of nc in various lipid environments using ssnmr, lsnmr, and molecular dynamics simulations apparently suggest that nc may form toroidal pores/detergent effects depending on the chain length of the lipids. further experiments on nc in presence of lipids using dsc, itc, ssnmr, oriented cd, ld, and confocal microscopy to determine the structure, thermal stability, orientation in lipid bilayers, and thereof the action of nc will help in proposing a comprehensive model for its mechanism of action in model membranes. dielectric method for measuring glass transition and denaturation temperatures of hydrated proteins g. e. thomas , s. bone , g. drago institute for bioelectronic and molecular microsystems, bangor university, gwynedd, uk., applied enzyme technology, pontypool, uk.the flexibility of protein structures is important in allowing the variety of motions, covering a wide range of magnitudes and frequencies, essential to biological activity. high frequency dielectric measurements can be used to study the flexibility of proteins by probing the relaxation of dipolar constituents of their structures. hydrated proteins exhibit a broad dielectric loss extending over the frequency range from mhz to ghz which can be decomposed into a number of constituent dispersions. one of these dispersions, with a relaxation time of ∼ ns, has been attributed to the relaxation of protein backbone peptide groups in the protein interior. in the work reported here, this dielectric dispersion was investigated as a function of temperature for the enzyme glucose oxidase. two critical temperatures were identified as the glass transition and denaturation temperatures, both of which were found to decrease with increasing protein water content. the results were consistent with a scheme in which the hydrated glassy protein undergoes a change in structural mobility at the glass transition temperature and experiences an irreversible change in conformation at a higher denaturation temperature. both glass transition and denaturation temperatures are key indicators of protein stability and are important in the production and storage of protein based pharmaceuticals. a. szymańska, k. kacprzyk, g.Ślósarek department of molecular biophysics, a. mickiewicz university, umultowska , - poznań, poland aggregation dynamics of proteins plays an important role in molecular biology and medicine as it permits explanation of several disease states. an interesting problem is to find out in which conditions the interactions of the protein molecules lead to formation of ordered structures and in which to disordered ones. in this study, dynamic light scattering, circular dichroism and also congo red dye experiments were performed to analyze various structural states of lysozyme induced under different concentration of ethanol solvent. at low ethanol concentration the attractive interaction between the protein macromolecules dominate. after addition of more ethanol solvent, the translational diffusion coefficient was much smaller than that for lysozyme solution at zero ethanol concentration. it can be explained by the structural transformation of the polypeptide chain leading to a partially folded conformation needed for oligomerization and fibrillation process. on the basis of the cd experiments we concluded that the ethanol solvent induces changes in secondary structure of lysozyme solution. on addition of % v/v ethanol solution the intramolecular hydrogen bonds were destabilized. above this ethanol concentration, β -sheet were the dominant secondary structure of lysozyme in solution. the phase diagram illustrating the formation of: monomers, oligomers at various structural states, protofilament formation state, protofilament and amyloid fibrils was constructed. key: cord- -ap n ijl authors: kopek, benjamin g; perkins, guy; miller, david j; ellisman, mark h; ahlquist, paul title: three-dimensional analysis of a viral rna replication complex reveals a virus-induced mini-organelle date: - - journal: plos biol doi: . /journal.pbio. sha: doc_id: cord_uid: ap n ijl positive-strand rna viruses are the largest genetic class of viruses and include many serious human pathogens. all positive-strand rna viruses replicate their genomes in association with intracellular membrane rearrangements such as single- or double-membrane vesicles. however, the exact sites of rna synthesis and crucial topological relationships between relevant membranes, vesicle interiors, surrounding lumens, and cytoplasm generally are poorly defined. we applied electron microscope tomography and complementary approaches to flock house virus (fhv)–infected drosophila cells to provide the first -d analysis of such replication complexes. the sole fhv rna replication factor, protein a, and fhv-specific -bromouridine '-triphosphate incorporation localized between inner and outer mitochondrial membranes inside ∼ -nm vesicles (spherules), which thus are fhv-induced compartments for viral rna synthesis. all such fhv spherules were outer mitochondrial membrane invaginations with interiors connected to the cytoplasm by a necked channel of ∼ -nm diameter, which is sufficient for ribonucleotide import and product rna export. tomographic, biochemical, and other results imply that fhv spherules contain, on average, three rna replication intermediates and an interior shell of ∼ membrane-spanning, self-interacting protein as. the results identify spherules as the site of protein a and nascent rna accumulation and define spherule topology, dimensions, and stoichiometry to reveal the nature and many details of the organization and function of the fhv rna replication complex. the resulting insights appear relevant to many other positive-strand rna viruses and support recently proposed structural and likely evolutionary parallels with retrovirus and double-stranded rna virus virions. positive-strand rna [(þ)rna] viruses contain messengersense, single-stranded rna in their virions; they represent over a third of known virus genera; and they include many important human, animal, and plant pathogens [ ] . a common, if not universal, feature of (þ)rna virus replication is the association of their rna replication complexes with infection-specific host intracellular membrane rearrangements [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . characterizing the features of these membrane-associated rna replication complexes should identify general principles and mechanisms of (þ)rna virus replication and could lead to broadly applicable control strategies for (þ)rna viruses including, e.g., hepatitis c virus and the sars coronavirus. for many (þ)rna viruses-including alphaviruses [ ] , other members of the alphavirus-like superfamily [ ] , rubiviruses [ , ] , flaviviruses [ ] , tombusviruses [ ] , and others [ , [ ] [ ] [ ] -rna replication occurs in association with ; - -nm diameter membranous vesicles or spherules that form in the lumen of specific secretory compartments or organelles. the similarity of these structures suggests that rna replication by such otherwise distinct viruses involves important conserved features related to membranes. for some viruses, the localization of viral replicase proteins [ , , , [ ] [ ] [ ] or viral rna synthesis [ , , ] suggest that such spherules may contain or comprise the viral rna replication complex. for brome mosaic virus (bmv) and some other viruses, twodimensional ( -d) electron microscopy (em) reveals that a fraction of such spherules have interiors that appear to be connected to the cytoplasm by membranous necks [ , , ] . however, limitations inherent in random sectioning and -d analysis prevent standard em from resolving many issues crucial to understanding spherule structure and function, such as the range of spherule diameter and volume, and whether all spherule interiors are connected to the cytoplasm or if some bud free from their adjacent bounding membranes. to resolve these and other issues central to the mechanism of rna replication, we used em tomography (emt) to provide the first, to our knowledge, three-dimensional ( -d) ultrastructural study of the membrane-bound rna replication complexes of a (þ)rna virus. emt generates highresolution, -d images or tomograms by digitally processing a series of - electron micrographs collected as a specimen is tilted in - increments on an axis perpendicular to the electron beam [ ] . similar -d emt analyses have been crucial to reveal many important features of complex cellular organelles such as the golgi apparatus [ ] [ ] [ ] [ ] , endoplasmic reticulum [ , ] , and mitochondria [ ] [ ] [ ] . we chose flock house virus (fhv), the best characterized member of the nodaviridae, as a (þ)rna virus with advantageous features for such studies. fhv has been used as a model to study rna replication [ , , [ ] [ ] [ ] , virion structure and assembly [ , ] , and genomic packaging [ ] [ ] [ ] [ ] [ ] . fhv has a . -kb bipartite rna genome in which rna ( . kb) encodes the capsid precursor [ ] whereas rna ( . kb) encodes an rna silencing inhibitor [ , ] and a multifunctional rna replication factor, protein a [ , , ] . protein a, the only fhv protein needed for rna replication, is directed by an nterminal targeting and transmembrane sequence to outer mitochondrial membranes, where it colocalizes by immunofluorescence with the sites of viral rna synthesis [ , ] . gradient flotation and dissociation assays showed that protein a behaves as an integral transmembrane protein [ ] . additionally, protease digestion and selective permeabilization after differential epitope tagging demonstrated that protein a is inserted into the outer mitochondrial membrane with the n terminus in the inner membrane space or matrix, while the majority of the protein a sequence is exposed to the cytoplasm [ ] . protein a also self-interacts in vivo in ways that are important for rna replication [ ] . like many other (þ)rna viruses [ , , , , [ ] [ ] [ ] [ ] [ ] [ ] , fhv infection induces the formation of ; -nm membranous vesicles or spherules, which, for the case of fhv, are found between the mitochondrial outer and inner membranes [ ] . here we use emt and multiple complementary approaches to provide -d visualization of a (þ)rna virus replication complex. among other findings, the results show that fhv spherules are compartments or mini-organelles for viral rna synthesis, which form by invagination of the outer mitochondrial membrane and communicate with the cytoplasm through ; -nm diameter necks. the results further indicate that each spherule contains, on average, ; membranespanning, self-interacting protein a molecules and that fhvinfected cells contain - genomic rna replication intermediates per spherule. these observations define a new level of understanding of the nature, structure, and organization of a viral rna replication complex, including principles that are likely relevant to many other (þ)rna viruses. protein a is the only fhv protein needed for rna replication and so must co-localize with viral rna replication complexes. prior immunofluorescence and immunogold labeling em localized protein a to the outer mitochondrial membrane in fhv-infected cells [ ] . however, in those prior attempts at immunogold labeling, fixation conditions needed to preserve spherule ultrastructure abolished protein a antigenicity for the polyclonal antibody used, hence blocking protein a localization relative to spherules. to overcome this, we identified a monoclonal antibody against protein a [ ] that was able to detect protein a under fixation conditions that sufficiently retained spherule ultrastructure. immunogold em with this protein a monoclonal antibody revealed that nearly all protein a was in or on mitochondrial spherules in fhv-infected cells ( figure ). over gold particles in different electron micrographs were counted and % % of the specific gold labeling density above background (see materials and methods) was associated with spherules. cytoplasmic labeling, presumably including protein a being translated and/or trafficked in the cytoplasm, was just % % above background labeling levels. the remaining % % of immunogold label was associated with mitochondria but not discernable spherules, including gold particles on the cytoplasmic face of the outer mitochondrial membrane where some protein a might have been localized that was not, or not yet, internalized into spherules. the clustering pattern of immunogold particles in a subset of spherules may be due to nonuniform epitope exposure or signal amplification by secondary antibodies. to avoid over-weighting the calculations due to such clustering effects, we also analyzed the same micrographs counting each cluster as one event. the resulting count of clusters gave a very similar pattern to the one described above ( % spherule associated). by immunofluorescence microscopy, we found that bromouridine (bru)-labeled fhv rna synthesis occurs exclusively at outer mitochondrial membranes in infected drosophila cells [ ] . to localize more precisely fhv rna synthesis in relation to spherules, we incubated mitochondria isolated from uninfected and fhv-infected drosophila cells with a nucleotide mix including -bromouridine '-triphosphate (brutp) and performed immunogold labeling em with an antibody recognizing bru incorporated into rna, but not unincorporated brutp. for mitochondrial preparations from fhv-infected cells, whereas cells store and replicate their genomes as dna, most viruses have rna genomes that replicate by using virus-specific pathways in the host cell. the largest class of rna viruses, the positive-strand rna viruses, replicate their genomes on intracellular membranes. however, little is understood about how and why these viruses use membranes in rna replication. the well-studied flock house virus (fhv) replicates its rna on mitochondrial membranes. we found that the single fhv rna replication factor and newly synthesized fhv rna localized predominantly in numerous infection-specific membrane vesicles inside the outer mitochondrial membrane. we used electron microscope tomography to image these membranes in three dimensions and found that the interior of each vesicle was connected to the cytoplasm by a single necked channel large enough to import ribonucleotide substrates and to export product rna. the results suggest that fhv uses these vesicles as replication compartments, which may also protect replicating rna from competing processes and host defenses. these findings complement results from other viruses to support possible parallels between genome replication by positive-strand rna viruses and two distinct virus classes, double-stranded rna and reverse-transcribing viruses. spherules were the major site of immunogold labeling ( figure a and b). of gold particles examined, % % were on spherules. the remaining % of gold particles that fell outside of spherules may include mature rna products released from spherules and nonspecific background labeling. for mitochondria from uninfected cells, background labeling levels were independent of the addition or omission of brutp and averaged % of the total immunogold labeling of brutp-treated mitochondria from fhv-infected cells. we found that using isolated mitochondria was advantageous for the brutp-labeling experiments because of low transfection efficiencies of brutp into whole drosophila cells. nevertheless, we were able to obtain some immunogold labeling results using intact drosophila cells, which also showed that spherules were the major sites of brutp-labeling ( figure c ). gold particles in the intermembrane space of the mitochondrion in the lower right are well within the distance ( nm) from spherules that may be spanned by the primary and secondary antibodies linking the immunogold particles to their target epitopes [ ] . having shown that spherules were the sites of protein a accumulation and fhv rna synthesis, we applied -d emt to provide a new level of analysis of spherule morphology and topology. as noted in the introduction, the -d nature of emt overcomes many serious limitations of -d em analysis to reveal possible connections to surrounding membranes and compartments, complete dimensions, and other funda-mental characteristics not accessible from conventional transmission em analyses of random sections. for example, along the z-axis parallel to the electron beam, standard transmission em projects a - -nm section into a single view, whereas emt allows computationally dissecting an entire ; -nm-thick sample volume into successively viewable planes spaced with a resolution of just a few nanometers [ ] . to produce -d reconstructions of fhv-infected cells including modified mitochondria, drosophila s cells were harvested h post infection (hpi) and fixed, embedded, and sectioned as described under materials and methods. for each reconstruction, a tilt series of images was collected by rotating a -nm-thick section of resin-embedded sample in increments between À to þ relative to the plane perpendicular to the beam, and was digitally processed to produce a tomographic reconstruction. using drosophila cells from three independent fhv infection experiments, five independent reconstructions were generated using a singletilt series technique ( figure c - d and additional unpublished data) and one reconstruction was performed using a double tilt technique ( figure a and b) to improve tomographic resolution further [ ] . representative results are shown in the figures. for one such tomogram, figure a shows the image of a computationally dissected, . -nm-thick virtual section, revealing an fhv-modified mitochondrion containing spherules in the mitochondrial intermembrane space. this -d image shows a typical view of randomly sectioned, fhv- modified mitochondria, in which some spherules appear to be light bulb-shaped invaginations attached to the outer membrane by small diameter necks (white arrowheads), whereas others appear to be free vesicles in the intermembrane space (asterisks). figure b shows another virtual section from the same tomogram, displaced down the perpendicular z-axis by ; nm to a point where those spherules that appeared to be free vesicles in figure a (asterisks) now show necked attachments to the outer membrane. to determine if all spherules were attached to the outer mitochondrial membrane, or if a population of spherules budded free of this membrane, we followed individual spherules through dozens of successive . -nmspaced adjacent planes perpendicular to the electron beam (a ''z-series'' of sections). when all six reconstructions were examined in this way, all ; spherules in all ; fhvmodified mitochondria examined were found to be connected to the outer mitochondrial membrane by a membranous neck observable in some plane of the sample. the red arrowhead in figure a points to a channel through the spherule neck that connects the interior of a spherule to the cytoplasm. thus, all spherules are necked invaginations of the outer mitochondrial membrane whose interiors remain connected to the cytoplasm, and sections in which a given spherule appears to be a free vesicle simply represent planes that did not pass through the smaller diameter neck linking the spherule membrane to the mitochondrial outer membrane. this is illustrated more dynamically in video s , which animates the progression through a z-series of sections of the tomogram of figure a and b. figure c - d shows two virtual sections from another tomogram, which are displaced ; nm down the perpendicular z-axis from each other. as shown in a video through this z-series (video s ), mitochondrion curves significantly in the space between these two sections, such that the plane of figure c sections mitochondrion spherules parallel to an axis through the spherule necks, whereas the parallel plane of figure d sections the spherules on another part of the figure , except that anti-bru antibody was used instead of anti-protein a. see figure c for an explanation of the nonstandard mitochondrial morphologies seen in the upper left and lower right of (c). the arrowheads and labeling of mitochondria, spherules, and cytoplasm are as in figure . in interpreting the immunogold localization, note that the primary-secondary antibody complex linking the gold particles to their target epitopes may span up to nm. doi: . /journal.pbio. .g plos biology | www.plosbiology.org september | volume | issue | e mitochondrion surface tangential to the axes through their necks. these two perpendicular views of similar spherules on the same mitochondrion are notable because figure c strongly resembles images of spherules induced by alphaviruses, nodaviruses, etc. [ , ] , whereas figure d resembles images of apparently distinct ''vesicle packets'' described for flaviviruses [ ] . thus, some apparently distinct membrane rearrangements and vesicle structures observed in connection with rna replication by different (þ)rna viruses may represent related structures distinguished in part by the perspective from which they were viewed. to generate -d surface maps of the virus-induced membrane rearrangements associated with fhv rna replication, we manually traced the inner and outer mitochondrial membranes (including spherules) over ; adjacent, . nm-spaced virtual sections of selected tomographic reconstructions, and we used a computer-generated mesh overlay to join these tracings into continuous surfaces ( figure ). figure a shows part of the relationship between the electron density of the mitochondrion in figure a and its -d map, and video s provides a much more dynamic visualization of this relationship and the complete -d map. for clarity, the cytoplasmic faces of outer mitochondrial membranes are colored blue, spherule membranes are white, and inner mitochondrial membranes are yellow. figure b shows a close-up view of a portion of the -d map in figure a that demonstrates the connection of the spherules to the outer mitochondrial membrane. this and other similar maps confirmed as noted above that the spherule membranes (white) are continuous with the outer mitochondrial membrane (blue). figure c is a rotation of figure b that shows a view looking down on the surface of an fhv-modified mitochondrion, with the outer membrane (blue) rendered translucent to reveal the spherules beneath (video s ). the necked channels connecting the interior of each spherule to the cytoplasm (red arrowhead) are clearly visible as circular openings in the outer membrane. for individual spherules in four mitochondria from four cells and three experiments, we measured the interior diameters of these neck channels as the distance between the two lipid bilayers, from inner leaflet to inner leaflet, at the point where the tomographic plane sliced through the center of the neck. the resulting distribution of neck diameters is shown in figure a . the average diameter of the neck channel was . . nm ( figure a ), which is more than large enough to allow import of ribonucleotides and export of rna products (diameter , nm). surface-rendered, -d maps of the two mitochondria from figure c are shown in figure d , illustrating also the inner mitochondrial membrane (yellow). using such surface-rendered maps (figure and other unpublished data), we also measured the interior volume and membrane surface area of spherules. as illustrated in figure c , the spherule volumes spanned a range of ; , to , nm . a range of spherule sizes is seen in figure e , which is a rotated view white arrowheads indicate the necks that connect spherules to the om. asterisks mark two spherules that appear to be free vesicles in (a) but are shown to have necked connections to the outer membrane in (b). a red arrow marks the ; -nm channel connecting a representative spherule interior to the cytoplasm. this red arrow also corresponds to the same spherule and connection as the red arrow in figure b - c. (c and d) images from another tomogram that are displaced from each other in the z-axis by ; nm. note the change in morphology of mitochondrion where spherules that appear to be connected to the outer mitochondrial membrane in (c) appear as a vesicle packet in (d). doi: . /journal.pbio. .g plos biology | www.plosbiology.org september | volume | issue | e of mitochondrion in figure d , with the outer membrane removed. the average spherule interior volume was ; , nm ( figure c ), and the average interior spherule membrane surface area was ; , nm ( figure b ). since both protein a ( figure ) and nascent fhv rna ( figure ) localized predominantly or exclusively to spherules, the relative numbers of protein a, rna replication templates, and spherules could provide important insights into the structure and organization of fhv rna replication complexes. accordingly, we measured the number of molecules of protein a and fhv rnas per cell in drosophila s cells at , , , and hpi with fhv. the numbers of positive-and negative-strand genomic rnas per cell were measured by quantitative northern blotting calibrated with known amounts of in vitro transcripts ( figure a and b) . the number of figure c . (e) a view of mitochondrion from (d) that has been rotated and has had the outer membrane removed to show the range of spherule sizes. doi: . /journal.pbio. .g plos biology | www.plosbiology.org september | volume | issue | e protein a molecules per cell was measured by quantitative western blotting calibrated with known amounts of coelectrophoresed, purified protein a standards ( figure e ). starting before hpi and continuing thereafter in fhv-infected drosophila cells, the primary mode of viral rna synthesis is (þ)rna synthesis from negative-strand rna [(À)rna] templates ( figure a- d ). the number of (þ)rna and (þ)rna per cell increased from ; , molecules of each rna species at hpi to ; - million each by hpi ( figure c ). such (þ)rna products primarily accumulate in the cytoplasm for translation and encapsidation, and only a minor fraction of (þ)rnas fractionate with the membrane-associated rna replication complex (p. van wynsberghe, p. ahlquist, unpublished data). by contrast to positive-strand export and accumulation in the cytoplasm, fhv (À)rnas appear to function only as rna replication intermediates and are completely membraneassociated (p. van wynsberghe, p. ahlquist, unpublished data). (À)rna thus is a key measure of a minimal rna replication complex, because every mature rna replication complex, active in (þ)rna synthesis, must contain at least one (À)rna template. therefore, the number of (À)rnas gives an estimate of the maximal number of replication complexes per cell. (À)rna accumulation plateaued by hpi at ; , copies per cell ( figure d ). (À)rna accumulation increased throughout the first hpi, although more slowly after hpi, reaching ; , molecules per cell by hpi ( figure d ). the number of protein a molecules plateaued by hpi (figure f ), which is consistent with prior results that protein a synthesis occurs early in infection and then declines [ ] . intriguingly, the peak level of protein a was ; million molecules per cell ( figure f ). protein a was thus present at dramatically higher levels than (À) rna templates were. the ratio of protein a to (À)rnas was relatively consistent over all time points examined, with averages throughout infection of and protein a copies per (À)rna and (À)rna , respectively ( figure g ). to understand the organization of the replication complex in relation to the spherules better, we compared the number of spherules per cell with the number of protein a and (À)rna molecules per cell. to measure the number of spherules per cell, we collected fhv-infected drosophila s cells at hpi, processed them for transmission em, and imaged randomly sectioned cell profiles. all spherules in each imaged cell section were counted and divided by the cell section volume, which was calculated by measuring the cell area using imagej (national institutes of health) and multiplying by the effective section thickness (see materials and methods). the number of spherules per cell was calculated by multiplying the resulting density of spherules by the average volume of the almost perfectly round, lm-diameter drosophila s cells [ ] (and our independent, matching measurements). these calculations revealed the average number of spherules per cell at hpi to be ; , , ( table ). the ratio of protein a per cell to spherules per cell revealed that on average, there are ; copies of protein a per spherule (table ) . further comparison to the figure data shows that, on average there are ; (À)rna and ; (À)rna molecules per spherule ( table ). the implications of these results for the organization of replication complexes are considered further in the discussion. to advance understanding of the crucial relationship between (þ)rna viruses and the intracellular membranes on which they replicate their rna genomes, we combined -d ultrastructural imaging with quantitative biochemical data and other results to model the architecture and organization of a nodavirus rna replication complex. immunogold labeling identified virus-induced membranous spherules as the sites of accumulation of the sole fhv rna replication protein, protein a, and of fhv rna synthesis (figures and ) . emt revealed that all fhv spherules maintain an open connection with the cytoplasm with a diameter of ; nm, which is wide enough to allow the exchange of ribonucleotides and rna products (figures c and a ). our stoichiometry measurements further revealed the presence of, on average, copies of the viral replicase protein a and - rna replication intermediates per spherule ( table ) . as discussed further below, these findings have substantial implications for the structure, assembly, and function of the fhv rna replication complex and likely also for the organization of many similar membrane-associated viral rna replication complexes. in addition to advancing understanding of viral replication mechanisms, such insights also should prove valuable for developing additional antiviral strategies or agents. protein a is a transmembrane protein in outer mitochondrial membranes [ ] and is ; % localized within spherules ( figure ). therefore, protein a must line the interior membrane surface of spherules. if protein a is similar to typical globular proteins, its volume would be ; nm , based on the protein a molecular weight of kda [ ] and the average partial specific volume of typical proteins [ ] . if globular, protein a then would have a diameter of ; nm and cover a surface area of ; nm . thus, the average spherule interior membrane surface area of , nm ( figure b ) provides enough space to accommodate at most ; protein a molecules, under a perfect close-packing arrangement. therefore, the measured value of ; protein a molecules per spherule (table ) is near saturation for the spherule interior membrane surface area. we modeled nm-diameter spheres representing protein a adjacent to the membrane surface within a tomographic model of half a typical spherule (figure ) to demonstrate how protein a may pack into the spherules. the resulting near-full occupancy of the interior membrane surface area by protein a (figure ) and the nature of protein a as a transmembrane protein whose self-interaction is required for rna replication [ , ] imply that the ; copies of protein a form an inner network or shell within the spherule ( figure b ). such a shell would explain the formation and maintenance of the high-energy membrane deformation of spherules. a shell of these dimensions appears reasonable, given that the main shell of a reovirus core is nm in diameter and is composed of copies of a slightly larger protein k ( kda) [ ] . the distribution of fhv spherule size spans a defined range of ; - -nm intramembrane diameter, suggesting some flexibility in the assembly of the protein a shell. other examples of highdensity protein shells of flexible size and shape include the capsids of retroviruses, influenza, and retrotransposons [ ] [ ] [ ] . for example, one species of ty retrotransposon forms virus-like capsids that have a - -nm range of diameters, similar to fhv spherules, and contain on average copies of a -amino acid protein subunit [ ] , a protein content very close to the fhv spherule average of copies of amino acid protein a. endocytic vesicles, secretory transport vesicles, and synaptic vesicles are further examples of protein-induced membrane vesicles that each have a range of variable sizes ( - -nm diameter), despite being formed by regular arrays of uniform proteins [ , ] . because ; % of protein a, the fhv rna polymerase (figure ), and ; % of newly synthesized fhv rna ( figure ) are spherule-associated, and essentially all fhv (À)rna templates are membrane associated (p. van wynsberghe, p. ahlquist, unpublished data), it appears likely that (À)rnas, and thus any double-stranded rnas (dsrnas) are within spherules. sequestration of dsrna within such a compartment may allow the virus to avoid, minimize, or delay dsrnainduced host-cell defense responses such as protein kinase, rna activated (pkr) and rnase l [ ] or rna interference (rnai) [ ] . such dsrna localization is consistent with earlier observations of virus-induced membrane spherules containing fibrils with salt-dependent nuclease sensitivity [ , ] . the ; protein a molecules per spherule (table ) would consume ; , nm of interior volume, leaving ; , nm within an average spherule to accommodate fhv rna. based on . nm per hydrated nucleotide for the crystal structure of duplex rna [ , , ] , the volumes of fhv rna , rna , and rna would be , , and nm , respectively. thus, in addition to ; protein a molecules, a spherule of average size has enough interior space to contain at most four single-stranded rna (ssrna) or two dsrna copies of all three fhv rna species. given this maximal occupancy, the estimate from biochemical data of an average of one (À)rna and two (À)rna templates per (table ) , together with at least one nascent (þ)rna progeny strand for each, appears fully reasonable. currently, it is not known if fhv rna and rna are replicated in separate or common spherules. if rna and rna were in separate spherules (i.e., % of spherules containing rna and % containing rna ), then the ratios of (À)rna and (À)rna to total spherules (table ) imply each rna -containing spherule would have two replication intermediates, and each rna -containing spherule would have approximately four replication intermediates. because rna ( . kb) is twice as long as rna ( . kb), the total rna content in both cases then would be nearly equal. if rna and rna were together in the same spherule, then each spherule would hold on average three replication intermediates (one rna and two rna ). the possibility of spherules containing both species of rnas is intriguing, considering the interactions of fhv rnas required for replication: fhv subgenomic rna , which is templated from rna , transactivates rna replication and, in turn, rna replication is suppressed by the resulting progeny rna [ ] . rna , and not its protein product, is responsible for transactivating rna [ ] . however, it is also possible that rna is produced in one spherule during rna replication and then exported to the cytoplasm prior to transactivating rna . membrane spherules similar to those of fhv are induced by many other (þ)rna viruses including alphaviruses [ ] , other members of the alphavirus-like superfamily [ ] , rubiviruses [ , ] , flaviviruses [ ] , tombusviruses [ ] , and others [ , [ ] [ ] [ ] . among these, one of the best-studied with regard to the localization and stoichiometry of rna replication complex components is bmv. bmv and fhv differ in many important respects including that bmv encodes a much larger complement of rna replication proteins [ , ] . nevertheless, although the understanding that we present here for fhv rna replication complexes is more advanced in many ways, the known characteristics of bmv rna replication complexes are strikingly similar to those for fhv. both bmv and fhv induce spherules of similar dimensions where viral rna synthesis and viral replication proteins are localized [ ] . bmv replication protein a, which is sufficient to induce spherules [ ] , is also a strongly membrane-associated [ ] , self-interacting [ , ] protein that is present at high copy number per spherule [ ] . similarly, whereas the ultrastructural organization of hepatitis c virus rna replication complexes has not been defined, recent results suggest that these may also involve a dramatic excess of nonstructural protein copies per (À)rna [ ] . in addition to the many (þ)rna viruses whose rna replication is associated with spherules, other (þ)rna viruses induce various, apparently distinct membrane rearrangements [ , , , , , , ] . although some or most of this variability reflects real ultrastructural differences, at least some of the perceived differences may be due to differences in perspective under conventional -d imaging. our tomography results demonstrated that equivalent fhv spherules appeared to vary in morphology and topological relation to adjacent membranes when viewed in two dimensions from different perspectives (figures c and d) . a greater understanding of the -d nature of membrane rearrangements associated with rna replication by other (þ)rna viruses may reveal shared features or common underlying principles. based on results with bmv, schwartz et al. identified potential parallels between the assembly, structure, and function of membrane-associated rna replication complexes and the cores of reverse-transcribing and dsrna virus virions, including the sequestration of genomic rna templates within a virus-induced compartment for replication [ , ] . the results presented here for fhv validate and extend these parallels by showing that all fhv spherules are figure , the spherule membrane is white and the contiguous outer mitochondrial membrane is blue. (b) schematic of likely protein a organization within a spherule. based on the average density of globular proteins, fhv protein a ( kda) is modeled in as a green sphere of ; nm in diameter. based on the average of ; protein a molecules per spherule (table ) , the figure shows the potential packing arrangement of protein a molecules in half a spherule. the spheres are shown lining the interior surface area of the spherule membrane because protein a is a transmembrane protein [ ] . (c) schematic representation of the structure and organization of the fhv rna replication complex. protein a (ptn a; green spheres) forms a shell within the mitochondrial membrane spherule within which rna synthesis occurs (n ¼ n terminus; c ¼ c terminus). protein a is also shown as possibly extending into the spherule neck, since it may be a determinant of the relatively constant -nm diameter neck. as noted in figure (white arrowheads), a small fraction of protein a may reside on the outer mitochondrial membrane external to spherules. the diagram shows (þ)rna synthesis (red arrow) from (À)rna templates (black segmented line), which is the predominant form of fhv rna synthesis throughout all but the earliest phases of fhv infection ( figure a- d) . doi: . /journal.pbio. .g membrane invaginations topologically equivalent to a budding, enveloped virion (figure ) , and that self-interacting, transmembrane protein a is present at levels sufficient to coat the inner spherule membrane in a multi-subunit shell similar to the capsids of retrovirus and dsrna virus cores ( figure ) . as with dsrna viruses, hepadnaviruses, and retroviruses, the high copy of protein a per spherule suggests that there may be threshold effects in replication protein expression to initiate replication. further analysis of the structure, interactions, and function of fhv rna replication complexes should provide additional insights into the basic mechanisms of (þ)rna virus replication and potentially identify new approaches for antiviral interference. cells and infection protocol. drosophila s cells were grown at c in gibco drosophila serum-free media (sfm). cells were dislodged by gentle scraping, pelleted, and resuspended at cells/ml. fhv was added at a multiplicity of infection of for all experiments. the cells and virus were incubated at c on a rotary shaker at , revolutions per minute (rpm) for h to let the virus attach. after the hour incubation, the cells were plated onto a tissue culture dish and further incubated at c. mitochondria isolation. mitochondria were isolated from drosophila cells as described by echalier [ ] . briefly, cells were recovered by scraping and centrifugation and resuspended in a hypotonic buffer that contained mm n- -hydroxyethylpiperazine-n'- -ethanesulfonic acid (hepes; ph . ), mm egta, and a protease inhibitor cocktail ( mm phenylmethanesulphonylfluoride, lg/ml pepstatin a, lg/ml chymostatin, mm benzamidine, lg/ml leupeptin, and . lg/ml bestatin). after a min incubation at room temperature, an equal volume of double isotonic buffer was added that consisted of the hypotonic buffer plus . m mannitol. the cells were lysed for min using a pre-chilled potter-elvehjem homogenizer fitted with a teflon pestle (kimble-kontes; www.kimble-kontes.com/) and attached to a stirrer motor spinning at rpm. the lysate was transferred to a dounce homogenizer fitted with a type b glass pestle (kimble-kontes) and disrupted manually for strokes on ice. unbroken cells and nuclei were removed by two min centrifugation steps at g at c. mitochondria were pelleted by centrifugation at g for min at c, resuspended in an isotonic buffer containing . m mannitol, and washed by a second centrifugation at g. brutp incorporation on the isolated mitochondria was performed at c for h as described previously [ ] . brutp transfection. drosophila cells were infected with fhv as above. at hpi, cells were treated with lg/ml actinomycin d for min. fugene (roche; http://www.roche.com) was diluted -fold in phosphate buffered saline ph . and mixed with brutp and actinomycin d to final concentrations of mm and lg/ml, respectively. the fugene/brutp/actinomycin d mix was incubated for min at room temperature then added to the cells and incubated at c for min. after the c incubation, the cells were moved to c for a -min labeling period and then immediately fixed and processed for em. monoclonal antibody. mouse monoclonal antibodies against fhv protein a have been described previously [ ] . mab clone - . . . . , which recognizes the protein a epitope between amino acids and , was used for immunogold em labeling. immunogold em labeling . brutp immunolabeling fixation was performed as described previously [ ] , except that samples were embedded in lr gold resin. samples were sectioned and placed on nickel grids. sections were blocked with a goat-blocking solution (aurion; http://www.aurion.nl), and incubated for h with an anti-bru antibody (prb- ; molecular probes; http://probes.invitrogen. com), diluted : in an incubation solution containing mm phosphate-buffered saline ph . and . % bsa-c (aurion). grids were washed six times in incubation solution without antibody, then incubated for h with a goat-anti-mouse antibody conjugated to an ultrasmall gold particle (aurion) that was diluted : in incubation solution, and washed six times again with incubation solution. silver enhancement was performed for min using r-gent se-em (aurion). protein a immunogold em was performed in the same manner using the mouse monoclonal antibody at a dilution of : . background labeling was determined using uninfected control cells. labeling density was determined by calculating the surface area of spherules, mitochondria, and cytoplasm using the point-hit method [ ] . specific labeling was determined by subtracting the background labeling density. northern blot analysis and quantitation of fhv rna. northern blotting was done as described previously [ ] . the number of molecules of fhv rnas was determined by comparison with a serial dilution of a known amount of in vitro transcripts representing a known amount of (þ)rna or (À)rna molecules. rna levels were quantitated with imagequant software (molecular dynamics; http:// www.mdyn.com/). western blot analysis and quantitation of fhv protein a. western blotting was done as described previously [ ] . the number of molecules of protein a was determined by comparison with a purified protein a standard. to generate the standard for quantitation, protein a was expressed in escherichia coli as described previously [ ] . to purify protein a, the hydrophobic transmembrane domain of protein a was deleted (amino acids - ), replaced with a c-terminal his tag, and purified by talon column (clontech; http://www. clontech.com) affinity chromatography. to further purify protein a, we performed preparative electrophoresis using a biorad miniprep cell. a %, . -cm gel was run at v for h with an elution speed of ll/min. fractions containing the purified, truncated protein a standard were collected and quantitated based on comparison with known standards of bovine serum albumin and bgalactosidase. we quantitated protein levels with lumi-imager software (roche). em. for conventional transmission em, cells were fixed and embedded as previously described [ ] . for electron tomography, cells were fixed in % parafomaldehyde and . % glutaraldehyde in . m sodium cacodylate, ph . , post-fixed in % osmium tetroxide with . % potassium ferrocyanide in sodium cacodylate buffer, stained with % uranyl acetate, dehydrated in a graded series of ethanol, and embedded in durcupan acm resin. to calculate the total number of fhv-induced mitochondrial spherules per cell, fhv-infected drosophila s cells were collected at hpi, processed for transmission em, and sectioned into -nmthick slices. for each of randomly selected cells imaged in these sections, we then counted all observable spherules with diameters larger than nm. the number of spherules counted for each cell then was divided by the relevant section volume, which was calculated by measuring the cell area using imagej (national institutes of health) and multiplying by the effective section thickness. the effective section thickness is a correction used to avoid overcounting spherules with centers outside of the -nm physical section, which would otherwise be counted twice if adjacent sections were analyzed. as previously used to calculate synaptic vesicles per cell [ ] , this effective section thickness is the thickness that would encompass the centers of all counted spherules. in this case, the effective section thickness was nm, based on adding nm (the distance from the spherule center to a radius-perpendicular plane bisecting the spherule to yield a -nm-diameter section) to each face of the nm section. the number of spherules per cell was calculated by multiplying the resulting density of spherules by the average volume of the almost perfectly round, lm-diameter drosophila s cells [ ] (and our independent, matching measurements). emt. three separate fhv infections produced samples for six independent tomograms. to survey the preservation quality and fhv-infection efficiency of the drosophila cells, thin-sectioned material (; nm thick) was examined using a jeol fx electron microscope. -d reconstructions of portions of the cell containing fhv-infected mitochondria were generated using current techniques of electron tomography [ ] . sections were cut with a thickness of ; nm from blocks exhibiting well-preserved ultrastructure. these sections were stained for min in % aqueous uranyl acetate, followed by min in lead salts. fiducial cues consisting of -nm colloidal gold particles were deposited on both sides of the section. for each reconstruction, a series of images was collected with a jeol ex intermediate-voltage electron microscope operated at kv. the specimens were irradiated before initiating a tilt series in order to limit anisotropic specimen thinning during image collection. pre-irradiation in this manner subjected the specimen to the steepest portion of the nonlinear shrinkage profile before images were collected. six tilt series were collected: five single-tilt and one doubletilt. ''fhv '' was the highest resolution single-tilt reconstruction and ''fhv '' was the high-resolution double-tilt reconstruction; the majority of the analyses were conducted on these two reconstructions. the single-tilt series were recorded at , magnification with an angular increment of from À to þ about an axis perpendicular to the optical axis of the microscope using a computer-controlled goniometer to increment accurately the angular steps. these single-axis tilt series were collected using a ccd camera with pixel dimensions , , . the pixel resolution was . nm. the illumination was held to near parallel beam conditions and optical density maintained constant by varying the exposure time. the imod package was used for generating the reconstructions [ ] . double-tilt tomography was performed by first collecting two tilt series of the same cellular region around orthogonal axes. after the first tilt series was complete using an angular increment of from À to þ , the specimen grid was rotated , and the second tilt series was acquired from À to þ . the imod software suite was used for fiducial mark tracking and alignment. the positions of gold particles were tracked in both tilt series. after alignment, the tomographic reconstruction was generated by a projective algorithm [ ] . volume segmentation was performed by manual tracing in the planes of highest resolution with the program xvoxtrace [ ] . the mitochondrial reconstructions were visualized using analyze (mayo foundation, rochester, mn, united states), imagej (national institutes of health), or the surface-rendering graphics of synu (national center for microscopy and imaging research, san diego, ca, united states) as described by perkins et al. [ ] . these programs allow one to step through slices of the reconstruction in any orientation and to track or model features of interest in three dimensions. measurements of structural features were made from planes within the reconstructed volume with the program imagej (national institutes of health) or within segmented volumes by the programs synuarea and synuvolume (national center for microscopy and imaging research). some -d maps, images, and videos were created using the software amira (mercury tgs; http://www.tgs. com). virus taxonomy cowpea mosaic virus -and -kilodalton replication proteins target and change the morphology of endoplasmic reticulum membranes expression of hepatitis c virus proteins induces distinct membrane alterations including a candidate viral replication complex rna replication of mouse hepatitis virus takes place at double-membrane vesicles biogenesis of the semliki forest virus rna replication complex markers for trans-golgi membranes and the 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plant cells infected by cowpea chlorotic mottle virus cytopathic structures associated with tonoplasts of plant cells infected with cucumber mosaic and tomato aspermy viruses open reading frame -a-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex identification of the hepatitis c virus rna replication complex in huh- cells harboring subgenomic replicons alphavirus rna replicase is located on the cytoplasmic surface of endosomes and lysosomes intracellular distribution of rubella virus nonstructural protein p electron tomography serial section electron tomography: a method for three-dimensional reconstruction of large structures golgi structure in three dimensions: functional insights from the normal rat kidney cell tomographic evidence for continuous turnover of golgi cisternae in pichia pastoris er-to-golgi carriers arise through direct en bloc protrusion and multistage maturation of 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large, multicomponent biological structures electron tomography of mitochondria after the arrest of protein import associated with tom depletion we thank randall massey and benjamin august of the university of wisconsin medical school electron microscopy facility for assistance with electron microscopy; priscilla van wynsberghe and billy dye for helpful discussions; dan lautenschlager, steve lamont, and jean yves-sgro for computer assistance; and johan den boon for critical reading of the manuscript.author contributions. all authors conceived and designed the experiments and analyzed the data. bgk, gp, and djm performed the experiments. bgk and pa wrote the paper.funding. this work was supported by nih grants gm to pa and p rr to mhe. pa is an investigator of the howard hughes medical institute.competing interests. the authors have declared that no competing interests exist. key: cord- -eo vjcmk authors: kielian, margaret; rey, félix a. title: virus membrane-fusion proteins: more than one way to make a hairpin date: journal: nat rev microbiol doi: . /nrmicro sha: doc_id: cord_uid: eo vjcmk structure–function studies have defined two classes of viral membrane-fusion proteins that have radically different architectures but adopt a similar overall 'hairpin' conformation to induce fusion of the viral and cellular membranes and therefore initiate infection. in both classes, the hairpin conformation is achieved after a conformational change is triggered by interaction with the target cell. this review will focus in particular on the properties of the more recently described class ii proteins. enveloped animal viruses are covered -or 'enveloped' -by a lipid bilayer that is derived from the host-cell membrane during virus budding. this membrane protects the genetic material of the virus until it is delivered into the cytoplasm of a new host cell to initiate infection. delivery takes place by fusion between the virus membrane and the membrane of the host cell. this membrane-fusion reaction is a crucial step in virus infection and is mediated by transmembrane (tm) proteins that are anchored on the virus surface. virus membranes are relatively simple compared to hostcell membranes, and the process of virus-host-cell fusion is devoid of the additional regulatory and recycling proteins that are present in many cellular fusion processes , . therefore, understanding the molecular mechanism of membrane fusion catalysed by viral membrane-fusion proteins is important not only for understanding virus infection, but also as a paradigm for cellular membrane-fusion reactions. virus membrane fusion can take place either at the plasma membrane or at an intracellular location following virus uptake by endocytosis , . the fusion reactions of viruses that fuse directly at the plasma membrane are triggered by virus-receptor interactions at neutral ph, as discussed below. by contrast, the fusion of many other viruses is dependent on their internalization by receptor-mediated endocytic pathways such as clathrin-dependent, caveolae-dependent uptake or nonclathrin-dependent, non-caveolae-dependent uptake , . viruses that use such routes frequently have fusion reactions that require exposure to mildly acidic ph within organelles of the endocytic pathway . although it is unclear why viruses use such a variety of entry pathways, it is possible that those viruses that have evolved clathrin a cellular protein that is composed of three heavy chains and three light chains. clathrin is the main component of the coat that is associated with clathrin-coated vesicles, which are involved in membrane transport in both the endocytic and biosynthetic pathways. specialized regions that contain the protein caveolin and form flask-shaped, cholesterol-rich invaginations of the plasma membrane. to fuse intracellularly gain a selective advantage from releasing their genomes at specific intracellular sites . virus membrane-fusion proteins drive the fusion reaction by undergoing a major conformational change that is triggered by interactions with the target cell. the specific trigger depends on the virus. for example, influenza viruses, alphaviruses and flaviviruses are classic examples of viruses that fuse upon exposure to low ph in the endocytic pathway or in the test tube , , , . by contrast, fusion of hiv- occurs at neutral ph and is triggered by the sequential interaction of the virus fusion protein with the receptor cd and a co-receptor such as ccr or cxcr , members of the -tm-domain chemokine receptor family , . other variations include viruses with fusion reactions which seem to be triggered by interaction with a single receptor , by the binding of the receptor to a separate attachment protein , by a combination of receptor binding plus low ph , , or by endosomal proteolysis . given the recently described variations on the pathways of fusion-protein activation, it is probable that there are additional interesting twists to be discovered. to date, two classes of virus membrane-fusion proteins (fig. ) have been defined, based on key structural features, as detailed below. viruses that are currently assigned to class i or class ii are listed in table . it is clear that for many viruses, the identity and/or functional features of the fusion protein have not yet been determined, making it difficult to classify them as class i or class ii, or perhaps even as the inaugural members of a new fusion-protein class (box ) . this review will focus primarily on recent developments in the structure and function of the class ii fusion proteins (fig. ) , using the alphavirus e glycoprotein as a paradigm. class i proteins will be introduced first a second receptor required for virus infection. in the case of hiv- , fusion is triggered by sequential interaction of viral gp with the cd receptor, followed by a co-receptor. cytokines involved in specific inflammatory responses. they are differentiated into cc or cxc chemokines on the basis of their primary sequence. a single-pass transmembrane protein that contains an n-terminal external domain and a c-terminal cytoplasmic domain. to establish our current understanding of these fusion proteins, as they provide important context for the studies of the class ii proteins. structure and function. the class i membrane-fusion reaction is mediated by the refolding of the fusion protein to a highly stable rod-like structure with a central trimeric α-helical coiled coil. such coiled-coil structures are emblematic of class i proteins, and physical demonstration or computer prediction of such a structure is frequently used to help define a fusion protein as belonging to class i . the class i proteins generally share several other important features, illustrated here by the example of the influenza-virus haemagglutinin (ha) and summarized in table and in refs , , . ha is a trimeric type tm protein that contains both the receptor-binding and fusion activities of influenza virus. it is synthesized as a fusion-inactive precursor, ha , which is processed by host-cell proteases to produce two disulphide-bonded subunits -ha ( amino acids), containing the receptor-binding site, and ha ( amino acids), the tm subunit responsible for fusion (fig. ) . ha is maintained in a 'metastable' state at the virus surface , . the n terminus of ha contains a conserved hydro phobic region known as the fusion peptide which inserts into the target membrane during fusion. the structure of the ha ectodomain reveals a trimer with a large globular head region that is composed mainly of ha , and a long α-helical coiled-coil stalk region . after processing, the overall structure of ha is almost unchanged, but the n-terminal fusion peptide of ha becomes buried at the trimer interface within the α-helical stalk (fig. ) . virus fusion is triggered by low ph, which destabilizes the ha trimer contacts at the head, causing the globular head domains to dissociate. this movement allows a loop-to-helix transition of a polypeptide segment of ha that was previously buried underneath the ha heads, projecting the fusion peptide ~ Å towards the target membrane, where it inserts irreversibly . this initial change is proposed to result in a 'pre-hairpin intermediate' , an extended structure that is anchored both in the target membrane by the fusion peptide and in the virus membrane by the tm segment . the c-terminal end of the long ha α-helix jackknifes back, reversing the direction of the viral-membrane-proximal segment of ha , which then interacts in an anti-parallel fashion with the groove formed by the n-terminal trimeric coiled coil . the final post-fusion conformation of ha is therefore a highly stable rod with the tm and fusion-peptide segments together at the same end of the molecule (fig. ) , a structure termed a 'trimer of hairpins' . the stability of the post-fusion conformation of ha is considerably higher than that of the metastable prefusion form, and class i membrane fusion is believed to be driven by the energy that is released during the conformational change , , , . hairpin formation by ha is postulated to force the target and virus membranes into close apposition to trigger fusion, which occurs by the initial mixing of the outer lipid leaflets (termed 'hemifusion'), followed by mixing of the inner leaflets, opening of a small fusion pore, and widening of the pore to give complete fusion. membrane insertion. viral fusion peptides generally tend to be apolar regions, conserved within a virus family, relatively rich in glycine and alanine residues, and containing several bulky hydrophobic residues . for influenza ha and several other class i proteins, the fusion peptide is located at the n terminus of the tm subunit, but other class i fusion peptides are located internally in the amino-acid sequence, and presumably insert into the membrane as a loop. structural studies of the ha fusion peptide indicate that it adopts a boomerangshaped structure formed by a kinked amphipathic α-helix lying roughly in the plane of the outer leaflet of the membrane at the polar-aliphatic interface . membrane insertion seems to be primarily regulated by the initial release of the fusion peptide from its buried position to permit its amphipathic interaction with lipid bilayers. cooperativity. fusion by ha is positively affected by protein density, suggesting cooperative ha interactions during fusion , . intriguingly, studies of cell-cell fusion mediated by influenza ha suggest that 'bystander' ha molecules outside the zone of cell-cell contact have effects on expansion of the fusion pore . the mechanism by which ha might interact during fusion and the role of bystander fusion proteins are not well understood. structural information on class i fusion proteins has suggested several approaches for inhibiting fusion-protein refolding and membrane fusion , . for example, the hiv- inhibitor t /enfuvirtide is a peptide that corresponds to part of the c-terminal helix of the envelope glyco protein gp (refs , ) . t blocks the interaction of the c-terminal segment of the fusion protein with the groove of the central coiled coil (fig. ) , and blocks virus fusion and infection. importantly, small molecules that target crucial sites of interaction in the trimer of hairpins have also been shown to act as potent class i fusion inhibitors . as small molecules can have higher bioavailability than peptides, they provide an important approach to develop more widely useful fusion inhibitors. although, to date, the class i inhibitors have mainly targeted viruses such as hiv- that are triggered at the cell surface, small molecules can also target viruses that fuse within endocytic compartments , . the alphaviruses and flaviviruses are members of the togaviridae and flaviviridae families, respectively , . these small spherically shaped viruses are composed of a nucleocapsid core that contains the single-stranded positive-sense genomic rna, surrounded by a membrane that contains the viral tm glycoproteins, which form an external icosahedral scaffold (fig. ) . the x-ray structures of the fusion proteins of the alphaviruses and flavi viruses, described below, revealed that the ectodomains of these proteins have remarkably similar secondary and tertiary structures. this result strongly suggests that, despite the lack of any detectable amino-acid-sequence similarity, the corresponding genes were derived from a common ancestor. the three-dimensional structure of these fusion proteins is radically different from that of the influenza virus ha, a finding that introduced the concept of a separate class of fusion proteins, class ii (table ) . class i and class ii fusion proteins share common features of target-membrane insertion of a hydrophobic fusion peptide and refolding/hairpin formation to drive the membrane-fusion reaction, whereas their structural characteristics are markedly different. what other classes of viral fusion proteins might there be? one possibility would be proteins that mediate fusion using the same general mechanism but with a structural basis that belongs to neither class i nor class ii. for example, rhabdoviruses such as vesicular stomatitis virus and rabies virus have a single homotrimeric membrane protein, g, which mediates fusion in a low-ph-dependent reaction that involves membrane insertion of a hydrophobic region of g and biochemically detectable conformational changes (reviewed in ref. ). however, unlike the class i and class ii proteins discussed in this review, g is not proteolytically processed or synthesized with a companion protein, the g-protein conformational changes induced by the fusion trigger (low ph) are reversible and, as yet, no clear structural similarities to class i or class ii proteins have been identified. it is also probable that different overall mechanisms of fusion remain to be defined. for example, herpesvirus fusion involves several virus membrane proteins and might represent a different model from the single fusion proteins of class i and class ii (reviewed in ref. ). likewise, recent data indicate that, in the case of the poxviruses, four proteins interact to induce fusion at low ph (see ref. and references therein). the many open questions on the fusion of complex viruses such as herpesviruses and poxviruses will continue to be important and interesting topics in the future. the non-enveloped viruses of the orthoreovirus genus include some viruses that cause cell-cell fusion of infected cells. this fusion activity is due to non-structural membrane proteins termed fast proteins (fusion-associated small transmembrane proteins), which range in size from ~ - kda . the ectodomains of fast proteins are small, in some cases comprising only ~ residues, and can contain short hydrophobic regions and/or n-myristic acid , . although their small size argues against hairpin formation, fast proteins are sufficient to mediate membrane fusion , and it will be important to determine how they fit into our growing knowledge of the mechanisms of protein-mediated membrane fusion. small opening that allows flux between two membranebound compartments. fusion pores form at an early stage of membrane fusion and widen when they lead to full fusion. biosynthesis and assembly. during biosynthesis, the alphavirus (e ) and flavivirus (e) fusion proteins fold co-translationally with a companion or regulatory protein, termed p (or pe ) for alphaviruses and prm for flaviviruses , (fig. ) . this heterodimeric interaction is important for the correct folding and transport of the fusion protein. both p and prm are cleaved by the cellular protease furin late in the secretory pathway, in a maturation reaction that is a crucial regulatory step for subsequent virus fusion [ ] [ ] [ ] [ ] . one important difference between these two groups of viruses is the budding site , . in alphaviruses, the p -e complex is transported to the plasma membrane, and the heterodimer interaction is maintained after p processing. new virions bud at the plasma membrane, in a process that is driven by lateral contacts between e -e heterodimers (e being the mature companion protein) to induce the required curvature of the lipid bilayer, in combination with interactions of the cytosolic tail of e with the nucleocapsid. budding results in formation of icosahedral enveloped particles of triangulation t = , containing trimeric e /e spikes , . by contrast, flavivirus particles bud into the endoplasmic reticulum as immature virions formed by trimers of prm-e. the immature particles have an organization similar to mature alphaviruses, with each trimer forming a spike in which prm covers the fusion protein e . the newly formed virions are then transported to the external milieu through the exocytic pathway. processing of prm generates the mature m protein with a short (~ residues) ectodomain. presumably because of the removal of a large portion of the prm ectodomain, the flavivirus surface dramatically reorgan izes after processing to give e-e homodimers arranged with icosahedral symmetry , [ ] [ ] [ ] . the mature flavivirus particles display a smooth, spikeless surface, with e dimers ordered in a characteristic 'herring bone' pattern. structure. crystal structures have been determined for the neutral ph ectodomains of the fusion proteins from the alphavirus semliki forest virus (sfv) , and the flaviviruses tick-borne encephalitis virus (tbe), dengue virus, and dengue virus - . the polypeptide chain of the class ii proteins follows a complex path, resulting in three globular domains -essentially constituted by β-sheets -organized so that the c terminus and the fusion peptide are found at the two ends of a rod-like molecule of ~ Å in length (fig. ) . domain i, which contains the n terminus and a conserved glycosylation site, is a β-barrel with an 'up-and-down' topology. two of the connections between adjacent strands in this barrel are long and elaborated, and comprise the 'finger-like' domain ii with the fusion loop at the tip of the molecule. domain iii, which lies at the opposite end of domain i, has an immunoglobulin-superfamily fold and is connected to the c terminus of domain i by an ~ amino-acid polypeptide. the tm domain of sfv e and the two tm domains of tbe were proteolytically removed to generate the soluble ectodomains. the sfv e ecto domain, referred to here as e *, retains part of the 'stem' region that connects domain iii to the tm domain, although this flexible region is not visualized in the neutral ph structure. the stem regions of the flavivirus fusion proteins have been completely removed in the crystallized forms. conformational changes during fusion. unlike the class i fusion proteins, which are trimeric in both their pre-fusion and post-fusion conformations, class ii fusion proteins undergo an oligomeric rearrangement during fusion [ ] [ ] [ ] , converting from the metastable prefusion dimer to a considerably more stable homotrimer conformation , (table ) . formation of the targetmembrane-inserted homotrimer is required for class ii virus fusion , . binding of protons in the acidic endosomal environment triggers a complete rearrangement of the surface of the class ii virus particles. the alphavirus e -e heterodimers or the flavivirus e-e homodimers dissociate , , resulting in disassembly of the icosahedral scaffold followed by a quaternary reorganization into e or e homotrimers inserted in the membrane by the fusion loops. importantly, as discussed below, the homotrimers seem to display lateral contacts between each other, forming a different type of surface lattice. in vitro studies using the ectodomains of both the alphavirus and flavivirus proteins showed that trimerization requires insertion of the fusion peptide into target membranes , . solubilization of the trimers from the liposomes using non-ionic detergent led to their crystallization , and to determination of their threedimensional structure [ ] [ ] [ ] . the crystal structures of the class ii homotrimers showed a fold-back arrangement strikingly reminiscent of that of class i fusion proteins, despite the different architecture of the proteins (fig. ) . the trimer is formed by a central parallel interaction of the subunits through roughly the n-terminal two-fifths of the molecule (including domains i and ii, with the fusion peptide at one end). the remaining c-terminal portion of the protein folds back along the sides of the core trimer, with domain iii moving ~ - Å towards the fusion loop. the portion of the stem region that is present in the sfv e protein interacts closely with the trimer core and extends towards the fusion loop. this hairpin organization is therefore the same as that observed for class i fusion proteins, resulting in a trimeric protein rod with the fusion peptide loops and the c-terminal membrane anchors together at the same end. (fig. ) . monoclonal antibodies to this region have been used to show that the fusion loop inserts into membranes and that insertion is required for fusion , , . the fusion loop is composed of relatively apolar and conserved residues, and fusion is blocked by substitution of negatively charged amino acids into this crucial region , . the receptor-binding subunit, e , is displayed as a sphere-filling model coloured grey, based on subtracting the e density from the cryo-electron microscopy reconstruction of the virus. the orange e fusion peptide (arrows) is buried at the e -e interface. c | shows the pre-fusion complex (e) of the flavivirus tick-borne encephalitis virus. the fusion peptide is buried at the e homodimer interface (arrows). all three panels are drawn at the same scale. the viral membrane would be tangential to a horizontal plane below the drawings. figure prepared using the ribbons program . ha e e interestingly, the process of insertion of the alphavirus fusion peptide into the target membrane seems to differ from that of the class i proteins. for example, although heterodimer dissociation exposes the sfv fusion peptide, this alone does not seem sufficient for membrane insertion. the fusion loop is solvent-accessible in the ph- form of the e * ectodomain . the crystal structure shows that in the monomeric form, the e fusion loop (which is longer than its flavivirus counterpart) folds back on itself, burying most of the bulky non-polar side chains. its insertion into the target membrane apparently requires specific triggering by low ph and target-membrane cholesterol , . this agrees with the cholesterol dependence of alphavirus membrane fusion, as discussed below. once e * has stably inserted into the target membrane, it then requires detergent for solubilization, similar to the membrane-inserted class i proteins. the structure of the sfv detergentsolubilized e * homotrimer shows that the fusion peptide unwinds, and the bulky non polar side chains are exposed, presumably within a detergent micelle in the crystal. liposome experiments show that alphavirus fusion is promoted by the presence of cholesterol and sphingolipid in the target membrane [ ] [ ] [ ] . flavivirus-liposome fusion is also enhanced by cholesterol, although the stringency of the requirement seems considerably less than that of the alphaviruses , . for both viruses, the action of cholesterol is more specific than its bulk effects on membrane fluidity or 'raft' formation, with the sterol β-hydroxyl group being particularly important for promoting membrane insertion, trimerization and fusion. interestingly, following membrane insertion, the sfv e * ectodomain is strongly associated with cholesterol-rich membrane microdomains . this could reflect a physical interaction of e with cholesterol, thereby coupling membrane insertion to the e conformational changes during fusion. in vivo studies show that membrane fusion and infection of the alphaviruses sfv and sindbis virus are decreased by - logs in cholesterol-depleted insect cells , . this system was used to select for sfv mutants that have increased cholesterol independence , . these srf (sterol requirement in function) mutants have single amino-acid changes in e , located in the ij loop at the tip (the srf- p s mutation) or in the hinge region (the srf- l f and srf- v a mutations) of domain ii (fig. ) . the molecular mechanisms by which the srf mutations alter alphavirus cholesterol dependence are not yet known. for srf- , the location in the ij loop suggests that the mutation could directly modulate the cholesterol dependence of the adjacent fusion loop. for srf- and srf- , the location in the hinge area suggests that these mutations might affect the inherent flexibility of this region, indirectly altering the angle of interaction of the fusion loop with the target membrane. to demonstrate the suggested membrane interactions of the proteins, the final fused membrane is diagrammed in cartoon form. the class i proteins are drawn such that the n-terminal half of the hairpin is blue and the c-terminal part is red, with the missing fusion peptides and transmembrane domains indicated for one of the three trimer subunits as blue and red stars, respectively. for ha , the residues are coloured as in the neutral ph form displayed in fig. , and a membrane-proximal extended c-terminal 'leash' interacts with the central coiled coil of ha. in hiv glycoprotein (gp ), the post-fusion structure is a six-helix bundle. the inhibitor t interacts at the site where the red helix is, effectively inhibiting the fusogenic conformational change. for the class ii proteins, an elongated red star indicates the predicted location of the tm segment, with a red arrow pointing to the c terminus of one of the subunits in the trimer of the crystallized class ii ectodomains, roughly showing the path of the missing 'stem' regions to complete the protein hairpin. figure prepared using the ribbons program . in addition to being the site of the srf- mutation, several features of the alphavirus ij loop indicated its possible importance in the membrane-fusion reaction. although the e homotrimer is highly resistant to proteolysis, under defined conditions protease cleavage sites were identified around the fusion loop . such protease cleavage releases the e * homotrimer from the target membrane. other than the cleavage in the fusion-loop region, the only other cleavage site was immediately n-terminal to histidine (h ) in the ij loop. sequence comparisons showed that, although the overall sequence of the ij loop is not highly conserved, a histidine residue is found in all of the reported alpha virus and flavivirus sequences . the role of h in sfv fusion and infection was addressed by constructing an h a mutation in the virus infectious clone . the resulting mutant virus is not infectious and is completely blocked in membrane fusion, including the initial steps of lipid mixing. however, h a virus undergoes apparently normal conformational changes upon exposure to low ph, including heterodimer dissociation and fusion-loop exposure, cholesterol-dependent target-membrane insertion, and formation of the e homotrimer. therefore, the h a mutation demonstrates a crucial role for this residue within the sfv ij loop, and identifies a novel late-stage inter mediate in the class ii fusion pathway. two important observations indicate that class ii trimers interact cooperatively during membrane insertion and fusion. first, electron-microscopy studies of the membrane-inserted ectodomains of sfv and tbe show that insertion is highly cooperative and produces characteristic arrays of trimers organized in rings of five or six to form a lattice covering the liposome surface , (fig. ) . liposomes fully decorated with inserted sfv e * display a fairly uniform diameter of ~ nm, considerably smaller than the mean diameter of control liposomes without the protein. the surface-protein lattice therefore seems to control the curvature of the coated liposomes. because hexagonal arrays of e * trimers result in a flat lattice, as shown in fig. , it is probable that rings of five e * trimers are required to introduce the necessary overall curvature to make a fullerene-like , , roughly spherical closed lattice at the liposome surface. second, the packing of sfv e * trimers in the threedimensional crystals largely occurs through two-fold interactions between the fusion loops of adjacent trimers . the trimer axes and the two-fold axis relating them intersect in space, making an angle close to the angle between three-fold and two-fold axes in an icosahedron. as in an icosahedron, the combination of these two operations (three-fold and two-fold about the corresponding symmetry axes) generates a five-fold axis also intersecting the three-fold and two-fold axes at the same point in space (at the centre of the body). the trimers in the crystals therefore interact through the fusion peptide in such a way that the contacts can be propagated -by repeating the observed trimer-trimer contacts through the fusion loops -to generate a closed ring of five e * trimers, all associated by their fusion loops, just like the rings observed by electron microscopy. the fusion peptides in the five-fold rings form a 'crater' ~ Å in diameter and Å deep, with a base composed of five fusion loops and a rim composed of the remaining ten fusion loops. as all three fusion loops in each trimer are believed to interact tightly with the lipid heads, the resulting arrangement of trimers is such that it creates a dome-like distortion in the target membrane, which is postulated to be important in fusion, as shown in the model in fig. . therefore, electron-microscopy data and the crystal contacts provide support for two types of cooperative homotrimer interactions, producing rings of five and six trimers. we hypothesize that during fusion, five-fold interactions will occur between target-membraneinserted trimers at the side of the particle closest to the cell membrane. such five-fold interactions would act at the fusion site to induce formation of a 'dome' in the target lipid bilayer, whereas the refolding of e to a hairpin would pull the tm regions toward the centre of the five-fold ring, forming an opposing dome in the viral membrane and inducing hemifusion (fig. ) . this 'lipid stalk' model for fusion is comparable to that proposed for the class i proteins , and in keeping with recent studies which show that alphavirus fusion progresses through a transient hemifusion intermediate . the six-fold trimer interactions might occur between trimers that do not directly interact with the target membrane and insert instead into the virus membrane. the more planar interactions of such trimers could have a role in the enlargement of the initial fusion pore by providing energy to flatten the viral membrane . although these models are currently speculative, they provide a starting point for thinking about the geometry of the fusion reaction. the left panel shows a negatively stained sample of e ectodomain homotrimers (e *ht) reconstituted at a low lipid-to-protein ratio to produce a planar hexagonal lattice. on liposomes, the e *ht is proposed to display a fullerene-like architecture, in which insertion of rings of five instead of six trimers allows the required curvature to form a closed sphere (see text). images of the planar lattice such as that shown on the left were used to generate a three-dimensional reconstruction into which the atomic model of the e *ht was fitted (right panel). direct lateral interactions between the trimer heads are observed from the fitting, as shown in this top view. interactions between the fusion loops of adjacent trimers could also occur through adjustment of the hinge region (indicated in fig. a) . figure how general is cooperativity? as discussed above, there is considerable evidence for fusion-protein cooperativity for members of the class i fusion proteins, particularly for the low-ph-induced influenza ha. interestingly, however, recent data indicate that some class i virus fusion reactions might be mediated by as few as one or two trimers. antibody-neutralization studies of hiv indicate that the target of neutralization is one trimer , and detailed studies using viruses containing mixtures of wild-type and dominant-negative envelope proteins showed entry of viruses containing a single active hiv env trimer . other class i viruses where fusion is triggered by receptor binding (murine leukemia virus) or by receptor plus low ph (avian sarcoma/leukosis virus) also showed entry properties that are suggestive of fusion mediated by a single trimer. by contrast, parallel studies of influenza ha fusion supported a requirement for eight or nine trimers . further investigations will be required to determine if viruses with fusion directly triggered by low ph generally require multiple fusion proteins, the importance of concerted and cooperative interactions of those proteins, and whether fusion proteins with triggers that involve receptor binding typically activate fusion autonomously. implications and future directions studies of the class i and class ii fusion proteins indicate that, although their structures are very different, both classes refold during fusion to give analogous hairpin conformations, with the fusion peptides or loops and the tm domains at the same end of a stable protein rod. these observations indicate that the overall mechanism of membrane fusion is probably the same for both class i and ii, indicating that there might be a universal membrane-fusion mechanism, applicable to proteins that are clearly not homologous. although the structural and functional studies summarized here suggest an overall picture for class ii fusion, many important questions remain and new questions can now be identified. for example, we do not understand the mechanism by which the class ii fusion loop inserts into the target membrane, or the role of cholesterol in insertion. the current data indicate that insertion and fusion can be modulated by the ij loop and hinge region, but their mechanistic effects are unclear. we have a picture of the fusionprotein ectodomain before and after fusion, but we do not know how the fusion loop and tm domains are disposed in the fused membrane or how they might interact with each other. a series of conformational changes in class ii fusion proteins can be proposed (fig. ) , but their order, kinetics and role in each of the steps of membrane fusion remain to be elucidated. we also do not understand how the refolding of the fusion protein to the trimer conformation takes place in the context of the whole virus particle, the fate of the companion protein (e in the case of alphaviruses) during this particle reorganization, and the role(s) of the observed five-fold and six-fold trimer interactions in the overall membrane-fusion process. an important question is whether the structure of the class ii fusion proteins can allow the development of inhibitors of specific steps in fusion. recent work indicates that exogenous domain iii blocks class ii membrane fusion and infection by binding to the fusion protein during the low-ph-induced conformational change . similar to the class i proteins, such inhibitors could prove important for dissecting the molecular mechanism of fusion and, ultimately, for developing novel antiviral strategies. clearly, there is much exciting work to be done, and we look forward to future developments and discussions on the fusion of the class ii viruses. membrane fusion intracellular and viral membrane fusion: a uniting mechanism the many mechanisms of viral membrane fusion proteins how viruses enter animal cells viral entry on the entry of semliki forest virus into bhk- cells sfv infection in cho cells: cell-type specific restrictions to productive virus entry at the cell surface changes in the conformation of influenza virus hemagglutinin at the ph optimum of virus-mediated membrane fusion structures and mechanisms in flavivirus fusion hiv- entry cofactor: functional cdna cloning of a seven-transmembrane, g protein-coupled receptor mechanisms of viral membrane fusion and its inhibition paramyxovirus fusion: a hypothesis for changes retroviral entry mediated by receptor priming and low ph triggering of an envelope glycoprotein sequential roles of receptor binding and low ph in forming prehairpin and hairpin conformations of a retroviral envelope glycoprotein endosomal proteolysis of the ebola virus glycoprotein is necessary for infection a spring-loaded mechanism for the conformational change of influenza hemagglutinin receptor binding and membrane fusion in virus entry: the influenza hemagglutinin mechanism of membrane fusion by viral envelope proteins conformational changes in the hemagglutinin of influenza virus which accompany heat-induced fusion of virus with liposomes influenza hemagglutinin is spring-loaded by a metastable native conformation structure of the hemagglutinin precursor cleavage site, a determinant of influenza pathogenicity and the origin of the labile conformation structure of the haemagglutinin membrane glycoprotein of influenza virus at Å resolution first structure of a viral membrane-fusion protein first structure of the 'post-fusion' conformation of a viral membrane-fusion protein evidence that the transition of hiv- gp into a six-helix bundle, not the bundle configuration, induces membrane fusion first demonstration that class i fusion is driven by hairpin formation membrane fusion machines of paramyxoviruses: capture of intermediates of fusion membrane structure and fusion-triggering conformational change of the fusion domain from influenza hemagglutinin membrane fusion mediated by the influenza virus hemagglutinin requires the concerted action of at least three hemagglutinin trimers synchronized activation and refolding of influenza hemagglutinin in multimeric fusion machines influenza hemagglutinins outside of the contact zone are necessary for fusion pore expansion the entry of entry inhibitors: a fusion of science and medicine a synthetic peptide from hiv- gp is a potent inhibitor of virus-mediated cell-cell fusion potent suppression of hiv- replication in humans by t- , a peptide inhibitor of gp -mediated virus entry targeting a binding pocket within the trimer-of-hairpins: small-molecule inhibition of viral fusion molecular mechanism underlying the action of a novel fusion inhibitor of influenza a virus structurebased identification of an inducer of the low-ph conformational change in the influenza virus hemagglutinin: irreversible inhibition of infectivity the fusion glycoprotein shell of semliki forest virus: an icosahedral assembly primed for fusogenic activation at endosomal ph prefusion structure of alphavirus e protein, showing unexpected similarity to flavivirus e protein, leading to concept of 'class ii' membrane-fusion proteins membrane fusion process of semliki forest virus ii: cleavage-dependent reorganization of the spike protein complex controls virus entry furin processing and proteolytic activation of semliki forest virus proteolytic activation of tick-borne encephalitis virus by furin cell-associated west nile flavivirus is covered with e+pre-m protein heterodimers which are destroyed and reorganized by proteolytic cleavage during virus release budding of alphaviruses a structural perspective of the flavivirus life cycle cryo-electron microscopy reveals the functional organization of an enveloped virus, semliki forest virus placement of the structural proteins in sindbis virus structures of immature flavivirus particles first description of mature flavivirus surface organization, showing the striking herringbone arrangement of fusion protein homodimers visualization of membrane protein domains by cryo-electron microscopy of dengue virus structure of west nile virus crystal structure of the semliki forest virus envelope protein e in its monomeric conformation: identification of determinants for icosahedral particle formation the envelope glycoprotein from tickborne encephalitis virus at Å resolution a ligand-binding pocket in the dengue virus envelope glycoprotein variable surface epitopes in the crystal structure of dengue virus type envelope glycoprotein conformational changes of the flavivirus e glycoprotein membrane fusion process of semliki forest virus i: low ph-induced rearrangement in spike protein quaternary structure precedes virus penetration into cells membrane fusion of semliki forest virus involves homotrimers of the fusion protein references and comprise the first reports of homotrimer formation for a class ii fusion protein oligomeric rearrangement of tickborne encephatitis virus envelope proteins induced by an acidic ph formation and characterization of the trimeric form of the fusion protein of semliki forest virus role of metastability and acidic ph in membrane fusion by tick-borne encephalitis virus mechanisms of mutations inhibiting fusion and infection by semliki forest virus mutational evidence for an internal fusion peptide in flavivirus envelope protein e the heterodimeric association between the membrane proteins of semliki forest virus changes its sensitivity to low ph during virus maturation membrane and protein interactions of a soluble form of the semliki forest virus fusion protein first demonstration that class ii ectodomain can form homotrimer when treated at low ph in presence of target membrane membrane interactions of the tick-borne encephalitis virus fusion protein e at low ph purification and crystallization reveal two types of interactions of the fusion protein homotrimer of semliki forest virus characterization of a membraneassociated trimeric low-ph-induced form of the class ii viral fusion protein e from tick-borne encephalitis virus and its crystallization conformational change and protein-protein interactions of the fusion protein of semliki forest virus structure of the dengue virus envelope protein after membrane fusion references , and are the first reports of low-ph-induced homotrimer structure of class ii fusion proteins multistep regulation of membrane insertion of the fusion peptide of semliki forest virus the fusion peptide of semliki forest virus associates with sterolrich membrane domains ph-dependent fusion between the semliki forest virus membrane and liposomes the role of cholesterol in the fusion of semliki forest virus with membranes membrane fusion of semliki forest virus requires sphingolipids in the target membrane membrane fusion activity of tick-borne encephalitis virus and recombinant subviral particles in a liposomal model system involvement of lipids in different steps of the flavivirus fusion mechanism cholesterol is required for infection by semliki forest virus the cholesterol requirement for sindbis virus entry and exit and characterization of a spike protein region involved in cholesterol dependence a single point mutation controls the cholesterol dependence of semliki forest virus entry and exit novel mutations that control the sphingolipid and cholesterol dependence of the semliki forest virus fusion protein molecular dissection of the semliki forest virus homotrimer reveals two functionally distinct regions of the fusion protein a conserved histidine in the ij loop of the semliki forest virus e protein plays an important role in membrane fusion visualization of the targetmembrane-inserted fusion protein of semliki forest virus by combined electron microscopy and crystallography physical principles in the construction of regular viruses graphitic cones and the nucleation of curved carbon surfaces protein-lipid interplay in fusion and fission of biological membranes class ii fusion protein of alphaviruses drives membrane fusion through the same pathway as class i proteins the protein coat in membrane fusion: lessons from fission stoichiometry of antibody neutralization of human immunodeficiency virus type stoichiometry of envelope glycoprotein trimers in the entry of human immunodeficiency virus type domain iii from class ii fusion proteins functions as a dominant-negative inhibitor of virus-membrane fusion photolabeling identifies a putative fusion domain in the envelope glycoprotein of rabies and vesicular stomatitis viruses reversibility in fusion protein conformational changes. the intriguing case of rhabdovirus-induced membrane fusion herpesvirus entry: an update the product of the vaccinia virus l r gene is a fourth membrane protein encoded by all poxviruses that is required for cell entry and cell-cell fusion a new class of fusionassociated small transmembrane (fast) proteins encoded by the non-enveloped fusogenic reoviruses reptilian reovirus utilizes a small type iii protein with an external myristylated amino terminus to mediate cell-cell fusion liposome reconstitution of a minimal protein-mediated membrane fusion machine we acknowledge the important work of the many researchers whose contributions were not fully covered owing to space constraints. we thank j. lepault for the electron microscopy the authors declare no competing financial interests. key: cord- - j stye authors: wang, xianfeng; ding, bin; sun, gang; wang, moran; yu, jianyong title: electro-spinning/netting: a strategy for the fabrication of three-dimensional polymer nano-fiber/nets date: - - journal: prog mater sci doi: . /j.pmatsci. . . sha: doc_id: cord_uid: j stye since , a rapid development has been achieved in a subject area, so called electro-spinning/netting (esn), which comprises the conventional electrospinning process and a unique electro-netting process. electro-netting overcomes the bottleneck problem of electrospinning technique and provides a versatile method for generating spider-web-like nano-nets with ultrafine fiber diameter less than nm. nano-nets, supported by the conventional electrospun nanofibers in the nano-fiber/nets (nfn) membranes, exhibit numerious attractive characteristics such as extremely small diameter, high porosity, and steiner tree network geometry, which make nfn membranes optimal candidates for many significant applications. the progress made during the last few years in the field of esn is highlighted in this review, with particular emphasis on results obtained in the author’s research units. after a brief description of the development of the electrospinning and esn techniques, several fundamental properties of nfn nanomaterials are addressed. subsequently, the used polymers and the state-of-the-art strategies for the controllable fabrication of nfn membranes are highlighted in terms of the esn process. additionally, we highlight some potential applications associated with the remarkable features of nfn nanostructure. our discussion is concluded with some personal perspectives on the future development in which this wonderful technique could be pursued. nanostructured materials, as the forefront of the hottest fundamental materials nowadays, provide one of the greatest potentials for improving performance and extended capabilities of products in a number of industrial sectors, which are gradually accessing into our daily life [ ] [ ] [ ] [ ] . as one of the most important nanomaterials, one-dimensional ( d) nanoscale materials have steadily attracted growing interest in the past decades because of their unique shape, fundamental properties, and potential applications in many different areas [ ] [ ] [ ] . a variety of advanced techniques have already been developed to fabricate d nanostructures in the form of fibers, wires, rods, belts, tubes, and spirals from various materials [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . nanofibers represent one of the most important d nanostructures standing at the leading edge of nanoscience and nanotechnology [ ] . in comparison with other methods of fiber fabrication such as template synthesis, drawing, and phase-separation, electrohydrodynamics (ehd) techniques [ ] [ ] [ ] [ ] (e.g. electrospinning) have emerged as straightforward approaches to the fabrication of nanofibers with high specific surface areas, high porosities, and controllable compositions for a wide range of applications [ ] [ ] [ ] [ ] [ ] [ ] . electro-spinning/netting (esn), as a recently developed advanced ehd technique, may be considered as a variant of electrospinning process [ ] [ ] [ ] [ ] . both of these techniques involve the use of a high voltage to induce the formation of a liquid jet. in addition to the formation of liquid jet, the esn process also comprises an electro-netting process, which defined as the phase separation-induced splitting of a small charged droplet in a high electric field [ , ] . therefore, the esn technique allows onestep fabrication of three-dimensional ( d) nano-fiber/nets (nfn) that are consisted of common electrospun nanofibers and unique ultrafine nano-nets. to be more precise, in d nfn membranes, the conventional electrospun nanofibers act as a support for the soap-bubble-like structured or spiderweb-like nano-nets comprising interlinked d nanowires [ , ] . nfn membranes possess the general properties and functions of conventional electrospun nanofibers as well as the fascinating features (e.g. extremely small diameter, high porosity, steiner tree network geometry, controllable coverage rate) that distinguish themselves from their counterparts [ ] [ ] [ ] . taking advantage of these intriguing characters, nfn materials are of huge interest for use in various applications. take for example the extremely small diameter, a large number of new opportunities could be realized by down-sizing currently existing structures to the nanometre scale [ , ] . the most typical examples can be seen in filtration, where ''smaller'' has always meant a greater performance and even promising as filters to intercept nano-sized viruses. moreover, nfn membranes are probably one of the safest nanomaterials currently used, since they are unlikely to become airborne and penetrate the body because of their fiber length and net area [ ] . there is no doubt that esn nfn nanomaterials has risen as a shining star in the horizon on the path of the scientists' searching for new materials for future environment, energy and healthcare applications. in the past few years, significant progresses have been made in terms of our fundamental understanding of the esn process, the controllable fabrication of nfn membranes based on various polymers as well as the exploitation of their applications [ ] [ ] [ ] [ ] , ] . in this article, we will provide a comprehensive review of the state-of-the-art research activities related to esn nfn nanostructures, including their fabrication, novel properties studies and potential applications. we begin with a brief introduction of electrospinning technique, description of their history and modern development, followed by the description of historical background of esn technique and three prevailing formation mechanisms of nfn materials. several fundamental properties of nfn nanomaterials such as extremely small diameter, high porosity, steiner tree network geometry, controllable coverage rate, and controllable density are also discussed. these properties have generated tremendous interest among material researchers. subsequently, we introduce the polymers and polymer composites that can be esn into nfn structures. the following chapter fully illustrates the strategies for the controllable fabrication of nfn membranes based on various parameters (solution, processing, and ambient). then, we highlight a range of applications associated with d nfn nanomaterials in various areas. finally, we draw conclusions regarding the research activities of this new technique and the prospects of future research directions. electrospinning, also known as electrostatic spinning, is a powerful, rather simple and highly versatile technique which allows fabricating micro-and nanoscale fibers from process solutions or melts using an electrically forced fluid jet ( fig. ) [ ] [ ] [ ] [ ] . the diverse properties of these fibers, based on various physical, chemical, or biological behavior, mean they are of interest for a multitude of applications ranging from filtration, wound dressing, drug delivery, tissue engineering, living membranes, sensors, and so on [ , [ ] [ ] [ ] [ ] . electrospinning is considered as a variant of the electrostatic spraying process (i.e. the behavior of electrically driven liquid jets) [ ] whose history can be traced back to more than years ago when bose used high electric potentials to generate aerosols from fluid drops in [ ] . the maximum amount of charge which is required to overcome the surface tension of a droplet was calculated in by rayleigh [ ] . subsequently, the first devices to spray liquids through the application of an electrical charge were patented by morton and cooley at the beginning of the th century [ , ] , and electrospinning as a physical phenomenon and an application to produce tiny fibers was first suggested. from to , formhals published a series of us patents, describing an experimental setup for the production of polymer filaments using an electrostatic force [ ] . in , taylor determined that an angle of . °is required to balance the surface tension of the polymer with the electrostatic forces [ ] . later on, focus shifted to study the structural morphology of nanofibers based on a wide variety of polymeric systems [ ] . despite these early discoveries, electrospinning was nearly forgotten until the late s. electrospinning technique has regained substantial attention triggered by a surging interest in nanotechnology, as ultrafine fibers or fibrous structures of various polymers with diameters down to submicrons or nanometers can be easily fabricated with this process [ , ] . several research groups (e.g. reneker group and wendorff group) revived interest in this process, have shown that a wide variety of polymeric nanofibers can be obtained via electrospinning [ ] [ ] [ ] [ ] [ ] [ ] . a lot of review papers have been published recently which provide an insight into the most prominent aspects of electrospinning [ , , , , ] . additionally, the popularity of electrospinning has increased sharply as clearly reflected from the fact that more than universities and research institutes in the world have engaged themselves in investigating various aspects of the electrospinning process. several companies (e.g. espin technologies, nanotechnics, and kato tech) are actively seeking to reap the benefits from the advantages offered by electrospinning, while companies such as donaldson company and freudenberg have utilized this technique to prepare air filtration products [ , ] . the preparation of nanofibers using electrospinning method has attracted worldwide attention due to its versatile maneuverability of producing controlled fiber structures, porosity, orientations and dimensions. generally, electrospun fibers are collected as nonwoven membranes with randomly arranged structures, which have greatly limited their applications in electronic devices or biomedical applications [ ] [ ] [ ] . in order to fully realize the potential of electrospun fibers, it is important to fabricate fibrous assemblies with controllable structures. recently, many groups have demonstrated that electrospun nanofibers could be collected as uniaxially aligned arrays by using specially designed collectors [ ] [ ] [ ] [ ] . for instance, li et al. [ ] reported that nanofibers could be uniaxially aligned by introducing insulating gaps into conductive collectors. matthews and co-workers [ ] demonstrated that aligned fibers could be fabricated by using a high-speed rotating roller; the mandrel rotation speed and fiber orientation strongly influence the properties of the electrospun nanofibers. moreover, it was possible to obtain various patterned architectures of the electrospun nanofibers by varying the design of electrode pattern [ , ] . one of the most interesting features associated with this approach is that this technique enables direct integration of nanofibers with controllable configurations into an electrode system, which will greatly facilitate the production of nanofiber-based devices [ ] . besides aligned and patterned fibers, recent demonstrations from a number of literatures indicate that electrospinning technique is also able to fabricate nanofibers with porous [ ] [ ] [ ] , ribbon-like [ ] , helical [ , ] , necklace-like [ , ] , firecracker shape [ ] , rice-grain shape [ ] , multi-channel tubular [ ] , multi-core cable-like [ ] , tube-in-tube [ ] , nanowire-in-microtube [ ] , core-shell [ , ] and hollow [ ] structures. additionally, this technique has become particularly significant when combining other remarkable features, such as tremendous surface-to-volume ratio and pore sizes of nanofibers, with unique physical, chemical, and mechanical functions provided by adding other components with ease and control [ ] . given the versatility of preparing various structured fibrous membranes based on different materials, electrospinning have been identified for use in different fields ranging from healthcare, biotechnology, and environmental engineering to defense and security, and energy generation [ , , , , ] . to fulfill these applications, several techniques such as multi-jet electrospinning [ , ] , tip-less electrospinning [ ] , electroblowing [ , ] and edge electrospinning [ ] have been developed to overcome the problem of low mass throughput. moreover, the electrospinning tip has also been modified to fabricate either side-by-side or coaxial multicomponent fibers and tubes [ ] . not surprisingly, the enhanced mass throughputs certainly will render electrospun nanomaterials commercially viable products. [ ] . a handle of ''fishnet-like nanowebs'' were highlighted by the red rectangle. notwithstanding the ability to generating micro-and nanoscale fibers of electrospinning which offer great potential in different fields, enormous researches in the past years have indicated that the large average diameter ( - nm) of common electrospun fibers prevented their further applications in ultrafiltration, ultrasensitive sensors, catalyst, etc. [ ] . simultaneously, recent studies have shown that properties such as surface area and porosity become more significant when the fiber diameter falls below nm [ ] . therefore, a current major challenge is to develop robust strategies for manufacturing large-scale and extremely small nanofibers (< nm). various approaches, such as decreasing the polymer concentration [ ] , elevating the solution temperature [ ] , and increasing the net charge density of the solution [ ] , have been utilized to reduce the diameter of electrospun nanofibers. however, these methods typically produce nonuniform fibers with poorly defined structures and the objective of reducing fiber diameter down to nm was rarely achieved. [ ] . Ó elsevier b.v.) and (b) horizontal set up of electrospinning apparatus (reprinted with permission from [ ] . Ó royal society of chemistry). the insets show drawings of the forces acting on the charged droplet and typical fe-sem images of nfn membranes. in , when ding and coworkers found a unique scanning electron microscopy (sem) image of electrospun polyamide- (pa- ) fibrous membranes in which existing a handle of ''fishnet-like nanowebs'' (supported by common electrospun nanofibers and comprised interlinked d ultrathin nanowires; highlighted with red rectangle) (fig. ) [ ] , they did not realize that they had discovered a material that caught much attention a few years later [ - ] . while they were excited about the discovery that might lead to a smaller, cheaper substitute for existing electrospun fibers, they were puzzled by the cause of this fascinating material and cautious about the future applications. until when surprisingly partly split polyacrylic acid (paa) nanowebs were observed, ding and coworkers preliminarily mastered the principles of structure control for nanowebs and proposed a possible formation mechanism of this versatile structure (section . . . ). the preliminary results were reported in the journal of ''nanotechnology'' in which they introduced the concept of ''electro-netting'' [ ] . electro-netting accompanies the electrospinning process and allows one-step fabrication of ultrathin nano-nets (i.e. nanowebs) in large quantities and with uniform size. here, the term of ''nano-nets'' was used to replace the older term ''nanowebs'' that was unsuitable in the representation of this d netlike structure, because a d electrospun fibrous structure can also be called ''nanowebs'' [ , , ] . the research object of electro-netting process is small charged droplets other than electrospinning jets. additionally, nano-nets were primitively regarded as a by-product caused by a high electric field that induces instability of suspended charged droplets during electrospinning rather than formed from the breaking jets [ ] . since then, the research on nano-nets including the exploitation of spinnable polymers, morphology control, functionalizing nano-nets and exploring the applications of nano-nets has grown exponentially [ , [ ] [ ] [ ] [ ] . the term ''electro-spinning/netting'', abbreviated as esn, represents the combination of ''electrospinning'' and ''electro-netting'', while the ''nano-fiber/nets'', abbreviated as nfn, represents the combination of nanofibers and nano-nets [ ] . fig. shows schematic diagrams illustrating the basic setup for esn process. currently, there are two standard esn setups, vertical (fig. a) and horizontal (fig. b) . like traditional electrospinning setup, an esn system also consists of three major components: a high voltage power supply, a spinneret (a metallic needle) and a grounded collecting plate (usually a metal screen, plate, or rotating roller) and utilizes a high voltage source to inject charge of a certain polarity into a polymer solution or melt, which is then accelerated towards a collector of opposite polarity [ , , ] . direct current (dc) power supplies are usually used for esn although the use of alternating current (ac) potentials is also feasible. in contrast to electrospinning, the esn process usually needs a higher voltage to enhance the instability of the taylor cone [ ] . with the use of syringe pump, the solution can be fed through the spinneret at a constant and controllable rate. the development of esn technology attracted the scientists who became eager to learn as much as they could understand the formation mechanism of nfn membranes. the knowledge on this issue turned out indispensable to select the spinnable polymer, control the manufacturing process, and regulate the final structure of resultant fibrous membranes. however, it is worth noting that the formation mechanism of the nfn structures is complicated and consensus on the formation mechanism has not been reached [ ] . one puzzling question is this: what happens to the charged polymer jets and droplets during their running in such small distance between needle and collector? more rigorous experimental and theoretical work need to be addressed to reveal the secrets behind this process. nevertheless, we are pleased to see that more and more researchers involve themselves in this domain and propose several mechanisms to explain the formation of the nfn structure [ , , [ ] [ ] [ ] . in this section, we will review current four predominant formation mechanisms of nfn structure which include phase separation of charged droplets, ions initiated splitting up of the electrospun fibers, intermolecular hydrogen bonding, and intertwining among branching jets. . . . . phase separation of charged droplets. the mechanism of phase separation of charged droplets generated during esn was proposed based on the experimental observation of defect paa films (fig. a) and partly split paa nano-nets ( fig. b and c) . based on the observation described above, ding et al. [ ] attributed the formation of nano-nets to the phase separation of charged droplets generated during electrospinning. they suggest that under a high electric force, the charged droplets deform significantly to a thin liquid film, which undergoes rapid phase separation with the solvent rich domains to transform into pores. the forces, including electrostatic force, drag force, gravity, coulombic repulsion force, surface tension and viscoelastic force, act on the charged droplet when it flights with a high speed in the electric field as shown in fig. a . the electrostatic force carries the charged droplet from capillary tip to collector. the drag force between the surrounding air and the charged droplet is the main cause that deforms the droplets into films. the coulombic repulsion force tried to expand the droplet. the surface tension and viscoelastic forces led the contraction of charged droplet [ ] . the electric field could be increased by increasing the applied voltage within a constant distance. consequently, the electrostatic and coulombic repulsion forces of charged droplet were reinforced with increasing of electric field. the increased electrostatic force further accelerated the moving of charged droplet, which led to an increased drag force. the distortion and expansion of charged droplet (i.e. from spherical-like to spindle-like) in the electric field during electrospraying was reported by grimm and beauchamp [ ] . the further expansion could happen when the electric field increased further to form thin films from droplets with the effect of increased coulombic repulsion and drag forces. moreover, the increased radial charge repulsion force also has a tendency to expand the charged films. as a result, the deformation of charged droplet was strongly affected by the electric field [ ] . fig. b shows the schematic diagram illustration of the possible formation mechanism of nano-net during esn. as shown in this figure, the defect films or nano-nets could be regarded as a by-product caused by a high electric field induced instability of suspended charged droplets during electrospinning [ ] . the microsized charged droplets [ ] could be generated together with the common electrospun fibers from the capillary tip with a high instability. during the flight of charged droplet from tip to collector, the microsized droplet was distorted and expanded into a thin film due to the comprehensive effects of the forces acting on it [ ] . the splitting of thin film into nano-net was ascribed to the rapid phase separation between polymer and solvent and to the fast evaporation of solvent at lower humidity. the fast phase separation led the spinodal or binodal types of phase morphologies within the fibers, and the solvent rich regions in the thin film were transformed into pores [ , ] . as the electrospun fibers and nano-nets were formed at the same time, the d nano-nets stacked into d fibrous mats in a layer-by-layer structure as well as d electrospun fibers. the improved formation frequency and area density of nano-nets could be achieved by increasing the instability of droplet at the electrospinning tip, such as increasing the applied voltage [ , ] . actually, the break-up of a jet and the formation of small charged droplets is a very old and interesting problem and the research can be date back to the ehd atomization process [ ] [ ] [ ] . in ehd atomization in the cone-jet mode a highly charged jet emerges at the apex of the liquid cone. due to its charge, the liquid is still accelerating, while breaking up into a number of primary or main droplets and a number of secondary droplets and satellites [ , ] . in order to model the jet break-up process with analytical relations, hartman et al. presented a physical numerical model to describe the harmonic perturbations on the jet surface based on a cylindrical coordinate system [ ] : where r is the radial component (m), h is the angular component (rad), z is the axial component (m), r s is the radius of the surface (m), r jet is the radius of the unperturbed jet, x is the growth rate of the perturbation (s À ), t is time (s), a is the amplitude of the perturbation at t = (m), m is a constant, k is the wave number of the perturbation (m À ), and k is the wavelength of the perturbation (m). depending on the value of m, three modes of jet break-up have been proposed (fig. a ). if m = , the jet break-up is independent of the angular component h, which is also called varicose instability. if m = , then the radius of the jet depends on angle h, which represents the whipping motion of the jet. this kind of lateral instability is also called kink instability. if m = , then the jet is no longer circular. this mode can only occur if the jet is highly charged (e.g. the esn process). if the surface charge is above a certain threshold value, then the electric stresses can overcome the surface tension. these electric stresses will transform the shape of the jet. the ramified jet is an extreme example of a jet in which this kind of perturbations occur [ ] . additionally, the formation of small charged droplets has also been demonstrated by kim et al. [ ] through a high-speed camera (fig. b ). ions initiated splitting up of the electrospun fibers. in addition to the phase separation of charged droplets, a recent study by kim et al. [ ] demonstrated that nano-nets can be prepared by adding strong ionic salts to the polar polymer solutions, such as pa- /formic acid. they believed that nanonets are formed by the joints between many fibers and the possible joints occur at the apex of taylor cone. to explain the differences between electrospinning of sol-gel and salt/polymer solution, they suggested a conceptual illustration to explain the mechanism of forming the nano-nets (fig. ) . in case of electrospinning sol-gel solution (fig. b) , the ionically-balanced sol-gel particles formed from the hydrolyzing and polycondensation of the utilized precursor was embed into the produced polymeric nanofibers, and therefore no nano-nets was observed. on the other hand, the ions randomly spread in the salt/polymer solution and might attach on the polymer chains (fig. c) . fig. d presents the corresponding behavior of this solution during the electrospinning process. the different charged electric poles generated in the nanofibers could lead to formation of the joints, which finally shaped the observed nano-nets within the nanofibrous membranes. to precisely confirm the proposed synthesizing hypothesis, they showed us a transmission electron microscope (tem) image that was obtained by placing a tem grid very close to the tip end for very short time (fig. e) . according to the tem image, they put forward that the joints do not only synthesized among the main nanofibers but also among pre-formed joints. in a further investigation, they attributed these joints to the splitting up of subnanofibers from the main fibers induced by the increased ionic strength [ ] . based on the investigation of pa- and methoxy poly (ethylene glycol) (mpeg) oligomer/pa- nfn membranes, kim and coworkers proposed the hydrogen bonds formation mechanism [ , ] . as shown in fig. which presents the possible hydrogen bond formation mechanism between nano-nets and main pa- nanofibers. in presence of high applied voltage during electrospinning, the protonated amide group of ionic molecule can effectively connect with oxygen atom of pa- molecule in main fiber and oxygen atom of ionic molecule can combine with hydrogen atom of amide of main fiber as usual, both of which can form the interconnected spiderweb-like nfn membranes (fig. ) [ ] . similarly, they also featured the formation of network with the help of possible hydrogen bond formation mechanism between active group of mpeg and nh or c@o group of pa- molecules when interpreted the formation of mpeg oligomer/pa- nfn membranes [ ] . reprinted with permission from [ ] . Ó elsevier b.v. intertwine among branching jets. recently, another plausible formation mechanism of nanonets has been proposed by tsou et al. [ ] . based on their argument, there were enormous tiny subsidiary jets and these jets underwent the whipping process as well as the main whipping jet. when many subsidiary jets with high ejecting speeds were intertwined in the chaotic whipping region, the short contact time between them diminished their mutual repulsive intrinsics. formation of a jet network became feasible after solvent removal, giving rise to nano-nets with connected nanofibrils. in other words, the formation of nano-nets is associated with the complex interaction between these subsidiary jets. the remaining challenge is that the diameter of subsidiary jets is too small to be seen using a high-speed camera with a frame rate of s À (fig. a) . the difficulty of clearly tracing a whipping jet with the proposed subsidiary jets is due to the high electric field experienced by this high conductive jet with a short l j (the distance between the needle end to the initiation of jet bending is denoted as l j ) (fig. b ). nfn possess the general properties and functions of conventional electrospun nanofibers and other d nanostructures fabricated using different techniques, as well as the impressive feature characters (e.g. extremely small diameter, high porosity, steiner tree network geometry, controllable coverage rate) that distinguish themselves from their counterparts, the properties donated by the polymer phase, and the d net-like geometry. these unique properties enable nfn membranes to have received a great deal of attention and been extensively investigated for wide applications. this chapter will highlight some remarkable features of nfn membranes mainly correlated with their existing nanostructures. achieving new properties through reducing the dimensions of a material is one major principle of nanotechnology [ ] . for instance, in semiconductor particles or films, a decrease in the diameter is often linked to new optoelectronic functions (quantum effects), and in magnetic materials, superparamagnetism appear [ ] . recently, growing attempts were reported to regulate the diameter or arrangement of electrospun fibers to achieve the designed functions [ ] [ ] [ ] [ ] [ ] . one attractive property of nanofibers associated with decreased diameter is the increased surface area, which in turn, has immediate repercussions on ultrasensitive sensors and other significant applications [ , ] . nfn membranes are consisted of conventional electrospun nanofibers and nano-nets, in which the electrospun nanofibers acted as a support for the nano-nets comprising interlinked d ultrathin nanowires. one of the most interesting aspects of the nfn membranes is that it contains enormous interlinked ultrathin nanowires. fig. a and b presents the typical field emission scanning electron microscopy (fe-sem) images of pa- nfn membranes, indicating that the resultant nanofibers were randomly oriented as d porous membranes. as shown in fig. d , the major distribution region (over %) of nanowire diameters is in the range of - nm with an average diameter of nm, which is one order of magnitude less than that of common electrospun nanofibers (fig. c) . additionally, nano-nets promote the surface area of pa- nfn membranes as evident from a high brunauer-emmett-teller (bet) surface area of . m /g compared to that of m /g for even porous pa- fibers [ ] . fig. shows the comparison of the diameters of nano-nets to those of biological and technological objects. it can be seen that the scale of nano-nets is particularly relevant for biological systems, because the dimensions of proteins and viruses fall in this size range. in comparison with the relatively wide range of electrospun nanofibers, nano-nets just span a narrow range from to nm. as well known, the microstructure plays the key role on the transport properties of porous materials [ ] . nfn membranes possess complex porous structures that mainly include the high density of pores formed because of entanglement of nanofibers and the enormous pores distributed among [ ] . Ó iop publishing ltd. several biological organisms that exhibit steiner minimal tree networks: (b) cell walls of sisal (reprinted with permission from [ ] . Ó springer-verlag.), (c) soap bubbles (reprinted with permission from [ ] . Ó royal society of chemistry.), (d) honeycombs, (e) spider webs (reprinted with permission from [ ] . Ó elsevier b.v.) and (f) nano-nets. nano-nets (fig. a) . the pores among the conventional electrospun nanofibers are relatively large in size and all pores are fully interconnected to form a d network. in contrast, the enormous pores formed in the nano-nets present more regular geometric structures with pore-width distribution ranged from several nanometer to hundreds nanometer, which is much less than that of pores among electrospun nanofibers (fig. a) . for example, the region of pore-width distribution of typical paa/ nacl nano-nets (fig. b ) ranged from to nm [ ] . the complex porous structures contribute two impressive advances for nfn membranes: ( ) the further enhanced specific surface area of the membranes that may improve the surface activity and hence gas sensitivity. ( ) porous structure may facilitate the air current transport in membranes and effectively reduce the air resistance when used as filters. therefore, the combination of these two kinds of pores and thus formed complex porous structure makes nfn membranes possess the great potential application in ultrasensitive sensors and filtration system for the removal of particles or viruses with a size to nanometer ranges [ , ] . the general problem of finding steiner minimal tree on a set of points is a very old and interesting problem and one which has been of considerable interest in network design and operations research [ ] . yin et al. [ ] proposed the geometric conservation laws and proved that the mechanically stable equilibrium network of biomembrane nanotubes or super carbon nanotubes (cnts) is geometrically equivalent to a steiner minimal tree. this law provides the geometric fundamental for the mechanics of biomembrane nanotube networks and super cnt networks. to construct a steiner minimal tree, simple geometric regulations were required, i.e. the angle between two neighboring branches should be °and the radii of the three branches should be equal (fig. a) . recently, li et al. [ ] further confirmed that steiner net-work is the geometric foundation for the mechanics of sisal fibers (fig. b) . besides the sisal fibers, nature also abounds with other mysterious biological organisms that exhibit steiner minimal tree networks, such as soap bubbles (fig. c ) [ ] , honeycombs (fig. d ) and spider webs (fig. e) [ ] . more exciting, we found that nano-nets present a clear geometric characteristic with ideal and weighted steiner networks, in which three neighboring nanowires form a three-way junction with angular symmetry and topological invariability (fig. f ) [ , ] . learning from nature gives us much inspiration to explain some mysterious phenomena. thus from pure geometric viewpoint, we can answer an important question that why do the nano-nets ''tend to'' be steiner geometry? the answer is very simple: once the jets or droplets phase are separated along with the steiner geometry, an optimization phase separation mode (i.e. the ''materials needed for architectures'' are minimal) is selected. minimal materials and maximum spaces are irresistibly attractive for the phase separation of the droplets. additionally, another possible reason may be ascribed to the minimal energy principle, which is of universality in nature [ ] , and is valid for the formation process of nano-nets. in another word, nano-nets tend to complete their phase separation processes by minimal energies. this means that a network with minimal energy is also a network with minimal length. then it can be concluded that the formation of nano-nets is dominated by steiner geometry. another important characteristic of nfn membranes is its controllable coverage rate, which is defined as the area ratio of nano-nets to the whole membranes. for accuracy, sem images with low magnification were randomly selected when we calculated the coverage rate. the coverage rate of nano-nets in d nfn nanostructures is of special interest since it may offer new functions or significant improvement of their filtration performance. this is for many cases the primary motivation to fabricate nanonets with large and uniform area. esn process represents a simple and straightforward method that can prepare nano-nets with controlled structures. large coverage scale of nano-nets was easily accessible by regulating the voltage [ ] , adding salts in the polymer solutions (salt content and stirring time of salt/polymer solution) [ ] , changing the polymer concentration or solvent components [ , ] , etc. for example, kim et al. [ ] reported the fabrication of nfn membranes by adding strong ionic salts to the polar polymer solutions and found that the stirring time had strong influence on the coverage rate of nano-nets ( fig. a-c) . as shown in fig. a , no nano-nets can be observed, however the salt nanoparticles are apparently attaching to the nanofibers. actually, the main reason behind this is that the utilized stirring time ( . h) is not enough to completely dissolve the salt in the polymer solution, which means that the stirring time is not enough to liberate ions on the solution. with relatively little long time ( h) the nano-nets start to appear but also some salt nanoparticles are present (fig. b ). however, with long stirring time (i.e. day), larger coverage rate nano-nets were formed within the main electrospun nanofibrous membranes and no salt nanoparticles could be observed (fig. c) . recently, nirmala et al. [ ] demonstrated the role of applied voltage for the coverage rate of pa- nano-nets ( fig. d-f ). there were no nano-nets when the applied voltage was kv (fig. d ). as the applied voltage was increased to kv, it was clearly visible that the formation of large-scale nano-nets was strongly bound in between the main fibers (fig. e) . however, at too high-applied voltage, the formation of nano-nets was reduced and somewhat loosely bound to the main fibers ( fig. f) . at lower applied voltage (e,g, kv), the electrospinning jet was too stable to form microsized droplets and thus no obvious nano-nets were formed. the larger area density of the nano-nets at higher applied voltage (e.g. kv) may be resulted from the improved formation probability of microsized droplets due to the increased instability of the taylor cone. further increasing the voltage can generate larger electrostatic forces on the droplets, which may stretch the jets fully for the favorable formation of completely split nano-nets (i.e. nano-nets were widespread in the fibrous membranes). therefore, the applied voltage plays an important role in controlling the coverage rate of nano-nets. the last but not the least interesting aspect of nfn membranes is their controllable density, which include the following two aspects: ( ) arranged density of nano-nets in a plane and ( ) stacking density of multi-layered nano-nets. controllable density of nano-nets in nfn membranes makes it attractive candidate for various significant applications, such as filters and reinforcement materials for composites. therefore, a few researchers have made efforts to regulate the density of nano-nets [ , , ] . for instance, an enhanced density of nano-nets has been demonstrated by increasing content of chitosan (cs) in cs/pa- composite nfn membranes [ ] . recent studies have also shown that the controllable density of nano-nets could be achieved by changing the esn parameters (i.e. solution, processing and ambient parameters) [ , ] , which will be discussed in the following section (section ). on the other hand, stacking density of multi-layered nano-nets can be easily realized by appropriately prolonging the esn time. it is worthy to mention that not the higher stacking density the better, there should be an optimal value that from the following two points to consider: ( ) higher stacking density of nfn membranes on the collector will certainly affect the electric field between the tip and collector and thus influence the morphology of subsequently deposited fibers; ( ) higher stacking density of nfn membranes will increase the air resistance in filtration applications. esn has been applied to several natural and synthetic polymers, including polymers soluble and spinnable from water, biocompatible and biodegradable polymers, polymer blends and polymers into which nanoparticles, salts, surfactants and other functional materials have been incorporated. similar to electrospinning technique, the formation of nfn membranes via esn also requires the materials to be fabricated to display electric conductivities in a limited range of values, specific viscous/visco-elastic properties as well as specific surface energies as obvious both from theoretical considerations and from experimental findings [ ] . although a broad range of polymers have been electrospun into micro-or nanofibers, it is worth noting that not all of those spinnable polymers can be esn into nfn structured membranes. a comprehensive summary of polymers that have been successfully esn into nfn structures to-date is listed in table for a quick and cursory review. also given in the table are the chemical formulas of these polymers, solvents that have been used and proposed or perspective applications of the corresponding fibrous membranes. fig. shows the typical morphologies of nfn membranes based on different polymer systems, such as paa, pa- , polyamide- (pa- ), poly(vinyl alcohol) (pva), gelatin, cs, and polyurethane (pu), which have been synthesized in our laboratory. in this chapter, we present some typical recently developed processes for the synthesis of nfn nanomaterials based on various polymers and their structural characterizations. additionally, spinnable polymers as supporter for functional materials have shown a great potential for various important applications. in the past few years, researchers have made successful attempts for functional materialpolymer composite nfn membranes [ , , ] . their functionalization can offer exceptional properties in composites and applications in the field of energy, environment and biomedical. this section will also review these composite nfn membranes, illustrating their fabrication and discussing their intrinsic fundamentals. . . polyamide- (pa- ) based nfn membranes . . . pa- pa- is a polymorphic, biocompatible and synthetic polymeric material which has good mechanical and physical properties [ ] . this polymer can be easily esn with a controlled manner for fabricating nfn membranes. this pa- nanostructure exhibits several interesting morphological characteristics, for example, a high surface area to mass or volume ratio, high porosity, vast possibilities for surface functionalization. these advantages render pa- nfn membranes good candidates for a wide variety of applications, including filters, composite reinforcements, drug carriers, and tissue-engineered scaffolds. at present, the most widely studied polymer for preparing nfn structured nanomaterials is pa- , with demonstrated lots of literatures. ding et al. [ ] firstly demonstrated the fabrication of fishnetlike pa- nfn membranes (fig. a ) and they reported that the formation of such kind of morphology was due to the phase separation of charged droplets. the nanowire diameter distribution of pa- nano-nets shown in fig. b exhibited that the major distribution region (over %) of nanowire diameters was - nm with an average diameter of nm. the standard deviation of the wire diameters in nano-nets was nm. pa- polymeric nfn membranes containing nano-net morphology have also been synthesized by the addition of metallic salt [ ] . nirmala et al. [ ] investigated the formation of nano-nets in pa- nfn membranes as a function of applied voltage ranging from to kv. a large amount of sub-nanofibers (< nm in diameter) in the form of spider-web-like structures were achieved by increasing the amount of solvent degraded polymer solution in the freshly prepared pa- solution ( fig. c ) [ ] . organically modified montmorillonite (o-mmt)/pa- nanocomposite nfn membranes ( fig. f) were also prepared by li et al. [ ] . the o-mmt layers were well exfoliated inside the nanocomposite fibers and were oriented along the fiber direction, which enhanced young's modulus and ultimate tensile strength of the composite membranes. recent study have also revealed that the incorporation of tio nanoparticles into the pa- solutions can form more spider-web-like nano-nets ( fig. d and e), which can lead to a remarkable increase in the number of reactive sites with a corresponding improvement in hydrophilicity, photocatalytic and antimicrobial activity [ ] . the composite nanofibrous membranes have unique physical and chemical properties and therefore, research interest in the formation of polymer composite nfn membranes has caught more attentions. kim and coworkers [ ] reported the preparation of a composite mpeg/pa- by esn the blend solutions containing mpeg oligomer and a viscous pa- supporting solution. mpeg/pa- composite nfn membranes showed highly interconnected spider-web-like structures with ultrathin nanowire diameter of ± nm (fig. a ). the mechanical strength of the pa- nanofibrous membranes with low mpeg content ( wt%) was greater than that of pure pa- and again decreased with increasing mpeg content (fig. b ). the enhanced mechanical strength up to wt% mpeg is due to the highly interconnected spider-web structure of mpeg with the larger pa- backbone nanofibers. upon increasing the mpeg content, the coverage rate of nano-nets was decreased, as can be observed in the fe-sem images (fig. a ). therefore, there was a decrease in mechanical strength. the results of contact angle measurement showed that mpeg/pa- nfn membrane with wt% mpeg was not only much more hydrophilic than pure pa- membrane but also more hydrophilic than a wt% mpeg membranes (fig. c ). it is probably due to the formation of more surface to volume ratio of well distributed nano-nets of hydrophilic mpeg throughout the wt% mpegn membrane than the others. therefore, strongly interconnected thin mpeg spider-web-like nano-nets with thick pa- nanofibers are responsible to increase mechanical strength and hydrophilic nature of pa- fibrous membranes, which make composite mpeg/pa- nfn membranes great potential in air filtration and different biomedical application. lecithin is a natural mixture of phospholipids and neutral lipids, which is a significant constituent of nervous tissue and brain substance [ , [ ] [ ] [ ] . it is a typical amphiphilic phospholipid with good biocompatibility and capable of mixing with different polymers, such as poly(lactic-co-glycolic acid) (plga), poly(lactic acid), poly(e-caprolactone) (pcl) and poly-l-lactic acid (plla) [ , ] . recently, lecithin blended in pa- nanofibers with spider-web-like nano-net structure ( fig. a-d) was successfully produced by esn technique for human osteoblastic (hob) cell culture applications [ ] . from the water wettability test, it was observed that the water droplets sink into the lecithin/pa- nfn scaffold ( fig. b-d) , indicating improved water wettability than that of the pristine pa- scaffolds (fig. a ). fig. e shows the fourier-transform infrared (ft-ir) spectrum of the lecithin/pa- nfn membranes, which indicated that the characteristic peaks of lecithin/pa- appeared at - cm À in the composite fibers. one prominent peak was observed for the lecithin/pa- at cm À (amide i), which is attributed to the amino groups of blended nanofibers. on the other hand, one intense extra peak at cm À was observed for the pristine lecithin, which is attributed to the c@o groups in lecithin. the ch group transmittance band in the - cm À region is slightly stronger than that of pa- , indicating that lecithin contains more ch groups. the broad peak between and cm À corresponds to a stretching of -oh and the intensity of this band was found to be decreased with increasing lecithin concentration in lecithin/pa- nfn membranes. the tga results show that pristine pa- nanofibers had higher onset temperature ($ °c) among all samples, while for pristine lecithin was around °c (fig. f) . the onset decomposition temperature monotonically decreased with increasing lecithin concentration in the in the composite nfn membranes, which demonstrated a significant difference in the thermal stabilities between the starting physical forms (i.e. lecithin and pa- ) of the composite nfn membranes. very recently, kim and co-workers reported on a one step preparation of cs/pa- composite nfn membranes with a single solvent system and investigated the electrical properties of the resultant membranes ( fig. ) [ ] . the resultant nanofibers are well-oriented and have good incorporation of cs. high density cs/pa- composite nano-nets with diameters of about - nm are bound in between main fibers ( fig. a-d) . current-voltage (i-v) measurements revealed interesting linear curve, including enhanced conductivities with respect to cs content (fig. e) , which could be due to the enhanced electrical pathways in the composite nfn membranes caused by the increased cs content. moreover, the sheet resistance of composite nanofibers was decreased with increasing content of cs (fig. f) , which could be attributed to the formation of highly denser ultrafine nano-net structures. furthermore, they also demonstrated that these novel nfn membranes could be used as nontoxic scaffold material for the osteoblast cell culture [ ] . conducting polymers constitute an attractive class of materials for electronic, magnetic, and optical applications [ , ] . among them, pani has received much attention due to its environmental stability, controllable electrical conductivity, and interesting redox properties [ ] . considering the well-known fact that the yield of electrochemical reactions occurring at an electrode is proportional to its surface area, it is expected that nanostructures of this polymer offer great opportunities for potential applications in electronic nanodevices like sensors [ ] [ ] [ ] . more recently, our group has presented continuous efforts toward the aim of generating pani-based nanostructured materials and for the first time fabricated composite pani/pa- nfn membranes (fig. a) , which were used as a platform for efficient sensing reaction by providing high specific surface area and porosity. evidence for the formation of pani/pa- composite nfn membranes comes from ft-ir spectral analysis (fig. b) . the bands at , , and cm - belong to c@o group, hydrogen bonded n-h stretch and -ch -group of pa- , respectively. the band corresponding to p-substituted chains of pani appears around cm - , and the bands corresponding to stretching vibration of n-b-n and n@q@n structure of pani appear around and cm - , respectively. furthermore, we showed that pani/pa- nfn membranes could be used as sensor strips for naked-eye colorimetric detection of cu + in water, achieving a detection limit as low as ppb by naked eye [ ] . acrylic acid monomer in a viscous supporting pa- solution was polymerized and fabricated simultaneously via an esn process by parajuli et al. [ ] . polymerization of acrylic acid was achieved via formic acid reduction during the esn process. in this process, formic acid acted as a reducing agent, which was activated by the applied voltage (fig. a ). this voltage removed the hydride ion to electrically excite acrylic acid and induced a nucleophilic chain reaction polymerization. this polymerization method defines the fiber morphology as a network of interconnected membranes (i.e. nano-nets). this network consists of smaller paa fibers (approximately nm in diameter) and larger pa- fibers (approximately nm in diameter) (fig. b) , which was demonstrated by the fe-sem image of paa/ pa- nfn membranes after washing with water (fig. c) . final observations of promoting experiments were taken several times and different feed ratios of acrylic acid monomer with a fixed viscous nylon solution remarkably enhanced mechanical properties along with the thermal behavior of paa/ pa- hybrid membranes. research and observation are underway. furthermore, they demonstrated smaller changes exhibited with an increase the volume ratio of acrylic acid (fig. d ). the close association of the charged polyelectrolytes in aqueous solutions and their counter ions is expected to play an important role in the conformation of polyelectrolyte molecules, thus molecular entanglement and solution viscosity, both of which are critical to fiber formation [ ] [ ] [ ] . furthermore, the investigation of the formation of polyelectrolyte fibers provides excellent models to understand how to generate fibers from proteins [ ] . therefore, it is of fundamental interest to understand the formation and properties of polyelectrolyte fibers from electrospinning and esn processes. paa is a high water absorbing and protein resistive hydrogel polyelectrolyte widely used in medical field [ ] . growing attempts were reported to regulate the morphology of paa fibers to achieve the designed functions [ , , ] . ding et al. [ ] firstly reported the formation of paa nano-nets, and concluded that nano-nets could be obtained by adjusting the solution properties and several parameters in the process of electrospinning (fig. a) . wang et al. [ , ] demonstrated that highly sensitive sensors could be prepared by esn deposition of paa nfn membranes on the electrodes of quartz crystal microbalance (qcm). additionally, in order to obtain paa nano-nets in large quantities and with uniform size, various additives, such as formic acid (fig. b) , agno (fig. c) , dodecylbenzene sulfonic acid (dbsa) (fig. d) , silica (fig. e) , and nacl (fig. f) , were incorporated into the paa solutions and their effects on the internal morphology of the electrospun nanofibrous membranes were also investigated [ ] . furthermore, the versatile nano-nets with small pore-widths create additional surface area, which would be particularly useful for applications such as ultrafiltration, ultrasensitive sensors, and catalyst supports. pva, a water-soluble polyhydroxy polymer, is the largest volume synthetic resin produced in the world. the excellent chemical resistance, physical properties, and complete biodegradability of pva resins have led to their broad practical applications [ , ] . nanomaterials can be rationally designed to exhibit novel and significantly improved physical, chemical, and biological properties because of their size [ ] . thus, engineering pva into nanomaterials has attracted extensive attentions. fig. a showed the morphology of the electrospun pure pva fibrous films. as a typical electrospun fibrous film, the pva fibers were randomly oriented as a porous film with a wide fiber diameter distribution. further investigations have demonstrated that pva nfn membranes could be prepared by changing the solution properties, such as adding zno [ ] , sio [ ] , and nacl [ ] into the polymer solutions. the sem image of composite pva/zno nfn membranes is shown in fig. b . it can be observed that the composite fibers have many junctions among the fibers, showing poor fiber uniformity compared with the pure pva fibers. the average diameter of pva/zno fibers ( nm) was larger than that ( nm) of pure pva fibers due to its increased viscosity (from to cps). additionally, the formation of nano-nets was observed among the fibers. the electrospun pva/zno fibers acted as a support for the spider-web-like nano-nets comprising interlinked d nanowires. the average diameter of the pva/zno nanowires ( nm) contained in this nano-net was about one order magnitude less than that of conventional electrospun fibers. another example for nano-net formation is the pva/sio nano-nets by esn the suspension of pva and sio nanoparticles (fig. c) . the formation of nano-nets probably was due to the increased instability by blending the high content nanoparticles in polymer solution. recently, barakat et al. [ ] revealed that the addition of the utilized salts to the pva solution resulted in formation of nano-nets (fig. c) . despite the success in fabrication of pva nano-nets, the small coverage rate of current pva nfn membranes restricts the use of pva in more extensive fields. extensive research should be performed on developing more proficient strategies to regulate the structure of pva nfn membranes, and thus further improving the quality of nano-nets. pu presents a class of thermoplastic polymer that possesses a lot of excellent properties, such as good mechanical properties, excellent elastomeric properties and high durability [ , ] . meanwhile, d nanostructure has achieved a tremendous amount of attention in recent years due to their fascinating chemistry and size-, shape-, and material-dependent properties. [ , , ] . electrospinning has been paid a considerable attention due to production of micro-or nano-sized fibers by applying high electric fields [ , , ] . many investigators have attempted to apply various degrees of control to this process in order to produce pu fibrous membranes with the designed functions [ , [ ] [ ] [ ] . however, the main hurdle for further applications in ultrafiltration, special protective clothing, ultrasensitive sensors, etc. is the larger fiber diameter ( - nm) of current electrospun pu fibers. therefore, the procedures for manufacturing large-scale and ultrafine nanofibers with diameter less than nm still require more investigation [ ] . recent investigations have revealed that different additives (e.g. multiwalled carbon nanotube (mwcnts), salts, and surfactants) incorporated into the pu solutions could generate spider-web-like nano-nets with ultrathin nanowires [ ] . for example, kimmer et al. [ ] reported the fabrication of the composite pu/mwcnts nfn membranes containing a handful of nano-nets (fig. a) . the presence of the mwcnts promoted the creation of net-like structures in comparison with the pu nanofibers without mwcnts. they suggested that these nano-nets were created because of strong secondary electric fields occurring between mwcnts during the electrospinning process. tem analysis of the pu/ mwcnt nfn membranes showed that individual and very well aligned mwcnts (except for a small curvature at the junction point) occurred in the outer surface of the main nanofiber from which nanonets was created (fig. b ). in addition of mwcnts, kim et al. [ ] added nacl into the pu solution in which tetrahydrofolate (thf)/n,n-dimethylformamide (dmf) were utilized as solvents and found that nano-nets with small coverage rate were produced (fig. c) . to obtain nano-nets in large scale, hu et al. [ ] demonstrated that the addition of sls surfactant at the critical micelle concentration (cmc) to the pu solution resulted in a significant improvement in the nfn morphology (fig. d) . ptt, as a new member in the linear aromatic polyester family, combines the desirable mechanical properties of poly(ethylene terephthalate) with the outstanding processing characteristics of poly(butylene terephthalate), which makes it a promising candidate in both fiber and engineering applications [ , ] . of particular interest is recently developed nanocomposite technology consisting of ptt and nanofiller (e.g. cnt) because the obtained nanocomposites usually show fascinating properties and hence, both the structure and the performance of ptt/cnt nanocomposites have been studied extensively with the aim of exploring their potential applications [ , ] . more recently, ptt/cnt composite nfn membranes were fabricated by esn the melt compounding solution (fig. a ) [ ] . carboxylic surface functionalized cnts are well embedded by the ptt and oriented along the fiber axis during esn process, leading to bead-free and uniform fiber morphology as well as spider-web-like nano-nets. they ascribed the formation of nano-nets to the self-governed network of cnts. however, it is noteworthy mentioning that the determination of the actual composition of nano-nets should be carried out, whether the cnt composition existing in the nano-nets. just as the authors said that ''this is interesting and worthy of further study'' [ ] . nevertheless, the formation feasibility of nano-nets based on ptt polymer system has been well revealed. further studies are needed to focus on the controllable fabrication of ptt nfn membranes. peo, as a nontoxic, fully biodegradable, and biocompatible polymer, has attracted tremendous attention in the biomedical field for controlled drug release and tissue engineering [ , ] . over [ ] . Ó american chemical society), (c) gelatin (reprinted with permission from [ ] . Ó elsevier b.v.), (d) cs (reprinted with permission from [ ] . Ó walter de gruyter gmbh & co. kg), and (e) bombyx mori silk/swcnt (reprinted with permission from [ ] . Ó american chemical society) nfn membranes. the past few years, extensive studies have been done on electrospun peo fibers produced from peo/ water solutions to study the effects of process conditions and solution compositions [ , [ ] [ ] [ ] . on the other hand, cnc, as one of the strongest and stiffest natural biopolymer, is a suitable candidate for tailoring mechanical properties of polymer materials [ ] . they have been successfully used as highly effective reinforcing nanofillers for various electrospun polymer nanofibers [ , ] . additionally, because cncs are nontoxic, biocompatible, and biodegradable, they have a significant potential in developing fully biodegradable polymer nanocomposites for biomedical applications [ ] . considering excellent dispersion of cncs in water, the exploitation of an all-aqueous system for esn based on a combination of cncs with water-soluble polymers offers advantages in avoiding the use of organic solvents. recently, peo/cncs nfn membranes have been fabricated when increasing wood-based cnc content to wt% in the polymer solutions (fig. b) . the resultant heterogeneous composite nfn membranes were composed of rigid-flexible bimodal nanofibers, consisting of the primary nanofibers and secondary ultrafine nanofibers. the authors attributed this behavior to the unstable jet flow arising from the unbalanced forces among the surface tension, viscoelastic, and electrostatic forces. furthermore, they have demonstrated that the cncs and nano-nets effectively improved the mechanical properties of the nfn membranes [ ] . gelatin is a natural polymer that is derived from collagen, and is commonly used for pharmaceutical and medical applications because of its biodegradability and biocompatibility in physiological environments [ , ] . generally, gelatin is of two types (i.e. type a and type b) depending on the hydrolysis condition of isolation from collagen [ , ] . gelatin is a kind of temperature sensitive polymer. temperature has a profound effect on gelatin aqueous solution properties. zhang et al. [ ] demonstrated the electrospinning of gelatin aqueous solution by elevating the spinning temperature from to °c. previous studies have also revealed that gelatin nanofibers could be prepared from various organic solvents, such as , , -trifluoroethanol [ ] , formic acid [ ] , cosolvent (acetic acid/ethyl acetate/water) [ ] and so on. through some posttreatment or blending with other biodegradable polymer, gelatin can be used to prepare nanofibrous membranes for tissue scaffolds, wound healing and health caring devices, and other biomedical applications [ ] . for this reason, gelatin has been electrospun into various forms for wide applications [ , ] . recent studies have demonstrated that d gelatin nano-nets could be fabricated by regulating the solution properties and several process parameters during esn process [ ] . the spider-web-like nano-nets that comprise interlinked d ultrathin nanowires ( - nm) are stacked layer-by-layer and widely distributed in the d porous membranes (fig. c) . moreover, they revealed that the occurrence of rapid phase separation on the splitting-film and the formation of hydrogen bond among gelatin molecules during esn process were responsible for the formation of these spider-web-like nano-nets. cs is a natural biopolymer derived by the deacetylation of chitin, possessing unique polycationic, chelating, and film-forming properties due to the presence of active amino and hydroxyl functional groups [ , , ] . as a natural polymer, cs intrinsically exhibits a unique set of characteristics such as biocompatibility, biodegradability, antimicrobial activity, physiological inertness, non-toxicity remarkable affinity to proteins, and its adequate absorption capabilities [ , ] . these significant biological and chemical characteristics make cs as a desirable biomaterial used in many different fields, including medicine, food and chemical engineering, pharmaceuticals, cosmetics, nutrition, and agriculture [ , ] . particularly, nanofibrous cs membranes have a great potential to be widely used in various applications derived from its biocompatibility and biodegradability [ , ] . earlier work had revealed that electrospun fibers from pure cs were difficult, until when direct electrospinning of pure cs was performed by some researchers using thf and acetic acid as a solvent [ , ] . cs nano-nets have been fabricated by esn cs solutions with wt% acetic acid aqueous solution as solvent [ ] . it is worthy to mention that the nano-nets bonded with polymer beads other than electrospun fibers in cs nfn membranes (fig. d) , which could be attributed to the lower concentration ( wt%) of the cs solution. in this case (i.e. polymer concentration was lower than critical entanglement concentration), insufficient chain entanglements are present to fully stabilize the jet, and thus bead-on-string structure was formed. taking advantage of the large surface areas, cs nfn membranes as the template on which modified with polyethyleneimine (pei) have been used as sensing materials on qcm for humidity detection [ ] . silk is a well-described natural fiber obtained from silkworms (bombyx mori and antheraea mylitta) and spiders and has been used in textile industries from thousands of years. recently, net-like structures (containing very fine fibers - nm in diameter) occurring together with fibers with much higher diameters ( - nm) have also been identified in natural biopolymers [ ] , such as collagen spider silk [ ] and b. mori silk [ ] . because of these structures mimic natural tissue structure; they can be used as scaffolds for biomedical tissue engineering. such nano-net structure can also lead to enhanced mechanical properties in fibers prepared via electrospinning [ , ] . lam [ ] explained that the formation of net-like structure might be a result of the inclusion of single wall carbon nanotubes (swcnts) into the fibers. during electrospinning, the silk/swcnt bundles were expelled from the polymer jet under extremely high force and velocity, which caused opening of the bundles to form the nano-net structure. similarly, ayutsede and coworkers [ ] demonstrated the feasibility of fabricating nanocomposite b. mori silk/swcnt nfn membranes through the esn process (fig. e) . additionally, they reported the reinforcement effects of swcnt on the mechanical properties of electrospun silk nanofibers, which showed significant enhancement in young's modulus (up to %) compared to the pristine electrospun silk fibers. consequently, by adding swcnt into the silk fibroin solution, strong and tough multifunctional nanocomposite fibrous membranes may be fabricated, which may open the gateway to producing multifunctional fibers that may be suitable for weaving into textiles. these results indicate that the swcnt incorporated into the polymer solutions plays an important role in the formation of the nano-nets. despite the rapid increase of research on polymer-based nfn nanomaterials as documented in this section, several challenges need to be overcome to realize overall development of esn nfn materials. . esn is a newly emerging interdisciplinary field in materials science, nanotechnology, ehd and engineering. the design and fabrication of nfn membranes is one of the most promising scientific and technological challenges in the coming years. according to preparation process and formation of current polymer nfn materials, we found that polymers with hydrogen bonds favored creating interconnect nano-nets [ , ] , adding zno [ ] , sio [ ] , nacl [ ] , cnt [ ] , cnc [ ] and swcnt [ ] into the polymer solutions could lead to nano-nets, and changing processing parameters could influence the morphology of nfn membranes. however, pursuing the universal law for explaining the formation of nano-nets is a complicated problem. revealing the process's secret can help people to look for additional spinnable polymers. . it is worthy to mention that current nfn membranes are all organic-based materials. on the other hand, inorganic materials are potentially important for those applications in many areas that include electronics, photonics, mechanics, and sensing [ ] . fabrication of inorganic nfn membranes is of significant importance, which of course is a complicated process. nfn membranes have attracted considerable attention due to their unique fundamental properties and diverse polymer compositions, making them model systems for the observation of novel ehd phenomenon and ideal building blocks for future applications. however, in practical point of view it is necessary, ( ) to create nfn structure in large quantities among the d fibrous membranes and ( ) to provide a universal principle to readily control the structure of nfn membranes. similar to electrospinning, the esn process is also influenced by many parameters, classified broadly into solution parameters, process parameters, and ambient parameters. solution parameters include concentration, conductivity, viscosity, surface tension, and solvent and process parameters include applied voltage and tip to collector distance [ , , , ] . each of these parameters significantly affects the nfn morphology (e.g. fiber and nanowire diameter, pore-width and coverage rate of nano-nets) obtained as a result of esn, and through proper manipulation of these parameters we can obtain nfn membranes with desired morphology and diameters. in addition to these variables, ambient parameters encompass the temperature and humidity of the surroundings, which also play a significant role in determining the morphology of nfn membranes [ , , ] . table overviews the ''global effects'' on the nfn morphology one can expect by changing the various parameters. the typical nfn membrane is composed of traditional electrospun fibers and d spider-web-like nano-nets. the effects of solution concentration on the morphology of common electrospun fibers have been extensively studied [ , , ] . solution concentration determines the limiting boundaries for the formation of electrospun fibers due to variations in the viscosity and surface tension [ , ] . low concentration solution forms bead-on-string fibers due to the influence of surface tension and as the solution concentration increases, the shape of the beads changes from spherical to spindle-like and finally uniform fibers with increased diameters are formed due to the higher viscosity [ , , , ] . therefore, there should be an optimal concentration range for the electrospinning process, and higher polymer concentrations lead to increased fiber diameters within this optimal range [ , ] . here, a question may come to mind, how morphology changes for nano-nets when we regulate the solution concentrations? does the universal law of electrospinning process can be applied to the esn process? fortunately, several researchers have paid their attention to solve these problems. previous studies have documented the esn fabrication of polymers like paa [ ] , pa- [ ] , and gelatin [ ] in certain concentration levels. ding and co-workers investigated the morphologies of paa nfn membranes formed with various concentrations of paa solution [ ] . it can be observed that all the samples exhibit incomplete splitting of defect films and the paa ( wt%) nfn membranes show the largest average pore-width and diameter deviation of nano-nets among the three samples (fig. a) . as the paa concentration is increased to wt% (fig. b) , the pore-width and shape of the nano-nets become uniform and the average pore-width is decreased compared to the sample in fig. a . moreover, as shown in fig. b , the shapes of the pores in the nano-nets are circular, triangular, quadrangular, pentagonal and hexagonal, while paa ( wt%) nfn membranes exhibited circular pores with the smallest average pore-width among the three samples (fig. c) . applied voltage " fiber and nanowire diameter ;, coverage rate and pore-width of nano-net " (within optimal range) [ , ] tip to collector distance " fiber and nanowire diameter ;, pore-width of nano-net " [ ] ambient temperature " coverage rate and density of nano-net " initially ( - °c), then ; (> °c) [ , ] ambient humidity " fiber and nanowire diameter ;, coverage rate ;, fiber-sticking and beads " [ , , ] ": increasing the parameter; ;: decreasing the parameter. by increasing the concentration of pa- solution, a morphological transformation of electrospun products is seen from the beaded fibers with diameters lower than nm at a concentration of wt% to the ribbon-like fibers with widths of - mm at high concentrations ( fig. d-g) [ ] . the ribbon-like fibers are initially discernible by electrospinning of the wt% nylon solution, and finally become the dominant feature in the products obtained from the wt% solution. for nano-nets, they are barely seen from pa- solutions with low and high concentrations ( fig. d and e) , but prevail as an intermediate concentration is used (i.e. the concentration of wt%) (fig. f) . in the presence of nano-nets, the nfn membranes consisting of bimodal fiber diameter distribution have the advantage of capturing a wider range of particle sizes in the ultra-filtration application. as is evident from the discussions, the concentration of the polymer solution influences the spinning of fibers and splitting of nano-nets as well as nfn structure and morphology. despite numerous discussions offered in the previous literatures on this subject, there remain several unresolved issues with the formation of nano-nets as a function of solution concentration. for example, the relationship between solution concentration and electrospun fiber diameter has been well established, however the theoretical model of this relationship for nano-nets need much concern. most of the polymers are conductive, and the charged ions in the polymer solution have a high effect on jet formation [ , , ] . the ions increase the charge carrying capacity of the jet, and thus subjecting it to higher tension with the applied voltage [ ] . solution conductivity is mainly determined by the polymer type, solvent used, and the availability of ionisable salts [ ] . the conductivity of the solution can influence the fiber morphology [ ] . highly conductive solutions will be extremely unstable in the presence of strong electric fields, leading to significant decrease in the fiber diameter and dramatic bending instabilities and thus a broad diameter distribution [ ] . additionally, the formation and the resultant morphology of nano-nets are also highly depending on the solution conductivity [ ] [ ] [ ] ] . increasing the conductivity of the solution could enhance the instability of the taylor cone, which would greatly improve formation probability of microsized droplets and thus dense nano-nets [ ] . kim et al. [ ] demonstrated the effect of ions by adding ionic salt on the morphology of pa- , pva and pu nfn membranes. the electrical conductivity is proportional to the salt amount in the solution, which can be explained as increasing in the amount of the free ions in the solution (fig. e) . they found that pa- fibers with the addition of ionic salts like nacl, kbr, and cacl produced multi-layers spider-web-like nano-nets within the electrospun nanofibers (fig. ac) , while the nano-nets became trivial when a weak metallic acid (h ptcl ) was used (fig. d) . additionally, yang et al. [ ] reported that the atomic radii of ions also exhibited effects on the structure of nfn membranes and they investigated the morphology of paa nfn membranes containing the same content of nacl, agno and cu(no ) . ions with smaller atomic radius have a higher charge density and thus a higher mobility under an external electric field [ ] . thus the elongational forces imposed on the jet with nacl should be higher than that with agno and cu(no ) , since na + and cl À have smaller radius than ag + , cu + and no À . therefore, the paa/nacl composite membranes possessed the smallest average fiber and nanowire diameter. concerning the nano-net formation within the electrospun nanofiber, increase the salt concentration had distinct impact on the internal morphology of the nfn membranes (fig. ) . fig. a -c compares sem images of paa/nacl nfn membranes formed with different nacl concentrations. the asprepared paa fibers have a diameter ranging from to nm, in which a few nano-nets with ultrathin nanowire ($ nm) was observed. in order to manufacture extremely small nanofibers in large quantities and a uniform size, salt was added into the esn paa solutions. fig. b provides the morphology of paa/nacl nfn membranes containing . wt% nacl. the fibers show a dramatically decreased average diameter ( nm) which corresponds to the sharply increased conductivity (table ) . a high-magnification fe-sem image in fig. b clearly shows that dense nano-nets, stacked layer-by-layer, almost completely covering the electrospun fibers and a strong bonding among them was also formed. further increase in the nacl concentration (from . to . wt%) resulted in a fibersticking morphology as well as an increase in the diameter of the spun fibers (fig. c, table ), in contrast to the conventional view that a more conductive solution causes a reduction in diameter [ ] . this surface morphology change could be attributed to the localized charge effects on the surface of the fibers [ ] , as well as changed chain conformation of paa (from the extended linear conformation to the coil one) with increasing the ionic strength of the solution [ ] , causing the fibers to stick together during the formation process. additionally, hu et al. [ ] revealed that the nacl concentration plays an important role in determining the morphology of the pu nfn membranes. with increasing the nacl concentration, the morphology of pu nfn membranes was changed from no nano-nets to dense nano-nets (fig. d-f ). for pa- system, the pristine pa- electrospun fibrous membranes did not contain nano-nets (fig. g) , and very clear spider-nets fastening the main nanofibers are observed when the salt concentration was . wt% (fig. h) , while microballs was obtained instead of the nano-nets when the salt concentration was increased to . wt% (fig. i ) [ ] . increasing content of nacl brings increase in electric conductivity, changes rheological behavior and considerably influences spinnability of the solution. as shown in fig. i , fiber morphology was getting worse with the further increased nacl salt concentration ( . wt%) and more non-fibrous bodies appear in the fibrous membranes. the increased electric conductivity meanwhile enhanced the instability of taylor cone, which may lead to the formation of micro-sized droplets. the droplets may form porous microballs due to the fast phase separation with the evaporation of the solvents. in addition to salts, the solvents used in polymer solution for esn process is strongly related to the final morphology of nfn membranes. addition of formic acid to the paa solution increased the number of free ions in the solution as well as the electrical conductivity of the polymer solution, and hence improved the formation of nano-nets [ ] . in another report, wang et al. [ ] compared the gelatin nfn membranes obtained from formic acid and acetic acid, respectively, and found that gelatin solution from formic acid possesses the larger conductivity due to the high dielectric constant of formic acid, which favors forming thinner fibers and dense nano-nets. surface tension, more likely to be a function of surfactants added into the solution plays an important role in the common electrospinning process and by reducing the surface tension of a polymer solution, defect-free nanofiber can be obtained [ , ] . recent studies have shown that surface tension was likely to be the primary factor that contributed to regulate the morphology of the nano-nets [ , ] . inspired by the soap bubbles, which exhibits a highly stable steiner networks, yang et al. [ ] considered the addition of dbsa into the esn paa solutions to decrease the surface tension and mimic the fascinating structure of soap bubbles. the surfactant was so effective that a concentration as low as . wt% was enough to produce nano-nets with large area and uniform size (fig. a) . high magnification images (insets of fig. a-c) reveal that nano-nets are more regular due to a more stable jet that encounters fewer perturbations from the surface tension effect of reducing surface area. additionally, the average fiber and nanowire diameter were both slightly decreased with increasing the contents of dbsa in the solutions, which could be attributed to the lower surface tension and increased solution conductivity [ ] . recently, hu et al. [ ] also demonstrated the effect of surfactant in improving the structure of nfn membranes at cmc of surfactant in polymer solutions (fig. d-f ). addition of sodium laurylsulfonate (sls) at cmc to the pu solution results in significant improvement in nanofiber morphology as evinced from reduced fiber-sticking phenomenon and uniform fiber diameter (fig. e) . by close observation of fig. e , it can be seen that nano-nets are more regular due to more stable jet that encounters fewer perturbations from the surface tension effect of reducing surface area. addition of small amounts of nonionic surfactant was found to no obviously change the nanofiber morphology (fig. d) . for sls concentration at higher level (>cmc), pu/sls nfn membranes displayed defected nanofiber morphology (fig. f) , which could be attributed to that surfactants may self-assemble to form colloidal aggregates above cmc. therefore, polymer-surfactant interactions may modulate the molecular structure and interactions of polymer molecules thereby altering rheological and interfacial properties of polymer dispersions, which are critical factors in the successful preparation of nfn membranes by esn. for the electrospinnability of a polymer solution, the solvent plays a major role, and thus its selection is a crucial step in the electrospinning and even esn process. according to the investigation by ding et al. [ ] , the formation of nano-nets from defect films was considered to be due to the fast phase separation of polymer and solvent in the charged droplet, which is thought to be caused by solvent evaporation during the flight inside the high electric field. therefore, the volatility of the solvent is very critical as it determines the process of the phase separation. paa fibrous membranes formed from various kinds of solvents (h o, h o/ethanol, and ethanol) have been investigated. several discontinuous irregular defect films with maximum lengths of up to lm are found among the electrospun paa fibers that are spun from the solvent of h o (fig. a) , while the defect films partly split into nano-nets due to the fast phase separation of polymer and highly volatile solvent in the charged droplet when the cosolvent of ethanol and h o is used (fig. b) . it is worth noting that the fibers formed from the cosolvent show a slightly increased diameter ( nm) which corresponds to the increased viscosity ( cps) and decreased conductivity ( ls m À ) ( table ). in comparison with the fibers formed from the solvents of h o or cosolvent, the fibers formed from the sole solvent of ethanol show an increased average diameter ( nm) due to the sharply decreased conductivity [ ] . more interestingly, the formation of a few nano-nets with soap-bubble-like structure (fig. c) is observed among the main fibers. besides paa system, our group also studied the structure of gelatin nfn membranes as a function of different solvents (i.e. formic acid and acetic acid), and the result showed that gelatin nfn membranes obtained from formic acid exhibited better morphology ( fig. d and e) [ ] . fig. and table also summarize morphological observations of the pa- fibrous membranes for the various solvent systems. from these results, it is interesting to note that well-aligned uniform electrospun nanofibers and nano-nets (diameters in the range from to nm) of pa- can be produced from formic acid and its mixture solutions (fig. f-i) . however, the other single solvent system (i.e. chlorophenol and hexafluoroisopropanol) can also be used as solvents for pa- , in which the expected nano-nets are not produced (fig. j and k) . consequently, the formation of nano-nets in pa- nfn membranes can strongly depend on the polyelectrolytic behavior in the solvent. as a result, the electrical conductivity of the polymer solution was increased due to the formation of enormous free ions. by changing the polymer concentration and solvent ratio one can obtain nano-nets with uniform diameters [ ] . the applied electric field strength plays important roles in the morphology of the esn nfn membranes. the influence was so significant that earlier investigators held the point that only higher reprinted with permission from [ ] . Ó springer-verlag. [ ] . high voltage is able to generate more charges to the solution or droplet surface located at the tip of the needle as well as larger electrostatic forces, both of which stretch the jets fully for the favorable formation of the thinner fibers and completely split nano-nets [ , ] . fig. a -c shows the variation in morphology as the paa/nacl nfn membranes formed at applied voltages of , and kv, respectively. it was seen that increasing voltage decreased the average diameters of nanofibers and nanowires, while enhanced the pore-width and end breakages of the nano-nets [ ] . hu and co-workers have reported that the pores of the pu nano-nets were less erratic at the lower voltage of kv, possibly due to lower instability of phase separation process for the thin liquid film. additionally, they also found that increasing the voltage decreased the average diameter of the nanofibers and nanowires, and increased the pore width of the nano-nets from nm ( kv) to nm ( kv) (fig. d-f ) [ ] . similar behavior of applied voltage on nfn morphology is also observed by ding et al. [ ] . they have showed the area density of nano-nets in pa- nfn membranes sharply increased along with the applied voltage and ascribed this result to the increase of bead defect density along with increasing applied voltage (fig. g-i) . the structure of esn nfn membranes is easily affected by the tip to collector distance because of their dependence on the deposition time, evaporation rate, and whipping or instability interval. recent investigations have demonstrated that a minimum distance was required to give the electrospun fibers sufficient time to dry before reaching the collector, otherwise with distances that are either too close or too far, beads would form [ ] . although the effect of tip and the collector distance on fiber morphology is not as significant as other parameters, the studies on this aspect have been done with esn of pa- , gelatin and pu. for example, ding et al. [ ] examined the morphological changes in esn pa- nfn membranes with variations in the distance between the tip and the collector (fig. ) . they showed that lesser tip-collector distance was responsible for the relatively large wire diameter of about - nm and strong bonding between fibers and nano-nets (fig. b) . as the distance increased to cm, the relatively sufficient expansion of nano-nets enabled them to form thinner wire diameter ( - nm) as well as larger pore-width at this long spinning distance (fig. d) . apart from solution and processing parameters, ambient parameters that include temperature relative humidity (rh), etc., also influence the structure and morphology of esn nfn membranes. investigations have been conducted to examine the effects of ambient parameters (i.e., temperature and humidity) on the esn process. wang et al. [ ] investigated the effect of low ambient temperature ( °c) on the morphology of gelatin nano-nets and found that the area density of nano-nets in membranes sharply decreased along with the decreased temperature from to °c (fig. a and b) . however, they did not investigate the morphology of nano-nets when the ambient temperature was at higher state. therefore, for a supplement, wang et al. [ ] investigated the formation of pa- /bacl nano-nets at much higher ambient temperature of °c (fig. d) . it is obvious that the coverage rate of nano-nets was decreased with increased temperature (from to °c), which indicated that °c was the optimal temperature ( fig. c and d) . additionally, the average diameter ( nm) of the nanofibers increased at higher temperature. increasing the temperature will accelerate the evaporation rate of the solvent and thus taking a shorter time for the jet to solidify, which favors the less elongation of the jet and the formation of thicker fibers. at a lower temperature, the evaporation rate of the solvent decreases exponentially with temperature. the polymer chains have more freedom to move at higher temperatures, resulting in lower solution viscosity. thus, these two effects may play negative role in nano-net formation and result in the decreased coverage rate of nano-nets. according to recent progress in controllable fabrication of polymer based nfn membranes, one can find that the rh plays an important role in formation of nano-nets. ding et al. [ ] observed that paa fibrous membranes electrospun in an atmosphere of rh more than % showed the fiber-sticking phenomenon without the appearance of nano-nets, and they attributed this structure to the solubility of paa in water and the retarded evaporation of solvents caused by the high rh. in another report, the effect of rh on the morphology of paa/nacl composite nfn membranes was also studied, and the results suggested that the microsized defect films were partly split into nano-nets at a rh of % (fig. a) and sticking ribbon-like fibers with small parts of nano-nets were observed when the rh was increased to % (fig. b) [ ] . besides of paa system, the variation in humidity while esn gelatin solutions has also been studied and shows that by increasing humidity there is an appearance of bead-on-string structured fibers in the membranes (fig. c) [ ] . the situation is much better for esn pu solution; it was possible to produce nfn morphology for the pu system even in a high humidity environment ( % rh) (fig. d) . in comparison with the samples obtained at lower humidity, the fibers and nanowires in the d network became thinner and a slight fiber-sticking structure could be observed [ ] . the past few years have witnessed significant progresses in applications of the electrospun nanomaterials, demonstrated by numbers of recent scientific publications on this area [ , , , [ ] [ ] [ ] . nfn membranes possess almost all the applications that traditional electrospun fibrous membranes have, and due to its much smaller fiber diameter and unique net-like structure, which bring esn nfn membranes highly attractive to some special area, such as ultra-fine filters to intercept viruses, ultrasensitive sensors, as well as biomedicine. a schematic diagram illustrating the perspective application areas are summarized in fig. . it should be realized that most of these applications have not reached their industry level, but just at a laboratory research and development stage. however, their promising potential is believed to be attracting attentions and investments from academia, governments, and industry all over the world. atmospheric and liquid environment pollutions caused by the uncontrolled development of industrial technologies, has now reached the dangerous point, which leads to the optimal selection of environmental filtration and separation technologies [ , , ] . it was estimated that future filtration market would be up to us $ b by the year [ , ] . nonwoven fibrous membrane as a kind of porous media has been widely used in different filtration applications, from indoor air filter to personal protective equipment (e.g. n respirator) [ ] . for filtration, the channels and structural elements of a filter must be matched to the size of the particles or droplets that are to be captured in the filter (fig. ) . to filter increasingly finer particles, a transition from micro-sized to nano-sized fibers is required. in addition, it is generally understood that the smaller the fiber diameter, the better are their filtration performance [ , [ ] [ ] [ ] . nanofibers provide dramatic increases in filtration efficiency at relatively small decreases in permeability [ , [ ] [ ] [ ] . additionally, nanofiber filter media also demonstrate improved filter lifetime and more contaminant holding capacity in many laboratory tests and actual operating environments [ ] . nfn possess the general properties and functions of conventional electrospun nanofibers, as well as the fascinating feature characters (i.e. extremely small diameter, unique geometric characteristic, controllable coverage rate) donated by the novel d nano-nets structure. it has been realized that esn technique is rising to the challenge of providing solutions for the removal of unfriendly particles in such submicron ranges. recently, our group has developed a new concept of two-tier filter by esn depositing pa- nfn membranes (as core filter medium) on the nonwoven polypropylene (pp) scaffold (fig. ) [ ] . it [ ] . reprinted with permission from [ ] . Ó royal society of chemistry. was found that the morphology of the nfn membranes and the filtration performance towards nacl aerosols (diameter $ nm) were both strongly dependent on the esn time and addition of chloride (fig. ) . fig. c compares the esn time dependence-filtration efficiency of pa- /pp and pa- / bacl /pp composite membranes. the filtration performance undergoes a dramatical rise and reaches a steady value after min. the filtration efficiency is increased with the prolonging of esn time, which is induced by the increased fiber mass and thickness. additionally, the pa- membranes doping with bacl show a better filtration performance (up to . %) than pristine pa- fibers, which further demonstrating the key role of nano-nets played in enhancing the filtration efficiency ( fig. a and b) . furthermore, the stacked nano-nets among the conventional electrospun fibers can greatly improve the porosity of pa- /bacl /pp composite membranes, which can account for the lower pressure drop compared with pa- /pp composite membranes (fig. d) . based on the fascinating features of nano-nets and the filtration mechanism, the nfn membranescontaining filters possess the following three outstanding advantages: ( ) a much higher efficiency and lower pressure drop for particulate filtration. considering the simple fiber filtration theory involving only a single fiber, smaller fiber diameter may lead to higher filtration efficiency. the nano-nets in nfn membranes played the role of ''second doorkeeper'' that could intercept particles smaller than its aperture size, which may further increase the filtration efficiency. meanwhile, the high porosity of nano-nets facilitated the flow of airflow through the membranes and thus resulted in decreased pressure drop; ( ) the two-tier filter allowed the particles captured at the surface other than deep inside, which facilitated the cleaning of nfn/pp composite filters through air back blowing and/or mechanical shaking and thus improved the filter lifetime [ ] ; ( ) in the event of the same filtration performance, the deposition amount of nfn membranes was much smaller than that of electrospun membranes, which significantly reduced the thickness of filtering membranes and made them more lightweight [ ] . [ ] . reprinted with permission from [ ] . Ó royal society of chemistry. even though nfn membranes have exhibited great advantages over conventional media in environmental applications, such as air and water filtration, there remain a large number of challenges. the challenges include the mass production strategies of high quality nfn membranes and nfn-based composites, the selection of suitable materials and of appropriate chemistry to introduce the desired functionality to meet specific needs [ ] . additionally, nfn membranes show the promising application as filters to intercept viruses and bacteria such as influenza a (h n ) virus, severe acute respiratory syndrome (sars) virus, and escherichia coli [ ] , however, further verify its feasibility by experiments is imperative. none of these challenges are trivial but they are also not insurmountable. developments of nanomaterials have provided researchers with an opportunity to construct electronic interfaces with components whose sizes are comparable to the size of molecules, potentially leading to a much more efficient interface [ ] [ ] [ ] . nanometer cross-section of nanomaterials gives them enhanced surface sensitivity, and allows them to utilize the benefits of size effects, such as quantization and single-molecule sensitivity [ , ] . taking advantages of comparatively large surface area and high porosity, nano-nets create enhanced interconnectivity and additional surface area and facilitate the diffusion of analytes into the nfn membranes, which make them attractive candidates for use in ultrasensitive sensors [ ] . this is one of the most desirable properties for improving the sensitivity of sensors because a larger surface area will absorb more analytes and change the sensor's signal more significantly [ ] . in this section, we will review recent progress in the development of esn nfn materials having applications in two predominant sensing approaches (i.e. qcm and colorimetric sensors), illustrate them with current examples showing how they have been applied, and discuss their intrinsic fundamentals and optimal designs. it will emphasize trends, but will also highlight gaps requiring further research. a comprehensive summary of sensors based on the esn nfn materials is illustrated in table . qcm as a mass sensitive device is widely used for detecting very small mass changes of even less than a nanogram deposited on electrode surface [ ] . through surface modification of the electrodes with sensitive materials, qcm devices have been used as highly sensitive sensors for environmental monitoring, chemical and biochemical analysis due to their sensitive interface measurement capability [ , ] . sauerbrey [ ] first derived the mass-frequency shift relation for quartz crystal resonators as follows: where df the measured frequency shift, f o the fundamental frequency of a bare qcm chip, dm the mass change per unit area, a the electrode area, q the density of quartz, and l the shear modulus of quartz crystal. up to now, enormous materials such as metals, ceramics, polymers, self-assembled monolayers, dendrimers, lipids, and waxes [ , ] , have been used as sensitive coatings on qcm to improve the sensor sensitivity and selectivity for chemical analytes. qcm sensors have been demonstrated to be versatile in various applications, whereas one major challenge lies in that the flat electrode surface limits the immobilization degree of the absorbing sites per unit area [ ] . to solve this problem, increasing attention has been paid to the development of nanostructured coatings on qcm to improve the sensor sensitivity, including nanotube [ , ] , nanofibers [ ] , nano-assembled thin films [ ] , and nanogold hollow balls [ ] . taking advantage of large specific surface area of the nanostructured sensing materials, the performance of the qcm sensors is greatly enhanced. in this part, we review recent progress in the development of esn nfn materials having applications in qcm sensing approaches. . . . . relative humidity (rh) sensing. humidity monitoring is significant to a wide variety of fields, such as meteorology, process controlling, agriculture, textiles, and manufacturing [ ] [ ] [ ] . it is well known that the sensitivity of chemical sensors towards a specific analyte is enhanced by increasing the specific surface area of the sensing materials [ ] . growing efforts have been put in the development of nanostructured materials based humidity sensors to further improve the sensing characteristics [ , ] . among various sensing strategies used to detect humidity such as impedance [ ] , capacity [ ] , and optics [ ] , qcm have been emerging as very stable sensors for humidity detection [ ] . on the other hand, many approaches for fabricating humidity-sensitive membranes such as spin coating [ ] , dip coating [ ] , layer-by-layer self-assembly [ ] , and ink-jet printing [ ] , have been reported. as a nanofabrication technique, esn has been identified as a remarkably robust and versatile method for fabricating fibrous membranes with net-like structure. the remarkable specific surface area and high porosity provide esn nanomaterials potential application in ultrasensitive and highly miniaturized sensors [ ] . wang et al. [ ] fabricated a humidity sensor by electrospinning and esn deposition of nanofibrous polyelectrolyte membranes as sensitive coatings on qcm. in their rh testing system, the controlled humidity environments were achieved using water injections into the chamber, which yielded the required humidity ranging from % to %. the sensing properties of the sensors to humidity were examined by measuring the resonance frequency shifts of qcm due to the additional mass loading. the results of sensing experiments shown in fig. c indicated that the response of the sensors increased by more than two orders of magnitude with increasing rh from % to % at room temperature, exhibiting high sensitivity and that, in the range of - % rh, the log(df) showed a good linearity (fig. c, inset) . the sensitivity of fibrous composite paa/pva membranes (fig. a) was two times higher than that of corresponded flat films at % rh. compared with fibrous paa/pva membranes, the paa nfn membranes (fig. b ) exhibited remarkably enhanced humidity sensitivity due to their high paa content and large specific surface caused by the formation of ultrathin nano-nets among electrospun fibers, which can facilitate fast mass transfer of water molecules to and from the interaction region [ ] . additionally, the developed sensors exhibited good long term stability (fig. d) . the measurements were repeated at °c every days for month. the variation of frequency shift is less than % at each humidity region. directly esn sensing materials onto qcm electrode provides one-step strategy to fabricate highly sensitive qcm sensors. however, this strategy is only appropriate for those materials that can be esn, which will inevitably limit the application of more sensing materials on qcm. therefore, it is urgent to explore other general platforms to meet these challenges. recently, a simple two-step strategy for fabricating rh sensing coatings on qcm has been developed, which include the esn deposition of nfn membranes on the surface of a qcm resonator followed by the surface modification process [ , ] . for instance, pei modified cs (pei/cs) nfn membranes were used as sensing materials coated on qcm for humidity detection (fig. ) [ ] . increasing the coating load of the membranes introduced more absorption sites in the sensing membranes, and hence greatly enhanced the sensor sensitivity (fig. a ). fig. b shows the response curve of sensor coated with pei/cs membranes with the rh changing from % to %. when the rh was increased from % to %, the response time were , , , , , , and s, respectively. additionally, the resultant sensors possessed good log(df) linearity ( - % rh), wide rh range of response, and small hysteresis, which exhibited great potential for humidity sensing applications at room temperature operations [ ] . although the pei/cs nfn membranes-based qcm sensors have presented good performance to rh sensing, a main drawback of using cs nfn membranes as substrate is the lower productivity of cs nfn membranes due to the low concentration ( wt%). therefore, a few researchers have made effort to esn deposition other polymer-based nfn membranes (e.g. pa- ) as substrates on qcm (fig. ) [ , ] . recent study of wang and coworkers revealed the pei functionalized pa- (pei/pa- ) nfnbased qcm sensor that exhibited high sensitivity and fast response/recovery time ( s/ s) to humidity, which outperform flat film-based sensors and even current porous structure-based sensors (fig. a) . the frequency changed approximately three orders of magnitude with rh varied from % to %. repeated humidity cycling tests demonstrated stable device operation and high repeatability of sensor response (fig. b) . additionally, the resultant sensors also presented relatively small hysteresis with a maximum humidity hysteresis rate of . % at % rh (fig. c) , indicating a good reliability of the humidity sensor. fig. d shows the possible humidity sensing mechanism of qcm-based pei/ pa- nfn sensor. for low rh levels, the response of the qcm sensor is dependent on water molecules adsorbed/desorbed masses on nfn membranes, whereas for increasing rh levels variations in interlayer expansion stress of nfn membranes derived from swelling effect becomes prevalent. several possible reasons may be responsible for the improved sensor properties: ( ) the spider-web-like nano-nets can provide an effective channel for the fast mass transfer of the water molecules toward and away from the interaction region, as well as greatly reduced interfacial areas between nanofibers and substrate; ( ) the piezoelectric qcm is an ultrasensitive mass-measuring device and has been used as a mass sensor in the air phase. therefore, the tiny mass of water molecules adsorbed in the fibrous membranes could be detected and calculated. this study demonstrates that nfn structured materials are of the potential applications for fabricating high performance humidity sensors [ ] . . . . . trimethylamine (tma) sensing. tma can cause headache and nausea, severe skin burns, as well as irritation to the eyes and the respiratory tract. safe exposure of ppm tma as a time weighted average concentration for up to a h workday has been standardized by the world health organization [ ] . on the other hand, tma has attracted considerable research attention in recent years as an indicator for food freshness, in particular for fish, seafood, meat, wine, and milk. therefore, the detection of tma is a very important problem in food industry and fish-processing industry. recently, it was demonstrated that paa/nacl composite nfn membranes could be used as sensing materials in a tma-selective qcm sensor [ ] . fig. a and b depicts the response of membranes coated qcm sensors with three kinds of structures, exposed to increasing tma concentrations ranging from to ppm. the response caused by the additional mass loading was primarily attributed to fig. . schematic diagrams illustration of (a) a gas testing system for rh detection and (b) sensing coating of pei/pa- nfn on qcm gold electrode. reprinted with permission from [ ] . Ó royal society of chemistry. the interaction between the tma molecules and the carboxyl groups of paa (fig. c, inset) . the rapid response time, approximately s to reach the % of the maximum value for each gas exposure, is comparable to similar tma exposure levels on polyaniline (pani)-tio nano-composite coated qcm sensor ($ s) [ ] . it is obvious from the figures that the gas sensor based on paa/nacl ( . wt%) composite nanofibrous membranes exhibits the best performance, which may be attributable to the abundant of active sites available for faster gas diffusion into the membranes because of the high surface and volume ratio of the nano-nets. additionally, the resultant sensors coated with paa/nacl nfn membranes exhibited an excellent selectivity to tma (fig. c) . according to the sauerbrey equation, df is directly proportional to the attached mass of gas molecules. the absorption behavior of tma molecule in nano-nets is a fickian process and fickian equation can thus be expressed as [ ] : where df t and df are the frequency changes at any time, t, and at equilibrium, respectively. therefore, the diffusion coefficient (d) can be determined from the initial slope of the absorption curve plotted in the form of df t /df versus t / /h (fig. d) . as a result, paa/nacl ( . wt%) composite nanofibrous membranes showed the largest d value ( . Â À cm /s), which may be due to enhanced interconnectivity and additional surface area induced by the large density of nano-nets. nanonets facilitate the diffusion of analytes into the membranes and significantly boosted the gas sensing properties [ ] . . . . . formaldehyde sensing. formaldehyde, one of the most serious indoor air pollutants among volatile organic compounds (vocs), can cause central nervous system damage, immune system disorders, as well as asthma and nasopharyngeal cancer [ ] . the detection of formaldehyde in air is of reprinted with permission from [ ] . Ó royal society of chemistry. significance for environmental monitoring and process control because of its high toxicity. the world health organization (who) has set a safe exposure limit of ppb ( min average) and the established limit by chinese environmental protection agency (epa) is ppb averaged over min [ , ] . considerable efforts have been made to develop formaldehyde-selective qcm sensors in the past few years [ , , [ ] [ ] [ ] . various sensing structures such as molecularly imprinted polymers films [ ] , electrospun fibrous pei/pva membranes [ ] , pei modified electrospun polystyrene (ps) (pei/ps) nanoporous fibers [ ] , pei modified bacterial cellulose [ ] have been deposited on qcm for formaldehyde detection. however, all of these qcm sensors have a limited ability to detect formaldehyde at low concentrations. therefore, it is very important to develop a rapid, simple and ultrasensitive device for detection of gaseous formaldehyde. taking advantage of several attractive features such as extremely large specific surface area, high porosity, large stacking density and strongly tight adhesive force to the devices, the nfn structured membrane (fig. e) can lead to enhancement in the facilitation of the analytes diffusion and oscillation transmission into the membranes and thus can detect formaldehyde at lower concentration level [ , ] . more recently, an effective formaldehyde detection system was developed by esn depositing pa- nfn membranes on qcm and then modifying sensing pei onto the nfn membranes (fig. a-d) . after pei modification, the d porous fibrous membranes with nfn structure were still maintained in the samples (fig. f ). when exposed to ppm of formaldehyde, qcm-based pei/pa- nfn ( kv) sensor has achieved the largest response value, approximately three times as much as pei flat film coated sensor. to detect formaldehyde at very low concentration, they developed a moisture-compensating-based detecting method, i.e., firstly injected the dilute formaldehyde aqueous solution ( wt%) into the chamber and then eliminated the influence of moisture. fig. a shows the responses of qcmbased pei/pa- nfn sensors upon injection of water and the dilute formaldehyde solution. the response of moisture-compensating qcm sensor exposing to formaldehyde was shown in fig. b , indicating that the pei/pa- nfn sensors exhibits a reduced detection limit for formaldehyde down to ppb and a rapid response time (< s). in comparison with previous works using qcm platform for formaldehyde sensing, the present qcm-based pei/pa- nfn sensor had not only the lowest detection limit but also the fastest time response (table ). additionally, the sensor showed the highest response to formaldehyde with a value of up to hz, while for other gases the response is less than hz, implying a high selectivity to formaldehyde and can be considered as an excellent candidate for gas sensing applications (fig. c) . furthermore, the pei/pa- nfn nanostructure reported here possesses a higher sensitivity compared with the pei-ps structure, which further demonstrated the advantages of the nfn structure (fig. d) . the lower sensitivity of the pei-ps sensor could be due to the energy dissipation during vibration transfer in soft ps membranes on qcm as well as the blocking phenomenon on the surface of porous fibers after pei modification. increasing public health and environmental protection demands have motivated researchers to look for more convenient detecting methods for low concentration of pollutants. among various alternatives, a considerable attention has been given to the colorimetric sensor array system due to its effectiveness, simplicity, low cost and allows assays to be detected with the naked eye [ , ] . the investigation of nfn nonmaterial-based colorimetric detection strategy is really a novel topic and only few reports have been developed so far [ ] . the determination of heavy metals in the environment has been an important worldwide concern since their dangerous effects on the ecosystem and human health depend on dose and toxicity. copper, one of the heavy metals, is also an essential trace element in the human body and plays pivotal roles in a variety of fundamental physiological processes in organisms. the epa gives an enforceable drinking water standard for cu + of . ppm to prevent potential health problems and a secondary cu + standard of ppm for aesthetic considerations. hence, sensitive, facile, and accurate detection of the levels of cu + in water is especially important from the practical point of view. ding et al. [ ] reported a novel, ultrasensitive, selective and flexible sensor strip based on pani/pa- nfn membranes for naked-eye colorimetric detection of cu + ions in water. the sensing mechanism involves the transformations between different oxidation and doping forms of pani. fig. a and b presents the changes of fe-sem and optical images that accompany the treatment of pani/pa- nfn membranes with hydrazine aqueous solution. during the process, pani emeraldine salt in nfn membranes reacted with the hydrazine, and the semiquinone segments in polymer backbone were reduced into benzenoid segments yielding the pani leucoemeraldine base, and thus the color changed from green to white vividly. upon exposure to cu + aqueous solution, the sensors exhibit two significant reflectance intensity decreasing bands at and nm which induce the color changes from white to blue dramatically ( fig. c and d) . this new sensor shows colorimetric response specifically to cu + ions (white-to-blue color change) over other possible interfering metal cations and allows for detection of cu + in aqueous solution with a low detection limit of ppb observing by naked eye. additionally, the colorimetric responses could be quantitatively visualized by using a color-differentiation map prepared form converted rgb (red, green and blue) values (fig. ) . with increasing cu + concentration, the three parameters were decreased within different rates; the r (red) parameter has the highest decreasing rate while the b (blue) parameter has the lowest one, resulting in the dark blue of the sensor strips (fig. a) . furthermore, the as-prepared pani/pa- nfn sensor strips could successfully combine with the color map (fig. b) , which suggested a promising analytical method as an economical alternative to traditional cu + sensors and also provided a new insight into the design and development of novel colorimetric sensing system based on the nfn platform. another colorimetric sensor based on nfn membranes has been developed for formaldehyde detection. the exploitation of highly efficient sensors for the detection of formaldehyde continues to be a significant scientific endeavor due to its possible threat to public health. up to now, numerous formaldehyde-induced color changes of sensors that are composed of arrays of metalloporphyrins [ , ] , ph indicators [ ] [ ] [ ] , chromotropic acid [ ] , and nanoporous pigments [ , ] have been successfully designed to detect formaldehyde with high selectivity. however, current colorimetric sensors for formaldehyde often use polymer film [ ] , cellulose tape [ , ] or even glass [ ] as platform for immobilization of colorimetric sensing elements, which will inevitably cause relatively slow response and low sensitivity. therefore, it is urgent to explore other general platforms to meet these challenges. wang et al. [ ] demonstrated a label-free colorimetric sensor strip for real-time formaldehyde detection based on methyl yellow-impregnated pa- nfn membranes (fig. ). when the sensor strip was exposed to formaldehyde, the methyl yellow on the tape reacted with sulfuric acid produced by the reaction of hydroxylamine sulfate with formaldehyde to produce a yellow-to-red color change (figs. and ) [ ] . the degree of color change could be recorded by measuring the intensity of reflecting light ( nm). this novel sensor strip not only maintains the high surface area and porosity of nfn structure, but also effectively improves the reflectance signal. taking advantage of extremely large specific surface area and high porosity of the nfn structured membrane, the as-prepared sensors offers easy handling, as well as ultrasensitive and selective color change for gaseous formaldehyde, achieving detection limit as low as ppb by naked eye (fig. a) . the kinetic sensing response was studied by continuously monitoring the reflectance spectra of sensor strips upon exposure to ppm formaldehyde as a function of time, which showed that the reflectance intensity kept decreasing with increasing reaction time and the reflectance intensity at nm varied nonlinearly with the exposure time between and min (fig. b) . moving forward, it is envisioned that this strategy will facilitate the design and development of other label-free colorimetric sensors toward various analytes in the future. knowing that the essential properties of protective clothing are high moisture vapor transport, increased fabric breathability, and enhanced toxic chemical resistance [ , ] , esn nfn membranes have been recognized as potential candidates for these applications [ ] . actually, as the supporter of nano-nets, conventional electrospun nanofibers have played an important role in defense and [ ] . reprinted with permission from [ ] . Ó elsevier b.v. security applications as protective materials. kang et al. [ ] developed waterproof-breathable fabric by applying electrospun web of pu directly onto the substrate fabric ( fig. a and b) . the electrospun pu fibers/fabric showed higher air permeability and vapor transmission properties than resin-coated fabrics, which could be attributed to the open porous structure of electrospun fibrous membranes. electrospun pp webs and laminates were developed using melt-electrospinning to explore an alternative way of manufacturing protective clothing materials for agricultural workers [ ] . the electrospun pp layer significantly enhanced barrier performance for challenge liquids. the potential of electrospun webs for future protective clothing systems has also been investigated. the u.s. army natick soldier center studied enhancement of barrier materials via a fine layer of electrospun fibers, focusing on preventing penetration of chemical warfare agents in aerosol form [ ] . they found that the electrospun fibrous membranes of pa- , polybenzimidazole, polyacrylonitrile and pu provided good aerosol particle protection, without a significant change in moisture vapor transport of the system. as mentioned above, pu have been used successfully in clothing and textile coatings for garments, such as rain coats and industrial safety clothing, and they are distinguished as being comfortable to wear and easy to care for [ ] . lee and obendorf [ ] prepared protective clothing materials by electrospinning pu fibrous membranes onto nonwoven fabric substrates. barrier performance of layered fabric systems with an electrospun pu fiber web layer was evaluated against challenge liquids of different physicochemical properties. the air permeability of layered fabric systems is higher than most protective clothing materials currently in use. the water vapor transmission rates of layered fabric systems were in a range comparable to typical woven work clothing fabrics [ ] . thandavamoorthy et al. [ ] demonstrated that self-assembled pu nanofibrous membranes with honeycomb-like structure are very useful in protective liners and filters for chemical warfare, because the honeycomb filters can trap and retain particles more efficiently, providing enhanced adsorption and filtration capabilities (fig. c) . similarly, here we can expect that pu nfn membranes interspersing with d soap bubble-like or honeycomb-like structured nano-nets can also be a potential candidate for protective clothing application (fig. d) [ ] . besides pu nfn membranes, pant and coworkers demonstrated the fabrication of spider-web-like pa- nfn membranes containing tio nanoparticles, with superior mechanical strength, high hydrophilicity, and good antimicrobial as well as uv blocking ability [ ] . these spider-web-like organicinorganic nanocomposite nfn membranes with good uv blocking capacity and antimicrobial properties would have great potentiality for different kinds of protective clothing. as shown in fig. a , the reflectance decreased as the amounts of tio in the composite mats increased because of the absorbance of uv light by tio nanoparticles for transferring the electron from the valance band to the conduction band, indicating that the novel pa- /tio nanocomposite nfn membranes can be used for uv blocking. additionally, the survival numbers of bacterial cells decreased with increasing amounts of tio in the composite nfn membranes, suggesting good bactericidal ability (fig. b) . via the esn technique, which produces very thin membrane-like webs with very small pore size, the obtained protective materials provide resistance to liquid penetration while allowing moisture [ ] . Ó springer-verlag. sem images of (c) self-assembled pu nanofibrous membranes with honeycomb-like structure (reprinted with permission from [ ] . vapor and thermal transport. in addition, controlling pore size by varying the area density of electrospun fiber and the pore-width of nano-nets would be useful for developing materials with different levels of protection and thermal comfort depending on the need and use [ ] . furthermore, combined with other functional materials through composite spinning or surface modification, multifunctional nanocomposite nfn membranes can be facilely prepared. the mechanical properties of fibrous membranes are highly depending on the fiber structure, geometrical arrangement of the fibers, individual fiber properties and interaction between fibers [ , , ] . recent investigations have demonstrated that the size of the fiber influenced their tensile response. for example, wong et al. [ ] determined the tensile deformation of individual pcl fibers and found that the mechanical properties (i.e. tensile strength and tensile modulus) were enhanced when the diameter of the fibers was reduced below the critical diameter (fig. ) . they ascribed the enhanced properties of finer diameter fibers to the gradual ordering of the molecular fig. . uv-visible spectra and antimicrobial efficiency of pristine pa- and different pa- /tio nanocomposite nfn membranes. reprinted with permission from [ ] . Ó elsevier b.v. fig. . plot of tensile modulus and tensile strength versus fiber diameter. tensile modulus increases with decreasing fiber diameter. these results can be attributed to the better molecular orientation and crystallinity in smaller fiber diameters. larger than lm, both tensile modulus and tensile strength appear invariant with fiber diameter. reprinted with permission from [ ] . Ó elsevier b.v. chains, modest increase in the crystallinity of the fibers, and the densely packed lamellae and fibrillar structures. based on these observations, nfn membranes containing ultrathin nanowires possess great potential as reinforcements for development of high strength/high toughness materials and materials with good thermal and electrical conductivity. from the point of view of nfn nature, the existence of spider-web-like nano-nets further enhanced the mechanical properties of nfn membranes, which could be attributed to the extremely small fiber diameter and the net-like structure. zhou et al. [ ] demonstrated that mechanical performances of heterogeneous -peo/cnc fibrous membranes with nano-nets were stronger than their homogeneous counterparts ( -peo/cnc fibrous membranes) for all compositions ( - wt% cnc contents). for the -peo/cnc nanofibrous membranes (fig. ) , the values of maximum tensile stress (r max ) and young's modulus (e) increased with increased cnc contents (from to wt%) from . ± . to . ± . mpa and . ± . to . ± . mpa, respectively, which correspond to the increased density of nano-nets in the nfn membranes. these results indicated that the especially heterogeneous network microstructure was beneficial to the mechanical properties of electrospun composite nanofibrous membranes. because the crystallinity of the peo/cnc composite nanofibrous membranes is lower than that of the peo nanofibrous membranes, their enhanced mechanical properties should be attributed to the efficient stress transfer from peo to cncs originating from the uniform dispersion and high alignment of cncs in the peo matrix. such reinforcements also benefited from the formation of strong hydrogen bonding between the peo matrix and the cncs [ ] . moreover, the sem-based morphologies of heterogeneous -peo/cnc- nfn membranes in different stages of the tensile process were also studied (fig. ) . before the yield point (i.e., the initial stage of tensile testing), nfn morphology (fig. a) did not show obvious changes, which was ascribed to the strong interaction among the nanofibers originating from the strong polar of peo and cncs. the improved young's modulus of heterogeneous nfn membranes (compared with homogeneous mats) could be attributed to the strong bonding and entanglement between nano-nets and primary nanofibers. increasing the tensile stress, most of the nanofibers in the nfn membranes gradually reached their tightened state and aligned along the tensile direction (fig. b) due to the destroyed bonding points between nanofibers. additionally, many nano-nets were also broken off from the primary fibers, which could defer the rupture of primary nanofibers and thus increase the elongation at break for nfn membranes. further increasing the tensile stress, most nanofibers were necked and fractured; leading to the final nfn membranes rupture (fig. c) . from the point of view of nfn reinforced polymer composites, nano-nets reinforced nfn membranes can significantly enhance the mechanical integrity of the polymer matrix compared to common electrospun fibers. additionally, nano-nets bring nfn structured membrane several advantages as reinforcement materials: ( ) the materials saving and the reduced brittleness of the matrix. to achieve the same reinforcement effect as electrospun fibers, smaller loadings of nfn in the matrix are required. ( ) the enhanced mechanical strength caused by the interlocking mechanism, the high percentage of porosity and irregular pores between the fibers can lead to an interpenetrated structure when dispersed in the matrix. ( ) the improved impact strength of the reinforced matrix induced by the large specific surface area between the nanofibers and the matrix promotes relaxation processes. despite these attractive advantages of nfn membranes used as reinforcement materials, the number of investigations on this topic is rather small. the research on nfn reinforcement is still at its beginnings. tissue engineering is a multidisciplinary field that contains both the principles of engineering and life sciences for the development of biological substitutes and also for restoration, maintenance, or improvement of tissue function [ , , [ ] [ ] [ ] . target tissue and organs include skin, bones, cartilage, lymph knots, arteries, muscles, as well as heart, lung, and liver tissue [ , , ] . nanofiber is an emerging material which plays a pivotal role in tissue engineering by serving as matrices for cellular in growth, proliferation, differentiation, and new tissue formation in three-dimensions due to their high surface area to volume ratios and high porosity of the fibers [ , , ] . an inherent feature of nanofibers is that they mimic the extracellular matrices (ecm) (i.e. a complex composite of fibrous proteins such as collagen and fibronectin, and glycoproteins) of tissues and organs [ ] . esn technique allows preparation of nanofibers and nano-nets in dimensions which resemble closely those of the cellular cytoskeleton or the diameter of collagen fibers within the ecm [ , , ] . nfn membrane is highly porous which is favorable for cell proliferation and the mechanical properties of the scaffold can be matched to those of the tissue to be replaced. in comparison with other fiber fabrication methods such as self-assembly and phase separation techniques, esn provides a simpler and more costeffective strategy to produce fibrous scaffolds with an inter-connected pore structure and fiber diameters in the sub-micron range. during the past few years, the field of application for esn nfn membranes has evoked much attentions [ , , ] ; a series of natural polymers, such as gelatin [ ] , b. mori silk [ ] , cs [ ] and synthetic polymers such as pa- [ , , ] , pa- [ ] , paa [ , ] , pu [ , ] , pva [ , ] , peo [ ] , and ptt [ ] have been esn into nfn structures. such esn nfn materials have great potential application in tissue engineering, immobilized enzymes, wound healing, artificial blood vessels, drug delivery, and so on. natural polymers (e.g. collagen, alginate, silk protein, hyaluronic acid, fibrinogen, cs) are often used for preparing nanofibrous scaffolds because of their enhanced biocompatibility and bio-functional motifs, as well as their blending into synthetic polymers can improve the overall cytocompatibility of the scaffold [ , ] . collagen, for instance, is usually used as a scaffold for cells since it makes up a major component of the ecm [ ] .gelatin is a natural polymer that is derived from collagen by controlled hydrolysis, and is commonly used for pharmaceutical and medical applications because of its biodegradability and biocompatibility in physiological environments [ , ] . li et al. [ ] demonstrated that electrospun gelatin nanofibrous scaffolds with diameter ranging from to nm could support attachment and growth of human embryonic palatal mesenchymal (hepm) cells (fig. a ). more recently, our group have demonstrated the large-scale fabrication of gelatin nfn membranes by regulating the solution properties and several process parameters during esn process [ ] . gelatin nano-nets that comprise interlinked d ultrathin nanowires ( - nm) are stacked layer-by-layer and widely distributed in the d porous membranes exhibited great potential application in tissue engineering (fig. b) , although this application was not investigated in this study. b. mori silk is another proteins found that have been esn into nfn structures. ayutsede and coworkers have demonstrated the feasibility of fabricating nanocomposite nfn membranes comprising b. mori silk and swcnt through the esn process [ ] . further study is needed for exploiting their applications in tissue engineering and other biomedical applications. a variety of synthetic polymer nanofibers have been considered for use as scaffolds for engineering tissues such as plga [ ] , plla [ ] , pcl [ ] , pa- [ ] , and pu [ ] . however, the limited properties of synthetic polymer nfn membranes with only one component restrict their application in biomedical engineering. therefore, the feasibility of incorporating natural polymers into polymer to form composite nfn membranes has revealed esn as an attractive technique to meet several specific functional applications. kim's group pioneered the application of nfn membranes in tissue engineering [ , , ] . they have demonstrated the one-step preparation of cs/pa- composite nfn scaffolds via esn technique for the osteoblastic cell culture applications [ ] . fig. a -d shows the fe-sem images of cs/pa- composite nfn membranes for the different concentration of cs. the diameter of the composite nanofibers were observed to be in the range of - nm, whereas the nano-nets consisted of regularly distributed very fine nanofibers with diameters of about - nm. the density of high aspect ratio fibers increases with increasing the concentration of cs. the adhesion, viability and proliferation properties of osteoblast cells on the cs/pa- blended nanofibers were analyzed by in vitro cell compatibility test. the results showed that the cells spread over the scaffold fibers, linked with fibers by cytoplasmic extensions (fig. e-h) . the mtt ( -[ , -dimethylthiazol- -yl] , -diphenyl tetrazolium bromide) and lactate dehydrogensae (ldh) level appeared to be increased with increasing cs concentration, and this was attributable to the density of nanofibers ( fig. i and j) . however, the mtt level of all the samples was observed to be higher than that of the negative control. the in vitro cytotoxicity evaluation of the cs/pa- blended nanofibers indicated that this scaffold material was nontoxic for the osteoblast cell culture. in another work, they used lecithin to replace the cs and successfully prepared lecithin/pa- homogenously composite nfn membranes. lecithin/ pa- nfn membranes showed non-cytotoxic behavior for the osteoblast cell culture [ ] . besides pa- based nfn membranes, they also investigated the preparation of pu nfn membranes blended with cacl for osteoblastic cell culture applications. the cacl was adapted in the composite scaffolds owing to its cell permeability and cellular metabolic activity. the mtt and ldh measurements of the composite nfn membranes demonstrated the cytocompatibility of this novel scaffold. because of good attachment and formation behaviors, there is a potential to utilize pu/cacl composite nfn scaffold for the bone regeneration applications [ ] . this article presents a comprehensive review of the progress in a novel nanomaterials fabrication technique (i.e. esn technique), starting from an overview about conventional electrospinning technique (the rudiment of esn technique), continuing with the illustration of esn technique (historical background and basic setup), and finishing with the discussion on the proposed formation mechanisms of nfn structures. additionally, we also present the fundamental properties (e.g. extremely small diameter, high porosity, steiner tree network geometry, controllable coverage rate, and controllable density) of nfn nanomaterials. the selection of polymers used for esn is outlined by the introduction of various polymer based nfn materials such as pa- , paa, pva, gelatin, cs, and pu. the following section fully highlights the state-of-the-art strategies for the controllable fabrication of nfn membranes based on the regulation of solution, processing, and ambient parameters. the remarkable level of a synthetic control for nfn nanostructures and their rich morphologies at the nanoscale has paved the way to the unique applications in the fields of environment, energy and healthcare. it has become evident that the fundamental properties of esn nfn membranes (e.g. extremely small diameter, high porosity, steiner tree network geometry, controllable coverage rate, controllable density) made it compelling for various engineering applications. intensive efforts have been devoted to exploring the fundamental structure control and outstanding applications. novel structure with d soap-bubble-like structured or spider-web-like nano-nets supported by conventional electrospun nanofibers have not been observed earlier from most conventional d electrospun nanomaterials. research publications in the past several years indicate that the importance of nfn nanomaterials might surpass traditional electrospun fibers in the development of filtration and ultrasensitive sensors. while electrospinning technique is at its mature stage to overcome the technological barrier, esn nfn nanomaterials are being extensively investigated as it overcomes the bottleneck problem of electrospinning technique. both esn and d nfn membrane as novel technique and new structured material still face many challenges ranging from formation theory and fabrication to the final practical applications and there are a number of areas where future work should be directed. the formation mechanism of the nfn structures is complicated, which relate to physical, chemical, ehd processes, etc., and thus consensus on the formation mechanism has not been reached currently. the main confused problem is this: what happens to the charged polymer jets and droplets during such small distance from tip to collector? theoretical modeling, calculation and prediction of this process, its collective behavior and inherent mutual interaction, have received little attention, which will potentially hinder the rapid process in this field. there have been so far only few publications dealing with the interpretation and theory background behind the observed phenomena of esn process. thorough understanding the formation mechanisms of nano-nets makes for preparing optimal nano-nets with ideal structure and extending the range of spinnable polymers. thus, more rigorous experimental and theoretical work is also required to consummate this novel technique. the design and fabrication of nfn membranes is one of the most promising scientific and technological challenges in the coming years. according to preparation process and formation of current polymer nfn materials, we found that polymers with hydrogen bonds favored creating interconnect nano-nets, adding zno, sio , nacl, cnt, cnc, and swcnt into the polymer solutions could lead to nano-nets, and changing processing parameters could influence the morphology of nfn membranes. however, pursuing the universal law for explaining the formation of nano-nets is a complicated problem. revealing the process's secret can help people to look for additional spinnable polymers. as described in the section of polymers and polymer composites used in esn process, as can be seen, current nfn membranes are all organic-based materials while the esn of materials other than polymers has not yet received much attention. particularly, inorganic materials are potentially important for those applications in electronics, photonics, mechanics, sensing, etc. as far as we know, fabrication of inorganic nfn membranes via esn process needs incorporation of inorganic materials or their precursors into the esn polymer solutions and followed by the calcinations process. in order to obtain the inorganic nano-nets after calcinations, the content of inorganic materials should be as much as possible, which will certainly influence the solution properties and final morphology of resultant membranes. therefore, several core issues such as the homogeneity of the composite solutions, the optimization of calcinations technique, and the brittleness of the inorganic fibrous membranes still require more investigation. we have shown in this review that nfn nanostructures have become one of the popular research pursuits due to the unique properties and wide range of potential applications. amongst various applications, the nfn based filters is very important as it can prevent harmful nanoparticles and even virus from getting into the environment and the human body, which fulfill the rising demand for healthier and safer living and working environments. however, there are still many unresolved issues, such as the mass production of nfn materials, the compounding technology between nfn membranes and nonwoven scaffold and the experimental validation of their filtration performance to viruses, which need to be investigated both experimentally and theoretically. additionally, fabrication of nanostructured sensing materials may significantly improve their performances in the existing devices or open the doors to new types of applications. the ability to narrow the compatibility gap between sensing devices and nanomaterials, however, remains a major challenge in the field. the past several years have witnessed a remarkable progress in 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nanofibrous tubes with controllable architectures magnetic-field-assisted electrospinning of aligned straight and wavy polymeric nanofibers electrospinning has nanofibers in alignment electrospinning of polymeric and ceramic nanofibers as uniaxially aligned arrays electrospinning of collagen nanofibers formation of nanofiber crossbars in electrospinning preparation and anisotropic mechanical behavior of highly-oriented electrospun poly(butylene terephthalate) fibers patterning of electrospun fibers using electroconductive templates collecting eectrospun nanofibers with patterned electrodes biomimetic super-hydrophobic micro/nanoporous fibrous mat surfaces via electrospinning direct fabrication of highly nanoporous polystyrene fibers via electrospinning investigation of silica nanoparticle distribution in nanoporous polystyrene fibers flat polymer ribbons and other shapes by electrospinning microscale polymeric helical structures produced by electrospinning controlled assembly of polymer nanofibers: from helical springs to fully extended fabrication of necklace-like structures via electrospinning large-scale synthesis of necklace-like single-crystalline pbtio nanowires firecracker-shaped'' zno/polyimide hybrid nanofibers via electrospinning and hydrothermal process rice grain-shaped tio mesostructures-synthesis, characterization and applications in dye-sensitized solar cells and photocatalysis bio-mimic multichannel microtubes by a facile method multi-core cable-like tio nanofibrous membranes for dye-sensitized solar cells oriented contraction: a facile nonequilibrium heat-treatment approach for fabrication of maghemite fiber-in-tube and tube-in-tube nanostructures nanowire-in-microtube structured core/shell fibers via multifluidic coaxial electrospinning zno hollow nanofibers: fabrication from facile single capillary electrospinning and applications in gas sensors direct fabrication of composite and ceramic hollow nanofibers by electrospinning thermo-responsive core-sheath electrospun nanofibers from poly (n-isopropylacrylamide)/polycaprolactone blends electrospun nanofibers as sorbent material for solid phase extraction formation of fibers by electrospinning fabrication of blend biodegradable nanofibrous nonwoven mats via multi-jet electrospinning multiple jets in electrospinning: experiment and modeling high throughput tip-less electrospinning via a circular cylindrical electrode electro-spinning and electro-blowing of hyaluronic acid edge electrospinning for high throughput production of quality nanofibers nanomanufacturing using electrospinning reducing electrospun nanofiber diameter and variability using cationic amphiphiles electrospun polymer nanofibres with small diameters electrospinning of polyacrylonitrile solutions at elevated temperatures fabrication and application of novel two-dimensional nanowebs via electrospinning the beat nano-web formation by the electrospinning at various electric fields preparation of atactic poly (vinyl alcohol)/sodium alginate blend nanowebs by electrospinning electrospun nanomaterials for ultrasensitive sensors a highly sensitive humidity sensor based on a nanofibrous membrane coated quartz crystal microbalance formation of electrospun nylon- /methoxy poly(ethylene glycol) oligomer spider-wave nanofibers large-scale fabrication of two-dimensional spider-web-like gelatin nano-nets via electro-netting electrospinning: a fascinating fiber fabrication technique buckling of jets in electrospinning studies on the electrospun nylon nanofibers from polyelectrolyte solutions: . effects of solution concentration and temperature carbon nanotube reinforced bombyx mori silk nanofibers by the electrospinning process polyurethane/multiwalled carbon nanotube nanowebs prepared by an electrospinning process ultrafine electrospun polyamide- fibers: effect of solution conditions on morphology and average fiber diameter dynamics of field-induced droplet ionization: time-resolved studies of distortion, jetting, and progeny formation from charged and neutral methanol droplets exposed to strong electric fields droplet dynamics changes in electrostatic sprays of methanol-water mixtures fabrication of spherical colloidal crystals using electrospray electrohydrodynamic atomization in the cone-jet mode physical modeling of the liquid cone and jet jet break-up in electrohydrodynamic atomization in the cone-jet mode electrokinetic transport in microchannels with random roughness electrostatic spraying of liquids in cone-jet mode charge and fission of droplets in electrostatic sprays time-resolved high-speed camera observation of electrospray electrospun nylon- spider-net like nanofiber mat containing tio nanoparticles: a multifunctional nanocomposite textile material effect of successive electrospinning and the strength of hydrogen bond on the morphology of electrospun nylon- nanofibers microstructural control and selective c h oh sensing properties of zn sno nanofibers prepared by electrospinning fabrication of continuous highly ordered mesoporous silica nanofibre with core/sheath structure and its application as catalyst carrier highly porous electrospun polyvinylidene fluoride (pvdf)-based carbon fiber nanofibrous lipid membranes capable of functionally immobilizing antibodies and capturing specific cells novel ultrathin composite membranes of nafion/pva for pemfcs predictions of effective physical properties of complex multiphase materials steiner minimal trees geometric conservation laws for perfect y-branched carbon nanotubes steiner trees'' between cell walls of sisal formation of high aspect ratio polyamide- nanofibers via electrically induced double layer during electrospinning effect of solvents on high aspect ratio polyamide- nanofibers via electrospinning preparation and electrical characterization of polyamide- / chitosan composite nanofibers via electrospinning lecithin blended polyamide- high aspect ratio nanofiber scaffolds via electrospinning for human osteoblast cell culture effect of the electrospinning process on polymer crystallization chain conformation in nylon- and nylon- effect of polymer molecular weight on the fiber morphology of electrospun mats formation and properties of nylon- and nylon- /montmorillonite composite nanofibers photocatalytic and antibacterial properties of a tio /nylon- electrospun nanocomposite mat containing silver nanoparticles phospholipid nonwoven electrospun membranes the reactivity of plasma phospholipids with lecithin: cholesterol acyltransferase is decreased in fish oil-fed monkeys functional electrospun nanofibrous scaffolds for biomedical applications construction of ureteral grafts by seeding urothelial cells and bone marrow mesenchymal stem cells into polycaprolactone-lecithin electrospun fibers biomedical modification of poly (l-lactide) by blending with lecithin preparation of polyamide- /chitosan composite nanofibers by a single solvent system via electrospinning for biomedical applications synthetic metals: a novel role for organic polymers (nobel lecture) about supramolecular assemblies of p-conjugated systems polyaniline: a polymer with many interesting intrinsic redox states electrospinning and alignment of polyaniline-based nanowires and nanotubes polyaniline: electrochemistry and application to rechargeable batteries polyaniline nanofiber gas sensors: examination of response mechanisms synchronized polymerization and fabrication of poly(acrylic acid) and nylon hybrid mats in electrospinning ultra-fine polyelectrolyte fibers from electrospinning of poly(acrylic acid) electrospun nanofibrous membranes coated quartz crystal microbalance as gas sensor for nh detection electrospinning and solution properties of nafion and poly (acrylic acid) the influence of branching and intermolecular interactions on the formation of electrospun fibers poly(vinyl alcohol)/poly(acrylic acid) hydrogel coatings for improving electrode-neural tissue interface polyaniline dispersions. . poly (vinyl alcohol) and poly (n-vinylpyrrolidone) as steric stabilizers preparation and characterization of a nanoscale poly(vinyl alcohol) fiber aggregate produced by an electrospinning method fabrication of a super-hydrophobic nanofibrous zinc oxide film surface by electrospinning mechanical behavior of electrospun polyurethane elastomeric electrospun polyurethane scaffolds: the interrelationship between fabrication conditions, fiber topology, and mechanical properties the synthesis and fabrication of one-dimensional nanoscale heterojunctions multilayer polypeptide nanoscale coatings incorporating il- for the prevention of biomedical device-associated infections fabrication of microropes via bi-electrospinning with a rotating needle collector melt dispersion and electrospinning of non-functionalized multiwalled carbon nanotubes in thermoplastic polyurethane electrospinning of polyurethane fibers d modeling of filtration process via polyurethane nanofiber based nonwoven filters prepared by electrospinning process mechanical properties of fibers made of polytrimethylene terephthalate electrospinning of poly(trimethylene terephthalate)/carbon nanotube composites crystallization behavior of poly (trimethylene terephthalate)/multi-walled carbon nanotube composites electrospinning bombyx mori silk with poly (ethylene oxide) electrospun polyethylene oxide/cellulose nanocrystal composite nanofibrous mats with homogeneous and heterogeneous microstructures the effects of solution properties and polyelectrolyte on electrospinning of ultrafine poly (ethylene oxide) fibers controlled deposition of electrospun poly (ethylene oxide) fibers preparation and evaluation of the electrospun chitosan/peo fibers for potential applications in cartilage tissue engineering cellulose nanocrystals: chemistry, self-assembly, and applications reinforcing poly (e-caprolactone) nanofibers with cellulose nanocrystals nanofiber composites of polyvinyl alcohol and cellulose nanocrystals: manufacture and characterization electrospun gelatin/polyurethane blended nanofibers for wound healing gelatin as a delivery vehicle for the controlled release of bioactive molecules electrospinning of gelatin fibers and gelatin/pcl composite fibrous scaffolds gelatin nanofibrous membrane fabricated by electrospinning of aqueous gelatin solution for guided tissue regeneration electrospinning and mechanical characterization of gelatin nanofibers characterization of gelatin nanofiber prepared from gelatin-formic acid solution evaluation of cross-linking methods for electrospun gelatin on cell growth and viability applications of chitosan for improvement of quality and shelf life of foods: a review selective antimicrobial activity of chitosan on beer spoilage bacteria and brewing yeasts preparation and characterization of stable chitosan nanofibrous membrane for lipase immobilization preparation and characterizations of anisotropic chitosan nanofibers via electrospinning electrospun chitosan-based nanofibers and their cellular compatibility electrospinning of chitosan electrospinning of chitosan dissolved in concentrated acetic acid solution quartz crystal microbalance-based nanofibrous membranes for humidity detection: theoretical model and experimental verification electrospinning of single wall carbon nanotube reinforced aligned fibrils and yarns fabrication of titania nanofibers by electrospinning transport properties of electrospun nylon nonwoven mats amphiphobic nanofibrous silica mats with flexible and high-heatresistant properties the change of bead morphology formed on electrospun polystyrene fibers electroluminescence in conjugated polymers experimental investigation of the governing parameters in the electrospinning of polymer solutions an introduction to electrospinning and nanofibers electrospinning: applications in drug delivery and tissue engineering structure and process relationship of electrospun bioabsorbable nanofiber membranes self-sustained thin webs consisting of porous carbon nanofibers for supercapacitors via the electrospinning of polyacrylonitrile solutions containing zinc chloride electrospinning of porous silica nanofibers containing silver nanoparticles for catalytic applications poly(acrylic acid) nanofibers by electrospinning electrospun nanofibers with associative polymersurfactant systems the charge effect of cationic surfactants on the elimination of fibre beads in the electrospinning of polystyrene electrospun polyacrylonitrile/zinc chloride composite nanofibers and their response to hydrogen sulfide experimental investigation of the governing parameters in the electrospinning of polymer solutions formation of high aspect ratio polyamide- nanofibers via electrically induced double layer during electrospinning electrospun silk biomaterial scaffolds for regenerative medicine tunable fabrication of three-dimensional polyamide- nano-fiber/nets for high efficiency fine particulate filtration gan nanofibers based on electrospinning: facile synthesis, controlled assembly, precise doping, and application as high performance uv photodetector mechanical and wettable behavior of polyacrylonitrile reinforced fibrous polystyrene mats deciphering glycan linkages involved in jurkat cell interactions with gold-coated nanofibers via sugar-displayed thiols developments in filtration: what is nanofiltration? high-flux thin-film nanofibrous composite ultrafiltration membranes containing cellulose barrier layer nanofibres in filter media filtration of nano-aerosol using nanofiber filter under low peclet number and transitional flow regime thin-film nanofibrous composite membranes containing cellulose or chitin barrier layers fabricated by ionic liquids zno@graphene composite with enhanced performance for the removal of dye from water high flux ultrafiltration membranes based on electrospun nanofibrous pan scaffolds and chitosan coating electrospun nanofibrous filtration membrane structure effects on electro-osmosis in microporous media nanofiber webs from electrospinning ultrasensitive and highly selective gas sensors based on electrospun sno nanofibers modified by pd loading bionanoelectronics with d materials the physical chemistry of materials seeing graphene-based sheets acoustic wave microsensor arrays for vapor sensing synchronized quartz crystal microbalance and nanoplasmonic sensing of biomolecular recognition reactions a layered mesoporous carbon sensor based on nanopore-filling cooperative adsorption in the liquid phase the use of quartz oscillators for weighing thin layers and for microweighing surface acoustic wave chemical sensor arrays: new chemically sensitive interfaces combined with novel cluster analysis to detect volatile organic compounds and mixtures cyclodextrin functionalized mesoporous silica films on quartz crystal microbalance for enhanced gas sensing the application of cnt/nafion composite material to low humidity sensing measurement adsorption of cationic dye (methylene blue) from water using polyaniline nanotubes base nanoporous polystyrene fibers functionalized by polyethyleneimine for enhanced formaldehyde sensing nano-assembled thin film gas sensors. iv. mass-sensitive monitoring of humidity using quartz crystal microbalance (qcm) electrodes. sensor actuat nanogold hollow balls with dendritic surface for hybridization of dna humidity sensors: principles and applications humidity sensitive properties of crosslinked and quaternized poly ( -vinylpyridine-co-butyl methacrylate). sensor actuat humidity sensor using mutually reactive copolymers containing quaternary ammonium salt and reactive function nanofibrous polyethyleneimine membranes as sensitive coatings for quartz crystal microbalance-based formaldehyde sensors high-sensitivity humidity sensor based on a single sno nanowire carboxylation multi-walled carbon nanotubes modified with liclo for water vapour detection highly sensitive and stable humidity nanosensors based on licl doped tio electrospun nanofibers a capacitive humidity sensor based on gold-pva core-shell nanocomposites a fibre-optic humidity sensor based on a porous silica xerogel film as the sensing element nano-polyaniline thin films as humidity sensors prepared using spin coating technique electrical properties of low-temperature polymeric humidity sensor prepared by dip coating method self-assembly of polyelectrolytic multilayer thin films of polyelectrolytes on quartz crystal microbalance for detecting low humidity humidity sensors fabricated with photo-curable electrolyte inks using an ink-jet printing technique and their properties g protein-coupled receptor mediated trimethylamine sensing polyaniline-tio nano-composite-based trimethylamine qcm sensor and its thermal behavior studies quartz crystal microbalance sensor for detection of aliphatic amines vapours evaluation of the health impact of lowering the formaldehyde occupational exposure limit for quebec workers sol-gel based sensor for selective formaldehyde determination highly sensitive gaseous formaldehyde sensor with cdte quantum dots multilayer films mist sensing of formaldehyde and toluene with quartz crystal microbalance coated with lipid film using ultrasonic atomizer a piezoelectric method for monitoring formaldehyde induced crosslink formation between poly-lysine and poly-deoxyguanosine the fabrication and characterization of a formaldehyde odor sensor using molecularly imprinted polymers formaldehyde sensors based on nanofibrous polyethyleneimine/bacterial cellulose membranes coated quartz crystal microbalance a colorimetric sensor array for odour visualization a colorimetric sensor array for organics in water interfacially polymerized metalloporphyrin thin films for colorimetric sensing of organic vapors detection of volatile organic compounds using porphyrin derivatives a simple and highly sensitive colorimetric detection method for gaseous formaldehyde an automatic monitor of formaldehyde in air by a monitoring tape method development of a monitoring tape for formaldehyde using hydroxylamine sulfate and methyl yellow colorimetric determination of formaldehyde in air using a hanging drop of chromotropic acid a colorimetric sensor array for identification of toxic gases below permissible exposure limits colorimetric sensor array for determination and identification of toxic industrial chemicals development of formaldehyde sensing element using porous glass impregnated with schiff's reagent. sensor actuat a facile and highly sensitive colorimetric sensor for the detection of formaldehyde based on electro-spinning/netting nano-fiber/nets. sensor actuat some fascinating phenomena in electrospinning processes and applications of electrospun nanofibers application of electrospun polyurethane web to breathable water-proof fabrics developing protective textile materials as barriers to liquid penetration using meltelectrospinning protective textile materials based on electrospun nanofibers use of electrospun nanofiber web for protective textile materials as barriers to liquid penetration self-assembled honeycomb polyurethane nanofibers thermal conductivity enhancement of carbon fiber composites lattice boltzmann modeling of the effective thermal conductivity for fibrous materials effect of fiber diameter on tensile properties of electrospun poly(epsilon-caprolactone) electrospinning of polymeric nanofibers for tissue engineering applications: a review carbohydrate engineered cells for regenerative medicine designing a binding interface for control of cancer cell adhesion via d topography and metabolic oligosaccharide engineering metabolic glycoengineering: sialic acid and beyond a novel size-tunable nanocarrier system for targeted anticancer drug delivery well-defined, reversible disulfide cross-linked micelles for ondemand paclitaxel delivery electrospinning approaches toward scaffold engineering: a brief overview in vivo compatibility and degradation of crosslinked gelatin gels incorporated in knitted dacron electrospun protein fibers as matrices for tissue engineering electrospinning of nano/micro scale poly (l-lactic acid) aligned fibers and their potential in neural tissue engineering human osteoblast cytotoxicity study of electrospun polyurethane/calcium chloride ultrafine nanofibers key: cord- - s ok uw authors: nan title: abstracts of the th annual symposium of the protein society date: - - journal: protein science doi: . /pro. sha: doc_id: cord_uid: s ok uw nan c-terminus glutamine-rich sequence deleted) to elucidate the role of metalloprotein hpn-like by fluorescence resonance energy transfer (fret) (figure ) [ ] . we found the selective coordination of ni(ii) and zn(ii) to the purified sensors and in e. coli cells. surprisingly, specific interaction between the fret sensors and bi(iii) was observed. our fret analysis confirmed the role of hpnl for ni(ii) storage and revealed the potential association of hpnl with bi-based antiulcer drugs in cells. pb- rna fate is controlled by highly-regulated rna binding proteins molecular mechanism that links the mrna-degradation pathway with extracellular signaling networks through the reversible unfolding of a rna binding domain (rbd). rna binding is also controlled by ph conditions. this finding becomes relevant for rbps such as t-cell intracellular antigen (tia- ), which shuttles between two cellular compartments (nucleus and cytoplasm) with slightly different ph values. in fact, rna binding by tia- is modulated by slight environmental ph changes due to the protonation/deprotonation of tia- histidine residues [ , ] . the ph dependence of the tia- /rna interaction provides a new insight into the function of tia- in recognizing new rna targets [ ] , like the ' terminal oligopyrimidine tracts ( tops) of translationally-repressed mrnas. along with tia- , the rbp hu antigen r (hur) is involved in the assembly/disassembly of cytoplasmic stress granules (sg), which arise as a protective mechanism by preventing mrna decay under stress situations. despite wide acceptance that rbps harboring aggregation-promoting prion related domains (prds), such as tia- , stimulate rapid self-association and formation of sgs, we propose that scaffolding sgs may be driven by rbds, since prd-lacking rbps, like hur, often form oligomers [ , , ] and are included in sgs. under continuous stress, the transition from the physiological to pathological aggregation of rbps in sgs may depend on post-translational modifications of rbds. rna-binding proteinopathies, characterized by the nucleation of irreversible sgs, are often found in neurodegenerative diseases. altogether, resulting insights into rna biology suggest that highly-regulated rbps determine mrna fate from synthesis to decay. a threat to million people in underdeveloped nations around the world, african trypanosomiasis (sleeping sickness) is a neglected tropical disease (ntd) caused by the protozoan parasite trypanosoma brucei (t. brucei). t. brucei is transmitted to humans via the tsetse fly, and replicates in the blood before crossing into the brain, causing death for the infected individual. current treatments that are available for african sleeping sickness are highly toxic and usually difficult to administer past the blood-brain barrier. it is our belief that coupling less toxic compounds with efficient drug delivery systems will contribute to the development of the most effective drug against african sleeping sickness. our goal was to determine a novel and effective chemical inhibitor with the potential to prevent the replication of t. brucei in the human body. the enzyme target for inhibition studied in this research was -phosphogluconate dehydrogenase ( pgdh), a cytosolic enzyme in the pentose phosphate pathway (ppp) of t. brucei. pgdh is essential in the ppp due to its ability to oxidize -phosphogluconate into ribulose- -phosphate, which is essential for the formation of nucleotides. primer overlap extension polymerase chain reaction (pcr) was used to synthesize the coding dna sequence of the pgdh gene, which was then cloned into a pnic-bsa inducible expression plasmid with an n-terminal histidine tag, by way of ligation independent cloning. the protein was then expressed in bl (de ) escherichia coli (e. coli) cells and purified via nickel column affinity and size exclusion fast protein liquid chromatography (fplc) to perform inhibition assays. through virtual screening, various ligands obtained from the chembridge library and nih clinical collection) were docked into the active site of the crystal structure of tb pgdh (pubchem identification pgj) using gold molecular docking software. the top scoring compounds were selected by utilizing parameters such as hydrophobic interactions, hydrogen bonds, and van der waals forces. the compounds with the best scores that also satisfied lipinski's rule of criteria for druggability were then tested in spectrophotometric enzyme inhibition assays monitoring the absorbance of nadph at nm. compounds that show inhibitory activity in the assays will be taken to higher levels of testing to determine their effect on t. brucei in other organisms. nmr studies of the structural influence of phosphopantetheinylation in nonribosomal peptide synthetase carrier proteins and impact on binding affinities andrew goodrich , dominique frueh nonribosomal peptide synthetases (nrpss) are modular enzymatic systems responsible for the production of complex secondary metabolites in bacteria and fungi. each module is comprised of (at least) three core domains whose combined action leads to the selection, activation, and incorporation of a single small molecule into a growing peptide. central to each module is the carrier protein (cp), which is first primed via attachment of a '-phosphopantetheine moiety (ppant arm) to a conserved serine to generate the active holo form. an adenylation (a) domain then covalently attaches an amino or aryl abstract acid onto the ppant arm via formation of a thioester. the cp then shuttles activated monomers and growing peptides between the active sites of catalytic domains in both the same and adjacent modules. during cp priming and peptide elongation, a cp thus exists in multiple different post-translational states and interacts with numerous catalytic domains. understanding how nrpss are able to efficiently orchestrate this series of sequential protein-protein interactions between a cp and its partner catalytic domains is key to unraveling the molecular mechanism of nrp synthesis. using a combination of isothermal titration calorimetry and nuclear magnetic resonance (nmr) titrations, we found that converting a cp from the apo to holo form alters its affinity for its partner a domain. this change in binding suggests a means by which directionality in protein-protein interactions is achieved in nrpss. however, we also found that a domain binding affects the same subset of residues in both the apo and holo forms. in order to identify the molecular features underpinning this difference in affinity, we solved the nmr solution structures of the apo and holo forms of the cp. here, we present the solution structures of an apo and holo cp and discuss them in light of their differential binding to an a domain. functional analysis of of conditional analog-sensitive alleles of essential protein kinases in the fission yeast schizosaccharomyces pombe. juraj gregan , mfpl/imp, the genome of the fission yeast schizosaccharomyces pombe encodes for protein kinases that are essential for viability. studies of the essential kinases often require the use of mutant strains carrying conditional alleles. to inactivate these kinases conditionally, we applied a recently developed chemical genetic strategy. the mutation of a single residue in the atp-binding pocket confers sensitivity to small-molecule inhibitors, allowing for specific inactivation of the modified kinase. using this approach, we constructed conditional analog-sensitive alleles of essential protein kinases in the fission yeast s. pombe. i will present the functional analysis of these mutants during meiosis. peptide conjugates: from self-assembly towards applications in biomedicine ian hamley university of reading, dept of chemistry self-assembling peptides and their conjugates offer exceptional potential in nanomedicine. i will present some of our recent work on nanoscale assembled peptides and their conjugates, focussing on lipopeptides [ , ] and peg-peptide conjugates [ ] . pegylation is an important technique in the development of conjugates for applications in therapeutics. it is found to greatly influence self-assembly of peptides and proteins -one example from our own work is a peptide which itself forms twisted fibrils but when peg is attached, self-assembly of the conjugate leads to spherical micelles [ ] . the conjugate can be enzymatically degraded using alpha-chymotrypsin, releasing the peptide. this nanocontainer delivery and release system could be useful in therapeutic applications. thermoresponsive telechelic peg/peptides with hydrophobic dipeptide end groups (di-tyrosine or di-phenylalanine) were developed, one of which shows a de-gelation transition near body temperature and which may be useful in bioresponsive delivery systems [ ] . examples from our recent work on self-assembling lipopeptides will also be outlined. our focus is to investigate potential relationships between self-assembly and bioactivity, in particular in the fields of regenerative medicine [ ] [ ] [ ] [ ] [ ] , antimicrobial systems [ , ] and immune therapies [ ] . been shown to become derivatized with argpyrimidine, a prominent nem that occurs on arginine residues [ ] , in certain human cancer tissues and cell lines [ , ] . this nem was linked to the elevated antiapoptotic activity of the protein [ , ] , whereby modification of arg- appeared to be of particular significance [ ] . in this work, hsp homogeneously modified with argpyrimidine at position is generated for the first time. using expressed protein ligation [ ] , the first semisynthesis of the unmodified protein is achieved as well. our approach, which combines organic chemistry, peptide synthesis and protein synthesis, enables complete control over protein composition and thus can provide previously unattainable insight into the properties of this vital chaperone following nonenzymatic modification. the synthesis of argpyrimidine-modified hsp and the progress towards structural and functional characterization of the protein will be presented herein. kunitz-type protease inhibitors belong to a widespread protein family present in many plant species and play an important role in plant defense against insect pests and pathogens. members of this family are typically inhibitors of proteases of serine class. interestingly, a few members were identified as inhibitors of proteases of cysteine class, however, they have not been functionally and structurally characterized. our study is focused on kunitz-type inhibitors of cysteine proteases (pcpis) from potato (solanum tuberosum). a series of kda pcpis was purified using a multi-step chromatographical protocol, and two most abundant and effective isoinhibitors named pci - and pci were characterized in detail. they were screened against a broad panel of model cysteine proteases and digestive cysteine proteases from herbivorous insects. pci - and pci exhibit different inhibitory specificity pattern and potency up to the nanomolar range. both isoinhibitors were crystallized and their spatial structures were solved and refined at . Å (pci - ) and . Å (pci ) resolutions. a position of reactive sites against cysteine proteases on the conserved b-trefoil fold scaffold was proposed. the work provides the first analysis of pcpis with respect to the structure-function relationships and evolution within the kunitz-type inhibitor family. role of the abcc transporter in the mode of action of the bacillus thuringiensis cry ac toxin in the diamond back moth plutella xylostella protonation pattern influence actively properties of molecules and play an essential role in biochemical mechanisms. for an accurate determination of the protonation equilibria, the absolute proton solvation free energy needs to be known. the determination of this energy represents one of the most challenging problems in physical chemistry. this is particularly difficult for protons solvated in water, where the solvation is dynamically performed by different water clusters and the proton is not attached to a single solvent molecule. the proton solvation is notably important in order to quantify mechanisms of proton transfer and such processes have been investigated for a long time based on different approaches, often leading to contradictory conclusions. a rigorous and accurate protocol for computing proton solvation in solvents of different nature is of prime importance for applied (pharmaceutical and material science) and fundamental sciences. in this study, proton affinities, electrostatic energies of solvation and pka values of a reference set of organic molecules are computed in protic and aprotic solvents. proportional to the free energy of proton dissociation, the pka value calculation is therefore strongly dependent on the free energy of proton solvation. such energy is then determined in acetonitrile (acn), methanol (met), water and dimethyl sulfoxide (dmso) in order to obtain the best possible match between measured and computed pka values. the computation of these values is based on a combination of quantum chemical (qc) and electrostatic approaches by using a thermodynamic cycle connecting gas-phase and solvent-phase of proton dissociation. the computed proton solvation energies in acn, met, water and dmso of the present study are very precise (rmsd much lower than ph value). they will be a basis for better understanding of proton solvation and help to predict pka values of organic compounds in different solvents more precise. biochemical characterization of two evolutionary distant ten-eleven translocation enzymes and their utility in -methylcytosine sequencing in the genomes at single-base resolution subtypes leading to an inability to perceive pain and painful neuropathies, respectively. however, as nav ion channels are intimately involved in almost all aspects of physiology, only the most selective inhibitors would be suitable as drug leads. disulfide-rich venom derived mini-proteins from cone snails and spiders are being actively pursued as novel therapeutics for pain, because of their high selectivity and potency at human ion channels, including sodium channels (nav). two main strategies of inhibition have been identified; blocking the pore and interacting with the voltage-sensor domains (vsd) surrounding the pore. the ion-conducting pore is highly conserved between all sodium channel subtypes whereas the voltage-sensor domain binding sites are less conserved. therefore, inhibition of a specific nav isoform is more achievable using inhibitors that modulate vsds than with pore blockers. gating modifier toxins from spider and cone snail venom inhibit nav . and nav . by interacting with the vsd. they appear to reach their target by partitioning into the lipid membrane surrounding the ion channel, thus enabling access to the vsd. toxin pharmacology may therefore not only be driven by the peptide-ion channel interactions, but also including the lipids surrounding the channel protein, a feature that is very much under explored. it is therefore apparent that peptide-lipid interactions in combination with peptide-channel interactions need to be considered when designing potent inhibitors. using a range of biophysical techniques, including surface plasmon resonance and nuclear magnetic resonance, we are studying the interactions underpinning the mechanism of action between toxins and membranes and toxins and ion channels. initial results show that the lipid composition surrounding ion channels play a major role in terms of toxin:lipid interaction and that these interactions can be used in combination with traditional structure-activity relationship studies to design selective and potent nav inhibitors, which will be discussed. we believe that our studies will ultimately delineate what drives toxin pharmacology and nav subtype selectivity and will lead to improve rationally engineering of novel therapeutics for the treatment of pain. micelles promote aß assembly into pore-forming oligomers montserrat serra-batiste , mariam bayoumi , margarida gair ı , mart ı ninot-pedrosa , giovanni maglia , nat alia carulla institute for research in biomedicine (irb barcelona), biochemistry, molecular and structural biology section, university of leuven, the formation of amyloid-b peptide (ab) oligomers at the cellular membrane is considered to be a crucial process underlying neurotoxicity in alzheime rs disease (ad). - therefore, it is important to understand how oligomers form within a membrane environment. using solution nuclear magnetic resonance (nmr) spectroscopy, combined with size exclusion chromatography (sec), we have studied the two major ab variants-ab and ab , the latter having a more prominent role in ad than the former-under carefully selected micelle conditions intended to mimic a membrane environment. our results indicate that after an incubation period, ab , but not ab , assembles into oligomers with specific structural properties, which we have named stabilized micelle oligomers (smos). smo complexes incorporate into lipid bilayers as well-defined pores, a feature linked to neurotoxicity. these results have important implications in the ad field as they provide a new perspective on how ab oligomers cause neurotoxicity. indeed, our findings constitute a first step towards the establishment of a new therapeutic target for ad. dimer formation. it should be noted that this nb peptide contains the autophosphorylatable ser- associated with phk activation, and phosphorylated nb; peptide was considerably less effective in promoting b-dimer formation than non-phosphorylated peptide. these results suggest a role for ser- autophosphorylation in mediating homodimeric b subunit interactions within the phk complex, and augment previous studies on the activation of phk by phosphorylation in which changes at the nterminus of b are critical in the activation of the catalytic g subunit. summing these results leads to a new model of activation. in this model, in the inactive state, the nonphosphorylated n-terminus of b interacts directly or indirectly with the regulatory c-terminal domain of the g subunit, inhibiting catalytic activity. upon phosphorylation of the n-terminus of b, three important events occur: ) the interaction between b and g is disrupted, ) the b subunits of the holoenzyme self-associate, and ) the catalytic domain is activated. thus, we envision that the n-terminus of b acts as an allosteric switch, with activation triggered by phosphorylation of this region, causing disruption of its previously inhibiting interactions with g and promotion of b b dimerization to stabilize the activated conformation of g . the research was supported financially by the university of kansas medical center biomedical research training program and nih grant dk . pb- hssb is involved in the cellular response to oxidative dna damage christine touma , nicolas paquet , derek j. richard , roland gamsjaeger , , liza cubeddu , school of science and health, university of western sydney, queensland university of te chnology, school of molecular bioscience, university of sydney cellular dna is subject to oxidative damage in the presence of reactive oxygen species. the , -dihydro- -oxoguanine ( -oxog) adduct is the most common form of oxidative damage and results in g:c to t:a transversions; these lesions are normally processed by the base excision repair (ber) pathway. singlestranded binding (ssb) proteins of the oligonucleotide binding domain family are heavily involved in dna repair processes, which involve the detection of dna damage and recruitment of repair proteins to the site of damage. using immunofluorescence we demonstrate that hssb (a novel human ssb) levels increase in response to oxidative damage (h ). cells depleted of hssb are hypersensitive to oxidative damage and are also unable to efficiently remove -oxog adducts. we show that hssb forms dimers and tetramers under oxidative conditions and that this oligomerisation is likely mediated by inter-domain disulfide bond formation. furthermore, using surface plasmon resonance, we also show that oxidised hssb binds to -oxo-g damaged ssdna with higher affinity than non-damaged ssdna, indicating a direct role for oxidised hssb in the recognition of -oxo-g lesions. as oxidative stress is associated with aging, cancer and alzheimer's disease, understanding the molecular mechanisms of how cells repair oxidative dna damage will be crucial in the development of potential therapeutic treatments. epidemic typhus, which is caused by the bacterial pathogen rickettsia prowazekii, is a menacing disease world wide that the nih lists as one of america's greatest biological weapons threats. this research seeks to find novel inhibitors of b-ketoacyl-acp-reductase (fabg), an enzyme that catalyzes one of the reactions in the fatty acid synthesis type ii system in bacteria. this pathway is essential for survival in bacteria. the fabg enzyme uses nadph as a substrate, which facilitates the binding of the second substrate, acetoacetyl-acp into the active site. the acetoacetyl-acp is subsequently reduced into b-hydroxyacyl-acp. the coding dna sequence for the rpfabg protein was cloned into a pnic vector and transformed into e.coli bl (de ), then the protein was expressed and purified using metal affinity and size exclusion chromatography methods. high throughput molecular docking software (gold) was used to screen a commercial library of ligands against the acetoacetyl-acp region of the active site. the ligands with the best gold scores were selected to be tested in vitro. spectrophotometric enzyme inhibition assays were performed to determine whether the drugs could inhibit rpfabg activity. chlorogenic acid, a previously known inhibitor of homologous fabgs, was tested along with the other potential drugs, and was determined to have moderate inhibitory effects on rpfabg. loop modeling using icm software was performed in order to create a prediction of the complete rpfabg structure, including the disordered loops that are not a part of the f i pdb structure. co-crystallization of rpfabg with both substrates was carried out in order to obtain a structure, but only nondiffracting crystals resulted. further inhibition assays and crystallography trials are being performed in order to continue the search for a novel inhibitor of rpfabg and ultimately a treatment for epidemic typhus. the university of hong kong bioconjugation of proteins has emerged as a useful tool in the study of biological systems. there is an increasing need to develop new synthetic technologies for the bioconjugation reaction of proteins, and metal-catalyzed site-selective modification of proteins has attracted considerable interest in recent years. we have developed a ruthenium glycosylated porphyrin-catalyzed carbenoid transfer reaction for the site-selective modification of proteins. we firstly applied the catalysis to the selective modification of the n-terminus of peptides. by using ruthenium glycosylated porphyrin as catalyst, the n-terminus of a number of peptides can be modified through carbenoid n-h bond insertion in aqueous media with moderate to excellent conversion. the reaction is highly selective, for example, the reaction with ytsssknvvr, which contains various types of oxygenhydrogen and nitrogen-hydrogen bonds possibly available for carbenoid insertion, catalyzed by the ruthenium glycosylated porphyrin gave the n-terminal-modified product with > % conversion and without the formation of other modified peptides including doubly modified and oxygenhydrogen bond insertion products. we next extended the n-terminal modification method to proteins. eventually success was attained in the modification of rnase a and insulin. the reaction of rnase a with a diazoacetate mediated by ruthenium glycosylated porphyrin gave corresponding n-terminal-modified protein with % conversion. we also achieved a bioconjugation to ubiquitin via ruthenium glycosylated porphyrin-catalyzed alkene cyclopropanation in aqueous solution in two steps: ( ) incorporation of an alkenic group by the reaction of n-hydroxysuccinimide ester with ubiquitin and ( ) cyclopropanation of the alkene-tethered lys ubiquitin with the fluorescent labeled diazoacetate in the presence of a catalytic amount of ruthenium glycosylated porphyrin. the corresponding cyclopropanation product was obtained with % conversion based on maldi-tof mass spectrometry. in conclusion, we developed a ruthenium porphyrin-catalyzed siteselective modification of peptides and proteins in aqueous media. the method provides an entry to new bioconjugation reactions for protein modifications using metalloporphyrins as catalysts. uridine monophosphate synthase: architecture versatility in the service of late blight control francisco tenjo castaño , , manuel garavito , , leonor garc ıa , , silvia restrepo , barbara zimmermann biochemistry and molecular biology research group, universidad de los andes., mycology and plan pathology laboratory, universidad de los andes uridine monophosphate synthase (umpase), a bifunctional enzyme in the de novo pyrimidine biosynthetic pathway, is a protein comprised of orotate phosphoribosyl transferase (oprtase) and orotidine monophosphate decarboxylase (odcase). different fusion orders of the two domains have been documented to exist in nature. in some organisms oprtase and odcase are monofunctional proteins, and act as a complex. here, umpase from solanum tuberosum (potato) and from phytophthora infestans (an oomycete) were examined. p. infestans causes late blight disease in s. tuberosum, destroying crops and increasing production costs. since pyrimidines are fundamental cellular components, we have proposed that umpase could serve as a target to control p. infestans infection. the enzymes from p. infestans and s. tuberosum differ in their fusion order of oprt and odc. the study of these two umpase could facilitate the design of species-specific inhibitors, and might shed light on the effect of fusing umpase domains in one order or the other. to this end we carried out bioinformatic and biochemical characterization of the enzymes. sequence analyses showed residue differences among the p. infestans umpase sequences from three strains: , and t - . strain t - was found to have a duplicated umpase, but neither sequence corresponded to the ones predicted previously from the genome. a recombinant umpase from strain was expressed in bacteria and purified but it showed low solubility and was inactive in vitro. the recombinant umpase from the strain complemented both oprtase and odcase deficient e. coli strains. a soluble, active, recombinant protein was expressed and purified in the presence of high salt and the product ump (specific activity . lmol min- mg- ). the sequence skq was found at the c-terminus of the p. infestans umpase sequences and resembles a peroxisome signal peptide (skl). the predicted hydrophobicity of this umpase and its architecture (oprt at the c-terminus and odc at the n-terminus) resembles that of the umpase from leishmania donovani, which has been localized to the peroxisome. we suggest that p. infestans umps could also be located in this organelle. in contrast to the oomycete enzyme, s. tuberosum umpase is highly soluble, and has a higher specific activity (vmax . lmol min- mg- ). we measured the kinetic parameters km(orotate) . lm, km(prpp) . lm, and found that it exhibited product inhibition by pyrophosphate. in conclusion, the different architectures of the two umps might be related to distinct biochemical characteristics, further supporting this protein as a good candidate for p. infestans control. we present computer simulation studies of three different antimicrobial peptides we have been studying by md computer simulation in collaboration with experimentalists. the first is daptomycin, a potent lipopeptide currently licensed to treat infections caused by multi-drug-resistent bacteria. the mechanism of action of daptomycin is currently not completely understood. we have solved the nmr structure of this molecule, and attempted to determine the size of its oligomer by small angle neutron scattering (sans) supported by computer simulation. feglymycin is a -amino-acid peptide with a high percentage of unusual amino acids such as -hydroxyphenylglycine and , -dihydroxyphenylglycine. feglymicin inhibits mura and murc enzymes which are involved in bacterial peptidoglycan synthesis, while also displaying anti-hiv activity by interaction with the viral envelope protein gp . a previous x-ray structure shows the molecule forming a dimer. here, the molecule was studied by nmr in water and dmso. in water, the molecule is clearly at least a dimer, while in dmso it is a monomer. we have performed noe refinement simulations in order to elucidate a structure, however, due to a lack of long-range noe contacts, a unique structure cannot be determined. labyrinthopeptin a is a lantibiotic that contains labionin, a unique carbacyclic posttranslationally modified amino acid that links the protein backbone in three different locations. labyrinthopeptin a has shown promising activity as a pain killer. starting from the x-ray structure, we present results from the first md simulation studies of this unique peptide. because of the extensive cross-linking, this peptide is observed to be highly rigid in its native form. simulation results of mutants are also presented. antibiotics with new mechanism of action are urgently required to combat the growing health threat posed by resistant pathogenic microorganisms. here we report the discovery of a new peptidomimetic antibiotic (l - ), which is active with a minimum inhibitory concentration (mic) in the low nanomolar range, only against pseudomonas sp., and with a non-membrane-lytic mechanism of action. a drug target identified both in a forward genetic screen for resistance determinants and by photoaffinity labeling is the ß-barrel protein lptd, which plays an important role in lps transport and the outer membrane biogenesis. the x-ray structure of lptd in complex with lpte from shigella flexneri shows a stranded b-barrel linked to a periplasmatic n-terminal jelly-roll domain. interestingly the homology model structure for lptd from pseudomonas shows a significant difference: an insertion of around amino acids in the n-terminal domain. the results of our attempts to purify and characterize this large outer membrane protein and to determine the binding site of the peptidomimetic antibiotic will be shown. the theory of how life on earth begun still remains unclear. nevertheless, according to some theories, at the beginning level proteins did not emerge as a complex globular forms as know today. at the times, when solely rna molecules stored both genetic information and catalyzed the chemical reactions in primitive cells, peptides acted as a proteins nowadays [ , ] . literature postulate that the possible role of primordial short peptides was to catalyze reactions in rna-world, as they possess an excellent ability to self-assemble into well-ordered nanostructures [ , ] . elementary functional loops (efls) can be considered as a small structures (blocks) having specific signatures and providing functional residues important for binding/activation as well as principal chemical transformation steps of the enzymatic reaction [ ] . p-loop efl is a widespread structure across vast majority of protein families such as motor domains, aaa , reca, pepck and many others. sequential alignment of these protein families reveals existence of a conserved p-loop motif, that is able to bind atp molecule. we investigated the structure and atpase activity of peptides, which sequences possessed strongly conserved gxgk[t/s] motif from ploop. the goal of our work was to check if peptides corresponding to the most conserved p-loop motif fragment are able to bind and hydrolyze atp molecule. all peptides under study were chemically synthesized and their structures was investigated by nmr spectroscopy. the ability to bind atp molecules was analyzed by using hplc chromatography. results of our study show, that peptides with conserved p-loop motif have a suitable structures to promote binding of the molecules with phosphate group, but cannot accelerate pyrophosphate hydrolysis process. conference participation for w. _ z. supported by the fp project mobi health (grant agreement no ). computational resources were provided by the informatics center of the metropolitan academic network (ic man task) in gdansk, poland. ck is a ubiquitous serine/threonine protein kinase, being one of the most pleiotropic of all protein kinases . ck plays a key role in cell growth, differentiation, cell death and survival, and become the therapeutic target in cancer treatment, since its level is significantly increased in cancer cells . halogenated ligands have been widely developed as potent inhibitors of protein kinases. among them , , , -tetrabromobenzoteriazole (tbbt) is one of the first potent and selective inhibitor of ck a, directed towards the conserved atp binding site . to assess contribution of electrostatic interactions to the specificity and strength of binding of multi halogenated inhibitors by a protein kinase, we have studied interaction between ck a and nine benzotriazole derivatives, representing all possible patterns of halogenation on the benzene ring. herein, we present results that support existence of two alternative regions that are involved in ligand binding. aspartic acid is known for its function in coordination of a mg ion, which is required for atp binding . asp has been identified in crystal structure of ck :tbbt complex (pdb j , fig. ) as the charged residue closest to tbbt. there is also lys proximal to tbbt, interaction with which may favor anionic form of ligands (pk for tbbt < ), however it is involved in the intramolecular salt bridge, and thus its mutation may significantly change stability of the protein. crystal structure of tbbt complexed with ck (pdb: j ). residues with a distance to tbbt (magenta) shorter than a are shown. red residue is negatively charged, blue ones are protonated. abstract comparison of kdiss values determined for ligands at ph and at ph shows that strength of the complex significantly varies upon deprotonation of the triazole ring. this confirms former hypothesis that a negatively charged ligands cluster at the atp binding site region proximal to lys , which is beneficial both to the specificity and to strength of the binding. we have also observed for the tested ligands variations in their binding to either wild type protein and its d n mutant (with less negative charge distributed over atp binding site). all ligands displaying higher pka for dissociation of the triazole proton bind to the mutant visibly weaker than to the wild-type protein. altogether reveals the predominance electrostatic intermolecular interactions. although, negatively charged ligands most probably cluster at the atpbinding site proximal to lys , beneficial for the strength of binding, the less dissociated forms are favored due to unfavorable interactions of the anionic form of ligands with asp . there are many virulence factors produced by these strains, many of which are encoded on mobile genetic elements. psms are of specific interest because these virulence factors are encoded on the core genome of the bacteria and therefore all strains of staphylococci bacteria produce some variation of psms with a variety of biological functions. the specific mechanism by which psms act as virulence factors has been poorly understood until recently. biological functions of psms include cell lysis, biofilm formation and the ability to kill neutrophils after phagocystosis. these toxins are of special interest to our research group due to their genetic similarities to certain bacteriocins, namely leaderless bacteriocins. both groups of peptides are ribosomally synthesized with a n-terminal formyl methionine and secreted from the bacteria by atp-binding cassette (abc) transporters without any leader sequence or signal peptide. abc transporters may also play a role in immunity towards psms and leaderless bacteriocins. these similarities led our group to investigate the solution structure of these peptides through nuclear magnetic resonance (nmr). isolating psms from the producer organisim, s. aureus, typically involves lengthy extractions and low yields. for these reasons, we opted to chemically synthesize the desired peptides using solid phase peptide synthesis (spps). utilizing a variety of spps techniques, psm a and psm a were successfully synthesized, however, due to the hydrophobic nature of psm b , an alternate genetic approach was devised to isolate psm b . formation of a fusion protein between psm b and the small ubiquitin like modifier (sumo) protein allowed for heterologous expression. upon cleavage of the fusion protein with sumo protease, and subsequent purification and isolation of the cut peptide, psm b was obtained. as previously reported, the psms were found to be alpha-helical in structure inducing solvents. a series of dimensional ( d) nmr experiments were ran to determine chemical shift assignments and to obtain noe data. importing the chemical shift assignments and noe data into the structure calculating software, cyana, we were able to elucidate the solution structure of psm a and psm a and we are currently working towards the elucidation of psm b . the synthesis, isolation, characterization and solution structures of the aforementioned psms will be discussed here. transition metals are critical for enzyme function and protein folding, but their excess can mediate neurotoxic oxidative processes [ ] . as, energy production involves oxidative phosphorylation, a process requiring a continuous flow of electrons, mitochondria are particularly vulnerable to oxidative damage [ ] . as such, mitochondria are the major sites of reactive oxygen species (ros) generation, which are produced as byproducts of the electron transport chain. since free iron and certain ros can engage into potentially deleterious processes such as fenton reaction, mitochondrial iron homeostasis must be tightly controlled, and dysregulation of iron metabolism in this organelle has been associated with various diseases, including friedichs ataxia (fa), alzheimer's, and other neurodegenerative disorders [ ] . engineering an efficient mitochondriatargeting, cell-permeable vector is a challenge due to the fact that mitochondrion is impermeable to a wide range of molecules. the development of delivery vectors has been made possible by a greater understanding of mitochondrial structure and chemical features of molecules that selectively localize within this organelle. from these findings, two generalized requirements for mitochondrial localization are delocalized positive charge and lipophilicity [ , ] . targeting iron in this organelle is proposed as a means to ameliorate fa symptoms. desferrioxamine (dfo) is a bacterial siderophore with high affinity for iron, but low cell penetration. we prepared conjugates of dfo with mitochondria penetrating peptides and studied their iron-binding characteristics in vitro. the lipophilic and charged peptides tat - (h-arg-lys-lys-arg-arg-gln-arg-arg-arg-oh) [ ] , a (h-cha-arg-cha-lys-cha-arg-cha-lys-nh ) [ ] , ss- (h-dmt-arg-phe-lys-nh ) [ ] and ss- (h-phe-arg-phe-lys-nh ) [ ] , are known to permeate cytosolic and mitochondrial membranes. they were prepared and conjugated to dfo in solid-phase [ ] , an alternative synthetic route. once detached from the resin, fully deprotected, purified and characterized by means of lc/ms and aminoacid analysis, it was observed that the dfo-conjugated peptides displayed iron-binding abilities identical to the free chelator dfo. dfo-conjugated peptides were also able to quench the iron-catalysed oxidation of ascorbate (a model of oxidative stress in plasma of iron-overloaded patients), as probed by a high throughput fluorimetric method [ , ] . these results indicate that our synthesis and conjugation strategy were successful in preserving the iron-binding moiety and the antioxidant ability of the free chelator dfo. the proteolytic activity and oligomerization status of the human htra protease functioning as a tumor suppressor of an n-terminal domain not required for proteolytic activity, a central serine protease domain and a cterminal pdz domain. the latter serves as a substrate or regulator binding domain and may participate in oligomerization. htra s, its short natural isoform, lacks the pdz domain which is substituted by a stretch of c-terminal amino acid residues, unique for this isoform. down-regulation of htra in tumors, shown by other groups and us, suggests htra s involvement in oncogenesis [ ] . htra acts as a proapoptotic protein and is suggested to function as a tumor suppressor. it promotes cytotoxicity of etoposide and cisplatin in lung cancer cell lines [ , ] . to date, htra has been poorly characterized from the biochemical point of view, mainly due to the fact that it is difficult to purify recombinant htra . we were able to express in bacterial system and purify htra in quantities sufficient to perform structural studies. the aim of this study was to characterize and compare the proteolytic properties and quaternary structure of the htra isoforms. both studied isoforms lacked the n-terminal domain. htra with the pdz domain removed (htra -dpdz) and htra s (htra s) were fully active at a wide range of temperatures and their substrate affinity was not impaired. this indicates that the pdz domain is dispensable for htra activity. as determined by size exclusion chromatography, htra formed stable trimers while both htra -dpdz and htra s were monomeric. this suggests that the presence of the pdz domain, unlike in other human htras (htra and htra ), influences htra trimer formation. the unique c-terminal sequence of dn-htra s appeared to have little effect on activity and oligomerization [ ] . cyclodextrins (cds) are cyclic oligosaccharides that have been recognized as useful pharmaceutical excipients. in aqueous solution cds are capable to form complexes with various ligands, hosting inside their cavity either a whole molecule, or part of a ligand. inclusion complexes with cds offers a variety of physicochemical advantages over the biologically active ligands, including the improved aqueous solubility, solution stability or an increase of bioavailability. ck is an ubiquitous, highly pleiotropic and constitutively active ser/thr protein kinase. halogenated benzotriazoles have been developed as potent and selective inhibitors of this enzyme. the interaction of the catalytic domain of human protein kinase ck with a series of brominated ligands, which represent all possible patterns of halogen substitutions to the benzene ring of benzotriazole, was previously studied by microscale thermophoresis (mst) [ ] . this method alloweddetermination of binding affinities for seven ligands, all of which were found consistent with the values determined independently by isothermal titration calorimetry (itc). however, a very limited aqueous solubility of some brominated benzotriazoles may decrease their bioavability, thus affectingtheir apparent activity [ ] . to overcome this limitation, the aqueous solubility of halogenated benzotriazoles in the presence of cyclodextrins has been tested. the formation of inclusion complexes with b-cyclodextrin (b-cd), hydroxypropylb-cyclodextrin (hp-b-cd) and g-cyclodextrin (g-cd) in aqueous solutions, followed by uv-vis spectroscopy, substantially improved the solubility of tbbt and its derivatives. the interaction between protein kinase ck and cyclodextrins, and also with their inclusion complexes with halogenated benzotriazoles, was followed with the aid of the microscale thermophoresis. the results obtained clearly demonstrate that the binding of halogenated benzotriazoles by ck is only moderately affected by cyclodextrins. oligonucleotide-based molecular circuits offer the exciting possibility to introduce autonomous signal processing in biomedicine, synthetic biology, and molecular diagnostics. here we introduce bivalent peptide-dna conjugates as generic, noncovalent, and easily applicable molecular locks that allow the control of antibody activity using toeholdmediated strand displacement reactions. employing yeast as a cellular model system, reversible control of antibody targeting is demonstrated with low nm concentrations of peptide-dna locks and oligonucleotide displacer strands. introduction of two different toehold strands on the peptide-dna lock allowed signal integration of two different inputs, yielding logic orand and-gates. the range of molecular inputs could be further extended to protein-based triggers by using proteinbinding aptamers. insights of a novel kind of cell wall binding domain that cleaves the peptidoglycan muropeptide: the cw_ motif noem ı bustamante , , manuel iglesias, noella silva-mart ın, isabel uson, pedro garc ıa, juan hermoso, marta bruix, margarita men endez institute of physical-chemistry 'rocasolano', csic, institute of physical-chemistry 'rocasolano', csic, ciber of respiratory diseases (ciberes), center of biological research (cib), csic, enzybiotics constitute a hopeful alternative to current treatments to fight against bacterial infections. phage endolysins are consider as enzybiotics due to their capacity to cleave the peptidoglycan (pg) of gram-positive bacteria in a generally species-specific manner and kill bacteria when exogenously added ( , ) . the cpl- endolysin, a lysozyme encoded by the pneumococcal cp- bacteriophage, is a remarkable exception among all the pg hydrolases produced by streptococcus pneumoniae and its bacteriophages due to its capacity of degrading pneumococcal cell walls containing either choline or ethanolamine ( , ) . this fact confers to cpl- the advantage of displaying a broader microbicide spectrum comparing to choline binding proteins ( ) . this behavior results from the acquisition of a cell wall binding module (cwbm) made of three identical repeats of amino acids each (cw_ motifs), with unknown specificity and totally unrelated with the choline-binding motives present in pneumococcal hydrolases. interestingly, cw_ repeats have been identified in many putative proteins potentially involved in cell wall metabolism (pfam entry: pf ) from different species of gram positive and gram negative bacteria, and some bacteriophages ( ) . preliminary studies of thermal stability in presence of a small cell wall structural-analogue (glcnac-murnac-l-ala-d-isogln) point to the muropeptide as the cell wall target recognized by cw_ motifs ( ) . in this communication we have gone in depth in the characterization of cw_ repeats. we present the first crystal structure of the cw_ motif, which reveals a three-helical bundle folding. using std_nmr spectroscopy the epitope of binding of the disacharide dipeptide to this repeats has been identified. interestingly, the b anomer of the murnac moiety, the form present in the peptidoglycan, seems to be preferentially recognized with respect to the a anomer. finally, a docking model of the complex cw_ /gmdp compatible with std results was built allowing to identify the major contacts between the protein and the muropeptide and to propose the relevant role of a conserved arginine residue in this interaction. energy-dependent aaa proteases carry out regulated proteolysis to ensure protein quality control and post-translational regulation of many cellular processes. control of proteolysis occurs primarily at the level of substrate recognition, which can be modulated by adaptor proteins. the clps adaptor protein enhances and inhibits degradation of different classes of substrates, and thus triggers a specificity switch in clpa. whereas the mechanism for substrate delivery by clps has been described in detail, the inhibition mechanism is poorly understood. we show that clps inhibits ssra substrate recognition and processing, instead of simply preventing substrate binding. we demonstrate that clpa engagement of the clps n-terminal extension (nte) is necessary, and may even be sufficient, for inhibition. in addition, we find that inhibition of substrate processing requires a longer nte, as compared to inhibition of substrate recognition. interestingly, the nte length required for inhibiting substrate processing is also necessary for suppression of the clpa atpase rate. furthermore, preliminary data suggests that clps slows down substrate translocation. these results support a model where there is an ssra•clpa•clps inhibitory complex in which the clpa pore engages the clps nte. this engagement of the nte causes suppression of atpase activity, and therefore slower substrate translocation and processing. this model illustrates how an adaptor protein can inhibit recognition of one type of substrate while efficiently promoting degradation of a different substrate. single-molecule assay development for studying human rna polymerase ii promoter-proximal pausing rna polymerase ii (polii) pausing has been shown to play a significant role in transcription regulation of elongating polii complexes in a large number of metazoan and mammalian genes ( ) . the traditional understanding of transcription regulation in mammals involved controlling polii recruitment to promoters and controlling initial steps at the promoter, including pre-initiation complex formation and promoter escape. most works investigating promoter-proximal polii pausing have employed chromatin immunoprecipitation followed by sequencing to determine polii localization or in vitro transcriptional assays using nuclear extracts analyzed with radio-active gel electrophoresis. in order to gain greater mechanistic insight into the regulation of promoter-proximal polii pausing, we have been developing a diffusion-based single-molecule method using alternating laser excitation on the micro-second timescale (msalex). the method detects rna transcripts generated by a reconstituted human polii system in vitro using complementary doubly dye-labeled single-stranded dna (ssdna) probes. the human gene hspa b for heat shock protein (hsp ) is used as a model system due to its extensive characterization in drosophila. the method would provide a rapid, sensitive and robust avenue to screen for protein factors regulating promoter-proximal polii pausing. controlling of the pic composition using the reconstituted system allows for dissection of the functional roles of different pic components in facilitating regulation of polii pausing. we have demonstrated the hybridization of double dye-labeled ssdna probe to complementary ssdna mimicking rna transcripts and to transcripts generated with bacterial rna polymerase. also, a functional reconstituted human polii system has been verified using radioactive polyacrylamide gel electrophoresis of transcripts from in vitro transcription assays. malaria is a major global health problem. in , there were an estimated million case of malaria and deaths, most of them children under years old [ ] . among the malaria species that affect humans, plasmodium falciparum is the most deadly form. since no efficient vaccine is available yet, the fight against malaria includes vector control, protection from mosquito bites and artemisinin combined therapy. however, resistances to all known treatments have been observed. therefore, new antimalarial strategies involving novel targets and new mechanisms of action are needed. during its life cycle, in erythrocytic stage, which causes all the malaria symptoms, plasmodium falciparum relies on phospholipids to build the membranes necessary for daughter cell development. approximately % of parasite phospholipids consist of phosphatidylcholine (pc) and phosphatidylethanolamine (pe) synthesized by the parasite through the de novo kennedy pathways. in the pathway of phosphatidylcholine biosynthesis, the second step catalyzed by ctp:phosphocholine cytidylyltransferase [ec . . . ] is rate limiting and appears essential for the parasite survival at its blood stage [ ] [ ] . we are focused on the structural characterization of this enzyme, the identification of effectors by fragment-based drug design approach (fbdd) and then their optimization to eventually design a lead. the first reported crystal structure of the catalytic domain of the enzyme target (pfcct) has been solved at resolution . Å, enzyme-substrates complexes (cmp-, phosphocholine-and choline-bound forms) at resolutions . - Å and an enzyme-product (cdp-choline) complex structure at resolution . Å that give detailed images of binding pocket, demonstrate conformational changes between apo-and holo-protein forms and provide the information about the mechanism of the catalytic reaction at atomic level. the fbdd method uses a library of small molecules (fragments) with molecular weight that does not exceed da to explore target binding sites. although fragments often have too low affinities to evoke a biological response, their probability of binding is high because they are small enough to prevent unfavorable interactions with target protein-binding sites. moreover, they represent more attractive and synthetically tractable starting points for medicinal chemistry compared to more complex compounds. as the affinity is low, fragment screening usually depends on detecting binding rather than inhibition. screenings of a fragment library ( molecules) has been performed by fluorescence-based thermal shift assay and nuclear magnetic resonance saturation transfer difference (nmr std) [ ] . this combination of techniques identified so far fragment hits that are currently evaluated for their binding modes and affinities. co-crystallization of the protein-fragments complexes is carrying out to provide accurate information on the molecular interactions. topology of interactions will be used to rationally monitor every iterative round of the optimization process allowing subsequent rational design. [ ] world health organization, world malaria report (who press, geneva, switzerland), http://www. who.int/malaria/publications/world_malaria_report_ /wmr- -no-profiles.pdf?ua protein scaffolds play a crucial role in signaling pathways by generating signal specificity and increasing signal efficiency and amplitude. engineered protein scaffolds can be used as key regulators for signal transduction in artificial signal transduction cascades where they can regulate in-and output of the network. in this research a - - protein scaffold is developed which induces dimerization of proteins mediated by the small molecule stabilizer fusicoccin. as proof of principle caspase is used to constitute proximity induced dimerization. dimerization of caspase leads to its activation and consecutively initiates the caspase cascade involved in the programmed cell death pathway. caspase does not naturally bind to - - proteins, therefore the caspase monomer is conjugated to a - - binding motif which is known to bind into the binding grooves of a - - dimer. this interaction can be stabilized by the small molecule fusicoccin. we showed that upon addition of small molecule fusiccocin caspase dimerization is induced, resulting in caspase activity which is measured using a synthetic caspase substrate. moreover the biphasic effect of the - - scaffold could be proven. additionally, the activated caspase is also able to cleave its natural substrate caspase , downstream in the caspase cascade. these results indicate that the - - platform is a versatile small molecule induced dimerization platform which can be used as tool for engineering of a synthetic signaling network. the g e variant of the apoptosis inducing factor, responsible of a rare encephalopathy, is hampered in nad /h binding luca sorrentino , laura rigamonti , mirvan krasniqi , alessandra calogero , vittorio pandini , maria antonietta vanoni , alessandro aliverti the apoptosis inducing factor (aif) is a highly conserved mitochondrial flavoprotein known to play two opposite roles in eukaryotic cells: in mitochondria it is required for efficient oxidative phosphorylation (oxphos), while, when released into the cytoplasm, it triggers caspase-independent apoptosis ( ) . the mechanism of aif-induced apoptosis was extensively investigated, whereas its mitochondrial role is poorly understood. there are many evidences of aif importance for mitochondrial correct morphology and functions and recently the discovery of its direct interaction with chchd , a key regulator of respiratory complexes subunits import and folding in mitochondria, was reported ( ) . a unique feature of aif, probably pivotal for its vital function, is the ability to form a tight, air-stable charge-transfer (ct) complex with nad and undergo dimerization. although some aspects of aif interaction with nad / h have been analyzed, its precise mechanism is not fully understood. we investigated the effect of the pathogenic g e replacement, associated with oxphos defect and neurodegeneration ( ) , to understand how it could alter aif properties at the molecular level. to do so, we analysed how the wild type and the g e forms of murine aif interact with nad /h and nicotinamide mononucleotide (nmn / h), finding that the pathogenic replacement resulted in a dramatic and specific decrease of the rate for ct complex formation and consequent protein dimerization only in the case of the physiological ligand. our results demonstrate that the adenylate moiety of nad /h is crucial for the ligand binding process and that the g e replacement causes an alteration of the adenylate-binding site of aif that drastically decreases the affinity for and the association rate of the ligand. in addition, we shed new light on the mechanism of the dimerization process, demonstrating that fad reduction rather than nad /h binding initiates the conformational rearrangement of aif that leads to quaternary structure transitions. taken together, our results contribute to define how aif works at the molecular level in binding nad /h and undergoing dimerization and also point out that the g e replacement, responsible of a rare neurodegenerative disease, has the selective effect of slowing down the formation of aif dimeric ct complex. dipartimento di bioscienze, universit a degli studi di milano, dipartimento di scienze veterinarie e sanit a pubblica, universit a degli studi di mical, from the molecule interacting with casl, indicates a family of conserved cytoplasmic multidomain proteins that catalyze a nadph-dependent f-actin depolymerization activity through their essential n-terminal fad-containing monooxygenase-like domain (mo) in response to semaphorin signaling [ ] . this domain is followed by calponin homology (ch) and lim domains, proline-and glutamate-rich regions and a c-terminal coiled-coil motif that mediate the interaction with various proteins (e.g: crmp, casl, plexin, g proteins, ndr) [ ] . to contribute to establish the catalytic properties of mical mo and their modulation by the additional domains and by the interacting proteins, we have produced and are characterizing the human mical (mical-fl) and forms containing the mo [ ] , mo-ch and mo-ch-lim domains. all mical forms contain stoichiometric amounts of fad in the mo domain and zn ions in the lim domain. mical-mo catalyzes a nadph oxidase (h o -producing) activity. the ch, lim and c-terminal domains lower its catalytic efficiency (kcat/km, nadph) mainly due to an increase of km for nadph. the kcat is similar for all forms excepted for mical-fl where a -fold drop is observed, in agreement with the proposed autoinhibitory function of the c-terminal domain [ ] . the ph dependence of the kinetic parameters of mo, moch and mochlim is complex suggesting that it does not reflect the ionization state of individual groups, but rather the overall protein charge. mical-mo, -moch and -mochlim catalyze a nadph-dependent f-actin depolymerization with a similar apparent km for actin. f-actin (but not g-actin) stimulates the rate of nadph oxidation by increasing kcat and lowering knadph. the extent of nadph oxidation exceeds total f-actin which is in contrast with the proposal of specific modification of actin met or met reported for drosophila and mouse moch [ ] [ ] , but it suggests that f-actin stimulates the nadph oxidase activity or a case of substrate recycling. accordingly, with hmical mo and moch several actin residues are oxidized beside met and met . thus, the ch and lim domains do not seem to be important for the mical-actin interaction and actin modification may be mediated by in situ h o production. in hek t and cos- cells mouse collapsin response mediator protein- (mcrmp ) interacts with mical inhibiting h o production [ ] , suggesting that crmp could be a hydroxylatable substrate of mical-mo. we have produced the same mcrmp form ( - aa) and we have shown that under conditions that limit non specific interactions a mild stimulation (up to %) of nadph oxidation is observed. f-actin reversed the effect of mcrmp suggesting their competition for mical. these results suggest that crmp , a major microtubules regulator, is not the substrate of the mo domain, but actin and microtubules cytoskeleton components may be linked through the formation of crmp-mical complex in response to semaphorin-plexin signaling. experiments are in progress to complete the characterization of mochlim and full length mical forms. green fluorescent protein (gfp), owing to its genetically encoded strong fluorescence, has become one of the most important tools in modern biology [ ] . enhanced gfp (egfp, f l/s t-gfp), frequently used variants of this protein, is thermodynamically more stable and -times brighter than gfp [ ] . due to the improved fluorescent properties, egfp is commonly used as a fluorescent intracellular marker in bio-imaging in vitro and in vivo. despite sustained interest of the scientific community and numerous practical applications, the actual biological role of gfp remains elusive. recent reports put forward a hypothesis of antioxidant and photo-protective functions of gfp [ ] . in this study, we focused on the photo-protective role of egfp against reactive oxygen species (ros) photo-generated by visible light in water suspensions of nano-particular nitrogen-doped titanium oxide (n-doped nano-tio ), that is in the system: 'n-doped nano-tio )/visible light'. n-doped nano-tio (sumitomo tp-s ) was chosen as a photo-catalyst, since it is widely accepted that nitrogen doping enhances visible light photoactivity of tio . -hydroxy- , , , -tetramethylpiperidine-n-oxyl (tempol), a paramagnetic water-soluble compound, belonging to the nitroxide class o superoxide dismutase (sod) mimetics, was used as a target for photo-generated ros. a solar simulator, with the flux output intensity of kw/m , was used as a visible light source. electron spin resonance (esr) was employed to monitor the changes in the paramagnetic signal of tempol exposed to the action of ros in the absence and presence of egfp. in the absence of egfp and after min of illumination, due to a combined action of superoxide (o •-) and hydroxyl (oh•) radicals generated by the system 'n-doped nano-tio )/visible light, the esr signal of um tempol decayed by %. moreover, the growth of a new signal, interpreted as -oxo- , , , -tetramethyl- -piperidinyloxy (tempone), resulting from the attack of oh•radicals on tempol, was also observed. in contrast, in the presence of egfp ( . um) , the ros-induced decay of the esr signal of tempol was markedly smaller, not exceeding %. concomitantly, the growth of the esr signal of tempone was also partially inhibited ( % smaller amplitude), as compared to the process performed in the absence of egfp. in summary, our results point to a significant inhibition of the photodecomposition of tempol in the presence of egfp and support the hypothesis of the protective role of this fluorescent protein against ros generated by the system 'n-doped nano-tio )/visible light'. school of chemistry, national university of ireland galway, school of biochemistry and immunology, trinity college dublin by studying a variety of anionic ligands and their interactions with cationic cytochrome c, we are building knowledge of protein recognition geared towards regulating activity. in previous work it was shown that psulfonatocalix [ ] arene selectively binds to, and encapsulates, three lysine side chains on cytochrome c . here, the binding of two small molecule ligands to cytochrome c was investigated. nmr spectroscopy was used and in one case, a crystal structure of the complex was obtained (fig ) . the calixarene bound to cytochrome c, reveals a crystal packing assembly that suggests it is a key mediator of crystal formation. nmr data analysis indicates the calixarene's binding site on cytochrome c. the pillararene, a relatively new class of compound, is a symmetrical arrangement with a p-rich cavity , related structurally to calixarenes. this suggests good host-guest complexation properties. previously, the carboxylatopillararene showed selective binding to arginine, lysine and histidine . with this ligand, an interaction with cytochrome c was observed and a complex formed. additionally, biphasic binding behaviour was observed through analysis of the chemical shift perturbations. this may indicate more than one binding event taking place. the data from these studies indicate that recognition is occurring and again that lysine side chains play an essential role. the enzyme dihydrofolate reductase (dhfr) is necessary for the growth and development of all organisms. the structure and function of escherichia coli dhfr have been characterized in buffer. however, dhfr exists in living cells, where the protein concentration can exceed g/l. we know that weak, non-specific chemical interactions with cytosolic proteins alter protein conformation and dynamics, , both of which are expected to influence dhfr catalysis. investigators have examined steady-state enzyme kinetics under crowded conditions, but conclusions can be conflicting. , here, the effects of crowding on e. coli dhfr catalysis are assessed through specific activity measurements in solutions of synthetic polymers. these kinetics studies are complemented by in-cell and in vitro f nmr data from fluorinated tryptophan residues. preliminary results suggest that the effects of polymeric crowders on dhfr activity are non-monotonic, which may arise from the polymer's transition from the dilute to semi-dilute regime. the data suggest that synthetic polymers are not a valid representation of the cellular interior. biotechnology department, university of verona calcium (ca ) is one of the most important second messengers in eukaryotes. ca binding proteins can be subdivided into two categories: "ca buffers" that modulate ca ion concentrations in cells, and "ca sensors" that decode ca signals in a wide array of physiological processes in response to external stimuli. calmodulin (cam) is the prototypical example of ca sensor proteins in both animals and plants. in addition to conserved cam, plants possess a unique family of cam-like proteins (cmls). many of these cmls still remain uncharacterized and the investigation of their biochemical and biophysical properties will provide insight into ca signalling in plants. herein, a detailed characterization of arabidopsis thaliana cml is reported. cml is a protein of amino acids with a theoretical molecular weight of , da and % amino acid sequence identity with atcam . cml is predicted to have one functional ca binding site despite the presence of three ef-hand motifs (prosite). we overexpressed cml in e. coli and analyzed its biochemical and biophysical characteristics, i.e. calcium affinity and stoichiometry and eventual changes in conformation, thermal stability and proteolytic susceptibility upon ca binding. isothermal titration calorimetry (itc) and nuclear magnetic resonance (nmr) spectroscopy identified one ca binding site in cml and showed that ca and mg compete for the same binding site. the kd values determined by itc established that cml has higher affinity for ca than for mg . our data were consistent with the sequence based prediction of one functional calcium binding site. differential scanning calorimetry (dsc) showed that ca and mg have the same stabilizing effects on protein folding. apo-cml undergoes two thermal unfolding transitions, but in the presence of ca or mg only one unfolding event at an intermediate temperature occurs. limited proteolysis experiments showed that ca binding affords protection against cml digestion by trypsin. surprisingly, cml exhibits very few conformational changes upon calcium binding, which were evaluated by ans fluorescence and stokes radius measurements in the apo-and ca bound-forms. these results suggest that cml does not show the characteristics of a classical ca sensor protein. to better understand the physiological role of cml in plants, in vivo analysis will be performed. pb- fbp controls the hepatocyte morphology through rho signaling jun zhang , mingming ling , qianying zhang , yunhong wang , deqiang wang the department of cell biology and genetics, the formin-binding protein (fbp ) widely expressed in eukaryotic cells was previously identified to play a role in morphological maintenance in hepatocyte, but the molecular mechanism keeps still unclear so far. in the present investigation, it was found that rho family proteins cdc /rac signaling was involved in the morphological regulation controlled by fbp . knockdown of endogenous fbp expression with rnai technique or dominant negative mutant of fbp could trigger the cell morphological remodeling from the epithelioid to fibroid following the significant down-regulation of cdc / rac activities and dephosphorylation of paxillin. while the rho protein specific activator could restore the cdc /rac activities, and in turn abrogated the silence effect. overexpression of wild type fbp could not result in any of the morphological transition. furthermore, withdrawal of the silence could induce morphological recovery when the fbp expression, cdc /rac activities and paxillin phosphorylation were restored to the normal level. the experimental evidences strongly indicated that fbp was implicated in morphological control probably via rho signaling pathway in hepatocyte. key words: fbp ; rho signaling; paxillin; morphological control; hepatocyte this work was supported by a grant from national natural science foundation of china (nsfc, no. cytochrome c oxidase (cco) is the final enzyme in the respiratory chain of mitochondria but also an integral part of the metabolism of many types of bacteria. in a complex, stepwise redox-reaction, cco catalyzes the reduction of molecular oxygen to water and utilizes the resulting free energy to pump protons across the membrane thereby creating an electrochemical gradient [ , ] . to investigate proton pumping spectroscopically it is possible to label the entrance of the proton entrance channel with fluorescein, a ph sensitive dye, which allows determining time resolved local changes in proton concentration at the cytoplasmic cco surface and related properties. it has already been shown that the redox state of copper and heme centers affects such properties at the cytoplasmic surface. [ ] this study is a theoretical approach to investigate changes of pka values of the fluorescein label at the entrance of the k-channel for different protonation pattern in both oxidized and reduced cco by performing molecular dynamics (md) simulations. further work is based on calculations of pka values of the fluorescein using software karlsberg [ , ] . methods for genetically and synthetically manipulating protein structure enable a greater flexibility in the study of protein function. we have shown that using inteins as traceless, cleavable purification tags enables the separation of full length unnatural amino acid (uaa) containing proteins from their corresponding truncation products. this method has been used to incorporate uaas in previously unattainable positions in a variety of proteins using a myriad of uaas, inteins, and purification tags. in other applications, we have used e. coli aminoacyl transferase (aat) to selectively modify the n-termini of proteins with uaas in denaturing conditions and conditions that maintain folding. applications of particular interest include overcoming the need for an n-terminal cys residue in expressed protein ligation, transfer of reactive handles for "click" chemistry labeling of proteins, and transfer of fluorogenic molecules for photophysical experiments. we have found that aat can transfer protected cysteine, homocysteine, and selenocysteine to expressed proteins. after ligation, these residues can be converted to met or ala, making the ligation traceless. we continue to develop variants of aat to broaden the substrate scope of both its transferred substrate and n-terminal recognition element. in addition, expressed protein ligation is being used to incorporate backbone modifications, such as the thioamide, into various positions in the protein calmodulin to determine how these modifications can impact the structure and function of an ordered protein. in general, by working at the interface of several protein modification technologies, we have made beneficial discoveries that might be missed by more focused approaches. function and modularity of cw_ motives in the c-terminal region of the endolysin cpl- encoded by the cp pneumococcal bacteriophage manuel iglesias-bexiga , , noelia bernardo-garc ıa , rub en mart ınez-buey , noem ı bustamante , , guadalupe garc ıa , , marta bruix , juan hermoso , margarita men endez , dept. of biological physical-chemistry, iqfr-csic, ciber of respiratory diseases (ciberes), department of crystallography and structural biology, iqfr-csic, bacteriophage lytic murein-hydrolases have been proposed as enzybiotics, an efficient way to fight bacterial infections. however, the use of these enzymes is normally restricted to gram-positive bacteria since the outer membrane of the gram-negative bacteria hampers the access of the hydrolases to the peptidoglycan substrates. all the murein hydrolases reported in the pneumococcal system, both from host or phage origin, depend on the aminoalcohol choline to be fully active. there is only a unique exception to this rule, the cpl- lysozyme. this hydrolase is encoded by the lytic pneumococcal phage cp- and, instead of the common cell wall binding module (cwbm) that recognizes choline, cpl- harbors a completely different cell wall binding structure. recent studies have revealed that reducing the net charge of the cwbm, from . to . , leads to an improvement in the antibacterial activity of cpl- ( ) . the cwbm of cpl- is composed by three identical repeats of amino acids, the cw_ motives, and it folds both in the presence and in the absence of the n-terminal catalytic module ( ) . this module shows the capacity of recognize the glcnac-murnac-l-ala-d-isogln muropeptide (gmdp), structurally related with the peptidoglycan basic unit ( ) . here, we report the high resolution structure of the cell wall binding module of the cpl- endolysin. each cw_ repeat is composed of a bundle of three a-helices with a highly negative electrostatic charge at the surface. the strong inter-repeat interactions and the high ionic strength used in the crystallization conditions allow them overcoming the electrostatic repulsions inducing a closed-packed structure with a three-fold symmetry. the module dimensions ( x x Å) and the repeat arrangement in the crystal structure are inconsistent with the gmdp binding characterization, the activity displayed by cpl- truncated variants with one or two cw_ repeats, or the experimental determined hydrodynamic properties. using the small angle x-ray scattering (saxs) technique and the atsas computational platform ( ), a different arrangement of the cw_ repeats is envisaged in solution (fig. ) , whose rather opened structure ( x x Å) is consistent with the experimental data. additionally, employing the saxs-based structure and the honeycomb structure proposed for the peptidoglycan, a model, where each cw_ repeat of the cell wall binding module fit in adjacent glycan chains, has been derived. in , the protein structure initiative (psi) was started as to determine three-dimensional structures of proteins within every family. once solved, structures are deposited into the protein data bank (pdb) and termed structural genomics (sg) proteins. as of june , there are over , sg proteins deposited in the pdb and most of them are of unknown or uncertain biochemical function. in addition, many of these sg proteins have a putative functional assignment based on their sequence and structural similarities with proteins of known function; such comparisons can be made against large databases using programs such as blast or dali. however, these putative functional assignments are often incorrect. this project analyzes members of the crotonase superfamily (cs). the cs consists of five diverse functional subgroups that are well characterized structurally and functionally, representing different types of reactivity, including hydrolase, isomerase, hydratase, and dehalogenase activities. this superfamily also contains at least sg proteins, so it is ideal to test predictions of protein function. our approach is based on local structure matching at the computationally predicted active site. first, partial order optimum likelihood (pool) is used to predict the functionally important residues of each sg protein and of the proteins of known function in the superfamily. next, structurally aligned local sites of activity (salsa) is used to align the predicted catalytic residues of the well-characterized members in the superfamily. from this analysis we generate chemical signatures for each functional subgroup and compare them to the sets of catalytic residues predicted for the sg proteins. we demonstrate based on these computational methods that the majority of the putative annotations in the cs superfamily are likely incorrect. currently, biochemical assays are being used to test these predictions. preliminary biochemical results show that one sg protein, thermus thermophilus q sls _thet , classified as a probable enoyl-coa hydratase, possesses hydrolase activity as predicted by our methods. the outcomes of this project will be to successfully classify the biochemical functions of sg proteins based on their local structure at the predicted active sites and to provide a conceptual framework for the functional classification of the remaining sg proteins within the pdb. this work is supported by nsf-che- . directly observing the synergistic dynamics in f-actin and microtubule assembly jun zhang , deqiang wang the department of cell biology and genetics, key laboratory of molecular biology on infectious disease although important in cellular activities, little attention was paid to the synergistic effects of actin and microtubule cytoskeleton assembly. with the time-lapse atomic force microscope (tl-afm), we directly observed the large-scale dynamic structure of actin filaments formed in the presence or absence of microtubulin in solution. in absence of microtubulin, the g-actin could be polymerized into ordered filamentous structures with different diameter from the slimmest filament of single f-actin to giant filament in tree-like branched aggregates. the polymerized actin filaments, to which our most intense attention was attracted, was discretely arranged and showed obvious polymorphism in structures completely distinct from those in the presence of microtubulin. the supra-molecular complex structures of the latter were mainly composed of single f-actin and/or multifilaments clearly consisting of several single f-actin and regularly cross-linked with the assembled microtubular bundles. the experimental results demonstrated that the f-actin dynamics could be coordinated by microtubule assembly. further analyses implied that the interactions between f-actin and microtubule could prevent the emergence of structural polymorphism of f-actin alone, and give rise to organization of specific complex structures instead. it was suggested that dynamic synergy between the f-actin and microtubule would be implicated in living cells. the adaptor protein - - is found in a diverse range of pathologically relevant protein-protein interactions (ppis). as - - is a hub protein with very diverse interactions, it is able to influence the intracellular localization of their binding partners and they are key regulators of signal transduction processes as well as regulators of cell cycle functions.nevertheless, there are only few examples of - - acting extracellularly. one of the extracellular targets for - - is aminopeptidase n (apn). apn is an extracellular trans-membrane enzyme that acts as a receptor for - - . binding to apn, - - excreted by keratinocytes can upregulate the excretion of matrix metalloproteinase- (mmp ) in fibroblasts. mmp , by breaking down collagens, is key in the remodeling of the extracellular matrix. modulation of the - - /apn interaction thereby may play a crucial role in the fundamental understanding and ultimately treatment of wound healing, respiratory diseases and tumor growth. in the eukaryotic cell, the - - dimer operates as an adapter platform for binding partners. a wide range of classes of (small) molecules, natural products and peptides has been used to modulate the ppis, providing either stabilization or inhibition of the interactions of - - with its binding partner. binding partner fragments or peptides are known to bind to the - - binding groove via arecognition motif containing a phosphorylated serine or threonine. making use of the dimeric structure of - - , novel small-molecule inhibitors may be tethered to exploit the bivalent effect. from a large virtual screening and experimental validation, a scaffold containing a phenyl phosphonic moiety was identified, showing inhibitory properties for - - ppis. potent derivatives of this scaffold were bridged by polyethylene glycol (peg) linkers of varying lengths, thereby facilitating the compound to reach both binding sites of the - - dimer and concurrently increasing the compound's solubility in aqueous solution. similar bivalent inhibitors have been proven to synergistically increase their efficacy. biophysical evaluation by means of fluorescence polarization (fp) inhibition competition assays, revealed an increase of the half maximal inhibitory concentration (ic ) from approximately lm for the monomeric phenyl phosphonate to approximately . lm for the bivalent inhibitor with a Å linker. this demonstrates a -fold increase of inhibitory effect towards - - and its binding partner peptide mimic. extensive thermodynamic, kinetic and structural analysis of the interaction is in progress.phosphonic moieties have been shown to pass the cell membrane poorly, due to their highly charged character. by being able to specifically inhibit the extracellular interaction between - - and apn, these inhibitors are prevented from interfering with the extensive intracellular - - interactome. hence, these bivalent phenyl phosphonate inhibitors provide a promising strategy towards extracellular application. the mre complex is an oligomeric assembly comprising of dimmers of mre and rad proteins in archea and additionally nbs subunit present in eukaryote. it is the central player in the dna damage response -a functional network comprising dna damage sensing, signal transduction, cell cycle regulation and dna double strand breaks (dsbs) repair [ ] . recent structural studies revealed that rad hinge domain is rather a short kink in the coiled-coil region and adopts unusual dimerization mode by intermolecular coordination of zn(ii) and formation of so-called zinc hook domain [ ] . to date, very limited structural data on the zinc hook domain have been reported, the only known structure was resolved for rad homologue from hyperthermophilic archaeon -p. furiosus. unusual zn(ii) coordination mode in zinc hook domain raises question of how zinc hook domain assembles to form interprotein zinc binding site with sufficient stability to function at low intracellular free zn(ii) concentrations [ ] . our study on minimal zinc hook domain fragment ( aa) indicated low femtomolar affinity towards zn(ii) [ ] . extended zinc hook domain fragment ( aa) reveals even zeptomolar affinity. therefore, our main goal was to probe the thermodynamic and structural effects that are hidden in the small interprotein interface and are responsible for the dimerization of the large and critical protein machinery. probing of those effects was achieved by detailed biophysical characterizations (including potentiometry, nmr, hdx ms and cd spectroscopy) of protein fragments of zinc hook domains with a number of point mutations. we showed that extremely high stability of zinc hook domain from p. furiosus is achieved by the formation of hydrogen bond network in b-hairpins and interprotein hydrophobic core. eindhoven university of technology dna-based molecular circuits have become a very attractive tool in molecular imaging, synthetic biology, molecular diagnostics and biomolecular computing. the highly modular and predictable nature of watson-crick base pairing allows the construction of complex circuits using a limited set of logic gates and building blocks. however, the lack of generic approaches to interface dna-based molecular circuits with protein activity limits their application in biomedicine and molecular diagnostics. here we present a new, highly modular approach to control the activity of a reporter enzyme based on the dna-directed assembly and disassembly of a complex between tem -b-lactamase and its inhibitor protein blip. both proteins are conjugated to a unique oligonucleotide, allowing the assembly of the enzyme-inhibitor pair and inhibition of enzyme activity by the addition of a complementary template strand. addition of an oligonucleotide that is complementary to a loop sequence in the template results in the formation of a rigid dsdna spacer that disrupts the enzyme-inhibitor complex, restoring enzyme activity. using this noncovalent approach allowed easy tuning of the template and target sequences with only a single set of oligonucleotide-functionalized enzyme and inhibitor. to show the modularity of the system, a panel of different template sequences were selected. only in the presence of their complementary viral dna sequences restoration of enzyme activity was observed. in addition to this excellent specificity the system showed to by higly sensitive towards its target, since the presence of as little as fmol of target resulted in an observable increase in enzyme activity. the use of a stable and well-characterized enzyme-inhibitor pair, complemented by the modular design of our reversible dna-directed protein switch make it an attractive system to implement in dna-based molecular circuits. several studies demonstrated important roles of human carbonic anhydrases (hcas) in a variety of physiological and pathological processes. consequently, in recent years the catalytically active hca isoforms have become an interesting target for the design of inhibitors with biomedical applications [ ] . derivatized sulfonamides of type r-so nh represent the class of ca inhibitors (cais) mostly used and best characterized. the large number of crystallographic studies so far available on these molecules clarified the main factors responsible for the binding of the sulfonamide moiety to the ca active site. in particular, it has been highlighted that even though these molecules generally behave as very potent cais, they do not show selectivity for the different isoforms. indeed, the sulfonamide moiety plays a predominant role in the interaction with the enzyme, while any change in the nature of the r substituent has generally a rather marginal effect on the enzyme-inhibitor affinity. these characteristics make difficult the design of sulfonamide derivatives selective for the different ca isoforms. consequently, much efforts were dedicated in last years to the development of new inhibitors that, although presenting lower affinity for the ca active site, would be able to be more selective toward the different isoforms. carboxylic acids have been recently investigated as cais, showing that these molecules can adopt different binding modes to the enzyme active site. in particular, they can coordinate directly to the zinc ion or be anchored to the zinc-bound water molecule. however, the structural reasons responsible of this peculiar behavior have not been clarified yet. in a general research project aimed at providing insights into the binding mode of these molecules to cas, we have undertaken the characterization of two carboxylic acids, namely an ortho-substituted benzoic acid [ ] and a saccharine derivative, by means of kinetic, crystallographic and theoretical studies. exploring the mechanism of fibril formation using fluorescently labelled human lysozyme variants ana bernardo gancedo 'exploring the mechanism of fibril formation using fluorescently labelled human lysozyme variants' human lysozyme is a widely characterised protein whose mutational variants misfold into fibrils that are associated with systemic amyloidosis ( ) . although the process of aggregation for human lysozyme has been well studied, the details of early events within this process are not fully characterised. single molecule fluorescence microscopy has been used to determine the oligomeric distributions present in the aggregation process of a number of disease-related intrinsic disordered proteins (idps) ( ) . recent advances in site-specific labelling of human lysozyme ( ) have made this protein amenable to these single molecule fluorescence studies. we have introduced alexa-fluorophores into the i t variant of human lysozyme and have demonstrated that the process of in vitro fibril formation is not significantly altered. using these fluorophore-labelled proteins we can apply single molecule fluorescence to study the early aggregation events within this system, allowing us to compare protein aggregation in a globular protein and with the aggregation process of idp's. abstract protein structure, folding and function, while specific interactions with lipid molecules can also contribute towards the biological activity of some membrane proteins. improving understanding of the interactions has resulted in the development of artificial lipid systems that allow the bilayer properties to be rationally manipulated in vitro to control protein behaviour. the bacterial transporter lacy is a well known integral membrane protein from the major facilitor superfamily, responsible for the protondriven uptake of d-lactose in e. coli. with a high resolution structure available and considerable understanding of mechanistic detail, and with observed changes to both structure and function in different bilayer environments, lacy is a good model system for examining the behaviour of a major class of membrane proteins in these lipid systems. purified lacy has been reconstituted into liposomes and droplet interface bilayer systems of varying lipid composition and the effect on protein function and bilayer properties examined. targeting abeta oligomers by trehalose-conjugated peptides: a molecular dynamics study alzheimer's disease (ad) is currently one of the most common and devastating forms of dementia correlated with beta-amyloid peptide (abeta) accumulation in human brain tissue [ , ] . inhibiting abeta selfoligomerization in brain tissue remains one of the main strategies to prevent or treat this disorder. as a consequence, in recent years much efforts have been spent in the understanding of the amyloid fibril growth process and its modulation by putative drug molecules. an interesting class of compounds able to prevent abeta fibrillogenesis, is represented by beta-sheet-breaker (bsb) peptides [ ] . although these molecules are thought to recognize in a self-complementary manner the abeta hydrophobic core region, however their precise mechanism of interaction is still unclear. in this context, we have studied the structural basis underlying the inhibitory effect of abeta( - ) fibrillogenesis explicated by two promising trehaloseconjugated bsb peptides (ac-lpffd-th (thct) and th-succinyl-lpffd-nh (thnt)) [ ] using an all-atom molecular dynamics (md) approach [ , ] . the pentameric nmr structure [ ] of abeta has been used to model amyloid protofibril, and the two protofibril ends have been investigated as putative binding sites. our simulations suggest that the interaction with the two protofibril ends occurs through different binding modes. in particular, binding on the odd edge (chain a) is guided by a well defined hydrophobic cleft, which is common to both ligands (thct and thnt). moreover, targeting chain a entails a significant structure destabilization leading to a partial loss of b structure and is an energetically favoured process, as assessed by mm/pbsa calculations. a significant contribution of the trehalose moiety to complexes stabilities emerged from our results. the basic structural unit of chromatin is the nucleosome, which is composed of histone proteins forming a scaffold with about base pairs of dna wrapped around. chromatin compacts eukaryotic genomes and regulates gene activity, which is mediated in part by posttranslational modifications (ptms) on the n-terminal tails of the histones. uncovering the detailed relationship between histone tail modifications and gene activity is a major topic of biomedical sciences and general techniques for generating nucleosomes with defined modification patterns in large numbers would greatly facilitate such investigations. to this end we are establishing a chemical toolbox for designer chromatin with defined histone ptm patterns. a protein semysinthesis approach is used that bases on "ligation-ready nucleosomes" with truncated histone h that can be ligated with the corresponding synthetic histone tail. we resorted to sortase-mediated ligation as chemoselective ligation method. here we report our recent developments in establishing the envisioned chemical toolbox for designer chromatin. evaluating cation-pi and pi-pi interaction in proteins using various biophysical methods in proteins the aromatic residues phenylalanine (phe), tyrosine (tyr), and tryptophan (trp) can be involved in aromatic interactions known as cation-pi and pi-pi interactions (dougherty ). compared to other non covalent interactions in proteins, like h-bonds, dipole-dipole, or van der waals interactions, relatively little is known about the pi-pi and the cation-pi interactions. the strength of both aromatic interactions is dependent on the pi-electron density in the aromatic residues. a lowering of electron density can be created by introducing strong electron-withdrawing substituents like fluorine atoms in the aromatic ring (dougherty ). in this way a nearly isosteric change in the aromatic system results in a marked change in electron density. substitution with methyl groups is known to slightly increase the electron density. the response to low cellular oxygen levels in humans and other animals is induced by the hypoxia inducible transcription factors (hifs). these transcription factors are regulated by hypoxia inducible factor prolyl hydroxylases (phds), which act as 'oxygen sensors' by hydroxylating hifs, thus leading to the proteomic degradation of the transcription factors. over the last years, there have been multiple reports that describe additional phd substrates other than hifs. among them are the large subunit of rna pol ii, several transcription factors, and components of signalling pathways. validating these reports is of major medicinal relevance given that phd inhibitors are now in the late stage phase clinical trials. in order to investigate the selectivity of phds, the reported proteins have been tested as substrates for hydroxylation by mass spectrometry, and as binders or competitors of the phds. initial work on peptides that contain the putative hydroxylation sites has indicated that the phds are much more selective for their well-established substrate hif. however, in ongoing work these initial results are going to be validated on protein level by co-expressing phds with the reported substrates. additionally, peptides of reported substrates were screened for their ability to alter the kinetics of hif-hydroxylation by phd . an inhibitory effect of at least two different peptides on phd was observed, suggesting that there is an interaction between the prolyl hydroxylase and these peptides. in order to investigate the mode of binding and inhibition, nmr studies have been carried out and binding of the two inhibitory peptides on phd has been shown. altogether, these results indicate that, although phds might be more selective for hif as a substrate as it was initially thought, the enzymatic activity of the prolyl hydroxylases is possibly influenced by a number of other proteins that can directly bind to phds. non-natural aminoacids via the mio-enzyme toolkit alina filip , judith h bartha-v ari , gergely b an oczy , l aszl o poppe , csaba paizs , florin-dan irimie biocatalysis and biotransformation research group, department of chemistry, ubb, department of organic chemistry and technology an attractive enzymatic route to enantiomerically pure to the highly valuable a-or b-aromatic amino acids involves the use of aromatic ammonia lyases (als) and aminomutases (ams). all these enzymes have in common an auto-catalically formed -methylene- , -dihydroimidazole- -one (mio) electrophilic prosthetic group, and show high structural and sequence similarities. the recent advances in improving the functional properties of these enzymes increased both their biocatalytic and therapeutic applications. we aimed to create a library of recombinant mio-enzymes consisting of the pals and pams with large substrate promiscuity in order to provide access to various non-natural aminoacids through enzymatic ammonia addition and/or ammonia elimination reactions of the substrate library already available in our researchgroup. the developed complementary substrate and enzyme library would provide the mio-enzyme toolkit useful for the synthesis of nonnatural aminoacids. the synthetic gene of the enzymes (pcpal, rtpal, avpal, papam) were cloned into pet b_j expression vector using xhoi and bpu i cloning sites. the plasmid dna was transformed to several e.coli host strains (rosetta, bl , origami ) in order to optimize the expression yields. the enzymes containing an n-terminal his -tag were purified with affinity chromatography, followed by ion-exchange or/and size-exclusion chromatography, obtaining pure and homogenous proteins, in their tetrameric, presumably native fold. the enzyme activity and the kinetic parameters of the purified enzymes was determined towards the natural substrate l-phenylalanine, as well as towards novel bulkier aromatic substrates (heteroaryl alanines, styryl alanines, biphenylalanines). furthermore to enhance their biocatalytic applicability we covalently immobilized the enzymes to carboxylated single-walled carbon nanotubes (swcnt cooh) using linkers with different lengths, and tested the activity and recycling of the immobilized enzyme. antibodies that bind protein antigens are indispensable tools in biochemical research and modern medicine. utilizing a phage display selection strategy, we have obtained synthetic antigen binders (sabs), based on a fab fragment of igg, to a wide array of proteins as distinct as membrane proteins, structural proteins, scaffold proteins and nuclear targets. here we demonstrate the applicability of the sabs towards the native, full-length proteins in cells. we show that the generated sabs are able to pull-down endogenous proteins from mammalian cell extracts along with their natural binding partners. we developed a method of utilizing our high affinity and specificity binders as fluorescently labeled tools to visualize target proteins in their native environment in the cells without the need of secondary antibodies or blocking reagents. our system also includes a method of efficient delivery of generated antibodies to living cells, where they can perform their function. the sabs have been successfully used for altering biological processes in a controllable manner. in vitro evolution from pluripotent peptide libraries with natural neurotoxin scaffolds to target receptors, proteases and trophic factors tai kubo , mohammed naimuddin , seigo ono national institute of advanced industrial science and technology (aist) in vitro evolution from pluripotent peptide libraries with natural neurotoxin scaffolds to target receptors, proteases and trophic factors small molecule natural products are precious resources for drug discovery. during millions of years of evolution, natural products must have been exposed to various selection pressures and have been refined in structure and function to obtain the present features. in some peptide neurotoxins, however, the basic molecular scaffold mainly configured by disulfide (s-s) bridges and/or alpha/beta structures, is strictly conserved within each family even under the evolution pressure. on the other hand the loop regions, which are not heavily involved in scaffold formation, are highly diverged. this mode of molecular evolution named 'accelerated evolution', is reasonable to quickly adapt to the vigorous change of the environment. the evolutionally selected scaffold is compact harboring both rigidity and flexibility in nature, and it may support a topology appropriate for target recognition and selective interaction. inspired by the system, we designed random peptide libraries from the peptide neurotoxins of the accelerated evolution. a three-finger ( f) shaped snake neurotoxin consists of huge family evolved by accelerated gene evolution. we prepared a f-peptide library by introducing random sequences in each fingertip. another random peptide library with an ick (inhibitor cystine knot) motif was prepared based on a neurotoxin gtx - from spider; originally identified as a t-type ca channel modulator. each library was subjected to in-vitro evolution directed to specific target molecules. for the f-peptide library cdna display method was applied to select binders. when interleukin- (il- ) receptors were targeted, the selected f peptides showed binding affinities (kd nm) comparable to the native ligand il- . when trypsin was targeted, peptides with serine protease inhibitor activities similar to sti and bpti (ki nm) were isolated. specific binders to a trophic factor vegf were also generated from the f library. to target membrane proteins, we developed a unique in-vitro evolution system, and named it as the periss (intra periplasm secretion and selection) method. in the system, target membrane proteins are expressed in inner membrane of e. coli and peptides are secreted to the periplasmic space, in between the inner and outer membranes; and the space is served for interaction and selection. the periss method enabled us to identify a peptide specific to muscarinic receptor m subtype from the ick peptide library. in conclusion, it was proved that the library designed from the scaffold of peptide toxin, which evolved in the mode of accelerated gene evolution, has pluripotency in target recognition, interaction and even bioactivity. phenylalanine ammonia lyase from petroselinum cripsum (pcpal) belongs to the class of enzymes containing -methylideneimidazole- -one (mio) as a prostetic group and it is responsible for the conversion of l-phenylalanine into trans-cinnamic acid. this reaction is reversibile under high ammonia concentration. we analyzed several factors that can influence the enantioselective synthesis of nitrophenylalanine mediated by whole cells as well as purified mio-containing and mio-less pcpals. first we investigated the behaviour of the enzymes depending on the ammonia concentration. we also inspected the influence of the ph on the pcpal catalyzed biotransformations. based on our results, we concluded that variation of ammonia concentration and the ph leads to decrease of enantioselectivity, suggesting that pcpal is able to catalyze the formation of both l-and d-enantiomers of electron-deficient structures. all microbial cellulase appears to have a conserved 'sg' amino acid sequence at an identical position in the n-terminal domain. the properties of the n-terminal amino acid sequence were also predicted computationally. this analysis showed that n-terminal sequence of the enzyme is unstable. the nterminal sequence also showed potential cleavage sites by different proteases which may contribute to its instability. the secondary structure analysis showed that the n-terminal sequence has % of the a.a. sequence in extended strand and % in random coil conformation. the n-terminal sequence was also analyzed for potential phosphorylation sites. while no potential serine and threonine sites were predicted, two tyrosine phosphorylation sites were predicted in the n-terminal sequence. the n-terminal sequence was also examined for the presence of kinase specific phosphorylation sites. the results showed the presence of one potential site which may be phosphorylated by pkc at position of the n-terminal sequence. the analysis for the prediction of the presence of oglcnac sites revealed that two such sites may potentially be present in the sequence. we have also predicted the ligand binding site in the n-terminal sequence of the protein. protein arginine methylation catalyzed by protein arginine methyltransferases (prmts), is a pivotal protein post-translational modification involved in a growing number of physiological and pathological processes including signal transduction, proliferation, differentiation and malignancy. prmt accounts for the majority of protein arginine methyltransferase activity in mammalian cells and, in consistence, a large amount of cellular substrates have been identified. several studies have reported that the activity of prmt changes upon stimulation in various cellular processes. in mammalian cells, prmt exists in a high molecular weight complex. the interacting partners of prmt , such as antiproliferative proteins btg and btg , protein phosphatase a, the orphan receptor tr , and ccr -associated factor (hcaf ) are shown to play a role in modulating the methyltransferase activity and the substrate selectivity of prmt . due to the pivotal roles of prmt in physiological and pathological conditions, intensive efforts have been put on the search of small synthetic chemical molecules which can efficiently modulate the activity of prmt for the potential development of therapeutics. in light of this, the intracellular small molecules that either transmit extracellular stimulation or act as cofactor to dictate the activity of prmts in cells are still poorly understood. our study focused on examining how cellular ions might affect the activity of prmt and found that divalent and monovalent ions differentially modulated the catalytic activity of prmt toward different substrates. oligomerisation properties of light-dependent protochlorophyllide oxidoreductase prothoracicotropic hormone (ptth) is one of the most important neuropeptide regulators for insect molting and metamorphosis. however, preparation of its recombinant protein has hardly been successful, because it is a homodimer protein with very complicated disulfide-bond structure. for example, silkworm ptth has three intramolecular disulfide bonds in its -residue polypeptide chain, and the two chains are further linked by an additional intermolecular disulfide bond to form the homomeric dimer. although the recombinant silkworm ptth was previously expressed in escherichia coli, the product was obtained only in precipitation fractions, and refolding of the precipitated protein provided the active dimer ptth in very poor yield. under such reductive conditions as in cytosol of the e. coli cells, formation of the correct disulfide-bond arrangement must be difficult. alternatively, for the heterologous expression of the silkworm ptth, we employed brevibacillus choshinensis (formally referred to as bacillus brevis), which has achieved good results in expression of various disulfide-bond-containing proteins. in this study, the silkworm ptth was expressed in the brevibacillus cells with an additional his -tag sequence at the c-terminus, for easier detection and purification. first of all, since the brevibacillus bacteria are equipped with a secretory system of the expressed proteins, a secretory signal sequence to be attached before the silkworm ptth was carefully selected. among four candidates in a commerciallyavailable kit, a signal sequence derived from an intrinsic cell-wall protein mwp gave better results in expression levels of the protein. second, incubation time of the cells was optimized, because an oligomerization state of the secreted ptth in the cell culture medium changed with the time. in the medium, various ptth oligomers including a monomer and a dimer were initially observed, but higher oligomers became a major portion of the secreted product after longer incubation than h. incubation for - h may be suitable for obtaining the native dimer form of the silkworm ptth. to remove the undesired monomer and higher oligomers, which mostly retained free sulfhydryl groups, the secreted proteins were treated with maleimide-peg -biotin. in the purification using a ni -nta column, the dimer of the his -tagged silkworm ptth was eluted with an imidazole gradient, separately ahead of other biotinylated proteins, probably due to interaction of the peg spacer with the ni -nta groups of the resin. after the reversed-phase hplc purification, the final product showed a single band on the nonreductive sds-page, and it had adequate ecdysone-releasing activity from isolated silkworm prothoracic glands. the brevibacillus bacteria are most promising host cells for the heterologous production of the insect ptth. role of the disulfide bridges in the transmembrane region of the insect prothoracicotropichormone receptor, torso torso is an insect cellular-membrane protein, which was recently identified as a receptor for prothoracicotropic hormone (ptth). although ptth is one of the important regulatory molecules in insect molting and metamorphosis, activation mechanism of torso by the ligand has not been elucidated yet. in this study, an oligomerization manner of the silkworm torso was examined, using heterologous expression in drosophila s cultured cells, because torso is a single-polypeptide receptor tyrosine kinase (rtk), and activation of such rtks is often triggered by the ligand-induced receptor dimerization on the cellular membrane. when activated with silkworm ptth, dimerization of the silkworm torso in the s cells was observed, using a cross-linking reagent bs , and the subsequent receptor autophosphorylation and downstream erk phosphorylation were also detected. surprisingly, however, the torso dimerization was revealed to occur even without the ligand stimulation, while the autophosphorylation and the erk phosphorylation were held in response to the stimulation. when fractionated by non-reductive sds-page, the silkworm torso showed an obvious dimer band, in addition to a faint monomer band, both with and without the ptth simulation, even though the receptor was not treated with the cross-linking reagent. this indicates that the torso protein is expressed originally as a disulfide-bond-linked dimer. in addition, by examining oligomerization states of several truncation and substitution mutants, cysteine residues in the transmembrane region were found to participate in the intermolecular disulfide bridges, linking the two receptor molecules in the dimer. when all of the three cysteines in the transmembrane region were replaced by phenylalanines, the disulfide-bond-linked torso dimerization was not observed, but spontaneous, ligand-independent association of the torso molecules was detected using the crosslinker bs . this spontaneous dimerization caused the apparent torso autophosphorylation, but it could not induce the downstream erk phosphorylation. consequently, without the intermolecular disulfide bridges, torso loses its responsiveness to the ptth stimulation. in conclusion, the disulfide bridges in the transmembrane region may play a role to preserve suitable relative position between the two torso molecules, which could induce ligand-dependent autophosphorylation leading to activation of the downstream signaling pathways in the cells. the yeast enzyme neutral trehalase (nth , ec . . . ) from saccharomyces cerevisiae hydrolyses the non-reducing disaccharide trehalose which serves as an energy source and a universal stress protectant in many different organisms. enzymatic activity of nth is enhanced by the yeast - - protein (bmh and bmh ) binding in a phosphorylation-dependent manner. nth activity is also regulated by ca binding to the ef-hand-like motif containing domain of nth [ ] .the native tbe page and analytical ultracentrifugation show that nth forms very stable complexes with bmh and bmh [ ] . to study the structure of nth alone and its complex with the - - protein we used circular dichroism, h/d exchange coupled to mass spectrometry, chemical cross-linking [ ] and small angle x-ray scattering (saxs) [ ] . at the same time protein crystallography of nth alone and its complex with bmh is performed.the low resolution structure of pnth :bmh protein complex revealed that binding of bmh induces a rearrangement of the whole nth molecule and that the region containing the ef-hand motif forms a separate domain which interacts with both bmh and catalytic domain of nth . we proved that integrity of the ef-hand motif is crucial for the bmh mediated activation of nth and ca binding. our data suggest that the ef hand-like motif functions as the intermediary through which bmh modulates the function of the catalytic domain of nth . these structural changes probably enable the substrate entry into the enzyme active site [ ] . our study of - - protein complex with the fully active enzyme nth offers a unique structural view of nth activation enabling us to better understand the role of the - - proteins in regulation of other enzymes. the assembly of self-regulating synthetic biochemical pathways in vitro has great potential as alternative catalysts for the high-yield production of low value/high volume commodity chemicals from biomass. high yields of low-value/high volume compounds that are required for economic viability is particularly difficult via traditional in vivo metabolic engineering of microbes due to competing biochemical pathways and toxicity. we have developed an alternative approach, called synthetic biochemistry, where the glycolysis pathway of central metabolism is reconstituted in vitro with an anabolic pathway that can produce useful compounds at high yield. in the specific synthetic biochemistry system described, reducing equivalents, atp, and carbon from glycolysis are funneled through the anabolic mevalonate pathway to produce the monoterpene limonene from glucose. the successful implementation of the in vitro pathway required development of a molecular purge-valve consisting of an nad and nadp specific reductase (ie wild-type and mutant pyruvate dehydrogenase), and nadh oxidase, noxe, to maintain proper nadp /nadph cofactor balance while allowing continuous carbon flux. we find that the purge-valve concept is readily transportable to other nad(p)h generating steps in central metabolism and can be used to convert glucose to limonene at high yield. chitinases (ec . . . ) are enzymes that randomly hydrolyze b- , glycosidic bonds of chitin and produce n-acetylchitooligosaccharide ((glcnac)n) that has various physiological functions such as immunostimulatory activity. most of fish takes crustacean such as shrimp and crab as food. therefore, the fish has chitinase in the stomach to chemically disrupt the chitinous envelope of crustacean. four chitinase isozymes ( - kda), pachia [ ] and pachib [ ] , and ptchia and ptchib, [ ] were purified from the stomach of silver croaker pennahia argentatus and threeline grunt parapristipoma trilineatum, by ammonium sulfate fractionation and column chromatographies, respectively. all the chitinases were stable and showed activity in the acidic ph range (ph - ). pachia and ptchia preferentially degraded the second glycosidic bond from the non-reducing end of (glcnac)n and pachib and ptchib had a preference for the third glycosidic bond of those. all the chitinases showed different substrate specificity toward insoluble long substrates. moreover, chitinase cdnas (pachi- and pachi- ) encoding pachia and pachib, and cdnas (ptchi- and ptchi- ) encoding ptchia and ptchib were obtained by cdna cloning using the rt-pcr and race method. the deduced amino acid sequences of all the chitinase cdnas contained n-terminal signal peptide, gh family catalytic domain, linker region, and chitin-binding domain. phylogenetic tree analysis of vertebrate chitinase revealed that fish stomach chitinases form unique chitinase isozyme groups, acidic fish chitinase- (afcase- ) including pachia and ptchia, and acidic fish chitinase- (afcase- ) including pachib and ptchib, which was different from an acidic mammalian chitinase (amcase) group. [ , ] the previously reported purified fish stomach chitinases [ ] can also be classified into two chitinase isozyme groups, afcase- and afcase- , by the n-terminal amino acid sequence. this study suggested that fish have excellent chitin degrading enzymatic system in which two different chitinases isozyme groups, afcase- and afcase- , with different degradation patterns are expressed in the stomach. recently, the enzymes produced by psychrophilic organisms have gained huge interest especially in the studies of temperature adaptation of the protein. previously, a cold-adapted yeast, glaciozyma antarctica pi was isolated from a marine environment in antarctica and the yeast was known to produce lipolytic and proteolytic enzymes. a gene encoding a unique recombinant bifunctional enzyme (lippi ) with cold active lipase with protease activity was successfully expressed, purified and characterized. temperature profile of the bifunctional lippi enzyme showed that the lipase functions optimally at c whereas the protease was more active at c. ph profile showed that both lippi lipase and protease were active at near neutral condition. activity of lippi lipase and protease were also activated in the presence of cacl but its protease counterpart seemed to be more active in the presence of zncl . effect of surfactants showed lippi lipase was activated by tween and sls and in contrast, lippi protease was almost deactivated in all surfactants tested. the presence of organic solvents did not affect both the lipase and protease activities. the lipase was more stable at solvents with higher log p value whereas the protease was slightly activated at low log p value particularly with dimethylsulfonyl. inhibitor studies revealed that lippi lipase was partially inhibited with edta and pmsf whereby the lippi protease was inhibited by pepstatin, edta and pmsf. lippi enzyme was successfully crystallized via vapour diffusion method. crystal of lippi enzyme was diffracted via synchrotron radiation. the three-dimensional structure of cold-adapted pi provided insight into cold adaptation and better understanding of the structural properties of lippi enzyme. the bifunctional properties of the enzyme could be potential candidate for low temperature industrial application. conformation-specific antibodies as enhancers and inhibitors of phosphatase activity of dep malgorzata nocula-lugowska , mateusz lugowski , anthony a. kossiakoff the university of chicago dep- (cd /ptp-h) is a transmembrane receptor-like protein tyrosine phosphatase (ptp) that has been implicated in the density-dependent regulation of cell growth, differentiation and transformation. it counteracts protein kinases by dephosphorylating a number of their substrates as well as the kinases themselves, thus potentially controlling the specificity of signals. for example egfr, vegfr , met, pdgf b receptor have been shown to be dephosphorylated by this phosphatase. dep- has been shown to act as a tumor suppressor and it has been proposed as a molecular target in antiangiogenesis therapy. as a result, both enhancers and inhibitors of dep- activity have the potential of elucidating pathways responsible for abnormal cell behavior. we generated synthetic antibodies against intracellular catalytic domain of dep- that act as modulators of the enzyme's phosphatase activity. by applying a combination of selection pressures an array of antibodies has been raised from phage display libraries of fab fragments which are capable of either enhancing or inhibiting dep- activity. in phosphatase assays with catalytic domain of dep- the antibodies demonstrate non-competitive or mixed kinetics. the crystal structure of dep- -inhibitor complex shows that this antibody binds to the part of the protein that is distant from the active site and acts by locking the enzyme in the nonnatural catalytically inactive state by hindering the closure of the wpd loop which is crucial for the reaction to occur. by contrast, as judged from the crystal structure of a complex of dep- with the antibody that enhances its phosphatase activity, this antibody seems to act by stabilizing the naturally found active state of dep- with wpd loop in the closed conformation. the antibodies are also able to recognize dep- in cells, as they stain dep- in immunofluorescence experiments. to test the applicability of raised antibodies in cells the activator was additionally used to pull down full-length endogenous dep- after being delivered to live cells. inhibition and enhancement of dep- activity by locking the enzyme in conformations which are either natural or imposed by allosteric binding of antibodies seems to be a mechanism that can be utilized to modulate activity of other tyrosine phosphatases. investigating acinetobacter baumannii pathogenesis: crystal structure of wbjb epimerase from a polysaccharide biosynthesis cluster oxygen homeostasis is regulated by hypoxia inducible factor, a transcription factor. when the oxygen level becomes too low (hypoxia), hypoxia-inducible-factor (hif- a) activates the expression of over a hundred genes, associated with angiogenesis, erythropoiesis, vegf (vascular endothelial growth factor), cell migration, and energy metabolism etc. hif- a cellular level is highly dependent on oxygen concentration and regulated by oxygen sensor enzyme, hif prolyl hydroxylase (phd plant sulphite reductase (sir) forms an electron transfer complex with ferredoxin (fd) for the reductive conversion of sulphite to sulphide. although previous studies have highlighted electrostatic interactions between oppositely-charged residues of the two proteins, detailed thermoenergetics of the intermolecular interaction for the complexation remains unknown. we herein carried out isothermal calorimetry of fd:sir complex formation at various nacl concentrations. driving force plot constructed from calorimetry showed that the complex was thermodynamically stabilized by both enthalpy and entropy through favourable electrostatic and non-electrostatic interactions. increasing nacl concentrations weakened interprotein affinity and contribution of the negative enthalpy changes became decreased, while no such significant decrease was found in the contribution of positive entropy changes. furthermore, a negative heat capacity change obtained from the enthalpy changes at distinct temperature indicated a contribution of hydrophobic interactions. these findings suggested that both electrostatic and nonelectrostatic interprotein interactions were energetically important for the complex formation. fddependent sir activity assay revealed a bell shaped activity curve with a maximum under a certain nacl concentration, while the methyl viologen-dependent assay of sir exhibited a profile of saturating curve, suggesting that an optimized interprotein interaction is a crucial factor in control of fd-dependent-sir activity. a residue-based nmr measurement of n-labeled fd upon complex formation with sir revealed that charged and non-charged residues were differentially contributed in the complex formation depending on nacl concentrations. we proposed that non-electrostatic forces were also critical for forming the fd:sir complex, and an optimized complex conformation for maximum enzymatic activity was achievable by a delicate balance among non-covalent intermolecular forces. these results may be extended for understanding of complexation between redox proteins containing biased charge clusters. ornithine transcarbamylase has a spatially extended active site as computationally predicted lisa ngu , kevin ramos , nicholas delateur , penny beuning , mary jo ondrechen understanding how an enzyme catalyzes a reaction is a fundamental problem in protein science. biochemical experimentation has revealed catalytic mechanisms of many enzymes; however these studies have focused almost exclusively on amino acid residues in direct contact with the reacting substrate molecule(s). here we report on the computational prediction and experimental verification of the importance of distal residues in enzyme catalysis, using e. coli ornithine transcarbamylase as an example. partial order optimum likelihood (pool), developed at northeastern university, is a machine learning technique that only requires the tertiary structure of a protein to predict important catalytic residues, based on computed, residue-specific electrostatic and chemical properties. pool has been shown to predict accurately the catalytic residues and to discern between compact and spatially extended active sites. dynamic conformational changes during catalysis and strong electrostatic interactions give rise to significant coupling between remote residues and the canonical active site residues of an enzyme. this suggests that at least some enzyme active sites are spatially extended, with second-and third-shell residues playing significant roles in catalysis. in this project, we focus on ornithine transcarbamylase (otc), for which dynamic processes are believed to play a role in its catalytic mechanism. otc is reported to undergo induced-fit conformational changes upon binding carbamoyl phosphate, which affects the subsequent binding of ornithine. residues predicted by pool to be catalytically important include five in direct contact with the substrate, r , h , d , c and r . pool also predicted remote residues to form a spatially extended, triple-layer active site. guided by computational predictions and using site-directed mutagenesis and kinetics assays of asp , his , glu and arg variants, we show that these pool-predicted remote residues, located in the second and third layers, are important for catalysis. alternative energy is a major focus of current research efforts. biodiesel, a mixture of fatty acid alkyl esters, is one of the most versatile alternative fuels currently in use. this is due to the fact that it is similar to gasoline and compatible with diesel engines found throughout the existing global infrastructure. biodiesel precursor lipids are abundant in cultivated feedstock organisms such as algae and bacteria. however, the standard process for converting oil to biodiesel is heat-intensive and requires complete removal of water, reducing the overall net energy gained in its production. our work constitutes an attempt to explore enzymatic synthesis of biodiesel from lipids such as those derived from emerging fuel crops. previous literature describes fatty acid alkyl ester formation in human patients with mrsa staphylococcus aureus wound lesions. these esters are formed by partially characterized esterase activity from an unidentified source. we have identified two mrsa enzymes responsible for this activity by using a combination of size exclusion chromatography, gas chromatography-mass spectrometry, and mass spectrometric protein sequencing. these two highly similar enzymes in the glycerol ester hydrolase (geh) family of proteins catalyze the synthesis of fatty acid alkyl esters in aqueous conditions at or near room temperature. we have demonstrated that other non-staphylococcal lipases do not exhibit this behavior. we have expressed these staphylococcal esterases in e. coli, and shown via gas chromatography that the expressed proteins catalyze the formation of fatty acid alkyl esters. based on sequence similarity to homologous proteins that have already been crystallized, we have predicted a structure for these enzymes and have engineered mutant fusions with higher rates of catalysis. our design hypothesis is that increased avidity for substrate molecules will yield a higher substrate concentration in the vicinity to the enzyme. to increase substrate concentration we have designed and expressed one of the enzymes as a chimeric fusion with the drosophila melanogaster alcohol-binding protein lush. gc-ms determination of biodiesel production rate indicates that the chimeric fusion has a lower-order rate constant with respect to ethanol. in other words, the fusion enzyme is less dependent on substrate concentration and is a superior catalyst at low ethanol concentrations. this result indicates that the rationally designed modification of binding avidity constitutes a potential avenue for improving the ability of enzymes to catalyze reactions with low-concentration or low-solubility substrates. functional elements of a human antizyme essential for binding and inhibiting human ornithine decarboxylase proteases are ubiquitous enzymes that catalyze the hydrolysis of peptide bonds within protein substrates; they have served as key model enzymes for studying the molecular basis for catalytic power and specificity. protease substrate specificity is most often defined in terms of linear sequence motifs that flank the cleavage site; however, the natural substrates of proteases are proteins with -dimensional shapes and complex conformational dynamics that are not well represented by -dimensional sequence alone. these structural and dynamical properties can impact recognition and binding of substrates by proteases, as well as the efficiency of catalysis itself. in this study, we explore the importance of substrate structure and dynamics for proteolysis using as our model the cleavage of the kunitz-bpti family of canonical serine protease inhibitors by mesotrypsin. bovine pancreatic trypsin inhibitor (bpti), an archetypal serine protease inhibitor of the kunitz family, has a high affinity interaction with trypsin, yet its peptide bond hydrolysis is many orders of magnitude slower than other peptide substrates. mesotrypsin, a trypsin variant, has been shown to hydrolyze kunitz family inhibitors at accelerated rates; this is especially true of human kunitz domain inhibitors. amyloid precursor protein inhibitor (appi) and amyloid precursor like protein- (aplp ), two human kunitz domain family members, are hydrolyzed by mesotrypsin several hundred times faster than bpti. here, we present a new, unpublished crystal structure of a cleavage intermediate aplp bound to mesotrypsin, refined to . Å resolution, revealing a dramatic substrate conformational change we hypothesize to be required during cleavage of a kunitz domain. using this structure along with published structures of appi and bpti complexes, we have modeled acyl-enzyme intermediates of mesotrypsin, and we have carried out molecular dynamic simulations that explore the transition of the initially formed native-like acyl-enzyme through the conformational transformation that allows the progression of the hydrolysis reaction. we further identify a specific hydrogen bond, present in bpti but not appi, which forms a stabilizing feature of the bpti scaffold. using site directed mutagenesis, we probe the contribution of this bond to the proteolytic stability of bpti. collectively our data for these highly structured substrates show that proteolysis rates are limited by a necessary conformational change in the substrate as the reaction progresses. rigid substrates possessing stabilizing features that render them highly resistant to this conformational change are proteolyzed more slowly than more flexible substrates of similar structure. lpmos are copper metalloenzymes that carry out the oxidative cleavage of the b- , -glycosidic bond, generating new chain ends that can subsequently be processed by cellulases, boosting the cellulose degradation. lpmos have a b-sandwich conformation with a flat binding surface, allowing for the enzyme to bind to crystalline cellulose. the cu ion, required for activity, is located in a so-called "histidine brace", in which the n-terminal histidine is highly conserved. regioselectivity according to the carbon atom being oxidized, lpmo types are identified: type and type oxidizing at the c and the c respectively, type lpmos oxidizing both the c and the c adjacent to the glycosidic linkage. we were able to express a type- lpmo (phanerochaete chrysosporium gh d) and a type- lpmo (trichoderma reesei cel a) in p. pastoris. this has proven to be very challenging, as lpmo activity requires a perfect cleavage of the signal sequence. after activity assays on pasc, characteristic hpaec-pad traces were obtained which will serve as a reference for engineering experiments. enzyme engineering using the dm database, a structure based multiple sequence alignment tool, it is possible to identify residues specifically conserved in subsets of protein sequences. by defining a subset for each lpmo type, we were able to identify residues contributing to regioselectivity. these positions are now being rationally engineered in subsequent rounds of mutagenesis, using trcel a as a template. the effect of the mutations will be determined by analyzing the hpaec-pad trace released from pasc. the main goal is to investigate the possibility of deleting the c specificity in a type lpmo. folding topology determines substrate binding order in the ribokinase superfamily alejandra herrera-morand e , victor castro-fern andez , madrid, españa ribokinase superfamily comprises three enzyme families: the adp-dependent sugar kinases family, the atpdependent coenzyme kinases family and the atp-dependent sugar kinases family. in all these families there is a large domain composed by a rossmann motif but only the atp-dependent enzymes have a b-meander motif in the c-terminal end. interestingly, these enzymes display an ordered kinetic mechanism where the substrate that will be phosphorylated binds first to the enzyme. the adp-dependent enzymes present a topological re-ordering of the secondary structural elements which produces an equivalent tertiary structure, which can be thought as a non-circular permutation (ncp) of the bmeander region. these enzymes also display an ordered kinetic mechanism but with an inversed order being the nucleotide the first substrate to bind to the enzyme. as this b-meander region of the proteins constitutes almost entirely the nucleotide binding site, and given that the permutation is the major structural difference between adp and atp-dependent kinases, it could the responsible for the nucleotide specificity. to test this hypothesis we introduce, by permutation, an atp-dependent topology in the homologous adp-dependent glucokinase from t. litoralis (pergk). size exclusion chromatography and circular dichroism spectra show that both the wild type and the permutated enzyme eluted as monomers with similar hydrodynamic behavior, and have the same secondary structure content. kinetic assays employing atp or adp as substrate demonstrate that even in the presence of mm atp, the pergk enzyme is not able to carry out the phosphoryl transfer. to test if the ncp has an impact in the kinetic constants and substrate binding order we determine the kinetic mechanism through classical protocols, involving initial velocity studies, product inhibition and dead end inhibitors. the results demonstrate that the pergk enzyme presents an altered substrate binding order compared to the wild type enzyme, where glucose was the first substrate to bind to the enzyme and glucose- -p the last product to be released. also, ligand-induced conformational changes were determined in the crystal structures. the apo, the enzyme-glucose and enzyme-glucose-adpbs structures were determined at . Å, . Å and . Å resolutions, respectively. structure analysis reveals that glucose binding provokes major conformational changes in the pergk enzyme, whereas adp binding does not cause further changes in the conformation of the protein. the results show that although the permutation has no effect on the nucleotide preference it provokes a change in the substrate binding order that correlates well with that those observed in the crystal structures. also, they demonstrate that during the evolutionary history of the ribokinase superfamily folding topology dictates the substrate binding order (fondecyt ). background: human ceruloplasmin (cp) is a circulating copper-containing glycoprotein produced in the liver and first described as a component of alpha -globulin fraction of human plasma. cp belongs to the multicopper oxidase family and it is nowadays regarded as a "moonlighting" protein, because it changes its function according to substrate, localization and expression. cp plays a key role in copper transport and iron metabolism and it is also a potent inhibitor of leukocyte myeloperoxidase (mpo) (kd nm), a major source of oxidants in vivo. the protein is extremely susceptible to proteolysis. in fact, cp is a structural homolog of coagulation factors v and viii, that are physiological substrates of thrombin (fiia). interestingly, thrombin participates in both haemostatic and inflammatory responses: in some focus of inflammation, such as rheumatoid arthritis (ra), the high activity of fiia has been documented. it was demonstrated that fiia can promote the chemotaxis of neutrophils and monocytes and their adhesion to endothelial cells, to increase vascular permeability. all these effect are mediated by par- interaction, that are abundantly expressed in inflamed rheumatoid synovial tissues. aims: in this study the interaction of cp with thrombin was investigated to confirm the participation of fiia in "spontaneous" proteolytic degradation of cp. in fact, in vivo the integrity of cp is essential for its role in the transport or metabolism of copper. results: our results indicated that thrombin cleaves cp in vitro at arg-ser and lys-val bonds, generating a nicked species that retains the native-like fold and the ferroxidase activity of the intact protein, whereas the mpo inhibitory function of cp is abrogated. analysis of the synovial fluid of ra patients reveals that cp is proteolytically degraded to a variable extent, with a fragmentation pattern similar to that observed with fiia in vitro, and that proteolysis is blocked by hirudin, a highly potent and specific thrombin inhibitor. we demonstrate that fiia has intrinsic affinity for cp (kd - nm), independently of proteolysis, and inhibits cp ferroxidase activity (ki nm). mapping of thrombin binding sites with specific exosite-directed ligands (i.e. hirugen, fibrinogen gamma-peptide) and thrombin analogues having the exosites variably compromised (i.e. prothrombin, prethrombin- , alpha-thrombin), reveals that the positively charged exosite-ii of thrombin binds to the negative upper region of cp, while the protease active site and exosite-i remain accessible. these results suggest that thrombin can exacerbate inflammation in ra by impairing via proteolysis the mpo inhibitory function of cp and by competitively inhibiting cp ferroxidase activity. an artificial pathway for isobutene production by direct fermentation: combining metabolic engineering and protein engineering benoit villiers , franc¸ois stricher the purpose of global bioenergies is to develop innovative metabolic pathways for the production of light olefins from renewable resources, by direct fermentation. light olefins (ethylene, propylene, linear butylene, isobutene and butadiene) are the core of the petrochemical industry. however, microorganisms do not naturally produce light olefins and no bioprocess to convert renewable resources to these molecules has been industrialized so far. global bioenergies has developed an artificial metabolic pathway including all the necessary enzymatic reactions from feedstock to isobutene. the metabolic route leading to isobutene can be divided in three parts, the first one being the use of natural reactions occurring in the host microorganism. second, heterologous natural reactions were introduced into the same host microorganism. finally, in contrast with most former approaches, non-naturally occurring reactions as enzymatic key steps were used, for example the decarboxylation of hydroxyisovaleric acid into isobutene. such non-natural critical steps were made possible by taking advantages of the natural catalytic and substrate promiscuity of exogenous enzymes. candidate enzymes are then evolved using systematic, random and semi-rational approaches in successive rounds in order to reach the desired catalytic efficiency. since all these reactions are enzymatic, isobutene can be obtained by direct fermentation, e.g. a process wherein all the chemical transformations are carried on by the host microorganism. the scale-up of this process began in november in a pilot plant installed in pomacle-bazancourt, france, with an annual capacity of tons of oxidation-grade isobutene. importantly, production of a volatile compound such as isobutene (and other light olefins) by direct fermentation presents two major advantages: first, the product is spontaneously removed from the culture broth, which alleviates the limitations linked with titer issues. second, the purification process is considerably easier and cheaper since no energy consuming methods such as distillation or phase separation are necessary to purify the end product. for the first time, batches of industrially produced isobutene from renewable resources have been obtained in the first half of . this isobutene has been in turn converted into isooctane, an additive currently used to improve gasoline quality, which could also be used as a standalone fuel. a demonstration plant is planned in leuna, germany, with an annual capacity of tons of polymer-grade isobutene and ibn-one, a joint venture with cristal union ( th european beet processor), has been formed to build and operate the first plant in france converting renewable resources into isobutene. finally, while the isobutene process is progressing towards industrial scale, global bioenergies is also developing new artificial metabolic pathways enabling direct bio-production of butadiene and propylene. the development of a coupled enzyme assay to detect isochorismate pyruvate lyase activity protein folding is typically defined in terms of the spatial arrangement of structural elements, i.e. helices, sheets and loops. we have, however, been developing an alternative and complementary paradigm based on conserved hydropathic interaction networks within proteins. these networks can be viewed as environments comprised of a mixture of polar and hydrophobic interaction fields, and may be the most important factor driving protein folding. this concept applies even to the lowest structural level within a protein: the sidechain conformations (or rotamers). exhaustive statistical analysis of existing crystallographic structures of proteins showed rotameric preferences and led to the creation of rotamer libraries frequently used in multiple aspects of structural biology, e.g., crystallography of relatively low-resolution structures, homology modeling and biomolecular nmr. however, little is actually known about the forces and factors driving the preference or suitability of one rotamer over another. in our study, tyrosine was analyzed since its sidechain has a comprehensive set of hydropathic properties that made it ideal as a proof of concept residue. construction of d hydropathic interaction maps of tyrosine residues in our dataset, reveals the environment around each, in terms of hydrophobic (p-p stacking, etc.) and polar (hydrogen bonding, etc.) interactions. after partitioning the tyrosines into backbonedependent bins, a map similarity metric based on the correlation coefficient was applied to each mapmap pair to build matrices suitable for clustering. notably, the first bin representing tyrosines, reduced to unique hydropathic environments with most diversity arising from favorable hydrophobic interactions with many different residue partner types. polar interactions for tyrosine include ubiquitous hydrogen bonding with the phenolic oh and somewhat surprisingly a handful of unique environments for the tyrosine backbone. all but one of the environments are dominated by a single rotamer, the exception being an environment defined by a paucity of interactions with the tyrosine ring and as a consequence its rotamer is indeterminate. this is consistent with it being composed of mostly surface residues. each tyrosine residue attempts to fulfill its hydropathic valences and thus, structural water molecules are seen in a variety of roles throughout these environments. alanine was analyzed using the same protocol as well. having the smallest sidechain (and small hydropathic interaction maps), alanine allowed us to investigate a significantly larger database, permitting us to examine the correlation between hydropathic maps and various structural features. in conclusion, the analysis of hydropathic environments strongly suggests that the orientation of a residue in a three-dimensional structure is a direct consequence of its hydropathic environment, which leads us to propose a new paradigm, interaction homology, as a key factor in protein structure. it is not the surrounding residues that direct sidechain conformations, but rather the hydropathic "field" of the surrounding atoms. folding studies of independent domains of lysine, arginine, ornithine binding protein (lao) protein folding problem has been addressed from the past years until nowadays, however, we still can not explain how proteins acquire their native structure from their amino acid sequence. different approaches has been taken in order to study protein folding, for example, the comparative study of folding mechanism between homologues proteins with high identity of sequence and structure, and the study of independent regions within a single protein. previously in our laboratory, thermodynamic and kinetic folding properties of lysine, ornithine, arginine binding protein (lao), a amino acid periplasmic binding protein (pbp), composed by two rossmann fold domains (one continuous and the other discontinuous) attached by a hinge region, has been studied. even there is a functional research about binding characteristics of histidine binding protei ns (his j) domains of when expressed independently (chu, b. ); there are no folding studies in these conditions for this or another pbps. it should be noted that his j shares % of sequence identity and tertiary structure (rmsd Å) with lao. in order to know the folding effect of encoding different domains in the same poly peptidic chain, as well as its influence in function, we are studying the thermodynamic and kinetic characteristics of folding of independently expressed lobes of lao, and comparing with those of native protein. by now, we expressed and purified the discontinuous domain. circular dichroism (cd) and fluorescence intensity spectra show that this independent domain has primary and tertiary structure. thermal denaturation has a single cooperative transition, which indicates this domain is folded. thermodynamic analysis of temperature and urea-induced experiments suggest that lao's folding characteristics are not just the addition of those from independent domains. furthermore, folding and refolding kinetics suggest the presence of a burst phase intermediate. a hypothesis to reconcile the physical and chemical unfolding of proteins a comprehensive view of protein folding is crucial for understanding how misfolding can cause neurodegenerative diseases and cancer. when using physical or chemical perturbations, nmr spectroscopy is a powerful tool to reveal a shift in the native conformation toward local intermediates that act as seeds for misfolding. high pressure (hp) or urea is commonly used to disturb folding species. pressure favors the reversible unfolding of proteins by causing changes in the volumetric properties of the proteinsolvent system. however, no mechanistic model has fully elucidated the effects of urea on structure unfolding, even though protein-urea interactions are considered to be crucial. here, we provide nmr spectroscopy and d reconstructions from x-ray scattering to develop the "push-and-pull" hypothesis, which helps to explain the initial mechanism of chemical unfolding in light of the physical events triggered by hp. in studying mpnep from moniliophthora perniciosa, we tracked two cooperative units using hp-nmr as mpnep moved uphill in the energy landscape; this process contrasts with the overall structural unfolding that occurs upon reaching a threshold concentration of urea. at subdenaturing concentrations of urea, we were able to trap a state in which urea is preferentially bound to the protein (as determined by nmr intensities and chemical shifts); this state is still folded and not additionally exposed to solvent [fluorescence and small-angle x-ray scattering (saxs)]. this state has a higher susceptibility to pressure denaturation (lower p / and larger dvu); thus, urea and hp share concomitant effects of urea binding and pulling and water-inducing pushing, respectively. these observations explain the differences between the molecular mechanisms that control the physical and chemical unfolding of proteins, thus opening up new possibilities for the study of protein folding and providing an interpretation of the nature of cooperativity in the folding and unfolding processes. zinc: a promoter or inhibitor for iapp aggregation? feng ding , praveen nedumpully-govindan zinc ions have been found to play an important and yet complex role in human islet amyloid polypeptide (hiapp) aggregation, which is associated with b-cell death in type-ii diabetes (t d). both concentration-dependent promotion and inhibition of iapp aggregation by zinc ions have been observed in vitro. similarly, at the population level, both positive and negative correlations were reported between the activity of a b-cell specific zinc transporter and t d risk. zinc ions are able to bind a single histidine in hiapp and coordinate the formation of zinc-bound hiapp oligomers. we hypothesize that the relative zinc/hiapp concentration determines the population of zinc-bound hiapp oligomers with different molecular weights. we have applied molecular dynamics (md) simulations to systematically study the structure and dynamics of a range of zinc-coordinated hiapp oligomers, including monomers, dimers, trimers, tetramers, and hexamers. our computational results suggest that different zinc-bound oligomers have distinct aggregation propensities. high-molecular weight oligomers ( peptides) have higher aggregation propensity than zinc-free and zinc-bound hiapp monomers at mm concentration in silico. therefore, our results provide a molecular insight into the complex role of direct zinc binding on hiapp aggregation. at low zinc/hiapp stoichiometry, zinc binding promotes aggregation. as the stoichiometry increases and zinc ions bind to single hiapp peptides, the aggregation of hiapp is inhibited due to electrostatic repulsion between the charged zinc ions. our computational study sheds light on the complex role of zinc on hiapp aggregation and t d development. biomolecules function in the densely crowded and highly heterogeneous cell, which is filled up to a volume of % with macromolecules [ ] . often, artificial macromolecular crowding agents are used to mimic these conditions in vitro and the excluded volume theory is applied to explain the observed effects [ ] . however, recent studies emphasize the role of further contributions aside from a pure volume effect including enthalpic and solvent effects [ , ] . we study cosolute effects at high molecular and macromolecular concentrations via a thermodynamic analysis of the thermal unfolding of ubiquitin in the presence of different concentrations of cosolutes (glucose, dextran, polyethylene glycol, potassium chloride) [ ] . in contrast to the excluded volume theory, we observed enthalpic stabilization and entropic destabilization forces for all tested cosolutes. the enthalpic stabilization mechanism of ubiquitin in macromolecular polysaccharide solutions of dextran was thereby similar to the effects observed in monomeric glucose. further, it remains unclear how such cosolutes reflect the physicochemical properties of the complex cell environment as a characterization of the in-cell crowding effect is lacking. thus, we developed a fret-based macromolecular crowding sensor to study the crowding effect in living cells [ ] . the averaged conformation of the sensor is similar to dilute aqueous buffer and cell lysate. we find that the in-cell crowding effect is distributed heterogeneously and can change significantly upon osmotic stress. the presented method allows to systematically study in-cell crowding effects and understand them as a modulator of biomolecular function. the stability of biomolecules under co-solvent conditions is dependent on the nature of the co-solvent [ ] . this can alter a protein's properties and structural features through biomolecular interactions between its functional groups and the co-solvent molecules. ionic liquids (ils) represent a rather diverse class of co-solvents. the design flexibility of these molten salts is an attractive feature, allowing the properties of the il to be tuned to meet the requirements of different applications [ ] . particularly, the modulation of reaction pathways between folding states, offering possibilities to control irreversibility in non-native protein aggregation [ ] . this has led us to investigate the impact of ils as co-solvents with the well-known protein denaturant urea. urea is considered to be a non-ionic chaotrope disturbing considerable the grid of hydrogen bonds with the protein backbone. urea interacts preferentially with the protein surface, mainly apolar residues and that dispersion, rather than electrostatic interactions, is the main energetic contribution to explain the stabilization of the unfolded state of the protein and the irreversibility of the unfolding process in the presence of urea [ ] . a large body of multidomain protein folding work has been devoted to study monomeric proteins. how do multidomain multimeric protein fold, avoiding accumulation of stable intermediate is yet to be studied in detail. our present study is focussed on understanding the folding and assembly of the domains of a homodimeric l-aspraginase from a hyperthermophile pyrococcus furiosus (pfa). each monomer of pfa consists of distinct n-and c-terminal domains (npfa and cpfa, respectively), connected by a linker. the folding mechanism of each domain with respect to full length protein was studied by mutating one out of two tryptophans, one in each domain. domains were purified and studied individually to obtain parallel account of the folding of each domain in isolation. subunit assembly was studied by analytical size exclusion chromatography (sec), multiangle light scattering and functional activity. through far uv cd, intrinsic trp fluorescence and sec, we demonstrated that domain folding and subunit association were intimately linked in full length pfa. interestingly, en route to its folding there was complete absence of hydrophobic intermediates as probed by ans fluorescence. folding of npfa was highly cooperative and, it provides interacting surfaces for cpfa to fold and also facilitates subunit assembly. the folding cooperativity of isolated domains was very less compared to the folding cooperativity of their full length counterparts, as indicated by equilibrium m values. to our surprise, during ph induced denaturation, at ph and , the dimer dissociates into highly hydrophobic folded monomers which readily underwent amyloidogenesis. we showed that at such extreme conditions, cooperativity in folding process in multidomain multimeric protein is not solely governed by the folding of individual domains, rather by concomitant folding and association of domains directly into a quaternary structure. in other case, where subunit folding occurred prior to association, protein readily underwent extensive aggregation. groel assisted folding of multiple recombinant proteins simultaneously over-expressed in e.coli megha goyal , tapan kumar chaudhuri aggregation prone recombinant proteins very often form inclusion bodies and also exhibits poor yield of functional protein during in vitro refolding process from chemically denatured form. bacterial chaperonin groel provides folding assistance to several proteins, when over-expressed with one of the recombinant proteins. there are instances that groel in presence of few other co-expressed chaperones like dnaj, dnak etc provides better yield of folded protein during homologous and heterologous expression. considering the ongoing events in the cells, it is known that molecular chaperone groel assists in the folding of various proteins in the cytoplasm. hence attempt to fold multiple recombinant proteins over-expressing simultaneously with the co-expression of chaperones can be worth trying. this approach may cut down various complexities in the functional recombinant protein preparation, including time and effective cost. keeping this view in mind, folding of two simultaneously expressed aggregation prone proteins, kda e.coli maltodextrin glucosidase (malz) and kda yeast mitochondrial aconitase have been investigated with the co-expression of groel and groes in e.coli cytosol. it has been previously reported that both the chosen proteins undergo co-expressed groel-groes assisted folding in e.coli cytosol, when they over-express alone. in this study we have optimized the overexpression of malz and aconitase simultaneously in e.coli. further optimisation was carried out to coexpress groel along with malz and aconitase. based on the basic philosophy that soluble protein mainly contains folded fraction, the event of groel/es assisted folding of simultaneously overexpressed proteins, malz and aconitase was monitored through the attainment of soluble proteins under various sets of conditions such as temperature. the major outcome of the present study is that, with the groel-groes assistance, the yield of soluble proteins (malz and aconitase) together constitutes higher percentage of folded protein in contrast to the percent yield when a single protein was overexpressed. significance of this type of study relies on the fact that the cells can over-produce higher amount of recombinant proteins, when multiple over-expression takes place. not only pushing up cell's capability of over-expression, co-expression of groel and groes efficiently assists in the folding of multiple proteins simultaneously over-expressed in e.coli. amyloid fibrils associated with serious diseases including alzheimer's, parkinson's, and prion diseases promoted the challenge of studying protein misfolding, leading to the development of amyloid structural biology. amyloid fibrils form in supersaturated solutions via a nucleation and growth mechanism. although the structural features of amyloid fibrils have become increasingly clearer, knowledge on the thermodynamics of fibrillation is limited. furthermore, protein aggregation is not a target of calorimetry, one of the most powerful approaches used to study proteins. here, with b -microglobulin, a protein responsible for dialysis-related amyloidosis, we show direct heat measurements of the formation of amyloid fibrils using isothermal titration calorimetry (itc). the spontaneous fibrillation after a lag phase was accompanied by exothermic heat. the thermodynamic parameters of fibrillation obtained under various protein concentrations and temperatures were consistent with the main-chain dominated structural model of fibrils, in which overall packing was less than that of the native structures. we also characterized the thermodynamics of amorphous aggregation, enabling the comparison of protein folding, amyloid fibrillation, and amorphous aggregation. in order to obtain general thermodynamic properties of protein aggregations, we further investigated aggregation of glucagon and insulin, two of the most famous amyloidogenic peptide hormones, using itc. we also observed characteristic heat of spontaneous amyloid fibrillation of both proteins after a lag time. taken all together, we showed that thermodynamic studies on amyloid fibrillation and amorphous aggregation were indeed possible by means of itc-based qualitative and quantitative calorimetric analyses. itc will become a promising approach for clarifying the thermodynamic properties of protein aggregates. the more case studies are required toward the establishment of thermodynamics of protein misfolding and aggregation when hydrophobic proteins are, for any reason, exposed to the cytosol they are rapidly captured by protective complexes which shield them from the aqueous surroundings and decide their fate (by either targeting them to their correct membrane homes or marking them for degradation by the ubiquitin/proteasome system). the bag holdase is a heterotrimeric protein complex, comprising bag , ubl a and trc , which works closely with the cochaperone sgta to triage hydrophobic proteins and pass them along the appropriate pathway. sgta also interacts with viral proteins and hormone receptors and is upregulated in numerous cancer types. these functions require further investigation to determine the scope of sgta as a therapeutic target. our lab has solved the solution structure of the n-terminal dimerization domain of sgta and characterised its interaction with two different ubiquitin-like (ubl) domains in the bag holdase (one from ubl a and the other from bag itself) using nmr chemical shift perturbation data and other biophysical techniques including isothermal titration calorimetry and microscale thermophoresis. at this meeting i will report on the progress we have made in structurally characterising further key players that participate in this quality control, with the aim of clarifying the intricate network of molecular interactions that governs these processes in health and disease. ensemble, ribbon and electrostatics spacefill views of the sgta dimerization domain structure. the final panel shows the structure overlaid with its yeast homologue. alpha synuclein is a small protein ( kda) expressed at high levels in dopaminergic neurons. fibrillar aggregates of a-synuclein inside the dopaminergic neuron are the major components of lewy bodies and lewy neuritis inclusion, which are considered as potential hallmark of parkinson's disease (pd). both in vitro as well as in vivo studies suggest that the soluble, oligomeric forms of a-syn are the more potent neurotoxic species, responsible for neuronal injury and death in pd. therefore, molecules that inhibit the toxicity of oligomers either by reducing their formation or by converting their more toxic oligomeric state to less-toxic fibrillar state would be effective agents for the drug development against pd. curcumin is one of the asian food ingredients which has shown a potential role as therapeutic agent against many neurological disorders including pd. however, the instability and low solubility makes it less attractive for use as potential therapeutic agent. the present work focuses on screening of the compounds similar to curcumin but having better effects on the morphology and toxicity of oligomeric and fibrillar assemblies of a-syn, which could be used as therapeutic agent preferentially over the naturally occurring curcumin. we synthesized and analyzed the effects of nine compounds, which are structurally similar to curcumin, on different stages of a-syn amyloid aggregation. here, we showed that curcumin and its analogs accelerate a-syn aggregation to produce morphologically different amyloid fibrils in vitro. however, there is no significant effect of curcumin and its analogs on the secondary structure of preformed a-syn fibrils. furthermore, these curcumin analogs showed differential binding affinities with the preformed a-syn aggregates, possibly due to difference in their chemical structures. the present data suggest the promising role of curcumin analogs in the treatment of a-synucleinopathy disorders. in vitro folding mechanisms determine the forces applied during co-translational folding there is currently much debate as to whether experiments conducted in vitro describe the folding of proteins in vivo. in particular, it is often suggested that the co-translational folding of nascent protein chains is dominated by the presence of the ribosome and associated chaperones, and that folding mechanisms will be affected by the vectorial nature of translation. here we use an arrest peptide assay to investigate the co-translational folding of a number of all-a spectrin domains that exhibit a range of thermodynamic stabilities and in vitro folding rates. our unexpected finding is that that the force exerted on the ribosome by these domains is not related to either the thermodynamic stability of the domain, or to the folding (loading) rate, but rather to the in vitro folding mechanism. we infer that the in vitro folding mechanisms of these domains are unaffected by the presence of the ribosome -even when part of the nascent chain is retained within the ribosome exit tunnel. there has been much work to date investigating the intermediates present in stalled translation complexes -but now, for the first time, we can begin to directly explore the rate limiting transition state in the co-translational folding of homologous proteins. can the structure of a protein (h . ) depend on the treatment of a solvent medium (explicit vs effective) in a coarse-grained computer simulation? ras pandey , barry farmer university of southern mississippi, air force research laboratory solvent medium plays a critical role in orchestrating the structure and dynamics of a protein. in computer simulation modeling of protein structure in a solvent medium, explicit, implicit, effectivemedium, approaches are often adopted to incorporate the effects of solvation. because of the complexity in incorporating all atomic and molecular details, the multiple components, reaching the large-scale, etc. implicit solvent or effective medium approach is generally more viable than the explicit solvent methods. some of the pertinent characteristics such as excluded volume of the solvent constituents, its concentration, and the underlying fluctuations which may be important in probing some issues are generally ignored in effective medium or implicit solvent approaches. using a coarse-grained approach, we investigate the structure and dynamics of a protein (a histone, h . ) in the presence of both effective as well as explicit solvent media over a range of temperatures with the monte carlo simulations. the protein is represented by a coarse-grained chain of residues whose interactions are described by knowledge-based residue-residue and hydropathy-index-based residuesolvent interactions. in effective medium approach, each empty lattice site around the protein structure acts as a solvent. only a fraction of lattice sites are occupied by mobile solvent constituents along with the protein chain in explicit solvent medium. large scale simulations are performed to analyze the structure of the protein for a range of residue-solvent interactions and temperature in both explicit and effective solvent media. we study a number of local (e.g. solvation and mobility profiles) and global (radius of gyration and structure factor) physical quantities as a function of temperature. we find that the response of the radius of gyration of the protein in explicit solvent is different from that in effective medium solvent. thus, the presence of fluctuations in explicit solvent approach have considerable effects on the structure and dynamics of protein h . . differences due to type of solvent on the response of some of these quantities as a function of temperature as well as general similarities will be presented. single-molecule vectorial folding and unfolding through membrane pores david protein folding and unfolding in vivo is frequently vectorial. for example, proteins are synthesized at the ribosome and emerge n-terminal first. as the polypeptide chain emerges from a nm wide pore is free to fold, interact with partners or misfold . in another example, proteins are unfolded at the proteasome by pulling from either the n or c terminus against a - nm wide pore, applying a tension on the residues surrounding the terminus of the protein . under this conditions, proteins may behave differently than when unfolded/refolded with temperature or urea. this may have important implications, as protein folding and unfolding in vivo is related to both function and disease. we noticed that vectorial folding is inherently linked to nanometer size pores. making use of nanopore technology we developed a method to monitor protein unfolding during membrane translocation at the single-molecule level . briefly, an oligonucleotide attached at either end of a protein threads a single protein nanopore inserted in a lipid membrane. in response to an applied membrane potential, the oligonucleotide pulls the protein through the pore and as it is forced to translocate it unfolds. analysing the ionic current we obtain the unfolding pathway and information on the polypeptide sequence. this methodology has shown that proteins unfold with different kinetics when pulled from one terminus or the other . remarkably, it is also possible to say whether the protein has been phosphorylated or not, and where . we have recently advanced our model system to study protein folding after translocation at the singlemolecule level . a single-protein molecule was translocated through a pore and forced to translocate back at predetermined times. we measured the stability of the refolded state at different times and we obtained the vectorial folding pathway of the protein. further, we observed that the protein was capable of co-translocational folding and that this premature folding contributed to the complete translocation of the protein. our results show that nanopore technology applied to proteins can be used to describe the vectorial folding and unfolding of proteins, providing insight to how these processes may work in vivo. further, single-molecule protein sequencing is a possibility that could revolutionise our knowledge on biological processes. thermodynamics studies of oligomeric proteins, which are the dominant protein natural form, have been often hampered because irreversible aggregation and/or slow reactions are common. there is not a single report on the reversible equilibrium thermal unfolding of proteins composed by (b/a) barrel subunits, albeit this "tim barrel" topology is one of the most abundant and versatile in nature. the eponymous tim barrel, triosephosphate isomerase (tim) is a ubiquitous glycolytic enzyme that catalyzes the isomerization of glyceraldehyde- -phosphate and dihydroxyacetone phosphate. the unfolding of several tims, mainly of eukaryotic organisms, has been extensively studied. regarding thermal unfolding, eighteen tims, mainly from eukaryotes, as diverse as amoebozoa, euglenozoa, ascomycota and chordata, have been studied. even though a full thermodynamic characterization has been hampered by irreversible aggregation and/or the presence of hysteresis in all of them, the activation parameters that describe the kinetic control of five eukaryotic tims have been reported. we characterized the structure, catalytic properties, association state and temperature-induced unfolding of the eponymous tim barrel, triosephosphate isomerase (tim), belonging to five species representative of different bacterial taxa: deinococcus radiodurans (drtim), nostoc punctiforme (nptim), gemmata obscuriglobus (gotim), clostridium perfringens (cptim) and streptomyces coelicolor (sctim). irreversibility and kinetic control were observed in the thermal unfolding of nptim and gotim, while for drtim, sctim and cptim, the thermal unfolding was found to follow a two-state equilibrium reversible process, a behavior not observed previously for others tims. shifts in the global stability curves of these three proteins are related to organismal temperature range of optimal growth and modulated by variations in maximum stability temperature and in the enthalpy change at that temperature. reversibility appears to correlate with low isoelectric point, the absence of residual structure in the unfolded state, small cavity volume in the native state structure, low conformational stability and a low melting temperature. furthermore, the strong coupling between dimer dissociation and monomer unfolding may reduce the possibility of aggregation and favor reversibility. it appears that there is a delicate balance between several contributions whose concerted interplay is necessary to achieve thermal reversibility in oligomeric enzymes. furthermore, the finding that the three reversible proteins come from organisms from different phyla suggests that unfolding reversibility may be more common than what is currently known supported by a critical step in the late phase of human immunodeficiency virus type (hiv- ) infection is targeting of the virally encoded gag proteins to the plasma membrane (pm) for assembly. prior to assembly, the hiv- gag polyprotein adopts a compact "folded over" conformation and exists in the monomeric or low-order oligomeric states. whereas it is established that the nucleocapsid domain of gag specifically recognizes motifs in the viral rna genome for packaging, there is compelling evidence that the myristoylated matrix (ma) domain also binds to cellular rna to prevent premature gag targeting to intracellular membranes. upon transport of gag to the pm, the interaction of ma with rna is exchanged for an interaction of ma with pm components. this molecular switch induces an extended conformation of gag, leading to formation of high-order gag oligomers on the pm. because gag is anchored and therefore captured by its interaction with the available phospholipids, the intracellular targeting of gag is likely to be determined by the relative strength of its interaction with the dominant lipids composing each membrane subcompartment. the key to understanding this essential molecular switch is elucidating at the molecular level the interaction of ma with specific pm components. for over two decades, biochemical, in vivo, in vitro and genetic studies have focused on factors that modulate binding of retroviral gag proteins to membranes but only recently the structural and molecular determinants of gag assembly have begun to emerge. in addition to the electrostatic interactions between a highly conserved basic region of ma and acidic phospholipids, it is now believed that the hydrophobicity of the membrane interior represented by the acyl chains and cholesterol also play important roles. we employ nmr methods to elucidate the molecular determinants of gag binding to the membrane. our structural studies revealed that phosphatidylinositol- , -bisphosphate (pi ( the production of functionally antibodies depends on the transition of immature b cells to mature plasma cells and is tightly linked to several "quality control" check points. during b cell development, the pre-b cell receptor (pre-bcr) is the first checkpoint which determines the viability and proliferation of the pre-b cell. the pre-bcr is composed of an immunoglobulin (ig) heavy chain molecule associated with an ig light chain-like molecule called the surrogate light chain (slc). the slc is composed by two proteins k and vpreb which possess a unique region at the n-or c-terminus, respectively. vpreb lacks a b-strand which is provided by the k protein allowing the non-covalent interaction essential for formation of the slc heterodimer. our understandings of the molecular mechanism of slc function and assembly are still at an early stage. in particular, we do not know how the slc associates and forms the pre-bcr for the selection of all heavy chains (hcs). our study focuses on dissecting the "fab fragment" of the pre-bcr to study the effect of the unexpected structural features of the slc to gain insight in hc selection. the analysis of the assembly of the slc revealed a significant difference between the single domains and the complexes in terms of stability and assembly. the folding behavior of the ch domain in the presence of the slc is key for the first quality control mechanism in the endoplasmic reticulum (er) prior to surface expression. our results show that the slc interacts with ch domain in a similar manner to the cl domain. thus, the folding of the naturally disordered ch domain upon interaction with the slc releases the hc retention in the er by bip. taken together, our study provides new insights into the folding and assembly of the "fab fragment" of the pre-bcr and paves the way for a detailed mechanistic understanding of hcs selection by the unique slc. though the - (sfgailss) region of human islet amyloid polypeptide (hiapp) has long been known to be crucial for amyloid fiber formation, lack of b-ordering of this region in structures of the final fiber as determined by both nma and x-ray has been puzzling. new evidence now suggests that the fgail region forms ordered b structures only in early intermediates. we present new dir studies on the fgail region of hiapp, with uniformly c o labeled amides, along with spectral and kinetic modelling. evolution of the peak frequency and d lineshape of the labeled region clearly present a transition from random coil to a stable b sheet, a conclusion which is substantiated by simulation of the d ir spectra. as determined from kinetic modeling, the fgail b-sheet creates a free energy barrier that is the cause of the lag phase during aggregation. these findings help to rationalize a broad range of previous fragment and mutation studies as well as provide a mechanism for fiber formation that has self-consistent kinetics and structures. the temperature dependence of protein stability in living cells studies addressing the consequence of crowding that exist in the interior of cells have reached an interesting stage. experimental data so far, predominantly from, small to medium sized proteins are indicating that, in general, natively folded proteins including, intrinsically disordered, gain structure and stability under conditions mimicking cell interior. however, on the other hand, a few studies on small proteins indicate destabilization of the native state. in very few instances, crowding resulted in compaction and aggregation of the unfolded and partially folded states. experimental data on the consequences of cell-like crowding situation on relatively large proteins with complex folding free energy landscape are absent. alpha subunit of tryptophan synthase, a kda tim barrel protein, provides a unique opportunity to address the consequence of crowding on the structure and stability of the native state and also on a partially folded state stable equilibrium intermediate populated in its (un)folding reactions. in the presence of increasing amounts the most commonly used crowding agent, ficoll- , a non-monotonous increase in the far uv-cd is observed for the native state. a steady increase up to mg/ml ficoll followed by a decrease in far-uv cd region is observed, indicating loss of structure at increased concentrations of the crowding agent. h- n hsqc nmr and fluorescence (fl) spectra confirm the of loss of structure at higher concentrations of ficoll- . loss of native base line in the urea induced unfolding reaction monitored by cd and fl clearly confirms the destabilization of the native state. similar to the structural changes observed for the native state, for the equilibrium intermediate state maximally populated at m urea also, non-monotonous changes in the far uv cd and fluorescence spectra are observed. the highly populated equilibrium intermediate shows an initial steady increase in the far uv cd signal followed by a sudden decrease. our results suggest that the structure of both native and partially folded states may be affected under crowding conditions. alpha- antitrypsin (aat) is a -kda serine protein inhibitor (serpin), which acts as an inhibitor of neutrophil elastase within the lungs. during inhibition, the protein undergoes a dramatic conformational change in which its exposed reactive centre loop (rcl) is cleaved and inserts into the central a-sheet as an extra beta-strand. this highly dynamic protein is also susceptible to mutations, resulting in misfolding and the accumulation of ordered polymers as intracellular inclusions within the endoplasmic reticulum of hepatocytes, where aat is synthesized. despite much knowledge of the folding and misfolding properties of aat as an isolated protein, very little is understood of how aat acquires its structure during biosynthesis. like all proteins, the biosynthesis of aat takes place on the ribosome, and protein folding occurs in a co-translational manner as the nascent polypeptide chain emerges from the ribosome's exit tunnel. this study aims to develop the biochemical and nmr structural strategies to characterize the co-translational folding characteristics of aat as it is being synthesized on the ribosome. for these studies, we have designed a series of secm-stalled ribosome nascent chain complexes (rnc) of aat of different lengths, which mimics the "snapshots" of the protein synthesis, capturing the folding process of the nascent chain during its emergence from the ribosome. using this library, we have recently developed a strategy to produce large quantities of the rncs both in vitro and in vivo within e. coli, a prerequisite for detailed biochemical and structural studies. using the aat-rncs, we are developing a suite of biochemical strategies to probe the capacity for aat nascent chains to adopt native structure on the ribosome. we have combined protease inhibition assays, western blot and native-page analysis to demonstrate that aat can fold while bound to the ribosome. in addition, we have employed a cysteine-based modification "pegylation" assay to probe lowresolution structural information of aat-rnc and this will guide our structural studies by nmr spectroscopy to provide a detailed understanding of aat folding on the ribosome at high resolution. thermodynamic properties of proteins vary with the environmental solvent condition (temperature, ions, ph, denaturants, etc.). although the effect of each environmental factor on proteins has been well studied, the complex effect of more than two environmental factors was not studied thoroughly. in this study, we investigate the simultaneous effect of urea denaturation (disruption of non-covalent bonds in proteins) and acid denaturation (titration of protein residues) on the nature of the folding transition for cyu protein. we performed the molecular dynamics simulations of bbl (pdb code: cyu) protein in various urea concentration at k. we calculated ph-dependent free energy landscape using the extended munoz-eaton model and described the phase diagram for the folding transition of bbl at various ph value and urea concentration. we mapped out the phase diagram of the folding transition of cyu, which clarifies the condition with which it undergoes the cooperative folding transition or the barrierless folding transition. biophysical analysis of partially folded states of myoglobin in presence of , , -trifluoroethanol paurnima talele , nand kishore the protein folding process involves one or more distinct populated intermediates. one such partially folded structure of particular importance observed during protein folding pathway is molten globule state. the properties of a molten globule state are intermediate between those of native and unfolded protein molecules. the importance of studying equilibrium molten globule is in its greater stability and flexible structure which has been shown to bind a variety of substrates and play a definite role in certain human diseases via aggregation, misfolding or some other mechanism. a protein must assume a stable and precisely ordered conformation to perform its biological function properly. the stability of a protein under specific conditions depends on its interactions with the solvent environment. therefore it is essential to understand protein folding intermediates, protein solvent interactions and protein stabilization. we have made attempts to thoroughly investigate the formation of stable molten globule state of the protein induced by alcohol using combination of calorimetric and spectroscopic techniques. the presentation will cover the topic on biophysical studies on partially folded states of myoglobin in presence of , , -trifluoroethanol. the thermal denaturation of myoglobin was studied in the presence of , , -trifluoroethanol (tfe) at various ph values using differential scanning calorimetry and uv-visible spectroscopy. the most obvious effect of tfe was lowering of the transition temperature with increasing concentration of tfe up to . mol•dm- , beyond which no thermal transitions were observed. the conformation of the protein was analyzed by a combination of fluorescence and circular dichroism measurements. at ph . and . , partially folded states of myoglobin were confirmed by cd spectroscopy. quantitative binding of ans to the tfe induced molten globule state of myoglobin was studied by using isothermal titration calorimetry (itc). the results enable quantitative estimation of the binding strength of ans with the molten globule state of myoglobin along with the enthalpic and entropic contributions to the binding process. the results also suggest occurrence of common structural features of the molten globule states of proteins offering two types of binding sites to ans molecules which has been widely used as a fluorescence probe to characterize partially folded states of proteins. modules. each cbr comprises a b-hairpin core followed by a short linker sequence. choline molecules are bound between two consecutive repeats through hydrophobic and cation-p interactions with aromatic side chains. apart from its biotechnological applications as an affinity tag for protein immobilization and purification, clyta is useful as a model for understanding the folding and stability of repeat proteins. in this sense, we proposed to get minimal peptides encompassing the sequence of a single cbr or even only its b-hairpin core able to maintain the native fold and the ability to bind choline. to that end, we first proceeded to analyze the peptide comprising the third b-hairpin core, denoted as clyt . based on cd and nmr data we demonstrate that the peptide clyt conserves its native bhairpin structure in aqueous solution, but forms a stable, amphipathic a-helix in detergent micelles and as well as in small lipid vesicles [ ] . considering the great differences in the distribution of hydrophobic and polar side chains shown by clyt b-hairpin and a-helix, we propose that amphipathic structures are stabilized in micelles or lipid vesicles. this "dual" behavior is the only up-to-now reported case of a micelle-induced conformational transition between two ordered peptide structures. to check whether other cbr repeats also undertake b-hairpin to a-helix transition in the presence of micelles, so that it represents a general tendency ascribed to all pneumococcal choline-binding modules, we will show new experimental evidences based on cd and nmr structural studies on peptides derived from the bhairpin cores of other clyta repeats, as well as in modified clyt peptides. continuing our studies of the effect of like-charged residues on protein-folding mechanisms, in this work, we investigated, by means of nmr spectroscopy and molecular-dynamics simulations, two short fragments of the human pin ww domain [hpin ( - ); hpin ( - )] and one single point mutation system derived from hpin ( - ) in which the original charged residues were replaced with non-polar alanine residues. results, for both original peptide fragments of hpin demonstrate the presence of ensembles of structures with a tendency to form a b-chain reversal. understanding the biology of huntington's disease via the pathogenic huntingtin monomer huntington's disease (hd) is caused by an abnormal extension of the polyglutamine (polyq) region within exon of the protein huntingtin from typically glutamines to over . disease onset correlates with the huntingtin misfolding and causing the formation of aggregates, however recent studies have postulated that pathogenic huntingtin monomer may form compact structures that are responsible for neuronal toxicity in hd. we sought to examine the conformation of huntingtin monomers, how polyq sequence length affects monomer structure and which protein-binding partners in the cell may exert a gain-of-toxic mechanism in pathology. hydrogen-deuterium exchange mass spectrometry was used to measure the degree of structure in both non-pathogenic ( q) and pathogenic ( q) huntingtin, with results showing that both forms exchanged % of potential nh hydrogen bond donors within seconds (n ), with little to no further exchange over the following ten minutes. this result suggested that the pathogenic conformations are not stabilized by slow exchanging hydrogen bonds. binding partners to the monomer were assessed in neuro a cell culture by immunoprecipitation and quantitative ms/ ms proteomics approaches after depletion of aggregates by pelleting. proteins that more prevalently co-precipitated with pathogenic huntingtin included fused in sarcoma (fus), glycine-trna ligase (gars), peroxiredoxin (prdx ), phosphatidylethanolamine-binding protein (pebp /rkip), and histone subunit hist h a, all of which were significantly enriched by two-fold or greater. rna-seq analysis indicated that none of these proteins had altered expression levels, suggesting that the binding interactions are not due to changes in background abundance. overall we found that the conformational differences are subtle, yet are sufficient to generate several specific proteome interactions that offer clues to a toxic gain-of-function mechanism in pathology. work is ongoing to probe the more subtle changes in conformation and the importance of these interactors to mediating mechanisms of dysfunction. hereditary tyrosinemia type i is an autosomal recesive disorder caused by deficiency of fumarylacetoacetate hydrolase (fah) enzyme. deficiency of fah leads to cellular accumulation of toxic metabolites which include mainly, succinylacetone (sa), maleylacetoacetate (maa) and fumarylacetoacetate (faa) in many body tissues. fah is mainly expressed in hepatocytes and renal proximal tubular epithelium. therefore, liver and kidney are the two primary organs affected by this disorder, and development of hepatocellular carcinoma is the major symptom. missense mutations leads to a loss of enzymatic efficiency which, in a high number of mutations, correlates with loss of kinetic and thermodynamic stability of the enzyme. in our ongoing project, we are trying to elucidate the molecular basis of tyrosinemia by means of biophisical and structural characterization of fah wild type along with its mutations. this knowledge should help us design new therapies based on the identification of pharmacological chaperones that could restore the altered enzymatic stability of the enzyme. human fah wild type and selected mutants were synthesized and inserted in an expression vector for e. coli. the proteins were purified in a fplc and, their thermodynamic and kinetic stability investigated using circular dichroism. our preliminary results confirm the loss of termodinamic stability of different mutants and its variability compared to wild type protein. repulsion between net charges of subunits during ferritin assembly daisuke sato , hideaki ohtomo , atsushi kurobe , satsuki takebe , yoshiteru yamada , kazuo fujiwara , masamichi ikeguchi department of bioinformatics, graduate school of engineering, soka university, jasri/spring- the organisms have a lot of spherical shell-shaped supermolecules consisting of identical or distinct subunits (e.g., ferritin, virus capsid, lumazine synthase and encapsulin). such multimeric proteins spontaneously assemble into their native structures from the subunits to acquire the specific functions. however, the assembly mechanism of such supermolecules has not been understood in detail. hence, to investigate the assembly mechanism is biologically important. escherichia coli non-heme ferritin (ftn) consists of identical subunits, which are assembled into a spherical shell-shape with / / symmetry. ftn is able to store iron inside cavity. the subunit includes a-d helices forming -helix bundle, a long bc-loop between b and c-helices and a short e-helix at the c-terminal. ftn dissociates into dimers at acidic ph. the dimer was shown to maintain the native-like secondary and tertiary structures by circular dichroism spectra and small angle x-ray scattering (saxs). the acid-dissociated ftn is able to reassemble into the native structure when ph increases. to clarify ftn assembly mechanism, we performed the stopped-flow time-resolved saxs (tr-saxs) experiments. the saxs profiles could be acquired every ms after the initiation of reassembly. the initial velocity calculated from the forward scattering intensity increment was proportional to the square of the protein concentration, implying that the reaction is second-order. we propose the sequential bimolecular reaction, in which two dimers bind to form tetramer, then another dimer attaches to the tetramer to form a hexamer, and so on. the assembly rate depended on ph and ion strength, indicating that the electrostatic interaction plays an important role in the assembly reaction. the assembly rate decreased with increasing ph in the range from . to . and increased with increasing nacl concentration. this indicates that there are repulsive electrostatic interactions between assembly units and that they increases with increasing ph from . to . . a possible interaction is the repulsion between net charges of dimers since pi of ftn is expected to be . . to test this possibility, we made several mutants with different net charges. as mutational sites, we selected charged residues that are far from the subunit interface. selected sites were glu , glu , glu , glu and glu . we constructed the mutants with one, two, three or four glu -> gln substitutions of selected sites. the structures of those mutants were similar to that of wild-type ftn. if aforementioned hypothesis is correct, the assembly rate is expected to increase with increasing the number of substitution. the result agreed well with this expectation and strongly suggested that the electrostatic repulsion between dimers is an important factor determining the assembly rate of ftn. improved modeling of protein unfolding rates and pathways through solvation and modeling of beta-barrels benjamin walcott , , lu ıs garreta , christopher bystroff , , department of biology, rensselaer polytechnic institute, center for biotechnology and interdisciplinary studies, department of computer science, universdad del valle, department of computer science, rensselaer polytechnic intitute an understanding of the folding and unfolding pathways of proteins is integral to improving our ability to associate the structural impact of point mutations and disease etiology. information gained here can also be used for protein structure prediction and design. to model unfolding pathways in proteins we utilize a computational method called geofold. this approach uses recursive hierarchical partitioning of protein structure and finite elements simulation. geofold considers three types of partitioning operations: translational motion (break), single point revolute joints (pivot), and rotation around two points (hinge). from these operations, a directed acyclic graph (dag) is constructed where nodes correspond to the substructures created by these operations and the edges represent the operations. for each operation in the dag, its dissociation and reassociation rates are determined as a function of solventaccessible surface area, hydrogen bonds, voids, and conformational entropy. finite element simulations are carried out to simulate the kinetics of unfolding. this model accurately predicts changes in unfolding pathways due to disulfides in a four-protein case-study, but it fails to produce a realistic pathway for b-barrel proteins such as green fluorescent protein (gfp). to better model these barrel proteins, a new partitioning operation is introduced involving the breaking of all contacts between an adjacent set of b-strands, called a seam. in addition, to improve the accuracy of kinetic modeling, several updates have been made to the energy function, including an improved solvation model and a contact-orderbased estimation of the reassociation rates. the predicted unfolding rates and pathways using this improved geofold are compared with experimentally measured values in kineticdb for proteins with multi-state unfolding kinetics, point mutations, circular permutations, and engineered disulfides. the presence of multiple domains in a protein can result in the formation of partially folded intermediates, leading to increased aggregation propensity. this can be reduced by cooperative, all-or-nothing folding of the multi-domain protein. in good agreement with ensemble folding experiments, a coarsegrained structure-based model of e. coli adenylate kinase (ake) folds cooperatively. ake has three domains, nmp, lid and core. we examine the role of the interfaces between these domains in facilitating folding cooperativity in ake. mutants in which these interfaces are deleted exhibit similar folding cooperativities as wild-type ake. on closer inspection, we observe that unlike a typical multi-domain protein in which one domain is singly-linked to its adjacent domain, nmp and lid are inserted into core, i.e. they are both connected to core by two linkers each. we create circular permutants of ake in which the inserted domains are converted to singly-linked domains, and find that they fold less cooperatively than wild-type ake. domain insertion in wild-type ake facilitates folding cooperativity even when the inserted domains have lower stabilities. the n-and c-termini of nmp and lid are constrained upon the folding of core and this facilitates their folding. thus, nmp and lid which undergo large conformational changes during catalysis can be smaller with fewer stabilizing interactions. in addition, inter-domain interactions need not be optimized for folding, and can be tuned for substrate binding, conformational transition and catalysis. analysis of protein domains using structural bioinformatics suggests several examples of multi-domain proteins in which domain insertion is likely to facilitate folding cooperativity. tuning cooperativity on the free energy landscape of protein folding pooja malhotra , jayant udgaonkar national centre for biological sciences, tata institute of fundamental research the mechanism by which a protein explores the free energy landscape during a folding or unfolding reaction is poorly understood. determining whether these reactions are slowed down by a continuum of small ( kbt) free energy barriers or by a few large (> kbt) free energy barriers is a major challenge. in this study the free energy landscape accessible to a small protein monellin is characterized under native-like conditions using hydrogen exchange in conjunction with mass spectrometry. cooperative and noncooperative opening processes could be directly distinguished from the mass distributions obtained in the ex limit. under native conditions, where the native state is maximally stable, the unfolded state is transiently sampled in an entirely non-cooperative and gradual manner. under conditions which stabilize the unfolded state or destabilize the native state of the protein, the slowest structure opening event becomes cooperative. the present study provides an understanding of the relationship between stability and folding cooperativity. it suggests that the cooperative transitions observed in unfolding reactions maybe a consequence of the changes in the stabilities of the unfolded state and the transition state. it also provides rare experimental evidence for a gradual unfolding transition on a very slow timescale. role of electrostatic repulsion between unique arginine residues on the assembly of a trimeric autotransporter translocator domain eriko aoki , kazuo fujiwara , masamichi ikeguchi haemophilus influenzae adhesin (hia) belongs to the trimeric autotransporter family. the autotransporter consists of an n-terminal signal peptide, an internal passenger domain and a c-terminal translocator domain. the signal peptide directs to export across the inner membrane via the sec system and is cleaved, the passenger domain is a virulence factor, and the translocator domain (hiat) is embedded in the outer membrane. the crystal structure of hia translocator domain (hiat) has shown that hiat forms a transmembrane b-barrel of b-strands, four of which are provided from each subunit. the b-barrel has a pore that is traversed by three a-helices, one of which is provided from each subunit. the protein has a unique arginine residue at . arg side chains from three subunits protrude from the b-strand toward the center of the barrel and are close to each other. these residues seem to have an unfavorable electrostatic effect on the assembly and decrease the trimer stability. to investigate the role of this residue on the trimer assembly and stability of hiat, we replaced this arginine with the neutral amino acid, methionine (r m) or the positively charged residue, lysine (r k), and properties of these mutants were investigated. hiat and two mutants were dissociated by formic-acid treatment, and they were able to reassemble in the presence of the detergent. to measure the time course of trimer reassembly, amounts of reassembled trimer and monomer were quantified by sds-page at different assembly times. although the neutralized mutation increased the rate of reassembly, the final amount of reassembled trimer decreased, especially at higher protein concentration. these suggest that the neutralized mutation cause the incorrect oligomer formation. the far-uv cd spectrum of reassembled wt hiat was nearly identical with that of the native wt hiat. however, the spectrum of the reassembled r m mutant was more intense that of the native r m mutant, although the proportion of trimer was much lower than that of the wt hiat. this suggests that the incorrect oligomer has a secondary structure different from the wt hiat. r k mutant showed assembly properties similar to those of the wt hiat. therefore, the repulsion between positively charged residues seems to be important for preventing hiat from misassembly. similar proximity of arginine residues is observed for hiv capsid protein, carboxysome shell protein, lumazine synthase and so on. the electrostatic repulsion between arginine residues may be a general mechanism for protein assembly. department of veterinary pathobiology, kagoshima university, institute for food sciences, hirosaki university, faculty of fisheries, kagoshima university, department of veterinary histopathology, kagoshima university, veterinary clinical training center, kagoshima university, department of veterinary anatomy, kagoshima university, sakamoto kurozu inc., the united graduate school of agricultural sciences, kagoshima university kurozu is a traditional japanese rice vinegar. during fermentation and aging of the kurozu liquid in an earthenware jar over year, solid residue called kurozu moromi is produced. in the present study, we evaluated whether concentrated kurozu or kurozu moromi could ameliorate cognitive dysfunction in the senescence accelerated p mouse. senescence accelerated p mice were fed . % (w/w) concentrated kurozu or . % (w/w) kurozu moromi for or weeks. kurozu suppressed cognitive dysfunction and amyloid accumulation in the brain, while kurozu moromi showed a tendency to ameliorate cognitive dysfunction, but the effect was not significant. we hypothesize the effect is caused by the antioxidant effect of concentrated kurozu, however, the level of lipid peroxidation in the brain did not differ in senescence accelerated p mice. dna microarray analysis indicated that concentrated kurozu increased hspa a mrna expression, a protein that prevents protein misfolding and aggregation. the increase in hspa a expression by kurozu was confirmed using quantitative real-time pcr and immunoblotting methods. therefore, the suppression of amyloid accumulation by concentrated kurozu may be associated with hspa a induction. however, concentrated kurozu could not increase hspa a expression in mouse primary neurons, suggesting it may not directly affect neurons. young-ho lee although amyloid fibrils are associated with a number of pathologies, their conformational stability remains largely unclear. we herein investigated the thermal stability of various amyloid fibrils. a-synuclein fibrils, freshly prepared at c at neutral ph, cold-denatured to monomers at - c and heat-denatured at - c. meanwhile, the fibrils of b -microglobulin, alzheimer's ab - /ab - peptides, and insulin exhibited only heat denaturation, although they showed a decrease in conformational stability at low temperature in the presence of chemical denaturants. a comparison of structural parameters with positive enthalpy and heat capacity changes which showed opposite signs to protein folding suggested that the burial of charged residues in the fibril cores contributed to the cold denaturation of a-synuclein fibrils. reinforced electrostatic repulsion at low temperatures may promote cold denaturation, leading to a unique thermodynamic property of amyloid fibrils. we propose that although cold-denaturation is common to both native proteins and misfolded fibrillar states, the main-chain dominated amyloid structures may explain amyloid-specific cold denaturation due to the unfavorable burial of charged side-chains in fibril cores. key structural differences between tbtim and tctim revealed by thermal unfolding molecular dynamics simulations angel piñeiro , miguel costas , andrea guti errez-quezada dept of applied physics, university of santiago de compostela, lab. of biophys. chem., dept of physical chemistry, fac. of chemistry, unam the thermal unfolding pattern obtained by differential scanning calorimetry for trypanosoma cruzi and trypanosoma brucei triosephosphate isomerase (tcim and tctim) are significantly different although the crystal structure of both proteins is almost indistinguishable and the sequences are highly homogolous. in order to explain these differences at molecular level a set of molecular dynamics simulations were performed at different temperatures between and k. the obtained trajectories were analyzed in detail and the residues that showed to be key in the unfolding pathway of each species were identified. a set of residues that behave significantly different between both proteins were selected and proposed for mutations. the general aim is to identify the minimum amount of residue mutations that allow providing tbtim with the behaviour of tctim and vice versa. experimental complementary work is also being performed on the same protein. repositioning som as a potent inhibitor of transthyretin amyloidogenesis and its associated cellular toxicity salvador ventura , ricardo sant'anna , maria ros ario almeida , nat alia reixach , raul insa , adrian velazquez-campoy , david reverter , n uria reig universitat aut onoma de barcelona, instituto de biologia molecular e celular, icbas, the scripps research institute, som-biotech, universidad de zaragoza transthyretin (ttr) is a plasma homotetrameric protein implicated in fatal amyloidosis. ttr tetramer dissociation precedes pathological ttr aggregation. despite ttr stabilizers are promising drugs to treat ttr amyloidoses, none of them is approved by the food and drug administration (fda). repositioning existing drugs for new indications is becoming increasingly important in drug development. here, we repurposed som , an fda-approved molecule for neurodegenerative diseases, as a very potent ttr aggregation inhibitor. som binds specifically to ttr in human plasma, stabilizes the tetramer in vivo and inhibits ttr cytotoxicity. in contrast to most ttr stabilizers, it exhibits high affinity for both ttr thyroxine -binding sites. the crystal structure of som -bound ttr explains why this molecule is a better amyloid inhibitor than tafamidis, so far the only drug in the market to treat the ttr amyloidoses. overall, som , already in clinical trials, is a strong candidate for therapeutic intervention in these diseases. neurometals as modulators of protein aggregation in neurodegenerative diseases s onia s. leal , joana s. crist ovão , cl audio m. gomes protein misfolding and aggregation is a hallmark across neurodegenerative diseases such as alzheimer's disease and amyotrophic lateral sclerosis (als). since these diseases are mostly sporadic, the formation of protein amyloids in the nervous system depends of chemical and biological triggers within the neuronal environment, such as metal ions [ ] . in this communication i will overview the metallobiology of neuronal calcium, zinc and copper, which are key players in brain function and have altered homeostasis in most neurodegenerative conditions. our recent work will illustrate how this allows establishing molecular mechanisms in neurodegenerative diseases [ ] [ ] [ ] [ ] [ ] . in the pursuit of this goal, in the last years we have been investigating superoxide dismutase (sod ), a cu/zn metalloenzyme that aggregates in the fatal neurodegenerative disorder als, as a model. in sod -als cases, this ubiquitous protein selectively aggregates in motor neurons, implicating a local biochemical factor in the process: interestingly, zn and ca levels are upregulated in the spinal and brain stem motor neurons of als patients, and increased ca triggers multiple pathophysiological processes which include direct effects on the sod aggregation cascade [ , ] . recently we established that calcium ions promote sod aggregation into non-fibrillar amyloid, suggesting a link to toxic effects of calcium overload in als [ ] . we showed that under physiological conditions, ca induces conformational changes on sod that increase sod b-sheet content and decrease sod critical concentration and nucleation time during aggregation kinetics. we also observed that calcium diverts sod aggregation from fibrils towards amorphous aggregates. interestingly, the same heterogeneity of conformations is found in als-derived protein inclusions. we thus hypothesized that transient variations and dysregulation of cellular ca and zn levels contribute to the formation of sod aggregates in als patients [ , ] . in a follow up study we combined experimental and computational approaches to show that the most frequent ligands for ca are negatively-charged gatekeeper residues located in boundary positions with respect to segments highly prone to edge-to-edge aggregation. calcium interactions thus diminish gatekeeping roles by shielding repulsive interactions via stacking between aggregating b-sheets, partly blocking fibril formation and promoting amyloidogenic oligomers such as those found in als inclusions. interestingly, many fals mutations occur at these positions, disclosing how ca interactions recreate effects similar to those of genetic defects, a finding with relevance to understand sporadic als pathomechanisms [ ] . the amino acid proline is well-known by its disorder promoting and helix breaking properties. prolines can be accommodated within transmembrane (tm) alpha-helices and participate in important biological tasks like signal transduction, ligand binding and helix-helix packing. x-ray crystallography and nmr indicate that proline residues in membrane proteins induce distortions of the helix geometry to different extents ranging from small bends to severe kinks. however, such studies provide essentially a static snapshot of membrane-embedded helices. therefore, the link between proline dynamics and function is not completely understood. in this work we have used singlemolecule f€ orster resonance energy transfer (smfret) and fluorescence correlation spectroscopy (fcs) to probe the structure and dynamics of the tm domain of human glycophorin a (gpa), a widely used model membrane protein for oligomerization studies. a fluorescent dye pair has been attached to both ends of the membrane-spanning region of gpa, which allowed monitoring the average distance and distance fluctuations between the attachment points. site-specifically double-labeled gpa has been reconstituted into two membrane-mimetic systems: sds micelles and phospholipid bilayers assembled into nanodiscs. using proline-scanning mutagenesis we have systematically evaluated the impact of proline residues in different positions along the membrane normal on transmembrane helix length and lateral packing. furthermore, we have investigated the distance distribution in tm helices containing native prolines, namely the insulin receptor and the nesprin protein. our results shed light into the relation between proline dynamics and the folding and function of tm helices. thermodynamic contributions of specific mutations of l e protein in the rna: protein interface region measured by analytical ultracentrifugation and gel shift assay bashkim kokona , , sara kim , margaret patchin , britt benner , susan white in saccharomyces cerevisiae, ribosomal protein l e acts as an autoregulator by inhibiting the splicing of its pre-mrna and translation of its mrna. the l e protein-rna binding site has been previously studied, revealing a rna kink-turn motif, which is characterized by a sharp bend in the phosphodiester backbone due to unpaired nucleotides and internal tertiary interactions. l e structural flexibility at the rna-binding interface makes such interaction an excellent model to explore the energetics of rna protein binding. we made l e k a, f a, and f w mutants to quantify the thermodynamic contributions of such interactions to the protein-rna complex. we used analytical ultracentrifugation sedimentation equilibrium (se) and sedimentation velocity (sv) to investigate conformational changes and protein-rna binding free energy changes due to mutations. our computed changes of binding free energy based on the sedimentation equilibrium experiments were consistent with the gel shift assay results. in addition, sedimentation velocity experiments on the l e wild type indicate that protein-rna interaction is highly dynamic and involves conformational changes of the kink-turn rna induced by l e protein. our results provide new insights on understanding the binding between ribosomal proteins and their rna molecules counterpart, which can be used to complement the x-ray structure. role of a non-native a-helix in the folding of equine b-lactoglobulin takahiro okabe , toshiaki miyajima , kanako nakagawa , seiichi tsukamoto , kazuo fujiwara , masamichi ikeguchi equine b-lactoglobulin is a small globular protein ( residues). although elg adopts a predominantly b-sheet structure consisting of nine anti-parallel b-strands (a-i) and one major a-helix in the native state, it has been shown that a non-native a-helical intermediate accumulates during the burstphase of folding reaction from the unfolded state in the concentrated denaturant. to ask whether the non-native helix formation is important for acquiring the native b-sheet structure, we determined first where the non-native a-helix is formed. a stable analogue of the burst-phase folding intermediate was observed at acid ph (a state). the amide hydrogen exchange experiment and proline-scanning mutagenesis experiment have shown that the non-native a-helix is formed at the region corresponding to the h strand in the a state. to investigate the role of this non-native a-helix on refolding reaction of elg, we constructed several mutant proteins, which were designed to destabilize the nonnative a-helix in the folding intermediate without perturbation on the native structure. a mutant, a t, fulfilled this requirement, that is, a t showed a native structure similar to that of the wildtype protein, and largely reduced cd intensity in the a state. then, the refolding kinetics were investigated by the cd and fluorescence stopped-flow method. a t mutation resulted in reduction of the burst-phase cd intensity, which confirmed that the non-native a-helix is formed around the h strand region. subsequent to the burst-phase, four kinetic phases were observed for a t and the wildtype protein. importantly, the folding rate constants of the four kinetic phases were similar between both proteins. furthermore, interrupted refolding experiments demonstrated that the native state was formed in the two parallel pathways in the two slower phases of the four kinetic phases. the relative amplitudes of the two pathways were similar between a t and the wild-type protein. these results clearly showed that the formation of the non-native helix has little effect on the folding rates and pathways, and suggested that the non-native helix formation may not be a severe kinetic trap for protein folding reaction. impact of the chaperonin cct in a-synuclein(a t) amyloid fibrils assembly ahudrey leal_quintero , javier martinez-sabando , jose mar ıa valpuesta , begoña sot centro nacional de biotecnolog ıa (cnb/csic)., centro nacional de biotecnolog ıa (cnb/csic)., centro nacional de biotecnolog ıa (cnb/csic)., centro nacional de biotecnolog ıa (cnb/csic) and fundaci on imdea-nanociencia cct is a eukaryotic chaperonin that uses atp hydrolysis to encapsulate and fold nascent protein chains. moreover, it has recently been shown that cct is able to inhibit amyloid fibers assembly and toxicity of the polyq extended mutant of huntingtin, the protein responsible of huntington disease. although this opens the possibility of cct being also able to modulate other amyloidopathies, this has not addressed yet. the work presented here intends to determine the effect of cct in the amyloid fibers assembly of a-synuclein(a t), one of the mutants responsible of parkinson disease. it is demonstrated that cct is able to inhibit a-synuclein(a t) fibrillation in a nucleotide independent way, suggesting that this effect is based on binding rather than on active folding. furthermore, using deletion mutants and assaying the interaction of cct with monomers, soluble oligomers and fibres, it has been possible to unravel the mechanism of this inhibition: cct interferes with fibers assembly by interacting with a-synuclein(a t) nac domain once soluble oligomers are formed, thus blocking the reaction before the fibers start to grow. amyloid-like aggregation of nucleophosmin regions associated with acute myeloid leukemia mutations daniela marasco , concetta di natale , valentina punzo , domenico riccardi , pasqualina scognamiglio , roberta cascella , cristina cecchi , fabrizio chiti , marilisa leone , luigi vitagliano department of pharmacy, cirpeb: centro interuniversitario di ricerca sui pepti, section of biochemistry, department of biomedical experimental and clinical scie, institute of biostructures and bioimaging nucleophosmin (npm ) is a multifunctional protein involved in a variety of biological processes and implicated in the pathogenesis of several human malignancies. npm has been identified as the most frequently mutated gene in acute myeloid leukemia (aml) patients, accounting for approximately % of cases ( ). the most frequent human npm mutations lead to variants with altered c-terminal sequences of the c-terminal domain (ctd) that, in its wild form, folds as a three helix bundle. aml modifications lead to (a) an unfolding of the ctd in the mutated protein and (b) its accumulation in the cytoplasm due to the loss of nuclear localization sequences with mutations of trp (mut e) and also of trp (mut a) ( ) . to gain insights into the role of isolated fragments in npm activities we dissected the ctd in its helical fragments. here we describe the unexpected structural behavior of the fragments corresponding to the helices h and h in both wild-type and aml-mutated variants. h region shows a remarkable tendency to form amyloid-like assemblies while only the muta sequence of h region is endowed with and b-sheet structure, under physiological conditions, as shown by circular dichroism, thioflavin t and dynamic light scattering. the aggregates of h , are also toxic to neuroblastoma cells, as determined by using the mtt reduction and ca influx assays ( ) . furthermore the effects of the local context on the different tendencies to aggregate of h and h were investigated and appeared to influence for the aggregation propensity of the entire ctd. since in aml mutants the ctd is not properly folded, we hypothesize that the aggregation propensity of npm regions may be implicated in aml etiology. these findings have implications to elucidate the pathogenesis of aml caused by npm mutants and aggregation phenomena should be seriously considered in studies aimed at unveiling the molecular mechanisms of this pathology. we report a resume of our study regarding the effects of microwaves in the range - mhz on a typical protein, myglobin. previous literature have concerned the effects on living and in vitro organic systems induced by high frequencies electromagnetic fields. we have focused our attention on a typical protein, myoglobin, because proteins are the simplest organic systems that are fundamentals in organic functions of livings. myoglobin is a protein found mainly in muscle tissue of vertebrates, consisting of a single protein chain with amino acids and one heme group that stores oxygen in the muscle cells. the physiological importance of myoglobin is mainly related to its ability to bind molecular oxygen. in particular, we focused our attention on the secondary structure of this protein in order to highlight whether exposure to microwaves unfold the protein producing transitions from a-helix component to b-sheet features. to this aim fourier transform infrared (ftir) spectroscopy have been used. the importance of this study is related to previous literature which indicated that transition from a-helix to b-sheet structure in a protein can be responsible for aggregation mechanisms that can lead to neurotoxicity and neurodegenerative disorders that can be considered as the first step to some pathologies [ ] [ ] [ ] . the aggregates consist of fibers containing unfolded proteins with a prevalent b-sheet structure termed amyloid [ ] . in our studies myoglobin in deuterium oxide (d o) solution was exposed for h to mobile phone microwaves at and mhz at a power density of w/m . ftir spectra were recorded by a spectrometer vertex v from bruker optics, following the protocol accurately described in [ ] [ ] [ ] . ftir spectroscopy analysis evidenced an increase in intensity of b-sheet structures and a significant shift to lower frequencies of about . cm- of the amide i vibration after exposure [ , ] . these results led to conclude that mobile phone microwaves induce proteins unfolding and formation of aggregates [ , ] . membrane proteins play a vital role in many biological processes, and yet remain poorly understood as they are frequently unstable in vitro. the goal of this project is to investigate the insertion and folding of membrane proteins into lipid bilayers, using a cell free expression system. we have used both e.colibased cell extracts (s ), and commercial translation systems (purexpress) in combination with synthetic liposomes of defined lipid composition. these studies will aid understanding of cooperative folding, folding intermediates, and the effects of the lipid bilayer on folding and insertion. model e.coli proteins have been investigated, as they can offer important insights into other proteins, and thus facilitate the further study of more biologically relevant proteins. it has been found that the rhomboid protease glpg spontaneously inserts into liposomes without the aid of an insertase such as secyeg. this spontaneously inserted glpg is functional, and is able to cleave bodipy-labeled casein, yielding a fluorescent product. the major facilitator superfamily (mfs) transport proteins lacy, galp and glpt have also been found to insert spontaneously into liposomes. it has been shown that the lipid composition of the liposomes has an effect on the amount of protein inserted into the bilayer, with all proteins tested to date preferring liposomes containing at least mol% dopg. ongoing and future work will involve the use of rare codons to alter the rate of translation, to investigate the effect this has on the final folded structure of the protein. preliminary work is also currently being done into whether the two domains of the mfs family transporters fold cooperatively or independently, thus aiding understanding into the folding and stability of membrane transport proteins. frederic greco , audrey toinon , nadege moreno , marie claire nicola€ ı rabies remains an important worldwide health problem that causes a fatal encephalomyelitis [ ] . currently, rabies in humans is under control in europe and north america following the use of efficient vaccines for dogs and wild animals. however, it still kills more than , people every year mainly in africa and asia [ ] . human vaccination prevents infection with very high efficacy. the vaccine contains an inactivated rabv produced on vero cells. rabv is an enveloped, negative single stranded rna virus which encodes five proteins, namely the nucleoprotein (n), the phosphoprotein (p), the matrix protein (m), the glycoprotein (g), and the viral rna polymerase (l) [ ] . the viral envelope is covered by trimer spikes of g-glycoprotein which is the most significant surface antigen for generating virus-neutralizing antibodies. here we illustrate the use of dsc (differential scanning calorimetry) to identify structural domains or proteins involved in thermal transitions. the dsc thermogram for intact beta-propiolactone inactivated rabv samples in pbs buffer reveals two major thermal transitions with a tm respectively at c and c. we have initially focused our investigations on one of the major proteins encode in rabv, glycoprotein g [ ] . glycoprotein g contains disulfide bridges on the ectodomain [ ] , is sensitive to bromelain cleavage [ ] and shows reversible conformation changes at low ph [ ] . considering these characteristics, our results provide evidence on the identity of one thermal transition observed by dsc. keywords: rabies virus, differential scanning calorimetry, protein unfolding domain swapping of the dna-binding domain of human foxp is facilitated by its low folding stability exequiel medina, sandro l. valenzuela, crist obal c ordova, c esar a. ram ırez-sarmiento and jorge babul departamento de biolog ıa, facultad de ciencias, universidad de chile, santiago, chile protein folding and dimerization (or oligomerization) are biologically relevant processes when reaching the quaternary structure is required for function. proteins that form dimers by exchanging segments or domains of their tertiary structure with another subunit, the so-called domain swapping phenomenon, are examples where folding and dimerization are tightly concerted processes. previous studies on domain swapping proteins, such as p suc and diphtheria toxin, have shown that, in general, a high kinetic barrier separates monomers and domain swapped dimers, and that this barrier can be lowered by promoting protein unfolding and refolding at high protein concentrations, thus favoring the swapped oligomer. recent crystal structures of the dna-binding domain of several human forkhead box (fox) proteins have shown that the p subfamily of these transcription factors (foxp) can form swapped dimers. the human foxp proteins are interesting models of domain swapping, because mutations of the dna-binding domain of these proteins are linked to diverse inherited disorders in humans, such as ipex and language deficits, and some of these mutations are located in the hinge region that connects the exchanged segment with the rest of the protein. moreover, foxp and foxp have been described to reach monomer-dimer equilibrium in solution after hours of incubation, suggesting that a low kinetic barrier separates both species. using foxp as a model of domain swapping, we analyzed the temperature and protein concentration effects on the dimer dissociation, obtaining the free energy change and enthalpy of the process by van't hoff analysis (dh of . kcal•mol- , ds of . kcal•mol- •k- and dg at c of . kcal•mol- ). these results indicate that the monomer-monomer association is an example of an enthalpy-driven process. to understand how foxp domains swap without protein unfolding, we performed equilibrium unfolding experiments using gndhcl as denaturant, showing that the wild-type protein has a low stability (dgu kcal•mol- , cm . m at c), in contrast to other domain swapping proteins with high kinetic barriers. we further explore the domain swapping mechanism of foxp through biased targeted molecular dynamics simulations, showing that the exchange process can occur by specific local destabilization and unfolding of the hinge region and helix h . to further corroborate that the low stability of wild-type foxp facilitates its domain swapping, we engineered a monomeric version of foxp through a single-point mutation in the hinge region, which has been previously described in the literature, and used this protein to visualize the effect of monomer stability in the dimer formation. comparison of the folding stability of the monomeric mutant a p and wild-type foxp shows that ddgu (mutant-wild-type) is . kcal/mol, concluding that the ability of foxp to domain swap rapidly can be explained through its low monomer stability and local unfolding of the exchange region. funding: fondecyt and . determining the coupled interactions that stabilize the structural framework of the ß-propeller fold loretta au , david green , , department of statistics, the university of chicago, department of applied mathematics and statistics, stony brook university, graduate program in biochemistry and structural biology, stony brook university, laufer center of physical and quantitative biology, stony brook university b-propeller proteins are a highly evolved family of repeat proteins that are involved in several biological pathways, such as signal transduction, cell-cycle modulation and transcription regulation, through interactions with diverse binding partners, despite having a similar fold. as for all repeat protein families, there is a consistent pattern in secondary structure for each repetitive region, in addition to the entire family. typically, four to ten propeller blades (each containing four anti-parallel b-sheets) are arranged in a toroidal shape, thus providing a large binding surface for ligands or other proteins. about % of known proteins adopt this distinctive fold, and although the requirements for tertiary structure and protein function are fundamentally encoded in primary structure, this relationship is not fully understood, and addressing it could provide insight on why the b-propeller fold is common. many techniques in comparative sequence analysis can successfully identify amino-acid conservation between closely related proteins, but molecular interactions between amino acids are often neglected, and further experimentation is still needed to determine the reasons underlying conservation. to explore how primary structure can dictate fold and function, we devised a computational approach to perform large-scale mutagenesis, by adapting the dead-end elimination and a* search algorithms (dee/a*), and also leveraged the structural conservation of each repeating region to understand how sequence variation influences protein fitness, defined here as a combination of stabilizing and binding interactions. dee/a* can evaluate low-energy protein sequences and their corresponding three-dimensional structures, and we used the bsubunit of a g-protein heterotrimer (pdb: gp , gia b g ) as a model system to demonstrate: ( ) how the multiple roles of individual amino acids in protein fitness can be deconvolved, and ( ) how epistatic interactions between them can contribute to structural stability. in doing so, we were able to identify important patterns in sequence complementarity between repeating regions that cannot be found using sequencebased methods alone. these results suggest that computational approaches can be used to determine important protein interactions, and help elucidate the prevalence of b-propeller proteins in biology. temperature induced conformational changes of the villin headpiece miniprotein stanislaw oldziej , wioletta _ zmudzi nska , anna hałabis the c-terminal subdomain of the actin-binding protein villin called hp (villin headpiece) has been used as a model protein in a number of studies of protein folding kinetics and protein folding mechanism [ , ] . the hp is a residue miniprotein with an alpha-helix bundle three-dimensional fold. the goal of our work was to determine conformational ensemble of polypeptide chain of the investigated miniprotein at a wide range of temperatures to get detailed information about how protein structure is influenced by temperature. d nmr spectra of the title miniprotein were registered at , and k. the three-dimensional structure of the hp based on restraints derived from nmr spectra registered at k is almost identical with structure deposited in the pdb database in the record f k [ ] . at higher temperatures ( and k) the general shape of the protein remains unchanged, with well packed hydrophobic core. however, with temperature increase alpha-helices start to melt. at k structure of the protein remains compact and in general shape similar to structure observed at k, but none of the alpha-helices could be observed. results obtained for hp protein are in agreement with previous observation for the trp-cage miniprotein [ ] , that with temperature increase regular secondary structure elements melt first before the break-up of the hydrophobic core of the protein. biological membranes provide a selective and chemically sealed barrier for cells. transport of ions and small molecules across the membrane is mediated by transporter proteins and the breakdown of a cell's ability to produce functionally folded membrane transport proteins can lead to dysfunction and has been implicated in many diseases . however little is known about the processes that govern the misfolding of a-helical integral membrane proteins, taking into account that these proteins fold and maintain functional structures within membranes of various organelles. the neurotransmitter sodium symporter (nss) protein family is an example of a-helical transporter proteins. the nss family encompasses a wide range of prokaryotic and eukaryotic ion-coupled transporters that regulate the transport of neurotransmitter molecules whose dysfunction has been implicated in multiple diseases and disor-ders . we have investigated the folding processes of prokaryotic homologue of the nss family leut responsible for the transport of neurotransmitters and amino acids to the sodium electrochemical gradient. previously folding processes of membrane transporters have mainly been characterised within detergent micelles. however, detergent micelles are not an accurate depiction of the environment of the membrane bilayer, with this in mind we have also attempted to investigate folding processes within a bilayer pd- nmr investigation of ph-induced unfolding of b domain of an escherichia coli mannitol transporter ii mannitol in the bacterial phosphotransferase system kim gowoon , yu taekyung , suh jeongyong the bacterial phosphotransferase system (pts) mediates sugar phosphorylation and translocation across the cytoplasmic membrane. cytoplasmic b domain (iib mtl) of the mannitol transporter enzyme ii mannitol, a pts family protein, delivers a phosphoryl group from a domain to an incoming mannitol that is translocated across the membrane. iib mtl is comprised of a four-stranded ß-sheet and three helices, representing a characteristic rossmann fold. we found that the iib mtl of escherichia coli unfolded at a mildly acidic condition. we made iib mtl mutants to investigate the mechanism of the ph-induced unfolding using nmr spectroscopy. we monitored backbone amide groups and side chain imidazole groups of histidine residues using d hsqc nmr, and pointed out a potential histidine residue that might be responsible for the unfolding. histidine residues may be generally important to the folding stability in response to environmental ph changes. can site-directed mutagenesis shed light on the refolding pattern of human glucose -phosphate dehydrogenase (g pd)? nurriza ab latif , , paul engel conway institute, univerversity college dublin, faculty of biosciences and medical engineering, universiti teknologi malaysia human glucose -phosphate dehydrogenase (g pd) is the first enzyme involved in the pentose phosphate pathway (ppp). this oligomeric enzyme catalyses the reaction of glucose -phosphate to form phosphogluconolactone with concomitant reduction of nadp to nadph. in erythrocytes nadph is important mainly for protection against oxidative stress. in connection with its role as the sole source of nadph, g pd deficiency commonly causes haemolytic disease and is known as the most common human enzyme deficiency globally. protein folding problems and instability are believed to be the major defects in the deficient enzymes. in this study, we employed site directed mutagenesis with hope to give more information on the role of -sh groups in the refolding of human g pd. two mutants were created: ) one in which all cys residues were replaced by ser and ) one in which only c and c were retained. the refolding of recombinant human g pd has been studied primarily by measuring the enzyme activity after refolding. we also used a combination of intrinsic protein fluorescence, ans ( -anilino- -naphthalenesulphonic acid) binding and limited proteolysis to look at the conformational change during the refolding. the results showed that gdnhcl-denatured recombinant human g pd wild type could be refolded and reactivated by rapid dilution technique. even though, as recombinants in e. coli, the mutants were well expressed and active, they remained inactive after attempts were made to refold them in vitro. the methods we applied may have provided some insights on the refolding pattern of this oligomeric protein, albeit qualitatively rather than quantitatively. a single aromatic core mutation converts a designed 'primitive' protein from halophile to mesophile folding connie tenorio , liam longo , ozan s. kumru , c. russell middaugh , michael blaber department of biomedical sciences, florida state university, department of pharmaceutical chemistry, university of kansas experiments in prebiotic protein design suggest that the origin of folded proteins may have favored halophile conditions. these results are consistent with salt induced peptide formation which shows that polymerization of amino acids is also promoted by high salt concentrations. as a result of various origin of life studies, a consensus on which amino acids likely populated early earth has emerged. these residues were synthesized by abiotic chemical and physical processes from molecules present in the surrounding environment. the properties of the consensus set of common prebiotic amino acids (a,d,e,g,i,l,p,s,t,v) are compatible with known features of halophile proteins, meaning these proteins are only stable in the presence of high salt concentrations. the halophile environment, thus, has a number of compelling aspects with regard to the origin of structured polypeptides. consequently, a proposed key step in evolution was, movement out of the halophile regime into a mesophile one commensurate with biosynthesis of "phase " amino acids -including the aromatic and basic amino acids. we tested the effects of aromatic residue addition to the core of a "primitive" designed protein enriched for the prebiotic amino acids (a, d, e, g, i, l, p, s, t, v) that required halophilic conditions for folding. the subsequent results show that the inclusion of just a single aromatic residue was sufficient for movement to a mesophile folding environment. thus, the inclusion of aromatic residues into the codon table could have conferred key stability to early proteins enabling adaptive radiation outside of a halophile environment. contact prediction methods that rely on sequence information alone, such as evfold, can be used for de novo d structure prediction and identification of functionally important residues in proteins. large multiple sequence alignments of protein families consisting of evolutionarily related and plausibly isostructural members reveal co-variation patterns that can be used to identify interactions between pairs of amino acids. we use a global probability model to disambiguate direct and indirect correlations. specifically, we use a maximum entropy approach called pseudo-likelihood maximization (plm) to distinguish causation (residue interactions) from correlation (correlated mutations) and compute evolutionary couplings (ecs). the inferred set of residue interactions can then be interpreted as physical contacts and used in de novo d structure prediction. furthermore, the interactions that are inferred can help guide experiments that measure the phenotypic consequences of protein substitutions, making the method useful for functional studies. the present work can be divided into three areas: (i) methodological improvements related to alignment, folding procedure, structure refinement and ranking; (ii) folding of proteins of known structure for benchmarking and prediction of proteins of unknown structure; and (iii) focused exploration of specific cases of interest. developing shuffle as a platform for expression and engineering of antibodies na ke , alana ali-reynolds , bryce causey , berkmen berkmen shuffle is a genetically engineered e.coli strain that allows disulfide bond formationin its cytoplasm with high fidelity. many proteins containing disulfide bonds have been successfully expressed in shuffle. in this study, we expressed, purified and characterized full-length monoclonal antibody igg in shuffle. for the first time, a fulllength igg can be functionally expressed in the cytoplasm compartment of an e.coli strain. in order to improve the folding and assembly of igg, we have investigated the expression of igg in various formats and vectors; we have co-expressed chaperones and other helper proteins with igg. several-fold increase in the yield of fulllength igg was observed. we characterized the shuffle produced igg and found it comparable to hybridoma produced igg. optimization of fermentation conditions for a large-scale production is in progress. we aim to develop shuffle as an easy, fast, robust platform for antibody engineering, screening and expression. experimental and computational studies of the effects of highly concentrated solutes on proteins: insights into the causes and consequences of quinary protein structure and cytoplasmic organization most studies of protein structure and function focus on pure, diluted samples; however, real-world biochemistry and typical biotechnological applications of proteins take place in complex media with very high concentrations of solutes ( - g/l) of varied size and chemical nature. on one side, this has recently fostered the study of proteins in vivo, in cell, or at least in media mimicking the native conditions. on the other hand, physical chemistry has for a long time studied the general effects of crowded and viscous conditions on proteins, looking mainly at coarse traits like diffusion and stability. but the general effects on traits relevant at atomic/residue resolutions have been less studied, and one fundamental issue remains unsolved: to what extent are proteins forced into interactions with highly concentrated solutes, and with what direct consequences? i will present here our ongoing efforts to dissect the fine effects of high solute concentrations and macromolecular crowding on proteins, based on nmr experiments and md simulations, two complementary techniques of high spatial and temporal resolutions. our results show that smaller solutes are prone to extensive interactions with proteins when at high concentrations while large solutes act chiefly through excluded-volume effects. overall, we observe location-specific perturbations of a protein's surface, its internal dynamics and internal dielectrics, and its hydration, all very dependently on the solute's size and chemical nature. our results support the growing notion that proteins should be studied in native-like media, adding that not only macromolecular crowders but also small molecules should be considered in these studies. last, the fact that high-concentration conditions affect far more than a protein's diffusion rate and stability suggests critical consequences of quinary protein structure and cytoplasmic organization on the regulation of proteins within cellular biochemistry. aldona jeli nska , anna lewandrowska , robert hołyst we developed an analytical technique for the study of interactions of ligands (e.g. cefaclor, etodolac, sulindac) with most abundant blood protein (e.g. bovine serum albumin) using the flow injection method. the experiments were conducted at high flow rates ( cm/s) in a long (> m), thin ( mm) and coiled capillaries. the compound of interest ( ml) was injected into carrier phase, which moved by the poisseule laminar flow. at the detection point we measure the concentration distribution of the analyte. the width of the final profile of the analyte concentration is inversely proportional to the effective diffusion coefficient of the analyte. from the differences between the widths of the concentration distribution of free and bound ligand we can determine value of the association constant. carbohydrate binding modules (cbms), which are defined as contiguous amino acid sequences within a carbohydrate-active enzyme, have been found in both hydrolytic and non-hydrolytic proteins and are classified into families, according to their primary structure similarity. the characterization of cbms by different methods has shown that these modules concentrate enzymes on the surface of polysaccharide substrates. it is thought that maintaining the enzyme in proximity with the substrate leads to more rapid degradation of the polysaccharide. therefore, the study of these kinds of modules or domains is relevant, since they are involved in multiple processes in organisms, like signaling, defense and metabolism; and some of them are involved in allergenic responses. in the present work we studied two different models: the first one is a cbm of the family from lactobacillus amylovorus (lacbm ) that binds starch these domains are present in a a-amylase like a repetitive tandem of five modules that are consecutive and do not present connectors. by means of itc and using a single recombinant lacbm domain we determined a ka . x m- for b-cyclodextrin and a ka . x m- for acyclodextrin. when the number of consecutive recombinant modules increased to three or five tandem modules, the ka values increased to m- ; however, these constants did not show an additive or a synergic effect. for these experiments we fitted the isotherms to different models and used different algorithms. additionally, we used circular dichroism in the uv-far region to determine if there existed conformational changes upon binding of the cyclodextrin molecules to the different tandem modules. we could only observe slight changes in a positive band centered around - nm, which has been explained in terms of p-p; interactions of the aromatic residues at the binding site. these cbms have been used as carriers for in vivo vaccine delivery and affinity tags. the second model is a hevein-like cbm of the family present in a chitinase-like protein from hevea brasiliensis (hbcbm ). hevein is a lectin from h. brasiliensis that shows a % identity with hbcbm . these cmbs are connected to the catalytic domain, in proteins such as chitinases, by a linker of approximately residues. in these experiments we used fluorescence techniques to determine the affinity constants for chitotriose. we previously reported a ka . x m- when using a hbcbm that has a met residue at the nterminal region. besides the aromatic residues at the binding site, the met residue also interacts with the ligand, as determined using crystallographic and docking techniques. the mutant hbcbm -r w that does not have the met residue showed a ka of . x m- with chitotriose, similar to the value reported for hevein using itc (ka . x m- ). interestingly, there exists an isoform of the hbcbm that has a connector between one cbm and a half cbm ( . xhbcbm ). this protein has a ka of . x m- with the same ligand. initiating vesicle formation at the golgi complex: auto-regulation and protein interactions govern the arf-gefs gea and gea margaret gustafson , j. chris fromme molecular decision-makers play critical roles in the effort to maintain efficient and accurate cellular functions. in the case of vesicular traffic at the golgi complex, the decision to initiate vesicle formation is made by a set of guanine nucleotide exchange factors (gefs) that activate the small gtpase arf , which is the master controller for the recruitment of cargos and coat proteins. saccharomyces cerevisiae possess three golgi arf-gefs, gea , gea , and sec , which work at distinct sub-compartments of the golgi to activate arf only when and where appropriate. in the case of sec at the trans-golgi network (tgn), this requires a positive feedback loop in which active arf relieves autoinhibition of sec , as well as recruitment to the golgi membrane and catalytic stimulation by signaling rab gtpases. we know far less about the decisionmaking process for gea and gea , which are responsible for retrograde traffic within the golgi and to the endoplasmic reticulum. i have found that both gea and gea can bind membranes weakly in vitro, an ability which is counteracted by their c-terminal hds domains. in addition, i have discovered membrane recruitment in vitro is aided by the rab gtpase ypt . however, these interactions cannot fully explain the distinct localization patterns of gea , gea , and sec , as all three have been shown to be recruited by ypt , which is found throughout the golgi. my work has revealed that in addition to the well-established distinct localization from sec , gea also occupies different golgi compartments from gea , so specific signals must exist which help the gefs decide where to go. my current efforts focus on understanding the roles of the other domains of gea and gea , identifying the signals which send them to different parts of the golgi, and unraveling the different roles they play in vesicle trafficking pathways. sequence variation in archaea through diversity-generating retroelements sumit handa , blair g paul , kharissa l shaw , david l valentine , partho ghosh department of chemistry and biochemistry, university of california san diego, marine science institute, university of california protein diversification is an essential tool for the survival and evolution for various species. diversitygenerating retroelements (dgr) in bacteria is known to generate massive variation in dna through an error prone reverse transcriptase and retrohoming, which leads to variation in protein sequence. recent discovery of dgrs in intraterrestrial archaeal systems have opened an opportunity to study this massive sequence variation in third domain of life (paul bg, et al. nat. comm.) here, we present the first crystal structure of variable protein from archaea with ligand-binding pocket is surface exposed. also, it has conserved c-type lectin (clec) fold, as shown by previous work on variable proteins, major tropism determinant (mtd) and treponema variable protein a (tvpa) which bind ligands through the clec fold. despite weak sequence identities ( - %) among these variable proteins, clec fold was found to be conserved. this variable ligand-binding site for archaea variable proteins can potentially generate variants. protein synthesis is a dynamic process mediated by a variety of proteins and enzymes. recent studies have shown that hydroxylation is a key post-translational modification involved in translation termination. in particular, the fe(ii)-and -oxoglutarate-dependent oxygenase, jumonji domaincontaining (jmjd ), regulates translation termination via the carbon hydroxylation of an invariant lysine residue, k , of the eukaryotic release factor, erf . in eukaryotes, translation termination is mediated by a release factor complex that includes erf . erf is comprised of three domains, and it is responsible for recognizing stop codons in mrna transcripts before triggering polypeptide release from the ribosome. the lysine residue hydroxylated by jmjd falls within the n-terminal domain and more specifically within the highly conserved niks motif. this motif has been identified by cross-linking and mutagenesis studies to play an essential role in stop codon recognition. while hydroxylation of k by jmjd has been found to increase translational termination efficiency, the exact molecular mechanism by which hydroxylation influences termination remains unclear. this work aims to understand how hydroxylation of erf affects translation termination by exploring the effect of hydroxylation on the structure, dynamics, stability, and binding of the n-terminal domain of erf (erf -n) using mass spectrometry, protein nmr spectroscopy, circular dichroism and differential scanning fluorimetry. in our efforts to understand the effect of hydroxylation, an additional jmjd -catalyzed modification, characterized by a da mass shift on k , was identified in vitro. the effect of this modification on erf was similarly explored. our findings suggest that hydroxylation has no effect on the in-solution nmr structure of erf -n, which experiences chemical shift changes localized to the target lysine residue. correspondingly, there are no significant differences in secondary structure content between wild type and hydroxylated erf -n. hydroxylation was also found to have no effect on protein stability or dynamics. interestingly however, the da modification appears to cause more significant chemical shift changes dispersed beyond the niks motif. this suggests a more global effect on the in-solution nmr structure despite the little differences observed in protein dynamics and secondary structure content. the da modification was also found to have a destabilizing effect on erf -n. neither hydroxylated nor da modified erf -n exhibited differences in rrna binding. while hydroxylation of erf was found to have little effect on protein structure, dynamics, stability, or binding, the da modification has marked effects on protein structure and stability. such differences suggest that this modification has the potential to play an important role in translation. functional and structural analysis of a gh ß-n-acetylglucosaminidase from the marine bacterium vibrio harveyi piyanat meekrathok , arthur t. porfetye , marco b€ urger , ingrid r. vetter , wipa suginta biochemistry-electrochemistry research unit, suranaree university of technology, max planck institute of molecular physiology vibrio harveyi b-n-acetylglucosaminidase (so-called vhglcnacase) is a new member of the gh glycoside hydrolase family responsible for the complete degradation of chitin fragments, with nacetylglucosamine (glcnac) monomers as the final products. however, the d structure of glcnacase is still unknown. in this study, crystal structure and function of glcnacase were investigated based on protein crystallography. size-exclusion chromatography and the native-page were employed to verify the protein state of glcnacase in a native form and the acidic active-site residues were mutated using sitedirected mutagenesis method. the effects of mutations on the binding and hydrolytic activities were studied by enzyme kinetics. to provide a structural basis of glcnacase, the wild-type enzyme was crystalized at k using a solution containing . m sodium acetate ph . and . m sodium malonate and recorded x-ray data. the wild-type enzyme was crystallized within days in the monoclinic crystal form, belonging to space group p , with unit-cell parameters a . , b . , c . Å. the crystal structures of v. harveyi glcnacase were solved and refined to highest resolution of . Å. structural investigation revealed that glcnacase comprises three distinct domains, designated as the n-terminal carbohydrate-binding domain, the a b topology domain and the tim-barrel catalytic domain. the substrate binding groove of glcnacase is a small pocket, which is suitable to accommodate a shortchain chitooligosaccharide. kinetic analysis revealed that a group of the adjacent d -h -e showed a significantly decreased activity as compared with the wild-type enzyme, and these residues might be important for enzyme catalysis. silencing the molecular timekeeper in human cancer alicia michael , stacy harvey , patrick sammons , amanda anderson , hema kopalle , alison banham , carrie partch university of california -santa cruz, university of oxford the circadian clock coordinates temporal control of physiology by regulating the expression of at least % of the genome on a daily basis. disruption of circadian rhythms through environmental stimuli (e.g. light at night) or genetic means can lead to the onset of diseases such as: diabetes, cardiovascular disease, premature aging and cancer. - the circadian clock orchestrates global changes in transcriptional regulation via the bhlh-pas transcription factor clock:bmal . pathways driven by other bhlh-pas transcription factors have a homologous repressor that modulates activity on a tissue-specific basis, but none have been identified for clock:bmal . we discovered that the cancer/testis antigen pasd fulfills this role to suppress circadian rhythms. pasd is evolutionarily related to clock and interacts with the clock:bmal complex to repress transcriptional activation. furthermore, deletion of one region, highly conserved with clock exon , alleviates repression by pasd to suggest that it utilizes molecular mimicry to interfere with clock:bmal function. structural and biochemical studies of the direct interaction of pasd with the clock:bmal complex using recombinant protein expression and biophysical techniques are currently underway. as a cancer/testis antigen, expression of pasd is natively restricted to gametogenic tissues but can be upregulated in somatic tissues as a consequence of oncogenic transformation. reducing pasd in human cancer cells significantly increases the amplitude of transcriptional oscillations to generate more robust circadian rhythms. our work suggests that mechanisms to suppress circadian cycling can be hard-wired in a tissue-specific manner and our data show that they can be co-opted in cancer cells to attenuate clock function. the scaffolding protein iqgap participates in various cellular functions such as cell-cell adhesion, cell polarization and migration, neuronal motility, and tumor cell invasion by binding to target proteins, including rac and cdc , two members of the rho family. to better understand the molecular basis of these interactions, we utilized in this study a novel time-resolved fluorescence spectroscopy to determine individual rate constants for iqgap interaction with fourteen different rho proteins. the results indicated that iqgap binds among rho proteins selectively to rac-and cdc -like proteins only in a gtp-dependent manner. moreover, the interaction of rho proteins with the c-terminal half of iqgap (grd-c), shorter fragment contains grd-gbd, only the grd and also grd-gbd with single and double phosphomimetic mutations s e and s d was performed. obtained results showed that, when both grd and gbd are existing, fluorescence changes is detected but for grd alone or in the case of s d or s e/s d no change was observed, suggesting that gbd and specifically, cysteine is critical for this interaction. furthermore, fluorescence polarization results showed that the grd-c interact with cdc and rac but not with rhoa, and interestingly the grd domain showed similar behavior, but with to folds lower affinity as compared with the grd-c. consistent with this, a gdp-bound form of cdc showed interaction with both grd and the grd-c in quiet comparable affinities. at last, competition experiments utilizing interacting partners of rac , e.g. tiam , p rhogap, plexin-b , p phox, pak and rhogdia, along with structural analysis, revealed two negative charged areas on the surface of rho-and rnd-like proteins, which might explain their inaccessible interaction with iqgap . the overlapping binding site of cdc and rac on the surface of iqgap together with the kinetic details of the selective interaction of iqgap with rac-and cdc -like proteins suggests that these interactions are most likely mediated via the same mechanism. ing dimerizes through its n-terminal domain, with a symmetric antiparallel coiled-coil structure , making it a bivalent reader of the h k me mark. ing is highly homologous with ing , but forms part of a different histone acetyl transferase complex . here, we show that ing is also a dimer and thus a bivalent reader of the h k me mark. however, the crystal structure of the n-terminal domain of ing shows an asymmetric dimer, different from the homologous ing domain. our nmr data (backbone assignment and paramagnetic relaxation effects) and saxs data indicate that the structure of the n-terminal domain of ing in solution is similar to ing , suggesting that the crystal structure of ing is likely a crystallization artifact. three point mutations in the n-terminal domain of ing have been described in oral squamous cell carcinoma: q r, i v, and c r . we have found that the n-terminal domains of the three mutants are dimeric coiled-coils but with different stability, as measured by thermal denaturation. while the q r mutant is as stable as the wild type, the i v and c r mutants are strongly destabilized, suggesting a role in cancer development at least for these two mutants. efforts so far, to combat alzheimer's disease (ad) have focused predominantly on inhibiting the activity of enzyme(s) that are responsible for the production of the main causative beta amyloid forming peptide. however, the inherent complexity associated with the network of pathways leading to the progress of the disease may involve additional targets for designing effective therapies. recent experimental findings have identified abelson's tyrosine kinase (c-abl), a non-receptor kinase involved in a variety of cellular functions as a new target for ad. in the present study we employed energy optimized multiple pharmacophore modeling strategy from multiple c-abl structures bound with ligands in the inactive atp binding conformation. virtual screening followed by docking of molecules from chembridge_cns database, and maybridge databases resulted in the identification of best scoring molecules. based on docking score and selectivity assessment and druggability parameters, four out of the molecules are predicted to show increased specificity for c-abl in comparison to closely related kinases. given the implied role of c-abl not only in ad but in parkinson's disease, the identified compounds may serve as leads to be developed as effective neurotherapeutics. rafael palomino , glenn millhauser , pietro sanna university of california santa cruz, the scripps research institute the central melanocortin system is recognized as a key regulator of energy balance and appetite. the hypothalamic melanocortin receptor, mc r, is a g-protein coupled receptor that is antagonized by the peptide ligand, agouti-related peptide (agrp), leading to increased feeding and weight gain. while much research has gone into how this ligand exerts its effects at the receptor, less is known regarding nonmelanocortin components of the pathway. syndecan- , a heparan sulfate proteoglycan, has previously been implicated in potentiating agrp antagonism, however details of this interaction are unclear. this work aims to investigate the role of syndecans at both a molecular level and in vivo. we hypothesize that agrp binds the glycosaminoglycan (gag) components of syndecans, and that this interaction increases the local concentration of the peptide near mc r. furthermore, we have previously shown that designed positive charge mutations to agrp lead to increased in vivo efficacy that is independent of mc r activity, and we hypothesize that this is due to greater affinity for the negatively charged gags. using isothermal titration calorimetry we have shown tight binding between agrp and heparan sulfate, the major gag component of syndecan- , and this affinity is strengthened by additional peptide positive charge. through nmr, we see that both positively charged and polar residues are necessary for binding various heparan sulfate polymers. these data implicate a specific region of agrp that is not required for mc r binding as being necessary in its role as a heparan sulfate binding protein. expanding on these findings, we are now using a syndecan knockout mouse line to explore the mechanism of differential feeding in our designed mutants. preliminary results indicate a reduction in weight gain in knockouts compared to their wildtype littermates post peptide administration. collectively, these data show that the physiologically relevant form of agrp, previously considered unable to interact with syndecans, is indeed a heparan sulfate binding protein. furthermore, our designed mutants have differential affinities for gags, with increased affinity correlating to increased feeding potency. finally, as the mc r pathway is thought to be a viable target for wasting disorders such as cachexia, we are interested in leveraging this data to improve the potency and stability of our designed agrp mutants. taken together, this work aims to develop new insights and probe the therapeutic potential of a critical metabolic pathway. evidence of a proteolytic phenomenon in the starch binding domain of the a-amylase from lactobacillus amylovorus zaira esmeralda s anchez cuapio , alejandra hern andez santoyo , sergio s anchez esquivel , romina rodr ıguez sanoja instituto de investigaciones biom edicas, universidad nacional aut onoma de m exico, instituto de qu ımica, universidad nacional aut onoma de m exico a-amylases are glycoside-hydrolases that catalyze the hydrolysis of internal a- , glycosidic bonds in starch and glycogen generating smaller oligosaccharides ( ). these multidomain proteins contain a catalytic barrel (b/a) and, in some cases, one or more non-catalytic domains whose function is generally described as carbohydrate binding module (cbm) and particularly as starch-binding domains (sbd). the sbd can bind granular starch increasing the local concentration of substrate at the active site of the enzyme and may also disrupt the structure of the starch surface ( ) . the a-amylase from lactobacillus amylovorus has a structure that consists of a catalytic domain (cd) and an unusual carboxy-terminal starch-binding domain with identical cbms (belonging to family ) in tandem ( ). each repeat acts as an independent fixing module with an additive or synergic effect between the units ( ). when we stored pure sbd from l. amylovorus we found multiple forms of low molecular weight with a constant pattern, which does not correspond to random degradation. interestingly, when the protein is stored at ph close to and edta is added, such proteolysis appears to decrease. so far, there is little information about the proteolytic process of amylases and the nature of it. here we show that divalent ions induce a proteolytic cleavage of the sbd, raising the possibility of an autoproteolytic activity. acknowledgments: this work is supported by grants papiit in - and conacyt . s anchez cuapio z is supported by a personal grant from consejo nacional de ciencia y tecnolog ıa, m exico. unnatural amino acid and related methods provided a special mechanism to implement site-specific spectroscopy active probe incorporation in a specific membrane protein in cells. the site specific incorporation resulted in a single signal during acquisition, resulting in unambiguous signal assignment. the protein specific labeling makes it possible for in situ membrane protein analysis using nmr or fluorescence detection. the f containing unnatural amino acid incorporation has been applied for dynamic studies of transporters in native lipid membrane, and the phosphorylation quantification analysis for tyrosine kinase in native lipid membrane with the aid of lipodisc. the fluorescent unnatural amino acid incorporation enabled the site-specific channel responses analysis upon ligand binding in a single cell. heather wiebe , noham weinberg , department of chemistry, simon fraser university, department of chemistry, university of the fraser valley the mechanism by which conformational changes, particularly folding and unfolding, occur in proteins and other biopolymers has been widely discussed in the literature. molecular dynamics (md) simulations of protein folding present a formidable challenge since these conformational changes occur on a time scale much longer than what can be afforded at the current level of computational technology. transition state (ts) theory offers a more economic description of kinetic properties of a reaction system by relating them to the properties of the ts, or for flexible systems, the ts ensemble (tse). the application of ts theory to protein folding is limited by ambiguity in the definition of the tse, although the experimentally observed first-order kinetics for folding of small single-domain proteins lends itself to interpretation by this theory. the pressure dependences of the folding rate constant can be used to obtain activation energies and activation volumes, which are rationalized as the properties of the folding tse. the large amount of activation volume data in the literature has gone largely uninterpreted at the quantitative level. we propose to utilize this data in conjunction with md-calculated volumetric properties to identify the tse for protein folding. the effect of pressure on reaction rates is expressed in terms of logarithmic pressure derivatives, known as activation volumes. according to ts theory, activation volumes can be identified as the difference in volume between the ts and reactant species: activation volumes dv ‡ have been experimentally determined for the folding of several proteins. the concept of activation volume can be extended to that of a volume profile, dv(y), which describes how the volume of a system changes along reaction coordinate y. if the position y ‡ of the ts along the reaction coordinate is unknown, it can be found by locating dv ‡ on the volume profile: such volume profiles can be built using our recently developed md-based displacement volume method.* using this method, volumes of single molecules can be calculated by taking the difference between the volume of pure solvent and solvent containing the desired solute. this method takes into account the strength and type of solvent-solute interactions as well as the geometrical configuration of the solute. in this work, we present the successful application of this method to several conformationally flexible systems. structure of the p paf/pcna complex and implications for clamp sliding on the dna during replication and repair to the canonical pip-box binding groove on the pcna front face. in contrast to other pcna interacting proteins, however, p paf also contacts the inside of, and passes through, the pcna ring. the mostly disordered p paf chain termini thus emerge at opposite faces of the ring, but remain protected from degradation by the s core proteasome. we also unveil a novel dna binding activity of p paf, both free and bound to pcna, which is mainly mediated by its conserved histone-like n-terminal tail. molecular modeling shows that a ternary complex with a duplex dna inside the pcna ring is energetically feasible and our electron micrographs show increased density inside the ring. we propose that p paf acts as a flexible drag that regulates pcna sliding along the dna, and may facilitate the switch from replicative to translesion synthesis polymerase binding upon dna damage. acknowledgements: this work has been mainly sponsored by mineco grant ctq - and juan de la cierva- contract to alfredo de biasio. metabolic syndrome (mets) is one of the leading causes of the death worldwide; however, exact pathophysiological mechanisms of mets remain largely unknown. growing evidence suggests that the increased availability of glucocorticoids at the tissue level play an important in mets development. one of the major determinants of glucocorticoid local action seems to be the enzyme b-hydroxysteroid dehydrogenase ( b-hsd ). this enzyme is a well-known member of the short-chain dehydrogenase/reductase (sdr) superfamily. it is an important carbonyl reducing enzyme that, besides its role fine-tuning of glucocorticoids actions, is involved in the biotransformation of drugs and in the development of lung cancer through metabolism of the tobacco specific carcinogen nnk. the phylogenetically closest relative of b-hsd is dhrs enzyme from the same superfamily. unlike b-hsd , dhrs is poorly characterized however it can be supposed at least partially overlapping function to b-hsd . moreover its possible association with similar pathological conditions in human as b-hsd has already been indicated by several studies. the aim of this study is the basic biochemical characterization of dhrs . the enzyme is a member of cluster of "classical" sdr; such members are considered to be retinoid and steroid metabolizing enzymes, so characterization the enzyme was based on this assumption. dhrs was prepared in recombinant form in the sf cell line. it was proved that this enzymes is an integral membrane-bound enzyme localized in the endoplasmic reticulum with luminal orientation, similarly to b-hsd . known substrates of b-hsd and related enzymes were tested also as substrates of dhrs . it was proved that dhrs is nadph-dependent reductase with important substrates as steroid hormones cortisone and androstene- , -dione, all-transretinal and also xenobiotics as , -naphtoquinone or carcinogen nnk at least in vitro. for better understanding of the catalytic function of dhrs its structural model was prepared and it is used also for the identification of additional substrates by ligand virtual screening. dhrs enzyme is expressed in several human tissues as adrenals, liver, prostate, small intestine and kidney. these brand new initial results point to the possible involvement of dhrs in important cellular processes that deserve further investigation. these results will lay the foundation for an understanding of dhrs role in human physiology resp. pathophysiology. this project was supported by grant agency of charles university ( /c/ ) and unce / ). structure-based functional identification of helicobacter pylori hp as a nuclease with both dna nicking and rnase activities bong-jin lee , ki-young lee hp is a conserved, uncharacterized protein from helicobacter pylori. here, we determined the solution structure of hp using three-dimensional nuclear magnetic resonance (nmr) spectroscopy, revealing that this protein is structurally most similar to a small muts-related (smr) domain that exhibits nicking endonuclease activity. we also demonstrated for the first time that hp is a nicking endonuclease and a purine-specific ribonuclease through gel electrophoresis and fluorescence spectroscopy. the nuclease activities for dna and rna were maximally increased by mn( ) and mg( ) ions, respectively, and decreased by cu ( ) ions. using nmr chemical shift perturbations, the metal and nucleotide binding sites of hp were determined to be spatially divided but close to each other. the lysine residues (lys , lys and lys ) are clustered and form the nucleotide binding site. moreover, site-directed mutagenesis was used to define the catalytic active site of hp , revealing that this site contains two acidic residues, asp and glu , in the metal binding site. the nucleotide binding and active sites are not conserved in the structural homologues of hp . this study will contribute to improving our understanding of the structure and functionality of a wide spectrum of nucleases. high-fidelity recombinant protein production in a silkworm bioreactor sungjo park , in-wook hwang , tatsuya kato , enoch park , andre terzic center for regenerative medicine, mayo clinic, laboratory of biotechnology, shizuoka university the domesticated silkworm, bombyx mori, is an attractive host naturally equipped with a proficient posttranslational modification machinery adequate to fulfill stringent demands of authentic recombinant protein production. silkworm-based protein expression has originally relied on a prototype baculovirus vector system that employs silkworm as a bioreactor in place of more traditional cell lines. recent development of the silkworm trophic b. mori nucleopolyhedrovirus (bmnpv) bacmid launches a second generation of silkworm-based protein production technology. introducing the recombinant bacmid dna into silkworms expedites heterologous protein expression by eliminating prior virus construction and amplification steps. salient examples of heterologous eukaryotic proteins produced in silkworms are acetyl-coa carboxylase , malonyl-coa decarboxylase, spot /mig heterodimer and a , -sialyltransferase with consistent high levels of protein expression. thus, equipped with a fail-safe post-translational modification machinery, eukaryotic proteins are readily bioengineered using a silkworm-based protein expression platform. studies exploring potential applications of synthetic antifreeze proteins in the frozen food industry ho zee (charles) kong , conrad perera , ivanhoe leung , nazimah hamid , viji sarojini school of chemical sciences, the university of auckland., school of applied sciences, auckland university of technology in nature, certain species of plants, insects and fish produce a group of antifreeze glycoproteins and polypeptides which enable them to survive the freezing temperatures of their natural habitat. these naturally occurring antifreeze proteins (afps) were first discovered in polar fishes such as antarctic notothenioids and winter flounder. these afps have the ability to bind to ice crystals and restrict their size and morphology; decrease the freezing point of water and inhibit the ice recrystallization processes. ice crystal formation is of primary concern to the frozen food industry, as ice crystal formation during freezing can be disruptive to and cause damage to the cellular structures in food. the unique properties of afps can be developed into a potential solution to minimize freeze-thaw damage to frozen food. a number of tailor made synthetic analogues based on the naturally occurring afps were successfully designed and synthesized. antifreeze activity studies of the afps were carried out using the clifton nanoliter osmometer attached with a microscope. the afps exhibited thermal hysteresis as well as modification of ice crystal morphology, confirming their antifreeze activity in vitro. the ability of these synthetic afps in preserving the texture and structure of frozen food was evaluated using the techniques of scanning electron microscopy. the afps showed great potential to preserve the cellular structures of frozen food samples during freeze-thaw process. additionally, secondary structure analysis of the afps was carried out using circular dichroism. this presentation will summarize our current results on the design, synthesis and anti-freeze activity analysis of the synthetic afps. invasive fungal infections remain a leading cause of death in immunocompromised patients. current antifungal agents have a host of issues including limited efficacy, host toxicity and an alarming increase in resistance. current research in our laboratories is focused on targeting the calcineurin signaling pathway that has been shown to be required for fungal pathogenesis. calcineurin is a highly conserved serine-threonine-specific ca -calmodulin-activated phosphatase important in mediating fungal pathogenesis and stress responses. it is a key regulator of a signal transduction network required for survival of the most common pathogenic fungi in humans, making it an ideal target for fungal drug development. calcineurin is a heterodimer of a catalytic (a) and regulatory (b) subunit. phosphatase activity requires association of the two subunits. calcineurin is also the target of the immunosuppressant fk , which functions as an inhibitor by first complexing with the peptidyl-prolyl cis-trans isomerase immunophilin, fkbp . the fkbp -fk complex subsequently binds to calcineurin in a groove between the a and b subunits and inhibits its activity. although fungal calcineurins are targeted by fk , it also targets mammalian calcineurin and is thus immunosuppressive in the host. in order to improve therapeutic efficacy, we have undertaken a unique effort that utilizes both structural biology and molecular mycology in an effort to overcome the fungal versus human specificity barrier. the nmr studies to be presented here have been focused on determining the resonance assignments and solution structures for the fkbp proteins from the pathogenic fungi candida albicans, candida glabrata and aspergillus fumigatus. notably, the x-ray crystallography structures of the wild-type candida albicans and aspergillus fumigatus fkbp proteins revealed an intriguing intermolecular interaction involving four residues in the 's loop including pro (in c. albicans) and pro (in a. fumigatus) which are stabilized in the cis conformation. these data suggest that the protein might use itself as an enzyme substrate. in efforts to establish if this interaction remains in a solution environment, we have determined the nmr structure and measured the t relaxation rates for the wild-type a. fumigatus fkbp protein and for the p g mutant variant that adopts a dramatically different orientation of the 's loop and does not form an intermolecular interaction in the crystal structure. the nmr chemical shift data indicate that, while the remainder of the protein structure remains unchanged, the 's loops in the two variants are indeed different. in addition, the t relaxation rates of the residues in this region are dramatically dissimilar in the two variants, but remain identical throughout the rest of the protein. we have also begun inhibitor binding studies of all of the fkbp proteins from each of the pathogens by titrating the fk inhibitor into native and mutant fkbp proteins in order to examine conformational changes associated in the protein upon complex formation. using this approach we plan to determine the relative kd values for binding of each inhibitor to the fkbp protein from each pathogen for comparison of binding proclivities. lupin (lupinus angustifolius l.) b-conglutin proteins: structure functional features, catalytic mechanism modeling and cross-allergenicity identification using protein threading and molecular docking methods lupin is an important pulse, which displays a wide range of benefits in agriculture, particularly these involved in possible plant pathogen suppression. furthermore, lupin seed proteins promote different positive health aspects, preventing cardiovascular disease, and reduction of glucose and cholesterol blood levels. "sweet lupine" seeds seem to be promising as a source of innovative food ingredients due to averaged protein content similar to soybean and an adequate composition of essential amino acids. thus, lupin seeds may be important source of proteins for human and animal consumption. however, and as drawback feature, the number of allergic people to lupin seed proteins is rising, becoming a serious and a growing problem in the western world, because of the rapid introduction of lupin seeds as new ingredients in traditional and novel foods. the goals of this study are the characterization the structure-functional properties of lupinus angustifolius l or narrow leafed lupin (nll) b-conglutin proteins, with a focus in its catalytic mechanism, and its molecular cross-allergenicity with other legumes, i.e. peanut, by extensive analysis using different computer-aided molecular approaches covering (i) physicochemical properties and functional-regulatory motifs, (ii) sequence analysis, -d and d structural (threading) modeling comparative study and molecular docking, (iii) conservational and evolutionary analysis, (iv) catalytic mechanism modeling, and (v) sequence, structure-docking based b-cell epitopes prediction, while t-cell epitopes were predicted by inhibitory concentration and binding score methods. b-conglutins (vicilin-like or s proteins) are seed proteins typically found in reserve tissues (endosperm and cotyledon). they belong to the cupin superfamily of proteins, containing a globular domain constituted by a conserved b-barrel. two barrels were found in all b-conglutin protein isoforms and an additional mobile n-terminal arm constituted bye a-helices. molecular modeling analysis has shown that one of this barrel contain a semi-conserved metal binding motive (hyx. . .r), typically found in oxalate oxidase (oxox) enzymes. interestingly, our results revealed considerable structural differences between b-conglutin isoforms, particularly affecting -d elements (loops and coils), and numerous micro-heterogeneities are present in fundamental residues directly involved in epitopes variability, which might be a major contributor to the observed differences in cross-reactivity among legumes. we also identified multiple forms of b-conglutins polypeptides ranging from - kda, with ige-binding characteristics in atopic patients. thus, b-conglutins might be considered as major allergen in different species of lupin, including the "sweet lupin" group, since several of these polypeptides were recognized by human iges, having the potential to trigger an immune response leading to allergy symptoms. influenza virus is one of the most prevalent pathogens causing respiratory illness which often leads to serious post influenza complications such as pneumonia and myocarditis. some viruses, as the avian influenza h n , are especially dangerous and draw special attention of who. this highly pathogenic virus spreads quickly among domestic poultry and wild birds resulting in high mortality. what is more distressing, the h n virus may be transmitted to humans. because of antigenic drift it is impossible to deliver an effective vaccine against all subtypes of the h n virus. moreover, traditional egg-based production of influenza vaccines is time-and cost-consuming, what makes it inadequate in case of a pandemic. hence, we have developed an efficient production process of influenza vaccine based on a recombinant hemagglutinin antigen (rha). recombinant vaccines underlay strict regulations and quality requirements. the purpose of this work was to develop a battery of analytical methods that allow to evaluate key quality attributes of rha on each stage of production. at first, we have focused on rha structure as a crucial issue for its activity. the primary structure of rha was confirmed by peptide mapping and tof/tof fragmentation (hplc, maldi tof/tof). furthermore, ftir analysis was used to evaluate the secondary structure of the protein. the disulfide bonds, which stabilize the tertiary structure, were assigned by peptide mapping. additionally, free thiols were measured using ellman's reagent. moreover, we have employed rp-hplc, sec-mals and dls to explore oligomerization of rha. these techniques appeared to be useful not only to confirm existence of native oligomers, but also to find and discard misfolded fraction, aggregates and truncated forms. in addition, two analytical methods (rp-hplc and cge) were developed to assess the purity of rha as required by ich guidelines. we also have determined isoelectric point and heterogeneity of rha by cief. afterward, developed methods were applied in the stability studies that provide a valuable insight into a chemical degradation process and conformational changes of rha during storage. this work was supported by innovative economy operational program, grant no. wnd-poig. . . - - / - as a part of project "centre of medicinal product biotechnology. package of innovative biopharmaceuticals for human and animal therapy and prophylactics." muscle cell atrophy via hsp gene silencing was counteracted by celastrol-mediated hsp overexpression molecular chaperone heat shock proteins (hsp) are known to assist protein quality control under various stresses. although overexpression of hsp was found to promote muscle mass retention in an unloading state, it is unclear whether muscle atrophy is induced by suppression of hsp expression and is counteracted by active hsp overexpression. in this study, we pre-treated hsp sirna to rat l cells for the hsp gene-silencing, and determined myotube diameter, hsp expression and anabolic and catabolic signaling activities in the absence or presence of triterpene celastrol (cel), the hsp inducer. relative to a negative control (nc), muscle cell diameter was reduced by % in the sirna-treated group, increased . -fold in the cel-treated group and remained at the size of nc in the sirna cel group. hsp expression was decreased % by sirna whereas the level was increased -to -fold in the cel and sirna cel groups. expression of foxo and atrogin- was increased . -to . -fold by sirna, which was abolished by cel treatment. finally, phosphorylation of akt , s k and erk / was not affected by sirna, but was elevated -to -fold in the cel and sirna cel groups. these results suggest that hsp downregulation by hsp gene-silencing led to muscle cell atrophy principally via elevation of catabolic activities. such anti-atrophic effect was counteracted by cel-mediated hsp overexpression. the centers for disease control and prevention report that at least million people in the united states will become ill due to antibiotic resistant pathogens leading to , deaths each year. in order to circumvent these resistance mechanisms, it is essential to quantitatively understand how the function of the protein(s) involved relates directly to resistance. integral membrane efflux pumps are known determinants of single-drug and multi-drug resistance in a wide variety of pathogenic organisms. these transporters are proteins whose characterization typically requires reconstitution in an artificial membrane. subsequently, these important proteins are difficult to characterize by traditional in vitro studies. my project aims to determine the physicochemical parameters of the efflux pump tetb utilizing molecular biology and mathematical modeling. tetb is composed of transmembrane (tm) alpha-helices and is found within the inner membrane of gram-negative bacteria. this protein allows for the efflux of tetracycline (tet), doxycycline (dox), and minocycline (mcn) antibiotics from the cytoplasm into the periplasm. these tetracyclines are a bacteriostatic class of antibiotics that inhibit protein synthesis by binding to the s ribosomal, therefore, blocking the binding of aminoacyl-trna. for cells grown in tetracyclines, the efflux mechanism of tetb decreases the cytosolic antibiotic concentration allowing for the rate of protein translation to increase. i have inserted a tet(b) expression system into the chromosome of an escherichia coli lab strain and have determined its growth profile under various concentrations of tet, mcn, and dox using a high-throughput -well plate format. the growth rate profiles correlate with tetb pumping rates for each drug. tetb more readily pumps out tet compared with dox and mcn and we observe that cells expressing tetb can grow at higher tet concentrations compared with dox and mcn. the shapes of the growth rate profiles produced in the different drugs give insight into the physicochemical mechanism of tetb. we have built a preliminary mathematical model that can simulate these growth profiles and predict efflux pump physicochemical parameters. we are currently working on understanding how efflux expression effects bacterial growth by testing ribosome binding site (rbs) sequences of varying strengths in our tet(b) expression system. future work is geared toward modeling more complex efflux pumps such as the tripartite pumps which traverse both bacterial membranes and cause multi-drug resistance. collectively, this project aims to build an in vivo system which will allow for the characterization of a variety of efflux pumps without the arduous tasks of protein purification and subsequent reconstitution. ( ) identified a small transmembrane region of both kcne and kcne that are essential for their unique modulation of the kcnq channel. by swapping a triplet motif in the transmembrane region of kcne and kcne , we can flip the primary function of these two proteins. while the key for kcne and kcne 's unique modulating is believed to lie in this triplet motif, the mechanism and structural changes involved in this modulation is not fully understood. by using nmr spectroscopy, biochemical studies, and computational docking, we aim to look at the structural and conformational differences between kcne and the triple mutant kcne substituted with the three essential kcne residues. we have expressed and purified n-labled kcne triple-mutant in sufficient quantities for nmr studies in lmpg detergent micelles and other membrane mimetics, and we have collected d nmr spectra using a trosy-based pulse sequence. partial backbone assignments of kcne triple mutant have been determined by aligning and transfer assignments of the wt kcne previous determined in our lab. with the structure of kcne triple mutant determined, we aim to computationally dock the triple mutant into a model of the full-length kcnq channel in the open and closed state. lastly, we will compare the known structure of kcne docked to a model of kcnq to that of the kcne triple mutant to determine key interactions, significant structural and conformational changes, and how the triple motif region gives rise to its specific structural and functional differences. with this information, we can begin to understand the mechanism of the functional diversity of the kcne family on kcnq potassium channel. biochemical characterization of brassica napus diacylglycerol acyltransferase and its regulatory domain .a) expressed in saccharomyces cerevisiae. purified bnadgat in n-dodecyl-b-d-maltopyranoside (ddm) micelles behaves as dimers, which can associate further to form tetramers. the acyl donor preference of the major dimeric form with sn- , -diolein as acceptor follows the following order: a-linolenoyl-coa > oleoyl-coa palmitoyl-coa > linoleoyl-coa > stearoyl-coa. the first residues of bnac.dg-at .a corresponding to a soluble regulatory region was expressed in escherichia coli and purified. truncation of this soluble domain reveals that the dimeric interface is located within residues - , while the first residues allow formation of tetramers. this n-terminal region was implicated as an allosteric exosite for acyl-coas as revealed by previous lipidex- binding studies. in the current study, circular dichroism spectroscopy and isothermal titration calorimetry were used to probe the binding kinetics and thermodynamics. dgat appears to shift between two oligomerization states, a phenomenon that may be related to regulation of enzyme activity and mediated by the n-terminal domain. alteration of lysine and arginine content as a strategy to modify such an interaction was found to increase the activity of rdrp in vitro. further, deletion of c terminal amino acid residues also resulted in increase in the polymerase activity that was comparable to the full length rdrp-p complex. it was proposed that the conserved c terminal disordered domain of rdrp was responsible for interaction with p and modulation of the activity. in the present study, role of the c terminal disordered domain was further investigated by determining the oligomeric status of the complex and the c terminal deletion mutants of rdrp and also by quantitating the rdrp-p interaction using surface plasmon resonance. size exclusion chromatography revealed that rdrp eluted in the void volume of the column whereas a significant fraction of the rdrp-p complex eluted at a position corresponding to the size of the : complex of rdrp and p ( kda). activity measurements indicated that the heterodimeric complex was more active than the aggregate eluting in the void fraction. interestingly, the c terminal deletion mutants of rdrp (c del & c del rdrp) were also found to be less aggregated as compared to full length rdrp and some of the protein eluted at a position corresponding to the respective monomers. these monomers were also more active than the aggregate fractions. these results demonstrate that the increase in activity observed either upon interaction with p or deletion of the c terminal domain could be due to the change in the oligomeric state of rdrp. in order to further analyze the interaction of rdrp with p surface plasmon resonance was used. rdrp and its deletion mutants were immobilized on biacore sensor surface and p protein was used as an analyte. full length rdrp and c del rdrp were shown to interact with p with kd values of . and um respectively. however, c del and c del rdrp did not show any binding with p . these results suggest that the region - from the c terminus of rdrp is essential for the interaction with p . further, the c del rdrp was inactive although c del rdrp continued to be active suggesting that residues - from the c terminus are crucial for rdrp activity. further studies are in progress to identify the residues within these motifs that may be essential for the activity or interaction with p . aggregation of androgen receptor in spinal bulbar muscular atrophy is a multistep process spinal bulbar muscular atrophy (sbma) is a member of the polyglutamine (polyq) expansion diseases, like huntington disease, and it is caused by a genetic expansion of the polycag tract in exon of androgen receptor (ar) that codes for the polyq region. sbma is a late onset disease, which involves a progressive degeneration of the motor neurons and consequent muscular atrophy. there is still no treatment available for this disease. ar is a nuclear receptor that responds to testosterone and that regulates the expression of the masculine phenotype. it is composed of an intrinsically disordered nterminal domain (ntd) that bears the polyq tract, a dna binding domain and a ligand binding domain. aggregates of ar protein with an extended polyq are observed in the motor neurons of sbma patients. in vitro studies showed that aggregation of androgen receptor takes place only in presence of testosterone and that the cleavage of the protein by caspase is a crucial event for cytotoxicity. however, there is no clear knowledge of the mechanism of aggregation, for this protein. an increasing body of evidence supports the hypothesis that the aggregation of these proteins is controlled by regions flanking the polyq tract, by regulating the rate of aggregation depending on their secondary structure. we have applied nuclear magnetic resonance (nmr) and circular dichroism for generating information on the secondary structure of the n-terminal cleavage product of ar by caspase and we have studied its aggregation with a set of biophysical methods, like dynamic light scattering, an hplc sedimentation assay and transmission electron microscopy. we have found that the polyq tract of ar presents a high degree of helicity. we attribute this conformation to the n-terminal flanking region, characterized by high helicity and we have tested this hypothesis by performing mutations. we have also observed that the rate of the first step of oligomerization is not dependent on the number of glutamine repeats, but instead is due to self interactions of a region n-terminal to and far from the polyq. its progression to fibril is dependent to the number of glutamines in the tract. we have therefore identified two steps in the aggregation process of ar, where a motif far from the polyq at its n-terminal drives the early oligomerization, followed by the interaction of the polyq chains that stabilize it and determine the progression to fibrils. these findings shed a light for possible interventions on the ar oligomerization process, thus suggesting a different strategy to study the onset of the disease in sbma patients. destabilizing the transient helical conformation of islet amyloid polypeptide hastens peptide self-assembly and potentiates cytotoxicity carole anne de carufel , phuong trang nguyen , alexandre arnold , isabelle marcotte , steve bourgault amyloidogenic polypeptides can be divided into two different structural classes: those that are intrinsically disordered and those that show a well-defined structure in their monomeric soluble state. natively folded proteins, such as transthyretin, have to unfold (or misfold), at least partially, to form amyloids. in contrast, intrinsically disordered polypeptides, such as the islet amyloid polypeptides (iapp) and abeta peptide, need to undergo conformational rearrangements allowing the formation of locally ordered structure(s) to initiate the amyloidogenic process. studies have shown that iapp and abeta adopt an alphahelix conformation in the initial steps of amyloidogenesis. this intermediate is believed to be on-pathway to fibril formation, although this hypothesis is still the matter of debate. in this study, we designed human iapp (hiapp) derivatives in which alpha-helix destabilizing substitutions were incorporated into the putative helical segment of iapp to probe the initial structural event in amyloid formation. using trifluoroethanol titration, we observed by cd spectroscopy that strategic incorporation of d-amino acids at positions and leads to an iapp derivative (diapp) that cannot fold into a helix. in homogeneous solution, hiapp and diapp show similar kinetics of fibrillization, as measured by thioflavin t fluorescence. although their amyloid fibrils display different characteristics by afm, iapp and diapp are able to self-associate to form amyloids when mixed together and when seeded with one another. studies in heterogeneous environment, notably in presence of glycosaminoglycans and model membranes of dopc/dopg ( : ), showed a helical intermediate for hiapp while only a beta-sheet secondary structure was apparent for diapp. while the rate of amyloid fibril formation was increased for both peptides, diapp was drastically affected by these anionic biomolecules with an absence of lag phase. the incapacity of adopting a transient helical conformation accentuates cell toxicity, supported by the caspase / activation level and the increase in intracellular calcium level. overall, this study indicates that the helical intermediate is offpathway to iapp amyloid formation and offers novel mechanistic insights for the development of molecular identities modulating peptide self-assembly and iapp-induced cytotoxicity. for an organism to survive, its proteins must adopt complex conformations in a challenging environment where macromolecular crowding can derail even robust biological pathways. the situation is perilous: many diseases arise from improper folding of just a single protein. to cope, cells employ a repertoire of molecular chaperones and remodeling factors that usher unfolded proteins into active conformations, sequester them, or target them for degradation. yet, not all aggregated proteins are the result of mis-folding. yeast prions are self-templating protein-based mechanisms of inheritance that rely upon chaperones for their propagation. the best studied of these is the prion domain (nm) of sup , which forms an amyloid that can adopt several distinct conformations (strains) that produce distinct phenotypes. using genetic, biochemical, spectroscopic, and solid state nmr techniques, we investigated the structural and dynamic underpinnings of sup amyloids and found that prion strains differ in both their atomic structure as well as their dynamic motions. interestingly, these mobility differences correlate with differences in the interaction with molecular chaperones in vivo. limitations on the specificity and sensitivity of biophysical techniques typically restrict structural investigations to purified systems at concentrations that are orders of magnitude above endogenous levels. therefore, i developed an approach to apply a sensitivity-enhancement technique for nmr, dynamic nuclear polarization (dnp), to investigate interactions between sup and molecular chaperones at endogenous concentrations in their native environments. critically, i found that the cellular environment induced structural changes in a region of sup that is intrinsically disordered in purified samples but known genetically to influence prion propagation from one generation to the next. this approach enables structural and mechanistic investigation of proteins in biologically relevant contexts. genetic instability within regions encoding repetitive proteins as a driver of adaptation stephen fuchs more than ten percent of all eukaryotic proteins contain within them a region of repetitive amino acid sequence. these repetitive domains range from short stretches of a single amino acid to multiple copies of longer, heterogeneous amino acid sequences and generally show lack of defined structure. they play diverse roles in cells including acting as structural proteins, promoting cell-cell interactions, and mediating the assembly of molecular machines. tandem repeat proteins are known to be variable in length within cellular populations although the mechanisms dictating this variability have not been elucidated. here we describe work uncovering specific features within the coding sequences of repetitive proteins that contribute to tandem repeat instability in yeast. furthermore, we demonstrate that cells will expand and/or contract repetitive regions in order to adapt to environmental stresses and describe a role for dna repair proteins in this process. lastly, we demonstrate how these mechanisms are likely conserved in higher eukaryotes, including humans. this study uncovers the molecular basis for an important aspect of natural protein evolution and describes a novel mechanism for adaptation in response to environmental changes. a proline-tryptophan turn in the intrinsically disordered domain of ns a protein is essential for hepatitis hepatitis c virus (hcv) nonstructural protein a (ns a) and its interaction with the human chaperone cyclophilin a (cypa), a peptidyl-prolyl cis-trans isomerase (ppiase), are both targets for highly potent and promising antiviral drugs that are in late stage of clinical development [ , ] . despite its high interest in the development of drugs to counteract the worldwide hcv burden, ns a is still an enigmatic multifunctional protein poorly characterized at the molecular level. ns a is required for hcv rna replication and is involved in viral particles formation and regulation of host pathways. thus far, no enzymatic activity or precise molecular function has been ascribed to ns a that is composed of a highly structured domain (-d ), as well as two intrinsically disordered domains (-d ) and (-d ). ns a-d structure has been solved by x-ray crystallography and ns a-d and -d have been characterized by nmr spectroscopy. these two last domains do not adopt a stable d structure but rather exist as an ensemble of highly dynamic conformers. using nmr spectroscopy, hcv ns a-d has been shown to establish a direct interaction with the human cypa and to be a substrate for the enzymatic ppiase activity of cypa [ ] . the cypa interaction site in ns a-d is composed of nearly residues that correspond to the most conserved region of the domain, with proline residues being strictly conserved among all hcv genotypes. whereas ns a-d is mainly disordered, some of its nmr resonances, corresponding to residues in the cypa binding site, display unexpected h and n nmr chemical shifts for an intrinsically disordered domain. thus we have further characterized this region by nmr spectroscopy. a short structural motif in the disordered ns a-d has been identified and we solved its nmr structure. in a cellular assay, we showed that this structural motif, a minimal pro -trp turn, is essential for hcv rna replication. we demonstrated that this pro-trp (pw) turn is required for proper interaction with the host cypa and influenced its enzymatic ppiase activity on residue p of ns a-d . this work provides a molecular basis for further understanding of the function of the intrinsically disordered domain of hcv ns a protein. in addition, our work highlights how very small structural motifs present in intrinsically disordered proteins can exert a specific function. [ ] [ ] . this -residue peptide also shows toxicity towards mammalian cells but at higher concentrations, suggesting its possible usefulness as a treatment for trypanosomiasis. here we present the peptide's relative cytotoxicity for bloodstream and procyclic forms of t. brucei and for mammalian cells, the fate of the peptide in t. brucei using fluorescently-labelled bt- , and its three dimensional structure using nmr spectroscopy.minimum inhibitory assays confirmed the peptide's selective toxicity towards both bloodstream and procyclic forms of t. brucei, demonstrating its potential to serve as a starting point for a trypanocidal drug. fluorescence spectrophotometric experiments, carried out using fluorescein labelled bt- , show that the peptide is released from the external surface of the parasite into the suspending medium under de-energized conditions but retained in energized cells. heteronuclear and homonuclear biomolecular nmr experiments (tocsy, noesy, h- c-hsqc, h- n-hsqc, etc) folowed by structural calculations (chemical-shift based as well as simulated annealing techniques) in the free state indicate that this peptide is mostly unstructured in aqueous solution, suggesting that there is a major conformational change upon binding to t. brucei that is required for uptake. we suggest that the evolutionary pressure that selected for the intrinsically disordered structure of this peptide was the advantage it conferred upon the host to bind to many different surface structures throughout the microbiological world. physikalische biologie, heinrich heine university, structural biochemistry (ics- ), research centre j€ ulich, chemistry and biotechnology, swedish university of agricultural sciences (slu) the misfolding and amyloid formation of proteins featuring intrinsically disordered regions is a pathological hallmark of several neurodegenerative diseases, including alzheimer's disease and parkinson's disease. engineered binding proteins targeting amyloidogenic proteins aid in the elucidation of the aggregation mechanism and suggest therapeutic strategies. we have constructed phage display libraries enriched in binders to amyloidogenic intrinsically disordered proteins, using zab , a protein with high affinity for the amyloid-beta peptide, as a scaffold. binding proteins selected from these libraries are termed beta-wrapins (beta-wrap proteins). the beta-wrapins as and hi exhibit nanomolar affinity for monomeric alpha-synuclein or islet amyloid polypeptide, respectively. as and hi potently inhibit in vitro amyloid formation and toxicity at substoichiometric concentration ratios, indicating that they interfere with the nucleation and/or elongation of amyloid fibrils. the nmr structures of the betawrapin:target complexes reveal beta-hairpin motifs in alpha-synuclein and islet amyloid polypeptide which are stabilized by coupled folding and binding. in the case of alpha-synuclein, the beta-hairpin is formed in the sequence region - which contains the beta-strand segments b and b of amyloid fibril models and most disease-related mutations. we show by disulfide engineering, biophysical techniques, and cell viability assays that intramolecular tertiary interactions between the b and b segments of alpha-synuclein interfere with its aggregation, and moreover inhibit aggregation of amyloid-beta peptide and islet amyloid polypeptide. our results reveal a common preference of different amyloidogenic proteins for formation of beta-hairpin motifs and demonstrate a critical role of hairpin conformers in the control of amyloid formation. interaction profiling through proteomic peptide phage display cecilia blikstad , moon-hyeong seo , norman davey , roland arnold , sachdev s sidhu , philip m kim , ylva ivarsson department of chemistry -bmc, donnelly centre a considerable part of the human proteome is intrinsically disordered. the disordered regions are enriched in short motifs serving as docking sites for peptide binding domains. domain-motif interactions are crucial for the wiring of signaling pathways. these interactions are typically transient and difficult to capture through most conventional high-throughput methods. we therefore developed a novel approach for the large-scale profiling of domain-motifs interactions called proteomic peptide phage display (prop-pd) ( ). in prop-pd we combine bioinformatics, oligonucleotide arrays, peptide phage display and next-generation sequencing. this allows the interrogation of domain-motif interactions on a proteome-wide scale and the de novo motif discovery.in our pilot experiment we generated two distinct phage libraries, one displaying all human c-terminal sequences and one displaying c-termini of known virus proteins. we used the prop-pd libraries to identify interactions of human postsynaptic density /discs large/zonula occludens- (pdz) domains. we successfully identified novel pdz domain interactions of potential relevance to cellular signaling pathways and validated a subset of interactions with a high success rate. recently, we created a prop-pd library that displays peptides representing the disordered regions of the human proteome. we validate our disorderome library against a range of peptide binding domains, which provides novel insights into their binding preferences and suggest interactions of potential biological relevance as will be presented here. prop-pd can be used to uncover protein-protein interactions of potential biological relevance in high-throughput experiments and provides information that is complementary to other methods. prop-pd is scalable and can be developed to any target proteome of interest. phosducin is a kda phosphoprotein that regulates visual signal transduction by interacting with the gtbg; subunit of the retinal g-protein transducin. the function of pdc is regulated by phosphorylation at ser and ser in a process that involves the binding of phosphorylated pdc to the regulatory - - protein, but the molecular mechanism of the regulation by - - protein is still unknown. pdc was also suggested to be involved in transcriptional control, the regulation of transmission at the photoreceptorto-on-bipolar cell synapse, and the regulation of the sympathetic activity and blood pressure [ ] [ ] [ ] . here, the solution structure of pdc and its interaction with the - - protein were investigated using small angle x-ray scattering, circular dichroism, quenching of tryptophan fluorescence, analytical ultracentrifugation, hydrogen-deuterium exchange coupled to mass spectrometry and nuclear magnetic resonance. we show that the - - protein interacts with and sterically occludes both the n-and c-terminal gtbg binding interfaces of phosphorylated pdc, thus providing a mechanistic explanation for the - - depedent inhibition of pdc function. the - - protein dimer interacts with pdc using surfaces both inside and outside its central channel. the n-terminal domain of pdc, where both phosphorylation sites and the - - binding motifs are located, is intrinsically disordered protein which remains likely highly flexible when bound to - - indicating the fuzzy-like character of this complex. in addition, it has been speculated that the - - protein binding decreases the rate of pdc dephosphorylation after a light stimulus through its interaction with phosphorylated ser and ser , thus lengthening the time that pdc remains phosphorylated after a light exposure. pdc is dephosphorylated in vivo by protein phosphatases known to cause neurodegenerative disease in a polyglutamine-length dependent manner. despite intense study, the molecular basis of polyq toxicity in hd or any of the other diseases has only partially been elucidated and potential routes to therapeutic intervention are sparse. the use of genetically tractable model organisms to identify the cellular pathologies caused by mutant huntingtin expression is essential to our understanding of the disease pathology in humans. in eukaryotes, many of the protein folding homeostasis pathways are highly conserved and yeast cells expressing a glutamine-expanded fragment of huntingtin exon exhibit a polyq length-dependent toxicity that recapitulates many of the basic protein folding defects associated with polyq diseases in neurons. taking an unbiased approach, we screened an overexpression library of the entire yeast genome for suppressors and enhancers of polyq toxicity and identified seven proteins with prion-like, q-rich domains that are strong suppressors in yeast. intriguingly, the q-rich domains of these proteins, and several other q-rich domains, suppress toxicity when expressed in isolation. these suppressors are also efficacious in mammalian cells and, strikingly, one suppressor was independently shown to alleviate polyq-expanded ataxin- toxicity in a drosophila model. in yeast, the suppressors co-aggregated with an otherwise highly toxic glutamine expanded huntingtin exon protein (htt q), resulting in a non-toxic aggregate and eliminating populations of diffusible oligomeric species. using a transcriptional sensor for protein coaggregation, we determined that yeast and human proteins that normally co-aggregated with htt q did not co-aggregate with these hetero-aggregates. thus, these q-rich domains may suppress htt q toxicity by two complementary mechanisms: trapping potentially toxic oligomers in larger aggregates and by limiting the interactome of the larger htt q aggregates. structuring disorder: the case of the intrinsically disordered unique domain of c-src mariano maffei about two thirds of eukaryotic proteins contain large intrinsically disordered regions. they represent a change of paradigm from "structure-function" to "information-function" (uversky, ; babu et al., ). structured proteins are information rich, but the current challenge is to discover how information is stored in disordered protein. regulation of c-src activity, the first discovered oncoprotein, by its intrinsically disordered n-terminal region has been recently demonstrated (perez et al., ). functional studies have revealed that mutations in the ulbr cause strong phenotypes when introduced in fulllength c-src and expressed in xenopus laevis oocytes (perez et al., ) or in human sw colorectal cancer cells (unpublished). however, the connection with the classical regulatory mechanisms is still missing. c-src domain structure consists of four "src-homology" domains: sh , sh , sh and sh , arranged in this order from the n-terminus to the c-terminus, with the intrinsically disordered "unique" domain separating the sh and sh domains. classically, the sh and sh domains are involved in regulation and the sh domain is the membrane anchoring site. we will present our recent results showing that the unique domain is part of a long loop closed by the interaction of the sh and sh domains (maffei et al., ). the conformational freedom of this disordered region is further restricted through direct contacts between the rt-loop of the sh domain and, primarily, residues located within the recently discovered unique lipid binding region (ulbr). the interaction between the unique and sh domains is allosterically modulated by a poly-proline ligand binding to the canonical binding site of the sh domain (maffei et al., ) . these results demonstrate a direct connection between classical c-src regulation involving the sh domain and the new regulation mechanisms involving the intrinsically disordered regions and provide new evidence of the functional importance and the underlying mechanism behind regulation of signalling pathways by intrinsically disordered domains. in mammalian cells, the golgi reassembly and stacking proteins (grasp and grasp ) are involved in the stacking of golgi apparatus cisternae and in the formation of the golgi ribbon. since grasps have been identified in many organisms, other roles for grasps have already been pointed out, such as chaperoning and transport of other proteins, involvement in cell apoptosis, cell migration, unconventional secretion, and in mitosis. in saccharomyces cerevisiae, it is observed that only % of the golgi cisternae are in stacks and do not form ribbon structures. this build yeast contains a single grasp, called grh , that is analogue to grasp . the structural differences of the golgi apparatus and the functional repertoire of grasps suggest a structural dynamic of these proteins. here, we used a combination of biophysical/biochemical methods to investigate the behavior of grh . bioinformatics and circular dichroism (cd) analyses of grh indicated a high percentage of either flexible regions or extended loops. the partial unfolded grh structure in solution folded into more ordered structures under temperature increasing, dehydration onto a surface and nonaqueous solvents as reported also by cd. hydration of the dehydrated folded protein is a reversible process that is accompanied by unfolding. furthermore, grh showed slow migration in sds-page, high susceptibility to proteases and low cooperativity of the chemical-induced unfolding process. fluorescence of trp residues along with cd data showed grh preserves a considerable amount of residual secondary structure, and the unfolding transition monitored by trp presented higher cooperativity. another cooperative transition was also reported by the extrinsic hydrophobic fluorescence probe ans upon chemical denaturation. these set of experiments indicate that grh behaves as a protein containing intrinsically disordered regions (idrs), characterized by unstructured regions of high polypeptide mobility experiencing many conformations. these findings suggest that an idp-like behavior may be the solution found by nature to account for grh functional need for interactions with several different partners in the cell. conformational changes governing dengue virus capsid protein function and its inhibition by pep andr e f. abstract dengue virus (denv) infection affects millions of people and is becoming a major global disease for which there is no specific treatment available. the interaction of denv capsid (c) protein with host lipid droplets (lds) is essential for viral replication. pep - , a peptide designed based on a denv c intrinsically disordered conserved region, inhibits this crucial interaction. combining bioinformatics and biophysics we determined pep - structure and ability to bind different phospholipids, in the context of denv c function. pep - becomes a-helical upon binding to anionic phospholipids. structure prediction of denv c n-terminal intrinsically disordered region reveals orientations that alternatively shield or expose denv c hydrophobic pocket, supporting a novel autoinhibitory role for this region. these findings pave the way for similar studies to understand disordered proteins and improved peptidomimetics drug development strategies against flaviviruses. topics intrinsically disordered proteins protein-lipid interactions pf- developing mechanistic insight into modulators of tau aggregation eri nakatani-webster , hannah baughman , shaylin higgins , abhinav nath the pathological self-association of microtubule-associated protein tau is implicated in a range of neurodegenerative disorders collectively called tauopathies, perhaps the most prominent of which are alzheimer's disease (ad) and chronic traumatic encephalopathy (cte). tau aggregation in vitro shares many features in common with fibril formation by other amyloid-forming proteins: a nucleationdependent polymerization reaction progressing via oligomeric intermediates into b-sheet-rich fibrillar aggregates, characterized by a distinctive sigmoidal kinetic. over the years, many investigators have advanced our understanding of how these time-courses might best be characterized and interpreted. in particular, elegant analytical and numerical approaches have been developed that supersede the empirical sigmoidal equations typically used to fit fibril formation traces. these modern approaches have enabled more rigorous insight into the mechanism of amyloid formation, and into how small molecules, protein chaperones, and other binding partners can modulate the process. an understanding of a modulator's effects on amyloid formation mechanism is necessary in order for us to predict and engineer its effects on amyloid pathology in a biological context. a given modulator may affect rates of primary or secondary nucleation, elongation, or fibril fragmentation to different extents. each of these perturbations, individually or in combination, can alter the kinetics of aggregation, the final state of the amyloid fibrils, and the sampled ensemble of oligomeric intermediates. unfortunately, fitting of mechanistic models to amyloid formation kinetics is an example of an "ill-posed problem", in that dramatically different combinations of elementary parameters can nevertheless generate very similar sigmoidal kinetic traces. this has typically necessitated global analysis of amyloid kinetic traces collected over a broad range of protein concentrations -a substantial expenditure of time, effort and material that must then be repeated in the presence of a modulator in order to gain insight into its effects. we propose an alternative approach: to fit amyloid formation traces to a large distribution of parameter sets, and determine how various aggregation modulators affect the distribution of parameters. this socalled "parameter distribution analysis" enables the inference of mechanistic effects from measurements at a single protein concentration. parameter distribution analysis based on numerical modeling has been made tractable by advances in computer hardware and software, and can be easily extended to include additional mechanisms or phases relevant to a protein or modulator of interest. here, we illustrate how parameter distribution analysis, complemented by fluorescence correlation spectroscopy (fcs), electron microscopy (em) and other biochemical techniques, can shed light on fundamental aspects of tau amyloidogenesis. we examine the disparate effects that natural products, pharmacotherapies and protein chaperones can have on the mechanism of aggregation, and also discuss the effects of heparin (widely used as an inducer of tau aggregation). these insights demonstrate the value of parameter distribution analysis as applied to amyloid formation and other ill-posed biochemical problems. new insights into amyloidogenesis of tau protein induced by enantiomers of polyglutamic acid amyloidogenesis of tau protein leads to the formation of amyloid fibrils (ordered fibrillar protein aggregates) which are accumulated in neurons of central nervous system during the course of neurodegenerative diseases called tauopathies. studying tau (a typical intrinsically disordered protein) amyloidogenesis has been challenging for many reasons. positive charge on the tau molecule must be compensated (e.g. in the presence of polyanions) in order to initiate the process. heparin (glycosaminoglycan) has been the most intensively studied charge-compensating agent in this context. on the other hand induction of tau aggregation by polyglutamic acid is poorly characterized. mechanisms responsible for the propagation of tau conformations has become an interesting research objective. prion-like features of tau amyloid can be studied in vitro also in the seed-induced regime of aggregation. tau amyloid seeds can act as nuclei for amyloidogenesis. such seeds can be obtained by fragmentation of amyloid fibrils by means of sonication. given that amyloidogenesis can proceed through various assembly pathways resulting in distinct amyloid 'strains' (self-propagating structural variants of amyloid) we have used poly-l-glutamic acid (plga) and poly-d-glutamic acid (pdga) to direct tau onto different amyloidogenic pathways. we have hypothesized that the chirality of the inducers could lead to fibril polymorphism. in our studies, we have used a recombinant human n r tau isoform. we have been using transmission electron microscopy (tem), sedimentation and kinetic measurment. firstly, we have characterized unseeded plga-/pdga-induced tau aggregation to find out that corresponding kinetics were significantly different. secondly, we have used sonicated fibrils to characterize the kinetics of seeded processes. both plga-/pdga-induced amyloid seeds were able to efficiently seed tau aggregation in the presence of plga, whereas in the presence of pdga the aggregation was much less effective. surprisingly, we found that pdgainduced amyloid seeds were able to catalyze fibrillogenesis of tau more clearly in the presence of soluble plga than in the presence of pdga -the primary inducer. we could not induce aggregation of tau in the absence of polyglutamic acids which indicates that positive charge on tau molecules must be unconditionally compensated in order to promote amyloidogenesis. thirdly, using tem we have characterized different morphologies of tau amyloid fibrils generated in unseeded and seeded processes. finally, to further characterize properties of the fibrils we have performed sedimentation experiments. fibrils induced by plga, pdga and heparin revealed different sedimentation properties. heparin-induced fibrils underwent sedimentation more readily than pdga-induced fibrils, whereas plga-induced fibrils remained in the supernatant. these results indicate distinct physicochemical properties of these fibrils. we believe that our findings will contribute to the current understanding of the molecular dynamics of tau amyloidogenesis. self-organizing structures of alpha-synulceins and its aggregates by a coarse-grained monte carlo simulation ras pandey , peter mirau , barry farmer alpha-synuclein (asn) consisting of residues, an intrinsically disordered protein, is linked to such neurodegenerative diseases as parkinson's disease (pd) and alzheimer disease via toxic clumping into abstract amyloid fibrils. we investigate the structure and dynamics of an asn chain as a function of temperature by a coarse-grained approach where a residue is represented by a node. in our coarse-grained approach, a residue is represented by a node. the basic idea is borrowed from the 'united atom' approach in polymer chain modeling that has been used extensively where the benefits and pitfalls of the method is explored for decades. such coarse-grained method has also been used protein chain modeling in recent years (e.g. aip advances , ( )). although the atomic scale structural resolution is sacrificed its specificity is captured via a set of unique knowledge-based residue-residue interactions matrix (e.g. classic miyazawa-jernigan matrix, macromolecules , ( )). a number of local and global physical quantities are analyzed such as contact map, neighborhood and mobility profiles, mean square displacement of protein, its radius of gyration and the structure factor. based on the mobility profile, we are able to identify three distinct segment of asn along its contour, i.e. sluggish nterminal ( - ) and c-terminal ( - , least mobile) separated by the central region ( - ), the nonamyloid component (nac) with higher mobility. contact profile shows that the probability of intrachain residue aggregation (clumping) is higher in the n-terminal region than the c-terminal with least aggregation in the nac region. we find that the radius of gyration (rg) decays monotonically with the temperature, consistent with the finding of allison et al. (jacs, , ( ) ). from the detail analysis of the structure factor we are able to predict the variation of the spatial mass distribution with the temperature as the residues in asn chain organize and disperse by evaluating its effective dimension d. we find the protein conforms to a globular structure (d ) at the low temperatures and to a random coil (d ) at high temperatures which is consistent with the estimates of uversky et al. (j. biol. chem. , ( )). in addition, we provide the estimates of d ( d ) for the intermediate structures as the protein chain makes a transition from globular to random coil. questions under-investigation includes what are the effects of mutations (e.g. b-and g-synuclein), how does the structure of an isolated asn chain change in presence of many interacting protein chains, and how do they organize over the multiple length scales? attempts will be made to address some of these issues as the data become available. tear down the wall: dismantling the biofilm scaffold of e.coli cesyen cedeno , nani van gerven , wim jonckheere , imke van den broek , han remaut , peter tompa csga is the major subunit of the so-called curli fiber system. this is an amyloid structure formed in the outer membrane on e.coli and acts as a scaffold for the biochemical machinery/matrix in the extracellular milieu (biofilms). extracellular matrices of this nature are robust platforms helping bacteria colonization; in this context csga becomes a key target in order to break the architecture within bacterial biofilms. chaperones are molecular machines able to stabilize misfolding prone proteins or even retrieve proteins trapped in non-physiological states. here we show how erd acts as a molecular chaperone inhibiting the formation of csga amyloid fibers in vitro. this work illustrates an alternative approach towards biofilm treatment at a molecular level. coupled folding and binding of transcription factors sarah shammas , alexandra travis , jane clarke intrinsic protein disorder is ubiquitous in transcription, particularly within transcription factors, which frequently fold into structures upon binding to partner molecules (dna or protein). the coupled folding and binding reactions that take place between individual transcription factors and the key hub co-activator proteins are crucial in determining the expression profile of the cell, and hence its phenotype. these interactions have been well studied by structural and equilibrium methods. here we present mechanistic insights into the process, gained through complementary kinetics experiments, for the binding of five separate transcription factors to a single prototypical co-activator (cbp kix). the transcription factors investigated belong to cellular (cmyb, mll, creb, e a) and viral (htlv- blz) classes. these reactions are remarkably fast; after removing the effect of long-range electrostatic rate enhancement the association rate constant is still approximately x m- s- , which is just above the typically quoted upper limit for diffusion-limited reactions between pairs of proteins ( - m- s- ), and is also the highest such value we have found reported. this, combined with the apparent insensitivity of the association rate to residual structure within the unbound state, indicates that binding preceeds folding (induced fit mechanism). interactions between kix and its transcription factors are additionally modulated by allostery between its two binding sites. we investigate the basis for this, finding it to be mediated by changes in protein flexibility. alternative hit finding strategies for intrinsically disordered proteins, exemplified by forkheadbox transcription factors harm jan (arjan) snijder , maria saline , tomas jacso , frank janssen , mattias rohman , tyrrell norris astrazeneca r&d, discovery sciences, se- ,pepparedsleden forkhead box o (foxo) proteins are emerging as key transcription factors in insulin and glucose metabolism, regulation of immune responses, and to balance cell proliferation, apoptosis and senescence. foxo proteins are predicted to be intrinsically disordered proteins (idps); idps are largely unstructured and often function as hubs mediating multiple interactions. idps are considered to be largely evasive from classical small molecule interference and lead-generation approaches, as they lack defined binding pockets. the available methods for addressing these targets have been lagging behind and needs to be developed to assess tractability of this target class. here we have evaluated the tractability of fragment screening on various domains of a forkhead box o member. we could confirm the intrinsically disordered character of foxo and used nmr screening to identify fragments that interact with foxo. one of these fragments was subsequently confirmed as a direct foxo binder in d hsqc-nmr spectroscopy and this fragment showed an effect in a foxo reporter gene assay. these results demonstrate that fragment screening may be a valuable approach for intrinsically disordered proteins although challenges remain to expand these fragments into more potent hits in the absence of detailed structural data. the characterization of amyloid-beta peptide (abeta) oligomer samples is critical to advance in the field of alzheime rs disease (ad). here we report a critical evaluation of two methods used for this purpose, namely sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page), extensively used in the field, and electrospray ionization ion mobility coupled to mass spectrometry (esi-im-ms), an emerging technique with great potential for oligomer characterization. to evaluate their performance, we first obtained pure cross-linked abeta and abeta oligomers of specific order. analysis of these samples by sds-page revealed that sds affects the oligomerization state of abeta oligomers, thus providing flawed information on their order and distribution. in contrast, esi-im-ms provided accurate information, while also reported on the chemical modifications and on the structure of the oligomers. our findings have important implications as they challenge scientific paradigms in the ad field built upon the sds-page characterization of abeta oligomer samples. coarse-grained simulation of protein association: application to rate prediction and implication for association mechanisms yinghao wu , the kinetics of protein binding is of paramount importance for understanding cellular functions. for instance, the binding kinetics between membrane receptors and their ligands control the speed of signal transduction after cells are exposed to stimulation. the experimentally measured association rates of protein binding span ten orders of magnitude, a range that was divided into two regimes. it was proposed that a fast association regime is limited by protein diffusion, while the other side of the spectrum is controlled by conformational changes. consequently, all previous simulation methods neglected conformational changes when calculating the association rate of a diffusion-limited regime. however, the most updated theory of protein binding suggests that a protein remains in a pre-existing equilibrium of unbound conformations. binding shifts the equilibrium toward its bound state. this highlights the importance of conformational factors for regulating protein binding. enlightened by this conformational selection model, we hypothesize that the conformational flexibility of protein structures regulates association more widely than previously anticipated. we develop a new coarse-grained model to simulate the process of protein association via the kinetic monte carlo (kmc) algorithm. each residue in this model is represented by its ca atom and a side-chain functional site. a simple physically based potential is used to guide the relative diffusion of two interacting proteins. given the size of the simulation box and the length of the simulation, the association rate constant can be derived by counting the frequency of dimerization among a large number of simulation trajectories. we further designed a prediction strategy that accounts for both the conformational and energetic factors of binding. our method is able to predict rates of protein association that are highly correlated with experimentally measured values. due to the coarse-grained feature, our model was further applied to several special cases of protein association. in one example, we studied the binding kinetics of proteins with flexible linkers. the interaction between thrombin and its functional inhibitor, rhodniin, was used as a testing system. we captured the conformational changes of flexible linkers from the all-atom molecular dynamic simulations. we found that the association with full-length flexible rhodniin was faster than its two individual domains and that their dissociation was more difficult, supporting a "flycasting" mechanism in which partial structures of an intrinsic disordered protein (idp) dock to the target first, while the remaining segments undergo conformational searches and sequentially coalesce around the target. in another example, we studied the binding kinetics of membrane receptors from cellular interfaces. the interaction between membrane proteins cd and cd , cell adhesion molecules known to mediate the activation of t cells and natural killer cells, was used as a testing system. the diffusive properties of these proteins on lipid bilayer were captured from all-atom molecular dynamic simulations. we showed that both d and d association rates could be simulated quantitatively with our method. the calculated values were close to the experimental measurements. we also provided detailed analysis of how molecular diffusions and membrane fluctuations affected d association. pf- (un)structure-function relationships on the ureg enzyme in the nickel-dependent urease system barbara zambelli , francesco musiani , stefano ciurli urease is an essential enzyme for many pathogens and soil microorganisms. its activity relies on the presence of nickel in the active site ( ) . the incorporation of this metal ion into the enzyme requires the formation of a supra-molecular chaperone involving four accessory proteins, named ured, uref, ureg and uree. uree is a metallo-chaperone involved in nickel binding and delivery into the enzyme active site. ureg is a gtpase essential for providing energy to the process of nickel site assembly. uref and ured form a complex that regulates the gtpase activity of ureg. the present work focuses on ureg, which exists in solution as an ensemble of inter-converting conformations ( ) . this observation made this protein the firstly discovered natural enzyme with an intrinsically disordered behavior, possibly allowing it to interact with different protein partners, such as uree ( , ) and uref ( ) and cofactors, such as metal ions ( ), in the urease activation network. ureg folding was studied perturbing protein conformation with temperature and denaturants, and investigating its folding response using circular dichroism, nmr and fluorescence ( ). a combination of light scattering, calorimetry, mass spectrometry, and nmr spectroscopy shed light on the effect of metal ion binding onto the conformational equilibrium of ureg ensemble ( ) . the results suggest that metal binding and solution conditions modulate affect the protein-protein interactions and enzymatic activity of ureg. nuclear inclusion protein a-protease (nia-pro) is a protease involved in processing of pepper vein banding virus (pvbv) encoded polyprotein to generate various intermediates and mature proteins at different stages of the viral life-cycle. nia-pro has two domains-n-terminal viral protein genome linked (vpg) and the c-terminal protease domain (pro).vpg belongs to the group of proteins that are intrinsically disordered, but attain stable structures upon interaction with other globular proteins. such proteinprotein interactions have a regulatory role on the function of the interacting partners. previously, the influence of vpg domain on the activity of pro was studied and it was shown that there was a substantial increase in the protease activity upon interaction with vpg (both in cis and in trans). in the present investigation, several deletion mutants of vpg and nia were constructed with a view to delineate the domain of vpg involved in interaction with pro. it was observed that deletion of residues from nterminus of vpg resulted in a decrease in the activity of pro in cis and in trans probably because of the abrogation of interaction between the two domains. interaction studies using spr (surface plasmon resonance) and elisa confirmed that the n-terminal residues of vpg are important for interaction with pro. the n-terminal residues of vpg are a part of the disordered region of vpg and their deletion resulted in the change in the secondary structure of the vpg and its oligomeric state. the ser and trp residues of pro domain were shown to be important both for the interaction of the two domains and for the activity of protease by mutational analysis earlier. these residues were identified to be a part of wc loop (w -c ) which relay the conformational changes to the active site catalytic triad (his , asp and cys ) leading to activation. however, mutations of these residues did not completely abolish the protease activity as well as the interaction with vpg. therefore, in the present study h and h which are observed to interact with trp and c (via non-covalent interactions) were mutated to alanine and the h a and h a mutants showed a drastic reduction in the activity of protease. molecular dynamics simulations of the wild type pro and the mutants revealed that trp -his -his -cys interaction pathway of the wild type pro was disrupted in the mutants and additional residues were involved in the interaction pathway, such alterations in the network of interactions could be responsible for the loss of activity. however, a change in the oligomeric status of these mutants was also observed as compared to the wild-type pro, suggesting that these residues are important for both the structural and functional integrity of pro and its interaction with vpg. thus, these results provide a molecular insight into the vpg-pro interactions and the modulation of their structure and function upon mutation of residues that are part of the interaction interface. transthyretin (ttr) is one of many proteins that are capable of forming amyloid fibrils in vivo. this protein is associated with two distinct amyloidosis: familial cardiac amyloidosis (fca) that causes a restrictive cardiomyopathy and familial amyloid polyneuropathy (fap) that affect peripheral nerves, they are hereditary and caused by mutations in the ttr gene. the non mutated protein can also aggregate in cardiac tissue in advanced age patients. the diagnosis was established at university hospital since due to a collaborative between our group and the center of amyloidosis antônio rodrigues de mello (ceparm). the only mutation found in brazil was v m in patients diagnosed in france. our group discovered new mutation not described in brazil and a novel mutation not described yet a d. the diagnosed patients are registered in transthyretin amyloidosis outcomes survey (thaos). the novel mutation a d causes a severe restrictive cardiomyopathy that is certainly related to a higher profile of aggregation observed for this mutant if compared to others amyloidogenic mutants of ttr. structural predictions using a bioinformatics tool called foldx showed that the insertion of the mutation cause a electrostatic clash that facilitates the dissociation and aggregation of protein. this mutant was purified heterologously and biophysical studies revealed that this protein is a dimer and not a tetramer as commonly the ttr structure. the crystallographic structure indicates that this mutant is structurally identical to wild type. biophysical studies revealed that this protein is a dimer and not a tetramer as commonly the ttr structure. the thermodynamic stability of a d is lower than the wild type ttr. the aggregation profile showed us that this protein can aggregate in a higher manner and with a fast kinetic to that observed for others amyloidogenic mutants of ttr, forming fibers in two hours of aggregation. heterotetramers of a d and wt are able to aggregate in the same fiber structure. the analysis of interface interaction of this mutant using the pdbsum showed modifications in the profile of hydrogen bonds and non bonded contacts. in addition the oligomers of a d are toxic for primary culture of cardiomyocytes from murine heart. the amyloidogenic profile displayed by this new mutant can be directly correlated with the aggressiveness observed in the disease developed by the identified patient. furthermore the recent consolidation of ttr diagnosis in our university hospital led to the identification of the rare a d variant in a brazilian patient, suggesting that other new, uncharacterized mutants could be identified in the coming years. multiple cellular proteins interact with ledgf/p through a conserved unstructured consensus motif [ ] . the ledgf/p -mll -menin complex was structurally characterized, but only partially [ ] . using nmr spectroscopy, we identified and mapped a novel mll -ledgf/ p interface. colony forming assays in mll -af leukemic cells expressing mll interactiondefective ledgf/p mutants revealed that this additional interface is essential for leukemic transformation. interestingly, the newly defined interface overlaps with the binding site of known ledgf/p interactor, the hiv integrase [ ] . while the pathophysiological interactions of ledgf/p are intensively studied, its physiological role remains unclear. since ledgf/p contributes to hiv integration and leukemic transformation and has become a new therapeutic target for drug development, it is crucial to study its physiological interactions. in addition to hiv in and mll -menin, the ledgf/p integrase binding domain (ibd) also interacts with several other proteins [ , ] . our recent data (manuscript accepted in nat. commun.) revealed structural details of ledgf/p interactions with physiological binding partners. the interaction with the ledgf/p ibd is maintained by an intrinsically disordered ibd-binding motif (ibm) common to all known cellular partners. based on the knowledge of this motif, we identified and validated iws as a novel ledgf/p interaction partner. naturally occurring single mutants, i t, f i, w r and d h of lysozyme in human, have been known to form abnormal protein aggregates (amyloid fibrils) and to accumulate in several organs, including liver, spleen and kidney, resulting in familial systemic amyloidosis. these human pathogenic lysozyme variants are considered to raise subtle conformational changes compared to the wild type. here we examined the effects of the aberrant mutant lysozymes i t, f i,w r and d h, each of which possesses a point mutation in its molecule, on a cultured human cell line, hek , in which the genes were individually integrated and overexpressed. western blot analyses showed lesser amounts of these variant proteins in the medium compared to the wild type, but they were abundant in the cell pellets, indicating that the modified lysozyme proteins were scarcely secreted into the medium but were retained in the cells. immunocytochemistry revealed that these proteins resided in restricted regions which were stained by an endoplasmic reticulum (er) marker. moreover, the overexpression of the mutant lysozymes were accompanied by marked increases in xbp s and grp /bip, which are downstream agents of the ire _ signaling pathway responding to the unfolded protein response (upr) upon er stress.rnai for the mutant lysozymes' expression greatly suppressed the increases of these agents. next, we addressed the interaction between amyloidogenic lysozyme and grp /bip as the former proteins were obtained by immunoprecipitation with the latter protein as well as colocalization of both proteins in the er. lysozyme composes of a-domain rich in helices and b-domain rich in sheet. two helices of a and a in the n-terminal region arrange in parallel and face to face where hydrophobic amino acids at the f, l , l , l , l and l allocate with equal interval there. in the back of dock, there is a core region of amyloid fibril formation, of which the side chain of i is exposed on the protruding. probably, these hydrophobic amino acids might be crucial for lysozyme folding. although mutated lysozymes undergo folding by grp /bip in such environment, the dissociation of the grp from lysozyme by failure of folding is likely inhibited and both proteins remain bound to, resulting in staying to the er. a part of aberrant lysozymes seem to remain bound to grp /bip during folding and insolubilize with aggregation, thus accumulate in the er accompanied with er stress. lysozyme amyloidosis might be caused by long-term accumulation in the endoplasmic reticulum of the abnormal protein. structural characterization of toxic oligomers that are kinetically trapped during alpha-synuclein fibril formation the accumulation of abnormally aggregated proteins within the body is a common feature of several medical disorders, such as alzheimer's disease, parkinson's disease and diabetes mellitus type . while the specific protein found to be the major component of such deposits varies from one disease to another, the formation of the pathological aggregates seems to occur via a common process of misfolding and self-assembly of a normally soluble polypeptide chain into a series of oligomeric intermediates and, ultimately, into insoluble amyloid fibrils that accumulate within specific organs and tissues. increasing evidence indicates that certain oligomeric protein species generated during the self-assembly of specific proteins into ordered fibrillar aggregates can be highly cytotoxic and are likely to be key players in the initiation and spreading of neurodegenerative diseases. however, little detailed structural information is currently available for these oligomeric species due to their often transient nature and, more importantly, because of their variability in terms of size and structure. we report here the isolation and detailed characterization of an ensemble of stable toxic oligomers of alpha-synuclein, the protein whose deposition is the hallmark of parkinson's disease. by defining and minimizing the degree of heterogeneity of these isolated alpha-synuclein oligomers which have accumulated during the process of amyloid formation, we have identified distinct subgroups of oligomers and determined their structural properties and three-dimensional molecular architectures. this characterization has been achieved by the application of a set of complementary biophysical techniques, including a variety of spectroscopic techniques along with analytical ultracentrifugation, atomic force microscopy, and electron microscopy. although these oligomers exist in a range of sizes, with different extents and nature of beta-sheet content and exposed hydrophobicity, all the oligomeric subgroups possess hollow cylindrical architectures with marked similarities to amyloid fibrils. this suggests that these types of oligomers are kinetically trapped during protein self-assembly and that the accumulation of at least some forms of amyloid oligomers is likely to be a consequence of very slow rates of rearrangement of their beta-sheet structures. our findings reveal the inherent multiplicity of pathways of protein misfolding and the key role the beta-sheet geometry acquired in the early stages of the self-assembly process plays in dictating the rates of structural conversions, and thus the kinetic stabilities and pathological nature of different amyloid oligomers. the results of this study provide the basis for a more complete understanding of the nature of the self-assembly of polypeptides into beta-sheet rich amyloid aggregates, and potentially contributes to efforts to identify specific targets for drug discovery. fish otoliths and mammalian otoconia, biominerals composed of calcium carbonate and organic matrix, are involved in the functioning of the inner ear, the sensory organ that plays an important role in hearing and balance [ ] . however, their developmental origins, growth, and the role of the matrix, especially the protein component, are still poorly understood. it has been shown that proteins involved in the formation of biominerals are usually very acidic. they often belong to the group of intrinsically disordered proteins (idps), a class of proteins devoid of a rigid tertiary structure [ , ] . the shape and polymorph selection of calcium carbonate otolith in danio rerio is controlled by the starmaker (stm) protein [ ] . recently, a gene was identified encoding the starmaker-like (stm-l) protein from oryzias latipes, a putative homologue of stm. it has been suggested that stm-l has a similar function as stm, although there is no sequence similarity between stm and stm-l [ ] . several methods, such as size exclusion chromatography, cd spectroscopy and analytical ultracentrifugation demonstrated that stm-l is an coil-like idp, with the tendency to form locally ordered structures [ ] . because stm-l was suggested to play a crucial role in calcium carbonate mineralization, it is possible that calcium ions may influence its conformation, as was previously shown for stm [ ] . however, other ions may also be involved in this process. the aim of this study was to investigate the effect of mono and divalent metal ions on the conformation of stm-l. we used single molecule f€ orster resonance energy transfer (smfret) and fluorescence correlation spectroscopy (fcs), which have shown that calcium ions compacts the proteins most efficiently, followed by magnesium and the monovalent ions. the difference in the effect of monovalent and divalent ions on the protein dimensions is likely to result from the different properties of the ions, like charge density and radius. cd experiments have shown that a high excess of calcium ions caused the formation of ordered secondary structure in stm-l, which may be crucial for the formation of calcium carbonate crystals, when the ratio of building ions to protein is high. it has been demonstrated that dmp is proteolytically processed into fragments, including k n-terminal region and k c-terminal region. as many proteins characterized to be engaged in biomineralization, dmp and its fragments belong to the group of intrinsically disordered proteins (idps). it has been suggested that dmp and its fragments can take a part in otoconia mineralization, as the protein is present in mouse otoconia, but the role of dmp and its fragments in the mineralization of calcium carbonate has not been examined until now. to determine the influence of the dmp fragments for otoconia development, k dmp protein was expressed in bacterial expression system, purified and used in in vitro biomineralization test of calcium carbonate. in particular, immobilized metal anion affinity chromatography (imac) was applied as a first step of purification procedure. because of high content of acidic amino acids, ion exchange chromatography with a mono q column was used as a next step. the development of insects is regulated by the combined action of ecdysteroids and juvenile hormones (jh). pulses of -hydroxyecdysone ( e) initiate each step of metamorphosis, while jh modulates its action and prevents precocious differentiation. the biological and molecular mechanism of e action is well described. in contrary, the way of the jh activity is still poorly understood. in wilson and fabian [ ] reported that drosophila melanogaster mutants lacking met are resistant to toxic doses of jh and its analogue methoprene. it has been proved, that met binds jh at physiological conditions. therefore met is believed to be a putative jh receptor. met may also be involved in a cross-talk between two hormonal signalling pathways, involving e and jh. the detailed structure of met is still unknown. therefore our main aim is to characterize structural properties of met. in silico analysis performed on a full-length met suggested, that n-terminal part of met contains three conserved domains characteristic for bhlh-pas transcription factors, whereas c-terminal part is most probably unstructured. )). capitalizing on self-and cross-amyloid interactions, we designed highly effective, peptide-based inhibitors of amyloid self-assembly of abeta and iapp. due to their favourable properties the designed peptides are promising leads for targeting protein aggregation in ad, t d or both diseases while the inhibitor design strategy should be applicable to other amyloidogenic polypeptides and proteins as well. apoptosis, the process of programmed cell death, must be carefully regulated in multi-cellular organisms to ensure proper tissue homeostasis, embryonic development and immune system activity. the bcl- family of proteins regulates the activation of apoptosis through the mitochondria pathway. dynamic interactions between pro-and anti-apoptotic members of this family keep each other in check until the proper time to commit to apoptosis. the point of no return for this commitment is the permeabilization of the outer-mitochondrial membrane (omm). translocation of the pro apoptotic member, bax, from the cytosol to the mitochondria is the molecular signature of this event. molecular interactions and conformational changes associated with this event have been difficult to obtain due to challenges associated with taking subtle measurements in the complex environment of live cells. to circumvent these challenges, we developed a novel method to reliably detect f€ orster resonance energy transfer (fret) between pairs of fluorophores to identify intra-molecular conformational changes and inter-molecular contacts in bax as this translocation occurs in live cells. in the cytosol, our fret measurements indicated that the c-terminal helix is exposed instead of tucked away in the core of the protein. this coincided with measurements using fluorescence correlation spectroscopy (fcs) that showed that cytosolic bax diffuses much slower than expected, suggesting possible complex formation or transient membrane interaction. we propose that this exposed helix allows for this contact to occur. cross-linking the c-terminal helix (a ) to helix a reduced the instances of these interactions while at the same time yielded fret measurements that are consistent with the a helix tucked into the core of the protein. after translocation, our fret measurements showed that bax molecules form homo-oligomers in the mitochondria through two distinct interfaces involving the bh domain (helix a ) and the c-terminal helix. these findings provide insight into the molecular architecture that may involve possible contacts with other bcl- proteins to permeabilize the omm, which would also be necessary for the regulation of apoptosis. abstract spatial resolution is especially advantageous for bacterial cells because of their small sizes. in the past few years the spatial organization and dynamics of a variety of bacterial cellular structures and protein macromachineries have been revealed with unprecedented details. as the field matures, it is now time to focus on the functional aspect of the observed spatial organizations and dynamics. are they essential in carrying out a specific cellular function? do they play a regulatory role in controlling the on and off of a certain cellular process? in this work i will present a few examples from our laboratory that examine the spatial and functional organization of macromolecules involved in bacterial cell division. transcription factors (tf) exert their function by interacting with other proteins and binding to dna. the nucleus is a compartmentalized space, and the spatial organization of tfs and their partners represents other step of gene expression regulation. we used the glucocorticoid receptor (gr) as a model of tf's mechanism of action. gr is a ligand-activated tf with a relevant role in physiology and a great variety of effects. it can be recruited to specific response elements on dna or interact with other tfs. also, the activity of gr is modulated by different co-regulators, e.g. tif /grip . gr and tif do not distribute homogeneously within the nucleus but accumulate in distinctive clusters. the functional role of this particular intranuclear organization remains unknown. we used advanced fluorescence microscopy techniques to study the dynamics of gr and tif in the nucleus of living cells with high spatial and time resolution. gr and tif fused to fluorescent tags were transiently expressed in newborn hamster kidney (bhk) cells and visualized by a confocal microscope. fluorescence correlation spectroscopy (fcs) experiments were carried on to measure the intranuclear mobility of both proteins. the method is based on the analysis of fluorescence intensity fluctuations due to the movement of fluorescent molecules in and out the confocal volume. the data could be fitted with a model that considers a free diffusion of tif and gr in the nucleus and their binding to fixed targets. we also studied the dynamics of different gr mutants in the presence of different ligands and our results suggest that the binding depends on dna. both gr and tif autocorrelation curves reveal an increase in the bound population upon gr activation by its agonist dexamethasone (dex). a cross-correlation analysis showed that, as expected, dex-stimulus increases the population of gr-tif complexes. without hormone, gr shows a homogeneous distribution and tif forms large clusters in the nucleus. upon dex-binding, gr accumulates in the nucleus, is rapidly recruited to tif foci and there is an important re-distribution of both proteins, that co-localize in the same pattern of small intranuclear clusters. the dynamics of gr and tif molecules at these clusters were studied by performing orbital-scanning measurements, tracking the clusters position in silico and analyzing the intensity fluctuations of the clusters along time. a positive cross-correlation between both channels indicates that dex-bound gr and tif interact at these foci and dissociate from them forming tif -gr hetero-complexes. in conclusion, advanced fluorescence microscopy methods allowed obtaining a dynamical map of gr distribution and function in the nucleus of mammalian living cells. assembly of membrane pores as a mechanism for amyloid cytotoxicity by the bacterial prionoid repa-wh cristina fern andez , rafael n uñez-ramirez , mercedes jimenez , germ an rivas , rafael giraldo amyloid fibril formation is associated with human neurodegenerative diseases. prefibrillar oligomers formed during the fibril assembly process, rather than mature fibrils are known to be central to disease abstract and may be responsible for cell damage. a commonly proposed mechanism for the toxicity of small oligomers is their interaction with the lipid bilayer of cell membranes, leading to loss of membrane integrity [ ] . recent studies from our laboratory have shown that repa-wh , a winged-helix domain from a bacterial plasmid replication protein, can assemble into amyloid fibrils in vitro. when expressed in escherichia coli repa-wh functions as a cytotoxic protein that shares features with the mammalian amyloid proteinopathies. these features have proved repa-wh to be a suitable synthetic model system to study protein amyloidosis [ , , ] . in this work, using the repa-wh bacterial model system, we have studied the interaction between the protein and model membranes (large and giant unilamellar lipid vesicles, luvs, and guvs respectively). repa-wh shows association and aggregation to membranes composed of anionic phospholipids. protein association in guvs did not result in lysis of the vesicles, suggesting the assembly of discrete protein pores as the mechanism for repa-wh membrane damage. to investigate the formation of pores we analyzed by electron microscopy the aggregation of repa-wh in the presence of a pre-formed e. coli lipid monolayer. the em images show the presence of pore-like particles on the monolayer. amyloid pores formation explains the permeabilization effect of repa-wh in vesicle models and is in agreement with observations for human amyloidogenic proteins. the approaches presented here provide a deeper insight into amyloid cytotoxicity towards membranes and will make possible the assay of inhibitors and effectors of amyloidosis under controlled conditions. references: b -adrenergic receptor (b ar) is a member of g protein-coupled receptors, which represent the single largest family of cell surface receptors involved in signal transduction. b ar recognizes a variety of ligands and communicates with cytoplasmic g-proteins by transmitting signals through the cellular membrane. thus, investigation of communication pathways for b ar may give important insights for understanding its allosteric mechanisms and identifying new target sites for more specific and efficient drug molecules to be used in the treatment of pulmonary and cardiovascular disease. in this study, various conformations from ms molecular dynamics (md) simulations and available crystal structures of human b ar were investigated to reveal alternative signaling pathways between its extra and intracellular regions. specifically, shortest communication paths connecting key residues (more than Å apart) at the orthosteric ligand binding site (d , s , t , f , n ) to either l or s located near the g-protein binding site were investigated. the conformers from previous md simulations [ ] include the intracellular loop (icl ), which especially affects the transmembrane collective dynamics but is lacking in x-ray structures. the protein was described as a graph composed of nodes linked by edges. nodes were placed at the alpha-carbon atoms and the edges were calculated based on the number of atom-atom interactions within a cut-off distance . Å for each residue pair. twenty shortest pathways were revealed using k-shortest path algorithm [ ] on the coarse-grained network. our results indicated that distinct signaling paths progressed most frequently on tm but alternative paths were also present, which passed partially through tm , tm , tm or tm depending on the conformation. among the critical residues that transmitted the signal between distant sites, f and n were detected, whose functional roles were reported in previous experimental studies. pathway shifting was observed depending on the open-to-closed transition of icl during md simulations. the sulfonylurea receptor (sur ) is an atp binding cassette (abc) protein that forms the regulatory subunit in katp channels found in the pancreas and the brain. mgatp binding and hydrolysis at the two cytosolic nucleotide binding domains (nbd and nbd ) in sur control gating of the katp channel pore. , proper regulation of katp channel gating by sur is critical. over mutations that lead to diabetes, hyperinsulinism and developmental delay have been identified in different domains of sur , including the nbds. therefore, molecular-level understanding of the structure and function of the nbds is essential for designing improved treatments for sur-related diseases. here we present biophysical and biochemical studies aimed at understanding the effect of disease-causing mutations on the conformation and nucleotide binding of sur nbd . specifically, we are investigating sur nbd mutations that cause neonatal diabetes (r w and h t) or congenital hyperinsulinism (c d, g v, r g, r d and k t). our nuclear magnetic resonance (nmr) data shows that the hyperinsulinism mutation k t causes chemical shift changes throughout the spectrum of nbd , implying overall changes in protein conformation that may affect mgatp binding and inter-domain interactions with other domains in the sur protein. size-exclusion data show that the other hyperinsulinism mutations (c d, g v, r g, r d) produce mostly aggregated protein, likely as a result of misfolding of nbd . misfolding of nbd may be the underlying cause of reduced katp trafficking seen with these mutations and hence decreased katp channel gating observed in hyperinsulinism. in contrast to the k t mutations, the congenital diabetes-causing mutations (r w and h t) cause few nbd nmr spectral changes. however, the congenital diabetes mutation r w decreases the affinity of nbd for mgatp, which is unexpected for congenital diabetes mutations. our fluorescence, circular dichroism and microscale thermophoresis data corroborate the results that we have obtained by nmr spectroscopy. our data provide molecular-level details on the effects of disease causing mutations in human sur . egfr increased stability: rmsf of the ca atoms during the md simulations suggest that glycosylation is associated with dampened motions, suggesting that the glycans stabilize the structure. subdomain iii is the most stabilized while subdomain i is stabilized largely in the proximity of the ligand. both dimer interfaces including the dimerization arm from domain ii and the tip of domain iv fluctuate less upon glycosylation. hydrogen bonding; persistent interactions seen for protein-glycan: in the disaccharide-containing system, we observed three highly occupied hydrogen bonds between the glycans and domain iii and iv of egfr. hydrogen bonds of domain iii involve the residue asp in which a sidechain oxygen interacts with oxygen atoms of the n-acetylglucoseamine linked to asn . in domain iv a hydrogen bond is seen between the cys backbone amide and the oxygen atom of n-acetylglucosamine linked to asn . in the oligosaccharide-containing system hydrogen bonds observed between the glycan attached to asn and domain ii. these hydrogen bonds form between the gln sidechain oxygen atom and cys backbone oxygen atom and the mannose linked to asn . the reduction in the mobility of these amino acids suggests that hydrogen bonds impart stability to both the sugars and to the interacting egfr. insects possess a complement-like immune response utilizing thioester-containing proteins, or teps. the only arthropod tep of known structure is anopheles gambiae tep , which is a key component in the natural immunity of this mosquito to malaria parasites (genus plasmodium). unlike vertebrate complement factors, agtep does not contain an anaphylatoxin domain which acts to regulate a massive conformational change accompanying activation of the protein. the mechanism of agtep must therefore involve an alternative mechanism for allosteric regulation of thioester activation. in place of a small internal domain, a large, heterodimeric complex of two leucine-rich repeat (lrr) proteins, lrim and apl c, have been shown to specifically bind and stabilize the active conformation of agtep . i will present my group's most recent work in this area. we have shown that different alleles of tep , which are known to influence the vectoral capacity of wild mosquitoes, differ significantly in their susceptibility to thioester hydrolysis. allelic variation is centered on residues at the protein-protein interface within tep containing the thioester bond. the lrim /apl c heterodimer is shown to form an extended and flexible ensemble in solution. two closely-related genes to apl c, apl a and apl b, can also form a complex with lrim , and apl b lrr domain can form a homodimer. we propose that a flexible and heterogeneous group ensemble of lrim /apl dimers interact with the active conformation of tep , thereby producing an array of immune complexes to protect mosquitoes from a diverse set of pathogens. human flap endonuclease- (hfen ) is an essential metallo-nuclease involved in okazaki fragment maturation and long-patch base excision repair. during these processes, bifurcated nucleic acid intermediates with ssdna '-flaps are generated by polymerase strand displacement synthesis and then cleaved one nucleotide into the downstream duplex by fen to create a nicked-dna that is a suitable substrate for ligase. until recently, how hfen achieves tremendous catalytic power (rate enhancements > exp ) and exquisite selectivity for the scissile phosphate had been understood poorly ( ) . in , the grasby and tainer labs solved the structures of hfen in complex with product and substrate. this study revealed that scissile phosphate selectivity is largely due to the substrate dna undergoing a novel di-nucleotide unpairing (dnu), which places the scissile phosphate diester in contact with the requisite divalent metal ions. in addition, by comparing the structures of hfen alone ( ) and in complex with substrate and product dnas ( ), grasby and tainer proposed a model, whereby protein conformational changes occur upon binding substrate resulting in placement of key basic residues that position and/or electrophillically catalyse hydrolysis of the scissile phosphate diester. further work using a cd-based assay showed that metals are absolutely required for dnu, whereas the key basic residues in the active site are not. surprisingly, perturbations to the protein structure that are much more distant from the fen active site (i.e., helical cap) prevent dna unpairing, implying that the fen protein actively participates in the unpairing process ( , ); however, how it does remains a mystery. the maximal multiple turnover rate of hfen reaction is rate-limited by enzyme product release, whereas hfen kinetics under substrate-limiting conditions ([e]<[s]<km) suggest that the enzyme is approaching catalytic perfection (i.e., almost diffusion controlled - exp m- s- ) ( ) . previous work has shown that a paralogue of hfen (i.e., t fen ) is not ratelimited by chemistry under single turnover (st) conditions, but rather some physical limitation such as conformational change ( ) . to ascertain whether the protein and/or dna conformational changes of hfen mentioned above are rate limiting under st conditions, we have initiated kinetic and nmr studies to determine the role of conformational dynamics in the catalytic cycle of hfen . owing to the latest advance in the computational technologies of microprocessor, high-speed inter-processor connection, and parallelization algorithm, all-atom molecular dynamics (md) simulations of microsecond time scale are getting popular to study the pharmaceutical target proteins. an antibody binds to its antigen with structure changes. a small molecule moves around the target protein and finds an entrance to get into the binding site. these phenomena can be observed in microsecond simulations, but the vital issue is whether the adopted force field is enough accurate to correctly describe the phenomena. we are developing a force field (fuji) based on general amber atom types and amber van der waals parameters in order to describe arbitrary organic molecules in a unified manner including proteins and nucleic acids. we use the first principle theoretical method to refine the force field in contrast to common empirical fittings to experimental data. the dihedral torsion parameters of protein backbone were determined to agree with the torsion energy profiles calculated by high-level quantum mechanical theory for the model systems of protein backbone. conformational preferences of dipeptides in water were measured by vibrational spectroscopy. comparing with the experimental distribution of ramachandran angles of dipeptides, how accurately molecular calculations predict the conformation distribution of dipeptides were investigated for various force fields and semiempirical quantum methods. fuji force field gave the best prediction score among the various methods (tzanov et al, ) . in this work we examine dynamical structure behaviors of pharmaceutical target proteins by microsecond md simulations with fuji force field. epiregulin (epr) is a transmembrane protein with residues belonging to the epidermal growth factor family. mature epr ( residues) binds to epidermal growth factor receptor (egfr), which stimulates the proliferative signaling in cancer cells. our epr antibody has a proline at the residue in the third complementarity-determining region (cdr) of the heavy chain, which has cis peptide bond in free state and trans one in the bound state with epr according to x-ray crystallography analysis. in order to clarify the structure changes in binding we performed extensive md simulations of our antibody (fab; residues) and epr. the total simulation time was about microseconds. we also performed md simulations for a protein kinase and a small molecule. we show how the complex system reaches thermal equilibrium states in simulations from the stiff x-ray crystal structure. the calculations are compared with experiments such as x-ray crystal structures and thermodynamic quantities measured by isothermal titration calorimetry (itc) and surface plasmon resonance (spr). this research has been supported by mext spire supercomputational life science (hp , hp ) and first kodama project in japan. structure-based recombination of drug resistance enzymes: structural and functional tolerance to new dynamics in artificially-evolved enzymes our understanding of the contribution of protein dynamics to function is still emergent. in a protein engineering context, do we need to take into account the dynamics in order to maximize the fitness and function of the resulting proteins? using high resolution crystal structures, nmr relaxation dispersion and ms molecular dynamics simulations, we compare two naturally evolved homologous class a b-lactamases, tem- and pse- which share a high degree of structural and functional conservation. we observed a conservation of dynamics on a catalytically relevant timescale. this is consistent with dynamics being an evolutionarily conserved feature. however, laboratory-engineered chimeric enzymes obtained by recombination of the two homologs exhibit striking dynamic differences, despite the function and structure being conserved. the laboratory-engineered chimeras are thus functionally and structurally tolerant to modified dynamics on the timescale of the catalytic turnover. this tolerance of blactamases to dynamic changes could be linked to the high fitness of the naturally evolved proteins and implies that maintenance of native-like protein dynamics may not be essential when engineering functional proteins. conformations solution. an immense advantage of our recombinant system is the possibility to perturb it by introducing unnatural amino acids for subsequent labelling with fluorescent dyes or to omit subunits from the multisubunit rnap. comparing free, nucleic acid-and factor-bound rnap complexes we found that the position of the clamp is modulated during the transcription cycle in a fashion exceeding the simplified closed-open picture drawn from crystallographic studies. we show that the clamp adopts at least two distinct conformations in both initiation and elongation complexes. moreover, we were able to link conformational states to the catalytic activity of the rnap. transcription factors tfe and spt / , which both bind to the rnap clamp domain and stimulate the activity of the enzyme, shift the equilibria towards one of the main conformations suggesting that both prompt an allosteric switch that influences the structure of the rnap. hence, our data provide a mechanistic rationale for the function of these factors and provide new insights into the complex dynamic behaviour of the transcriptional machinery. solvent models for protein simulations -the good, the bad and the applications duy hua , amitava roy , he huang , carol post the solvent environment plays a crucial role in determining the structure, dynamics and function of a biomolecule. in order to utilize molecular dynamics (md) simulations to examine the structural changes abstract of biomolecules, it is imperative that the solvent environment be accurately modeled. implicit solvent models (isms), in which water molecules are absent and the solvent effect is estimated using an energy function, offer a low-cost approach to describe the solvent environment in md simulations. isms have been used for a variety of biological studies; yet, the performance of implicit solvation methods in capturing the structural characteristics of proteins has not been rigorously demonstrated. in this work, three isms, namely gbmv ii, facts, and scpism, were evaluated for their abilities to emulate the solvent environment and its effect on the structures, dynamics and electrostatic interac- thioredoxins (trx) are ubiquitous proteins that play a key role in redox state regulation of the cell. they interact with multiple targets in the cell. our group solved the structure of s. cerevisiae (ytrx ) and measured its dynamics in different timescales (pico to seconds). it is well established that the asp is the proton acceptor in the reduction process of the target protein. we proposed that asp also works to couple hydration and motion of the interacting loops. we studied conformational motions of the residues of water cavity and interacting loops. the water molecules in water cavity of the protein play a vital role in the redox activity of thioredoxin. the degree of mutational frustration explains in a great deal the multiple timescales observed in the protein dynamics in ytrx and the mutant d a. the ytrx displays a complex equilibrium between the ground state and several excited hydration states. this excited states are more permeable to water and it is key to understand the proton transfer and activation of cys . in this current study, we examined the mutant d a, and observed that this small hydrophobic residue induces conformational exchange in residues exposed to the water cavity, such as cys . along with molecular dynamics simulations and calculation of mutational frustration levels, we were able to describe conformational details of the water cavity. it is formed by three independent contiguous lobes which we called lobes a, b and c. lobe b is the central lobe that contains the catalytic important residues cys and asp . it closed upon mutation d a. lobe a and c remains open upon mutation, meaning that their hydration are independent. the nmr structures for the oxidized and reduced state of the mutant d a has been studied. in-vitro and in-silico studies of ligand binding to the nuclear receptor ppargamma using fret and md narutoshi kamiya , gert-jan bekker , takuma shiraki , haruki nakamura ipr, osaka university, kinki university the nuclear receptor ppargamma is a ligand-dependent transcription factor which regulates gene expression related to glucose homeostasis and insulin sensitization. it is a potential therapeutic target for metabolic syndrome, cancer, and inflammatory diseases. crystal structures have revealed that its ligands bind deep inside the pocket. however, how the ligand recognizes ppargamma and penetrates inside the pocket remains unclear. we have measured the binding kinetics using the fret method and have calculated potential dissociation paths via molecular dynamics (md) simulations. the binding kinetics were measured using fret between the ligand, c -bodipy, which is a fluorescent pigment structurally similar to one of its agonists -oxo-eicosatetraenoic acid, and cyan fluorescent protein (cfp) which is fused to ppargamma. fluorescence of c -bodipy is observed when it is close to cfp when cfp is excited by light at nm. the ligand was simultaneously added to the ppargamma-cfp solution using the stopped-flow method. time-resolved fluorescence signals of c -bodipy and cfp were also monitored. two time constants were observed by the fret experiment, which correspond with the landing and docking processes. we used our new md program, psygene-g [ ] , which utilizes the gpu for acceleration of the non-bonded interactions, such as electrostatic interaction which uses our original zero-dipole summation method [ , ] . previously we were able to attain similar thermodynamics with respect to the particle mesh ewald method in membrane proteins and dna-water-ion systems [ , ] and have applied it to the thermodynamic study of dynein [ ] . we have introduced random accelerated md (ramd) into the psygene-g program to predict a ligand's dissociation path from the initial crystal structure (pdb id: zk ). in ramd, an external random-directional constant force is added to the ligand. we executed -ramd simulations with different random seeds for the initial pull directions. twelve ramd trajectories managed to dissociate within ns, while the remaining simulations did not manage to disassociate within this time-frame. in all twelve cases the ligand went past r , which is located at the surface of ppargamma. interestingly, a somatic mutant (r h) is related to colon cancer. we concluded that r plays a role of a gate keeper to assist ligand binding. sumoylation is a post-translational modification involved in various cellular processes, such as nuclearcytosolic transport, transcriptional regulation, apoptosis, protein stability, response to stress, and progression through the cell cycle. sumoylation is a conjugation of glysine residue of sumo protein with lysine residue in target(rangap ) protein through the influence of enzyme e (ubc ) and e (ranbp ). sumo proteins modify the function of target protein in the cell. but the kinetic mechanism of sumoylation is not well-understand. we traced out the full kinetic process of sumoylation of the sumo -ubc -rangap -ranbp complex in atomic detail via molecular dynamic simulation. we uncovered the kinetics of sumoylation mechanism and verified the important residues which play a key role in the sumoylation process. the comparison of our results with the experimental one are also discussed. human potassium channel kcnq is expressed in several tissues including inner ear, heart muscle, lung, intestine, and stomach, each requiring a unique current profile for proper function. to tune its output current, kcnq complexes with several accessory proteins from the kcne family, each kcne family member modulating kcnq distinctly: kcne causes the channel to delay opening and become more conductive in the open state, kcne causes the channel to be constitutively conductive, and kcne closes the channel. the purpose of this study is to uncover the atomic-scale, mechanical mechanism of how kcne modulates kcnq . to do this we modeled the interaction between kcnq and kcne with a hybrid experimental-computational approach. our strategy is to determine the nmr structure of kcne alone in bilayer-mimicking bicelles, build a homology model of the open-state kcnq , and dock the structure of kcne onto the model of kcnq using electrophysiology-based restraints to validate and refine the resulting model complexes. paramagnetic relaxation enhancement, residual dipolar coupling and sequence analysis suggest the single span transmembrane helix of kcne is significantly curved. hydrogen-deuterium exchange, nuclear overhauser effect cross peaks, and amber simulation suggest kcne carries a significant amount of water in the c-terminal side of the transmembrane helix. the h- n two dimensional nuclear magnetic resonance spectra of a known phenotype-changing mutation in the center of the transmembrane helix, v t, suggests the helix adopts multiple conformations when the extra side-chain hydroxyl group is present. a docking funnel corroborates a binding pocket suggested by the in vivo electrophysiology data where kcne wedges between a helix near the potassium pore and two helices in the voltage sensitive element of the channel. within this pocket, we believe the conformational sampling and structural rigidity-or flexibility, in the context of the v t mutant-of the accessory protein kcne directly influence the modulation profile. watching conformational changes in proteins by molecular dynamics simulations kresten lindorff-larsen proteins are dynamical molecules and their ability to adopt alternative conformations is central to their biological function. examples include motions that underlie allosteric regulation or ligand binding, or protein dynamics in enzymes that can modulate the overall catalytic efficiency. protein motions can often be described as an exchange between a dominant, ground state structure and one or more minor states. the structural and biophysical properties of these transiently and sparsely populated states are, however, difficult to study, and an atomic-level description of those states is challenging. in an attempt to determine how well molecular dynamics simulations can capture slow, conformational changes in protein molecules we have studied two different protein systems which are known to undergo conformational exchange on the millisecond timescale, and for which structural information is available for both major and minor states. using enhanced-sampling all-atom, explicit-solvent molecular simulations, guided by structural information from x-ray crystallography and nmr, we show that current force fields and sampling methods allow us to sample experimentally-determined alternative conformations with surprisingly high accuracy. in particular, we find that we can reversible sample both the ground state and minor state, and that the simulations capture the structure of the minor states also. our simulations enable us to calculate the conformational free energy between the two states, and comparison with relaxation dispersion nmr experiments demonstrates a high accuracy. thus, we show for two distinct proteins that we can map the free energy landscapes and that both the structure and energetics are in excellent agreement with nmr experiments. our simulations provide insight into the structural and biophysical properties of transiently populated minor states, and help reinterpret previous experimental measurements. further, our results demonstrate that, at least in the two cases we have studied, modern simulation methods enable us to examine these otherwise "invisible" states of proteins and describe their structural, functional and thermodynamic properties. our results in this way help demonstrate how simulations can now add to our knowledge of transiently formed, "hidden" states of proteins. cell signaling is a fundamental process for all living organisms. g protein-coupled receptors (gpcrs) are a large and diverse group of transmembrane receptors which convert extracellular signals into intracellular responses primarily via coupling to heterotrimeric g proteins. in order to integrate the range of very diverse extracellular signals into a message the cell can recognize and respond to, conformational changes occur that rewire the interactions between the receptor and heterotrimer in a specific and coordinated manner. by interrogating the energetics of these interactions within the individual proteins and across protein-protein interfaces, a communication network between amino acids involved in conformational changes for signaling, is created. to construct this mapping of pairwise interaction energies in silico, we analyzed rhodopsin gpcr coupled to a gai b g heterotrimer. the structure of this g protein complex was modeled in the receptor-bound and unbound heterotrimeric states as well as the activated, monomeric ga(gtp) state. from these tertiary structural models, we computed the average pairwise residue-residue interactions and interface energies across ten models of each state using the rosetta modeling software suite. here we disseminate a comprehensive analysis of all critical interactions and create intra-protein network communication maps. these networks represent nodes of interaction necessary for g protein activation. structure and dynamics of the polymyxin-resistance-associated response regulator pmra in complex with the promoter dna yuan-chao lou , yi-fen kao , tsi-hsuan weng , yi-chuan li , chwan-deng hsiao , chinpan chen institute of biomedical sciences, academia sinica, institute of molecular biology, academia sinica pmra, an ompr/phob family response regulator (rr), takes part in the two-component system that manages genes for polymyxin resistance through a phosphorylation-dependent regulation. phosphorylation of ompr/phob rr induces the formation of a two-fold symmetric dimer in the n-terminal receive domain (rec), promoting c-terminal dna-binding domains (dbds) to recognize tandem repeat dna sequences on the promoter to elicit adaptive responses. recently, narayanan et al. presented the complex structure of active-like kdpe in complex with dna, which reveals a unique asymmetric rec-dbd interface that is necessary to form stable complexes for transcription activation. in this study, we report the . Å resolution crystal structure of bef -activated pmra in complex with promoter dna as well as its dynamics in solution by nmr. unlike the case of kdpe, pmra-dna complex structure reveals a different asymmetric rec-dbd interaction. interestingly, nmr assignments and dynamics study suggest that rec and dbd do not form stable contacts in solution; instead, domains tumble separately in the absence or presence of dna. relaxation dispersion experiments on methyl groups further show that several rec-dbd interfacial residues exhibit similar slow dynamics in the presence of dna. it is hence highly possible that the slow dynamics observed on these interfacial methyl groups are related to the formation of asymmetric rec-dbd interaction, which reduces the mobility of pmra-dna complex and promotes crystallization. in solution, two domains tumble independently and have diverse orientations, which together with the dbd-dbd interface may facilitate searching best interactions with rna polymerase holoenzyme for transcription activation. time-resolved x-ray observations of nano-scale protein assembly networks yufuku matsushita , hiroshi sekiguchi , noboru ohta , keigo ikezaki , yuji goto , yuji sasaki , the university of tokyo, graduate school of frontier science, advanced materials science, spring- , osaka university, institute for protein research protein aggregation and network formation in diverse protein species had been studying in various experimental approaches such as absorbance spectrophotometry, dynamical light scattering and x-ray scattering. however, these conventional methods are only able to observe averaged information on bulk conditions and also these techniques have a limitation of time resolved observations. recently, development of single molecular observation techniques are remarkable. diffracted x-ray tracking (dxt) is one of the notable single molecular observation methods to capture detailed intramolecular dynamics of an individual single protein molecule by the labeling x-ray method. in this study, we present an innovative method for observing the protein assembly networks of nano-scale size on the bulk solution using a dxt that possess pico-meter scale positional accuracy and micro-second time resolution. this method is founded by detecting angular rotational displacement of a coexisted and dispersed single gold nanocrystal (approximately nm) on target solution. for target sample, we choose a crystal precursor metastable state of mg/ml lysozyme solution (hen egg white lysozyme: . g/ mole on . m sodium acetate - w/v nacl, ph . buffer) and mg/ml of lysozyme solution such as stable condition. for reference sample, we prepare mg/ml and mg/ml of ribonuclease a solution (ribonuclease a (bovine): . g/mol, . m sodium acetate - w/v nacl, ph . buffer). the experiment was carried out at spring- bl xu. from dxt analysis, we obtained logarithmic rotational velocity distribution of mg/ml, mg/ml lysozyme solution and mg/ml, mg/ml of ribonuclease a solution during ms and we processed regression analysis of gaussian fitting in each distribution. from this result, mg/ml of lysozyme solution (stable) and mg/ml, mg/ml (reference) have definitely regressed by single peak normal gaussian distribution that are positional peaks at . mrad and . mrad and . mrad, respectively. in contrast, mg/ml of lysozyme solution consisted of double peak logarithmic distribution at . and . mrad. this peak separation tendency from dxt are typically observed in a supersaturated condition of the sodium acetate solution which coexisting nano-scale solute networks (dxt and small angle x-ray scattering (saxs) experiment). from this study, dxt measurement results and detailed analysis process for a protein solution such as crystal precursor metastable state of lysozyme solution are concerned that this technique is a powerful tool for observing nano-scale protein network's existence and its local dynamics in bulk solution. at the present time, we will demonstrate the detailed analysis and processing from dxt raw data and the conformity of the results from another experiment confirmation such as saxs and darkfield microscopy. finally, we present a detailed protein network model of lysozyme metastable solution from dxt result. increasing evidence suggests that conformational fluctuations experienced by proteins play a key role in promoting function. however, the experimentally derived dynamic behavior of discrete protein systems has yet to provide clear evidence on the evolutionary conservation of motional events between structural and functional protein homologues. in this work, we used a statistical coupling analysis (sca) approach to analyze the role of co-evolving amino acids in protein function and flexibility. sca facilitates the identification of distinct residue sectors and provides an understanding of the correlation between amino acid co-evolution and biological function. as a model system, we used the ribonuclease a (rnase a) family, which includes canonical members that are structurally similar, but perform diverse activities such as angiogenesis, anti-pathogenicity and neurotoxicity, while preserving the common ribonucleolytic function. analysis of sequences with sca . allowed the determination of five independent components (ics) corresponding to amino-acid sectors with distinct functional, structural and/ or dynamical roles within the family. while most ics correspond to residues critical for protein stability and catalytic function, ic correlates with residues experimentally determined to be highly dynamic on the catalytic timescale in several rnase a homologues, as we confirmed by nmr n-cpmg experiments. additionally, ic residues form a hydrogen-bonding network shown to allosterically modulate and coordinate catalytically productive motions. these results provide a direct observation between the role of residue sectors in stability and motions relevant to function in the rnase a family. this study highlights the role of concerted action of multiple residues towards a common function, which can guide experiments for engineering proteins to enhance function such as catalysis. halophilic archea are a type of extremophiles that thrive in hypersaline conditions. in order to avoid the osmotic shock, their cytoplasm is maintained with higher ionic strength up to - m. such a high ionic strength of intracellular environment protects the cells from desiccation or salting out. however, one wonders how such high salt concentrations keep the cell functional and active throughout all the cellular reactions (i.e. enzyme catalysis). the answer lies in the evolution of the halophiles that has left the halophilic proteins with a certain preference for amino acids composition [ ] (prefers more acidic residues and overall decrease in hydrophobic side-chains). it's also known that the salt concentration can affect the activity and the underlying mechanism [ ] of the halophilic enzymes. in order to further understand the haloadaptation, we have chosen to study a model system, dihydrofolate reductase (dhfr) from haloferax volcanii (hvdhfr). dhfr is a very well understood enzyme and known to bound variety of ligands that can modulate the dynamics and the energy landscapes of the enzyme [ ] . we have probed the ls-ms dynamics of hvdhfr bound to the cofactor nadph (holoenzyme) and other substrates (dhf, thf) using relaxation dispersion nmr. in order to understand the salt contribution to the dynamics and catalytic mechanism, the experiments were done in the increasing order of salt concentration, shedding light on the conformational ensembles involved in the catalytic mechanism. the nmr relaxation dispersion analysis of different enzyme complexes in the presence of m and m kcl has shown more conformational fluctuations than compared to the enzyme with m kcl. this result suggests that the enzyme preferably is highly active & samples more flexible conformers in the hypersaline environment. further, quantification of these conformers/invisible-states and their salt-dependence will provide new insights into the mechanism of the enzyme-catalysis. also, it will facilitate designing novel enzymes that can be used in the bioprocess industrial set-up under extreme conditions. . tadeo we have identified the extracellular protein hbps in the cellulose degrader streptomyces reticuli and shown that it specifically binds iron ions, heme and aquo-cobalamin. based on d crystal structures, structural alignments, sequence comparisons, mutagenesis, and comparative biochemical investigations, we identified the coordination sites for iron, heme and aquo-cobalamin, and binding kinetics were elucidated [ , ] . hbps interacts with the membrane-embedded sensor kinase sens. while under nonstressed conditions hbps inhibits sens autophosphorylation, under oxidative-stressing conditions activates it. sens in turn phosphorylates the response regulator senr that activates the transcription of anti-oxidative genes [ ] . we crystallized hbps and solved its d crystal structure that revealed an octomeric assembly which is required for interaction with sens [ ] . using mutagenesis, fret, cd spectroscopy, fluorescence spectroscopy and site-directed spin labelling combined with pulse electron paramagnetic resonance spectroscopy, we demonstrated that ironmediated oxidative stress induces both secondary structure and overall intrinsic conformational changes within hbps. we additionally showed that hbps is oxidatively modified, leading to the generation of highly reactive carbonyl groups and tyrosine-tyrosine bonds [ ] . we concluded that these molecular events are responsible for the hbps-mediated activation of the sensor kinase sens. this presentation will focus on the hbps structure and dynamics within the protein. indian institute of technology bombay for (fop-related protein of kda), a centrosomal protein, is conserved across all ciliated eukaryotes. it has been shown to be involved in ciliogenesis in rpe cells and in the s-phase progression in hela cells. it localizes to the pericentriolar satellite and at the centrosome. the localization of for at the centrosome has been found to be throughout the cell cycle. in paramecium, for is involved in docking of the basal body to the cell membrane and in the assembly of the transition zone. we found that for colocalizes with ciliated microtubules in nih t cells. since, the localization of for to the pericentriolar satellite is dependent on microtubules and the cilium itself is a microtubule-based structure; we sought to investigate the role of for in microtubule assembly. the over-expression of gfp-for in hela cells led to the depolymerization of microtubules while only the gfp expressing hela cells showed typical microtubule network. in addition, the microtubules of gfp-for expressing hela cells were found to be more sensitive towards nocodazole, a microtubule-depolymerizing agent, suggesting that for destabilizes microtubules. the effect of for on the polymerization of tubulin was monitored by light scattering, sedimentation assay and electron microscopy. in vitro, for inhibited the rate and extent of polymerization of tubulin. for example, , , and mm for inhibited the polymerization of purified tubulin by , , and %, respectively. further, the sedimentation assay suggested that for inhibited the polymerization of tubulin. in addition, electron microscopic analysis of the assembly mixture showed that for inhibited microtubule formation in vitro. the binding of for to purified tubulin was monitored by several complimentary techniques. using size exclusion chromatography, for was found to be co-eluted with tubulin indicating that for binds to tubulin. further, the interaction between for and tubulin was analyzed by surface plasma resonance technique and the result showed that for binds to tubulin with a modest affinity. the data together suggested that for regulates microtubule assembly through a direct interaction with tubulin. amide hydrogen exchange (hx) is widely used in protein biophysics even though our ignorance about the hx mechanism makes data interpretation imprecise. notably, the open exchange-competent conformational state has not been identified. based on analysis of an ultra-long molecular dynamics trajectory of the protein bpti, we propose that the open (o) states for amides that exchange by subglobal fluctuations are locally distorted conformations with two water molecules directly coordinated to the n-h group. the hx protection factors computed from the relative o-state populations agree well with experiment. the o states of different amides show little or no temporal correlation, even if adjacent residues unfold cooperatively. the mean residence time of the o state is ps for all examined amides, so the large variation in measured hx rate must be attributed to the opening frequency. a few amides gain solvent access via tunnels or pores penetrated by water chains including native internal water molecules, but most amides access solvent by more local structural distortions. in either case, we argue that an over-coordinated n-h group is necessary for efficient proton transfer by grotthuss-type structural diffusion. differences in redox reactions with nadp /h between ferredoxin-nadp oxidoreductases from bacillus subtilis and rhodopseudomonas palustris daisuke seo , hidehiro sakurai , pierre s etif , takeshi sakurai graduate school of natural science and technology, kanazawa univ., research institute for photobiological hydrogen production, kanagawa university, ferredoxin-nad(p) oxidoreductase (fnr) is a ubiquitous enzyme that catalyze the redox reaction between soluble small iron-sulfur protein ferredoxin and nadp /h. fnrs from photosynthetic green sulfur bacterium chlorobaculum tepidum (ctfnr), low-gc content gram-positive bacterium bacillus subtilis (bsfnr) and purple non-sulfur bacterium rhodopseudomonas palustris (rpfnr) are homodimeric proteins containing one fad prosthetic group per subunit. crystal structure analyses of ct-and bsfnrs have revealed their significant structural homology to nadph-thioredoxin reductase from eschelicia coli, which is distinct from the monomeric fnrs from plastids of higher plants, cyanobacteria, g-proteobacteria and putidaredoxin reductase. previous studies on steady-state reaction of bs-and rpfnrs with nadp /h by diaphorase assay have demonstrated that nadph oxidation rates of bsfnr and rpfnr were comparable to those of the fnrs from plastid and cyanobacteria. but affinities toward nadp /h and rates for oxidation of nadph differed significantly between bsfnr and rpfnr, despite their high amino acid sequence homology. in this work, we report pre-steady state reactions of bsfnr and rpfnr with nadp /h by a stopped-flow spectrophotometry. mixing oxidized bsfnr with nadph yielded a rapid formation of charge transfer complexes (ctcs) followed by a reduction of the enzyme. bsfnr was almost fully reduced at equilibrium. mixing photochemically reduced bsfnr with nadp also rapidly provided an absorption spectrum of ctc but followed reoxidation of reduced bsfnr was very slow. the amount of oxidized bsfnr after equilibrium depended on nadp concentration. kinetic analyses indicated that the rate-determining steps were the hydride-transfer reactions in both directions and the rate for the forward direction was much faster than that for the reverse direction. mixing oxidized rpfnr with nadph exhibited a rapid ctcs formation followed by a slower reduction of the enzyme. increase in nadph concentration reduced the observed rate, suggesting redox potential of rpfnr was similar to that of nadp /h couple in the presence of excess nadph. mixing photochemically reduced rpfnr with nadp rapidly provided ctcs. the decay corresponding to the reoxidation of reduced rpfnr with nadp involved two distinctive kinetic phases, which would be due to the similarity in hydride transfer rates of forward and reverse directions, and the presence of excess amount of nadp . kinetic analyses indicated that the rate-determining steps were the hydride-transfer reactions in both directions and the rate for the forward direction was close to that for the reverse direction. obtained data suggested bsfnr and rpfnr are optimized for different direction of the reaction and may play different physiological roles. stephan k€ ohler , , friederike schmid , giovanni settanni , institute of physics, johannes gutenberg university mainz, germany, graduate school materials science in mainz, fibrinogen is a serum multi-chain protein which, when activated, aggregates to form fibrin, one of the main components of a blood clot. fibrinolysis controls blood clot dissolution through the action of the enzyme plasmin, which cleaves fibrin at specific locations. although the main biochemical factors involved in fibrin formation and lysis have been identified, a clear mechanistic picture of how these processes take place is not available yet. this picture would be instrumental, for example, for the design of improved thrombolytic or anti-haemorrhagic strategies, as well as, materials with improved biocompatibility. here, we present extensive molecular dynamics simulations of the fibrinogen complex which reveal large bending motions centered at a hinge point in the coiled-coil region of the complex. this feature, likely conserved across vertebrates according to our analysis, suggests an explanation for the mechanism of exposure to lysis of the plasmin cleavage sites on fibrinogen coiled-coil region. it also explains the conformational variability of fibrinogen observed during its adsorption on inorganic surfaces and it is supposed to play a major role in the determination of the hydrodynamic properties of fibrinogen. in addition the simulations suggest how the dynamics of the d region of fibrinogen may contribute to the allosteric regulation of the blood coagulation cascade through a dynamic coupling between the a-and b-holes, important for fibrin polymerization, and the integrin binding site p . membrane curvature -the assembler of proteins mijo simunovic , , gregory voth , patricia bassereau chemistry department, the university of chicago, physico-chimie curie, institut curie many biological phenomena require the membrane to change its shape. this process is often mediated by curvature-generating proteins, most notably by those containing one of many bar domains. at the same time, membrane curvature controls the way proteins interact with one another and so it acts as a vital signaling mechanism in the cell. we combine theoretical modeling with microscopy imaging techniques to study the driving force underlying the reshaping of biological membranes induced by n-bar proteins. in particular, we employ a combination of coarse-grained molecular dynamics with field-theoretical simulation methods to study the assembly of proteins on the membrane at molecular resolution. this approach allowed us to elucidate the precise mechanism by which cell membranes rapidly and from large distances recruit proteins to membrane-reshaping sites. it also let us identify a surprising role of membrane tension in directing the strength and geometry of protein-protein interactions. by complementing our simulations with fluorescence and atomic force microscopies, we study the way molecular assembly of proteins affects the membrane morphology at a more macroscopic level. we demonstrated how largescale ordering of the proteins on the membrane is mediated by a strikingly long-range interaction that is driven by membrane fluctuations. in sum, our combined theoretical and experimental approach gives vital clues on how the mechanical properties of the membrane may regulate protein dynamics in living cells. adnan sljoka , alexandr bezginov kyoto university, university of toronto understanding how a protein functions depends in critical ways on predicting which parts are rigid and which are flexible. using rigidity-theoretical techniques, a number or programs such as first, proflex or abstract kinari can rapidly decompose a protein into flexible and rigid regions. in this study we extend this technique and develop a novel computational approach for detecting protein allosteric interactions. it is widely believed that the binding of a ligand at the allosteric site triggers a conformational change that is transmitted through the protein to cause a rearrangement and alteration of the shape of the active site. allostery was first described more than years ago; however, the underlying allosteric mechanism is still not well understood. we will introduce a rigidity-based allosteric mode of communication together with an algorithm which can detect transmission of rigidity and shape changes between two (or more) distant binding sites in allosteric proteins. our algorithm is also used to predict and identify regions in the protein that are critical for the coupled communication between distant sites (i.e. allosteric pathways). starting with a set of known gpcr -dimensional structures, we apply our methods and show how binding of an activating ligand (i.e. agonist) triggers small rigidity changes which propagate to the critical g-protein binding regions. in contrast, in the inactive gpcr structures no such rigidity allosteric communication is observed. detailed predictions and analysis on activated (agonist-bound) and inactive adenosine receptors is discussed and results are compared with experimental evidence. these results show that rigidity-based allosteric model and algorithm is a powerful new tool for detecting allostery in gpcrs. comparing the intrinsic dynamics of multiple proteins using elastic network models reveals global similarities based on their overall shape sandhya tiwari , , nathalie reuter , with increasing protein structural data, we find ourselves with the opportunity to study the dynamical similarities within large ensembles in order to understand the role of protein dynamics at various levels. is there evolutionary pressure to conserve dynamics? is this necessary to retain functionality, as it has been suggested? we performed strategic comparative analysis using the elastic network model, which has proven to be highly efficient and informative (fuglebakk et al., bmc bioinformatics, ) to investigate how the dynamics of related proteins change when their functional or oligomeric state shifts, and if it is conserved within protein families. in our work, we have found that proteins with different ligandbinding states, as in adenylate kinase (tiwari et al., bmc bioinformatics, ), or oligomeric states, as in the pyrr family (perica et al., science, ), can be classified based on the global similarity of their intrinsic dynamics. moreover, our recent analysis on functionally distinct protein families with the tim barrel fold indicates that the overall similarity in shape dictates similarity in intrinsic dynamics regardless of sequence and functional conservation or evolutionary links. we suggest that since shape dictates a large part of dynamical similarity in proteins, the changes in local flexibility play a strong role in differentiating various functional states. oxygen-affinity and cooperativity of hemoglobin introduction: the widely-held mechanism of allostery of hb has been that the changes from the r-quaternary/tertiary structures of oxy-hb to the t-quaternary/tertiary structures of deoxy-hb exert certain constraints on the coordination structure of the heme group, leading to a lower o -affinity of the hemes and thus that of hb [ ] . however, we found that there is no causal correlation between the static t/r-quaternary structures and the low/high o -affinities of hb, respectively [ ] . we explore an alternative mechanism of the regulation of the ligand-affinity and cooperativity in hb. results and discussion: the o -affinities of free fe[ii]-protoporphyrin ix-nitrogenous base complexes in organic solvents are very low (p > torr), whereas the apparent o -affinities of these metalloporphyrins, which are incorporated in apo-myoglobin, apo-hb, serum albumin, etc., increase substantially to p < - torr, though their coordination structures are apparently unchanged [ ] . such substantial increases in the apparent ligand-affinities of metalloporphyrin-containing proteins are accomplished by preventing/inteferring with the dissociation of the ligand by protein matrix, since the interior of globin is nearly fully packed by protein matrix. in hb, the dissociation process of the ligand proceeds through the "caged" state [ ] [ ] [ ] , which can be produced by cryogenic photolysis of the ligated-states at . k and in which the metal-ligand bond is broken and the un-bonded ligand is trapped near the bonding site within the globin moietiy. this "caged" state has spectral features distinct from those of either deoxyor ligated states of the respective hemoproteins. the apparent ligand-affinities of hb are regulated by heterotropic effectors without detectable changes in either static quaternary/tertiary structures of the globin moiety or the coordination/electronic structures of the metalloporphyrin moiety and thus the ligand-affinity of the metalloporphyrins themselves [ ] [ ] [ ] . the reduction of the apparent ligand-affinities of hb may be caused by increases in the migration rate of ligands through globin matrix from the "caged" state to solvent, resulting from the effector-linked, enhanced high-frequency thermal fluctuations which increase the transparency of the globin matrix toward small diatomic ligands [ ] [ ] [ ] . conclusion: the ligand-affinity of hb is regulated through protein dynamics by heterotropic effectors, rather than static quaternary/tertiary structural changes. thus, the "caged" state of hb acts as a critical transition state in regulation of the affinity for small diatomic ligands in hb [ ] . the role of metal ions in the regulation of life processes is extremely important. they act as signal transducers, protein configuration stabilizers, enzymatic cofactors, oxygen transport supporters and many others. for example, subtle perturbations in calcium homeostasis may lead to mental disabilities and are linked to diseases such as autism spectrum disorders (asd). in this study we focus on complex protein systems, mainly those present in the brain. we search for dimers mediated by the presence of metal ions, and determine the impact of the presence or absence of the latter on the structure and energetic properties of the complex in the protein-protein interface. we investigate ions' influence on the interface stability using classic molecular dynamics methods (md), including steered md. moreover, we apply a novel suite of enhanced md-based methods recently developed by our team (rydzewski & nowak) to explore ion diffusion pathways in protein fragments of the synapses. finally, we describe specific inter-protein ion binding motifs with the most important interactions, collating them with various structures deposited in the protein data bank [ ] . the binding of integrins to collagen plays a critical role in numerous cellular adhesion processes including platelet activation and aggregation, a key process in clot formation. collagen is an unusually shaped ligand, and its mechanism of recognition and role in selectivity and affinity are unique, and at this stage not well understood. the i-domain of the integrin protein binds to collagen specifically at multiple sites with variable affinities, however the molecular mechanism of integrin i-domain (ai) regulation remains unknown. using nmr, along with isothermal titration calorimetry, mutagenesis, and binding assays we are developing a novel integrated picture of the full recognition process of the integrin a i binding to collagen. the adhesion of the a b integrin receptors to collagen is cation-dependent with collagen binding a mg(ii) ion that is located at the top of the extracellular integrin a i-domain (a i). our results show evidence for a regulatory effect of the mg(ii) ion on a i affinity, by inducing allosteric ms-ms motions of residues distant from the binding site. we propose a novel model of a i recognition to collagen, comprising a two-step mechanism: a conformational selection step, induced by mg(ii) coordination, and an induced-fit step caused by collagen binding. hydrogen-deuterium exchange experiments show that the induced-fit step is facilitated by the reduced local stability of the c-terminus. we propose that the conformational selection step is the key factor that allows discrimination between high and low affinity collagen sequences. cytochromes p (cyp) are heme containing enzymes involved in the metabolism of endobiotics and xenobiotics, such as drugs or pollutants. [ ] in humans, cyps are attached to the biological membranes of endoplasmic reticulum or mitochondria by n-terminal transmembrane anchor and they are partially immersed by their catalytic domain to different level. [ ] generally, the composition of lipid membrane may significantly affect behavior of protein embedded in respective membrane e.g. the cholesterol in membrane alters membrane properties such as: thickening of the membrane, changing the stiffness or enhancing ordering of the membrane. furthermore, the increasing amount of cholesterol in membrane may also alter interaction with membrane proteins and affect solute partitioning between membrane and water molecules. [ ] cholesterol is also known to noncompetitively inhibit the most typical drugmetabolizing cyp -cyp a , [ ] however the mechanism was unknown. for this reason, we prepared the set of simulations of cyp a embedded in dopc lipid bilayers with various cholesterol concentrations ( , , , and % wt; figure ) and the ns long md simulations were carried out. md simulations showed the formation of funnel-like shape of the lipids close to the catalytic domain of cyp. in addition, the cholesterol molecules have tendency to accumulate in the vicinity of membrane-attached f/g loop. the catalytic domain sunk deeper into the membrane with cholesterol and also the number of amino acids in contact with membrane was bigger than in the pure dopc bilayer. in contrast, the presence of higher amount of cholesterol affected the pattern of channel opening effectively blocking the access to the active site from the membrane, which in turn may affect the substrate preferences and catalytic efficiency. [ ] finally, we study the effect of different lipid types on membrane-attached cyp a . anti-( -hydroxy- -nitrophenyl)acetyl (np) antibodies are one of the most widely analyzed type of antibodies, especially with respect to affinity maturation [ ] [ ] [ ] . affinity maturation is a process in which b cells produce antibodies with increased affinity for the antigen during the course of an immune response, and is like "evolution" in term of increasing antigen-binding affinity. during the course of affinity maturation, the structural dynamics of antibodies, which are closely correlated with the binding function, can change. to analyze the structural dynamics at atomic resolution and the single-molecule level, we tried to express and purify single-chain fv (scfv) antibodies against np. using scfv antibodies, we can also analyze the effects of key residues on affinity maturation via site-directed mutagenesis. as the first step, we have succeeded in generating a sufficient quantity and good quality of scfv of affinity-mature anti-np antibody, c , with a linker composed of four repeats of gggs. the scfv protein was expressed in the insoluble fraction of e. coli, and solubilized using m urea, followed by refolding by step-wise dialysis to decrease the urea concentration. the final step of purification using an antigen column indicated that approximately % of the solubilized protein was correctly refolded and possessed antigen-binding ability. the analytical ultracentrifugation (auc) analysis showed that the purified c scfv exists in the monomeric state with little oligomeric contamination. the secondary structure and thermal stability of c scfv were analyzed using circular dichroism (cd). the far-uv cd spectra of c scfv indicated typical b-sheet-rich structures. upon antigen binding, the far-uv cd spectrum remained unchanged, but the thermal stability increased by approximately oc. the antigen-binding function of c scfv was analyzed using a surface plasmon resonance (spr) biosensor, biacore. the binding affinity and kinetics of c scfv for np conjugated to bovine serum albumin immobilized on the sensor chip were similar to those of intact c . taken together, the results of auc, cd, and spr indicated that c scfv could be refolded successfully and would possess its functional structure. next, to analyze the structural dynamics of c scfv in the absence or presence of antigen, experiments involving diffracted x-ray tracking (dxt) were performed [ ] . c scfv with an n-terminal his-tag was immobilized on substrate surfaces using tag chemistry, and au-nanocrystals were labeled on the surface of scfv as tracers. the motions of c scfv were analyzed in two rotational directions representing tilting (u) and twisting (v) mean square displacement (msd) analysis from more than trajectories showed that the slope for c scfv without antigen, especially in the u direction, was greater than that for c scfv with antigen, suggesting that the motion of scfv was suppressed on antigen binding. the antibiotic resistance enzyme aph( '')-ia confers antimicrobial resistance to aminoglycoside antibiotics in staphylococci and enterococci. this kinase phosphorylates aminoglycosides such as gentamicin and kanamycin, chemically inactivating the compounds. we have determined multiple structures of the enzyme in complex with nucleoside and aminoglycoside substrates and cofactor magnesium. introduction of aminoglycoside to crystals of aph( '')-ia induce gross conformational changes in crystallo, illustrating several important stages of the catalytic cycle of the enzyme. an interaction between nucleoside triphosphate and an amino acid residue on a conserved loop has also been identified that appears to govern a conformational selectivity and modulates the enzyme activity when no substrate is present. comparisons between multiple protein molecules both within and between crystal structures allow us to infer functional states of the enzyme as it carries out catalysis. these structures collectively highlight an enzymatic flexibility that not only allows the binding of diverse aminoglycosides, but also appears to transition from a stabilized, inactive enzymatic state to a catalytically active enzyme with an active site geometry identical to distantly-related eukaryotic protein kinases. mechanistic insight gained from these studies begin to demystify a widespread staphylococcal resistance factor, and provide a starting point for the development of anti-infectives toward this important antimicrobial resistance machine. ryan godwin , william gmeiner , freddie salsbury wake forest university -department of physics, wake forest university health sciences -department of cancer biology the zinc-finger of the nf-jb essential modulator (nemo) is a ubiquitin binding domain, and an important regulator of various physiological processes including immune/inflammatory responses, apoptosis, and oncogenesis. the nominally functioning residue monomer ( jvx) is represented by a bba motif, with a cchc active site coordinating the zinc ion. here, we investigate the effects of a single point mutation that has been linked to the disease states associated with ectodermal dysplasia. the single mutation of the last binding cysteine (residue ) to a phenylalanine ( jvy) distorts the available conformation and dynamics of the protein, as shown via microsecond, gpuaccelerated molecular dynamics simulations. we examine these two proteins in various states of zinc-binding and coordinating cysteine protonation. in addition to destabilization of the alphahelix induced by the cysteine to phenylalanine mutation, prominent conformations show the bsheets turned perpendicular to the alpha-helix, providing a possible mechanism for the induced disease state. , catalytic ( - aa) and c-terminal ( - aa)) were expressed in e. coli. several truncated in variants containing amino acids - , - , - and - were also prepared. a full-size ku with a gst-tag on its n-terminus was purified from e. coli. all the experiments performed showed that neither n-terminal nor c-terminal domains of hiv- in are essential for its binding with ku despite a weak binding capacity retaining to the c-terminal domain. the catalytic core ( - aa) as well as the mutant lacking c-terminal domain ( - ) both demonstrated affinity to ku comparable to the affinity of the full-size in, whereas its truncated variant ( - aa) bound to ku protein only weakly. we also expressed a c-terminal ha-tagged full-length in and its - variant in hek t cells together with a wt ku - flag and showed that both in variants are stabilized by co-expression with ku by approx. twofold. we hypothesize that the binding surface within in lies in the region from to a.a. that is a long a-helix. we have shown that a homologous integrase from prototype foamy virus that lacks this structural element does not bind to ku . it is worth noting that ku does not affect the interaction of in with its major cellular partner -ledgf/p as well as its interaction with the dna substrate. this work was supported by an rfbr grant - - and by an rscf grant - - . the nadph-dependent cytochrome p oxidoreductase (cypor) is large amino-acid long microsomal multidomain enzyme responsible for electron donation to its redox partner cytochrome p (cyp) involved in drug metabolism. electron transfer (et) chain is mediated by two riboflavin-based cofactors -flavin mononucleotide (fmn) and flavin adenine dinucleotide (fad) within their respective domains and nicotinamide adenine dinucleotide phosphate (nadph). during this electron transfer cypor undergoes several structural changes in open and closed state of both domains in different degree of contact. in spite of the fact that cyp-cypor complexes play a key role in drug metabolism, the atomistic mechanism of structural rearrangements during complex electron transfers is still lacking. here, we present the results of our study on structural changes during cypor multidomain complex movement between individual electron transfers using classical molecular dynamics (md) and metadynamics (mtd) simulations with cofactors of nadph, fad and fmn in resting state. homology model of human cypor in both forms (opened and closed) were embedded into pure dioleoylphosphatidylcholine (dopc) bilayer. after system equilibration (figure ), structural changes of protein, anchor and cofactor movement were studied. we were able to select possible cypor-membrane orientation which would allow interaction with cytochrome p . in addition, spontaneous closing of open cypor was observed. however structural changes between crystal structures and structures obtain from md simulations lead us to the use of metadynamics in order to speed up the process. fmn and fad cofactor remained in close van der waals contact during the -ns long simulation stabilized by p stack interaction of fad with trp , whereas continual movement of nadph continually weakens its p stack interaction with fad. after ns of classical md additional metadynamics simulations were performed in order to investigate internal motion of cofactors during electron transfer. atoms c n (nadph) and n (fad) which are responsible for et were able to move closer to the distance of Å after adding biasing potential. this distance is more than sufficient for electron transfer to occur. after switching back to classical md cofactors got into resting positions ( Å) again. our results show that cypor undergo several structural changes and internal motions of cofactors in order to transfer electrons to its redox partner -cyp. research & utilization div., jasri/spring- , grad. school frontier sci., univ. tokyo, grad. sch. sci., univ. hyogo, japan, national institute of advanced industrial science and technology, japan, pentameric ligand-gated ion channels (plgics) are a major family of membrane receptors that open to allow ions to pass through the membrane upon binding of specific ligands. plgics are made up of five identical (homopentamers) or homologous (heteropentamers) subunits surrounding a central pore. structural information about their multiple allosteric states, carrying either an open or a closed channel, has become available by recent studies by x-ray crystallography. however, dynamic information are needed to understand their mechanism of gating, notably the long-range allosteric coupling between the agonist binding site and the ion channel gate. here we used the diffracted x-ray tracking (dxt) method ( ) to detect the motion of the extracellular and transmembrane domain two plgics: the nicotinic acetylcholine receptor (nachr) and a proton-gated bacterial ion channel from gloeobacter called glic. dxt is a powerful technique in biological science for detecting atomic-scale dynamic motion of allosteric proteins at the single molecular level and at tens of micro seconds timescale resolution. the dynamics of a single protein can be monitored through trajectory of a laue spot from a nanocrystal which is attached to the target protein immobilized on the substrate surface ( , ). dxt detects two kinds of rotational motions of nanocrystal, tilting and twisting, based on x-ray incident beam axis. dxt analysis with . ms/f time resolution showed that tilting motion of the transmembrane domain of glic and both tilting and twisting motions of the extracellular domain of glic and nachr were enhanced upon application of agonists (lowering the ph for glic, and binding of acetylcholine for nachr). the detailed dynamic information, including size effect of gold nanocrystal to the motion of them, is discussed. [ proteins possess unique structure-encoded dynamics that underlie their biological functions. here, we provide experimental evidence for an evolutionary mechanism driven solely by long-range dynamic motions without significant backbone adjustments, catalytic group rearrangements, or changes in subunit assembly. crystallographic structures were determined for several ancestral gfp-like proteins that were reconstructed based on posterior sequence predictions, using members of the stony coral suborder faviina as a model system. the ancestral proteins belong to the kaede-type class of gfps, a group of proteins that undergoes irreversible green-to-red photoconversion and is therefore frequently employed in superresolution microscopy. surprisingly, we find that the structures of reconstructed common green ancestors and evolved green-to-red photoconvertible proteins are very similar. therefore, we analyzed their chain flexibility using molecular dynamics and perturbation response scanning. we find that the minimal number of residue replacements both necessary and sufficient to support lightinduced color conversion provide for increased fold stiffness at a region remote from the active site. at the same time, the allosterically coupled mutational sites appear to increase active site conformational mobility via epistasis. these data suggest that during evolution, the locations of fold-anchoring and breathing regions have been reversed by allosteric means. therefore, we conclude that the green-tored photoconvertible phenotype has arisen from a common green ancestor by migration of a knob-like anchoring region away from the active site diagonally across the beta-barrel fold. based on titration experiments, we estimate that at ph , . % of the protein population harbors neutral side chains for his and glu , residues that form an internal salt bridge near the chromophore. we propose that this reverse-protonated subpopulation constitutes the catalytically competent state. in the electronically excited state, light-induced chromophore twisting may be enhanced, activating internal acid-base chemistry that facilitates backbone cleavage to enlarge the chromophore. in this way, a softer active site appears to be coupled to a mechanism involving concerted carbon acid deprotonation and betaelimination. dynamics-driven hinge migration may represent a more general platform for the evolution of novel enzyme activities by tuning motions in the active site. the binding of an agonist to a gpcr causes a conformational change in the receptor that leads to its activated functional state. rhodopsin, the membrane receptor responsible for photoreception in the vertebrate retina, is a prototypical gpcr and has been extensively used in structural, biochemical and biophysical studies of this class of receptors. different small molecules have been described to be capable of binding to rhodopsin. in addition, mutations in rhodopsin have been associated with retinal diseases and efforts have been carried out in order to find potential ligands that can offset the effect of these mutations. cyanidins, a group of flavonoids within the larger family of polyphenols, have been reported to stimulate chromophore regeneration of rhodopsin by means of the formation of regeneration intermediates. the aim of the current study was to evaluate the effect of the flavonoid quercetin on the conformational properties of both native bovine rhodopsin and heterologously expressed recombinant rhodopsin. rhodopsin was purified from bovine retinas by immunoaffinity chromatography, and photobleaching, thermal stability, metarhodopsin ii decay and chromophore regeneration assays were carried out in the absence or in the presence of mm quercetin. for recombinant rhodopsin, a plasmid encoding wild-type opsin was transfected into mammalian cos- cells, in the absence or in the presence of mm quercetin, harvested, regenerated with -cis-retinal, or -cis-retinal, and subsequently purified in dodecyl maltoside solution. no differences in photobleaching behavior, upon illumination, could be detected in the purified quercetin-containing samples compared to those in the absence of this flavonoid. in the case of rhodopsin, and the recombinant wild-type protein regenerated with -cis-retinal, quercetin did not significantly alter the thermal stability and rate of regeneration of the purified proteins under our experimental conditions. however, a two-fold increase in the thermal stability and a % increase in chromophore regeneration were observed for the recombinant wild-type protein regenerated with -cis-retinal in the presence of quercetin. in contrast, the presence of quercetin did not alter the electrophoretic and basic spectroscopic properties of rhodopsin, or those of the recombinant wild-type protein, suggesting no important structural alterations as a result of quercetin binding to the receptor. the positive effect of quercetin on the stability, and chromophore regeneration of rhodopsin, could be potentially used to counteract the effect of naturally-occurring misfolding mutations in rhodopsin. thus, quercetin could help stabilizing rhodopsin mutants associated with retinal diseases such as retinitis pigmentosa. furthermore, docking of the ligand, carried out on the crystallographic structure of rhodopsin (entry gzm), reveals several favorable sites for quercetin binding. one of this would be compatible with -cis-retinal suggesting a complementary binding to the receptor of this isomer which would not be compatible with -cis-retinal binding. identification of prospective allosteric sites of p by computational methods protein function is intrinsically associated with structural flexibility, so that understanding the functional properties of proteins requires going beyond the static picture produced by x-ray diffraction studies. structural flexibility can also be interpreted as a dynamic exchange between different conformational states with low energy barriers at room temperature. allosterism is a mechanism to regulate protein function associated with the plasticity exhibited by proteins. allosteric sites can be considered transient cavities that can be occupied by a small molecule with the subsequent modulation of the protein plasticity. occupation of these sites may modify the affinity of the protein for its native substrate that can be positive when the affinity increases or negative when the affinity decreases. allosterism can be used for the design of non-competitive ligands as new therapeutic agents. this mechanism of activity modulation is particularly interesting for those targets that use a common substrate for activation, like in the case of kinases to search for selective compounds. proteins can be viewed in solution as an ensemble of diverse energy accessible conformations. binding of an allosteric ligand produces a redistribution of the population of the diverse conformational states, which at the end modulate the affinity of the native substrate. allosteric sites can be characterized using computational methods by ensemble docking. it consist of characterize a set of structures that represent the accessible conformations of a protein that can then be used to perform virtual screening. in the present work we have studied prospective allosteric sites of p using computational methods. the protein is a member of the mitogen-activated protein kinases (mapks), a highly regulated group of enzymes that control a variety of physiological processes, including mitosis, gene expression, apoptosis and metabolism movement among others. the conformational profile of p was assessed using a us trajectory of accelerated molecular dynamics as sampling technique in explicit solvent. we used as starting structure the apoform of p in its inactive conformation (entry p ). the conformational features of the protein were assessed through the analysis of the variance of the most flexible regions of the protein using principal component analysis. the snapshots of the trajectory were projected onto the two principal components. subsequent cluster analysis permitted us to select a few structures for further studies. specifically, prospective biding sites were identified using a hydrophobic probe as implemented in the sitemap program. the results show previously described regulatory sites and some new prospective ones. hydrogen/deuterium exchange-mass spectrometry provides clues on the mechanism of action of min e maria t. villar , kyung-tae park , joe lutkenhaus , antonio artigues cell division in most bacteria is initiated by the formation of the z ring, an essential cytoskeletal element that serves as a scaffold for the cytokinesis machinery, at the mid body of the cell. in e coli the spatial location of the z ring is regulated by the min protein system, comprised by three major proteins: minc, mind and mine. the dynamic interaction between these proteins results in the formation of an oscillating protein gradient between the poles of the cell. this oscillation determines the position of the formation of the z ring. many aspects of this simple mechanism are beginning to be understood. in particular, the conformational changes associated with the interaction of the three min proteins between them and with the cell membrane, are of especial interest. hydrogen/deuterium exchange mass spectrometry (hdx ms) is a sensitive technique for the detection of changes in protein conformation and dynamics. the main advantages of this methodology are the ability to study native proteins in solution, the requirement for low protein concentrations, the potential to discriminate multiple coexisting conformations, and the lack of an upper limit to the size of protein to be analyzed. here we use hdx ms to analyze the dynamics of the wild type mine and of its inactive double mutant d a d a. our results show significant differences in the rates of exchange and in the total amount of deuterium exchanged at the end of the reaction between these two forms of mine. the wild type protein exchanges most of the amide hydrogen during the first few seconds of initiation of the exchange reaction. on the other hand, the mutant protein exchanges only % of the total amide hydrogen atoms during the first seconds of initiation of the exchange, and the remaining % amide hydrogen atoms are exchanged more slowly during the next few minutes of the reaction. our data are consistent with the existence of a highly flexible structure for the wild type protein and the coexistence of at least two rigid conformations for the double mutant that are undergoing a cooperative transition. interestingly, the central b-sheet forming the interface between the two subunits is protected against exchange on both proteins. these results provide insights into the conformational changes that mine undergoes during its interaction with mind. biased signalling and heteromization of the dopamine d receptor in schizophrenia and parkinson's disease pablo herrera nieto , james dalton _ , jes us giraldo _ universidad aut onoma de barcelona biased signalling and heteromization of the dopamine d receptor in schizophrenia and parkinson's disease as a significant component of dopamine signalling in the brain, the dopamine d receptor (d r), a member of the class a gpcr family, is an important target in the treatment of neurological conditions such as schizophrenia and parkinson's disease. d r shows a variety of signalling pathways through g proteins, including adenylyl cyclase inhibition, gbgpotentiation of adenylyl cyclase , and erk kinase activation, in addition to b-arrestin recruitment,. these pathways are differentially activated by some agonists and it has been suggested that d r ligands with gai/o antagonist and b-arrestin agonist activity may have anti-psychotic behavioural activity with reduced extra-pyramidal side effects. d r has also been found to form homodimers or higher-order hetero-oligomers with other gpcrs, which may modulate d r conformation and activity, thus constituting an additional form of allosteric receptor regulation. based on these findings, we have computationally modelled the full-length structure of d r, including its long intracellular loop (icl ) that is residues in length and absent in all homologous gpcr crystal structures. using state-of-the-art tools, such as rosetta for ab initio protein folding and acemd for micro-second molecular dynamics (md) simulations we have successfully de novo folded icl , which primarily consists of extensions to transmembrane helices (tmh) and and an intervening disordered histidine/proline-rich region, which is highly flexible. the latter is observed to interact with other receptor intracellular loops (icl and icl ) and appears to restrict access to the g-protein binding-site. in addition, we have docked a structurally diverse collection of ligands (biased agonists, antagonists and allosteric modulators) into our d r model and observed characteristic binding patterns suggestive of different biased signalling mechanisms. finally, through protein-protein docking with rosettadock, we have generated a complete heterodimer model of d r with the adenosine a a receptor (aa ar), where a mutual interface is formed between their respective tmhs and , as well as an association between the c-terminus of aa ar and icl of d r. this may be a particularly relevant biological complex in the treatment of parkinson's disease where antagonists of aa ar have been shown to ameliorate disease effects, potentially through direct interaction with d r. bis-ans as a tool to monitor conformational changes upon assembly of binary and ternary complexes of eif e, e-bp inhibitory protein, and the mrna 'cap specific recognition of the mrna ' terminal cap structure by the eukaryotic initiation factor eif e is the first and rate-limiting step in the cap-dependent translation. small e-binding proteins, e-bp , e-bp , and e-bp , inhibit the translation initiation by competing with eif g initiation factor for the same binding site, and by blocking the assembly of the translation machinery [ ] . our recent studies revealed intricate cooperativity between the cap and e-bp binding sites of eif e [ ] . here, we applied a fluorescent dye, , '-dianilino- , '-binaphthyl- , '-disulfonate (bis-ans) to investigate conformational changes upon assembly of binary and ternary complexes composed of human eif e, e-bp , and the mrna 'cap analogue, m gtp. the fluorescence quantum yield of bis-ans increases significantly upon binding to hydrophobic sites of proteins, making the probe a convenient tool to determine the accessibility to hydrophobic surfaces, and to monitor structural reorganisation of macromolecules [ ] . we characterised the interaction of bis-ans with eif e and e-bp by fluorescence titration. the association processes takes up to several hours until the saturation of the fluorescence signal is achieved, reflecting high flexibility of the protein structures. the association constants kas of eif e/bis-ans complexes are very high for the non-specific interaction. the kas values for eif e/bis-ans and eif e/ e-bp /bis-ans are similar ( m ), whereas the presence of m gtp results in ca. -fold weaker binding of the probe to eif e. the affinity of bis-ans for e-bp is -fold lower than that for eif e. we found no effect of either m gtp or e-bp on the fluorescence of bis-ans in complex with eif e, thus indicating lack of conformational changes around the probe on eif e/m gtp or eif e/ e-bp complex formation. it also testifies that bis-ans does not bind to the cap-binding site, despite the hydrophobic nature of this eif e region. on the contrary, addition of m gtp to the eif e/ e-bp /bis-ans complex causes an increase of the probe fluorescence, which indicates differences in the structural reorganisation in the binary, m gtp/eif e, compared with the ternary, m gtp/eif e/ e-bp , complexes, and confirms the spatial cooperation between the cap and e-bp binding sites. we also observed an increase of fluorescence for bis-ans bound to e-bp in the presence of eif e, pointing out that e-bp partially folds upon association with eif e. in summary, our results provide a deeper insight into the structural aspects of the molecular interaction at early stages of the cap-dependent translation. acknowledgements: this work was supported by the bst /bf project from university of warsaw background: beta -glycoprotein (b gpi) is a protein abundantly present in human plasma and highly conserved in all mammals. b gpi has been identified as the major antigen in the antiphospholipid syndrome (aps), a severe thrombotic autoimmune disease. despite its importance in the pathogenesis of aps, the physiological role of b gpi is still elusive. in a previous work we have demonstrated that b gpi significantly prolongs the clotting time in fibrin generation assays, and inhibits aggregation of gel-filtered platelets (ic . um), either isolated or in whole blood, by inhibiting cleavage of par on intact platelets (ic . um) and in solution. importantly, b gpi does not alter the ability of thrombin (fiia) to generate the anticoagulant protein c, with or without thrombomodulin added. hence, we concluded that b gpi inhibits the key procoagulant properties of fiia, without affecting its unique anticoagulant function. we also proposed that b gpi, together with other more efficient anticoagulant pathways such as thrombomodulin-fiia -protein c and antithrombin iii-fiia, may function as a mild anticoagulant in vivo especially in those compartments were the efficacy of thrombomodulin is limited, as in the large vessels, or is even absent, as in the brain vasculature. aims: lacking the threedimensional structure of b gpi-thrombin complex, the aim of this work is to identify the peptide regions either on thrombin and b gpi involved in complex formation. results: data obtained by fluorescence and surface plasmon resonance (spr) indicated that b gpi interacts whit fiia whit physiological affinity (kd nm). kd values calculated by reverting the interacting systems are very similar to each other (kd nm), suggesting that b gpi in the mobile phase has a conformation which is competent for the binding to immobilized fiia. the affinity of fiia for immobilized b gpi is markedly decreased by increased ionic strength (i.e. kd increases by -fold going from . m to . m), suggesting the electrostatic interactions play a key role in fiia -b gpi recognition. filling/inactivation or perturbation of fiia active site does not alter the affinity of fiia for immobilized b gpi, confirming that the active site is not involved in the interaction. mapping of thrombin binding sites with specific exosite-directed ligands (i.e. hirugen, gpibalpha, hd aptamer) and thrombin analogues having the exosites variably compromised (i.e. prothrombin, prethrombin- , alpha-thrombin), reveals that the positively charged exosite-ii of fiia plays a key role in b gpi binding. from the docking model of the bb gpi-thrombin complex, we identified a highly negatively charged segment - in domain v of b gpi interacting with positively charged pathes in thrombin exosite ii. the synthetic peptide b gpi( - ) was able to bind to fiia with an affinity (kd nm) comparable to that of full-length b gpi, deduced from fluorescence or spr measurements and to compete in spr measueremnts with the binding of full-length b gpi to thrombin. hence, combining experimental and theoretical data, we obtained a reliable model of the b gpi-thrombin complex. metalloproteases are one of the most diverse types of proteases, presenting a wide range of folds and catalytic metal ions. in the case of the merops ma clan, where most of the known metalloproteases are grouped based on the consensus hexxh sequence motif, a single catalytic zinc ion and common fold architecture [ ] . despite these common features, members from distinct families present distinct domain composition and topology. given our interest in developing new tailor-made metalloproteases for bioengineering applications, an in-depth understanding of the factors governing their function is required. protein internal dynamics includes the space of functionally-relevant structural changes occurring during an enzymatic reaction, and there is an increasing understanding on how it relates with protein sequence and structure evolution. therefore, we have recently assessed how the structural heterogeneity of metalloproteases relates with the similarity of their dynamical profiles [ ] . first, the dynamical profile of the clan ma type protein thermolysin, derived from the anisotropic network model, was evaluated and compared with those obtained from principal component (pc) analysis of a set of crystallographic structures and essential dynamics (ed) analysis of a ns molecular dynamics simulation trajectory [ ] . a close correspondence was obtained between normal modes (nm) derived from the coarse-grained model and experimentally-observed conformational changes (rmsip between nm -nm and pc of . ), corresponding to functionally-relevant hinge bending motions that were shown to be encoded in the internal dynamics of the protein (cumulative overlap of ed -ed and pc of . ). next, dynamics-based comparison methods that employ a related coarse-grained model (b-gaussian elastic network model) was made for a representative set of ma clan members [ ] , allowing for a quantitative description of its structural and dynamical variability. although members are structurally similar ( % pairs with dalilite z-score > . ), they nonetheless present distinct dynamical profiles ( % of pairs with aladyn p-value > . ), with no identified correlation between structural and dynamical similarity. for cases where high dynamical similarity was observed, the respective modes corresponded to hinge-bending motions encompassing regions close to the active site. further inspection of the produced alignments indicates that for ma clan metalloproteases, conservation of internal dynamics has a functional basis, namely the need for maintaining proper intermolecular interactions between the protein and respective substrate. previously unnoticed dynamical similarity between clan members botulinum neurotoxin type a, leishmanolysin and carboxypeptidase pfu was also found. together, these results suggest that distinct selective pressure mechanisms acted on metalloprotease structure and dynamics through the course of evolution. this work shows how new insights on metalloprotease function and evolution can be assessed with comparison schemes that incorporate additional information of protein dynamics. glucokinase from antarctic psychrotroph pseudoalteromonas sp. as- (psgk) has a higher specific activity at low temperatures and a higher thermal stability than its mesophilic counterpart from e. coli (ecgk). in order to elucidate the structural basis for cold-adaptation and thermal stabilization of psgk, we have determined the crystal structure of psgk at . Å and compared it with the ecgk structure. psgk is a homodimer of the subunit of amino acid residues. each subunit consists of two domains, a small a/b domain (residues - and - ) and a large a b domain (residues - ). the active site is located in a cleft formed between the two domains. the identity of amino acid sequence between psgk and ecgk was %, but three dimensional structures of them are very similar to each other, having the conserved catalytic residues and substrate-binding residues. the analysis of the mainchain temperature factors revealed that the regions of small domain and the hinge region connecting two domains of psgk showed higher temperature factors with a lower number of intramolecular hydrogen bonds and ionic interactions than the corresponding regions of ecgk. however, the large domain regions of psgk showed lower temperature factors with a higher number of intramolecular hydrogen bonds than ecgk. furthermore, the atomic temperature factors of catalytic asp on the small domain were higher, but those of glucose-binding glu , his , and glu on the large domain were lower than ecgk. these results suggest that highly flexible hinge region and the catalytic residue on the small domain of psgk may contribute to its cold-adaptation, namely higher activity at low temperatures, whereas a more rigid structure of the large domain of psgk stabilizes its overall structure more strongly than ecgk. nowadays non-waste technologies in synthetic chemistry become more and more popular. such processes are often carried out using different enzymes. dehydrogenases represent the large group of enzymes, which are widely used in synthesis of chiral compounds and other useful molecules. such enzymes need nadh or nadph as a cofactor and due to high cost of reduced coenzymes a cofactor regeneration system is an obligate part in such kind of processes. it was shown that formate dehydrogenase (fdh, ec . . . .) is one of the best enzymes for nad(p)h regeneration. fdh catalyses the reaction of formate oxidation to carbon dioxide coupled with reduction of nad(p) to nad(p)h. the main advantages of fdh are the irreversibility of catalyzed reaction, low price of formate ion and wide ph optimum of activity. our laboratory has the largest collection of formate dehydrogenases from different sources. many fdh genes from bacteria, yeasts and plants were cloned and enzymes were expressed in active and soluble forms. mutant formate dehydrogenases from bacterium pseudomonas sp. show the highest thermal stability as well as activity in comparison with other reported formate dehydrogenases. now we have focused on eukaryotic genes. the recombinant enzymes from soya glycine max (soyfdh), arabidopsis thaliana (athfdh), moss physcomitrella patens (ppafdh) and yeast ogataea parapolymorpha (opafdh) were obtained by genetic engineering methods. it was revealed, that soyfdh has the best michaelis constants among all known fdhs, but it's less thermally stable compared to other fdhs. new mutant forms of soyfdh with excellent catalytic characteristics and high thermal stability were obtained by protein engineering. other enzymes (athfdh, ppafdh and opafdh) are comparable in their stability with majority of bacterial enzymes (but not with psefdh), so all the new obtained fdhs can be successfully used for cofactor regeneration. marmara university, wellesley college, antibiotics are essential therapeutic drugs widely used in the treatment of bacterial infections. unfortunately, misuse of these drugs resulted in the development of bacterial defense mechanisms. blactamase synthesis is among these mechanisms that renders b-lactam antibiotics ineffective. understanding the dynamic behavior of this enzyme is an important step in controlling its activity. in a former study, the importance of highly conserved w in modulating the hinge type h motion was reported. in the light of this information, mutant tem- b-lactamase enzymes with w a, w f and w y substitutions were constructed. wild-type and mutant tem- b-lactamases purified with ni affinity chromatography were subjected to enzyme assay using centa as the substrate. with w f and w y mutations, the remaining activity was approximately % of the initial activity. however with the w a mutation, activity was totally lost. structural studies of the w a mutant with cd and florescence spectroscopy indicated that there was no major change in the overall structure. however this mutation disrupted the interactions of w which resulted in an increase in the flexibility of this region of the protein. this project was supported by t € ub _ itak project no m . light-switchable zn binding proteins to study the role of intracellular zn signaling stijn aper , maarten merkx zn plays an important catalytic and structural role in many fundamental cellular processes and its homeostasis is tightly controlled. recently, free zn has also been suggested to act as an intracellular signaling molecule. to get increased understanding of the signaling role of zn we are developing light-switchable zn binding proteins to perturb the intracellular zn concentration using light. these protein switches consist of two light-responsive vivid domains and the zn binding domains atox and wd , linked together with flexible peptide linkers. in the dark, zn is tightly bound in between the two zn binding proteins. light-induced dimerization of the vivid proteins disrupts this interaction and thus results in zn release. the fluorescent proteins cerulean and citrine were attached to the vivid domains to allow the different conformational states of the protein switch to be monitored using fret. zn titrations revealed a -fold decrease in zn affinity going from dark-to light-state for the initial design, which was further improved to -fold by optimizing the linkers between the protein domains. in addition, the zn affinities of both states were tuned to be optimal for intracellular applications. switching between the high affinity dark-state and the low affinity lightstate was found to be reversible for at least two light-dark cycles. following the in vitro characterization, we are currently assessing the performance of this genetically encoded 'caged' zn in mammalian cells. proteins as supramolecular building blocks: engineering nanoscale structures school of biological sciences, university of auckland, school of biological sciences, victoria university proteins hold great promise in forming complex nanoscale structures which could be used in the development of new nanomaterials, devices, biosensors, electronics and pharmaceuticals. the potential to produce nanomaterials from proteins is well supported by the numerous examples of self-assembling proteins found in nature. we are exploring self-assembling proteins for use as supramolecular building blocks, or tectons, specifically the n-terminal domain of a dna binding protein (nterm-lsr ) and a typical -cys peroxiredoxin (hsprx ). non-native forms of these proteins have been designed undergo selfassembly into supramolecular structures in a controllable manner. self-assembly of nterm-lsr is initiated via proteolytic cleavage, thereby allowing us to generate supramolecular assemblies in response to a specific trigger. we will show that the degree of oligomerisation can be controlled by variations in environmental conditions such as ph and protein concentration. furthermore, via protein engineering, we have introduced a new "switch" for oligomerisation via enteropeptidase cleavage. the new construct of nterm-lsr can be activated and assembled in a controlled fashion and provides some ability to alter the ratio of higher ordered structures formed. hsprx has been shown to oligomerise into dimers, toroids, stacks and tubes in response to specific triggers such as ph and redox state. in this work we have utilised the histidine tag to further control the assembly of this versatile protein tecton. we will show that minute variations in ph can induce oligomersation of hsprx toroids into stacks and tubes. furthermore, by utilising the histidine tag as a ligand we can bind divalent metals to these supramolecular structures. this not only drives the formation of higher ordered oligomers but also provides a facile route which may facilitate the functionalisation of these protein nanoscale structures after they have been assembled. danielle basore , , rajesh naz , scott michael , sharon isern , benjamin wright , katie saporita , donna crone , christopher bystroff , , biological sciences, rensselaer polytechnic institute, cbis, rensselaer polytechnic institute, chemical and biological engineering, rensselaer polytechnic institute, computer science, rensselaer polytechnic institute, obstetrics and gynecology, west virginia university, unintended pregnancy is a worldwide public health concern, with million pregnancies being classed as unintended in . the magnitude of this number clearly indicates an unmet need in terms of contraception. methods that are currently available are effective, but exhibit many problems. side effects, ease of use, cost, and availability are all concerns. we propose a contraceptive vaccine that would be safe, effective, long-lasting, cheap, and reversible. our vaccine would prevent pregnancy by targeting sperm with antibodies raised in the woman's body. several approaches have been taken to developing a contraceptive vaccine in recent years. the most successful so far has been using human chorionic gonadotropin (hcg), a hormone produced during pregnancy, as an antigen . the hcg vaccine progressed to phase clinical trials, but only displayed an % efficacy, which is insufficient for a contraceptive. our lab uses a structure based approach to the design of an anti-sperm antigenic protein. we believe this will raise a more vigorous immune response that will produce a longer lasting titer. the catsper complex is a heterotetrameric calcium channel found in the tail region of sperm . each subunit of the complex contains an exposed loop known as the p-loop. the p-loop is unique on the surface of sperm because it is not glycosylated, allowing antibodies to potentially recognize and bind it. ylp is a twelve residue peptide that mimics the glycans in the glycocalyx of sperm . ylp is a member of the flitrx library, and in mice, produced protective titers that were reversible both voluntarily and involuntarily. our designs will introduce these two potential antigens into a loop of the l protein of human papilloma virus. l spontaneously assembles into virus like particles, and will aid in the production of a robust immune response. protein carriers for passage of the blood-brain barrier sinisa bjelic medical solutions that help protein therapeutics accumulate into the brain are crucial for future treatment of neurological disorders. biodrugs have a tremendous potential to treat disorders of the nervous system, but their efficiency has been severely restricted. to reach the brain all drugs must traverse the blood-brain barrier (bbb) -a permeable wall that separates blood from the brain -whose main function is to protect the nervous system from environmental influences of bacteria and toxins. unfortunately the bbb is also the culprit that effectively blocks access to therapeutics required for treatment of neurological diseases. a way to boost exposure of therapeutics across the bbb is to piggyback onto the transferrin receptor, a multidomain protein anchored in the membrane, which is involved in the physiological facilitation of iron uptake. here i present research that aims at successfully developing potent protein carriers for transferrin receptor-mediated passage of the bbb by using computational protein design in combination with yeast display methodology for hit validation and optimization. the longterm goal is to couple therapeutics -as for example drugs against alzheimer's -to the designed carriers to increase the brain uptake and cure neurological disorders. medium-throughput multistep purification of coagulation factor viia jais r. bjelke , gorm andersen , henrik Østergaard , laust b. johnsen , anette a. pedersen , tina h. glue there is a need of medium-to-high throughput purification of low-titre recombinant protein variants for screening to identify the final biopharmaceutical lead. such proteins include coagulation factors to be used for treatment of haemophilia and other bleeding disorders. at novo nordisk we have established a platform for production of recombinant coagulation factor viia variants, which include a spectrum of single-point mutations to large domain insertions. the variants were produced using transiently transfected hek f, hkb or choebnalt (qmcf technology) suspension cells. harvest cultivations were typical in the range of . -to l. a -step continuous, multistep purification method was implemented on € aktaxpress systems (ge healthcare). the interlinked process steps include capture using an immunoaffinity column, polish, concentration and buffer exchange using an anion-exchange column and proteolytic activation of the zymogen variant forms using a coagulation factor xaimmobilized column. buffers were designed such that elution from the capture column was aligned with binding conditions on the polish column to avoid a desalting step in-between. the following and final enzymatic activation was optimized with regards to flow rate to ensure full conversion while minimizing unwanted secondary cleavages in factor viia. the final products were fractionated in sharp chromatographic peaks ready for characterization. hplc and sds-page analyses showed a solid quality of the produced variants and more than variants have been produced in sub mg scale using the outlined method. biomimetic sequestration of co : reprogramming the b domain of protein g through a combined computational and experimental approach esra bozkurt , ruud hovius , thereza a. soares , ursula rothlisberger ecole polytechnique f ed erale de lausanne, federal university of pernambuco protein engineering is a powerful tool to generate highly specific enzymes for biomimetic production of chemicals. among many applications, the development of enzymes to accelerate carbon dioxide fixation is a possible route to limit co emission. in this project, we are inspired by the ancient enzyme carbonic anhydrase which efficiently catalyzes the reversible hydration of carbon dioxide in the presence of a zinc ion active site. to create an efficient biocatalyst, the engineered gb domain containing a his cys zn (ii) binding site was used as a starting point. in subsequent work, b domains comprising of his wat zn (ii) binding sites have been rationally designed to produce carbonic anhydrase mimics. the re-engineering was accomplished through a series of mutations to orient the zinc bound reactive species to form a hydrogen bond network in the active site while retaining the native secondary structure. we performed classical molecular dynamics (md), quantum mechanics/molecular mechanics (qm/ mm) simulations and metadynamics, with the aim to explore potential catalytic roles of the reengineered b domains and to elaborate the reaction mechanism. briefly, we introduced novel zn (ii) binding sites into thermostable b domain. in parallel, experiments are underway. wild-type protein was expressed and purified. structural and mutagenesis studies are ongoing. the results emphasize the power of theoretical work to enable the mimicking of nature's enzymes for desired catalytic functions. the roles of entropy and packing efficiency in determining protein-peptide interaction affinities diego caballero , , corey o'hern , , , , lynne regan , , physics, yale university, integrated graduate program in physical and engineering biology, yale university, mechanical engineering and materials science, yale university, applied physics, yale university, molecular biophysics and biochemistry, yale university, chemistry, yale university despite many recent improvements in computational methods for protein design, we still lack a quantitative and predictive understanding of the driving forces that control protein stability, for example, we do not know the relative magnitudes of the side-chain entropy, van der waals contact interactions, and other enthalpic contributions to the free energy of folded proteins. in addition, we cannot reliably predict the effects of point mutations on enzyme specificity or sequence tolerance in ligand binding sites. the tetratricopeptide repeat (tpr) motif is a common and versatile protein system that has been used as a model to study protein-protein interactions. for example, recent studies have experimentally measured the binding affinity and specificity for different tpr binding pockets and peptide ligands and generated a ranking of the protein-peptide pairs with the highest affinity. to gain a fundamental understanding of the interplay between atomic close packing and fluctuations of side-chain conformations in protein-peptide binding pairs, we performed all-atom langevin dynamics simulations of key residues near the binding interface of tpr proteins and their cognate peptides. the langevin dynamics simulations enabled us to calculate the entropy and potential energy of side chain conformations in the presence of backbone fluctuations for each protein-peptide pair. we compile rankings of the stability and affinity of mutant tpr-peptide structures to those obtained from experimental studies. this research has enhanced our ability to rationally manipulate protein-peptide interfaces. advances from this research will enable the design of tpr modules that specifically recognize biologically important proteins. monitoring protein-protein interactions using tripartite split-gfp complementation assays protein-fragment complementation assay (or pca) is a powerful strategy for visualizing protein-protein interactions in living cells. previously described split-gfp based sensors suffer from the poor solubility of individual pca fragments in addition to background signal originating from their spontaneous selfassembly ( ). we developed a new encoded genetic reporter called "tripartite split-gfp" for visualizing protein-protein interactions in vitro and in living cells. the assay is based on tripartite association between two twenty amino-acids long split-gfp tags, gfp and gfp , fused to interacting protein partners, and the complementary gfp - detector. when proteins interact, gfp and gfp selfassociate with gfp - to reconstitute a functional gfp ( ). using coiled-coils and frb/fkbp model systems we characterize the sensor in vitro and in escherichia coli. we extended our studies to mammalian cells and examine the fk- inhibition of the rapamycin-induced association of frb/fkbp . the small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence and for screening modulators of complex formation in cell-based assays. aldehyde dehydrogenases (aldhs) catalyze the oxidation of aldehydes to their corresponding acids using nad(p) as coenzyme. these enzymes are responsible for the detoxification of lipid peroxidation products, which have been involved in the etiology and pathogenesis of different diseases involving increments in oxidative stress. recent data from our group, showed that aldh a is resistant to inactivation by lipid peroxidation products, even at concentrations - times higher than those required to inactivate aldh a and aldh . the amino acids sequence of the aldehyde-binding site of the three enzymes was analyzed, and it was found that the enzymes susceptible to the effect of lipid peroxidation products (aldh a and aldh ), have cys residues flanking the reactive cys (position ), based on this criteria and considering that these aldehydes react preferentially with cysteine, a mutant of aldh was generated changing the cys residues adjacent to cys . the mutant aldh -cys thr-cys val, was resistant to the inactivation by acrolein and -hne, even at concentrations -fold higher than those required to inactivate aldh . however, the mutant presented values of km , and -fold higher for acrolein, propionaldehyde and acetaldehyde, respectively, compared to the wild type enzyme, but showed a catalytic efficiency similar to the parent enzyme. these data revealed that cys residues near to the reactive cys in aldh are important in the inactivation process induced by lipid aldehydes, but also participate in determining the specificity for the substrates in this enzyme. small molecule-assisted shutoff: a widely applicable method for tunable and reversible control of protein production h. kay chung , conor jacobs , yunwen huo , jin yang , stefanie krumm , richard plemper , , roger tsien , michael lin department of biology, stanford university, department of pediatrics, stanford university, department of pharmacology, university of california san diego, department of pediatrics, emory university, institute for biomedical sciences, georgia state university, department of chemistry and biochemistry, university of california san diego, howard hughes medical institute, university of california san diego, the ability to quickly control the production of specific proteins would be useful in biomedical research and biotechnology. we describe small molecule-assisted shutoff (smash), a technique in which proteins are fused to a self-excising degron and thereby expressed in a minimally modified form by default. degron removal is performed by a cis-encoded hepatitis c virus (hcv) protease, so that applying clinically available hcv protease inhibitors causes degron retention on subsequently synthesized protein copies and suppresses further protein production. we find that smash allows reversible and dosedependent shutoff of various proteins with high dynamic range in multiple cell types, including yeast. we also successfully use smash to confer drug responsiveness onto a rna virus for which no licensed drug inhibitors exist. as smash does not require permanent fusion of a large domain, it should be useful when control over protein production with minimal structural modification is desired. furthermore, as smash only uses a single tag and does not rely on modulating protein-protein interactions, it should be easy to generalize to multiple biological contexts. top, a protein of interest is fused to the smash tag via a hcv ns protease recognition site. after protein folding, the smash tag is removed by its internal ns protease activity, and is degraded due to an internal degron activity. bottom, addition of protease inhibitor induces the rapid degradation of subsequently synthesized copies of the tagged protein, effectively shutting off further protein production. vaccine development has emerged, epitope-focused immunogens, but in the past these have failed to deliver the expected outcome. here, we employed a new computational design methodology (rosetta fold from loops or ffl) to design epitope-focused immunogens. ffl was devised to insert structurally defined functional sites into protein scaffolds. throughout the ffl stages the structure of the scaffold is folded and its sequence designed to stabilize the desired functional conformation of the inserted site. we used ffl to design epitope-focused immunogens for the respiratory syncytial virus (rsv), for which despite the intense research we are still lacking an approved vaccine. we designed three-helix bundles harboring an rsv epitope, that was previously co-crystallized with the neutralizing antibody motavizumab. the designs were thermodynamically stable (tm > ˚c) and showed extremely high affinities to motavizumab (kd pm). structural characterization through x-ray crystallography of antibodybound and unbound scaffolds showed good agreement to the computational models in the overall structure (rmsd - . Å) and exquisite mimicry of the epitope region (rmsd - . Å), when compared to the peptide-epitope in complex with motavizumab. the designed immunogens were used to immunize non-human primates (nhp), and approximately % of the cohort developed rsv neutralizing activity, in some instances with high potency. to evaluate the therapeutic relevance of the elicited neutralization activity, we compared the nhp neutralization titers to those of human sera after natural rsv infection, which generally yields protective levels of antibodies. the neutralization potency of the best nhp responders was comparable to that of the human sera. to better understand the features of the antibodies elicited, we isolated several rhesus monoclonal antibodies (rhmabs) from the animal that exhibited the most potent neutralization. two of the rhmabs bound to the immunogen with very high affinity (kd pm) and were potent rsv neutralizers. interestingly, these rhmabs were approximately fold more potent than the fda-approved prophylactic antibody palivizumab. our results provide the first proof-of-principle for epitope-focused vaccine design, and demonstrate the power of the ffl figure . schematic of nucleotide binding, exchange and hydrolysis in tubulin, and its coupling to mt assembly. exchange of gdp (orange) for gtp (magenta) at the e-site in b-tubulin (blue) happens in the unpolymerized dimer (left). the active, gtpbound tubulin dimer adds to a growing mt (right). interaction of the incoming a-tubulin (green) with the e-site nucleotide at the plus end of a mt (with b-tubulin exposed) results in gtp hydrolysis. the mt cartoon (bottom right) shows an oversimplified representation of a gtp cap as it first grows by tubulin addition and then shrinks by polymerization-coupled gtp hydrolysis (here b-tubulin that is bound to gtp is shown in red and that bound to gdp is shown in blue). cryo-em density map (emdb- ) and atomic model (pdb: jak) for an eb -decorated mt bound to gtpgs. a-tubulin, b-tubulin and eb are colored green, blue, and orange, respectively. computational methodology. we anticipate that ffl will be useful for a variety of other challenges in the computational design of functional proteins. designed repeat proteins as templates for photoactive molecules and fluorescent nanoclusters sara h. mejias , , antonio aires , , javier l opez-andarias , pierre couleaud , , begoña sot , , carmen atienza , nazario mart ın , , aitziber l. cortajarena , imdea nanoscience, c/faraday, , ciudad universitaria de cantoblanco , cnb-csic-imdea nanociencia associated unit "unidad de nanobiotecnolog ıa", departamento de qu ımica org anica i, facultad de qu ımica, universidad complutense self-assembly of biological molecules into defined functional structures has a tremendous potential in nanopatterning, and the design of novel bionanomaterials and functional devices. molecular selfassembly is a process by which complex three-dimensional structures with specified functions are constructed from simple molecular building blocks. we present first the study and characterization of the assembly properties of modular repeat proteins, in particular designed consensus tetratricopeptide repeats (ctprs), and their application as building blocks in order to generate functional nanostructures and biomaterials. ctpr proteins can be assembled into self-standing thin films, and thin nanometer fibers in solution. in this work, we show the use of the designed consensus repeat proteins as scaffolds to template: ( ) photoactive organic molecules, and ( ) fluorescent nanoclusters. .we explore the potential of ctpr proteins to arrange donor-acceptor pairs for electro-active materials. in particular, porphyrin rings arranged by ctprs in a defined distance and orientation for favoring face-to-face orientation which should lead to an improvement in the optoelectronic properties. our results confirm the successful ability of ctpr proteins to be used as scaffold for ordering organic chromophores, while preserving their structure. the unique self assembly properties of ctpr scaffolds have been exploited to generate ordered conductive films of the protein-porphyrin conjugates. these results open the door to fabricate hybrid protein-based solid devices. .we show results on the ability of ctpr to encapsulate and stabilize fluorescent gold nanoclusters. we investigated the influence of the protein sequence in the final properties of the nanoclusters. the structural and functional integrity of the protein template is critical for future applications of the protein-cluster complexes. therefore synthetic protocols that retain the protein structure and function have been developed. as a proof of concept, a ctpr module with specific binding capabilities has been successfully used to stabilize nano clusters. biohybrid photoelectrochemical cells have been developed by functionalizing the hematite photoanode with the light-harvesting cyanobacterial protein c-phycocyanin (pc) yielding a substantial enhancement of the photocurrent density. photoelectrochemical cells combining light-harvesting proteins and inorganic semiconductors have potential for the use in artificial photosynthesis. in this work we present processing routes for the functionalization of hematite photoanodes with pc, including in situ co-polymerization of pc with enzymatically-produced melanin and using a recombinantly produced pc . moreover, recombinant forms of the light-harvesting protein c-phycocyanin from synechocystis sp. pcc were engineered to carry a peptide with affinity for hematite. similarly, a bacterial laccase was engineered to acquire affinity for hematite. results obtained from the different approaches to hematite functionalization and the advantages offered by protein engineering will be presented. minimizing a suitable free energy expression is arguably the most common approach in (ab initio) protein structure prediction. the achieved accuracy depends crucially on the quality of the free energy expression in use. here, we present corrections to existing free energy expressions which arise from the thermal motion of the protein. we (i) devise a term accounting for the vibrational entropy of the protein, and (ii) correct existing potentials for 'thermal smoothing'. (i) vibrational entropy is almost always neglected in free energy expressions as its consideration is difficult. this practice, however, may lead to incorrect output because distinct conformations of a protein can contain very different amount of vibrational entropy, as we show for the chicken villin headpiece explicitly [ ] . for considering vibrational entropy, we suggest a knowledge based approach where typical fluctuation and correlation patterns are extracted from known proteins and then applied to new targets. (ii) at ambient conditions, timeaveraged potentials of proteins are considerably smoothened due to thermal motion where the strength of this effect varies strongly between atoms. distinguishing these inhomogeneities by introducing new atom species regarding their locale environment can therefore increase the precision of time-averaged potentials [ ] . extraction of general principles from the continually growing protein data bank (pdb) has been a significant driving force in our understanding of protein structure. atomistic or residue-level statistical potentials, secondary-structural propensities, and geometric preferences for hydrogen bonding are among the classical insights that arose from observations in the pdb. given the magnitude of structural data available today, it is likely that many quantitative generalizations remain to be made. here we hypothesize that the pdb contains valuable quantitative information on the level of local tertiary structural motifs (terms), with term statistics reflecting fundamental relationships between sequence and structure. we define a term to be the structural fragment that captures the local secondary and tertiary environments of a given residue, and put our hypothesis through a series of rigorous tests. first, we show that by breaking a protein structure into its constituent terms, and querying the pdb to characterize the natural ensemble around each, we can estimate the compatibility of the structure with a given amino-acid sequence through a metric we term "structure score." considering submissions from recent critical assessment of structure prediction (casp) experiments, we find a strong correlation (r . ) between structure score and model accuracy, with poorly predicted regions readily identifiable. this performance exceeds that of leading atomistic statistical energy functions. next, we show that by considering the terms of a structure that are affected by a given mutation, and mining the pdb to characterize sequence statistics associated with each, we are able to predict mutational free energies on par with or better than far more sophisticated atomistic energy functions. finally, we ask whether term statistics are sufficient to enable the design of proteins de-novo. we demonstrate that given a native backbone conformation, term considerations alone with no input from molecular mechanics correctly predict roughly the same fraction of amino acids from the corresponding native sequence as state-ofthe-art computational protein design methods. knowledge-based energy functions have already put pdb statistics to good use by parsing structural environments into geometric descriptors, generally assuming their conditional independence. our results suggest that it may now be possible to instead consider local structural environments in their entirety, asking questions about them directly. if this is the case, then the pdb is an even larger treasure trove of information than it has been generally known to be, and methods of mining it for term-based statistics should present opportunities for advances in structure prediction and protein design. comprehensive understanding of a protein fold is intertwined with successful design. recent advances in designing de novo structures have shown that proteins can be designed for a few globular and helical folds. however, designing all-b structures and barrels remains challenging because loops and intricate long range interactions that are important in these topologies are difficult to control. for designing novel catalysts, the (a/b) -barrel (or tim-barrel) fold is one of the most important examples, for it is the most common topology for enzymes. for almost year, attempts in designing de novo tim barrel structures have all resulted in poorly folded proteins. here we describe the successful design of a -fold symmetrical (a/b) barrel directly from geometrical and chemical principles. designed variants with a wide range of stabilities from being molten globules to cooperatively folded proteins were experimentally characterized, and the results revealed the importance of sidechain-backbone hydrogen bonding for defining the characteristic a/b-barrel. the residue tim barrel structure is among the smallest tim-barrels and has a fully-reversible melting temperature of c. the x-ray crystal structure shows atomic-level agreement with the design model. despite this structural similarity, psi-blast searches do not identify sequence similarities to known tim-barrel proteins. more sensitive profile-profile searches suggest that the design is sufficiently distant from other native tim-barrel superfamilies to be in a superfamily of its own, further implying that nature has only sampled a subset of the sequence space available to the tim-barrel fold. the ability to de novo design tim-barrels opens new possibilities for custom-made enzymes. university of texas southwestern medical center, biofrontiers institute, university of colorado creation of new molecular sensors and actuators based on fluorescent proteins relies on methods for identifying complex photophysical phenotypes and subsequently performing separations on cell populations. we developed a microfluidic flow cytometry approach tailored to interrogating the performance of genetically-encoded fluorophores and present the results of studies employing this technology. the system screens cell-based libraries on the basis of multiple photophysical parameters relevant to imaging, including brightness, photostability, and excited-state lifetime (i.e. a proxy for fluorescence quantum yield) at a rate of up to cells/sec. in a first generation of experiments, molecular dynamics-guided design was used to create a library of mcherry mutants that was screened with this system, resulting in the identification of a variant with a higher stability b-barrel and improved photostability but with a decreased brightness due to reduction in the fluorescence quantum yield. to avoid inadvertent decreases in this important performance criterion, subsequent rounds of selection were performed on the basis of both photostability and excited-state lifetime as sorting criteria. in these second generation selections, mutations were designed to target pathways of oxygen access through the bottom of the bbarrel in addition to a position that directly interacts with the chromophore. furthermore, subsequent rounds of screening were used to improve folding and maturation. the multiparameter sort identified multiple clones with up to -fold improved photostability and up to double the excited-state lifetime of the parent mcherry fluorescent protein. the best mutant we identified produces one order of magnitude more photons before photobleaching compared to mcherry, at excitation conditions characteristic of confocal fluorescence microscopy. our results demonstrate the utility of combining moleculardynamics-guided library design with technology for photophysics-based selections. we anticipate that the new fluorescent proteins obtained in this work will find use in low-copy-number and long-duration imaging live cell imaging applications in cell-lines created by genomic editing techniques. targeted protein degradation achieved through a combination of degrons from yeast and mammalian ornithine decarboxylase rushikesh joshi , ratna prabha c. the maharaja sayajirao university of baroda targeted protein degradation achieved through a combination of degrons from yeast and mammalian ornithine decarboxylase targeting the over accumulated protein in the cell for degradation using specific degrons is an emerging research area. the degradation of the vast majority of cellular proteins is targeted by the ubiquitin-proteasome pathway. but in the case of ubiquitin independent protein degradation, odc/az system is more effective in achieving targeted protein degradation than other types of degradation . ornithine decarboxylase (odc) is key regulatory enzyme in the biosynthesis of polyamines. the protein has two domains namely, n terminal a/b barrel domain and c-terminal b-sheet domain. degradation of odc is mediated by polyamine inducible protein, antizyme (az). antizyme interacts with odc on n-terminal region, which results in degradation of odc by proteasomes. in mammalian odc the c-terminal has an unstructured tail of residues, which pulls odc into proteasome for degradation. it was reported earlier by coffino's group that the unstructured tail acts as a degron in chimeric fusion with gfp . in yeast, same function is achieved by n-terminal residues . present study focuses on accomplishing targeted protein degradation in saccharomyces cerevisiae by adding these two degradation signals or degrons of yeast odc and mammalian odc as tags to a reporter protein. we have selected two degrons namely, n terminal a/b barrel domain of yeast odc and c-terminal residues of mouse odc and grafted them to n and c-terminus of the reporter protein yegfp. degradation of yegfp and yegfp fusion with degrons of odc (degron-yegfp) were monitored by western blot using anti-gfp antibody and fluorescence spectroscopy. initially, the amount of degron-yegfp fusion protein was very low compared to control yegfp. it means that the chimeric protein underwent rapid degradation in the cells. after inhibition of proteasome, increase in the level of degron-yegfp was observed, confirming that the degrons cause rapid degradation of reporter protein through proteasome. earlier, we have also tagged ubiquitin from yeast with last residues of modc and observed enhanced degradation of ubiquitin in saccharomyces cerevisiae. therefore, both the degrons of odc alone and in combination are capable of decreasing stability of reporter protein in the cells. however, the combination of degrons is more effective than either of them in isolation. enzymes fold into unique three-dimensional structures, which underlie their remarkable catalytic properties. the requirement that they be stably folded is a likely factor that contributes to their relatively large size (> , dalton). however, much shorter peptides can achieve well-defined conformations through the formation of amyloid fibrils. to test whether short amyloid-forming peptides might in fact be capable of enzyme-like catalysis, we designed a series of -residue peptides that act as zn dependent esterases. zn helps stabilize the fibril formation, while also acting as a cofactor to catalyze acyl ester hydrolysis. the fibril activity is on par with the most active to date zinc-protein complex. such remarkable efficiency is due to the small size of the active unit (likely a dimer of -residue peptides), while the protein is at least -fold larger in molecular weight. the observed catalytic activity is not limited to ester hydrolysis. we have designed copper binding peptides that are capable oxygen activation. these results indicate that prion-like fibrils are able to not only catalyze their own formation -they also can catalyze chemical reactions. thus, they might have served as intermediates in the evolution of modern-day metalloenzymes. these results also have implications for the design of self-assembling nanostructured catalysts including ones containing a variety of biological and nonbiological metal ions. rational design of the cold active subtilisin-like serine protease vpr with improved catalytic properties and thermal stability abstract proteinase vpr, from a psychrophilic vibrio species and its thermophilic structural homologue, aqualysin i (aqui) from thermus aquaticus, we set out to design a mutant of vpr which would be more thermostable, but would retain the high catalytic activity of the wild type enzyme. our starting protein template was a previously stabilized mutant containing two inserted proline residues close to the nterminus of vpr (n p/i p). this vpr_n p/i p mutant was shown to have a significantly increased thermal stability but displayed a concomitant tenfold loss of catalytic efficiency. from our previous studies we selected two mutations, one which increased catalytic activity (q k) of the enzyme significantly and another which stabilized the protein against thermal denaturation (n d). the n d mutation had been shown to introduce a salt bridge into the structure of the cold adapted proteinase, yielding higher stability but without negative effects on activity. the q k exchange had been shown to double the turnover number (kcat) to that of the wild type enzyme. insertions of these selected mutations into the vpr_n p/i p mutant were according to predictions; the q k increased the kcat tenfold, and the n d mutation increased the thermal stability. in the combination mutant, vpr_n p/i p/n d/q k, thermal stability was increased by c and c, in terms of tm and t %, respectively. furthermore, the catalytic activity of the mutant was somewhat higher than that of the wild type enzyme. critical peptide stretches may not serve as faithful experimental mimics for protein amyloidogenesis bishwajit kundu , dushyant garg certain amino acid stretches are considered critical to trigger the amyloidogenesis in a protein. these peptide stretches are often synthetically produced to serve as experimental mimics for studying amyloidogenesis of the parent protein. here we provide evidence that such simple extrapolation may be misleading. we studied the amyloidogenesis of full length bovine carbonic anhydrase ii (bcaii) and compared it with those formed by its critical amyloidogenic peptide stretch - (pepb). under similar solution conditions and initial monomeric concentrations, we found that while amyloid formation by bcaii followed aggregation kinetics dominated by surface-catalyzed secondary nucleation, pepb followed classical nucleation-dependent pathway. the afm images showed that bcaii forms short, thick and branched fibrils, whereas pepb formed thin, long and unbranched fibrils. atr-ftir revealed parallel arrangement of cross b sheet in bcaii amyloids, while pepb arranged into antiparallel b sheets. amyloids formed by bcaii were unable to seed the fibrillation of pepb and vice versa. even the intermediates formed during lag phase revealed contrasting ftir, far uv cd signature, hydrophobicity and morphology. we propose that for any polypeptide, the sequences flanking a critical region are equally effective in modulating the initial nucleation events, generating prefibrillar and finally fibrillar species with contrasting characteristic. the results have been discussed in light of amyloid polymorphism and its importance in the design of therapeutic strategies targeting such toxic regions. aksana labokha , ralph minter all approved biological drugs target extracellular proteins and not the majority of the expressed human genome, which resides within intracellular compartments. included in the latter category are many important, disease-relevant targets which cannot be easily addressed by small molecule approaches, such as the oncology targets c-myc and k-ras. although bacteria and viruses have evolved strategies to deliver biological material to the cell cytoplasm and nucleus, our ability to engineer recombinant proteins to replicate this is somewhat limited by (i) our nascent understanding of protein uptake and trafficking pathways and (ii) the ability to easily quantify cell delivery to the cytoplasm and cellular organelles. the aim of my project is to address these challenges by developing an effective assay for cytoplasmic uptake and then using it to measure the delivery efficiency of recombinant proteins which mimic natural delivery strategies e.g. cell penetrating peptides fusion, exotoxin mimics, and supercharged proteins (proteins with high surface charge which can enter cells). i also intend to explore the influence of the rab superfamily, which are the master regulators of protein trafficking, to influence and control both the kinetics and final subcellular destination of exogenous proteins. protein engineering: what's next? with the growing industrial need for engineering enzymes for the deconstruction and transformation of plant biomass in biorefineries, there is a want for the development of new approaches for designing special purpose biocatalysts. techniques, such as directed evolution, which mimic the natural selection process by evolving proteins towards the improvement of a given property, have unquestionably demonstrated their value and are routinely used in large industrial companies. nevertheless, the brute force employed in these methods, could significantly gain from an all-atom description of the underlying catalytic mechanisms, to center the efforts on more limited areas of the protein. in the last years, we have developed computational tools, which combine the electronic structure description of qm/mm methods with the potential to model long time scale processes of pele, to study the details of a variety of reactions. examples, which will be discussed, include rationalizing the selective oxyfunctionalization of steroids using fungal enzymes and the study of the effect of point mutations on the oxidation efficiency of laccases. these methods have shown their potential not only at the descriptive level but, more importantly, through their high predictive capability that opens many opportunities for their use in biotechnology. in this talk, we will show how recent advances in in silico approaches are setting new grounds for future computer guided directed evolution. several orthogonal bioreactions take place simultaneously within membrane bound organelles in eukaryotes and proteinaceous microcompartments in bacteria. these subcellular structures contain sets of enzymes co-involved in metabolic pathways. towards the goal of creating artificial protein microreactors, we seek to develop an artificial organelle that emulates the metabolic activity of the carbon fixating organelle of autotrophic bacteria, the carboxysome. here, we show that the two key carboxysomal enzymes, ribulose- , -bisphosphate carboxylase/oxygenase (rubisco) and carbonic anhydrase (ca), can be efficiently co-encapsulated using our previously reported encapsulation system which is based on a bacterial capsid formed from the protein lumazine synthase (aals- ). our preliminary results suggest that the enzymes can act in tandem and that the co-encapsulation of ca with rubisco in the capsid is necessary for enhanced rubisco activity in vitro. we attribute this observation to the high local concentrations of the rubisco substrate, co , produced by ca within the capsid. we are developing a theoretical model of a minimal carboxysome using the kinetic rate constants of our rubisco and ca variants and aals- as the shell to complement these experiments. next, we will incorporate our minimal carboxysome within an expression host such as e.coli, opening up the possibility of further optimization through directed evolution. in the past targeting and engineering of chemokines has led to several interesting drug candidates. [ ] amongst them, met-rantes, a met-ccl with high g protein-coupled receptor (gpcr) affinity but no subsequent signal transduction, as well as mutants addressing the interaction with the so-called glycosaminoglycans (gags) seem to be the most promising candidates. both, gag knockout as well as gag affinity matured chemokine isoforms have been considered as anti-inflammatory drug candidates, out of which an il- mutant with modifications reached clinical phase where it was profiled for acute neutrophil-related exacerbation in copd. [ ] cxcl (ip- ) is a proinflammatory chemokine released by various cells following stimulation by interferon g (ifn-g) . it is therefore considered as a late chemokine being responsible for the attraction of different lymphocytes. [ ] any therapeutic indication is consequently related to chronic and multiple applications. we have therefore engineered cxcl very conservatively at positions to ultimately generate dominant-negative mutants with a mildly improved gagbinding affinity and an entire knock off gpcr activity. the first steps of our engineering approach were in silico modelling of the mutants and the establishment of a suitable upstream-and downstreamprocessing protocol. next we generated a fluorescently engineered cxcl variant for our fluorescence-based affinity studies which was subjected to biocomparability investigations relative to the native, non-fluorescent protein. compared to the wild type, the fluorescently engineered mutant exhibited similar biological, chemotactic and gag-binding properties. next we started to produce sufficient amounts of the members of our nascent mutant library which were tested with respect to their biophysically behavior as well as to their knocked out chemotactic potency on cells. these experiments included gel electrophoresis and western blot analysis to determine identity and purity; circular dichroism (cd) and chaotrope-induced unfolding to approximate structure; isothermal fluorescence titration (ift); surface plasmon resonance (spr) and isothermal titration calorimetry (itc) to quantify gagbinding affinity and boyden chamber experiments to determine the chemotactic activity. our results show that we are able to tune the gag binding strength along with the gpcr activity of human cxcl which could lead to therapeutic applications in the future. nanodiscs are composed of a nanometer-sized phospholipid bilayer encircled by two a helical, amphipathic membrane scaffold proteins (msps). these particles provide a unique detergent free lipid bilayer model enabling biochemical and biophysical characterization of membrane proteins in a physiologically relevant medium. previously, the largest diameter reported of a nanodisc assembled using msps was about - nm. here we present a method to create large nanodiscs (up to nm in diameter) assembled with covalently circularized msps (cmsp). we can observe the homogeneity in nanodiscs diameter as a narrow distribution using negative-stain em. using our method, we have created nm nanodiscs and used them to study poliovirus ( nm diameter) entry and rna translocation. a nm nanodisc is sufficiently large to accommodate multiple copies of the cd receptor (also known as the poliovirus receptor), and has enough surface area to act as a surrogate membrane for the rna translocation complex during viral uncoating. the nm nanodiscs functionalized with the his-tagged ectodomain of poliovirus receptor, cd , were generated by adding lipids derivatized with a nta nickel- chelating head group to the lipid mixture during nanodisc assembly. cd receptor was added to the already assembled nanodiscs and incubated for minutes at room temperature. the receptordecorated nanodisc complex was purified by size exclusion chromatography. the purified complex was then incubated with poliovirus for minute at c, and then heated to c for minutes to initiate receptor-mediated viral uncoating. virus binding to nanodisc-cd complex and subsequent insertion of viral components into and across the membrane were confirmed by negative-stain electron microscopy (figure c) . to obtain a high-resolution structure for the rna translocation complex we conducted single-particle cryo-em studies using a polara f microscope. unlike liposomes, generating a reconstruction of samples containing nanodiscs is less complicated since the nanodiscs are more homogenous in size, and allow for thinner ice. also, the viral rna can be visualized more easily. the method for making large nanodiscs as well as the negative stain and cryo-em data will be will be presented and discussed. parametric design of alpha-helical barrels and pore-like assemblies with very high thermodynamic stabilities computational design of novel protein structures and enzymes with new functions is a promising tool to create superior biological materials with tailor-made properties, new pharmaceuticals, complex fine chemicals or renewable fuels. it also challenges our understanding of protein folding, protein evolution, molecular recognition and catalysis. here we present a procedure for designing proteins with backbones produced by varying the parameters in the crick coiled-coil generating equations [ ] . combinatorial design calculations using the software suite rosetta identify low energy sequences for alternative helix supercoil arrangements. after that, loop modeling is applied to connect the designs with lowest energy. the extent to which the designed sequences encode the designed structures is evaluated using large-scale structure prediction calculations, as well as symmetric and asymmetric protein-protein docking calculations. subsequently, synthetic genes are generated for sequences that converge strongly on the designed structure for experimental characterization. we applied this approach to monomeric three and four helical bundle structures as well as a pentameric five-helix bundle structure using idealized coiled-coil geometries [ ] . recently we expanded this approach to higher complexity backbones, which resulted in the de-novo design of monomeric, antiparallel six-helix bundles with untwisted, left-and right-handed geometries. circular dichroism (cd), size-exclusion coupled multi-angle light scattering measurements (sec-mals), negative stain electron micrographs (em) and small angle x-ray scattering (saxs) of these designs suggest that they indeed form the designed structures. in addition, we used rosetta protein-protein interface design functionality to computationally design oligomers out of our previously published three and four helix bundle structures to generate self-assembling pore-like structures with the potential use as channels or transporters. again, experimental validation of these designs by cd, sec-mals, em and saxs show that the designs are correct. we are currently undertaking further structural investigation of all these designs by x-ray crystallography. the designs described above can act as templates for protein or small molecule binding, holding a catalytic machinery or for scaffolding enzymes in reaction cascades. some of these applications are currently under investigation, including a self-sufficient redox system employing two copper-centers, binding of heme-moieties as a prosthetic group and tailoring the pore-like geometries to be used in nanopore sequencing. university of washington, university of california, san francisco, repeat proteins are an example of how evolution proceeds by building on existing structures and functions, but also a source of modular protein scaffolds for molecular recognition and biomaterials. however, it is unclear whether the limited number of folds and families that we know today is the result of the intrinsic limitations of polypeptide chains or the consequence of the path followed by evolution. we explored this hypothesis by computational design of repeat proteins based on modular units formed by two alpha helices and two loops of variable lengths, without relying on information from available repeat protein families. the automated sampling of the conformational space resulted in a large number of architectures from which de novo designs were selected for experimental characterization. % of the proteins were stable up to c and monodisperse and designs were structurally validated by small angle x-ray scattering. crystal structures were solved for of them, with root mean square deviation from the models between . Å and . Å. the designs differ from known proteins both at the sequence and structure levels and cover a broader range of geometries than observed in naturally occurring repeat protein families, indicating that existing architectures represent only a small fraction of what can be achieved. our results show that it is possible to expand the range of repeat protein architectures beyond the naturally occurring families, and that computational design can provide new scaffolds and enable the design of proteins tailored for specific applications. the serpin family of proteins consists of over members, all with a highly conserved native structure that is metastable ( ). serpins use this metastability to control the activity of proteases, via a specific inhibitory process. the serpin binds to its target protease through specific residues within the reactive centre loop, the protease cleaves the loop and results in a large conformational change causing the protease to become distorted and catalytically inactive whilst the serpin becomes much more stable ( , , ) . the metastable nature of aat is therefore required to facilitate the rapid and gross conformational changes required for its inhibitory function ( , ) . several disease-causing mutants of aat have been identified, the most common of them being the z-variant ( ). the z-variant has an increased propensity to polymerize in the endoplasmic reticulum of hepatocytes leading to cell death and liver damage ( ) . during the past fifteen years, many groups have unsuccessfully screened a number of serpins and a vast range of solution conditions to identify a combination of serpin and conditions that will enable the folding reaction of a serpin to be characterized. we have now taken an alternative approach and designed a synthetic "model" serpin that folds reversibly to its native state. in order to do this, we used a consensus design approach, analysing a sequence alignment of serpin sequences and determining the prevalent amino acid residue at each position, we termed this serpin conserpin (consensus serpin). here we present the structural, biophysical and functional characterisation of conserpin. combined crystallographic and folding studies reveal the characteristics of conserpin that likely dictate its unique stability and folding behaviour, whilst retaining activity as a serine protease inhibitor. the development of enhanced protein binding scaffolds is a key for engineering protein inhibitors and biosensors with advanced characteristics. utilizing the structural variability and designability of repeat proteins offers a means for designing protein binders where the overall shape is customized to optimally match a target molecule. we developed a computational protocol for the design of repeat proteins with a predefined geometry. by combining sequence optimization of existing repeats and de novo design of capping structures, we designed leucine-rich repeat (lrr) proteins where the building blocks assemble into a novel structure. the suggested design procedure was validated by engineering an artificial donut-like ring structure, which is constructed from ten self-compatible repeats. characterization of several designed constructs further suggests that buried cysteines play a central role for stability and folding cooperativity in certain lrr proteins. this effect could provide a means for selectively stabilizing or destabilizing specific parts of an lrr-based protein binder. the computational procedure may now be employed to develop repeat proteins with various geometrical shapes for applications where greater control of the interface geometry is desired. engineering apobec g enzymes for altered specificity and processivity louis scott , muhammad razif , aleksandra filipovska , , oliver rackham , harry perkins institute of medical research, school of chemistry and biochemistry, the university of western australia apobec g (a g) is a host-encoded protein involved in the defense against hiv- and other retroviral infections. a g is a cytidine deaminase with a ' to ' processive nature, causing targeted c to t mutations along a dna strand. the catalytic and processive activity of a g leads to the hypermutation of nascent retroviral cdna, resulting in premature termination codons and dysfunctional proteins. ultimately, the action of a g inhibits viral replication. the ability of a g to jump and slide along a dna strand, deaminating at targeted sequences, makes it an interesting candidate for protein engineering. engineered a g enzymes for increased activity, altered specificity, and altered processivity are attractive options for expanding the dna modifying enzyme toolbox. mutation of catalytic residues, residues thought to affect its processive nature and those thought to be involved in target recognition, can create novel a g enzymes. using structure guided selection, residues in key functional sites that are amiable to mutation will be chosen. individuals from the resulting libraries of mutants will be selected by directed evolution for desired characteristics. the resulting a g enzymes will be examined for the relationship between their structure and function. such engineered a g enzymes could be targeted to catalyse the reversion of deleterious genetic mutations. furthermore, engineered a g enzymes could be used in mutational studies that call for targeted deamination along a dna strand, or mutational studies that call for unspecific and high throughput dna deamination. engineering porous protein crystals as scaffolds for programmed assembly thaddaus huber , luke hartje , christopher snow a key motivation for nano-biotechnology efforts is the creation of designer materials in which the assembly acts to organize functional domains in three dimensions. crystalline materials are ideal from the validation perspective because x-ray diffraction can elucidate the atomic structure. relatively little work has focused on engineering protein crystals as scaffolds for nanotechnology, due to the technical challenges of coaxing typical proteins into crystallizing, and the likelihood of disrupting the crystallization process if changes are made to the monomers. we have circumvented these limitations by installing guest protein domains within engineered porous crystals ( nm pore diameter) that have been rendered robust using covalent crosslinks. the retention of the scaffold structure despite changes to the solution conditions and macromolecule uptake can be validated through x-ray diffraction. we have engineered scaffold crystals for the non-covalent and covalent capture of guest macromolecules. by controlling the reversible loading and release, we can prepare "integrated" crystals with spatially segregated guest loading patterns. as assessed using confocal microscopy, such host-guest crystals are highly stable. ultimately, the resulting crystals may serve as a robust alternative to dna assemblies for the programmed placement of macromolecules within materials. engineering ultrasensitive protein probes of voltage dynamics for imaging neural activity in vivo francois st-pierre , , michael pan , , helen yang , xiaozhe ding , , ying yang , , thomas clandinin , michael lin , department of bioengineering, stanford university, department of pediatrics, stanford university, nervous systems encode information as spatiotemporal patterns of membrane voltage transients, so accurate measurement of electrical activity has been of long-standing interest. recent engineering efforts have improved our ability to monitor membrane voltage dynamics using genetically encoded voltage indicators. in comparison with electrophysiological approaches, such protein-based indicators can monitor many genetically defined neurons simultaneously; they can also more easily measure voltage changes from subcellular compartments such as axons and dendrites. compared with genetically encoded calcium indicators, voltage sensors enable a more direct, accurate, and rapid readout of membrane potential changes. however, several challenges remain for in vivo voltage imaging with genetically encoded indicators. in particular, current voltage sensors are characterized by insufficient sensitivity, kinetics, and/or brightness to be true optical replacements for electrodes in vivo. as a first step towards addressing these challenges, we sought to develop new voltage indicators that further improve upon the performance of the fast voltage sensor accelerated sensor of action potentials (asap ). in asap , voltage-induced conformational changes in a natural voltage-sensing domain perturb the fluorescence emission of a covalently linked green fluorescent protein (gfp). using a structurebased approach to guide mutagenesis, we discovered several amino acids that tune the kinetics and voltage sensitivity of asap . these residues are not only located in the voltage-sensing domain, but also in the fluorescent protein and in the linkers bridging sensing domain and gfp. our most improved variant, asap , exhibits improved sensitivity to voltage transients such as neuronal action potentials and subthreshold depolarizations. we sought to characterize the ability of these new voltage sensors to monitor neural activity in vivo using laser-scanning two-photon microscopy, a technique that allows imaging with lower autofluorescence and deeper tissue penetration. we report that asap sensors were able report stimulus-evoked voltage responses in axonal termini of the fly visual interneuron l . asap sensors enabled voltage imaging with dramatically improved temporal resolution compared to three recently reported calcium and voltage sensors. overall, our study reports novel voltage indicators with improved performance and highlights how specific amino acids can tune the performance of a proteinbased fluorescent sensor. we anticipate that these results will pave the way for further engineering of voltage sensing proteins, and that our new sensor asap will facilitate current and future efforts to understand how neural circuits represent and transform information. assembly of armadillo repeat proteins from complementary fragments erich michel , randall watson , martin christen , fabian bumback , andreas pl€ uckthun , oliver zerbe demonstrated that complementary fragments of a designed consensus armadillo repeat protein (armrp) recognize each other [ ] . the two fragments ym : ma, in which y, m and a denote the n-cap, internal repeats and the c-cap, respectively, form a : complex with a nanomolar dissociation constant, which is essentially identical to the crystal structure of the continuous ym a protein. we further demonstrate that structurally intact armadillo repeat protein complexes can be reconstituted from fragments obtained at various split sites -essentially after every repeat but also within repeats. the fragments display variable affinities towards each other, depending on the split site. the low affinity of some complementary pairs can be dramatically increased upon addition of peptide ligands. while a number of proteins are known that can be reconstituted from fragments we believe that the fact that armadillo repeat proteins can be reconstituted from various complementary fragments is novel and opens new interesting perspectives and applications in biochemistry. a reliable method for generating optically controllable proteins would enable researchers to interrogate protein functions with high spatiotemporal specificity. we recently engineered a tetrameric fluorescent protein, dronpa n, that undergoes light-induced monomerization, then developed a general architecture for lightinducible proteins based on this light-induced transition. we created proteins whose active sites were blocked by fused dronpa n domains in the dark, but would become unblocked by light. here we present further two extensions to this concept that together enabled the generalization of this method to additional classes of proteins. first, we engineered a photodissociable dimeric dronpa (pddronpa) with tunable affinity, faster photoswitching speed, and decreased level of protein aggregation, enabling better performance of fusion proteins. second, we introduce the concept of caging a protein active site by insertion of dronpa domains into loops rather than strictly at the protein termini. we use the pddronpa system to impose optical control on kinases and the cas endonuclease. the resulting light-inducible mek kinase, raf kinase, and cas endonuclease showed high caging efficiency of protein activities in the dark, and robust protein activation upon light illumination. we believe that our efforts on further improving and generalizing this method would bring the power and benefits of light control to a broad community of biologists. exploring the evolution of folds and its application for the design of functional hybrid proteins saacnicteh toledo patiño , birte h€ ocker the structural diversity of proteins may appear endless, nevertheless even large protein complexes can be decomposed into protein domains and smaller sub-domain sized fragments. only recently, we could identify such fragments employing sequence-based comparisons of different folds, as the tim-barrel and the flavodoxin-like fold (farias-rico et al., ) . as an extension of this work, we compared all a/b proteins and identified several fragments shared by different folds illustrating how nature may have achieved structural and functional diversity from a reduced set of building blocks. inspired by this combinatorial concept, we searched for homologous fragments bearing active sites to engineer a functional fold-chimera. we extracted the vitamin-b binding part from methylmalonyl coa mutase, which belongs to the flavodoxin-like fold (fl) and used it to replace the corresponding fragment in uroporphyrinogen iii synthase, which belongs to the hemd-like fold (hdl). the new hybrid resulted in a stable and well-folded protein whose structure was determined by x-ray crystallography. moreover, cobalamin-binding function was successfully transferred to the new protein from the fl parent, which shows the advantage of using this approach for the design of new functional proteins. in addition, profile alignments revealed sequence and structural evidence that suggested an evolutionary path for hdl from fl by gene duplication. to test this hypothesis, we expressed a modified c-terminal half of uroporphyrinogen iii synthase and solved its structure by nmr spectroscopy, thereby confirming the predicted fl architecture. altogether, our approach facilitates the detection of common ancestry among different folds contributing to our understanding of protein development. furthermore, our results show how new complex proteins can be designed using fragments of existing proteins that serve as building blocks in a lego-like manner. we believe that combining fragments containing existing properties will provide a successful method for the design of novel functionalities in the future. [ ] . the active site cysteine plays a key role in the reaction mechanism and we investigated this residue in more detail by exchanging this moiety with selenocysteine (sec) and homocysteine (hcy). the sortase mutants were generated by semisynthesis using expressed protein ligation (epl). the resulting cys-, sec-and hcy-sortase enzymes were characterized and showed a moderate - -fold reduction of activity for sec-sortase. the activity of hcysortase was barely detectable with less than % of wildtype activity. the alkylation efficiency of the active site nucleophiles correlated with the expected pka values of sec, cys and hcy. analysis of the ph dependency of the transpeptidation reactions showed that the activity optimum of sec-sortase was shifted towards more acidic conditions. these investigations provide further insights into the reaction mechanism of sortase a and the semisynthetic enzymes may provide new tool for further biochemical studies. propanediol oxidoreductase from escherichia coli (fuco) uses nadh/nad as cofactors to catalyze the conversion of s-lactaldehyde to s- , -propanediol and vice versa. fuco is an attractive enzyme in the search for possible biocatalysts producing a-hydroxy aldehydes, which are important for the synthesis of natural products and synthetic drugs. enzymes catalyzing these types of reactions are unique in catalytic power and stereoselectivity. the usage of fuco in synthetic industry is limited by the restricted substrate scope, which makes fuco inactive with larger phenyl-substituted alcohols. we used reengineering and directed evolution to enable fuco to catalyze the regio-and enantioselective oxidation of arylsubstituted vicinal diols, such as phenylpropanediols, into a-hydroxy aldehyde products. we mutated amino acids considered to restrict the entry into the active site, and modeled the mutants that were most active with the substrates phenylacetaldehyde and s- -phenyl- , -propanediol and performed docking studies with them. as expected, our experimental and in silico results show that the mutations enlarge the active site cavity and enable the mutant enzymes to accommodate the new substrates. we also found specific amino acids in the active site, which need to be conserved to allow the substrates to make stabilizing interactions. interestingly, an asparagine residue makes the mutant enzymes able to discriminate between phenylacetaldehyde and s- -phenyl- , -propanediol. in conclusion, we successfully re-engineered the specialist enzyme fuco to accept also bulkier molecules as substrates, thereby making it more useful for industrial purposes. one way to gain insight into the sequence-structure-function relationship in proteins is to de novo design artificial proteins. despite impressive successes in de novo protein design, designing a folded protein of more than amino acids still remains a challenge. using this approach, an idealized (beta/ alpha) fold protein was designed leading to the production of a protein of amino acids (octarellin v). this protein showed a low solubility and stability. through directed evolution we produced a soluble variant, octarellin v. . the biophysical characterization of octarellin v. shows a well folded monomeric and thermostable protein with a tm over c. however, after several screenings, we could not find crystallization conditions for this protein. as an alternative, we decided to co-crystallize octarellin v. with a protein partner that helps the crystallization process. we used protein partners: alpha-reps and nanobodies. the first one is characterized to interact through a large surface contact, whereas the second is characterized to recognize an specific small epitope. crystallization of both complexes was performed successfully by vapor diffusion and the structures were solved. the experimental structures correspond to the first for an artificial protein of this size and it will allow to criticize the computational design of the octarellin v. generation of synthetic antibodies against membrane proteins in nanodiscs for use in structural biology methods. here, we describe a robust strategy for generating a class of high performance antibodybased affinity reagents that have proven useful in determining the structures of relevant functional states of membrane proteins. these reagents are fab fragments that are generated by phage display from fully synthetic libraries and are called synthetic antibody fragments, or sabs. we have developed phage display sorting strategies that can trap a desired conformational state, making it accessible to structural analysis, or target a particular epitope on the protein surface. however, to maximize this technology for membrane proteins, several limitations of phage display sorting in detergent formats had to be overcome, the greatest being that using detergents can produce non-native conformational biases. we sought to address these limitations by embedding membrane proteins into nanodiscs, soluble lipidfilled discoidal particles, to better mimic the native membrane environment. nanodiscs stabilize the membrane protein and allow it to respond to conformation-inducing stimuli such as ligands, ions and ph during phage display selections. we have established and validated an improved protocol using two membrane protein systems: ) mj , an archaeal membrane protein of unknown function, and ) cora, a pentameric magnesium ion channel. using mj , we compared the nanodisc protocol with the standard method performed in detergent, and as an important byproduct, we characterized the influence of the membrane protein environment on the apparent affinity of sabs to their cognate antigen. using cora, we developed a more sophisticated sorting strategy resulting in a variety of sabs specific to either the open or closed conformation of the channel. finally, using sabs as crystallization chaperones we obtained the structure of mj at . Å resolution, and crystallized cora in several new conditions. lipocalin-type prostaglandin d synthase (l-pgds) is a member of the lipocalin superfamily, and binds a large variety of small hydrophobic molecules. using this function of l-pgds, we have already reported the feasibility of l-pgds as a novel drug delivery vehicle for the poorly water-soluble drugs [ ] . sn- , -ethyl- -hydroxy-camptothecin, is a semi-synthetic analogue of anti-cancer alkaloid camptothecin that targets dna topoisomerase i. despite of the potent anti-tumor activity, however, sn- was not used directly in a clinical practice due to its poor water solubility. thus, irinotecan hydrochloride (cpt- ), which is the water-soluble prodrug of sn- , is used for the cancer treatment. however, cpt- shows approximately . % cytotoxic activity of sn- against the various cancer cell lines in vitro, and its metabolic conversion rate is % of the original volume of cpt- . here, we show the development of the drug delivery system utilizing l-pgds, which enables a direct clinical usage of sn- . first, we investigated the effect of l-pgds on the solubility of sn- . in the presence of mm l-pgds, the concentration of sn- was . mm, which was , -fold as compared with that in pbs. then, we carried out isothermal titration calorimetry measurements to investigate the detailed binding mode of sn- to l-pgds. as a result, it was revealed that l-pgds binds three molecules of sn- , and the dissocia- control over the sensitivity with which artificial biomolecular receptors respond to small changes in the concentration of their target ligand is critical for the proper function of many cellular processes. such control could likewise be highly useful in artificial biotechnologies in which highly responsive behavior is of value, such as biosensors, genetic logic gates, and "smart" materials and delivery devices. in nature, the control of molecular responsiveness is often achieved using "hill-type" cooperativity, a mechanism in which sequential binding events on a multivalent receptor are coupled such that the first enhances the affinity of the next, producing a steep, higher-order dependence on target concentration. here we use an intrinsic-disorder-based mechanism that can be implemented without requiring detailed structural knowledge to rationally introduce this potentially useful property into several normally noncooperative biomolecules. to do so we fabricate a tandem repeat of the receptor that is destabilized (unfolded) via the introduction of a long, unstructured loop. the loop spatially separates the two sets of the two halves of the binding sites, preventing a complete binding site that enables target molecule binding without prior closure of the loop. thus, the first binding event requires the energetically unfavorable closing of this loop, reducing its affinity relative to that of the second binding event, which, in contrast occurs at a pre-formed site. using this approach we have rationally introduced cooperativity into three unrelated aptamers, achieving in the best of these a hill coefficient experimentally indistinguishable from the theoretically expected maximum. the extent of cooperativity, and thus the steepness of the binding transition, are, moreover, well modeled as simple functions of the energetic cost of binding-induced folding, speaking to the quantitative nature of this design strategy. essential and non-essential amino acid species for an ancestral protein satoshi akanuma the translation system is an essential element for life because it links genetic information embedded in genes to functional molecules, proteins. the modern genetic code, which encodes the standard amino acids (and three terminations) using triplet codons, is shared by most of the extant organisms on the earth. a number of theories have been proposed for the origin and evolution of the genetic code, and these theories suggest that only a fewer amino acids were used in primitive proteins and later the amino acid repertoire gradually increased up to through the course of evolution. if so, one would wonder how many number of and which types of amino acids were involved in the primitive proteins. i have begun to address this issue experimentally. i first resurrected several ancestral proteins and then restricted the amino acid usage of one of the resurrected proteins. i targeted nucleoside diphosphate kinase (ndk) that catalyzes the transfer of a phosphate from a nucleoside triphosphate to a nucleoside diphosphate. ndk may have arisen early because at least one gene that encodes ndk is present in most extant organisms. the first step in the reconstruction of ancestral ndk sequences is to prepare multiple amino acid sequence alignments using homologous sequences of ndk from extant species. then, phylogenetic trees were built. ancestral sequences of ndk that represent the last common ancestors of archaea and of bacteria were reconstructed using the information contained in the predictive phylogenetic trees. the reconstructed ancestral kinases are extremely thermally stable [akanuma et al., ] . then, using the most thermally stable ancestral ndk, arc , as the starting molecule, i restricted its amino acid usage. arc does not contain any cysteine residue and therefore consists of amino acid species. i completely replaced one of the amino acid species by other amino acid species and thus created proteins each of which consisted of amino acid species. then, i evaluated the stabilities and activities of the resulting arc variants to assess the individual contributions of the amino acid species. as the result, i found that the amino acid species do not equally contribute to the stability and activity of arc and that some amino acid species can be easily lacked but others are important or essential for its stability and function. the result clearly shows that the full amino acid species are not necessarily essential and supports the hypothesis that proteins in the early stage of evolution were made from a reduced amino acid set. the protein surface recognition for protein-protein interactions (ppi) is involved in signal transduction, immune reaction, and creation of the nanostructures in living cells. the methods for rational designing of ppi that could provide non-antibody scaffolds and nanostructured materials are required for the therapeutic and nanotechnological applications. although there have been some successful rational designs with computational methods, it is still difficult to design freely the ppi onto arbitrary proteins. the reason for this limitation is decreased solubility in the designed protein due to the additional hydrophobic residues in order to drive ppi. another reason is a limited set of design modes by which proteins can interact, because the target proteins have individual surface structures. therefore, many methods of constructing an interface for numerous target scaffold proteins without loss of their solubility are necessary. surface exposed a-helices are often observed in natural globular proteins. moreover, there are many examples for naturally occurring oligomeric proteins where an a-helix from each subunit interacts to form an intermolecule coiled coil. further, the works related to designing of artificial helical bundle reported by the several other groups have provided information about how to generate and tune the interaction between a-helices. therefore, a surface exposed a-helix would be a good target for designing a de novo interface onto the scaffold protein. here we engineered two different proteins, sulerythrin and cys-larfh, to form the cys-larfh-sulerythrin dimer-cys-larfh heterotetramer via an intermolecular helix-helix interaction. wild-type sulerythrin forms a dimeric eight-helix bundle. cys-larfh is a designed monomeric protein that forms four-helix bundle containing interhelical s-s bonds. both sulerythrin and cys-larfh are extremely thermostable. to design protein-protein interfaces onto the individual proteins, we first introduced six leucines to the two a-helices of sulerythrin and three leucines to a a-helix of cys-larfh. as expected, the introduction of the hydrophobic amino acids reduced their solubilities. to recover the solubility, we then introduced six aspartates or glutamates around the hydrophobic surface of the sulerythrin (hereafter referred to as l d or l e). similarly, three arginines were introduced around the artificial hydrophobic surface of the cys-larfh (hereafter referred as iv- l r). the solubilities of the mutants with the hydrophobic interface and additional charged residues were recovered their solubility. in addition, the sulerythrin mutants l d and l e exist mainly as dimer. the cys-larfh mutants iv- l r, also exists as monomer. we then examined the interaction between l e or l d and iv- l r. a pull-down experiment, in which co beads bound to either his-tagged cys-larfh and iv- l r were used to pull down wild-type sulerythrin, l d, or l e, demonstrates that l d or l e specifically interacts to iv- l r. furthermore, when analysed by size exclusion chromatography, the dominant peaks of the mixture of l d and iv- l r appeared at the volume expected for the heterotetrameric complex. thus we successfully created the de novo ppi by using a very simple concept involving hydrophobic interaction in combination with charge interactions. in vitro selection of liposome anchoring peptide by cdna display naoto nemoto , ryoya okawa , yuki yoshikawa , toshiki miyajima , shota kobayashi a liposome-anchoring peptide (la peptide) was selected against liposomes composed of dioleoyl-snglycero- -phosphocholine (dopc) by in vitro selection using cdna display method. the selected peptide la peptide consists of the n-terminal region (hydrophobic) and the c-terminal region (basic) in a characteristic manner. thus, la peptide was synthesized chemically and the interactions between la peptide and particular types of liposomes were investigated and confirmed by confocal laser scanning microscopy. designing of a novel platinum-binding amino acid sequence on a protein surface asumi kaji , hiroya niiro , satoshi akanuma , tetsuya uchida , akihiko yamagishi designing of a novel interaction between a metal and a protein is a key to create hybrid materials between organic and inorganic materials. for example, in a glucose biosensor, which is widely used for measuring glucose concentration in blood, glucose oxidoreductase molecules are immobilized on a platinum electrode by polyacrylamide gel. a metal-binding tags that is added to the n-or cterminus of a protein is also used for fix the protein to a metal. however, a technique to create a metal binding site on a desired position of a protein has not been invent. if such a technique would be established, the technique would contribute to developing and improving biosensors and to producing new bionanoelectronic materials. in this study, we created a platinum-binding site on a loop located at a protein surface. we used an artificial protein, larfh, that had been synthesized by connecting four identical alpha helices originated from the c-terminal segment of the escherichia coli lac repressor with three identical loops. we randomized the ser, gly, gln, gly, gly, ser sequence within one of the inter-helical loops and then selected for binding to platinum by a t phage display system. most of the selected larfh variants contained the tyr, lys, arg, gly, tyr, lys (ykrgyk) sequence in the randomized segment. we then evaluated the affinity of the larfh variant to platinum by means of quartz crystal microbalance analysis. we found that the variant binds to platinum more strongly than does the original larfh. in the annual symposium, we will also report about the affinity of the isolated ykrgyk sequence to platinum and about the crucial role of the first tyrosine in binding to platinum. engineering of an isolated p a subunit of pi ka permits crystallization and provides a platform for structure-based drug design pi ka remains an attractive target for development of anticancer targeted therapy. a number of p a crystal structures in complex with the nsh -ish fragment of p regulatory subunit have been reported, including a few small molecule co-crystal structures, but the utilization of this crystal form is limited by low diffraction resolution and a crystal packing artifact that partially blocks the atp binding site. taking advantage of recent data on the functional characterization of the lipid binding properties of p a, we designed a set of novel constructs allowing production of isolated stable p a subunit missing the adapter binding domain (abd) and lacking or featuring a modified c-terminal lipid binding motif. while this protein is not catalytically competent to phosphorylate its substrate pip , it retains ligand binding properties as indicated by direct binding studies with a pan-pi ka inhibitor. additionally, we determined apo and pf- bound crystal structures of the p a ( - ) subunit at . Å and . Å respectively. comparison of isolated p a ( - ) with the p a/p complex reveals a high degree of structural similarity, which validates suitability of this catalytically inactive p a for iterative sbdd. importantly, this crystal form of p a readily accommodates the binding of non-covalent inhibitor by means of a fully accessible atp site. the strategy presented here can be also applied to structural studies of other members of pi kia family. identification of structural determinants involved in the differential conformational changes of ef-hand modules emma liliana arevalo salina , joel osuna quintero , humberto flores soto , gloria saab rinc on instituto de biotecnolog ıa, universidad nacional aut onoma de m exico identification of structural determinants involved in the differential conformational changes of ef-hand modules calcium signals are regulated by several proteins, most of which belong to the ef-hand superfamily. the ef-hand motif is formed by a helix-loop-helix that binds calcium through its loop . these motifs occur in adjacent pairs, forming a single globular domain which is the basic structural and functional ca binding unit. the proteins in this family can be classified as calcium sensors or modulators, according with their function. the first group undergoes a major conformational change upon calcium binding, while the second one remains practically unchanged , . to explain the biophysics behind the different behavior of these proteins upon ca binding, we have sought to identify structural determinants that could account for these features, especially for the difference in the conformational change. we examined the primary structure from two ef-hand motifs: a sensor ef-hand from chicken troponin c (sciii) and a modulator ef-hand from bovine calbindin d k (clbn). the main differences were in the binding ca loop and a group of charged residues in the h helix of the modulator ef-hand. then, we constructed chimeric clbn motifs containing the loop or the loop and h from sciii motif (h clbnsciii and h h clbnsciii). these constructs were analyzed using a reporter system that discriminates ef-hand-sensor motifs from signal-modulators at the single-motif level. this reporter is based on the fusion of genes codifying for the ef-hand and the prephenate dehydrogenase from e. coli (tyra), a protein which is active only as a dimer. isolated ef-hand motifs have the ability to homo-dimerize and in the fusion can stabilize and activate tyra. the sensor motif exhibits a conformational change by binding calcium and in doing so, destabilizes the dimeric conformation of tyra and virtually eliminates its activity. in the modulators, on the other hand, the rather small conformational change only gives rise to a decreased tyra activity. both constructed chimeric ef-hand fusions showed a loss of activity upon ca binding, indicating that the residues connector of the sensor ef-hand from sciii is sufficient to confer the conformational change. in addition we used cd and extrinsic fluorescence spectroscopies to analyze any conformational change in the h h clbnsciii and h clbnsciii isolated modules, not finding any difference between the ca free and ca bound chimeras, suggesting that the change in activity of the reporter protein is due to a change in the orientation of the helices in the ef-hands induced by calcium. the effect of ca binding of the chimeras in the context of the entire calbindin d k protein is under investigation. mapping side chain interactions at the n-and c-termini of protein helices nicholas e newell, independent researcher interactions involving one or more amino acid side chains near the ends of protein helices stabilize helix termini and shape the geometry of the adjacent loops, contributing to supersecondary structure. side chain structures that have been identified at the helical n-terminus include the asx/st n-caps, the capping box, and hydrophobic and electrostatic interactions. at the cterminus, capping is often achieved with main-chain polar groups, (e.g. the schellman loop), but here also particular side chain motifs clearly favor specific loop geometries. key questions that remain concerning side chain interactions at helix termini include: ) to what extent are helix-terminal motifs that include multiple amino acids likely to represent genuine cooperative interactions between side chains, rather than chance alignments? ) which particular helix-terminal loop geometries are favored by each side chain interaction? ) can an exhaustive statistical scan of a large, recent dataset identify new side chain interactions at helix termini? in this work, three analytical tools are applied to answer the above questions for both n-and c-termini. first, a new perturbative least-squares d clustering algorithm is applied to partition the helix terminal structures in a large ( , example), low-redundancy pdb dataset by loop backbone geometry. the clustering algorithm also generates a set of structural exemplars, one for each cluster, that is used to represent the most important loop geometries at each terminus. next, cascade detection (newell, bioinformatics, ), an algorithm that detects multi-amino acid cooperativities by identifying overrepresented sequence motifs, is applied to each cluster separately to determine which motifs are most important in each loop geometry. finally, the results for each motif are displayed in a capmap, a d conformational heatmap that depicts the distribution of motif abundance and overrepresentation across all loop geometries by projecting these quantities onto the structural exemplars generated by clustering. the capmap reveals the loop conformations most favored by a motif. actual structures from the clusters corresponding to these favored conformations are then examined in a structure browser to characterize the side chain interaction associated with the motif. this work identifies a 'toolkit' of side chain motifs which are good candidates for use in the design of synthetic helix-terminal loops with specific desired geometries, because they are used in nature to support these geometries. highlights of the analysis include determinations of the favored loop geometries for the asx/st motifs, capping boxes, big boxes, and other previously known and unknown hydrophobic, electrostatic, h-bond, and pi-stacking interactions. a goal of future work is to make these results available in a structurally-addressable database that would enable researchers to immediately retrieve the side chain interactions most compatible with a desired loop geometry. generation of fluorescent protein-tagged gp mutants to analyze the intracellular distribution of hiv- envelope protein shuhei nakane , zene matsuda green earth research center, green earth institute co., ltd., res ctr for asian infect dis, inst of med sci, the univ of tokyo, lab of struct virol and immunol, institute of biophysics, cas hiv- is a causative enveloped virus of aids. its envelope protein (env) has two non-covalently associated subunits, gp and gp , which are proteolytically processed from a gp precursor. the gp subunit is a surface protein and gp is a transmembrane protein. the gp and gp subunits are responsible for the receptor recognition and membrane fusion, respectively. the cytoplasmic tail (ct) of gp is about amino acids long and is believed to play a critical role in intracellular trafficking of env. to visualize dynamic trafficking, the c-terminus of gp has been tagged with fluorescent proteins such as gfp. however, tagging of ct may cause a concern to affect the interactions between the ct and cellular proteins that are involved in intracellular trafficking. to avoid this problem, here we tried to insert gfpopt, a gfp variant, into five variable regions of gp . we have analyzed the phenotypes of env mutants, such as the cell surface expression, processing of gp , membrane fusion activity, and virion incorporation. among variable regions of gp , the v region was most sensitive to insertion. v /v region was less sensitive than v . consistent with the recently revealed structure, exteriorly located v and v were highly tolerant to insertion. we used the mutant with the gfp insertion in the v region to analyze the intracellular distribution of env with and without ct. we found that deletion of ct increased the presence of vesicles colocalized with late endosome markers. this is consistent with the hypothesis that the ct region contains a motif regulating intracellular trafficking. our results showed that env with gfpopt insertion in its gp subunit is a useful tool for the study of intracellular dynamics of hiv- env. these mutants would also be useful to trace the fate of virus particles during infection. pi- ngs-guided phage panning: comparison to conventional panning strategy buyung santoso , dorain thompson , john nuss , john dwyer phage display is a powerful tool for generating binders to a target protein. multiple rounds of panning with conventional phage display strategies typically result in a number of hits, which are then individually screened using in vitro assays. clones screened at this stage are a combination of specific binders, sequences that are selected due to amplification bias, and non-specific binders. if the number of specific clones is low relative to the non-specific sequences, a larger number of clones have to be screened to ensure sufficient diversity of early leads. with the advent of next generation sequencing (ngs) technology, we aim to test whether we can increase the diversity of specific hits and decrease the number of non-specific sequences. in our experiment, four rounds of conventional panning produced ten peptide binders to target protein. ngs analysis after two rounds of panning was done in parallel, yielding more than ten thousand sequences, ranked by abundance. all ten binders from conventional panning were found in the top most abundant ngs hits. more importantly, additional hits were found in ngs analysis but not in conventional panning, highlighting this strategy as a promising alternative for hit discovery with the significant upside of more diverse and higher affinity leads. numerous processes in pharmaceutical development, including construct screening, structural genomics, protein engineering and expression optimization among others, require the use of higher throughput plasmid dna purification. the majority of issues encountered in mini, midi, and maxiprep purification kits involve flocculate removal following alkaline lysis, and there is currently no easy way to produce large amounts of plasmid dna without the addition of complicated and time consuming clarification steps. the existence of a hassle-free automated system that is not restricted by sample size would significantly help in cutting time and costs during the initial processing steps of plasmid purification. the autoplasmid mea instrument provides a fully automated solution to traditional problems faced in plasmid purifications, allowing mini, midi, and maxiprep plasmid purifications to be performed on a single instrument. the data presented here on plasmid yield, purity, and suitability for sequencing and transfection/transformation illustrate a new strategy for automated plasmid preps. by eliminating traditional clarification methods, cell culture volumes between - ml can be processed leading to yields ranging from - lg. this flexible system was developed in order to satisfy a wide variety of concentration and yield requirement, while eliminating the time consuming steps previously needed to obtain similar results. the ability to perform fully automated mini, midi, and maxi plasmid preps on one instrument allows for a customized all-in-one purification system that is not restricted by traditional clarification methods, eliminating manual intervention, and streamlining the purification process. the modular nature of protein architectures suggests that proteins have evolved through duplication and fusion to give rise to modular, often symmetric forms, which later diversified under the influence of evolutionary pressure. we have developed a computational protein design method termed reverse engineer evolution (re volution) to create symmetrically self-assembling protein building blocks. we have used this method to design a perfectly symmetric b-propeller protein called pizza. subsequently, we have engineered a metal binding site into this pizza protein. this new pizza variant carries two nearly identical domains per polypeptide chain, and forms a trimer with three-fold symmetry. the designed single metal ion binding site lies on the symmetry axis, bonding the trimer together. two copies of the trimer associate in the presence of cadmium chloride in solution, and high resolution x-ray crystallographic analysis reveals a nano-crystal of cadmium chloride, sandwiched between two trimers of the protein. this nano-crystal, containing seven cadmium ions lying in a plane and twelve interspersed chloride ions, is the smallest reported to date. our results indicate the feasibility of using rationally-designed symmetrical proteins to biomineralize nano-crystals with applications in bionanotechnology. bacillus licheniformis trehalose- -phosphate hydrolase structures suggest keys to substrate specificity chwan-deng hsiao , min-guan lin , long-liu lin , yuh-ju sun institute of molecular biology, academia sinica, department of applied chemistry, national chiayi university, depaertment of life science, national tsing hua university trehalose- -phosphate hydrolase (trea) of the glycoside hydrolase family (gh ) catalyzes the hydrolysis of trehalose- -phosphate (t p) to yield glucose and glucose- -phosphate. products of this reaction can be further metabolized by the energy-generating glycolytic pathway. here we present the crystal structures of bacillus licheniformis trea (bltrea) and its r q mutant complexed with p-nitrophenyl-a-d-glucopyranoside (r q/ ppng) at . Å and . Å resolution, respectively. the overall structure of bltrea is similar to other gh family enzymes. however, detailed structural comparisons revealed that the catalytic groove of bltrea contains a long loop adopting a different conformation from those of gh family members. unlike the homologous regions of bacillus cereus oligo- , -glucosidase (bcogl) and erwinia rhapontici isomaltulose synthase (nx- ), the active site surface potential of bltrea exhibits a largely positive charge, contributed by the four basic residues his , his , lys and lys . mutations at these residues resulted in significant decreases of bltrea enzymatic activity. strikingly, a hhlk motif and the lys residue played critical roles in bltrea substrate discrimination. crystal structure of engineered lrrtm synaptic adhesion molecule and a model for neurexin binding anja paatero , katja rosti , alexander shkumatov , cecilia brunello , kai kysenius , prosanta singha , henri huttunen , tommi kajander institute of biotechnology, university of helsinki, helsinki, finland, dept of pharmaceutical and pharmacological sciences, ku leuven, leuven, belgium, neuroscience center, university of helsinki synaptic adhesion molecules are key components in the development of the brain, and in the formation of neuronal circuits, as they are central in the assembly and maturation of the chemical synapses. several families of neuronal adhesion molecules have been identified such as ncams, neurexins and neuroligins, and in particular recently several leucine rich repeat protein families, e.g. netrin g-ligands, slitrks and lrrtms. the lrrtms form a family of four proteins. they have been implicated in excitatory glutamatergic synapse function, and were specifically characterized as ligands for neurexins in excitatory synapse formation and maintenance. in addition, lrrtm and lrrtm have been found to be ligands for heparan sulphate proteoglycans. we report here the crystal structure of a stability-engineered mouse lrrtm , with a tm c higher than the wild type protein, while retaining its function. we localized the neurexin binding site to the concave surface based on protein engineering, sequence conservation and prior information on the ligand interaction with neurexins, allowing us to propose a tentative model for lrrtm:neurexin interaction compex. cell culture studies and binding experiments show that the engineered protein is functional and capable of forming synapse-like contacts. small angle x-ray scattering data suggests that the wild type protein forms transient dimers, which may have importance for the function. the structural and functional data presented here provide the first structure of an lrrtm protein, and a model for molecular mechanism of lrrtm function in adhesion. computational design of phenylalanine binder olga khersonsky , gil benezer , sarel fleishman recently, abdesign algorithm was developed in our lab for de novo design of antibodies ( ). it is guided by natural conformations and sequences, and exploits the modular nature of antibodies to abstract generate an immense space of conformations, which can be used as scaffolds for design of stable highaffinity binders. we have used abdesign to design a binder of phenylalanine. , antibody scaffolds were obtained by splicing h and l fragments into a template (pdb id brr), and subsequent optimization of vh and vl orientation. phenylalanine binding site, based on native phenylalanine binders, was introduced into the scaffolds with rosettamatch ( ), and the sequences were subsequently optimized by rosetta enzyme design protocol ( ) . designs were experimentally tested by yeast display for binding of biotinylated phenylalanine ligand. several designs were found to bind the ligand, and we plan to further characterize this affinity and improve it using directed evolution techniques. in collaboration with the group of prof. johnsonn, the resulting phenylalanine binder will be incorporated in a bio-luminescent (lucid) sensor for phenylalanine ( ) . phenylalanine monitoring device would be of primary importance for patients with phenylketonuria, a genetic disease with phenylalanine metabolism problem. cold-adapted enzymes are interesting because of their higher catalytic activity compared to mesophilic and thermophilic homologues. alkaline phosphatase (ap) from a psychrophilic vibrio marine bacteria (vap) has an unusual large surface loop that extends from each of its monomers to stabilize a homodimeric structure ( ). in many cold-adapted enzymes, the loop regions are longer compared to proteins of mesophilic organisms and our aim was to study the functional and structural role of this loop. three substitutions (r l, y f and f y) were introduced within the large surface loop as directed by microsecond molecular dynamics (md) simulations. with the r l mutation, two hydrogen bonds were broken that connect the loop to residues on the adjacent subunit, and further two hydrogen bonds broken with the adjacent q . as a consequence, r l displayed a % higher kcat compared with wild-type and a slight decrease in the km value. overall, the catalytic efficient improved by %. the global heat stability (tm) and the active site sensitivity to heat (t %) were reduced by c and c, respectively. md simulations showed that hydrogen bonds to arg are important for longrange communication to the active site. certain rotamers of two important residues in the catalytic site, ser and arg , were favored, presumably toward states more competent for catalysis upon the replacement of arg with leu. in the y f variant, removal of one hydrogen bond between the loop and the other subunit caused a small drop in stability parameters, whereas both kcat and km were reduced by about half, giving similar kinetic efficiency (kcat/km) to that of wild-type. finally, we changed a residue at the root of the large loop (f y) such that one new intersubunit hydrogen bond could form. this variant maintained the wild-type characteristics. in conclusion, removing hydrogen bonds connecting the major loop of one subunit to the protein surface of the other subunit in vap produced higher catalytic activity and this shows functional connections between loop mobility and the active site. our study also demonstrates that interactions between residues in the large disordered loop and the opposite subunit in the dimeric vap are determinants of its stability. thus, we managed to show that loosening of interface contacts between the two vap subunits by replacement of crucial residues provides a way to orchestrate structural and kinetic dynamics in a productive way. the de novo design of artificial proteins arises as a stringent test of our understanding of the relationship between sequence, structure, and function. examples include the design of a four a-helix bundle, a new protein topology called top , and a series of artificial (ba) -barrels called octarellins. however, de novo design has proven difficult for larger proteins with more than amino acids. here we present two methods to generate the backbone and to perform the de novo design of (ba) -barrel proteins through the use of the software rosetta; both have different advantages and limitations. the first method for generating the backbone is knowledge-based, with a first analysis of a non-redundant database of natural (ba) -barrel proteins in order to obtain statistical analysis on preferred secondary structure element length and amino acidic propensities. with this information we use the rosetta cm software to create more than models which are then ranked in term of rosetta energy. the second method is performed with the parametricdesign package of rosetta, in which only geometrical information are requested (number of strands and helices, radius of the b-and a-barrels, degree of inclination, orientation of the side chains, among others). both methods contain a step of loop refinement and multiple steps of sequence design with the package rosetta design, in order to find low scoring amino acid sequences for each of the starting backbone conformations. thousands of models will be generated by both methods and then analyzed in term of sequence similarity, secondary and tertiary structure prediction, and stability by molecular dynamics simulations. the best candidate sequences will be selected for the experimental verification. in order to identify a putative successfully design, we added a metal binding site during the design step. all the proteins will be expressed in e. coli. the solubility of the designed proteins inside bacteria will be determined thanks to the fusion to green fluorescence protein (gfp). solubility, stability, secondary structure, and cooperativity of folding will be assessed for each protein before determination of their three-dimensional structure. construction of protein capsule possessing drugs controlled release ability shota shimizu , masatoshi nakatsuji , keisuke yamaguchi , yuya sano , yuya miyamoto , takashi inui most compounds that exhibit anti-tumor activities are water-insoluble, thus limiting their clinical use. chemical modification of these compounds and the use of solubilizing agents such as organic solvents, surfactants and ph modifiers improve their solubility. however, chemical modification of compounds decreases their potency, and the use of solubilizing agents causes toxicity in many cases. thus, drug delivery systems (dds) for poorly water-soluble anti-tumor drugs which exploit liposomes, cyclodextrins, and lipid nanoparticles have been studied intensely. in these dds, the controlled release of drugs from the delivery vehicle is one of the most important functions. selective release in target cells leads to adequate therapeutic efficacy with few side effects. in our laboratory, we have already demonstrated that lipocalin-type prostaglandin d synthase (l-pgds), an intravital transporter protein, is a novel and valid drug delivery vehicle for sn- , a poorly water-soluble anti-tumor drug. in this study, we generated l-pgds-based protein capsules with a controlled-release function by introducing a disulfide bond into the upper part of the drug-binding cavity of l-pgds. the intracellular concentration of glutathione ( . mm) is known to be substantially higher than the extracellular concentration ( mm). therefore, it is expected that in the extracellular oxidative environment the disulfide bonds in the protein capsule remain stable, avoiding premature release of the internal drugs during circulation of blood, after reaching the target cells, the disulfide bonds are cleaved in the intracellular redox-environment, and then the internal drugs are released. we generated three kinds of protein capsules which have disulfide bonds in different positions, w c/w c, k c/h c, k c/w c, based on tertiary structure information of human l-pgds (pdb id: o y). firstly, we performed circular dichroism (cd) measurements to confirm the structure of each capsule. the cd spectra of three protein capsules were similar to that of wild-type l-pgds in the far-uv region. therefore, the secondary structures of three protein capsules were not changed from wild-type l-pgds by introducing the mutations. quantitative analysis of the free thiol group in the protein capsule by dtnb assay revealed that the intermolecular disulfide bond was formed by h o -induced oxidation and cleaved by dithiothreitol-induced reduction. in addition, to investigate the solubility of sn- in the presence of protein capsules, we mixed the protein capsule of reduced-form with sn- suspension, and stirred at c for hours. the resulting concentrations of sn- in pbs with mm w c/w c, k c/h c, and k c/w c were mm, mm, and mm, respectively. these values were approximately -fold higher than without protein capsules. sds-page analysis showed that the bond formation decreased in a time-dependent manner, and that new intermolecular disulfide bond was not formed in the protein capsules after hours' incubation. from the above, we succeeded in generating drug delivery vehicles possessing openable and closable lids that are responsive in an oxidation-reduction environment. takaaki miyamoto , mai kuribayashi , satoshi nagao , yasuhito shomura , yoshiki higuchi , , shun hirota graduate school of materials science, nara institutte of science and technology, graduate school of science and engineering, ibaraki university, department of life science, graduate school of life science, university of hyogo, domain swapping has been of interest as a mechanism of protein oligomerization, where a secondary structural region or a domain of one protein molecule is replaced with the corresponding region or domain of another protein molecule. we have previously shown that c-type cytochromes and myoglobin form oligomers by domain swapping. , in this study, we show that a four-helix bundle protein cyt cb , in which the heme of cyt b is attached to the protein moiety by insertion of two cys residues, forms a domain-swapped dimer. dimeric cyt cb was more stable than dimeric cyt b at c, showing that attachment of the heme to the protein moiety stabilizes the domain-swapped structure. absorption and cd spectra of dimeric cyt cb were similar to the corresponding spectra of the monomer, showing that the active site and secondary structures were similar between the dimer and monomer. the redox potential of dimeric cyt cb was also similar to that of its monomer. the dissociation temperature of dimeric cyt cb was c, and its dh on dissociation to monomers was . kcal/mol (per dimer). according to x-ray crystallographic analysis, dimeric cyt cb exhibited a domain-swapped structure, where the two helices in the n-terminal region (helices and ) in a protomer and the other two helices in the c-terminal region (helices and ) of the other protomer interacted between each other. the heme coordination structure of the dimer was similar to that of the monomer. we have previously shown that domain-swapped oligomers of horse cyt c form through intermolecular hydrophobic interaction between the n-and c-terminal a-helices at the early stage of folding. it has been suggested that helices and form first at the initial stage of folding in wild-type apo cyt b . therefore, we propose that cyt cb forms a domain-swapped dimer when helices and interact intermolecularly at the initial stage of folding, whereas the intramolecular interaction of helices and results in formation of a monomer. a highly buried and conserved tryptophan residue close to the dimer interface in a cold-adapted phosphatase is phosphorescent and important for activity. jens g. hj€ orleifsson and bjarni asgeirsson. department of biochemistry, science institute, university of iceland, dunhagi , reykjavik, iceland. alkaline phosphatase (ap) from vibrio g - is a cold-adapted dimeric enzyme with one of the highest catalytic efficiency reported for known aps. it contains five intrinsic tryptophan (trp) residues and one additional trp located on the c-terminal streptag used for expression and purification. in this study, we made several single trp-substitutions to determine the role of each of the trp in the fluorescence emission spectrum. we also determined their solvent exposure by acrylamide fluorescence quenching. the results indicate that trp , trp and trp are mostly responsible for the fluorescence emission. quenching experiments with acrylamide indicated that all the trp residues were about equally accessible for quenching, except trp which was shown to be highly buried in the core of the protein. interestingly, the enzyme was found to be highly phosphorescent at c, having two phosphorescence lifetimes. the longer lifetime is due to trp . trp is located close to the dimer interface and points towards a helix in the active site where his binds an active-site zinc ion. in other aps, an aromatic amino acid is conserved in the location occupied by the trp residue. in most cases for cold-adapted aps it is indeed a trp. interestingly, the mutation of the trp to a phenylalanine affected both stability and activity of the enzyme. kcat/km was -fold lower than for wild-type. overall, this study reveals that trp can be used as a phosphorescent probe of local dynamics and could possibly also serve to study the dimer-monomer equilibrium due to proximity to the dimer interface, an area clearly crucial for enzyme activity and stability. modulating protein-protein interaction with a molecular tether helen farrants , oliver hantschel , kai johnsson ecole polytechnique f ed erale de lausanne (epfl) high-affinity scaffolds for protein-protein interactions, such as monobodies and darpins can be engineered in vitro to bind to protein targets. we speculate that the affinity for the target protein can be modulated by incorporating these evolved scaffolds and a synthetic intramolecular tether into protein switches, in a protein construct of composed of snap-tag, a monobody and a circular permutated dihydrofolate reductase. the tether, attached to the construct via snap-tag, was composed of a linker and trimethoprim, which interacts reversibly with the circular permutated dihydrofolate reductase. we have investigated the affinity between the n-sh domain of the phosphatase shp and an evolved monobody in such a protein construct using a fret assay. when the intramolecular tether was bound the circular permutated dihydrofolate reductase ("closed" conformation), there was an increase in the affinity of the construct to the target n-sh . in the presence of a small molecule competitor ("open" conformation) the affinity of the monobody construct to its target was reverted to the value reported in the literature. the intramolecular tether in these protein constructs combined with engineered scaffolds for protein-protein interactions may be a general approach towards protein switches. because most proteins are long polymers of amino acids with twenty or more chemically-distinct sidechains, there are an enormous number of potential protein sequences. here, we report the construction of biologically active proteins with minimal chemical diversity. transmembrane domains of proteins can specifically interact with other transmembrane domains to modulate the folding, oligomerization, and function of transmembrane proteins. for example, the bovine papillomavirus e protein is a -amino acid transmembrane protein that transforms fibroblasts to tumorigenicity by binding directly to the transmembrane domain of the platelet-derived growth factor b receptor (pdgfbr), resulting in ligandindependent receptor activation and cell transformation. these studies showed that a free-standing transmembrane domain could fold properly in cells and act in trans to modulate the activity of a larger transmembrane protein target. because of the relative chemical simplicity of transmembrane domains and this ability to act even when not linked to more complex soluble protein domains, we reasoned that short transmembrane proteins could be used to define the minimal chemical diversity sufficient to construct biologically active proteins. to accomplish this, we infected cultured mouse cells with a retroviral library expressing -amino acid proteins consisting of an initiating methionine followed by a randomized sequence of leucines and isoleucines, two hydrophobic amino acids that differ only by the position of a single methyl group, and selected rare proteins with transforming activity. we isolated numerous proteins consisting of diverse sequences of leucine and isoleucine that cause morphologic transformation, escape from contact inhibition and focus formation, and growth factor independence. genetic and biochemical analysis of these proteins indicate that like e they interact with the transmembrane domain of the pdgfbr to specifically activate the receptor and transform cells. mutational analysis of individual proteins identified specific leucines and isoleucines required for transforming activity, and insertion of a single isoleucine at a particular position in a stretch of leucines is sufficient for activity. these proteins identify the minimal chemical diversity required to generate a biologically active protein and have important implications for biochemistry, protein evolution, protein engineering and synthetic biology. yusuke azuma , donald hilvert virus-like particles that are precisely loaded with functional cargo are an important tool to study the effect of spatial confinement and create novel entities with application in biotechnology and medicine. by genetic fusion to a positively supercharged green fluorescent protein (gfp( )), an enzyme retroaldolase (ra) was efficiently targeted to the negatively charged lumen of an engineered protein cage, aquifex aeolicus lumazine synthase variant (aals- ). the encapsulation is quantitative under mild aqueous condition up to a mixing ratio of guest enzymes per host cages. the chromophoric tag is used for precisely quantifying the enzyme concentration, which allows detailed characterization of the effect of encapsulation on the enzyme activity. the generality of the encapsulation system was examined with structurally different enzymes. introduction and purpose: in the immune system, high affinity antibodies are generated by selection of b cells activated by antigen-stimulation followed by additional optimization through somatic hyper mutation of antibody genes. in the artificial antibody libraries, such as phage libraries, selection of specific antibody clones from the library is performed by in vitro selection process called biopanning and the subsequent binding screening. however, in spite of high efficiency of enrichment in biopanning, there is a possibility that we overlook the minor antigen-specific clones in the screening because of the limitation of the number of clones employed for screening. in recent years, high-throughput analysis of dna sequences by the next-generation sequencer (ngs) has become available not only for genomic analysis of organisms but also repertoire analysis of antibodies. in this presentation, we report the successful isolation of a variety of antigen specific antibodies from patients-derived antibody phage library by a combination method of high throughput sequence analysis on ngs and biopanning. method: we constructed two kinds of human single chain fv (scfv) antibody libraries from pooled mrna of five cancer patients and of a wheat allergy patient, respectively. after biopanning against a cancer antigen or wheat allergy antigen "gluten", the phagemid vector dna prepared from the pooled phages before or after biopanning was used for pcr amplifications of vh genes, adding the index and adapter sequences for ngs analysis. the high throughput sequencing was performed on miseq (illumina) using miseq reagent kits v . after discarding the short sequences and low quality data, '-and '-reading sequences were unified by a merge program. the frequencies (%) of all vh sequences were evaluated using a program based on usearch . clustering software and the changes of the frequency (%) of each sequence between before and after panning were assigned as amplification rate. results and discussion: vh sequences at each round of pooled phages after biopanning against cancer antigen were analyzed on ngs. after three rounds of biopanning, three clusters of antibody sequences were specifically enriched suggesting these are specific binders. to check this, scfv gene were regenerated by pcr using h-cdr specific primers and scfv-displaying phages reconstructed were subjected to binding analysis. all three phages showed a clear specific binding to cancer antigen in elisa. subsequently, to test the usefulness of this method, we applied it to identify allergen-specific scfv from allergy patient-derived antibody phage library. the phylogenetic tree analysis of vh sequences which showed the amplification rate higher than . by a single round of biopanning elucidated total eleven clusters of vh sequences. the vh sequences in the two clusters with the highest amplification factor were selected and the regenerated scfv-displayed phages were tested for binding analysis. the prepared scfvdisplayed phages and also scfv proteins showed a clear binding ability to allergen. thus, it is suggested that the analytical method of vh sequences on ngs before and after biopanning is very useful to isolate a variety of disease related antigen-specific novel antibodies quickly with high degree of certainty. biochemical analysis of the recognition helix of z-dna binding proteins: roles in conformational specificity yang-gyun kim , xu zheng , so-young park conversion of right-handed b-dna into left-handed z-dna is one of the dramatic structural transitions in biological processes including gene regulation and chromatin remodeling. z-dna binding motif, zalpha (za), was first discovered from human adar . subsequently, with sequence and structure similarity to the hzaadar , families of proteins including viral e l, interferon-induced protein dai (zbp ) and pkz has been identified to have za domain(s). interestingly, the za domain of the e l protein from vaccinia virus (vvzae l) was confirmed to have the ability of z-dna-binding, but it does not have the b-to-z conversion activity. here, we showed that the replacement of the a -helix of vvzae l (vvzae l-a ) with that of hzaadar results in acquiring the ability to converting b-dna to z-dna. the detailed biochemical analysis of the a -helix mutants of vvzae l further suggested that the contribution of positively charged residues in the c-terminal part of the a -helix is crucial during the b-to-z transition. in addition, hydrophobic residues of the n-terminal part of the vvzae l-a also influence on the b-to-z conversion activity, possibly through forming a tightly-packed structure. in conclusion, our results revealed the previously-unknown contribution of amino acid residues existed in the a -helix of the za domains to the b-to-z conversion. moreover, it strongly implies that such residues may play important roles in initiating conformational changes of dna structure during the b-to-z conversion event. the ability of switching the activity of proteins at will is of great interest from an application point of view. one promising approach utilizes a protein modification with an organic photochromic molecule. linking two protein side chains with the photochrome that undergoes a light induced conformational change, protein secondary and tertiary structure can be stabilized or destabilized and thus the structure dependent activity can be switched "on" and "off" by light irradiation. for this the photochrome must fulfil several requirements. foremost, it must possess two states of comparable stability that differ significantly in their geometry. it must further be water soluble and non-toxic, and should not experience fatigue phenomena upon multiple irradiations. there are two classes of molecules that fulfil those requirements: azobenzenes and spiropyrans. we are pursuing two different strategies for the design of photoswitchable proteins. in the first approach we attach an azobenzene compounds to side chains of the alpha-helical antifreeze protein type i. the end to end distance of the photochromic molecule is sterically compatible with the folded helix only in one form, photoisomerization therefore switches the folding state between an active helical state and an inactive unfolded form. in a second, more general approach we use the trp-cage domain as a switching unit. the trp-cage is the smallest known folded protein ( amino acids). its folding is induced by hydrophobic interactions of a tryptophan side chain in a short helical segment. after modification with a photochromic molecule in appropriate positions, its structure is rendered sensitive to the state of the chromophore. by creating protein chimera of such a trp-cage and biologically active peptides with helical propensity, we aim at conferring the light-dependent fold of the cage to the attached peptide moiety. salt-bridges are electrostatic interactions between groups of opposite charges. net interaction energy (ddgnet) of a salt-bridge is partitioned into bridge (ddgbrd), desolvation (ddgdsolv) and protein (ddgprot) energy-terms of which estimation of ddgdsolv and ddgprot are only possible by computational means. thus, general purpose poisson-boltzmann equation solver: "delphi" (in commercial package of insight-ii) and "apbs" (open-source) are popularly used to determine these energy-terms. nevertheless, the computation-method is highly involved one than other uses of these solvers. moreover, protein-specific saltbridges, grid-points, center, hydrophobic-isosteres-mediated mutation-files of original charge-radius file and others are to be worked out prior to the computation. this might answer as to why only limited numbers of structure files ( % of crystal-structure-database) are worked out till date. at this juncture, an efficient fully automated all-in-one-procedure that could analyze large dataset in a single run would be useful. to the best of our knowledge, such procedure is truly lacking in public domain. at this end, our fully automated all-in-one procedure: adsetmeas (available freely at http://sourceforge.net/projects/adsetmeas/along with detailed documentation) uses "apbs" method to compute component as well as net energy-terms of salt-bridges and redirect compact output in excelformat. further, micro-environments of salt-bridges are also been reported based on the presence of polar, dipolar, acidic, basic and hydrophobic side-chains in their proximity. the procedure provides versatility to users in choosing a] model for computation of energy-terms to-date available in the literature and b] method (default or advanced) for parametric optimization in "apbs" calculations. it works in unix like environment including cygwin. it processes all proteins present in the working directory with any number of salt-bridges in them. a pre-released version of the procedure was successfully applied for energy-terms on salt-bridges from halophilic proteins. overall, our adsetmeas provides intricate details on salt-bridge energetic from crystal structures and find application in the field of computational structural biology. these and other results will be discussed in the conference. next generation analgesics -targeting ion channels with antibody-drug conjugates (adcs) anna wojciechowska-bason , clare jones , chris lloyd postdoctoral fellow, adpe, medimmune, cambridge, ria, medimmune, cambridge, adpe ion channels are common targets for chronic pain therapies. small molecule analgesics are widely used therapeutically, but due to poor specificity they often cause a wide range of side effects. as a result, efficacy of existing treatments is very limited. we believe that to achieve the required specificity and efficacy, a novel and innovative approach is required that would combine the potency of the small molecule with the selectivity of an antibody. therefore, we propose to apply antibody-drug conjugates (adcs) to deliver small molecules or peptides to ion channels in order to specifically modulate pain signalling pathways. voltage-gated sodium channel nav . has a well characterised role in the perception of pain. here we present the activity of the peptide huwentoxin-iv (hwtx-iv) and small molecule inhibitors ptc-a, ptc-b and ptc-c on voltage gated sodium channels nav . and nav . . in novel findings, we report that these inhibitors show little selectivity between the voltage-gated sodium channel family members, nav . and nav . , and that the ic values and the impact on channel biophysics (voltage-dependence of activation and fast inactivation) of the inhibitors are largely similar for both channel types. therefore, the use of hwtx-iv and other small molecule inhibitors of nav . for pain therapy could be dose-limited due to side effects mediated by the inhibition of channel nav . . in conclusion, we propose that hwtx-iv and the investigated small molecule inhibitors could be used for the treatment of pain as part of a nav . antibody-drug conjugate (nav . -adc), establishing nav . specificity and minimising side effects. maria antonietta carillo , daniel varon silva malaria is one of the most infectious diseases caused by plasmodium species parasites. the merozoite surface protein (msp ) is the most abundant protein on the surface of the plasmodium species merozoite stage, which plays an important role during the erythrocytes invasion process [ ] . msp is synthesized as a -kda glycosylphosphatidylinositol (gpi) anchored protein precursor which is processed at the end of the schizogony into four different fragments. the primary processing step produces a complex of four fragments that are present on the merozoite surface. the secondary processing step at erythrocytes invasion results in the detaching of the complex from the surface, except for the cterminal -kda domain (msp ), which remains anchored to the parasite surface by the gpi moiety. in human malarial infections, the gpi is considered to be a toxin that causes the expression of various host genes and induces a pro-inflammatory immune response, making it a valuable candidate for the development of anti-malarial drugs. in order to study the function of the gpi and evaluate the effects, msp fragment has been expressed, purified and anchored to the synthetic gpi molecule using protein trans-splicing strategy based on the split intein method [ ] . the role of the gpi moiety will be studied through protein folding experiments and the effect of the anchored protein will be evaluated in vitro in order to understand the function of the gpis. assessment of uch-l substrate selectivity using engineered ubiquitin fusions with varying linker lengths peter suon, mario navarro, john love san diego state university, san diego state university, assessment of uch-l substrate selectivity using engineered ubiquitin fusions with varying linker lengths peter suon, mario navarro, and john j. love san diego state university the ubiquitin proteasome system (ups) is a complex system composed of multiple structural and functional elements that play key roles in cellular processes such as signal transduction, cell cycle regulation, apoptosis, and protein degradation. proteins destined for degradation are first tagged with the protein, ubiquitin, which is covalently attached to internal lysine residues. once the target has be degraded by the proteasome; the enzyme ubiquitin carboxy hydrolase l (uch-l ) is believed to prepare ubiquitin for additional rounds of ubiquitination by cleaving small peptides and chemical adducts from the ubiquitin c-terminus. previously in our laboratory, protein substrates of uch-l were engineered and used to characterize uch-l substrate selectivity. the engineered substrates consisted of n-terminal monoubiquitinated test variants derived from streptococcal protein g (protein gb ) and staphylococcal protein a (spab). the thermal denaturation temperatures (tm) of the fusion proteins were measured using circular dichroism and span a range of over c. more importantly, the rate of hydrolysis for the fusion proteins is demonstrated to be directly correlated to the tm of the test variant fused to the c-terminus of ubiquitin. previously, the engineered substrates were designed to emulate natural ubiquitin fusions and thus did not contain any 'linker' residues between the c-terminus of ubiquitin and the n-terminus of the test protein. to explore the effects of linker length on uch-l hydrolysis we are engineering new uch-l substrates that contain an unstructured amino acid linker between ubiquitin and the test protein. to further explore the catalytic efficiency of uch-l we will revisit diubiquitin (ub-ub), which is not hydrolyzed by uch-l , and will make mutations in the hopes of generating a hydrolysable substrate. using rational design, the new variants will be engineered to destabilize the c-terminal ubiquitin to determine if this results in hydrolysis of the new ub-ub construct. the thermal stability of these new fusion protein substrates will be measured using circular dichroism spectroscopy (cd) and uch-l hydrolysis rates will be characterized using existing assays. our goal is to continue the use of engineered substrates to further explore the catalytic properties of uch-l activity and the potential role in protein trafficking and degradation within living cells. we present a biophysical study of a suite of helical proteins that have been modified to contain and -amino acid additions on their termini that impart increased resistance to degradation in e. coli abstract recombinant expression systems. the b domain of staphylococcal protein a (ab) and the homeobox dna-binding domain from d. melanogaster engrailed (en) are small -helix bundles. these domains do not appreciably accumulate in the e. coli bl (de ) cytoplasm when expression in a pet vector is chemically induced. this is likely due to host protein degradation/recycling factors that function to efficiently degrade these two proteins. addition of sequences encoding either of two amino-terminal beta-hairpins to either the n-or c-terminus of ab and en results in the accumulation of large amounts of these new chimeric proteins. additionally, destabilization of the ab or en sequence does not abolish the expression enhancement effect of the beta-hairpin addition. we have investigated the biophysical origins and effects of the beta-hairpin additions using circular dichroism (cd) spectroscopy, and have determined that the added sequence does not significantly perturb the secondary structure of ab or en, nor does it significantly influence the unfolding temperature (tm). while investigation into the origin of the accumulation effect is ongoing, we hypothesize that the addition of the sequence is disruptive to recognition events in the native protein degradation machinery in e. coli. thus, this approach represents both a biotechnological tool for expressing helical peptides recalcitrant to expression, as well as a system well-suited to probing mechanisms of protein recycling and homeostasis. a special class of these proteins are lipidated proteins containing a glycosylphosphatidylinositol (gpi) glycolipid moiety at the c-terminus. the lipid chains of the gpi anchor molecule are responsible for the membrane association of the attached protein. a unique feature of gpi-anchored proteins is that after isolation they can be reinserted into the membrane of recipient cells with the retention of the biological function. accordingly, the exogenous introduction of fluorescent gpi-anchored protein analogues into cell membranes is a useful method for visualizing the cellular traffic of membrane associated proteins and for engineering cell surfaces. we have recently shown that cholesterol can be applied for anchoring proteins to the plasma membrane of live cells without perturbing the membrane. in order to introduce proteins containing covalent modifications that are not genetically encoded, an enzymatic method was considered and fused with the c-terminal cholesterylation method. the usefulness of the method is demonstrated via the preparation of multimeric model proteins of kda monomers, that is an appropriate representation of the ligation of domain size proteins. transmembrane domain dimerization drives p ntr partitioning to lipid rafts irmina garc ıa carpio , marc¸al vilar sociedad de biof ısica de españa. sbe p neurotrophin receptor (p ntr), is best known for its role in mediating neuron cell death during development or after injury but it also regulates cell proliferation, axon guidance or survival. the key to understand its signaling could rely in its structure and conformational states. it has been described that p forms disulfide-linked dimmers through the cys in the transmembrane domain which are essential for its ngf mediated signaling. previous studies have shown that p is present in lipid rafts, where it interacts with intracellular adaptors to activate different signaling pathways. we design several p mutants in the tm domain that impairs dimerization and study the role of tm domain dimerization in lipid rafts recruitment. our analysis suggests that p tm domain dimerization influences lipid raft partitioning. these results could be a key role to understand its signaling and processing pi- bioluminescent sensor proteins for therapeutic drug monitoring of the monoclonal antibody cetuximab martijn van rosmalen , remco arts , brian janssen , natalie hendrikse , dave wanders , maarten merkx therapeutic drug monitoring (tdm) -adapting the drug dosage scheme to the individual patient's pharmacokinetic and pharmacodynamic characteristics -is still uncommon for therapeutic monoclonal antibodies, despite preliminary studies showing its potential benefits. one of the factors impairing tdm implementation is the lack of equipment and trained personnel to regularly measure drug concentrations in patients receiving treatment. point-of-care diagnostic devices which could be used by patients themselves or by their general practitioners would greatly advance the feasibility of tdm. here we present a biosensor for the therapeutic monoclonal antibody cetuximab. we developed a series of cyclic peptides that specifically recognize cetuximab, covering a fourfold range of affinities, and incorporated these cyclic peptide sequences into a set of luminescent sensor proteins. the sensors translate cetuximab concentrations into a change in emission color that can be read out using a mobile phone camera. together, these sensors can quantify cetuximab levels within the relevant therapeutic concentration range and we propose that they can be used for therapeutic drug monitoring applications. genetically encoded biosensor for cell permeability of inhibitors of the p -hdm interaction silvia scarabelli , thomas vorherr , kai johnsson ecole polytechnique f ed erale de lausanne, the evaluation of the permeability across the cellular membrane is a key step in the development of therapeutics, since it affects the distribution and the efficacy of the latters. reliable and versatile techniques for the determination of structural permeability determinants of molecules and information about the entry kinetics are still missing. we introduced in the past a class of semi-synthetic ratiometric sensor proteins (snifits) that has been shown to be suitable for the measurement of intracellular metabolites concentrations. here we describe a totally genetically encoded sensor based on the snifits modular design for the assessment of the cell permeability of small molecules and peptides inhibitors of the protein-protein interaction between p and hdm . we show that our sensor detects the presence of hdm -binding stapled peptides in vitro, and, when expressed in mammalian cells, it responds to the perfusion of the known small molecule hdm inhibitor nutlin- a. moreover, experiments made with an automated microscope show that the sensor is suitable for measuring and comparing the kinetics of entry of different kinds of inhibitors in the cytosol of living cells. in parallel, we are developing an hcaii-based sensor protein for the sensing of sulfonamides and eventually their peptide derivatives. we show that the sensor responds to the presence of different kinds of hca-inhibitors in vitro and in perfusion experiments. this second sensor would broaden the range of molecules and peptides whose permeability can be studied with our tools beyond the family of the hdm -binders. our sensors overcome the limitations of the already existing techniques for measurements of permeability while offering a simultaneous measurement of the cell permeability and of the binding efficiency of small molecules and peptides of interest. archer: predicting protein function using local structural features. a helpful tool for protein redesign. jaume bonet , javier garcia-garcia , joan planas-iglesias , narcis fernandez-fuentes , baldo oliva structural bioinformatics lab, grib, upf, division of metabolic and vascular health, university of warwick, the advance of high-throughput sequencing methodologies has led to an exponential increase of new protein sequences, a large proportion of which remain unannotated. the gap between the number of known proteins and those with assigned function is increasing. in light of this situation, computational methods to predict the function of proteins have become a valid and necessary strategy. here we present archer, a server that exploits archdb's hierarchy of super-secondary structures to map go and enzyme functions upon protein regions and, thus, infer the function of a protein. the server relies on either the sequence or structure of the protein of interest and returns the mapping of functional subclasses extracted from archdb. moreover, it computes the functional enrichment and significance of each subclass, combines the functional descriptors and predicts the function of the query-protein. combining the functional enrichment analysis of the super-secondary structures with the structural classification of archdb, users can select variants of the target sequence that swap the region of a supersecondary structure by another that putatively fits in the same scaffold minimizing the effect on the global tertiary structure. only variants that modify the predicted function are offered for selection, thus providing a rational, knowledge-based, approach for protein design and functionalization. the archer server is accessible at http://sbi.imim.es/archer. phytochromes are natural photoreceptors known to regulate photosynthesis in plants, fungi and bacteria. phytochromes found in bacteria share common architecture and consist of a pas-gaf-phy photosensory core and a c-terminal output module, responsible for biological function. a bacterial phytochrome, bphp , from rhodopseudomonas palustris undergoes reversible conversion from the farred absorbing state (pfr) to the red-absorbing state (pr) followed by the conformational change upon nm light irradiation. as most of bacterial phytochromes, bphp forms a dimer. it was shown that nm light causes a protomer swapping between the bphp dimers; and likely, the output module is involved in this process. however, the mechanism of the light-induced swapping is poorly studied. we tested an ability of the protomer swapping between bphp dimers using pull-down biochemical assay. for this, strep-tagged bphp was immobilized on strep-tactin sepharose beads in the presence of untagged bphp fused to mruby at different concentrations. after incubation, the proteins were eluted and visualized in sds-gel using a zinc-induced fluorescence assay. an amount of the bound to beads protein was estimated by densitometry. it was found that more than % of heterodimers (streptagged-bphp and bphp -mruby ) form within . h of incubation under nm light at -fold excess of one of the interacting partners. in darkness, the swapping was much slower. in the similar setup we checked the amount of heterodimers after , and min of incubation. no difference was observed for different time points, suggesting that the protomer swapping is relatively fast process. next, a role of the c-terminal effector domain of bphp in the light-induced interaction was studied. for this, kinetics of the pfr-to-pr transition was analysed by measuring of absorbance at nm and nm for full-length bphp and a bphp mutant with the deleted c-terminal domain. while full-length bphp showed the normal pfr-to-pr transition, absorbance of the mutated bphp at nm did not raise. however, nm absorbance changes were similar for both proteins; and surprisingly, the similar dark relaxation kinetics was observed. we propose that the impaired pfr-to-pr transition is caused by restricted pr conformation in the mutant rather than by fast pr-to-pfr relaxation. understanding the mechanisms of the bphp light-induced structural changes and the protomer interaction should advance engineering of bacterial phytochromes into fluorescent probes and optogenetic tools. antibody detection is an integral part of many diagnostic strategies, most crucially so when infectious diseases are involved. currently used assays, such as elisa or spr, enable detection of antibodies in the laboratory with high sensitivity, yet a translation of these technologies to an application outside of the laboratory setting is far from trivial. problematically, the burden of disease for many infectious diseases is carried precisely by those countries where access to laboratory facilities is severely limited. we therefore developed a novel, one-step assay that allows the detection of antibodies directly in solution using a luminescent sensor protein. our strategy is based on the use of a bright luciferase, nanoluc, tethered to a green fluorescent protein (mneongreen) via a semi-flexible linker containing two epitope sequences. crucially, two small helper domains were fused to the protein termini. these domains keep nano-luc and mneongreen in close proximity in the absence of antibody, enabling efficient bioluminescence resonance energy transfer (bret). binding of antibody to the epitopes in the sensor proteins linker domain pulls the bret partners apart, effectively changing the color of emission from green to blue. the assay allowed the detection of picomolar amounts of anti hiv -p antibodies directly in solution, both under optimized buffer conditions and in blood plasma. in principle. the modular sensor architecture should allow detection of any antibody with a well-defined epitope of sufficient affinity. to demonstrate this, the hiv-epitopes were substituted for two ha-tag epitopes, yielding a sensor that enabled the detection of picomolar amounts of anti-ha antibodies. the simple optical readout provided by the sensor system allowed us to record the emitted signal with a conventional mobile phone camera. a simple software application that analyzes the image based on rgb values sufficed to interpret the recorded image vis-a-vis the presence of antibody. bearing in mind the eventually envisioned application in a point-of-care diagnostic setting, this combination of sensor recording and interpretation using nothing more than a mobile phone and a software application holds considerable diagnostic potential. beyond point-of-care diagnosis of infectious diseases, a simple assay to detect and quantify antibodies directly in solution could also have a substantial impact in other fields. antibodies are ubiquitous in biotechnology, and this is reflected by the plethora of potential sensor applications, which range from a role in microfluidic circuits or monitoring the biotechnological production of antibodies, including validation of bispecificity, to veterinary applications, diagnosis of autoimmune diseases and monitoring the success of vaccination campaigns. the continually growing protein data bank (pdb) has been a key resource for general principles of protein structure. for example, parsing structural observations in the pdb into simple geometric descriptors has given rise to statistical energy functions. here we present a novel strategy for mining the pdb on the basis of local tertiary structural motifs (term). we define a term to be the structural fragment that captures all local secondary and tertiary structural environments of a given residue, and query the pdb to obtain quantitative information for each terms. first, we show that by breaking a protein structure into its constituent terms, we can describe its sequence-structure relationship via a new metric we call "structure score." using submissions in recent critical assessment of structure prediction (casp) experiments, we find a strong correlation (r . ) between structure score and model accuracy -a performance that exceeds leading atomistic statistical energy functions. next, we show that querying terms affected by point mutations enables the quantitative prediction of mutational free energies. our simple approach performs on par with state-of-the-art methods fold-x and popmusic on mutations, and provides superior predictions in certain cases where other methods tend to fail. in all, our results suggest that the data available in the pdb are now sufficient to enable the quantification of much more sophisticated structural observations, such as those associated with entire terms, which should present opportunities for advances in computational structural biology techniques, including structure prediction and design. exploiting natural sequence diversity for protein crystallization sergio mart ınez-rodr ıguez , valeria risso , jos e m sanchez-ruiz , jos e a. gavira , departamento de qu ımica-f ısica, universidad de granada, laboratorio de estudios cristalogr aficos, iact-csic-ugr granada during the last decade, different rational and high-throughput approaches have been successfully applied in the protein crystallography field to widen thejjso-called "protein crystallization bottleneck" [ , ] . despite the enormous efforts carried out by our community, the statistics presented by structural biology consortiums [ ] suggest that so far only the easy-to-pick fruit has been attained; thus, new approaches are necessary to further expand the crystallization limiting step to relevant targets. on the basis of previous hypothesis suggesting that the difficulties found in protein crystallization might be a result of evolutionary negative design [ ] , we have used two different protein engineering approaches exploiting natural sequence diversity using beta-lactamase as toolbox: i) ancestral reconstruction and ii) consensus approach [ ] . both approaches resulted in hyperstable and promiscuous ancestral derivatives. furthermore, our initial crystallization results also suggest that both approaches increased the crystallizability of the resulting enzymes when compared to the extant tem- beta-lactamase. the adipocyte-derived hormone adiponectin has become a key player for the understanding of overweight related diseases like obesity, diabetes, atherosclerosis or the metabolic syndrome. one of its abstract major functions are the insulin sensitizing effects, which are mediated by the activation of ampk, p -mapk and ppara ( ). furthermore adiponectin is involved into glucose regulation and fatty acid oxidation. recently, three adiponectin receptors adipor , adipor and t-cadherin have been described while an unknown fourth receptor is hypothesized ( ) . for only two of them (adipor and adipor ) the signaling transduction via adiponectin has been confirmed ( ). in order to find new binding partners or co-receptors, we cloned and expressed full length adiponectin as a fusion protein with a c-terminal intein and a chitin binding domain (cbd) as well as an n-terminal his -tag. by using the impactsystem, the fusion protein was cleaved to form the corresponding thioester. to separate the starting materials as well as the cleaved intein chitin binding domain, the purification was performed with chitin beads. furthermore, the product was concentrated by ni-nta-affinity chromatography. accordingly, the obtained adiponectin thioester was reacted with a tamra-or a biotin labeled peptide, respectively, to receive the corresponding ligation product. finally the functionalized adiponectin was purified by size exclusion chromatography. further studies will allow screening for interacting molecules in cell and tissue derived samples. departamento de quimica fisica, facultad de ciencias university of granada, dpto. de quimica fisica biologica. instituto de quimica fisica rocasolano, departamento de quimica organica, facultad de ciencias university of granada, rational design of non-natural enzyme activities has proved challenging. here, we report the introduction of catalysis of the kemp elimination (a model of proton abstraction from carbon) in scaffolds corresponding to precambrian nodes in the evolution of the antibiotic resistance protein b-lactamase. we used a single-mutation, minimalist approach based on chemical intuition, and obtained catalysis levels similar to those reported in the literature for computational kemp-eliminase designs involving multiple mutations. remarkably, the approach was unsuccessful when performed on modern b-lactamases. we provide experimental evidence that enhanced conformational flexibility contributes to the success of the minimalist design in the ancestral scaffolds. this work has implications for the understanding of function emergence in protein evolution and demonstrates the potential of ancestral protein resurrection in enzyme engineering and design. exploring the importance of dimerization for dj- function through engineered domain fusions sierra hansen , jiusheng lin , mark wilson parkinson's disease is a progressive neurodegenerative disease that affects approximately . million people worldwide and is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. dj- (park ) is one of several genes that are mutated in rare forms of familial parkinsonism. dj- is a dimeric cytoprotective protein that defends against oxidative stress and preserves mitochondrial function. dimerization of dj- is thought to be essential for this function, as some diseaseassociated mutations cause poor folding and disrupt the dj- dimer. however, recent reports suggest that dj- may be functional as a monomer. to test this, we have engineered a non-dissociable dj- dimer that is a fusion of two human dj- domains. this construct cannot dissociate into monomers and thus will provide a stringent test of the importance of monomeric dj- . our engineered construct is modeled on plant dj- homologs, which feature naturally occurring duplicate dj- domains separated by a small ( amino acid) linker region. using x-ray crystallography, we confirmed that this engineered non-dissociable human dj- dimer has identical structure to the naturally occurring dimeric protein. we have investigated the influence of enforced dimerization of the pathogenic effects of the parkinsonian l p and l p mutations. cd spectroscopic analysis reveals that single and double l p mutations in the non-dissociable dj- dimer maintain a higher degree of structure than l p mutations in the native protein. additional characterization of the protective capacity and subcellular trafficking of this non-dissociable dj- dimer is underway. the purification, crystallization and preliminary characterization of sdre from s. aureus the purification, crystallization and preliminary characterization of sdre from s. aureus staphylococcus aureus (s.aureus) is an important human opportunistic pathogen which colonizes about % of the human population persistently [ ] . surface proteins of s.aureus can excretion a kind of sortase, which represents a surface organelle responsible during the pathogenesis of bacterial infection the host circulation [ ] . sdr proteins were a component of cell wall anchored family proteins, including sdrc, sdrd and sdre [ ] . sdre could combine with the complement regulatory protein factor h to escape the alternative pathway of complement [ ] . to further investigate the functions of sdre, we have expressed and purified the adhesive domain (residues -' : ), and crystallized the recombinant protein. in addition, we also constructed the mutant s.aureus, and the cell experiments confirmed that sdre gene participate in the bacteria invasion. bacterial microcompartments (bmcs) are proteinaceous organelles that sequester key metabolic reactions to increase enzymatic efficiency or to prevent the loss of volatile or toxic intermediates. there is an increasing desire to engineer bmcs for non-native enzymatic processes. it is thought this will increase multi-enzyme pathway efficiency and allow the expression pathways that may produce toxic or volatile intermediates in bacteria. the mechanisms of small molecule transport and retention of toxic intermediates by bmcs remain poorly understood. better understanding of the bmcs pores critical to engineer bmcs for these non-native pathways. in order to better understand the bmc pore we have undertaken structure-guided modifications of the the hexameric pdua shell protein of the , -propanediol utilization microcompartment (pdu mcp). these modifications include pore mutations in an attempt to alter substrate specificity and permutations of pdua to allow more drastic alterations to the structure of the protein. crystal structures of pdua pore mutants, solved to atomic resolution ( - . Å) provide evidence of the pore residues that confer specificity. further, a pdua permutation (pduap) has resulted in a closed icosahedral cage. this novel pduap cage shows a ph and salt dependent assembly and may serve as a reaction vessel or be utilized for cargo delivery. ( , ) . anm-mc is used to identify targeted transition pathways and intermediates between open and closed states of proteins. at each step of this iterative technique, the protein is deformed along the collective anm mode showing the best overlap with the target direction and its energy is minimized via short mc run. in this work, optimization of simulation parameters (number of mc moves and their perturbation strength, anm deformation factor in each cycle and force constant for backbone bonds) was performed in order to increase the efficiency of this technique. as a result, this technique can now be applied to much larger systems and conformational changes. the transition pathway between apo and dna-bound conformations of the yeast rna polymerase, which is a hetero- -mer with more than residues, will be presented here. moreover, the pathway intermediates for more than diverse proteins were analyzed in terms of changes in local strain energy and backbone torsional angles during apo-to-complex transitions. certain residues interacting with the ligand are detected to exhibit large changes with respect to any of these two parameters for more than half of the proteins in our dataset. department of chemistry and chemical biology, harvard university, howard hughes medical institute, harvard university, transgenic crops have radically reshaped the agricultural landscape. since their introduction in the late s, transgenic crops have affected economic gains greater than us$ billion globally due to reduced production costs and increased yield gains. crops modified to produce biological insecticides derived from the soil bacterium bacillus thuringiensis (bt) are among the most robust methods of pest control. bt toxins offer many advantages over traditional insecticides, chiefly their inability to affect human biology and exquisite selectivity for defined pest species. however, the evolution of resistance to bacillus thuringiensis oendotoxins (bt toxins) in insects has been widely observed in the field, and greatly threatens the use of this mechanism of pest control in the future. we developed a phage-assisted continuous evolution (pace) platform for the rapid generation of high-affinity protein-protein interactions and validated the system by evolving known high affinity antibody mimetics in < days of pace. we applied this system to the evolution of the bt toxin protein cry ac to recognize a non-cognate cadherin-like receptor from trichoplusia ni, a pest for which bt toxin resistance has been observed in both the laboratory and the field. the resulting evolved cry ac variants exhibits high affinity for the target receptor, and kill insect cells more potently than wild-type cry ac. our findings establish that the directed evolution of novel receptor recognition in bt toxins can be used to target resistant pests, and has far-reaching implications for biological reagents and therapeutics. optimization of a designed protein-protein interface brian maniaci , collin lipper , john j. love san diego state university, university of california protein-protein interactions play key roles in practically every biological process. protein-protein interactions vary with composition, affinity, and lifetime of the complex. studying designed protein-protein interactions will provide insight into the underlying principles of complex assembly and formation. computational protein docking and amino acid sequence design were used previously to generate protein dimers from monomeric proteins. the normally monomeric b domain of streptococcal protein-g (gb ) was computational docked to itself, followed by optimization of the interfacial side chains. two variants, monomera and monomerb, were computationally derived as a result of a designed protein-protein interface. these designed proteins were characterized using analytical ultracentrifugation and heteronuclear nmr techniques. this design resulted in a pair of protein monomers that formed a heterodimer of modest binding affinity. a tetrahedral metal-templated interface design strategy was implemented in an attempt to strengthen the monomera-monomerb complex by introducing cross-monomer metal coordination. another advantage of using the metal-templated interface is the ability to control the protein-protein interaction both temporarily and spatially. a number of newly engineered variants of monomer a and monomer b with metal coordination sites were designed, produced, and tested for increased affinity of the protein-protein complex. while the generation of a metal-templated monomera-monomerb complex was unsuccessful, we were able to obtain monomera variants that form a homodimer assembly only in the presence of zinc (ii) ions. the crystal structures of metal-templated monomera variants in the presence of zinc provide an explanation for the observed dimer formation. the crystal structure indicates that the protein-protein interaction is not driven by the designed protein interface, but rather non-specific association via edge-strand interactions. new variants were designed with the goal of engineering a high affinity homodimer in a helix-to-helix orientation as the originally designed protein-protein interface. current evaluation of monomera variants for self-association via metal coordination are being evaluated using size exclusion chromatography with a multi-angle light scattering detector for oligomerization state quantification. the results of this protein design project should lead to a greater understanding of the biophysical parameters that drive natural protein-protein interactions. continuous evolution of site-specific recombinases with highly reprogrammed dna specificities the ability to precisely modify the genome of human cells has enormous potential as a novel therapy and a powerful research tool. in contrast to reprogrammable nucleases, such as talens or a cas / sgrna pair -which specifically cleave dna but then rely on stochastic host cells processes to effect gene insertion -site specific recombinases directly catalyze genomic integration with high efficiency. a major limitation of this approach is that recombinases, such as cre, natively bind with high specificity to long dna target sequences (loxp in the case of cre) that do not exist in the human genome. previous attempts at evolving cre resulted in modest changes to its specificity, or required hundreds of rounds of manual protein evolution. we developed and validated a phage assisted continuous evoluiton (pace) selection for rapidly altering the dna specificity of cre recombinase towards a site present in a human genomic safe harbor locus. the pace experiments resulted in cre variants capable of recombining a substrate with nearly % of the nucleotides altered compared to loxp. we successfully used one of these variants to integrate exogenous dna into the genome of unmodified human cells. we are currently using sequencing methods to determine the specificity of the new recombinase clones. aleardo morelli , burckhard seelig generation of comprehensive deletion libraries mediated by in vitro transposition analysis of protein enzymes and ribozymes from nature, and from in vitro evolution, revealed that deletions of up to dozens of amino acids (or nucleotides) can be structurally tolerated. furthermore, shortened variants can exhibit better stability and increased catalytic activity. in order to investigate the effects of deletions, we developed a new procedure based on in vitro transposition to build libraries of more than , deletion mutants in three to four days. we tested our procedure on dna sequences coding for an artificial rna ligase called ligase c. we used the generated library for an mrna display selection, and isolated two active mutants containing and amino acids n-terminal deletions. structural characterization of ppsc, a multi-domain polyketide synthase from mycobacterium tuberculosis using a fragment-based approach alexandre faille , nawel slama , anna grabowska , david ricard , annaik qu emard , lionel mourey , jean-denis pedelacq polyketide synthases are of great interest in numerous scientific fields. they are composed by multiple domains, each having a different role to play in the catalysis of sequential reactions including condensation, reduction and esterification. their reaction products, named polyketides, represent a large variety of chemical compounds, from antibiotics to immunosuppressors or even anticancer drugs. ppsc is a kda polyketide synthase, organised into six catalytic domains (ks-at-dh-er-kr-acp) with singular functions. along with other type i polyketide synthases, ppsc is responsible for the biosynthesis of an essential polyketide for the virulence of mycobacterium tuberculosis (mtb) and thus is a target of choice for the design of inhibitors. to date, no structural information of any type i polyketide synthase in its entire form has been described. main reasons are the length of these large size enzymes and the flexibility imposed by the linkers between domains, thus making them very difficult to crystallize. numerous questions about domain-domain interactions, spatial arrangement of this complex machinery, substrate specificity and stereochemistry are still unanswered. addressing the structural and functional characterization of ppsc would then help answering these questions and provide valuable information for drug design. to overcome the length-and flexible-dependent problem originating from the presence of multiple domains and linkers, we decided to study domains expressed alone. for this purpose, we used our domain trapping strategy to identify soluble fragments representing a single domain from ppsc [ ] . it has the advantage of not relying on the bioinformatically designed domain boundaries and can even sometimes include parts of linkers to obtain more soluble fragments. using this strategy, we were able to identify relatively small and highly soluble fragments representing each domain of ppsc, thus facilitating the downstream structural and functional characterization. more than fragments have been submitted to crystallization trials. among these, gave crystals and allowed us to determine the x-ray structure of ppsc at, er, in addition to the dh domain in complex with a substrate analog for which activity was confirmed in vitro. the computational design of proteins that bind small molecules remains a difficult challenge in protein engineering. the ability to computationally design native-like interactions with high accuracy and efficiency would be an asset towards therapeutic development, enzyme design, and engineering functional proteins. we have developed a systematic approach to designing interfaces. we first identify ligands with naive binding affinity to our protein scaffold, then use rosettaligand to computationally dock the ligand while designing the interface for a tighter interaction. this way, we are taking a 'shot in dim light' for design as opposed to a 'shot in the dark', allowing us to more thoroughly investigate the successful and not-so-successful designs, and improve the computational methods. of ligands screened, we identified weakly-binding hits in the range of - mm. thus far, rosettaligand has successfully designed one tighter protein-ligand interface, from mm to mm. in progress experiments include designing and experimentally validating more designed interfaces. structural studies of human acidic fibroblast-growth factor (fgf ) mutants with a probable anticancer activity lectins are carbohydrate-binding proteins ubiquitously present in nature. they play a role in biological recognition phenomena involving cells and proteins. the interaction lectin-carbohydrate is highly specific, and can be exploited for the development of nanoparticles containing on their surface lectins specifically directed to carbohydrate residues present only on malignant cells and absent on healthy ones ( ) . lectins have been found to possess anticancer properties and they are proposed as therapeutic agents, binding to cancer cell membranes or their receptors, causing cytotoxicity, apoptosis and inhibition of tumor growth. some lectins are able to prevent the proliferation of malignant tumor cells because they recognize the t-antigen (gal b - galnac) found specifically on the surface of tumor cells ( ) . the main problem is that their use as a detection agent for the t-antigen in clinical studies is not possible because the immune system can recognize them as foreign molecules and develop an immune response. previous studies with x-ray crystallography made in our laboratory have characterized a lectin found in mushrooms called bel b-trefoil which has antiproliferative activity on tumor cell lines, because it contains three binding sites for the t-antigen. unlike other lectins with this property, bel b-trefoil shows structural homology with a human protein, acidic fibroblast growth factor (fgf ) ( ). superposition of their structures suggests that the human protein could be mutated to contain at least one of the binding sites for the t-antigen. such mutations should create in fgf the potential capacity of recognizing tumor cells with less immunogenicity than the fungal protein. fgf is mitogenic and chemotactic, and mediates cellular functions by binding to transmembrane receptors, which are activated by ligand-induced dimerization requiring heparin as co-receptor. to reach our purpose, the fgf cdna was cloned into a bacterial plasmid and then mutated in five different positions to eliminate its mitogenic activity and to engineer in the protein the t-antigen binding capacity. attempts to crystalize the mutants of fgf were made using the hanging drop technique with the final aim to carry out their structural characterization by x-ray diffraction analysis of the crystals. the de novo synthesis of proteins in response to the activation of cellular signaling pathways is a crucial element of many high-level biological processes, including the synaptic plasticity underpinning memory formation in the brain. while of fundamental biological importance, there has been a shortage of tools with which to specifically target pools of newly synthesized proteins of interest for study. thus, we have developed timestamp and smash, methods for drug-dependent tagging, or destruction, respectively, of newly synthesized copies of proteins of interest. both methods rely on protein tags that remove themselves by default via an internal hepatitis c virus (hcv) ns protease, but which are retained in the presence of cell-permeable small molecule protease inhibitors. the timestamp tag contains split yfp halves and epitope tags which are reconstituted and preserved, respectively, on proteins of interest following drug application, whereas the smash tag contains a strong degron which remains attached to proteins of interest following drug application, resulting in their clearance. one limitation of time-stamp and smash is that they can only be used to independently manipulate one protein of interest at a time. furthermore, the application of timestamp and smash to study endogenous protein pools in mammals has not yet been explored. here, we report on efforts to extend these techniques by reengineering ns proteases which can be inhibited by two different drugs orthogonally to one another. by incorporating different drug resistance mutations into two ns protease variants, we engineered ns protease domains that are inhibitable either by asunaprevir only, or by telaprevir only. we found that these tags permit simultaneous and independent control over the newly synthesized pools of two proteins of interest within the same population of cells. we also report the development of transgenic knock-in mouse strains incorporating timestamp and smash tags, which allow the interrogation of newly synthesized pools of specific endogenous synaptic proteins in the context of their endogenous regulatory elements, and without relying on overexpression. infectious diseases are often diagnosed by the presence of specific antibodies that are produced in response to the invading pathogen. one example are antibodies that are present in patient blood after infection with the dengue virus serotype and that are directed against an epitope on the virus' nonstructural protein (ns- ). traditional antibody diagnosis relies on time-consuming multi-step assays that require sophisticated equipment in a laboratory environment. a promising alternative are protein switches that are based on bioluminescence resonance energy transfer (bret). these switches comprise a luciferase (nanoluc) and a green fluorescent protein (mneongreen), which are connected via a semiflexible linker. the linker contains two epitope sequences of ns- to which the antibodies bind specifically. if no antibodies are present nanoluc and mneongreen are held in close proximity via two helper domains and bret can occur; thus green light originating from mneongreen is visible. if antibodies are present, they bind to the specific epitopes in the linker of the switch and cause stretching of the linker and therewith break the interaction of the helper domains. as a result, nanoluc and mneongreen are separated in such a way that bret cannot occur anymore; thus only blue light originating from nano-luc remains visible. using this principle, monoclonal anti-ns- antibodies were detectable in a controlled buffer system and in spiked plasma samples. furthermore, the developed antibody switch was applied to plasma samples of macaques after a primary infection with dengue virus serotype . signal readout was possible using a laboratory-based plate reader as well as the camera of a standard smartphone. we demonstrate that this bret-based protein switch can quickly detect antibodies in solution in a single-step assay format using simple equipment for signal readout, such as a standard smartphone. this simplified antibody detection platform has the potential to be carried out outside of a laboratory, thus in areas with limited laboratory infrastructure and a high number of diverse infectious diseases. proteins expressed from more than two-thirds of the human genome reside within intracellular compartments. of these proteins many are important disease-related targets such as kras and c-myc which cannot be easily addressed by conventional small molecule approaches. some of the weaknesses of small molecules can be addressed by biologic drugs, for example high target specificity and inhibition of protein-protein interactions. the challenge for biologics is how to engineer recombinant proteins to access the intracellular space. one strategy is to use systems evolved by bacteria and viruses to deliver material inside the cells. an example of such pathway is used by pseudomonas exotoxin a (pe). the modularity of pe allows the catalytic domain to be replaced with a biologic payload against desired intracellular target. an additional benefit of pe-based delivery is a possibility of targeting the drugs only to relevant cells in the body by modifying the cell-targeting domain of the pe. the aim of this project is to deliver functional payloads against k-ras and c-myc into the cell using a pseudomonas exotoxin a translocation domain. we used phage and ribosome display to select antibody mimetics that bind k-ras and c-myc. here, we present their activity in biochemical assays and the initial results on generation of pe-based constructs. ( ) . hemagglutinin is synthesized as ha molecule assembled as noncovalently bound homotrimers on the viral surface. this precursor protein is cleaved by trypsin-like proteases to yield two subunits ha and ha linked by a single disulphide bond ( ) . ha is also post-translationally modified by n-glycosylation ( ). it is well established that the virus hemagglutinin is the main antigen, inducing the neutralizing antibodies. in the attempt towards developing influenza vaccine production (the egg-based manufacturing lasts several months) that would be faster and safer the utilization of recombinant antigen alone is currently being observed. recently we demonstrated that yeast produced influenza h protein although cleaved into two subunits induced strong immunological response in mice ( ) . in this report, we describe the biochemical and immunological characterization of the h antigen, based on hydrolytic domain of the h n gene, with deletion of multibasic cleavage site and expressed in yeast system. the ha encoding gene from h n virus with deletion of nucleotides was cloned into ppiczac vector. rha fusion protein with his -tag was secreted into the culture medium and was purified to homogeneity in one step using ni-nta agarose. the efficiency of the antigen purification was mg/l. glycosylation sites of rha were determined using lc-ms-ms/ms. analysis of the n-linked glycans revealed that the rha is glycosylated at the same sites as the native ha in the vaccine strain. next we investigated if the hemagglutinin with deletion of the cleavage site oligomerize into higher molecular forms. to determine the oligomeric forms of the recombinant antigen various approaches were applied e.g. native-page, size exclusion chromatography or dynamic light scattering. as a final experiment to measure the size of oligomers in a protein sample a combined technology sec-mals was conducted, using multi angle light scattering (mals) as a detector. the immunological activity of rha was tested in chicken and mice, where antigen elicited high immune response. the data presented here demonstrate that new influenza antigen produced in p. pastoris is highly immunogenic and might be consider as a candidate for subunit vaccine. structural motifs capture redundant patterns that frequently occur in proteins. motifs associated with contiguous fragments of structure (i.e., secondary structural motifs) are well studied and have been successfully used to capture "rules" describing sequence-structure relationships in protein design and structure prediction. we have extended this concept to motifs that capture tertiary information-(i.e., tertiary structural motifs or terms. we have discovered that a relatively small alphabet of terms describes the known structural universe (all secondary, tertiary and quaternary information in the pdb) at sub-angstrom resolution. this alphabet of universal motifs reveals the remarkable degeneracy of the protein structure space, with just a few hundred terms sufficient to accurately capture half of the known structural universe. we have begun to demonstrate the considerable promise this structural alphabet has for applications such as protein design, structure prediction, and docking. we have developed a novel protein design framework that selects amino acid sequences, given a desired structure, using solely information from the universal terms. we show that given a native backbone, this framework recovers the native sequences to a level on par with state-of-the-art atomistic protein design methods, indicating that the motifs capture the salient structural rules governing native proteins. further, predicted sequence distributions agree closely with observed evolutionary variation. given the apparently high degeneracy among even complex features of protein structure, methods based on mining the pdb for tertiary information should provide ample opportunities for advancement in problems of computational structural biology. sortase-mediated synthesis of protein-dna conjugates for sensitive biosensing bedabrata saha , marieke op de beeck , remco arts , maarten merkx in recent years, semisynthetic protein-dna conjugates have emerged as attractive biomacromolecules for different applications in bio-nanotechnology, biosensing, diagnostics and therapeutics. in protein-dna conjugates, synthetic oligonucleotides allow the construction of desired molecular architecture with high specificity, while maintaining the original functionality of the protein molecules for desired application. however, the synthesis of site-specific and stoichiometric protein-dna conjugates can be challenging. due to the diversity in composition and physico-chemical properties of the proteins, few generic strategies are available for conjugation of protein molecules to a dna scaffold. a common approach is to use thiol-based covalent conjugation, but the introduction of additional cysteines can lead to the formation of intermolecular disulfides or interfere with the formation of native disulfide bonds. as an alternative, here we have developed a site-directed protein-dna conjugation strategy based on sortase mediated trans-peptidation reaction. the sortase recognizes a 'sorting motif' (i.e. lpxtg, x any amino acid), which is recombinantly introduced by site-directed mutagenesis at the cterminal end of the protein molecule. the sortase cleaves the t-g peptide bond and catalyzed the formation of a new amide bond between the lpxt peptide and the n-terminal amine of any molecule bearing an n-terminal oligoglycine motif. for this purpose, a triglycine motif was introduced at the 'end of single-stranded dna (ssdna). on-column synthesis of triglycine modified ssdna, protected on a controlled pore glass beads, simplified the purification process and enhanced the yield of triglycinemodified ssdna (> %). we used this conjugation strategy in several biosensing applications. for example, we used the method to conjugate ssdna linkers at the c-termini of a range of single-chain antibody fragments (scfv) and applied these constructs to allow oriented display of capture molecules on biosensor surfaces. ssdna-scfv were using an excess of triglycine modified ssdna, we achieved % conversion scfv-ssdna conjugate, which can be further purified by in two step purification process consisting of ni-nta affinity column and ion-exchange chromatography. we also extended this sortase-based conjugation strategy to develop a bioluminescence based assay for sensitive target oligonucleotide detection. in this regard, the ' and ' end triglycine-modified ssdna molecules were successfully conjugated with a bret protein pairs, nanoluc luciferase and mneongreen fluorescent protein. the introduction of a c-terminal sortase-his tag and and n-terminal strep-tag allowed efficient purification of theseprotein-ssdna conjugates from excess oligonucleotides and unreacted protein. mass spectrometry based proteomics to identify the protein differences in human breast milk from breast cancer patients and controls devika channaveerappa , roshanak aslebagh , kathleen f. arcaro , costel c. darie breast cancer is the second leading cause of cancer death in women. about % women in the us develop breast cancer. death rates due to breast cancer have been declined over the years due to advancements in mammography and treatment. although, mammography helps in the early detection of breast cancer, it has few limitations. dense breast tissue makes mammogram less accurate. breast milk can be assessed to evaluate the risk of one getting breast cancer by comparing the proteomes of breast milk from healthy and breast cancer suffering individual. this study makes use of mass spectrometry based proteomics to identify the differences between the control and cancerous samples which would further help in identifying potential biomarkers for breast cancer. firstly, sds-page was used to separate the proteins from the whole milk sample. the gel bands for each sample was then excised and cut into small pieces. the gel pieces were washed and trypsin digested in order to extract the peptides. peptide mixtures in the solution were cleaned using c zip-tipp and then analyzed by liquid chromatography-tandem mass spectrometry (lc-ms/ms). minutes and minutes gradient were used for lc-ms/ms analysis. raw data obtained were converted to pkl files using proteinlynx global served (plgs version . ). raw data were then submitted to mascot database search for protein identification. the mascot results were then exported as .dat files and further analyzed using scaffold version . software. three breast cancer milk samples were investigated against healthy control milk samples. in the sds-page gel, after coomassie staining, the protein patterns did show minor differences. after lc-ms/ms analysis, the proteins identified by mascot database search were imported into the scaffold software and compared for the relative ratio between the proteins from the milk sampled from control donors and the donors with breast cancer. there were significant differences identified in the proteomes of the two sets of samples. some of the proteins were upregulated in the breast cancer samples and some were down regulated when compared with the controls. additional investigation of more breast milk samples is ongoing. this study focuses on identifying biomarkers directly in the milk of donors with breast cancer. leukolike vectors: leukocyte-inspired nanoparticles claudia corbo , , alessandro parodi , , roberto palomba , , roberto molinaro , michael evangelopoulos , francesco salvatore , , ennio tasciotti the houston methodist research institute, fondazione irccs sdn, nanomedicine aims to improve drug efficiency by enhancing targeting and biocompatibility, and reducing side effects. multiple surface modifications have been proposed to provide nanocarriers with these features, based on complex synthesis processes and very often inefficient in contemporary providing biological tolerance and targeting properties [ ] . bio-inspired approaches based on surface coatings developed from the purified cell membrane of immune cells represents a new paradigm shift for the development of carrier enable of prolong circulation and proper tumoritropic capabilities. we showed that nanoporous silicon (nps) particles coated with leukocyte cellular membranes -leukolike vectors (llvs) -possess cell-like properties [ ] . llvs can escape macrophage uptake, delay sequestration by the reticulo-endothelial system, target tumor inflamed vasculature and accumulate within the cancer parenchyma [ ] . llvs were fully characterized for their shape, size, surface charge and coating through dynamic light scattering and scanning electron microscopy. in addition we characterized the content and function of the leukocyte's proteins transferred onto the llvs coating through high-throughput proteomic analysis and the results revealed the presence and the correct orientation of several important markers of leukocytes: cd , cd and mhc-i were identified as key players in determining llvs biocompatibility, while leukocyte associated function- (lfa- ) and mac- contributed to the llvs targeting ability and bioactivity towards inflamed endothelium [ ] . recent investigation showed that the coating induced the formation of a singular protein corona (i.e. the protein adsorption layer) on the surface of the nanoparticles compared to negative control following in vivo injection. in addition, the proteolipid coating favored active extravasation of the llvs in the tumor vasculature by molecular mechanisms similar to those used by tumor infiltrating leukocytes. this work shows that is possible to transfer biologically active leukocyte membrane proteins onto synthetic nanoparticles, thus creating biomimetic carriers retaining cell-like functions that are not affected by the protein corona effect that occurs in vivo. the targeting of the inflamed endothelium can be applied to a broad range of diseases and the approach used to formulate the system could open new avenues for the fabrication of the next generation of personalized treatments by using as cell membrane source the immune cells of patients. references: [ ] alessandro parodi, claudia corbo, armando cevenini, roberto molinaro, roberto palomba, laura pandolfi, marco agostini, francesco salvatore, ennio tasciotti. enabling cytoplasmic delivery and organelle targeting by surface modification of nanocarriers. nanomedicine uk. accepted. steroid hormone receptors are intracellular receptors that initiate signal transduction in response to steroid hormones, including oestrogen and androgens. generally, the binding of the steroid to the nuclear receptor induces the protein to form a dimer and relocate onto the chromatin, although the order of these events may vary. the location of receptor binding on the chromatin is defined by specific hormone response elements (hre). once located, the receptor promotes gene activation by the recruitment of other co-factors. it is this process that makes the complex of receptor protein and co-factors play a pivotal role in the regulation and activation of genes. the failure to regulate this process correctly is a key step in the development of several endocrine-driven cancers. for example: oestrogen receptor positive (er ) breast cancer is one of the most common forms of cancer and accounts for % of all breast cancer cases. in er tumours, the oestrogen receptor (er) drives the tumour growth and cell proliferation. understanding the interactions of the er with other proteins, either directly or indirectly, can provide vital insight to the regulation of the system that drives this cancer. the progesterone receptor (pr) has also been implicated in breast cancer, and the androgen receptor (ar) is a known driver in the majority of prostate cancers. to meet the challenges of elucidating these systems, we have developed methods to purify and analyse cross-linked regulatory complexes bound to dna by mass spectrometry (chip-ms). this allows for the enrichment of proteins involved in gene regulation. chip-ms, combined with tandem mass tags (tmt), makes it possible to realise a quantitative method to investigate the dynamic network of interactions between proteins within complexes that undertake the regulation of biological systems. chip-seq is a well-established method for identifying where these protein complexes are bound to the genome. this work focuses on how to combine these technologies with my previous development of cross-linking coupled mass spectrometry techniques (xcms) to provide a strategy for visualising the dynamic organisation of the proteins on the chromatin. global kinetic analysis of caspase protein substrates in cell lysate reveals selective roles and target specificity olivier julien , min zhuang , arun wiita , james wells caspases are cysteine proteases that play important roles in development, cell differentiation and cell death. however, the limited number of known caspase substrates hinders our understanding of caspase function. here we performed a non-biased identification and kinetic analysis of caspase- and caspase- proteolytic substrates in cell lysate, using an enzymatic n-termini enrichment approach followed by mass spectrometry. we identified and potential substrates for the initiator caspase- and putative executioner caspase- , respectively. our results not only confirm known substrates but also identify many more new substrates with the precise location of proteolysis. given the emerging roles of caspases- and in inflammation and neurodegeneration, these new substrates may provide molecular insight into the progression of related diseases. the sequence consensus logo of caspase- targets was very similar to a classical executioner caspase motif (devd), while caspase- revealed a vevd motif. using selected reaction monitoring (srm), we quantified the kinetics of proteolysis of a large subset of these substrates by measuring the appearance of the caspase cleavage product over time. in the end, we measured and kcat/km values for individual substrates cut by caspase- and caspase- , respectively. by comparing these data with our previous analysis of caspase- , , and , we found that substrates that are shared between caspases are often cleaved at rates that differ by orders of magnitude. thus, despite having nearly identical primary sequence motifs, the caspases exhibit remarkable substrate specificity that may reflect their specialized roles within the cell. the rockefeller university, new york university school of medicine, johns hopkins university school of medicine line- (l ) retrotransposons are catalysts of evolution and disease whose sequences comprise a significant proportion of the human genome. despite tremendous influence on genome composition, l rnas only encode two proteins. consequently, l particles include a combination of permissive host factors that are essential to their lifecycle as well as repressive factors that constitute defenses against l 's mutagenic activity. we previously characterized host proteins associated with synthetic and natural human l retrotransposons, as expressed in cell culture, using a combination of techniques including metabolic labeling and affinity proteomics. to build on these analyses, we have implemented a series of d separations and post-purification treatments to produce a multi-dimensional interactomic characterization of affinity isolated l s. these studies have revealed the presence of at least two populations of putative transposition intermediates that may exhibit distinctive intracellular localizations. we report a comprehensive, quantitative survey of the proteins partitioning within these distinct l populations and their associated in vitro activity. our observations provide a basis for the classification of l interactors with respect to their physical and functional links, facilitating hypotheses to direct in vivo experimentation. polyubiquitin recognition by continuous ubiquitin binding domains of rad probed by modeling, small-angle x-ray scattering and mutagenesis sangho lee , trung thanh thach , namsoo lee , donghyuk shin , seungsu han , gyuhee kim , hongtae kim rad is a key protein in double-strand break dna damage response (ddr) pathways by recognizing k -linked polyubiquitylated chromatin proteins through its bipartite ubiquitin binding domains ubz and lrm with extra residues in between. rad binds k -linked polyubiquitin chains as well as k linked ones and mono-ubiquitin. however, the detailed molecular basis of polyubiquitin recognition by ubz and lrm remains unclear. here, we examined the interaction of rad ( - ), including ubz and lrm, with linear polyubiquitin chains that are structurally similar to the k -linked ones. rad ( - ) binds linear polyubiquitin chains (ub , ub , ub ) with similar affinity to a k -linked one for diubiquitin. ab initio modeling suggests that lrm and the extra residues at the c-terminus of ubz (residues - ) likely form a continuous helix, termed 'extended lr motif' (elrm). we obtained a molecular envelope for rad ubz-elrm:linear ub by small-angle x-ray scattering and derived a structural model for the complex. the rad :linear ub model indicates that elrm enhances the binding of rad with linear polyubiquitin by contacting the proximal ubiquitin moiety. consistent with the structural analysis, mutational studies showed that residues in elrm affect binding with linear ub , not monoubiquitin. in cell data support that elrm is crucial in rad localization to dna damage sites. specifically e seems to be the most critical in polyubiquitin binding and localization to nuclear foci. finally, we reveal that the ubiquitin-binding domains of rad bind linear ub more tightly than those of rap , providing a quantitative basis for blockage of rap at dsb sites. taken together, our data demonstrate that rad ( - ) forms continuous ubiquitin binding domains, comprising ubz and elrm, and provides a structural framework for polyubiquitin recognition by rad in the ddr pathway at a molecular level. optimization of a protein extraction method for the proteomic study of pozol cynthia teresa leyva-arguelles , carmen wacher , rosario vera , romina rodr ıguez-sanoja instituto de investigaciones biom edicas, unam., facultad de qu ımica, unam., instituto de biotecnolog ıa, unam key words: proteomics, fermentation, pozol pozol is a mexican traditional no alcoholic beverage elaborated by various ethnic groups in the southeastern of mexico. pozol is obtained from the natural fermentation of nixtamal (heat-and alkali-treated maize) dough. the main carbohydrate in maize dough is starch ( - %), because others such as sucrose, glucose and fructose are mostly lost during nixtamalization; so, the starch remains as the major carbohydrate available for fermentation [ ] . a wide variety of microorganisms have already been isolated from the fermentation of pozol; these microorganisms include fungi, yeasts, lactic acid bacteria, and non-lactic acid bacteria [ ] . however, only few bacteria are amylolytic in this fermentation and all of them are weakly amylolytic [ ] . in an attempt to explain how a very low content of soluble sugars can support a diverse and abundant microbiota, a proteomic approach was designed to understand the fermentation of pozol [ ] . nevertheless, the extraction of proteins from pozol remains a limiting step in proteomic analysis mainly due to the complexity of the sample. on the basis of the aforementioned reasons, the aim of this work was to obtain a suitable extraction method of proteins for proteomic analysis. therefore, the fermentation of pozol was continued for h and samples were taken at , , and h. for each sample, the total sugar content was determined by the dubois et al. method [ ] and protein extraction was performed by two methods: a) direct extraction from the dough [ ] and b) initial extraction of microorganisms and soluble proteins (this work). comparison between the two protein methods was performed on two-dimensional gels with silver stain. then, gels underwent to image analysis by the image master d platinum software. comparing the d-gels, more proteins spots were obtained with method b than that with method a, indicating a more efficient protein extraction with method b. although, using method a higher concentration of total proteins was observed, they were mostly maize proteins, that in turn overlap and reduce the efficiently extraction of the microbial low abundant proteins. then, method b allows a better extraction of those low abundant proteins and removes sample components that may interfere with the determination. these results could help us to find the proteins involved in carbohydrate metabolism of the microbiota and finally elucidate the dynamics of pozol fermentation. proteomics has been applied to the enology field for numerous purposes including fermentation control, improvement of fermentation processes, ensuring wine quality, etc. according to rodriguez et al., ( ), the information provided by wine proteomics is not only useful for these intentions, but also offers excellent prospects for innovation and diversification of winemaking processes in the near future. in this context, our group has focused research on the identification of proteins that might be important for yeast survival under typical wine elaboration conditions (standard fermentation, sherry wine biological aging and sparkling wine second fermentation) as well as proteins that configure the content of metabolites which are ultimately responsible for wine quality. by using novel proteomic (offgel fractionator and ltq orbitrap xl ms) and metabolomic techniques (sbse-td-gc-ms) we have identified a high amount of up-regulated proteins involved in processes like oxidative stress response (in biological aging) or protein biosynthesis (in second fermentation) as well as thirty-three proteins directly involved in the metabolism of glycerol, ethanol and seventeen aroma compounds excreted by the yeast under biological aging conditions. further, in order to validate proteome data; null mutants of genes codifying proteins up-regulated in the biological aging condition were constructed. analyses of correlated phenotypes are in progress. this technique and its combination with metabolomics within the enology context will provide enough knowledge to design or choose yeasts or conditions that satisfy wine production and/or wine characteristics such as color/aroma/texture/flavour profile demands of winemakers and consumers. additional binding sites for cytochrome c on its redox membrane partners facilitate its turnover and sliding mechanisms within respiratory supercomplexes blas moreno-beltr an , antonio d ıaz-quintana , katiuska gonz alez-arzola , alejandra guerra-castellano , adri an vel azquez-campoy , miguel a. de la rosa , irene d ıaz-moreno ibvf, ciccartuja, universidad de sevilla -csic, bifi -iqfr (csic), universidad de zaragoza, departamento de bioqu ımica y biolog ıa molecular celular, universidad de zaragoza, gliding mechanisms of cytochrome c (cc) molecules have been proposed to shuttle electrons between respiratory complexes iii and iv within plant and mammalian mitochondrial supercomplexes, instead of carrying electrons by random diffusion across the intermembrane bulk phase [ ] [ ] . in this work, the binding molecular mechanisms of the plant and human cc with mitochondrial complexes iii and iv have been analyzed by nuclear magnetic resonance and isothermal titration calorimetry. our data reveal that both cc-involving adducts possess a : stoichiometry -that is, two cc molecules per adduct -. the presence of extra binding sites for cc at the surfaces of complexes iii and iv opens new perspectives on the mitochondrial electron transport chain, where membrane respiratory complexes can be either in independent, free diffusional motion or forming macromolecular assemblies. in the latter context, such new binding sites for cc facilitate the turnover and sliding mechanisms of cc molecules within supercomplexes. indeed, the accommodation of several cc molecules between complexes iii and iv in supercomplexes provide a path for cc diffusion from complex iii to iv. such path could have physiological significance in the electron flow, which is controlled in supercomplexes to optimize the use of available substrates [ ] [ ] [ ] . can bio-functionalities be deciphered from protein sequence information using computational approaches? background: the processes of uncovering bio-functionalities such as pharmacological activities, disease processes, physiological and structural properties by means of clinical approaches are irrational. this is because they are resource and time consuming. sometimes, they involve sophisticated and expensive equipments, reagents and animal tissues. contrarily, sequence information-based computerized approaches are rational and have become relevant in assessing bio-functionalities. they include geno pheno [coreceptor] [ ] , position-specific scoring matrix (pssmsi/nsi and pssmcxcr /ccr ) [ ] , and informational spectrum method (ism)-based phylogenetic analysis (istree) [ ] . aim: this presentation demonstrates how bio-functionalities could be deciphered from sequence information using computational approaches. method: ism procedure and peptides, vipmfsals and capagfail are engaged. results: protein sequences of the peptides are converted into bio-functionality (affinity). affinity between the two peptides is demonstrated as significant amplitudes at the point of common interaction also referred to as consensus frequency, signifying remarkable affinity. discussions: bio-functionalities of bio-molecules are known to be expressed in one or two genes, which have been found to provide as much biological information as the bio-molecules. this indicates that biological characteristics, represented in these genes and proteins can now be extracted from their sequence information. for example, multi-drug resistances arising from a variety anti-microbial agent from several classes including alkaloids, flavonoids, etc can be retrieved from the sequence information of their encoding genes (mdr and mdr ). similarly, translation of hiv infection to aids disease can be extracted from the protein sequence alterations in the hiv gp . similarly, effectiveness of anti-retroviral agent, maraviroc on the hiv isolate h bx and ndk can be deciphered from the sequence information of their v observed at the predicted sequences. these positions are important as they surround the cleavage site in the three-dimensional structure, and are probably less tolerant to change. moreover in previous studies, cys at p position has been shown to be the dominant determinant for cleavage efficiency, while cys, pro and glu at p position have also been shown to be correlated with increased cleavage efficiency of ns / a protease. for adv cysteine protease, on the other hand, bsst produces similar significant results for both type (xgx-g) and type (xgg-x) consensus cleavage sites, where p and p ' positions have gly with highest percentage in type (xgx-g) while p and p positions have gly in type (xgg-x). these indicate that the bsst seems to provide a powerful methodology for predicting the substrate specificity for the hcv ns / a serine protease and adv cysteine protease, which are targets in drug discovery studies. protein plasticity improves protein-protein binding description chiara pallara , juan fern andez-recio an accurate description of protein-protein interactions at atomic level is fundamental to understand cellular processes. however the current structural coverage of protein-protein interactions (i.e. available experimental structures plus potential models based on homologous complex structures) is below % of the estimated number of possible complexes formed between human proteins. , for these reasons, computational docking methods aim to become a complementary approach not only to solve the structural interactome but also to elucidate the basis of the protein-protein association mechanism. in spite of the advances in protein-protein binding description by docking, dealing with molecular flexibility is a major bottle-neck, as shown by the recent outcomes of the capri (critical assessment of prediction of interactions) experiment. this data clearly confirms that the protein dynamics plays a key role in protein-protein association. the use of conformational ensembles generated from unbound protein structures in combination with computational docking simulations might represent a more realistic description of protein-protein association. here, we present the first systematic study about the use of precomputed unbound ensembles in docking, as performed on a set of cases of the protein-protein docking benchmark . . the primary aim of our work is to understand the role of the protein conformational heterogeneity in protein-protein recognition. to do this, small conformational ensembles were automatically generated starting from the unbound docking partners, and then an extensive analysis of their binding properties was performed in the context of pydock docking scheme. the results show that considering conformational heterogeneity of interacting proteins can improve docking description in cases that involve intermediate conformational changes in the unbound-to-bound transition. more interestingly, we found that protein plasticity increases chances of finding conformations with better binding energy, not necessarily related to bound geometries. the relevance for future docking methodology development and for understanding protein association mechanism will be discussed. purpose of the research: there is increasing interest in the development of protein scaffolds that can be used to develop affinity reagents that are alternatives to antibodies. the affimer scaffold is based on the cystatin protein fold. the affimer scaffold is biologically inert, biophysically stable and capable of presenting a range of designed or random binding surfaces defined by peptides inserted at different loops. the result is highly specific, high affinity interactions with a wide range of targets including ones that are inaccessible to antibodies. affimers are designed to work in the same way as the very best antibodies, but with a number of key advantages. affimers are quick to develop (typically weeks) without using animals. they contain no disulphide bonds, are expressed easily in e. coli and have no batch to batch variability. affimers are small molecules ( aa, kda), robust and stable (resistant to ph range, thermally stable and not sensitive to edta). affimers can be a direct replacement for antibodies -no process or workflow change required -and perform identically to antibodies in assays such as elisa, facs, ihc, western blots, affinity purification, microarray and potentially therapeutics. we describe some applications of the technology in regards of affimer development for custom targets on one hand and for the biomarker discovery workflow using affimer microarrays on the other. main results: by screening of our very large ( x ) library against yeast sumo protein we identified affimers with high affinity allowing their use for elisa. moreover, no cross-reactivity was observed when affimers were used on western blots leading to a unique band specific to yeast sumo when compared to human proteins. a library of , random affimers, expressed in e. coli, was printed on glass microscope slides and challenged with plasma from children (n ) with sepsis and from healthy children (n ). unsupervised hierarchical clustering based on the , affimers allowed differentiation between the control and patient samples. affimers were found to differentially bind proteins between the groups with a > fold change. the affimer arrays identified a strong signature of sepsis and roc curve analysis allowed confident prediction of disease (auroc of . ). affinity purification and preliminary mass spectrometry analysis identified known biomarkers of sepsis and also potentially novel biomarkers not previously associated with this disease. major conclusions: this work demonstrates the scope of affimer affinity reagents to develop alternative binders to antibodies, where affimers perform identically in most assays without the disadvantages associated with antibodies. moreover, affimers enable a new protein microarray-based biomarker-discovery workflow and we predict that array-based validation of signatures identified using discovery arrays prior to affinity purification and mass spectrometry will offer a cost-and time-effective methodology compared to purely mass spec-driven workflows. tau pathologies, called 'tauopathies', are related to several neurodegenerative diseases including alzheimer disease (ad). in ad, tau protein is observed hyper-phosphorylated and aggregated as paired helical filament (phf). the neuronal tau protein is an intrinsically disordered proteins (idps). nuclear magnetic resonance spectroscopy (nmr) is here used to study the tau protein phosphorylations and protein-protein interactions (ppis). in in vitro assays, tau phosphorylation by rat brain extract is considered as an hyperphosphorylation model that was furthermore pointed out to enable tau aggregation [ ] . in a first step, we have identified all the phosphorylation sites of rat brain extract phosphorylated-tau, using the analytical capacity of nmr. we showed that the protein is modified at ser/thr sites. among the kinases that we have characterized so far using tau as substrate, only the extracellular signal-regulated kinase (erk ) shows an ability to modify in vitro tau protein on so many sites. we have indeed identified phosphorylated ser/thr-pro motifs out of potential phosphorylation sites in the sequence of full length -residue tau. in addition, we showed using transmission electron microscope (tem) a similar in vitro aggregation capacity of erk-phosphorylated tau protein compared to that of rat brain extract phosphorylated-tau. this shows that phosphorylation by the erk kinase generates an hyperphosphorylated tau. given the high efficiency of erk towards tau, we have next looked into the mechanism of tau recognition. erk kinase possesses two well-characterized docking domains: d recruitment sites (drs) and f recruitment sites (frs), which recruit complementary docking sites and increase the specificity and efficiency of the interaction with both its upstream regulators and downstream substrates [ ] . as the interaction between tau protein and erk kinase is analyzed by nmr spectroscopy, multiple sites of interaction are observed along the tau sequence, similar to drs docking sites, all located in the so-called microtubule binding domain of tau. these sites are short sequences loosely matching the reported consensus for d sites w - uxu (w, u, and x refer to positively charged, hydrophobic, or any intervening residues, respectively) [ ] , and also the reverse sequence uxuw - .to confirm the mapping of the interaction, two tau recognition sites were produced as recombinant peptides of about amino-acid in fusion with an n-terminal his-tag sumo. interaction assays using d [ h, n] hsqc spectra of the peptides confirm their binding to erk kinase. the potential of these peptides to inhibit erk activity with tau as substrate is now being investigated. while rigid-body docking has become quite successful for predicting the correct conformations of binary protein complexes, determining whether two given proteins interact remains a difficult problem. successful docking procedures often give equally good scores for pairs of proteins for which there is no evidence of interaction. studies investigating what we define as the 'pre-docking' problem via in silico approaches have only recently become feasible with the help of supercomputers and gridcomputing systems. in a previous work, on a restricted set of protein complexes, we showed how predictions of interacting partners could be greatly improved if the location of the correct binding interface on each protein was known. experimentally identified complexes are found to be much more likely to bring these two interfaces into contact, at the same time as yielding good interaction energies. we present data from a complete cross-docking (cc-d) study of a database of proteins, including the treatment of more than , potential binary interactions. the performance of the interaction index we developed to predict binding probability compares well with other methods. by studying the interaction of all potential protein pairs within a dataset, cc-d calculations can also help to identify correct protein interaction interfaces. the present large-scale study also reveals the influence of various protein families (enzyme-inhibitor, antibody-antigen, antigen-bound antibody, etc.) on binding specificity, showing, in particular, the distinctive behavior of antigenic interfaces compared to enzymes, inhibitors or antibodies. the performance of our approach is encouraging. although identifying interaction interfaces significantly helps in the identification of interacting proteins, further refinements will be necessary to make in silico cross-docking a viable alternative to high-throughput experimental methods. whole-protein mass spectrometry reveals global changes to histone modification patterns in hypoxia sarah wilkins , kuo-feng hsu , christopher schofield chemistry research laboratory, oxford university cells respond to limiting oxygen availability (hypoxia) by altering the gene expression profile. this primarily involves changes at the level of transcription via the activity of hypoxia-responsive transcription factors, although increasing evidence suggests that changes in chromatin structure (i.e. from a condensed 'silent' state to a more open or 'active' state) are required in order for transcription to take place. in particular, post-translational modifications (ptms) to histones have an important regulatory function in gene expression under hypoxic conditions. the n-terminal tails of histone proteins are accessible to a set of enzymes capable of 'writing' and 'erasing' ptms including acetylation, methylation, ubiquitylation, sumoylation and phosphorylation. to date, studies in hypoxia have employed antibody-based methods to investigate changes in histone modifications, and so have focused on individual marks in isolation. the interplay between coexisting ptms is thought to be much more important than the effect of any single mark. therefore, a global view of the histone modification profile is essential to gain a complete understanding of the function of histone ptms and their roles in gene regulation. in this study, we apply whole protein mass spectrometry to investigate hypoxia-induced changes in histone marks. this 'top-down' approach provides insight into combinational modification patterns that are difficult to establish by antibody-based methods or peptide ms analysis. we investigated changes in the global ptm profiles of histones from a range of human cell-lines and tissues under severe hypoxia (< . % o ). we find that hypoxia causes a shift in the overall profile towards a more highly modified state, with significant changes in methylation and phosphorylation. marked changes in histone ptms were also observed following treatment of cells with epigenetic inhibitors and commonly used hypoxia mimetics, including several iron chelators currently in clinical trials for the treatment of anaemia. finally, we show that this method can be used to identify the histone variant h ax, whose phosphorylation at serine is an indicator of double-stranded dna breaks in cancer. overall, these data provide important insights into the epigenetic changes associated with hypoxia in normal and disease contexts. we hope to further develop this method in combination with different labelling strategies to enable quantitative analysis of histonemodifications in cells. mass spectrometry-based protein biomarker discovery in neurodevelopmental disorders interactions. there is currently no biological diagnosis or known cause of asd. slos is characterized by a cholesterol deficiency due to a mutation on the dhcr gene. approximately / , babies are born with slos. diagnosis is achieved by measuring cholesterol and -dehydrocholesterol ( dhc) levels in the blood, however, there is currently no proven treatment for slos. because of this, research is increasing to determine biomarkers for these disorders. here, samples from people with asd (sera and saliva) and slos (saliva), and matched controls were analyzed using a combination of gel electrophoresis (tricine-page, sds-page and blue native page), in gel digestion or insolution digestion and nanoliquid chromatography-tandem mass spectrometry (nanolc-ms/ms) to investigate differences between the proteomes of people with these neurodevelopmental disorders and matched controls. several alterations in protein expression were identified. these differences may lead to potential biomarkers for diagnosis, possible therapeutic targets and an altogether better understanding of the disorders. understanding protein recognition using structural features protein-protein interactions (ppis) play a crucial role in virtually all cell processes. thus, understanding the molecular mechanism of protein recognition is a critical challenge in molecular biology. previous works in this field show that not only the binding region but also the rest of the protein is involved in the interaction, suggesting a funnel-like recognition model as responsible of facilitating the interacting process. further more, we have previously shown that three-dimensional local structural features (groups of protein loops) define characteristic patterns (interaction signatures) that can be used to predict whether two proteins will interact or not. a notable trait of this prediction system is that interaction signatures can be denoted as favouring or disfavouring depending on their role on the promotion of the molecular binding. here, we use such features in order to determine differences between the binding interface and the rest of the protein surface in known ppis. particularly, we study computationally three different groups of protein-protein interfaces: i) native interfaces (the actual binding patches of the interacting pairs), ii) partial interfaces (the docking between a binding patch and a non-interacting patch), and iii) back-to-back interfaces (the docking between non-interacting patches for both of the interacting proteins). our results show that the interaction signatures in partial interfaces are much less favoured than the ones observed in native and back-to-back interfaces. we hypothesise that this phenomenon is related to the dynamics of the molecular association process. back-to-back interfaces preserve the exposure of the real interacting patches (thus, allowing the formation of a native interface), while in a partial interface one interacting patch is sequestered and becomes unavailable to form a native interaction. structural characterization of the cytoplasmic mrna export platform laboratory of cellular and structural biology, the rockefeller university., laboratory of mass spectrometry and gaseous ion chemistry, the rockefeller univ., university of california, san francisco, the new york structural biology center, department of biochemistry, faculty of medicine, university of montreal mrna biogenesis is an intricate process that begins within the nucleus and culminates with the remodeling and nuclear export of the mrnp particles through the nuclear pore complex (npc). defects in this conserved mechanism have been shown to cause serious human diseases. the protein assembly that performs the last steps in mrnp biogenesis and export is located at the cytoplasmic face of the npc and is formed by different proteins, organized into several subcomplexes whose arrangement and molecular architecture are poorly understood. in this study we applied an integrative approach, combining cross-linking and mass spectrometry (cx-ms), electron microscopy and available high-resolution structures, to describe the molecular architecture of the endogenous npc cytoplasmic mrnp export machinery. we generate a hybrid, close-to-atomic structure of the yeast native nup complex, the core of the assembly. our map also reveals how the nup complex organizes the entire cytoplasmic mrnp export machinery, and how this in turn docks into the architectural core of the npc. mapping of phenotypic profiles into our structures allows us to generate a first functional map of the ensemble. we expect that our map will serve as a framework to understand the molecular mechanisms underlying this key step of mrnp biogenesis. study of candidate proteins to pore associated with p x receptor in different cell types carla oliveira , anael alberto , mônica freitas , luiz alves laborat orio de comunicac¸ão celular -fiocruz, centro nacional de ressonância magn etica nuclear -ufrj aim: the p x r is a purinergic receptor, which differs from others subtypes due to its structural and pharmacological characteristics. when exposed for extended time or to high concentrations of its agonist (atp), promotes an increase in membrane permeability, allowing the passage of molecules up to da. there is a controversy among several authors that leave in doubt if this receptor needs a second protein for the pore formation and which protein could be. we select five pore-forming proteins: trpv , trpa , connexins- (cx- ), pannexin- (panx- ) and vdac. we believe that different mechanisms and proteins could be associated with p x r, depending on the cell type and their microenvironment stimuli. in this context, our main goal is identify possible proteins that could be associated with the p x r pore in different cells and species. methods and results: we started with rt-pcr technique of cell lines: j .g , n a, u , u , hek- and primary cells from wistar mouse and swiss mice. we used different primers and pcr cycle for each target at different species. we observed that the p x , panx- and cx- are the most abundant and are present in all cell types except the absence of p x in u cells and panx- in mice macrophages and u cells (n> ). however, trpv was seen at n a and u cells and trpa in and primary cells from mouse and mice and in j .g cells (n> ). regarding to the vdac, it is present in mouse macrophages, j .g and hek- cells (n> ). the further steps, we verified if those proteins could be physically associated with the p x r. we coimmunoprecipitated the p x r of j .g (with or without atp), mice macrophages, hek- and u cells. the samples were applied in two separated . % bis/acrylamide gels: one destined to mass spectrometry (ms) and the other to western blot. at this point, we confirmed the presence of p x r, and observed several others proteins associated to p x r at different cell conditions, mainly when we exposed, j .g cell, to mm atp (n ). at this condition, we found by ms, hsp , , and ; alpha and b tubulin; myosin va; alpha, b and g actin; malate and lactate dehydrogenase (n ). although u and hek- had not received atp treatment, we found several proteins associated to p x . the next step was to immunoprecipitated those proteins in j .g (treated or not with atp) and use it to verify if p x are physically associated to them. as result we saw the p x associated to panx- in j .g cells. conclusion: we conclude that the p x r activated by extracellular atp triggers the recruitment of variety different proteins. at this condition, we can suggest that maybe there is a conformational change, regardless of the numerous recruitment structural proteins. in addition, apparently, the pore-forming protein pannexin- is associated with p x r, and the others pore forming proteins (vdac, cx- , trpv , trpa ) seems not be linked to p x r at j . recently, we developed a series of molecular modeling tools for structure-based studies of protein functions and interactions. these tools are publicly available as web servers that are easily operated even by non-specialists: cabs-fold server for protein structure prediction [ ] ; cabs-flex server for modeling of protein structure flexibility [ ] ; aggrescan d server for prediction of protein aggregation propensities and rational design of protein solubility [ ] ; and cabs-dock server for prediction of peptide binding sites and peptide docking [ ] . the web servers are freely available from the laboratory website: http://biocomp.chem.uw.edu.pl/tools sandy on , pinghui feng university of southern california, keck school of medicine, developing a technique to detect deamidated proteins and peptides using rig-i sandy on, pinghui feng university of southern california, norris comprehensive cancer center, department of microbiology, and molecular biology, los angeles ca perhaps the most notable type of post-translational modification of proteins and peptides into a higher order structure is deamidation of asparagine and glutamine. deamidation occurs when an amine group is removed, degrading the molecule for purpose of regulating intracellular levels. previous studies have demonstrated that this notable post translational modification has been uncovered over time for use in dna recombinant technology as well as use as a biological clock to facilitate the rapid turnover of biologically important components of the cell. while the effects of this non-enzymatic chemical reaction have been widely studied, the method to uncover modification sites over a large quantity of proteins remains an issue. one of the most common types of deamidation is of asparagine and glutamine residues. at this time, most researchers will depend on mass spectrometric based proteomic techniques for identification of these post-translational sites. the issue is that mass spectral analysis of deamidated proteins and peptides is complication and can lead to misassigned identification attributed by an overlapping of c peak of the amidated form with the deamidated monoisotopic peak; these two peaks are only separated by . mda. while these issues can be mediated by using a mass spectrometer with a high mass measurement accuracy, and high resolving power, it is essential to establish simpler methods for identifying substrates that have undergone deamidation. if deamidation is present, different protein bands will be exhibited in the western blot, which will be compared to a triple mutant rig-i, which resists deamidation, to observe the location of this modification on the protein. with enough testing, i will determine specific sites of digestion and use this information to make conclusions of unknown proteins. i will make results regarding whether the protein has been modified based on the digestion sites. i will use mass spectrophotometry analysis to compare the proteins on a wider scale and double check my results. i have narrowed it down to a couple of different digestion sites that indicate deamidation. though the analysis work can be tedious, it is crucial to ensure the sites we isolate are accurate in order to establish this technique. from my research, we can apply this method for wider scale use such as in clinical settings. in areas of inflammation of parkinson's' patients, we can review specifically the infected cells versus uninfected and isolate the proteins, usually deamidated, responsible often smaller in size and more specific. in addition, research articles have already shown that suppressing modification of certain cells such as bcl-xl playing a major in leading the regulation of cancer cell death by apoptosis. by leading the discovery of a simpler methods to uncovering deamidation in cells, researchers will more easily and quickly be able to scan through various proteins, some of which discovered eventually may play pivotal roles in cancer research. influenza virus (iv) hemagglutinin (ha) is a homotrimeric integral membrane glycoprotein that mediates receptor-binding and membrane fusion. it constitutes the prominent viral surface antigen and a main target for neutralizing antibodies. bacterial, recombinant ha-based vaccines indicate high potential to confer protection against highly pathogenic (hp) avian iv (aiv) h n and arise as alternative for the traditional egg-or cell culture-based manufacturing. relatively short time of bacterial has production can be of great importance in case of a pandemic. escherichia coli produced protein, based on the ha sequence of a/swan/poland/ - v / (h n ) hpaiv*, has been successfully expressed in the form of inclusion bodies at institute of biotechnology and antibiotics. refolded and purified antigen was obtained in a soluble form, isolated by reversed phase hplc and identified with peptide mass fingerprinting using matrix-assisted laser desorption/ionization time of flight mass spectrometry (maldi tof/tof ms). the performed research in a great extent allowed to confirm the amino acid sequence of the recombinant ha (rha) assumed based on the cdna and allowed to establish the location of a total of six disulfide bridges. however, during purification and storage of the rha, apart from desired higher order rosette-like structures of the protein, other non-native species resulting from posttranslational modifications, misfolding, aggregation and degradation may occur what results in reduced vaccine potency. here, besides the properly folded monomers, we indicate non-native aggregates induced by disulfide crosslinking. moreover, several free cysteine residues and unexpected intrachain s-s were identified in rha tryptic peptide maps. cys was found most susceptible to formation of disulfide bridges between the distinct chains of rha. the above findings allow to assume that not all rha particles fold to form the native structure. reduced cys residues exhibit tendency to undergo oxidation and uncon- new strategies and approaches to understand how antibodies recognize and neutralize snake toxins represent a challenge to improve the antivenoms. the neurotoxic activity of micrurus venom is carried majority by two distinct proteins families, ftx and pla . the conserved structural folding of these toxins can be appreciated as model to generate inhibitors against them. in this regard, monoclonal antibodies (mabs) can be used as tool to find hot spots for inhibit the toxins and represent the first step in order to develop recombinant neutralizing molecules. in this work our goals were analyse a set of monoclonal antibodies against the most toxic components of m. altirostris venom by proteomics approaches. the venom was fractionated; its major toxic proteins identified by in vivo tests based on murine lethal toxicity analyses (approved by the ethical committee for animal experimentation from center of health and science of the federal university of rio de janeiro -no. . / - ). the toxic components were used to generate a panel of five monoclonal antibodies. elisa and antivenomics results allowed us identify the specificity of all mab and their neutralizing efficacy was measured by in vitro tests. three mabs showed reactivity towards ftx and two against pla . all monoclonal antibodies against ftx lack a broad recognition. however, we identified a pair of monoclonal antibodies able to recognize all pla molecules of m. altirostris venom and showed a synergism to inhibit the catalytic activity of them. moreover, we challenge monoclonal antibodies against to micrurus venom for inhibit the pla activity of naja naja, specie taxonomically out of micrurus cluster. our results showed that pla of m. altirostris venom share a pair of conserved antigenic regions and draw attention to use these epitopes to miming antigen to generate antibodies for antivenom production. moreover, face to the cross reactivity and the pla activity inhibition capability by mabs towards the naja naja venom, our results highlight the conservation of neutralizing epitopes across the elapidae family. protein-protein interactions are known to play key roles in the most important cellular and biological processes such as signaling, metabolism, and trafficking. one major goal of structural biology is the structural characterization of all protein complexes in human and other organisms. these efforts can be complemented by computational approaches. in this context, computational docking attempts to predict the structure of complexes from their monomeric constituents. the docking problem presents two main challenges: the generation of structural poses or sampling, and the identification of the correct structures with a scoring function (sf). docking methods can be successful if the interacting partners undergo small conformational changes. however, in a general situation, these algorithms generate a large number of incorrect predictions, and therefore the predictive success strongly depends on the accuracy of the sf used to evaluate the docked conformations. a variety of strategies have been developed to score putative protein-protein docked complexes. they are usually based on atomic level potentials, residue level potentials, or a combination of both. in current work, we have evaluated different sf, taken from cchappi server, on the results of different rigid body docking methods, ftdock, zdock, and sdock, using the docking benchmark . and a docking set built from capri scorers experiment. our results show sf that showed better or similar success rate than the in-built sf. some of these sf increase the docking success rates especially for flexible or weak-binding cases, which are the most challenging for docking. of them are residue level sf robust enough to detected solutions in cases with large conformation change. in particular we found two sf that shows outstanding robustness, one designed for protein modeling and shared among docking methods, and the other is for protein docking which is also the best success rate in the top ranking in the capri scorers set. the other atomic level sf display high success rate to find a solution within weak binding proteins. the most successful sf are shared between the docking methods and display high success rate in the hard cases of the benchmark . and in the capri scorers set. the difference between them in the resolution level at which they work, one being atomistic the other residue-based. we found that they success rate vary according to the docking method chosen, allowing them to explode different properties of the sampling used. this way to characterize a protein complex can help to develop new combined scoring functions in protein docking or a new ranking strategy to enhance the success rate. multi-ptk antibody: a powerful tool to detect a wide variety of protein tyrosine kinases (ptks) isamu kameshita , noriyuki sueyoshi , yasunori sugiyama the eukaryotic protein kinases consist of large families of homologous proteins and play pivotal roles in various cellular functions. these enzymes are classified into two major groups; protein serine/threonine kinases and protein tyrosine kinases (ptks). ptks are believed to be involved in various cellular events such as cell cycle, proliferation, differentiation, apoptosis, and cell adhesion in multicellular eukaryotes. as many as ptk genes have been identified in the human genome and many of these ptks are known to be closely correlated with various diseases such as cancer. therefore, it is important to elucidate the expression profiles of the entire ptk family in cells and tissues. to investigate the expression profiles of the cellular ptks, we produced an antibody that detects a wide variety of ptks. for production of the antibody, antigenic peptides corresponding to amino acid sequences of a highly conserved region (subdomain vib) of ptks were synthesized and immunized to balb/c mice. among various antigens, a peptide with amino acids, cyvhrdlraan, efficiently produced a polyclonal antibody with a broad reactivity to ptks. we established a hybridoma cell line producing a monoclonal antibody, yk , which appeared to cross-react with various ptks. at least ptks could be detected by yk antibody, as evidenced by its reactivity with the recombinant src tyrosine kinases whose subdomain vib had been replaced by those of the other ptks. when differentiated hl- cells were analyzed by western blotting after two-dimensional electrophoresis with yk antibody, we observed significant changes in the immunoreactive spots in hl- cell extracts along with the changes in the morphology of the cells. these results suggest that the multi-ptk antibody, yk , will be a powerful tool for the analysis of a variety of cellular ptks. analysis of the siglec- and hvap- interactions leonor carvalho , vimal parkash , heli elovaara , sirpa jalkanen , xiang-guo li , tiina salminen structural bionformatics laboratory, department of biosciences, medicity research laboratory, department of pharmacology, drug development and therapeutics, sialic acid-binding immunoglobulin (ig)-like lectins (siglec) are type i transmembrane proteins. siglec- has an n-terminal v-set domain followed by two c -set domains in the extracellular region. it contains an immunoreceptor tyrosinebased inhibitory motif (itims) in its cytoplasmic tail and can function as an inhibitory receptor by dampening the tyrosine kinase-driven signaling pathways. these proteins are expressed primarily on leukocyte subsets and, thus, are thought to be involved in regulation of leukocyte functions during inflammatory and immune responses. recently, phage display screening experiments identified siglec- as leukocyte surface ligand for human vascular adhesion protein (hvap- ; aoc gene product) and their interaction was confirmed by cell adhesion and enzymatic assays (kivi et al., ; aalto et al., ) . based on our preliminary data, hvap- sugar units with sialic acid (sa) might mediate interactions with the v-set domain in siglec- . furthermore, it is known that the siglec peptides binding to hvap- are located in the ce loop of the second c -set of domain (siglec- _c ). based on current hypothesis an arginine in siglec- _c interacts with the tpq residue in the active site of hvap- . the ce loop of siglec- _c has two arginines (r and r ) and, therefore, the interacting arginine is unclear. we will now study the interaction mode of hvap- and siglec- in silico to predict the role of the arginines in the c domain and the role of sa-binding using the d model of the full-length ectodomain of siglec- and the hvap- crystal structure. the in silico analysis will be conducted in parallel with experimental site-specific mutational studies and the result will be combined to elucidate the mechanism of hvap- -siglec- interaction. adam middleton , catherine day attachment of ubiquitin to substrate proteins regulates almost all cellular processes, including protein degradation and cell division. ubiquitylation involves a cascade of three families of proteins: ubiquitin activating (e ), ubiquitin conjugating (e ) and ubiquitin ligase (e ) enzymes. the . kda protein can be attached as a monomeric moiety or as a polyubiquitin chain, and the type of modification spells out the 'ubiquitin code' that directs the fate of the substrate. polyubiquitin chains can be formed via eight different linkage types, and the arrangement of chain formation is typically directed by the e enzymes. forming a polyubiquitin chain involves binding of two molecules to the e : the donor (ubd) and acceptor (uba) ubiquitin. ubd is linked to the e via a thioester bond between its c-terminal gly and the active site cys of the e , and when primed for catalysis it interacts with a particular face of the e . in contrast, coordination of uba by e s is transient and cannot be easily measured; however, uba binding defines the linkage type of polyubiquitin chains. the e , ube k, directs lys chain synthesis, which results in modified proteins being degraded by the proteasome. we generated a stable form of the ube kub conjugate and crystallized it, and showed that both ube k and its ubiquitin conjugate are monomeric. using molecular docking, we modelled the position of both ubd and uba and investigated the interfaces with site-directed mutagenesis. these experiments led to a molecular model that revealed how ube k can synthesise lys -linked ubiquitin chains. this molecular explanation provides a foundation for understanding how other e s generate lys -linked polyubiquitin chains. the two chromophorylated linkers of r-phycoerythrin in gracilaria chilensis marta bunster, francisco lobos-gonz alez, jos e aleikar v asquez, carola bruna, jos e mart ınez-oyanedl fac de cs biol., universidad de concepci on the two chromophorylated linkers of r-phycoerythrin in gracilaria chilensis. francisco lobos-gonz alez, jos e aleikar v asquez, carola bruna, jos e mart ınez-oyanedel, marta bunster. departamento de bioqu ımica y biolog ıa molecular, facultad de ciencias biol ogicas, universidad de concepci on. phycoerythrin is a phycobiliprotein present in phycobilisomes in gracilaria chilensis as a complex with chromophorylated linker proteins. our interest is to discover the role of these linkers in the function of phycobilisomes. phycobilisomes(pbp) are auxiliary light harvesting protein complexes in charge of channeling energy towards photosystem ii in alga, cyanobacteria and cryptophyta. this is possible thanks to fluorescent proteins called phycobiliproteins (pbp) and the chromophores (phycobilins, open-chain tetrapyrrols) attached to specific cysteines. phycobiliproteins share a common general structure; they are organized as (alfab) heterodimers which themselves assemble as trimers(alfab) or hexamers (alfab) ; this complexes are organized in high order structures to form the core and the rods. besides pbps, pbs have linker proteins in charge of the assembly and stabilization of the complex, and also it has been proposed that they collaborate in the fine tuning of the energy transfer steps between chromophores. these linkers are located within the rods, the rod-core interface, the core and the core-membrane interface. although most linker proteins are colorless, chromophore bearing linkers have been described, which suggest its participation in the energy transfer process. two of them, g and g are associated to r-phycoerythrin in gracilaria chilensis, nevertheless the information available on these linkers in eukaryots is still limited. to understand how these linkers collaborate with the function of the phycobilisome, we need structural information, especially the coordinates of all the chromophores present in the complex; we have sequenced both linkers from the genomic dna, performed sequence analysis and also we have purified the linkers by anion exchange, molecular sieve and hpl chromatography. the characterization was performed by denaturant electrophoresis, absorption and emission spectroscopy and by mass spectrometry. the results show that they have molecular masses as predicted, with a peptide signal for chloroplasts, an internal sequence repeat; residues - with residues - for g and residues - with residues - for g , and the presence of conserved cysteine residues putative sites of chromophorylation. the spectroscopy shows that they have different composition of phycobilins and a very short t / . a preliminary model for both linkers shows that they belong to aa structural class and that they share a common fold (heat like motifs) frequently involved in protein-protein interactions. dept. of phys., chuo univ., grad. sch. of inform. sci. and eng., tokyo tech, rigid-body docking algorithms are useful for predicting tertiary structures of near-native protein complexes. however, this algorithms generate many protein complex poses including false positives. then, near-native poses are searched in a post-docking process. there are many computational softwares with rigid-body docking algorithms, for example, zdock. we developed a high-performance protein-protein interaction prediction software, megadock, which is basically used on supercomputing environments for a large scale and network level in this work, we then tried to use these docking softwares and the profile method for understanding mechanisms of protein-protein interactions. we focused on some physicochemical properties, electrostatic and hydrophobicity, of a set of protein complex poses generated by a rigid-body docking process. from these poses, we obtained sets of possible interacting amino acid pairs. a set of interaction profiles has some information of docking spaces. from the view of a network prediction, the docking spaces of a set of protein complex poses are one of the properties for discriminating native protein-protein pairs from non-native pairs. in this work, ensemble docking process is performed by megadock ver. . and zdock ver. . . . cluster analysis is used with profiles of physicochemical properties. we used a dataset composed of typical monomer-monomer protein pairs and will discuss mainly differences between native and non-native protein pairs. the structural studies of the two thermostable laccases from the white-rot fungi pycnoporus sanguineus marta orlikowska , grzegorz bujacz institute of technical biochemistry, lodz university of technology, poland laccases (ec . . . , benzenodiol oxygen oxidoreductases) are enzymes that have the ability to catalyze the oxidation a wide spectrum of phenolic compounds with the four-electron reduction of molecular oxygen to water [ ] . it has been found that the active site is well conserved in between laccases from different organisms. it contains four copper atoms: one paramagnetic type cooper (t ) that is responsible for their characteristic blue color and where the oxidation of the reducing substrate occurs, one type cooper (t ) and two type coopers (t ) that conform a trinuclear cluster in which molecular oxygen is reduced to two molecules of water [ ] . laccases are present in many different species and they have been isolated from plants, fungi, prokaryotes, and arthropods in most cases laccases are monomeric glycoproteins of around amino acids with molecular weights in the range of - kda. the various functions carried out by those enzymes include the antagonistic ones such as their involvement in lignin biosynthesis (in plants), lignin degradation, pigment production, fruiting body formation, pathogenesis (in fungi) and spore protection against uv light (in bacteria) [ , ] . the diversified functions of laccases make them an interesting enzyme for study from the point of view of their structure, function and application. laccases of white-rot fungi (wrf) are of special interest because one of its role is to degrade lignin and most of them are extracellular enzymes helping purification procedures [ ] . during the last two decades, there has been an increasing interest in the genus pycnoporus for its ability to overproduce high redox potential laccases as the ligninolytic enzymes. we present the crystal structures of two thermostable lacasses produced by strain pycnoporus sanguineus cs (laci and lacii). the molecular weights of laci and lacii, determined by sds-electrophoresis, is and kda, respectively [ ] . both isoforms shows high amino acids sequence similarity ( %) between them and high thermal stability, at c and c. they remained active at high concentration of organic solvent (acetonitrile, ethanol or acetone). the unique properties make them promising candidates for industrial applications in wasterwater treatment. laci exerted a higher thermal and ph stability, tolerance against inhibitors and was a more efficient catalyst for abts and dmp (laccases substrate) then lacii [ ] . based on the structures we would like to understand the isoforms differences that confers laci a markedly better performance than lacii in ph and thermal stability as well as better resistance to inhibitors. analysis of liver proteome in cystathionine ß-synthase deficient mice using d ief/sds-page gel electrophoresis, maldi-tof mass spectrometry, and label-free based relative quantitative proteomics izabela bieli nska , Łukasz marczak , hieronim jakubowski , institute of bioorganic chemistry, polish academy of sciences, rutgers university, new jersey medical school homocysteine (hcy) arises from the metabolism of the essential dietary protein amino acid methionine. levels of hcy are regulated by remethylation to met and transsulfuration to cys. cystathionine bsynthase (cbs) catalyzes the conversion of homocysteine to cystathionine (first step of transsulfuration reaction). human cbs deficiency is a recessive inborn error of homocysteine metabolism that casues severe hyperhomocysteinemia (hhcy) and diverse clinical manifestations, including fatty liver disease [ ] . although the causes of fatty liver disease in cbs deficiency have been studied the underlying mechanism is not understood. we hypothesize that cbs deficiency induces changes in gene expression that could impair liver homeostasis. to test this hypothesis and gain insight into hepatic functions of cbs we analyzed the liver proteome of cbs -/-and cbs / mice [ , ] using d ief/sds-page gel electrophoresis and maldi-tof mass spectrometry (n ) we identified twelve liver proteins whose expression was significantly altered as a result of the cbs gene inactivation. expression of three proteins was upregulated and of nine down-regulated by the cbs-/-genotype. two up-regulated liver proteins are involved in iron metabolism (ftl and fth). those proteins are associated with oxidation stress and inflammation. third up-regulated liver protein (cbr ) is related to oxidation-reduction process. the downregulated protein are involved in the hydrolysis of n-acylated or n-acetylated amino acids (acy ), regulation of endopeptidase activity (a at ), cholesterol biosynthetic process (fpps), amino acid degradation (huth), cellular calcium ion homeostasis and l-ascorbic acid biosynthetic process (rgn). using label-free based relative quantitative proteomics (n ) we identified fourteen liver proteins whose expression was significantly altered as a result of the cbs gene inactivation. expression of four proteins was up-regulated and of ten proteins was down-regulated. the down-regulated liver proteins are linked with regulation of bone mineralization and inflammatory response (ahsg) or regulation of mrna splicing (roa ). the up-regulated liver proteins are involved in tricarboxylic acid cycle (suca), oxidation-reduction process (cy ), cholesterol metabolic process, iron ion homeostasis (fech), fatty acid metabolic process (ssdh; eci ) and response to oxidative stress (lonm). our findings suggests that cbs interacts with diverse cellular processes, including lipid metabolism, that are essential for normal liver homeostasis. deregulation of genes involved in lipid metabolism provides a possible explanation for fatty liver disease associated with cbs deficiency. transcription factors play central roles in coordinating developmental processes, as evidenced by the increasing number of transcription factor-related developmental disorders being uncovered by nextgeneration sequencing and genome-wide studies of copy number variation. the action of a transcription factor in regulating gene expression depends on interactions with other transcription factors, coactivators/co-repressors and chromatin modifying and remodeling complexes. transcription factors are commonly regulated by post-translational modifications. however the study of protein-protein interactions and post-translational modifications of transcription factors by common techniques such as coimmunoprecipitation and mass spectrometry is hampered by the difficulty in preserving interactions and modifications through cell lysis. to circumvent this issue, we developed a bioluminescence resonance energy transfer (bret) assay, which allows protein-protein interactions to be observed in live cells. in this assay, a protein of interest is expressed as a fusion with luciferase from renilla reniformis, and its putative interaction partner as a fusion with yellow fluorescent protein (yfp). upon addition of a cell-permeable substrate, the distance-dependent non-radiative transfer of energy from luciferase to yfp is quantified by measurement of light emission at two wavelengths to assess the interaction between the two fusion proteins. to validate the utility of this assay for investigating transcription factor interactions, we confirmed homodimerization of the foxp transcription factor, haploinsufficiency of which causes a rare and severe speech and language disorder, as well as interaction of foxp with other members of the foxp family. we also confirmed the interaction between foxp and multiple candidate interactors identified through yeast two-hybrid assays, including the autism-related transcription factor tbr , the co-repressors ctbp and ctbp , and post-translational modification enzymes of the pias family. the role of pias enzymes in sumoylation -the covalent modification of proteins with small ubiquitin-like modifier (sumo) proteins -led us to further explore this process, which is notably difficult to investigate because of the dynamic and labile nature of the modification, which is also typically present on only a minor fraction of molecules of a given protein. combining the bret assay with gel-shift techniques we demonstrated that foxp is sumoylated. finally, we used the bret assay to examine the effects of etiological foxp variants in speech and language disorder on protein-protein interactions and post-translational modification. in summary, the bret assay is a sensitive, reliable and potentially high-throughput technique for exploring protein biology in the context of live cells. we have demonstrated applications of the assay in validating putative protein-protein interactions, assessing posttranslational modifications, and investigating functional effects of protein variants identified in patient cohorts. these investigations have provided novel insights into the function of the foxp transcription factor in neurodevelopment and into the etiology of foxp -related speech and language disorder. the directly interaction between pres of human virus b and human heat shock protein (hsp ) deqiang wang , chen ke , jun zhang key laboratory of molecular biology on infectious disease, the department of cell biology and genetics the directly interaction between pres of human virus b and human heat shock protein (hsp ). hepatitis b virus (hbv) has infected billion people worldwide, and million of them are chronically infected. the chronic virus infection, a major public health problem worldwide, leads to bout two-thirds of hepatocellular carcinoma (hcc). the hbv envelope consists of the large (l), middle (m) and small (s) envelope proteins, which contain pres -pres -s, pres -s, and s domain alone, respectively [ ] . the pres domain is believed to mediate virus attachment to the high-affinity receptor. yan et al employed a novel technique to propose sodium taurocholate co-transporting polypeptide (ntcp) as the candidate hbv receptor, and consequently, ntcp is a target for a new family of anti-hbv agents [ ] . whereas, it remains a query to clarify that ntcp is the only or major hbv receptor in vivo. to illuminate if other host proteins cooperatively participate the hbv infection, we detect the interaction between pres and many candidate host proteins. fortunately, we have found that the human heat shocking protein (hsp ) could directly interact with the pres domain of the hbv virus protein. both the pull down and the size exclusion chromatography experiments verify that the grp have the ability binding to pres . whereas, whether the interaction between hsp and pres relates to the hbv infection need further experiments to clarify. the member sponsorship in the vast world of naturally occurring peptides, where more than peptides are known and approximately peptide therapeutics are currently being evaluated in clinical trials (fosgerau & hoffmann, ), the rapid and accurate determination of their physicochemical properties is key in peptide drug discovery. among these properties, hydrophobicity is crucial for understanding molecular recognition and biomolecular aggregation. hence, there is a great interest in determining hydrophobicity scales for amino acid structures. in this work, octanol/water partition (log p) and octanol/water distribution (log dph, fig. ) of n-acetyl-l-amino-acid methyl amides were determined by means of quantum mechanical ief-mst solvation calculations taking into account the intrinsic conformational preferences of each amino acid according to dunbrack's libraries (dunbrack & karplus, ; ) . the results reveal log d . differences for a-helical and b-sheet conformations in arg, lys, hid, asn, gln, met, cys, leu and ile. furthermore, by decomposing the octanol/water transfer free energy into electrostatic and non-electrostatic components, we estimated that the non-electrostatic cost of transferring the amino acid side chain amounts to . . cal/mol.Å , in agreement with previous estimates reported in the literature. comparison of our scale with other theoretical and experimental hydrophobicity scales yields satisfactory results, leading to correlation coefficients ranging from . to . . additionally, the mstderived hydrophobicity scale led to significant correlations with the rp-hplc retention factors measured for eight decapeptides (r . ) and for influenza virus hemagglutinin -mer (ac-ypydvp-dyaslrs-amide) peptides (r . ). finally, the hydrophobicity scale was able to reproduce the experimental log p for random neutral peptides (r . ) and log d . for ' : random charged peptides (r . ), fig. . future studies will address the application of this methodology to nonproteogenic amino acids, the prediction of peptide hydrophobicity at global and atomic level in peptides, and the scoring of peptide-protein interactions. docking-based tools for discovery of protein-protein modulators docking-based tools for discovery of protein-protein modulators. protein-protein interactions (ppis) play an essential role in many biological processes, including disease conditions. strategies to modulate ppis with small molecules have therefore attracted increasing interest over the last few years. although protein-protein interfaces (ppifs) are considered difficult to target with small molecules given its lack of well defined cavities. successful ppi inhibitors have been reported into transient cavities from previously flat ppifs. recent studies emphasize on hotspots (those residues contribute for most of the energy of binding) as promising targets for the modulation of ppi. pydock algorithm is one of the few computational methods that use energy of solvation to predict protein-protein interfaces and hotspots residues. we present an approach aimed at identifying hotspots and transient pockets from predicted proteinprotein interfaces in order to find potential small molecules capable of modulating ppis. the method uses pydock to identify ppifs and hotspots and molecular dynamics (md) techniques to propose putative transient cavities. we benchmarked the protocol in a small set of protein-protein complexes for which both structural data and ppi inhibitors are known. the method applies to the unbound proteins of the complexes the fast fourier transform algorithm, followed by the energy-based scoring from pydock to calculate the normalized interface propensity (nip) values derived from rigid-body protein docking simulations to predict the ppifs and hotspots residues without any prior structural knowledge of the complex. then we used md to describe the possible fluctuations of the interacting proteins in order to suggest transient pockets that could be useful as targets of small molecules for the modulation of ppis. finally, we evaluated by ligand docking, the validity of predicted hotspots and pockets for in silico drug design. we found that the nip-based method from pydock protein-protein docking identifies hotspots residues that are located within the binding site of known inhibitors of ppis. predicting ppifs from a three dimensional structure is a key task for the modulation of ppis. the use of the nip-based hotspots prediction method improve the identification of transient cavities from md simulation when compared to known binding cavities. this approach can be extremely useful in a realistic scenario of drug discovery targeting ppifs, when there is no information at all about the protein-protein complex structure. protein complexes are the fundamental molecular organizations that assemble multiple proteins to achieve various biological processes. identification of protein complex membership should provide a genotype-phenotype map to elucidate human gene-disease associations. it has been routinely assumed that network clusters with dense connections inside and sparse connections outside would form functional protein complexes. therefore, searching highly modular subgraphs in protein-protein interaction networks was explicitly or implicitly implemented in the algorithms to find protein complexes. however, to our surprise, we found a large portion of complexes with a medium-to-low modularity from the analysis of experimentally confirmed protein complexes. we also discovered that these complexes have cellular functions enriched in highly time-and space-dependent expression, such as signal transduction or subcellular localization. we further developed an algorithm to find such complexes by weighing network connections to capture transient interactions with intrinsically disordered regions. we confirmed that our method improved the identification of biologically relevant members of protein complexes and covered more complexes with a medium-to-low modularity. furthermore, newly discovered subunits in protein complexes could explain more disease-gene associations, indicating its utility to expand current genotype-phenotype map of human diseases. expanding template-based protein-protein complex prediction using ab-initio docking sergio mares-s amano , luis angel rodr ıguez-lumbreras , juan fern andez-recio structural characterization of protein-protein interaction (ppi) networks is crucial for understanding the underlying molecular mechanisms whereby life processes and disease arise. however, due to inherent limitations of experimental techniques, such characterization only covers an extremely reduced fraction of the human ppi network (interactome). recent studies have shown that although available structural templates may suffice to model a significant proportion of the interactome, model accuracy and binding specificity remain unsolved problems. consequently, improving the ability to predict ppis structurally will help to provide a better d profile of the known interactome, which may ultimately lead to the development of new therapeutic applications. here we show a novel approach that combines templatebased modeling with protein-protein computational docking to the structure-based prediction of ppis. our approach samples different protein-protein structural models derived from docking simulations. models are subsequently ranked using a function that incorporates an energy-based scoring term and a structural template similarity score. the energy-based scoring function includes electrostatics, van de waals and desolvation calculations, whilst the template similarity score accounts for the degree of structural similarity of models against a high-resolution and diverse dataset of structural templates. our approach highly improved the predictive success rate over individual ab-initio docking and templatebased techniques across a large benchmark dataset, including protein-protein complexes. when compared to the performance of the ab-initio docking algorithm, we found that the approach increased consistently the success rate, by approximately %, for the top , top and top solutions. the success rate improvement was even more notorious when the comparison was performed against the predictions from the traditional template-based docking. though incorporating ab-initio docking expands considerably the scope of the template-based docking method, challenges remain for interacting proteins in which high conformational changes occur upon binding and also the size and diversity of the repertoire of structural templates needs to be increased. is essential for the development of multicellular organisms. in mammalian cells, early events in pcd involve the release of cytochrome c (cc) from mitochondria to the cytoplasm, so letting cc play a key role in assembling the apoptosome and triggering apoptosis. in plants, pcd is part of a general process -the so-called hypersensitive response -in which mitochondrial cc is likewise released into the cytosol but its further role and cytoplasmic partners remain veiled. such a coincidence in cc release made us think of a common link for pcd in such evolutionarily distant species along evolution. to go deeper in understanding the pcd-dependent role of cc, a proteomic approach based on affinity chromatography with cc as bait was run using human and plant cell extracts. upon combining this approach with bimolecular fluorescence complementation (bifc), a total of eight and nine unknown proteins interacting with cc under pcd conditions were identified in human and plant cells, respectively [ , ] . such novel cc-partners -which are located in the cytoplasm and even in the nucleus -are involved in protein folding, translational regulation, oxidative stress, dna damage, energetic and mrna metabolism [ ] . strikingly, some of the novel human cc-partners are closely related to those for plant cc, so indicating that the evolutionarily well-conserved event of cc release from mitochondria could involve a common signalosome consisting of a wide range of common targets [ ] . to also understand such a promiscuity of cc from a structural point of view, the cc surface residues involved in complex formation with each one of its counterparts were mapped by using nmr spectroscopy. the resulting data shows that the heme crevice of cc is at the cc-partner interface in most of the complexes, which is in agreement with the vast majority of known redox adducts of cc. in contrast, however, to the high turnover number of the redox cc adducts inside the mitochondria, the complexes formed by cc under pcd conditions lead to the formation of rather stable nucleo-cytoplasmic ensembles. altogether, these findings suggest that extra-mitochondrial cc interacts with nuclear and/or cytoplasmic pro-survival, anti-apoptotic proteins in both humans and plants so as to lead living cells to dye. keywords: cytochrome c, programmed cell death, signalosome. post-translational phosphorylation often modulates the function of proteins. in particular, they affect the role that cytochrome c (cc) plays in cell life and death [ ] . cc is phosphorylated in vivo in tyr and tyr residues [ , ] , but recently, two new phosphorylation sites have been described at positions and [ ] . hence, we aim at understanding the structural and functional changes induced by thr and ser phosphorylation cc. for this purpose, we designed two phosphomimetic mutants of cc by replacing either thr or ser by the canonical amino acid aspartic acid (t d and s d). as control, two other mutants at the same two positions (t a and s a) were analyzed so as to differentiate the effects due to the presence of a negatively charged residue. remarkably, the s a mutant is significantly less stable than the wild-type species. we found that phosphorylation at position thr diminishes the redox potential and oxygen consumption. in addition, t d mutation affects the ability of cc to bind the distal site pcc , thereby suggesting that phosphorylation at this position affects the electron carrier capacity of cc. mass spectrometry (ms) is widely used techniques to gain knowledge about biomolecules [ , ] . it produces a high amount of data which is often presented as a list containing thousands of proteins. that list usually contains few hits interesting for our research. the pocess to select those proteins may include integrating experimental with annotation data. it requires spending some time in both, performing calculus and searching in databases. in this poster we present msbiodata analysis tool, a web service thought to deal with this tedious work. with this tool, researchers can set rules to select the most interesting hits in his lists using both, experimental data and gene ontology [ ] annotation. the data can be upload to the web using an excel spreadsheet or a flat files in a mztab format, and rules are easily constructed by means logical sentences. those sentences are composed by one or more terms linked by logic operators (and and or). each term in the logical sentence indicates to our program the conditions that selected hits must meet. once the alysis is finished, the results are delivered by email. msbiodat analysis tool do not requires any programming knowledge to be used and is freely available at: http://msbiodata.innomol.eu keywords bioinformatics/data analysis/proteomics/data mining/ mass spectrometry. beside the rate of protein synthesis, the regulation of protein degradation plays a crucial role in the white muscle protein accumulation and overall fish growth. intracellular proteolysis in salmonid species, such as atlantic salmon, salmo salar l. and rainbow trout, oncorhynchus mykiss walb., was studied to evaluate the basic mechanisms of protein degradation that could possess a potential target to regulate the body mass accumulation in farmed fish. a number of white muscle proteases such as cathepsins b, l, and d, proteasomes, and calcium-dependent proteases (m-and m-calpains), was studied in the juvenile specimens of different size-and age-groups both wild and farmed salmonids. the correlations between the protease activity and expression levels and morphometric characteristics of fish were found. the size-and age-related differences in intracellular protease activity revealed in fish muscles indicate both general role of proteolysis regulation in salmonid growth and the specific role of the individual proteolytic enzymes as well. the data on negative correlation of cathepsin d and calpain activity in muscles and the rate of weight increase in juvenile salmonids were obtained. a revealed positive correlation of cathepsin b activity and morphometric parameters in fish young presumably indicates its primary contribution to non-myofibrillar protein turnover. ubiquitin-proteasome system seems to contribute to background protein turnover as the proteasome activity was not corresponded with growth rate. summarizing the data obtained the autophagy-lysosomal and calpain-related protein degradation pathways were recognized to be directly involved in body growth and muscle protein retention in salmonid fish. the work was carried out using technical facilities of ib karrc ras equipment centre and financially supported by the russian science foundation, grant no. - - "salmonids of the north-west russia: ecological and biochemical mechanisms of early development". solving the proteomic organization of fitness-related genes in uropathogenic escherichia coli in life threatening sepsis. nowadays, complete genomes for almost all major bacterial pathogens are available, helping researchers to identify virulence factors. however we still ignore how these genes are organized at the proteome level and how this association influences bacteria pathogenicity. we integrated available databases on upec e. coli (strain cft ) to investigate the genomic and proteomic organization of genes related to upec fitness in the host. intriguingly, we found that most fitnessrelated genes have orthologs not only in other pathogenic strains but also in non-pathogenic bacteria such as e. coli k- . these genes are organized in clusters and operons with similar structure. by integrating protein-protein interaction data we observed that genes with high impact on fitness also display a highly clustered organization when compared to other genes. overall, our results show that proteinprotein interaction clusters associated to upec fitness in the host represent a promising target for the design of new antibiotics. elucidating the molecular mechanisms by which the hnh endonuclease gp activates the terminases in bacteriophage hk ( ) . hnh endonucleases are characterized by two highly conserved his residues and an asn residue( ). gp is essential for phage head morphogenesis, likely because gp enhances the activity of the hk terminase enzymes toward the cos site ( ) . notably, enhancement of the terminase-mediated cleavage of the phage cos site requires the presence of an intact hnh motif in gp . mutation of the canonical metal binding his in the hnh motif abrogates gp mediated-terminase activity. although phages are widely studied, there is no definitive structural or mechanistic evidence as to how the hnh endonuclease within gp functionally interacts with the adjacent terminase enzymes to facilitate phage morphogenesis. previous work on hnhcontaining bacteriophage proteins does not address explicitly how the requirement for divalent metal binding at the hnh endonuclease site induces interaction with the terminase enzymes that are so crucial for phage dna packaging during morphogenesis ( , ) . in addition, gp possesses no sequence similarity to hnh proteins for which the structure has been determined ( ), making structural studies of gp necessary. toward these ends, we use nuclear magnetic resonance (nmr) spectroscopy to probe metal and terminase binding of gp in the wild type state and bearing metal binding mutations. we also report backbone resonance assignment of gp . our nmr studies have elucidated residues within gp required for metal binding and terminase activity. these data are being used to assess the role of specific gp residues in phage morphogenesis. together, this work will identify the enigmatic role describing how metal binding in hnh endonucleases is crucial in the replication and morphogenesis of phages. meat production from pigs for human consumption is a resource heavy process, indeed every part of the animal that is not used constitutes a protein food-chain loss, which is neither economically nor environmentally viable. the goal of this project is to better harness slaughterhouse waste such as the keratin rich pig bristles and nails through microbial conversion. instead of using identified single microorganisms, it is the goal to define microbial consortia where microorganisms synergistically show the ability of efficient keratin degradation/conversion. candidate consortia have been obtained by selecting for microorganisms growing on enriched media that contains milled pig bristles as sole carbon and nitrogen source. by using mass spectrometry and various biochemical analyses to investigate keratinolytic enzymes, methods will be established for identifying and characterizing suitable consortia. protein families likely to be involved are keratinases, which are specialized proteases including serine, cysteine and metallo proteases, as well as systems capable of reducing or otherwise breaking disulfide bonds which are highly abundant in hair and nails. furthermore, interactions and symbiosis of microorganisms in a consortium will be investigated at the meta-proteomics level. the project will lead to development of biotechnological degradation of keratin rich fibers, and provide new insights into functional dynamics and efficacy of microbial consortia. a comprehensive protein domain analysis to map cancer-type-specific somatic mutations interpretation of the genome-wide association studies (gwas) of cancer patients to find cancer-typespecific biomarker is challenging due to the mutational heterogeneity of cancer types. network approaches to find cancer-type-specific variants and biological pathways are increasing since genes tend to act together to display phenotypic or disease outcomes. phenotype similarity has proven to reflect the relationship of functionally related genes. we applied phenotype similarities between various diseases for expanding molecular connections of cancer-type-specific variants to discover cancer-type-specific modules. specifically, cancer-type-specific variants of cancer types from the cancer genome atlas (tcga) were analyzed to find phenotype-inferred relationships among the variants. we find that cancer variants that cause the similar disease phenotypes tend to be linked as a cluster of biological pathways or functions. moreover, cancer-type-specific modules could explain the underlying pathogenicity of specific symptoms which manifest in particular cancer types. cancer-type-specific modules and pathways found from phenotype similarity/dissimilarity based on cancer symptoms improved the discrimination performance to sort cancer-type-specific variants to accurately predict patient groups. our method will be further developed to find genetic biomarkers for the diagnosis or prognosis of specific cancer types pk- engineering a stable, symmetric membrane protein scaffold amanda duran , jens meiler computational protein engineering has the potential to contribute to various fields including drug design, protein therapeutics, and materials science. protein-ligand interface design and the construction of large, stable proteins rely on stable scaffolds. symmetry is a great tool for protein stability both in protein engineering and nature. several membrane protein structures exhibit pseudo-symmetry and are proposed to be the result of gene duplication, fusion and diversification events originating from a monomeric gene. aquaporins (aqp) are a class of membrane proteins that exhibits a two-fold inverted pseudo-symmetry. the escherichia coli aqp glycerol facilitator protein (glpf) was originally computationally engineered to be perfectly symmetric in sequence and presumably in structure. the symmetric gene was assembled, cloned, and expressed. however, after facing many challenges experimentally, the computational study has been expanded to aqps of known structure for a more extensive symmetric backbone search. mammoth structural alignment was used to align the structures to their inverted counterparts. cutpoints were calculated based on a-carbon distance. finally, the rosetta protein modeling software suite was used to refine and energetically minimize the symmetric backbones. from over generated symmetric backbones, candidates were chosen for experimental verification. these studies are ongoing.currently, the symmetric backbone models have scored to be more stable than the wild-type proteins. experimental verification of these symmetric backbones will provide valuable information for the current state of membrane protein modeling and design using computational methods. intrinsically disordered proteins drive heritable transformations of biological traits daniel jarosz , james byers , sohini chakrabortee , sandra jones , amelia chang , david garcia stanford university, whitehead institute for biomedical research, rockefeller university the transmission of information from one generation to the next generally occurs via nucleic acids. the only known protein-based molecular memories are prions, which drive heritable biological traits based upon self-templating changes in protein conformation. these protein-based genetic elements have previously been identified systematically, but at least three do not share the sequence biases or structural characteristics that have informed such studies. here we employed a comprehensive library of yeast proteins to examine the breadth of protein-based inheritance. transient overexpression of more than forty proteins created new traits that were heritable and beneficial. some shared properties of known prions, but most employed distinct genetic and biochemical mechanisms to act as elements of inheritance. traits with these characteristics were common in wild yeast strains and could also be elicited using orthologous mammalian proteins. the inducing proteins were strikingly enriched in intrinsically disordered sequences that have been widely conserved across evolution. intrinsically disordered proteins are associated with human disease and with dosage sensitivity in yeast, flies and worms. our results suggest another widespread role for such intrinsically disordered sequences: induction of heritable epigenetic switches that transform phenotypic landscapes and drive adaptation to stressful environments. prediction of binding affinity in protein complexes: contacts do matters almost all critical functions in cells rely on specific protein-protein interactions. understanding these is therefore crucial in the investigation of biological systems. despite all past efforts, we still lack a thorough understanding of the energetics of association of proteins. here, we introduce a new and simple approach to predict binding affinity based on functional and structural features of the biological system, namely the network of interfacial contacts. we assess its performance against a protein-protein binding affinity benchmark and show that both experimental methods used for affinity measurements and conformational changes have a strong impact on prediction accuracy. using a subset of complexes with reliable experimental binding affinities and combining our contacts-and contact types-based model with recent observations on the role of the non-interacting surface in protein-protein interactions, we reach a high prediction accuracy for such a diverse dataset outperforming all other tested methods. free radical oxidation -a new method for obtaining stable protein coatings on magnetic nanoparticles magnetically targeted nanosystems (mtnss) are now considered to be applicable in different areas of biology and medicine such as hyperthermia, magnetic resonance imaging, immunoassay, cell and molecular separation, a smart delivery of drugs to target cells. proteins are promising materials for creation of coatings on magnetic nanoparticles (mnps) due to their biocompatibility, an ability to protect magnetic cores from influence of biological liquids and prevent agglomeration of mtnss in dispersion, their possible functional activity as therapeutic products and biovectors. the creation of stable protein coatings with retention of native properties of molecules is still an important biomedical problem because of disadvantages of the commonly used methods such as formation of a polydisperse ensemble of particles, nonselective linking of proteins leading to cross-linking of macromolecules in solution, and desorption of coatings. a novel method in obtaining stable single-layer coatings assembled from protein molecules on the surface of magnetite nanoparticles has been developed. it is based on protein liability to free radical modification, leading to the formation of intermolecular covalent cross links. free radicals are locally generated on the surface of nanoparticles via the fenton reaction thereby proteins adsorbed on the surface are subjected to the cross-linking. o-phenylenediamine was used for detection of free radical generation initiated by nanoparticles. the proteins drastically differing in their structure and properties, namely, serum albumin, thrombin and immunoglobulin g were selected for creating the protein coatings. the properties of the obtained coatings and their stability have been studied with the help of dynamic light scattering (dls), uv/vis spectrophotometry, antibody-antigen test and the method of spectral-fluorescent probes. albumin molecules in mnps coatings have been shown to retain their capability of binding with a dye and be conformationally stable. the dye , '-di-(g-sulfopropyl) , 'diphenyl- -ethiloxacarbocyanine-betaine interacting with albumin with a growth of fluorescence and with partial cis-trans conversion of the dye has been used. it has been proven that coatings composed of protein macromolecules are ) stable, ) formed around individual nanoparticles and ) have several nanometers in thickness. the free radical linking of thrombin and immunoglobulin g on the surface of nanoparticles has been shown to almost completely keep native properties of the protein molecules. the free radical linking method reveals new possibilities for design of single-layer multiprotein polyfunctional coatings on the surfaces of all the nano-, micro-and macroobjects containing metals of variable valence (for example, fe, cu, cr). the spectral-fluorescent investigation was supported by the russian foundation for basic research, project nos. - - and - - mol_a. regulation of neuronal snares by accessory proteins shrutee jakhanwal , reinhard jahn regulation of neuronal snares by accessory proteins shrutee jakhanwal and reinhard jahn department of neurobiology, max planck institute of biophysical chemistry, fassberg, goettingen, germany- . synaptic vesicle exocytosis lies at the heart of the process of neurotransmitter release. and, the family of proteins that is central to the process of synaptic vesicle exocytosis is the family of snare proteins. there are three kind of neuronal snare proteins namely syntaxin, snap and synaptobrevin. these three snare proteins interact through their snare-motifs to form a highly stable four-helix bundle, which in turn, pulls two membranes together to mediate fusion. years of work in this field have established that the four-helix bundle is critical for the membrane fusion to occur. however, the process of regulation of snare-mediated fusion remains very poorly understood. the major regulatory proteins involved in the process are munc , munc , synaptotagmin and complexin. the major aim of my project is to obtain a closer look at the regulation process of snare-mediated fusion by focusing on the interaction between the snare proteins and the regulatory proteins. to achieve this objective, i express and purify the different proteins involved in the process of snare-mediated fusion and thereafter subject them to appropriate biochemical characterization. in order to assess the role of the purified proteins in the process of fusion, i reconstitute them into liposomes and perform in-vitro lipidmixing assays. these assays are based on f orster resonance energy transfer (fret). based on the discretion of assessing the protein-protein or protein-lipid interactions, either the proteins or the lipids can be fluorescently labeled. also, the lipid compositions can be varied in order to assess the effect of lipid on the function of the respective protein. fluorescence-based anisotropy measurements can also provide information about the degree of freedom of a protein, indirectly providing information about the kinetics of a reaction. employing these techniques, i observe that munc - leads to displacement of syntaxin from a complex of syntaxin and snap . also, a complex of syntaxin and munc is resistant to the action of the aaa-atpase, nsf and its co-factor asnap, implicating this complex as a strong candidate for acting as the starting point for the process of neurotransmitter release. munc also appears to enhance lipid-mixing by interacting with the snare-complex. further investigations on the same lines can provide very useful insights into the process and can help us unravel the secrets that underlie the beauty of the exquisitely regulated process of neurotransmitter release. binding of thymidine nucleotides to a viral thymidine monophosphate kinase aldo a. centro de investigaci on en alimentaci on y desarrollo theme: biochemistry there is great interest in the evolution and activities of fish trypsins, since they appear to have evolved into different families. the cdna for trypsin iii from the monterey sardine (sardinops sagax caerula) was obtained and its deduced amino acid sequence matched its identity with a purified protease from the fish by mass spectrometry analysis. molecular modeling of sardine trypsin iii compared to other homologs showed a typical trypsin fold with all the cognate components for catalysis, and specific amino acid distribution that are possible factors that explain the cold adaptation. from phylogenetic analysis, sardine trypsin iii belongs to the novel y family, which is proposed to have evolved for cold adaptation. the obtained recombinant trypsin iii showed a low catalytic efficiency, but it remained active at cold temperatures, similar to other cold-adapted trypsins. the cold-adaptation of sardine trypsin iii opens a wide range of biotechnological applications for this protease and is also interesting from the serine protease structure-function relationship point of view. fungicidal mechanism of scolopendin , a cationic antimicrobial peptide from centipede heejeong lee , dong gun lee drastically (from . x - to x - colonies) upon deletion of this residues domain from the full length trai. we are investigating the structure and function of this very c-terminal end of trai using nmr spectroscopy. for the backbone assignment we used slice-selectively homonuclear broadband decoupled spectra along with standard experiments. three-bond scalar coupling constants were obtained through real-time j-upscaling experiments. with the backbone assignments, we have the first hand evidence which shows that his domain is for the most part intrinsically disordered, but contains short a-helical regions. structural development, interaction studies to find the binding partner and transition of disorder to order orientation of this domain will be further investigated in this project. here we investigated a model system where mab aggregation is induced by increasing the ionic strength (nacl) at low ph. the aggregation depends both on protein and sodium chloride concentration. with nanoparticle tracking analysis (nta) and micro flow imaging (mfi) the aggregation formation was further characterized. aggregation can be partially reverted by lowering the ionic strength as determined by soluble monomer concentration measurement using se-hplc: parts of insoluble aggregates could be solubilized as soluble aggregates, dimers or even monomers. a quasi equilibrium is formed in between the subtypes. the whole aggregation process was examined by ftir and cd-spectroscopy to identify structural changes of the mab. screen of protective additives: the effect of osmolyte additives on aggregation kinetics and final aggregate concentration is investigated, revealing protective effects in both cases. in a screen with more than compounds not only the aggregation propensity was studied but also structural changes. the aggregation index (quantity for colloidal stability) and the melting point (quantity for conformational stability) measured by differential scanning fluorimetry were determined. the used mtp format screen has potential for buffer optimization and formulation development. structural biology and protein dynamics tetraspanin cd has a broad range of cellular functions, such as integrin association forming tetraspanin-enriched domains, synapse formation between b and t cells, cell adhesion, motility, invasion and signalling. furthermore, cd is one of the four receptors involved in the cell entry of hepatitis c virus (hcv) and therefore infection onset, one of the major causes for chronic liver disease resulting in cirrhosis and hepatocarcinoma. human cd large-extracellular-loop (hcd lel) is composed of a "stalk" and a "head" subdomain; with the latter interacting with hcv-e glycoprotein. we present four novel hcd lel crystal forms. analysis of the fourteen independent observed hcd lel high-resolution x-ray structures suggests that the dynamism of the hcd lel head-subdomain is an inherent molecular property, an observation supported also by molecular dynamics (md) studies. we classify the conformations in three distinct clusters (closed, intermediate and open) , which are seen both in the crystal structures and in the molecular dynamics simulations. the md simulations also show that conformational variability is modulated by ph changes, with distinct probability for each cluster at acidic and neutral ph. furthermore, in silico docking of the recent e core structure with three of the major types of hcd lel head-subdomain clusters highlights hydrophobic interactions as the major forces in the e core: hcd lel recognition mechanism. we propose that the flexibility of the hcd lel is exploited by hcv at different stages of cell entry from virus attachment to internalization and fusion with the endosomal membrane. our results provide important insights on the basic mechanism governing hcv binding to hcd , and can help structure-based drug design of entryinhibitors of hcv. allophycocyanin of gracilaria chilensis: from gene to function jorge dagnino-leone , jos e martinez-oyanedel , marta bunster-balocchi universidad de concepci on theme: structure-function relationship of proteins the phycobilisomes (pbs) are auxiliary photosynthetic complexes that allow cyanobacteria and red algae to enhance the energy uptake in the range of - nm. in gracilaria chilensis, an eukaryotic red algae, pbs is composed of phycoerythrin (pe), phycocyanin (pc) and allophycocyanin (apc); these proteins possess chromophores which capture energy and then transfers it to photosytems. pbps are oligomers of a ab heterodimer; it oligomerizes into a trimer (ab) , this trimer has discoidal shape and it is associated in hexamers (ab) , several of this hexamers forms cylinder-like structures. pbs has components: antennas and core. the antennas are composed of pe and pc, whose function is to capture energy between - and - nm respectively and transfer it to the core. the core is formed by apc, which can absorb energy in the - nm range. apc emission allows transferring energy to the photosystems with high efficiency. pbs is also composed by linker proteins which allow the correct assembly of pbs and possibly regulate the energy transfer. the main goal in our group is to build an atomic model of the gracilaria chilensis phycobilisome. we have solved the crystal structure of pe and pc and created an antenna model. at present we are working in apc and the chromophorilated linker proteins. the objective of the present work is to create a model of the core of gracilaria chilensis; to achieve these we have used molecular biology, biochemistry and bioinformatics techniques. we designed oligonucleotides primers for the four allophycocyanin subunits genes and for the globular domain of the apce linker. these primers were used in pcr experiments to obtain the genes sequences. the sequences were translated to a aminoacid sequences and used to build a d model for apc subunits and trimers using the software modeller. on the other hand we purified and analyzed the spectroscopic properties of apc from gracilaria chilensis using absorption and fluorescence spectroscopy. we also determined apc oligomerization state using gel filtration. molecular docking using the cluspro server was performed to obtain a hexamer and apc cylinder models. based on electron micrographs obtained by our lab a tri-cylindric core model was built. all the models were submitted to a molecular dynamics using gromacs software. finally we determine possible energy transfer pathways in the core model applying the extended forster equation, spectroscopic data from literature and the transition dipole moments of each of the chromophores present in the core. as conclusion of this work we built the first atomic model of gracilaria chilensis phycobilisome core and propose energy transfers pathways inside the core in the context of a phycobilisome. novel practical strategies to access artificial metalloenzymes marco filice , jose miguel palomo departamento de biocat alisis, instituto de cat alisis, csic protein chemistry and engineering since the first report, the design of artificial metalloenzymes has rapidly been converted into an important topic in biological and inorganic chemistry due to their potential applications in synthetic chemistry, nanoscience and biotechnology. the combination of a catalytically active organometallic moiety with a macromolecular host has permitted the creation of biohybrids, a new kind of heterogeneous catalytic entities combining the attractive features of both homogeneous and enzymatic systems. presenting our most recent achievements in this research area, here we describe two novel powerful and promising approaches focusing the practical synthesis and large scale production of heterogeneous artificial metalloenzymes showing chimeric activity. the first strategy is based on the in situ synthesis of noble metal nanoparticles and their supramolecular assembly with a microbial lipase from candida antarctica (fraction b) finally creating an ultra-active organometallic-enzyme heterogeneous nanobiohybrid. in the second approach, combining different protein engineering protocols (molecular biology, orienting immobilization, solid-phase bioorganic modification and bioinformatic tools), an orthogonal solid-phase strategy creating novel unnatural catalytic sites was designed and optimized. the application of such a strategy onto the structure of the lipase from geobacillus thermocatelunatus permitted the generation of a heterogeneous artificial metallolipase with chimeric activity. as proof-of-concept, the combinatorial library of generated artificial metalloenzymes obtained by both strategies was successfully assessed in a set of different synthetic reactions (selective c-c bond formation as suzuki, heck or diels-alder reactions) and also combining both activities (metallic and enzymatic) in cascade processes such as dynamic kinetic resolution of amines or production of arylamines. the obtained results were excellent in all cases. extending this strategy to other enzymes, proteins and catalytic metals, we envisage the creation of a combinatorial library of programmable artificial enzymes useful for a wide set of applications (i.e. fine organic and medicinal chemistry, bioremediation or biomedicine). proteomic examination of the yeast nuclear pore complex dynamics protein turnover and exchange nuclear pore complexes (npcs) are proteinaceous assemblies situated in nuclear envelopes of eukaryotic cells. the main function of the npc is the selective transport of macromolecules. npcs also partake in other functions, such as nuclear organization and gene regulation. the core scaffold of the npc is thought to be a stable structure, while the peripheral components exchange at various rates. however, these phenomena have not been elucidated in detail. the recent findings that yeast daughter cells get a higher proportion of the old npcs and the core scaffold hardly turns over raise the possibility that the exchange of the peripheral nucleoporins can be a repair mechanism. yeast provides a useful organism for the interrogation of nucleoporin exchange, as it performs closed mitosis; hence the only mixing of npc constituents is due to exchange. we have developed a panel of genetic tools providing for conditional induction and repression of nucleoporins. by combining these switches with stable isotope metabolic labeling and affinity capture, cross linking coupled to mass spectrometry, we are able to distinguish between pre-existing and newly synthesized proteins and quantify their relative amounts in the npc. our preliminary findings are in agreement with results obtained in other organisms: the core scaffold of the npc (inner ring, outer ring) appears to be stable, however does exchange slowly over time, while peripheral components exchange faster. by looking at the exchange rates of yeast nucleoporins we hope to gain insight into the npc biology of actively dividing eukaryotic cells. active site clustering identifies functional families of the peroxiredoxin superfamily angela harper , janelle leuthaeuser , patricia babbitt , jacquelyn fetrow department of physics, wake forest university, department of molecular genetics and genomics,-wake forest university, departments of physics and computer science, wake forest university bioinformatics understanding the relationships between proteins is vital to increasing our knowledge of the protein universe. while there are large databases of sequence information, the massive data influx over the past decade has prevented adequate classification of proteins at the molecular function level. however, it has been previously suggested that a protein's active site information may correlate with these known molecular functional differences; thus, active site profiling was developed to use residues around the active site of a protein to relate proteins. subsequently the deacon active site profiler (dasp) was developed to create these active site profiles and search them in a database, such as gen-bank, in order to find proteins with similar active site environments. by using dasp to computationally cluster proteins based on the similarity of their active site profiles, the peroxiredoxin (prx) superfamily was analyzed through active site similarity methods. the residues from the active site of each prx structure were extracted and clustered, and these profiles were iteratively searched in genbank through a multi-level iterative sequence searching technique (misst). the prx superfamily has been studied by experts, allowing the results of these searches to be compared to a well-annotated group of proteins. while previous sequence based evolutionary methods have been unable to identify functional differences between some subgroups of the prxs, notably the ahpc-prx and prx subgroups, misst discretely separates these subgroups. classifying prx proteins into functionally relevant groups using computational active site similarity methods lays the foundation for an automated process for identifying protein functional groups beyond the prx superfamily. synthesis and conformational studies of glycoprotein n homolog of bovine herpesvirus (bhv- ) by using cd, nmr and molecular modelling it serves as a chaperone for viral glycoprotein m and, in its gm-unbound form, acts as an inhibitor constraining the transporter associated with antigen processing (tap). the ul . /gm complex formation is required for the maturation and proper trafficking of both viral proteins. in the absence of gm, ul . blocks transport of antigenic peptides by tap and their mhc i-restricted presentation. the molecular mechanism of ul . activity still remains elusive. in order to investigate the structural requirements for biological function ul . study was conducted using cd, nmr and molecular dynamics methods. the data obtained with the use of high purity synthetic peptides encompassing ul . confirmed the presence of an alpha-helix structure, formed preferentially in the presence of dodecylphosphocholine (dpc) micelles as a membrane-like environment. in order to determine the three-dimensional structure of ul . protein in the present work its nmr solution structure in the presence of membrane-like environment was performed. the nmr data were used as a set of restraints for a simulated annealing protocol that generated dstructures of the colin johnson , sara codding membrane proteins resealing of tears in the sarcolemma of myofibers is a necessary step in the repair of muscle tissue. defects in this repair process are responsible for muscular dystrophy and cardiomyopathy. the repair pathway is triggered by the influx of calcium through lesions in the membrane, which result in membrane fusion and patching of the wound. recently dysferlin has been identified as a calcium binding protein essential for sarcolemma repair, as well as other snare mediated exocytotic events including cytokine and acid sphingomyelinase secretion. in this presentation we demonstrate a direct interaction between dysferlin and the snare proteins syntaxin and snap- . in addition, fret and in vitro reconstituted lipid mixing assays indicate that dysferlin accelerates snare heterodimer formation and snare mediated lipid mixing in a calcium sensitive manner. our results suggest a model whereby dysferlin acts as a calcium sensing snare effector for exocytosis and membrane fusion. exploring the therapeutic potential of a peptide derived from a poxviral immune evasion protein: nmr determination of the solution structure of viper and its inactive mutant toll-like receptors (tlrs) have a role in viral detection leading to cytokine and ifn induction, and as such they are targeted by viruses for immune evasion. the poxviral protein a has been identified to inhibit tlr signaling by interacting with tir domain-containing proteins of the receptor complex to collectively inhibit all tlr adaptor proteins that positively regulate transcription-factor activation ( ). one aa peptide (kysf-klilaey) termed viper (viral inhibitory peptide of tlr ) was reported to retain the inhibitory properties of full length a against tlr signaling. a r homopolymer delivery sequence at the c-terminus provided delivery of the peptide into cells. structural comparisons are presented between r-viper, which is active in preventing tlr -dependent cytokine induction in cell culture, and a mutant that exhibited loss of function ( r-viper l a,e a), through solution nmr spectroscopy. we find that despite a relatively minor sequence difference, the loss of hydrophobicity as well as negative electrostatic interactions result in subtle but potentially significant differences in the region of the peptide proposed to interface with tlr . reference: wake forest university, wake forest university, university of california san francisco protein function prediction the elucidation of protein molecular function lags far behind the rate of highthroughput sequencing technology; thus, it is essential to develop accurate and efficient computational methods to define functional relationships. protein clustering based on sequence similarity has emerged as a simple, high-throughput method for defining protein relationships, but sequence-based techniques often inaccurately define molecular function details. active site profiling (asp) was previously developed to identify and compare molecular details of protein functional sites. protein similarity networks were created using both active site similarity and sequence similarity for four manually curated superfamilies, and results demonstrate that asp-based clustering identifies detailed functional relationships more accurately than sequence-based clustering. building on this, two iterative pipelines were developed using active site profiling and profile-based searches to cluster protein superfamilies into functional groups. first, the two level iterative clustering process (tulip) utilizes active site profiling and iterative pdb searches to divisively cluster protein structures into groups that share functional site features. across eight superfamilies, tulip clusters exhibit high correlation with expert functional annotations. subsequently, the multi-level iterative sequence searching technique (misst) utilizes iterative profile-based genbank searches to identify protein sequences that belong in each tulip group. the results indicate that these asp-based methods accurately and efficiently identify functionally relevant groups through a process that can be applied systematically and on a large-scale. moreover, the approach can be applied more quickly than detailed manual curation, suggesting its value in guiding annotation efforts. dept. biochemistry and molecular biology. university of valencia, lab of peptide and protein chemistry. centro de investigaci on pr ıncipe felipe membrane proteins changes in the equilibrium between pro-survival and pro-apoptotic members of the b-cell lymphoma- (bcl- ) protein family at the mitochondrial outer membrane (mom) induce structural changes that committed cells to apoptosis. bcl- homology- (bh )-only proteins participate in this process activating pro-apoptotic effectors and promoting permeabilization of the mom. the membrane association of bh -only proteins is a controversial issue due to the lack of a canonical carboxyl-terminal (c-terminal) transmembrane (tm) domain. we used an in vitro transcription/translation system to study the insertion capacity of these hydrophobic c-terminal regions of the bh -members bik, bim, noxa, puma and bmf into microsomal membranes, and an escherichia coli complementation assay to validate our results in bacterial cells. furthermore, we have fused these hydrophobic regions to gfp to investigate the subcellular sorting. these results will allow further refinement in the elaboration of the bcl- protein-protein and protein-membrane interactome network. alexis peña , flaviyan jerome irudayanathan , shikha nangia syracuse university, dept. of biomedical and chemical engineering computational modeling, biostatistics, biomedical and chemical engineering tight junctions (tj) are vital intracellular barriers that are responsible for regulating paracellular transport. claudins, a family of abstract small transmembrane proteins with approximately members, are an integral part of the tj strands. tight junctions provide molecular-level protection and prevent infection and toxins from entering the body; in the same sense tjs allow nutrients and vital solutes to pass through. claudins are associated with various diseases including metastatic cancer as well as an entry point for many viruses. despite their importance and abundance in all cell membranes and their ubiquitous nature, the exact -d structure of claudins has remained elusive to traditional x-ray crystallographic and nmr studies. in this investigation, a computational approach was used to determine the claudin structure of claudin - . homology modeling, molecular dynamic simulations, and reverse mapping were employed to predict the protein structures with relative accuracy. understanding structure of claudin proteins and its interaction at the molecular level can lead to effective drug delivery technology. determination of optimal conditions for an isothermal titration calorimetry essay to obtain kinetic parameters of trypsin i from pyloric caeca of monterey sardine (sardinops sagax caerulea) idania emedith quintero reyes , francisco javier castillo y añez , enrique fernando vel azquez contreras , roc ıo sugich miranda , david octavio corona mart ınez , aldo alejandro arvizu flores , ivet cervantes dom ınguez protein kinetics determination of optimal conditions for an isothermal titration calorimetry essay to obtain kinetic parameters of trypsin i from pyloric caeca of monterey sardine (sardinops sagax caerulea) trypsin is the most studied alkaline protease and it s very common to found isoforms from this protein as the case for monterey sardine (sardinops sagax caerulea); as it shows an expression of trypsin i and trypsin iii according to the cdna characterization. trypsin i was determine to be a cold adapted enzyme as it shows a higher catalytic efficiency (kcat/km) than the mesophilic counterparts. the kinetic parameters were obtained by spectrophotometric essays, which are not fallible for all the enzymes because native, recombinant or mutant enzyme activity could be below the detection limit of the assay, opaque or turbid solutions interfere with spectrophotometric detection, etc. alternative tools as the isothermal titration calorimetry (itc) can measure enzyme kinetics using thermal power generated by the enzymatic conversion of substrate to product; were the rate of reaction is directly proportional to thermal power. the objective of this study was to stablish the optimum conditions to obtain kinetic parameters of trypsin i from pyloric caeca of monterey sardine using itc. to reach the objective trypsin i was purified from viscera of monterey sardine using molecular exclusion and affinity chromatography obtaining a yield of . mg/ml. at c kcat and km of tryipsin i form monterey sardine were . s- and . mm respectively. at c were . s- and mm (kcat and km) and at c kcat was . s- and km . mm. the kinetic parameters obtained by spectrophotometric assay at c were kcat and km s- and . mm respectively. at c the kcat was . s- and km . mm and at c kcat s- and km mm. comparing the values obtained for kcat with the spectrophotometric essay were higher fold than those obtained by itc and the values in km were similar by both methods. even though the differences in kcat, we can reassert the psychrophilic behavior of trypsin i as the catalytic efficiency is higher by both methodologies. in the understanding that the kinetic behavior of enzymes is important to not only understanding biochemical pathways and catalytic mechanisms but is again a fruitful area for drug discovery and development; so the itc provides a universal approach to determining the kinetic behavior of enzymes and can yield in a single experiment a complete set of kinetic parameters for an enzyme-catalyzed reaction that can be applied for the different alkaline proteases from pyloric caeca of monterey sardine (sardinops sagax caerulea). mysterious world of stress-responding sigma factors in bacillus subtilis olga ramaniuk protein-dna interaction bacterial transcription is mediated by the rna polymerase holoenzyme containing sigma factors -essential proteins for the initial step of transcription that recognize and bind to promoter dna. the primary sigma factor is essential in exponential phase of growth while alternative sigma factors are active during transcription under stress conditions. this project has three main aims. the first aim is to explore the binding properties of b. subtilis alternative sigma factors; specifically, whether sigma factors lacking the autoinhibitory domain . can bind to promoter dna in the absence of rnap. the second aim explores whether rnap associated with alternative sigma factors is regulated by the concentration of the initiation nucleoside triphosphate. the third aim is to define the regulon of sigma i. in order to achieve our aims, out of alternative sigma factors were successfully purified using affinity chromatography and ion exchange chromatography. we set up in vitro transcription system with selected sigma factors and initiated experiments with sigma i regulon determination. results named above and our future findings will help to better understand gene expression regulation on the level of transcription initiation. this work was supported by grant no. p - -g from the czech science foundation. assessing the costs and benefits of protein aggregation protein aggregation and cell fitness protein aggregation has been associated with numerous diseases but also with important cellular functions such as epigenetic inheritance. here we present a population genetics approach to infer the costs and benefits of protein aggregation on cell fitness. this information is crucial to understand how cellular systems tolerate the formation of protein deposits and which factors modulate this event. using our experimental system, we measured different protein aggregation effects (deleterious, neutral or beneficial) within the same genomic background. single cell analyses, within the same population, showed stochastic variability in the aggregate's size and in its effect on cell fitness. our data indicates that, in certain conditions, protein aggregation can enhance population variability and survival expectancy. overall, these results suggest that the presence and formation of protein aggregates could be almost harmless whereas the associated gain and loss of function are critical for the cell. revealing the key role of negatively charged residues of heme sensor proteins involved in geobacter sulfurreducens' signal transduction pathways marta a. silva , telma c. santos , teresa catarino , carlos a. salgueiro ucibio-requimte, departamento de qu ımica, fct-unl., instituto de tecnologia qu ımica e biol ogica, unl signal transduction proteins bacterial chemotaxis systems sense and regulate the microbe mobility in response to environmental conditions. such mechanisms constitute a striking example of cell motility to gain advantages for cell survival and permit the bacteria to fill important niches in a diversity of anaerobic environments [ ] . geobacter sulfurreducens (gs) is an anaerobic bacterium with a considerable respiratory versatility whose genome encodes for an unusual family of methyl-accepting chemotaxis proteins (mcp), each containing at least one heme c-binding motif [ ] . these sensor proteins, gsu and gsu , are involved in signal transduction pathways mediated by chemotaxis-like systems [ ] . the thermodynamic and kinetic characterization of the sensors gsu and gsu by visible spectroscopy and stopped-flow techniques, at several ph and ionic strength values revealed that sensor gsu midpoint reduction potentials are lower than those of gsu at all ph and ionic strength values and the same were observed for the reduction rate constants [ ] . the origin of the different functional properties of these closely related sensor domains are rationalized in the structural terms showing that gsu has two extra negatively charged residues in the vicinity of the heme group, which have no counterpart in gsu : glu and asp . residue asp is less exposed compared to glu and it was suggested that its carboxylic group might have a role in the modulation of the heme reduction potential of gsu . to investigate this, both residues were replaced by a positively charged amino acid (lysine) and by a neutral one (asparagine or glutamine). for the mutants with enough expression, a functional characterization was carry out, using several spectroscopic techniques, including uv-visible and cd, together with kinetics and potentiometric measurements. significant changes on the reduction potential values are observed when a negative charge is replaced by a positive one at position or . therefore, the decrease of the reduction potential in asp and glu mutants reinforces the hypothesis that the higher reduction potential observed for heme sensor domain gsu is related with the less negative electrostatic surface around the heme. this work provides, for the first time, evidence for the co-existence of two similar methyl-accepting chemotaxis proteins functioning in different working potential ranges. these proteins are responsible to allow geobacter sulfurreducens triggering an adequate cellular response in different anoxic subsurface environments. national autonomous university of mexico, faculty of medicine, national autonomous university of mexico, faculty of chemistry, national autonomous university of mexico, institute of chemistry molecular evolution the glycolytic enzyme triosephosphate isomerase (tim) is an oligomeric (b/alpha) barrel that catalyses the interconversion of d-glyceraldehyde -phosphate and dihydroxyacetone phosphate in a diffusion-limited reaction. although each subunit has its own active site, naturally occurring monomeric tims have not been reported; in fact, monomer association is very tight. tim topology is well conserved among the three domains of life. nevertheless, their folding mechanism and inhibition properties vary across species. comparative studies of proteins have proved to be very useful in understanding the relationship between sequence and physicochemical properties, however, they lack the capacity to give a more integrative and evolutive correlation. in order to elucidate how the catalytic properties, the oligomerization state and the stability of extant tims arose, in this work we examined the molecular history of eukaryotic tim through ancestral protein reconstruction methods (maximum likelihood) and the subsequent physicochemical characterization of the resurrected enzymes. we first characterized in detail the protein corresponding to the last common ancestor of animals and fungi (tim ). the cd and fluorescence spectra of tim are similar to those of extant tims. secondary structure is lost in a cooperative transition with tm . c. the enzyme loses activity upon dilution suggesting that only the dimer is active. dilution experiments followed by isothermal titration calorimetry indicate that dissociation enthalpy is small; moreover the heat capacity change observed is three times higher than the one predicted for a rigid body dissociation process, suggesting partial unfolding of the monomers. when compared with extant tims, the catalytic efficiency of tim is reduced -fold, whereas binding of pgh, a transition-state analogue, shows a similar thermodynamic signature. these data indicate that although monomer association may have been less tight in ancestral tims, catalysis has been always linked to oligomerization. analysis of the crystal structure of tim , obtained at . Å resolution, suggests that the lack of four salt bridges observed in the interface of extant tims is responsible for the low dimer stability. in order to test this hypothesis we also studied the stability of four younger reconstructed ancestors that acquired the salt bridges in two different phylogenetic lineages. we found a correlation between the appearance of stabilizing interactions in the interface, dimer stability and catalysis; suggesting that these salt bridges are partially responsible for extant dimer stability and shed light on the dimeric nature of extant tims. receptor protein-tyrosine phosphatases: dimerization, receptor kinase interaction and allosteric modulation elizabeth dembicer , damien thevenin department of chemistry, lehigh university theme: receptor tyrosine kinase and receptor protein phosphatase signaling many cell-signaling events are regulated through reversible tyrosine phosphorylation of proteins, which is controlled by the counterbalanced actions of two key enzyme families: protein tyrosine kinases and protein tyrosine phosphatases. interestingly, both families include transmembrane receptor-like enzymes, namely the receptor tyrosine kinases (rtks) and the receptor-like ptps (rptps). while the regulation and actions of many rtks are well characterized, the mechanisms controlling the enzymatic activity of rptps and how they interact with their substrates remain to be fully explained. thus, understanding how these receptors function and interact will give fundamental insights into how tyrosine phosphorylation is finely tuned in cells, and how it can be modulated. increasing evidence indicates that rptps, like rtks, are regulated by homodimerization. however, it appears that homodimerization inhibits the activity of most rptps. even though the transmembrane (tm) and the juxtamembrane domains have been proposed to be involved in this process, there is no clear structure-based proposal for the role of these regions. moreover, several rptps have been identified as candidate regulators of rtks. in particular, the receptor-type tyrosine-protein phosphatase eta (ptprj; also known as dep or cd ) is capable of attenuating egfr tyrosine phosphorylation. physical interactions of egfr with ptprj at the cell surface have been documented, but the basis for these interactions is unknown. here, using a dominant-negative transcriptional activator-based assay (dn-aratm), and mutagenesis analysis, we show that: ( ) ptprj has a strong tendency to homodimerize, ( ) ptprj heterodimerizes with egfr through tm-tm interactions, ( ) these interactions are mediated by specific residues, and can be modulated by the delivery of peptide binders. this work represents the first structure-function study of rptp-rtk interaction, and may not only result in significant progress towards a better understanding of the basic biology of rptps in cancer cells, but also offer new possibilities for targeting protein tyrosine phosphatases for therapeutic modulation of egfr in oncology. inhibiting egfr dimerization and signaling through targeted delivery of juxtamembrane domain peptide mimics using phlip anastasia thevenin , kelly burns , janessa guerre-chaley , damien thevenin regulating receptor tyrosine kinase signaling the elevated phosphorylation of key regulatory tyrosines on oncogenic signaling proteins that result from aberrant protein tyrosine kinases activity plays well-abstract established roles in promoting tumorigenesis and in the high frequency with which resistance arises to existing therapeutic treatment. for instance, this is the case for the epidermal growth factor receptor (egfr). thus, there is a clear need for novel specific targeting methods to inhibit the activity of receptor protein tyrosine kinases, such as egfr, in cancer. egfr becomes activated upon ligand binding to the extracellular domain, leading to receptor dimerization. the juxtamembrane (jm) domain of egfr is critical for intrinsic tyrosine kinase activity and receptor dimerization by stabilizing the active conformation of egrr through the formation of a antiparallel helical dimer. therefore, peptides mimicking the jm domain -if specifically delivered to cancer cells -have the potential to prevent egfr dimerization, receptor activation, downstream signaling, and thus to attenuate aberrant egfr activity in cancer cells. here, phlip (ph low insertion peptide), a peptide that can selectively target cancer cells and tumors based solely on their extracellular acidity, is used to selectively translocate the jm domain of egfr in cancer cells to prevent egfr dimerization. at ph above , phlip is soluble and unstructured, however, when exposed to lower ph such as observed in tumors, phlip inserts as a transmembrane (tm) alphahelix, allowing the direct translocation of cargo molecules into the cytoplasm. using the dominant negative arac-based transcriptional reported assay (dn-aratm), which assesses jm and tm domain interactions in cells membranes of e. coli, we show that phlip-jm is able to disrupt egfr dimer by %. current work is focused on testing the ability of such phlip-jm peptide conjugate to perturb egfr homodimerization and decrease downstream signaling through soluble kinases, such as akt and erk, in cancer cells. the thumb subdomain of yeast mitochondrial rna polymerase is involved in processivity, transcript fidelity and mitochondrial transcription factor binding gilberto velazquez , luis brieba , rui sousa universidad de guadalajara, langebio cinvestav, university of texas healthsscience center at san antonio dna protein interaction abstract single subunit rna polymerases have evolved two mechanisms to synthesize long transcripts without falling off a dna template: binding of nascent rna and interactions with an rna:dna hybrid. mitochondrial rna polymerases share a common ancestor with t-odd bacteriophage single subunit rna polymerases. herein we characterized the role of the thumb subdomain of the yeast mtrna polymerase gene (rpo ) in complex stability, processivity, and fidelity. we found that deletion and point mutants of the thumb subdomain of yeast mtrna polymerase increase the synthesis of abortive transcripts and the probability that the polymerase will disengage from the template during the formation of the late initial transcription and elongation complexes. mutations in the thumb subdomain increase the amount of slippage products from a homopolymeric template and, unexpectedly, thumb subdomain deletions decrease the binding affinity for mitochondrial transcription factor (mtf ). the latter suggests that the thumb subdomain is part of an extended bindingsurface area involved in binding mtf . design principles of membrane protein structures vladimir yarov-yarovoy , diane nguyen membrane protein structure membrane proteins play key role in cellular signaling and ion transport. statistical analysis of expanding database of high-resolution membrane protein structures in protein data bank (pdb) provides useful information about membrane protein structure and function. we used rosettamembrane software (yarov-yarovoy v et al ( ) proteins) to analyze unique alpha helical membrane protein structures in pdb and derive knowledge based energy function for membrane protein structure prediction, membrane protein-protein docking, and membrane protein design. the rosettamembrane residue environment energy term is based on amino acid propensities in hydrophobic, interface, and water layers of the membrane and depends on the residue burial state -from being completely buried within a protein environment to being completely exposed either to the lipid or water environments. residue buried state is determined from the number of residue neighbors within and Å spheres. the rosettamembrane residue-residue interaction term is based on the propensities of amino acid pairs to be in close proximity to each other within hydrophobic, interface, and water layers. results of our statistical analysis reveal fine details of favorable and unfavorable environments for all amino acids types in all membrane layers and residue burial states. we find that large hydrophobic amino acids are favorable facing the hydrophobic core of the lipid bilayer. small amino acids are favorable facing the protein core within the hydrophobic layer of the membrane. aromatic or positively charged amino acids and favorable facing the lipid head groups. residue-residue interactions are often favored between polar and charged amino acids and also between some of small and large hydrophobic amino acids inside of the protein core within the hydrophobic layer of the membrane. these data will be useful for rational design of novel membrane protein structures and functions. coordinated gripping of substrate by subunits of a aaa proteolytic machine ohad yosefson , andrew nager , tania baker , robert sauer protein quality control' or 'protein degradation' hexameric aaa protein-remodeling machines use conserved loops that line the axial pore to apply force to substrates during the mechanical processes of protein unfolding and translocation. an open question in the aaa field is whether pore loops from different subunits of the hexameric ring grip the substrate coordinately (all six subunits involved), independently (one subunit at a time involved), or partially coordinated (two or three subunits at a time). to answer this question, we studied covalently linked hexamers of the e. coli clpx unfoldase bearing different numbers and configurations of wild-type and mutant pore loops and challenged these variants with protein substrates with a broad range of stabilities. we find that successful unfolding of increasingly resistant substrates requires the coordinated action of a greater number of wild-type pore loops. our results support a mechanism in which a power stroke initiated in one subunit of the clpx hexamer results in the simultaneous movement of all six pore loops, which coordinately grip and apply force to the substrate. structure and function of the toc m-domain, and its role in targeting the preprotein receptor to the chloroplast outer envelope membrane matthew smith , shiu-cheung lung , prem nichani , nicholas grimberg , j. kyle weston , shane szalai , simon chuong deartment of biology, wilfrid laurier university, department of biology, university of waterloo chloroplast biogenesis and function rely on the import of thousands of nucleus-encoded preproteins from the cytosol. preprotein import is supported by the toc and tic (translocon at the outer and inner envelope membranes of chloroplasts) complexes, which work cooperatively to translocate preproteins across the double-membrane envelope to the chloroplast interior. toc is one of the preprotein receptors of the toc complex, is also encoded in the nucleus and post-translationally targeted to the chloroplast, and is comprised of distinct domains: ) the intrinsically disordered n-terminal acidic (a-) domain; ) the central gtpase (g-) domain; and ) the c-terminal membrane (m-) domain that anchors the protein to the chloroplast outer membrane (com) through an unknown mechanism. the m-domain has no known homologues and does not contain a predicted trans-membrane domain, but does contain intrinsic chloroplast targeting information at the extreme c-terminus. the m-domain also contains a predicted b-helix motif, which may be important for anchoring the protein to the com. we are interested in characterizing the structure of the m-domain and determining the nature of its association with the com, as part of our larger goal of understanding the role toc plays in protein import into chloroplasts. we are also interested in defining the precise nature of the targeting information contained within the extreme c-terminus of toc , elucidating the targeting pathway that is used, and whether other com proteins use this pathway. we will present our most recent data on the structure, function and targeting of the toc m-domain. structural investigation of nlpc/p protein acquired by trichomonas vaginalis through a lateral gene transfer event jully pinheiro , , augusto simoes-barbosa , david goldstone microbiology, school of biological sciences, university of auckland, structural biology, school of biological sciences, university of auckland trichomonas vaginalis is an extracellular flagellated protozoan parasite that causes the most common non-viral sexually transmitted disease, with approximately million cases worldwide annually. nevertheless, the biochemical processes behind t. vaginalis infection and its interaction with the vaginal microbiota are still not well defined. in the draft genome sequence of trichomonas vaginalis strain g was described, identifying , protein-coding genes. of these, nine genes encode nlpc/p -like members. this superfamily is widely represented in the different kingdoms of life and has diverse enzymatic functions, such as amidases, endopeptidases and acetyltransferases. previous studies have shown that members of this superfamily hydrolyze specific peptide linkages in bacterial cell walls affecting germination, vegetative growth, sporulation and division or cell lysis/invasion. as a typical eukaryote, the protozoan parasite t. vaginalis does not have a cell wall itself. previous studies suggest that the t. vaginalis nlpc/p genes were acquired via lateral gene transfer from bacteria and must have an important function, possibly controlling the vaginal microbiota and aiding parasite invasion and infection. to investigate the function of the nlpc/p family of proteins in t. vaginalis we have expressed, purified and crystallized a member tvag_ and report its three-dimensional structure, determined at . Å resolution, by x-ray diffraction. the structure of the protein reveals a typical papain-like fold resembling peptidoglycan hydrolases from the nlpc/p family with a conserved cysteine and histidine; forming the catalytic residues. the protein contains two bacterial sh domains at the n-terminus. this domain acts as a general binding domain and is likely to aid the interaction of the nlpc/p domain with substrate components. combined with biochemical and enzymatic characterization, the structure of this nlpc/p protein will help to elucidate the molecular origin of its hydrolase activity and to decipher their putative role in the parasite infection. novel dna polymerases from red sea brine-pools: new potential polymerases for pcr application masateru takahashi , etsuko kimura , mohamed salem , ulrich stingl , samir hamdan protein biotechnology the polymerase chain reaction (pcr) is a key tool in medical and biological research. the most common pcr reaction relies on the thermal cycling method that consists of repeated cycles of heating and cooling steps for dna melting and extension by the dna polymerase, respectively. the introduction of new dna polymerases to the market is a major area of development that tremendously helped in improving the performance and quality of pcr. nonetheless, pcr still requires optimization of salt and metal ion concentrations leaving a room in the market for introducing new dna polymerases that are robuster in their salt and metal ion concentration dependence. in this study, we will present the characterization of a novel archaeal dna polymerase from the red sea brine-pool (termed br ) and demonstrate how its enzymatic activity reflects on every aspects of the environment of the brine-pool -high tolerance to concentrations and types of salts and metal ions including utilization of zn ions in its active site. these results suggest that the brine-pool microorganisms are likely to contain novel chemical pathways to deal with its exterior harsh conditions. we will further show the mechanism of br polymerase how it was adjusted to be active in harsh condition. structural basis for the identification of the n-terminal domain of coronavirus nucleocapsid protein as an antiviral target ming-hon hou , shing-yen lin , chia-ling liu , yu-ming chang , jincun zhao , stanley perlman institute of genomics and bioinformatics, national chung hsing university., institute of biological chemistry, academia sinica., department of microbiology, the university of iowa drug discovery coronaviruses (covs) cause numerous diseases, including middle east respiratory syndrome and severe acute respiratory syndrome, generating significant health-related and economic consequences. covs encode the nucleocapsid (n) protein, a major structural protein that plays multiple roles in the virus replication cycle and forms a ribonucleoprotein complex with the viral rna through the n protein's nterminal domain (n-ntd). using human cov-oc (hcov-oc ) as a model for cov, we present the d structure of hcov-oc n-ntd complexed with ribonucleoside '-monophosphates to identify a distinct ribonucleotide-binding pocket. by targeting this pocket, we identified and developed a new coronavirus n protein inhibitor, n-( -oxo- , -dihydrophenanthridin- -yl)(n,n-dimethylamino)acetamide hydrochloride (pj ), using virtual screening; this inhibitor reduced the n protein's rna-binding affinity and hindered viral replication. we also determined the crystal structure of the n-ntd-pj complex. on the basis of these findings, we propose guidelines for developing new n protein-based antiviral agents that target covs. thermal and structural stability of ß-glucosidases gh maira artischeff frutuoso departamento de bioqu ımica do instituto de qu ımica da universidade de são paulo enzymology we compared the stability of thermophilic b-glucosidases gh to mesophilic ones in the presence of denaturants as urea and high temperature by following the transitions between the native and unfolded states by tryptophan fluorescence, enzymatic activity and differential scanning fluorimetry (dsf). the bacterial b-glucosidases (bgla) and (bglb) of the mesophile paenibacillus polimyxa and bglucosidase (bglthm) of the thermophile thermotoga maritima were expressed as recombinant proteins in novablue (de ) and purified by affinity chromatography (ni-nta resin). these recombinant enzymes have very similar folding type structure (b/a) barrel, as shown in crystal structures and exhibited a characteristic peak between and nm in the tryptophan fluorescence spectra, indicating that those proteins are folded. circular dichroism analysis in the far-uv region ( nm to nm) also showed typical spectra of folded proteins with secondary structure composition of % of a-helix and % of b-sheets for bgla, % of a-helix and . % of b-sheets for bglb and % of a-helix and % of b-sheets for bglthm. the average degree of accessibility to the exposed tryptophan residues in the native enzyme to increasing concentrations of the acrylamide suppressor (stern-volmer constant -ksv) is greater to bgla ( . ), but similar to bglb ( . ) and bglthm ( . ). the thermal stability determined by dsf was higher for bglb (tm . c) than for bgla (tm . c) . the bglthm was stable at c and remained stable for up to h at c. in addition the thermal inactivation kinetics at c evaluated by the relative remaining activity showed that bgla denaturation (kinactivation of . s- ) is faster than bglb (kinactivation of . s- ). on the other site, bglthm inactivation at c was a two-step process, which exhibited an initial fast step (kinactivation of . s ) followed by a slow step (kinactivation of . s- ). the chemical denaturation by urea followed using tryptophan fluorescence showed a transition pl- covalent structure of single-stranded fibrinogen and fibrin oligomers cross-linked by fxiiia. the influence of free radical oxidation anna bychkova , vera leonova , alexander shchegolikhin , marina biryukova , elizaveta kostanova , mark rosenfeld n. m. emanuel institute of biochemical physics, russian academy of sciences protein structure and function native fibrinogen is a key blood plasma protein whose main function is to maintain hemostasis by virtue of producing the cross-linked fibrin clots under the effect of thrombin and fibrin-stabilizing factor (fxiiia). fxiiia-mediated isopeptide g-g bonds are known to be produced between g polypeptide chains of adjacent fibrinogen or fibrin molecules. but there are apparently conflicting ideas regarding the orientation of g-g bonds. in this study several peculiarities of self-assembly of fibrin(ogen) and induced oxidation of the proteins have been studied with the aid of elastic and dynamic light scattering, uv-, ftir-and raman spectroscopy methods. in the presence of fxiiia both the non-oxidized and oxidized fibrinogen molecules has been shown to bind to each other in the "endto-end" fashion to form the flexible covalently cross-linked fibrinogen homopolymers. to identify the orientation of g-g bonds in fibrin protofibrils a novel approach based on self-assembly of soluble cross-linked fibrin protofibrils and their dissociation in the urea solution of moderate concentrations has been applied. the results of elastic and dynamic light scattering coupled with analytical ultracentrifugation indicated the protofibrils to exhibit an ability to dissociate under increasing urea concentration to yield single-stranded structures entirely brought about by g-g bonds. the results of this study provide an evidence to support the model of the longitudinal g-g bonds that form between the g chains end-to-end within the same strand of a protofibril. since fibrinogen is known to be sensitive to ros the mechanisms of fibrinogen and fibrin self-assembly under induced oxidation have been investigated. in both cases the polypeptide chains of the oxidized fibrin(ogen) proved to be involved in the enzymatic cross-linking more readily than those of unaffected molecules. the enhancing role of the d:d interaction under oxidation could be considered as an compensatory mechanism in the assembly of fibrin when the d:e interaction is impaired. the experimental data on fibrinogen and fibrin oxidation acquired in the present study, being combined with our earlier findings, make it reasonable to suppose that the spatial structure of fibrinogen could be evolutionarily adapted to some ros actions detrimental to the protein function. the study was supported by the rfbr, research projects - - mol_a and - - a. structural and thermodynamic analysis of co-stimulation receptor cd phosphopeptide interactions with grb , gads, and pi -kinese sh domains in addition to the signaling produced by the binding of antigen-major histocompatibility complex to tcell receptors, co-stimulatory signals from other receptor-ligand interactions are required for full activation of t-cells. the cd receptor on the t-cell surface has been well characterized, and the binding of ligand to cd is critical for producing co-stimulatory signals. cd has no enzymatic activity and its cytoplasmic region consists of amino acids that contain the sequence ymnm, in which the tyrosine residue is phosphorylated by kinase. the phosphorylated sequence, pymnm, is recognized by src homology (sh ) adaptor proteins, such as growth factor receptor binding protein (grb ), grb related adaptor downstream (gads), and the phosphatidylinositol -kinase (pi -kinase) regulatory subunit, p . the consensus sequence for the binding of grb sh and gads sh is pyxnx, and that of p n-terminus sh (nsh ) and c-terminus sh (csh ) is pyxxm. we reported the high-resolution crystal structure of grb sh in complex with the cd phosphopeptide [higo et al., plos one , e , ] , and recently determined those of gads sh , p nsh , and p csh . these data along with the results of binding thermodynamics analyzed using isothermal titration calorimetry, helped to elucidate the molecular recognition mechanisms of cd by adaptor proteins. the sh proteins were overexpressed in escherichia coli, and were purified using affinity and gel-filtration chromatography. the cd phosphopeptides, -residue (octp) and -residue (ddcp ), were synthesized using the solidphase supported technique, and were purified using reversed-phase chromatography. the crystals were obtained by the hanging-drop vapor diffusion method. x-ray diffraction data were collected at synchrotron radiation facilities, and the structures were determined by the molecular replacement method. the models of grb sh , gads sh , p nsh , and p csh in complex with octp were refined at . , . , . , and . Å resolutions, respectively. the crystal structures showed that the phosphotyrosine phosphate moiety directly interacted with the side-chain of arginine in sh , which is common in all complex structures. in the grb sh and gads sh complexes, the side-chain of asparagine at the py position forms a pair of hydrogen bonds with the main-chain amide and carbonyl groups of lysine in sh . alternatively, in the p nsh and csh complexes, the side-chain of methionine at the py position is located in hydrophobic pockets of nsh and csh , in which the hydrophobic interactions of csh would be stronger than those of nsh . this idea is supported by the observed binding thermodynamics. the binding affinity of csh to ddcp , because of a favorable enthalpy change, is about -fold higher than that of nsh . the binding affinity of grb sh to ddcp is similar to that of gads sh to ddcp , and is about -fold lower than that of nsh to ddcp . these results indicate that the contribution of hydrophobic interactions of nsh and csh at the py position are stronger than those of hydrogen bonds of grb sh and gads sh at the py position. novel kinetochore protein complex from silkworm holocentric chromosomes takahiro kusakabe , hiroaki mon , jaeman lee the kinetochore, which consists of centromere dna and a multilayered protein complex, plays important roles in chromosome organization and segregation. interactions between chromosomes and spindle microtubules allow chromosomes to congress to the middle of the cell, and to segregate the sister chromatids into daughter cells in mitosis, which is followed cytokinesis. in contrast to monocentric chromosomes, in which the centromere is normally present at a single region on each chromosome, the holocentric chromosomes have centromeric activity along the entire length of the chromosome. it has been known that the silkworm, bombyx mori, has holocentric chromosomes since s, none of silkworm kinetochore proteins, however, have been identified so far. here we report the identification of a novel set of genes for outer kinetochore proteins in silkworm by using bioinformatics and rna interference-based screening. under the hypothesis that depletion of essential kinetochore genes causes cell cycle arrest in mitosis, we performed rnai in the silkworm cell line, bmn -sid , targeting a set of candidate genes. knockdown of five genes caused significant cell cycle arrest at the g /m phase. we also found that these five proteins make a complex, and that all of them are localized along the chromosome arms, indicating that the silkworm kinetochore extends along the chromosome. inactivation of bine aldehyde dehydrogenase from spinach by its physiological substrate bine aldehyde to contend with osmotic stress caused by drought, salinity, or low temperatures some plants synthesize the osmoprotectant glycine bine (gb) from bine aldehyde (bal). the last step-the irreversible nad dependent oxidation of bal-is catalyzed by aldh enzymes that exhibit bine aldehyde dehydrogenase (badh) activity. we here report that the spinacia oleracea badh (sobadh) is reversibly inactivated by bal in the absence of nad in a time-and concentration-dependent mode to approximately % of the original activity. inactivation kinetics are consistent with a partial reversible, two-steps mechanism that involves the formation of an active non-covalent enzyme•bal complex before formation the inactive enzyme-bal complex. crystallographic evidence indicates that in the enzyme previously inactivated by bal the aldehyde forms a thiohemiacetal with the nonessential cys (sobadh numbering) located at the aldehyde-entrance tunnel, thus totally blocking the access to the catalytic cysteine. accordingly, bal does not inactivate the c s sobadh mutant. two crystal structures of the inactivating enzyme-bal complex showed that the trimethylammonium group of bal is inside the active-site aromatic box, as in the productive way of binding. this explains why the inactivation of the a i mutant-where the binding of the trimethylammonium group is hindered-requires non-physiologically high bal concentrations, while the a c mutant-where the binding is allowed-is inactivated similarly to the wildtype enzyme. cys- is conserved in most plant aldh enzymes of known sequence, and in all of them with proven or predicted badh activity. inactivation by bal appears therefore to be a common feature of plants badhs. this short-term regulation may be of great physiological importance since the irreversibility of the badh-catalyzed reaction would unbalance the nad /nadh ratio if the aldehyde concentrations are high, the nad concentrations low and the reaction is not slowed down. plants badhs are prone to this situation since they work under osmotic stress conditions, when high bal concentrations are required for the synthesis of high levels of the osmoprotectant gb. the partial nature of the decamer possesses a donut shaped structure with calcium ions on the surface available for interactions with carbohydrate molecules. binding specificity was evaluated for carbohydrates using differential scanning fluorimetry (dsf) that showed bjcul interacts with galactose and lactose but less with glucose and sacarose. surprisingly, high levels of thermostabilization of bjcul was achieved with the antibiotic aminoglycosides geneticin (g ) and gentamicin in a calcium concentration dependent manner, but not kanamycin. intriguingly, while lactose and galactose inhibited erythrocyte agglutination by bjcul, g and gentamicin did not affect hemagglutination implying a second site of binding. dsf analysis also suggested the presence of a second binding site for the antibiotics and crystallization of the complexes are in progress in order to understand fully this new binding mechanism of c-type lectin with antibiotics. ab initio modelling of structurally uncharacterised antimicrobial peptides mara kozic institute of integrative biology, university of liverpool ab initio modelling of structurally uncharacterised antimicrobial peptides mara kozic * institute of integrative biology, biosciences building, university of liverpool, crown street, liverpool l zb, united kingdom * mara.kozic@liverpool.ac.uk antimicrobial resistance within a wide range of infectious agents is a severe and growing public health threat. antimicrobial peptides (amps) are among the leading alternatives to current antibiotics, exhibiting broad spectrum activity. an understanding of the structure of a protein can lead us to a much improved picture of its molecular function. furthermore, an improved understanding of structure-function relationships facilitates protein design efforts to enhance their activity. currently, the d structures of many known amps are unknown. to improve our understanding of the amp structural universe we have carried out large scale ab initio d modelling of structurally uncharacterised amps. such ab initio modelling is facilitated by the typical small size of amps as well as their tendency to contain disulphide bonds, these providing valuable additional information to simulations. preliminary results reveal unexpected similarities between the predicted folds of the modelled sequences and structures of well-characterised amps. for example, lacticin q was revealed to contain a helical bundle fold that bears a striking resemblance to enterocin a. we also found a remarkable similarity between the predicted structure of silkworm peptide and b-hairpin amps such as tachyplesin i. our results improve the understanding of the structure-function relationship of amps. surface aggregation-propensity as a constraint on globular proteins evolution susanna navarro , marta diaz , pablo gallego , david reverter , salvador ventura institut de biotecnologia i biomedicina and departament de bioquimica i biologia, institut de biotecnologia i biomedicina, universitat aut onoma de barcelona in living cells, functional protein-protein interactions compete with a much larger number of nonfunctional interactions. theoretical studies suggest that the three-dimensional structures of present proteins have evolved under selective pressure to avoid the presence of aggregation-prone patches at the surface that may drive the establishment of anomalous protein contacts. however, no experimental evidence for this hypothesis exists so far. the a-spectrin sh domain (spc-sh ) has been used as a protein model to decipher the sequential aggregation determinants of proteins. here we use it to address the structural determinants of protein aggregation and their link to protein evolution. to this aim we exploit aggrescan d (a d), a novel algorithm developed by our group, which takes into account both protein structure and experimental data to project aggregation propensities on protein surfaces. we used a d to design a series of spc-sh variants with progressively stronger aggregationprone surfaces and characterized their thermodynamic, structural and functional properties. our data support evolution acting to constraint the aggregation propensities of globular protein surfaces in order to decrease their potential cytotoxicity and the protein quality control machinery acting to buffer this negative selective pressure. utilizing d structure for the annotation of structural motifs in the conserved domain database narmada thanki-cunningham , noreen gonzales , gabriele marchler , myra derbyshire , james song , roxanne yamashita , christina zheng , stephen bryant , aron marchler-bauer , farideh chitsaz conserved domain database, structure group cbb/ncbi/nlm/nih the conserved domain database (cdd) is a protein classification and annotation resource comprised of multiple sequence alignments representing ancient conserved domains. cdd protein domain models are curated by ncbi and use d protein structure explicitly to define domain extent and the location of conserved core structures, and to provide accurate alignments between diverse family members via structure superposition. cdd also imports external collections such as pfam and tigrfam. recently, a novel class of annotation labeled as "structural motifs" has been introduced to supplement current capabilities. these annotations define compositionally-biased and/or short repetitive regions in proteins, which are difficult to model as functional domains conserved in molecular evolution. structural motifs include transmembrane regions, coiled coils, and short repeats with variable copy numbers. for many types of short tandem repeats, a few position-specific score matrices (pssms) suffice to annotate more than % of the known instances of that structural motif. unfortunately, a lack of sequence similarity within coiled-coil regions prohibits the development of only a few generic models; therefore, models for coiled-coil regions in the context of specific families have been developed using the spiricoil database as a reference. increased coverage of coiled-coil regions in cdd, specific site annotations of these structural motifs as well as their representation on the webpages will be discussed. specific in vivo ultrasound imaging of e-selectin expression in tumors using a microbubble contrast agent covalently attached to the peptide ligand iellqar, known to bind to e-selectin [ ] . however, it was observed that this probe has a limitation in the imaging of cardiovascular diseases where higher shear stresses prevent microbubbles from remaining attached to the target. therefore, peptides with higher eselectin affinity are needed to design probes capable of imaging these diseases. in this context, automated docking and molecular dynamics methodologies were combined and applied to different e-selectin binding peptides. these studies predicted the energetically more favorable binding mode as well as the key interactions between the peptide ligands and the e-selectin receptor. some of these peptides were prepared by solid-phase peptide synthesis and their interactions with e-selectin analyzed by surface plasmon resonance technique. the results showed that these peptides have different affinities for e-selectin. these data were correlated with the computational studies and evaluated to obtain crucial information of the key recognition elements needed for higher e-selectin affinity. these recent results will be presented. burkholderia pseudomallei is the causative agent of melioidosis, a serious invasive disease of animals and humans in tropical and subtropical areas. sedoheptulose- -phosphate isomerase from b. pseudomallei (bpgmha) is the antibiotics adjuvant target for melioidosis. in general, bpgmha converts dsedoheptulose- -phosphate to d-glycero-a-d-manno-heptopyranose- -phosphate (m p). this is the first step of the biosynthesis pathway of ndp-heptose responsible for a pleiotropic phenotype. therefore, this biosynthesis pathway is the target for searching novel antibiotics increasing the membrane permeability of gram-negative pathogens or adjuvants synergistically working with known antibiotics. the crystal of this enzyme has been solved at . Å resolution. there is an active site pocket where a putative metal binding site is located. to find out inhibitors of bpgmha, in-silico virtual screening with zinc, a free database of commercially-available compounds, has been performed. tens of thousands of chemical compounds were docked into the active site of bpgmha. a number of putative bpgmha binding compounds better than m p were found using surflex-dock included in the sybyl software package. characteristics of these compounds were surveyed and classified to identify common binding properties with bpgmha. mapping the structure of laminin using cross-linking and mass spectrometry gad armony , toot moran , yishai levin , deborah fass weizmann institute of science, department of structural biology, weizmann institute of science, israel center for personalized medicine laminin, a kda heterotrimer, is a major element in the extracellular matrix (ecm). within the ecm, laminin contributes to the adhesion and migration of cells, both in health and disease. the laminin trimer was observed by rotary shadowing electron microscopy to be cross shaped: the three short arms of the cross are formed by the amino-terminal halves of the three subunits, whereas the long arm of the cross holds the three chains together in a long coiled coil. the narrow and flexible arms of the laminin cross complicate studying its structure to high resolution by crystallography or electron microscopy single particle reconstruction. to advance our understanding of this remarkable quaternary structural assembly, we have used cross-linking and mass spectrometry to analyze the organization of the laminin trimer. this technique was validated by known crystal structures of isolated laminin domains. in all cases the crystal structure distances agree with the cross-linker length. the identified cross-links were particularly helpful in assigning the register and the subunit order of the long coiled coil due to the high content of cross-linkable residues in this region. using known x-ray crystal structures, homology modeling, and distance restraints provided by two cross-linker chemistries, a clearer picture of the laminin quaternary structure is obtained. non-sequential protein structure alignment program mican and its applications shintaro minami , george chikenji , motonori ota dept. of info. sci., nagoya univ., dept. of comp. schi. & eng., nagoya univ. in some proteins, secondary structure elements are arranged spatially in the same manner, but they are connected in the alternative ways. analysis on such non-sequential structural similarity in proteins is important because it provides a deeper understanding of the structural geometry of protein. this can be also observed even in the homologous proteins, indicating the non-sequential structural similarity is significant in the protein evolution. however, the non-sequential structural similarity in proteins is less investigated. we developed a novel non-sequential structural alignment program mican, which can handle multiple chains, inverse direction of chains, c$lpha$models, alternative alignments, and non-sequential alignments. we performed comprehensive non-sequential structural comparison among homologous proteins in the same scop superfamily by using the mican program. based on the result, we found that approximately % of superfamilies include at least one protein pairs showing non-sequential structural similarity. % nonsequential structurally similar pairs are aligned in a simple way, e.g. circular permutation, $$strand flip/ swap, but % are complicated. interestingly, most of such complicated non-sequential similarities can be explicable by combination of - simple non-sequential relationships. this result indicates that accumulation of simple structural changes in the course of protein evolution produces completely different fold homologs. as early as , ritter surmised that the cell's molecules cooperate to form a "special apparatus and an organised laboratory". despite supporting evidence from srere, mcconkey and others, efforts to understand molecular organisation in vivo are still in their infancy. however, important aspects of the cell interior have already been revealed. for example, weak molecular interactions structure the cytoplasm into time-evolving, functional zones. weak interactions are difficult to capture and can preclude protein detection in cells by many biophysical techniques, including nmr spectroscopy. , we explored the effects of cell-like milieus on the cytochrome c (cyt c)-flavodoxin (fld) interaction. these oppositely charged proteins interact weakly with a number of cognate partners. neither cyt c nor fld is detectable by nmr in escherichia coli confirming their "sticky" nature ( figure a) . the cyt c-fld interaction was assessed in buffer, % polyacrylamide gels and in solutions containing g/l of macromolecular crowders ( figure b) . h, n hsqc nmr revealed that the interaction was transient in buffer, proceeding via the known binding site for both proteins. substantial line broadening was effected in crowded and confined solutions suggesting that the cyt c-fld complex is stabilised under native-like conditions. the stabilising effect of macromolecular crowders was also observed by native gel electrophoresis and crystallization. these findings coincide with spitzer and poolman's model for cytoplasmic structuring, emphasising the role of charge-charge interactions and crowding in the formation of macromolecular "clusters". the implications for cytoplasmic structuring will be discussed alongside related investigations of cationic protein interactions in e. coli extracts. , detergent:protein ratio. the transmembrane b-barrel of bama is folded in either micelles, bicelles or nanodiscs, however an n-terminally attached single potra domain is flexibly unfolded, due to the absence of stabilizing contacts with other protein domains. measurements of backbone dynamics show distinct time scales of dynamic behavior for bama b-barrel and parts of its extracellular loop l , revealing high local flexibility within the the lid loop. this work presents the first high-resolution d solution nmr spectra of the bama barrel and establishes improved biochemical preparation schemes, which will serve as a platform for structural and functional studies of bama and its role within the bam complex. protein arginine methylation is a widespread and important posttranslational modification in eukaryotic cells, shown to be involved in the activation or repression of transcription, modification of the splicing machinery, signaling, and dna repair. mammalian protein arginine methyltransferases include a family of nine sequence-related enzymes that transfer one or two methyl groups onto the terminal guanidino groups on arginine residues, producing monomethylarginine only (mma, type iii), symmetric dimethylarginine (sdma) and mma (type ii), or asymmetric dimethylarginine (adma) and mma (type i). while prmt , , , , , and have been characterized as type i enzymes, and prmt as a type ii enzyme, the role and activity types of the two final members of this family of enzymes, prmt and prmt , had been unclear due to conflicting results in the literature, and the substrates for these enzymes had been elusive. both prmt and prmt are distinct members of the family with two methyltransferase or methyltransferase-like domains and containing acidic residues in otherwise well-conserved substrate double e binding motif, features not seen in the other prmt enzymes. recent work in our laboratory confirmed prmt as the only type iii mma-forming enzyme in the group, with a unusual low temperature optimum for activity, and a heretofore not seen preference for a basic stretch of residues in an r-x-r sequence for methylation. mutations of the acidic residues in the substrate-binding motif results in a loss of the specific r-x-r activity and the appearance of a g-r-g specificity typical of many of the other prmts. the physiological substrate of prmt has yet to be confirmed, although histone h b is an effective in vitro substrate. prmt , on the other hand, had no reported activity, until immunoprecipitation from hela cells showed it pulled down two splicing factors, sf b and sf b , in a complex. amino acid analysis showed that prmt methylates sf b to produce both mma and sdma, thus making it the second type ii enzyme in mammals. prmt knockdown results in modulation of alternative splicing events. this enzyme appears to be relatively specific for the sf b protein; a peptide containing the methylatable arginine residue was not found to be a substrate, and typical substrates of other prmts are not recognized by prmt . we found that the position of the methylated arginine residue in sf b is important, and the acidic residues in the substrate-binding motif also play an important role in substrate recognition. thus, prmt and prmt represent unique members of the mammalian prmt family. hydrogen peroxide levels, endogenous hormones (cytokinins, salycilic acid, as well as jasmonic acid and its conjugates), polyphenolics and terpenoids in a model system of a. alba in vitro with inhibition of rootng and stimulation of callusogenesis by means of individual and combined cytokinin and cytokinin/ auxin treatments. results: it was established that inhibition of rooting and stimulation of callusogenesis caused by benzyl adenine (ba) or combinations of ba and indole- -butiric acid (iba) in vitro were related to elevation of sesquiterpenoids in the essential oils, as well as polyphenolics content, accompanied by a drop of stress hormones, bioactive cytokinins and preservation of oxidative stress and lipid peroxidation levels, as compared with non-treated control. individual treatments with either iba or ba, also increased the sesquiterpenoid content in the essential oil of the plant, in a concentration related manner, this effect being more profound after ba treatment. in addition, ba treated plants exhibited a drop of protein levels of the aerial samples, as well as profound differences of enzymatic activity in the callus tissues, as compared with callus of plants treated with different combinations of ba and iba. conclusion: the results of the present work indicate that alterations of endogenous phytohormonal levels, caused by exogenous plant growth regulators treatment, might be the mediator between primary and secondary metabolism by means of affecting protein levels and activity of key enzymes in vitro. three different additives (( . % (v/v); formic acid, acetic acid, ammonium format with formic acid) have been investigated in response to ion intensity of esi-ms for individual hnp - in saliva. kinetex v r column separation efficiency was evaluated using two different column dimensions ( x . mm and x mm.) and two different stationary phases (c and c ). kinetex v r column (homogenous porous shell) performance was also compared to new ultra ace v r (encapsulated bonded phase) column. sample optimisation revealed that the spe method removes interference from salivary glycoproteins and consequently yields larger peak area ( - %) for all hnps. hnps were extracted by spe with a recovery of - %. the meoh: h o: acetic acid ( . %) provided enhanced (p> . ) hnp - ion intensities. the kinetex v r c ( x . mm, . mm) column facilitated a better separation efficiency of the four hnps as compared to the ultra core super c ace v r ( x . mm, mm) column, the kinetex v r c ( x . mm, . mm) and the kinetex v r c ( x . mm, mm) column. the relative levels of the hnps were determined in healthy volunteers before and after a rigorous exercise regime: it is possible that prolonged strenuous exercise will affect oral innate immunity and therefore also the level of salivary defensins. hnp - are traditionally detected in an enzyme-linked immunosorbent assay (elisa) which does not discriminate between the different hnps due to their structural similarities. there has therefore been a need to develop a mass spectrometry method that will discriminate between the defensins. as part of the method validation, the hnp - level was determined by elisa and the data was compared with the lc-ms data. here we present this cross-validation; the data revealed no significance difference between the two methods (r . ) which confirms that the developed lc-ms method is and equal sensitive method for the detection of these potential antimicrobial markers. this method can easily be adopted for similar molecular weight of peptides as hnps and also for any other biological matrix. moonlighting proteins: relevance for biotechnology and biomedicine luis franco serrano , sergio hern andez , alejandra calvo , gabriela ferragut , isaac amela , juan cedano , enrique querol institut de biotecnologia i biomedicina. universitat aut onoma de barcelona, laboratorio de inmunolog ıa, universidad de la rep ublica regional norte-salto multitasking or moonlighting is the capability of some proteins to execute two or more biochemical functions. the identification of moonlighting proteins could be useful for researchers in the functional annotation of new genomes. moreover, the interpretation of knockout experiments, in which the result of a gene knocking does not produce the expected results, might be enhanced. the action of a drug can also be facilitated because it might have an off-target or side effect with somewhat hidden phenotypic traits. it would be helpful that bioinformatics could predict this multifunctionality. in the present work, we analyse and describe several approaches that use protein sequences, structures, interactomics and current bioinformatics algorithms and programs to try to overcome this problem. among these approaches there are: a) remote homology searches using psi-blast, b) detection of functional motifs and domains, c) analysis of data obtained of protein-protein interaction databases (ppis), d) matches of the sequence of the query protein to d databases (i.e., algorithms like pisite), e) mutation correlation analysis between amino acids using algorithms like mistic. remote homology searches using psi-blast combined with data obtained from interactomics databases (ppis) have the best performance. structural information and mutation correlation analysis can help us to map the functional sites. mutation correlation analysis can only be used in very specific situations because it requires the existence of a multialigned family of protein sequences, but it can suggest how the evolutionary process of second function acquisition took place. we have designed a database of moonlighting proteins, multitaskprotdb (http:// wallace.uab.es/multitask/). from this database we determine the frequencies of canonical and moonlighting coupled functions (being an enzyme and a transcription factor the highest), the percentage of moonlighting proteins involved in human diseases ( % of the human moonlighting proteins in the database) and the percentage of moonlighting proteins acting as a pathogen virulence factor ( % of the moonlighting proteins in the database). correlation between potential human neutrophil antimicrobial peptides (hnp - ) and stress hormones in human saliva nadia ashrafi , frank pullen , birthe nielse , cris lapthorn , fernando naclario university of greenwich (faculty of engineering and sciene), university of greenwich (centre of sports science and human performance) numerous studies have investigated the effect of exercise on mucosal immunity but the focus has mainly been on salivary immunoglobulins lysozymes and hormones (cortisol, testosterone). this is not surprising given that iga and igg are the predominant immunoglobulins in saliva and there is a relationship between mucosal immunity and upper respiratory illness. it is well known that physical and mental stress provoke the release of cortisol from hypothalamic pituitary adrenal axis, by which stress can modulate various immune responses. in general, cortisol and growth hormones helps to induce the activation of neutrophils. to date, this study represents the first study that investigated the correlation between human neutrophil alpha defensins family against cortisol (stress hormone) and testosterone (growth hormone) in human saliva before and after exercise or training. twelve resistance trained athletes volunteered to participate in the study. participants consumed supplements during exercise and the hnp - , cortisol and testosterone response was investigated pre, post and minutes of the workout. the correlation between salivary antimicrobial peptide (hnp - ) and stress hormone (cortisol and testosterone) has been investigated using elisa. cortisol showed no significant (p . ) difference for (pre to min post) between cho and pl (cho: . . ng/ml; pl . . ng/ml) conditions but a strong trend (p . ) was observed for (pre to min post) post (cho: . . ng/ml; pl . . ng/ml) condition. testosterone showed no significant (p . ; p . ) difference for (pre to min post) between cho and pl (cho: . . ng/ml; pl . . ng/ml) and for (pre to min post) post (cho: . . ng/ml; pl . . ng/ml) condition. hnp - showed no significant (p . ) difference for (pre to min post) between cho and pl (cho: . . ; pl . . ) conditions but significant difference (p . ) was observed for (pre to min post) between cho and pl (cho: . . ; pl . . ) condition. the present findings suggested that there is no correlation between salivary hnp - and cortisol for (pl: r . and cho: r . ); hnp - and testosterone (pl: r . and cho: r . ). a worth note from previous study which suggested that using murine skin model (an increase in endogenous glucorticoids (cortisol) by physiological stress reduced mrna levels of antimicrobial peptide (cathelicidin). it is not clear that the correlation between hormones and antimicrobial peptide has been affected by the time interval of the exercise. both cortisol and antimicrobial peptide demonstrated a transient increase after exercise but it is surprising that they are not correlate to each other. one of the hypothesis from the present finding could be cortisol responses slow and it will be interesting to do further research with longer interval. the second hypothesis demands a further investigation to determine the synergism between substances. school of biomolecular and biomedical science, conway institute, ucd., king saud university, sciences, biochemistry department. the crystal structure of a human glucose -phosphate dehydrogenase (g pd) shows that each subunit has two nadp sites; in addition to a catalytic site there is a "structural" site which is distant from the catalytic coenzyme site. mutations causing severe deficiency tend to cluster round and close to the dimer interface and the structural nadp , indicating that the integrity of these areas is important for enzyme stability and therefore for maintenance of activity. in order to understand the molecular basis of g pd deficiency, and to have a clearer indication about the role of some features of the threedimensional structure, a fuller study of the second, "structural" nadp binding site is needed. human g pd controls the first committed step in the pentose phosphate pathway. it catalyses the oxidation of glucose -phosphate to gluconolactone -phosphate, generating nadph which is essential, amongst other things, for protection against oxidative stress. the human enzyme can be active in dimer or tetramer forms. human g pd of "structural" nadp per subunit of enzyme. this tightly-bound nadp can be reduced by g p, probably following migration to the catalytic site. the importance of nadp for stability is explained by the structural nadp site, which is not conserved in prokaryotes. after removing the tightly bound "structural" nadp the enzyme is still active but not stable. the effects of different nadp fragments on the stability of human recombinant g pd have been investigated. nadp is crucial for the long term stability of human g pd, and only one of nadp analogues which is adenosine diphosphate ribose - '-phosphate was able to slightly promote the stability of enzyme. . molecular characterization of specific positively selected sites in mammalian visual pigment evolution miguel a. fern andez-sampedro , eva ramon , brandon m. invergo , jaume bertranpetit , pere garriga grup de biotecnologia molecular i industrial., visual rhodopsin is a member of the g-protein coupled receptors superfamily. this membrane protein consists of a -cis-retinal cromophore bound to a seven transmembrane protein, opsin, by means of a protonated schiff base linkage. it has an important role as a dim light photoreceptor in the retina of the eye. by statistical models, where episodic selection in rhodopsin is tested on one branch of the phylogeny against a background of neutral or purifying selection on the rest of the tree, we have found some significant evidence of specific positively selected sites in early mammalian divergence. we have chosen the three amino acid sites identified with the highest posterior probability of having been targets of positive selection to perform experimental studies, i.e. (positively selected from m to f), (positively selected from r to q) and (positively selected from s to a). we have constructed, expressed, immunopurified and functionally characterized the proposed candidates, f m, q r and a s rhodopsin mutants located at the n-terminus, the transmembrane domain and the c-terminus region of the protein respectively. from the analysis of the molecular features of the f m mutant, we conclude that position is very important for protein folding and also for proper protein glycosylation, since we only could observe cromophore regeneration after its rescue in the double cysteine (n c/ d c) mutant background that stabilizes the n-terminal extracellular domain of the protein. our results also show that mutants q r and a s alter the g-protein activation rate, and hydroxylamine susceptibility in the dark-adapted state. in the case of q r, disrupting critical interactions with the neighbouring y of the conserved d/ery motif, critical in gt activation, could cause the lower gt activation ability. the mutant a s would create a potential additional phosphorylation site in the protein which could affect rhodopsin phosphorylation after photoactivation and, in turn, could affect the binding affinity of arrestin, a regulator of rhodopsin deactivation. this extra phosphorylation site could provide an evolutionary explanation for the enhanced response observed in the case of gt activation. in conclusion, these results highlight the importance of molecular investigations of positive selected sites in rhodopsin evolution and the relevance of structural and functional analysis of these sites in unravelling the molecular basis of visual pigment evolution. natural evolution sheds light on modern drug resistance in protein kinases marc hoemberger , christopher wilson , roman agafonov , dorothee kern the anti-cancer drug imatinib exhibits highly specific binding to the human kinase and oncogene abl with a three thousand fold weaker affinity for the structurally and functionally very similar kinase src. it has been shown recently that the major difference in binding of imatinib to abl and src stems from an induced fit after binding of the drug. to further understand the mechanism of imatinib binding to its target we used ancestral sequence reconstruction (asr) and resurrected enzymes along the node from the common ancestor of abl and src up to the extant kinases. we show that imatinib affinity is gained towards the evolution of extant abl while it is lost towards evolving src. the combination of asr and crystallographic data of the ancestors in addition to kinetics data allowed us to identify a subset of residues involved in imatinib specificity sufficient to switch from an intermediate binder to a tight binder. preliminary data shows that a network of hydrogen bonds and packing interactions stabilize the kinked p-loop conformation for tight binders thus allowing for more interactions between the kinase and the drug. strikingly, many of these residues were identified in human cancer patients as "hot spots" for the development of resistance mutations. further investigation into the identified subset of residues in combination with these commonly found imatinib resistance mutations will allow us to understand emerging drug resistances better. an evolutionary view of the cold adapted catalysis of enzymes vy nguyen , christopher wilson , dorothee kern the diversity in protein function that we see today arose as a result of life adapting to a cooling earth. how did enzymes, the catalysts of many crucial cellular processes, achieve this cold adaptation? this is a challenging question to answer because ancient sequences of proteins that existed billions of years ago are not available. to address this question we used ancestral sequence reconstruction to create adenylate kinase (adk) enzymes from the divergence of anaerobic and aerobic firmicutes towards modern day thermophilic, mesophilic and psychrophilic organisms. adk is a phosphotransferase that catalyzes the conversion of two adp molecules into atp and amp. we make the following observations. first, all ancestral enzymes are active with optimal catalytic rates linearly corresponding to the temperature of the environments where these proteins would have been found. most strikingly, the catalytic rate of our oldest adk ancestor exhibits a higher enthalpy of activation at low temperatures as compared to the modern thermophilic adk. this suggests a large enthalpic penalty had to be paid for reactions to occur at cold temperatures in an ancestor that existed in a hot environment. second, several high resolution crystal structures of extant proteins that we solved ( . Å - . Å), show that the oldest ancestors were more rigid than the modern adks due to an intricate salt-bridge network. this work, thus shows for the first time, the molecular and thermodynamic determinants of cold adaptation in an enzyme over a time period that spans billions of years. induced oxidative modification of plasma and cellular fibrin-stabilizing factor anna bychkova , tatiana danilova , alexander shchegolikhin , vera leonova , marina biryukova , elizaveta kostanova , alexey kononikhin , anna bugrova , evgeny nikolaev , mark rosenfeld n. m. emanuel institute of biochemical physics, russian academy of sciences, institute for energy problems of chemical physics, russian academy of sciences the main function of plasma fibrin-stabilizing factor pfxiii is to catalyze the formation of the intermolecular covalent cross-links between both gand afibrin polypeptide chains. the crosslinking crucially affects mechanical strength of fibrin and its resistance against fibrinolysis. the precise role of cellular fibrin-stabilizing factor cfxiii remains poorly understood. pfxiii is a heterotetramer (fxiii-a b ) consisting of two single-stranded catalytic a subunits (fxiii-a ), and two identical single-stranded inhibitory/ carrier b subunits (fxiii-b ). the subunits are held together by weak non-covalent bonds. contrary to plasma fxiii, cfxiii is a dimer (fxiii-a ) devoid of b subunits. as well as many other proteins circulating in the bloodstream, pfxiii is known to be a target for reactive oxygen species (ros) causing processes of protein oxidative modification. since the conversion of pfxiii to the active form of the enzyme (fxiiia) is a multistage process, ozone-induced oxidation of pfxiii has been investigated at different stages of its enzyme activation. the biochemical results point to an inhibition of enzymatic fxiiia activity depending largely on the stage of the pfxiii conversion into fxiiia at which oxidation was carried out. uv-, ftir-and raman spectroscopy demonstrated that chemical transformation of cyclic, nh, sh and s-s groups mainly determines the oxidation of amino acid residues of pfxiii polypeptide chains. conversion of pfxiii to fxiiia proved to increase protein susceptibility to oxidation in the order: pfxiii < pf-xiii activated by thrombin < pfxiii in the presence of calcium ions < fxiiia. with the aid of massspectrometry it has been demonstrated that oxidation leads to decreasing fxiii-a and fxiii-b coverage both in the forms of zymogen and in the presence of calcium ions. a group of amino acid residues involved in oxidation modification of pfxiii is identified in this study. the oxidation of either cfxiii or cfxiiia has revealed an almost complete loss of enzyme activity caused by dramatic changes in the primary and secondary structure of the proteins detected by the ftir data. taking into account these new findings, it seems reasonable to assume that the inhibitory/carrier fxiii-b subunits can serve as scavengers of ros. hypothetically, this mechanism could help to protect the key amino acid residues of the fxiii-a subunits responsible for the enzymatic function of fxiiia. the study was supported by rfbr, research project no. - - - a. mass spectrometry study was supported by the russian scientific foundation grant no. - - . performance and quality. making microcalorimetry simple with microcal peaq-itc natalia markova , ronan o'brien , mark arsenault microcal, malvern instruments ltd. dynamic interactions involving biomolecules drive and regulate all biological processes. studies of biomolecular interactions are fundamentally important in all areas of life sciences. data provided by isothermal titration calorimetry (itc) enables scientists in academia and industry to directly and quantitatively characterize these interactions in solution. microcal peaq-itc, the latest generation of microcal itc instrumentation, offers a whole range of solutions for addressing current bottlenecks associated with interaction analysis. among the most recognized challenges are the needs to adequately address a broad range of binding affinities and to reliably interpret binding data complicated by the presence of inactive protein fraction or inherent uncertainty in the concentration of a ligand. consistently high performance of microcal peaq-itc enables increased confidence and data resolution when measuring low heats at low or uncertain sample concentrations and complex binding modes. the new microcal peaq-itc analysis software allows for utomated data analysis, minimizing analysis time and user subjectivity in assessing data quality. data quality is determined and advanced fitting performed in a few seconds per experiment allowing for analysis of large data sets of or more experiments in a matter of seconds. glutamine-rich activation domain of transcription factor sp -biochemical activity and structure jun kuwahara , chisana uwatoko , emi hibino , katsumi matsuzaki , masaru hoshino faculty of pharmaceutical sciences, doshisha women's university, graduate school of pharmaceutical sciences, kyoto university transcription factor sp is ubiquitously expressed in a mammalian cell, activates reasonably large subset of mammalian genes, and is involved in the early development of an organism. the protein comprises two glutamine-rich (q-rich) regions (a and b domains) located in its n-terminal half, while three tandem repeats of c h zinc finger motif at its c-terminus binds directly to a gc-rich element (gc box) of dna. in general, q-rich domain is one of the typical motifs found in trans-activation domain of transcription factors together with acidic and proline-rich domains. transcriptional signal of sp are transmitted via interaction between q-rich domains of sp and different classes of nuclear proteins, such as tata-binding protein (tbp) associated factors (tafs) in components of basic transcription factor complexes (tfii). in addition, self-association of sp via q-rich domains is also important for its regulation of transcriptional activity. it has been considered that an sp ! molecule bound to a 'distal' gc-box synergistically interacts with another sp molecule at a 'proximal' binding site. although formation of multimers via q-rich domains seems functionally important for sp , little is known about relevance between biological activity and structural nature of q-rich domains. we analyzed nature of glutaminerich domains of sp by biochemical and physicochemical methods. we found that q-rich domains do not have clear secondary structure whereas they can indicate biochemical activity. detailed analysis of nmr spectra indicated interaction between the domains. the q-rich domains of sp might be one of the intrinsically disordered proteins (idp). chipping away at the yeast proteome: redesigning an e ubiquitin ligase for targeted protein degradation michael hinrichsen , lynne regan one of the central goals of synthetic biology is to exploit biological systems in order to produce compounds of therapeutic or industrial value . often, these efforts are complicated by the many natural biochemical pathways in cells that can compete for the same small molecule precursors. currently, the most common solution is to simply delete the genes coding for the competing enzymes . while such an approach has been successful, it is only applicable to nonessential genes and can produce unintended off-target effects such as decreased cell viability . an alternative strategy is to instead target proteins directly for degradation. using this strategy, scientists would first grow cultures of engineered cells to high densities under permissive conditions (i.e. targeted proteins are stably expressed). then, once sufficient cell density has been reached, enzymes of competing pathways would be rapidly degraded, resulting in the rapid production of high concentrations of the compound of interest. we propose to create such a tool by reengineering the c-terminus of hsp interacting protein (chip), an e ubiquitin ligase. chip recognizes substrate proteins through a short c-terminal peptide tag on target proteins . we have shown that fusing this tag to non-native substrates is sufficient for ubiquitination in vitro (data not published). cellular assays have also been performed in s. cerevisiae, a model organism commonly used in metabolic engineering applications . as a number of native yeast proteins possess c-termini similar to that of chip's native substrates (data not published), it was necessary to develop an orthogonal chip-peptide pair. this was achieved by replacing chip's natural tpr ligand-binding domain with a ligand-binding domain engineered previously in the regan lab . the altered chip construct has been shown to be active both in vitro and in vivo, and produces an altered growth phenotype when targeted against an enzyme involved in uracil biosynthesis. future work will focus on further kinetic characterization of the engineered enzyme, increasing its activity, and introducing the system into a proof of concept synthetic biology application. advances in modern sequencing techniques have resulted in an explosion of genomic data. correctly classifying this new wealth of information can be daunting not only because of the sheer volume of sequence data, but also because the propagation of erroneous and less-than-ideal names and functional characterizations in the current databases gets in the way of functional classification by mere sequence similarity. we are investigating the extent to which protein domain architecture can be utilized to define groups of proteins with similarities in molecular function, and whether we can derive corresponding functional "labels", starting with some of the most common domain architectures found in bacteria. to this end, we have developed an in-house procedure called sparcle ('specific architecture labeling engine') that lets us track and examine specific or sub-family domain architectures, resulting from annotating protein sequences with domain footprints provided by the conserved domain database (cdd), which includes hierarchical classifications for many common domain families. we will discuss how the proteins are grouped into specific architectures, our successes in assigning functional labels, and the major limitations we have encountered to date. while we will be able to assign functional labels to a large fraction of protein models derived from genome sequences, this effort has the added benefit of pointing out insufficient coverage and resolution of the current protein domain model collections that constitute cdd. we will also discuss alternative procedures that utilize pre-computed domain annotation for clustering protein sequences at a level that is well suited for functional labeling. we hope that this preliminary study will help to identify approaches that facilitate rapid and accurate annotation of genomes with a minimum of manual intervention. pegylated amyloid peptide nanocontainer delivery and release system self-assembly of telechelic peg end-capped with hydrophobic dipeptides collagen stimulating effect of peptide amphiphile c -kttks on human fibroblasts self-assembly of palmitoyl lipopeptides used in skin care products bioactive films produced from selfassembling peptide amphiphiles as versatile substrates for tuning cell adhesion and tissue architecture in serum-free conditions influence of elastase on alanine-rich peptide hydrogels interaction between a cationic surfactant-like peptide and lipid vesicles and its relationship to antimicrobial activity self-assembled arginine-coated peptide nanosheets in water toll-like receptor agonist lipopeptides self-assemble into distinct nanostructures approved drugs containing thiols as inhibitors of metallo-ß-lactamases: a strategy to combat multidrug-resistant bacteria references leukotriene a hydrolase -an envolving target. inflammatory diseases -immunopathology, clinical and pharmacological bases the bifunctional enzyme leukotriene-a, hydrolase is an arginine aminopeptidase of high efficiency and specificity lipoxygenase and leukotriene pathways: biochemistry, biology, and roles in disease a critical role for lta h in limiting chronic pulmonary this work was supported by the czech science foundation (project p / / ) and czech academy of sciences the tn antigen-structural simplicity and biological complexity bel b-trefoil: a novel lectin with antineoplastic properties in king bolete (boletus edulis) mushrooms acknowledgements: cynthia leyva-arg€ uelles is supported by a personal grant from conacyt, mexico. this work is supported by conacyt grant ' : and papiit grant in sequence information-based deciphering of biofunctionalities using ism-based techniques has fetched calculation of biological functionalities, designing of biomedical device called computer-aided drug resistance calculator, the understanding of the mechanism of hiv progression to aids [ ], and others. they have compared the efficacies of drugs and vaccines, which formed the basis for the innocentive award (id ) for assessing vaccine potency. conclusions: deciphering biological features without engaging reagents, equipments and animal tissues but biological data such as sequence information is one novel, feasible genotypic hiv-coreceptor tropism prediction with geno pheno [coreceptor]: differences depending on hiv- subtype a reliable phenotype predictor for human immunodeficiency virus type subtype c based on envelope v sequences available: http:// istree.bioprotection.org signal processing-based bioinformatics methods for characterization and identification of bio-functionalities of proteins an empirical framework for binary interactome mapping estimating the size of the human interactome coming to peace with protein complexes? th capri evaluation meeting pj- cabs-dock web server for protein-peptide docking with significant conformational changes and without prior knowledge of the binding site while other docking algorithms require pre-defined localization of the binding site, cabs-dock doesn't require such knowledge. given a protein receptor structure and a peptide sequence (and starting from random conformations and positions of the peptide), cabs-dock performs simulation search for the binding site allowing for full flexibility of the peptide and small fluctuations of the receptor backbone cabs-flex: server for fast simulation of protein structure fluctuations cabs-fold: server for the de novo and consensus-based prediction of protein structure cabs-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site mechanism of folding and binding of an intrinsically disordered protein as revealed by ab initio simulations modeling of protein-peptide interactions using the cabs-dock web server for binding site search and flexible docking cabs-fold: server for the de novo and consensus-based prediction of protein structure cabs-flex: server for fast simulation of protein structure fluctuations aggrescan d (a d): server for prediction of aggregation properties of protein structures cabs-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site staphylococcal pathogenicity island dna packaging system involving cos-site packaging and phage-encoded hnh endonucleases the etiology of asdis unknown, but it is believed that it involves genetic and environmental components. the purpose of this work is to assess the possible involvement of food contaminants, such as mycotoxins, in the etiology of asd. the hypothesis is that the mycotoxins ingested with the diet could bind to proteins and expose the entire organism,including cns, to the negative effects of xenobiotics, in genetically predisposed patients. in this study some possible protein targets for the mycotoxinswere identified to evaluate if the bond between any protein target and the mycotoxin in exam could play a role in asd. twelve mycotoxins were selected (ochratoxin a, gliotoxin, aflatoxin b , aflatoxin b , aflatoxin m , aflatoxin m , aflatoxicol, a-zearalanol, b-zeralanol, zearalenone, deoxynivalenol, patulin),which are contaminants of milk and cereals.for each of these molecules,possible protein targets were searched by a reverse docking approach using the idtargetserver[ ].from the results given by idtarget, human protein targets expressed in the brain or involved in brain diseaseswere selected. subsequently, a direct docking was made using auto-dock . [ ], in orderto verify the strength of the interaction between selected proteins and each mycotoxin, and to identify the mycotoxins' binding site on each of the selected protein. finally, the bond of some mycotoxins to selected protein targets has been experimentally tested. for each mycotoxin, idtarget returned thousands of possible protein targets,and only those with the best binding energy were selected and evaluated. among them, human protein targets that are expressed in the brain or that are involved in cerebral diseases,have been selected; moreover the protein targets that were not human but that idtargetselected for five or more mycotoxins, were replaced with their human counterparts. at the end of the procedure, nineteen protein targets have been identified for the following direct docking approach. from the docking results, eight proteins have been selected for experimental tests, having a predicted binding energy lower than kcal/mol. finally, the interactions between acetylcholinesterase (ache), b-secretase (bace ) and neuroligin- , x-linked (nlg x) with aflatoxin b , aflatoxin b , gliotoxin, ochratoxin a and deoxynivalenol, were evaluatedusing fluorescence spectroscopy and microscale thermophoresis. these experiments confirmed the presence of an interaction between bace and aflatoxin b idtarget: a web server for identifying protein targets of small chemical molecules with robust scoring functions and a divide-and-conquer docking approach the calculation of spatial structure and "assembling" of the whole protein from the obtained peptide structures were performed by using molecular dynamics of the protein in the fully hydrated -palmitoyl- -oleoyl-sn-glycero- -phosphatidylcholine (popc) [ ]. the obtained structural model may contribute to identification of ul . active sites and elucidation of its mode of action nmr structural studies of membrane proteins acknowledgments: polish national centre for research and development -grant number pl- functional and mechanistic studies of dysferlin, an essential protein in cell membrane repair references moreover the 'm' parameter, which represents the denaturant effect on the protein stability, is cal•mol- for bgla and cal•mol- for bglb albert einstein college of medicine protein structure modeling, protein-protein interaction computational modeling of ini /smarcb and novel insights into its interaction with hiv- integrase savita bhutoria epsteain bar virus, nuclear antigen) . ini /smarcb has no known structural homologues, and its amino-acid sequence yields little insight into its function. a detailed understanding of structure-function relationships is hampered by the lack of structural information for ini . computational methods that model protein/peptide structures with sufficient accuracy to facilitate functional studies have had notable successes. we carried out combination of sequence analysis ab initio structure modeling and dynamics studies of integrase binding domain of ini and found it to be similar to that of phospholipase a activating protein, plaa. structural similarity with this distant protein suggests divergent evolution of the two proteins. the modeled structure sheds light on various protein-protein interactions of ini . by integrating the experimental studies about the binding, we have shown through docking, how a fragment of ini binds to the hiv- in. molecular docking and experimental studies indicated that two proteins bind tightly through charged/polar residues surrounding a hydrophobic cleft. these studies provide first modeled structure of ini /smarcb or any component of the swi/snf complex, and provide structural basis for in-ini interactions. this molecular interpretation of the intermolecular interactions is expected to facilitate design of inhibitors as novel class of anti-hiv- therapeutic agents ): e . with their catalytic activity towards rna substrates, other biological properties have been reported and evolution studies suggest an ancestral host-defence function in vertebrates. indeed, genetic studies confirmed a rapid molecular evolution within the family, a distinctive trait for host defence proteins exposed to a changing pathogen environment. previous studies from our laboratory characterized the wide spectra antimicrobial activity of two highly cationic human rnases: the eosinophil rnase and the skin derived rnase ribonucleases and have antimicrobial function in the human and murine urinary tract structural determinants of the eosinophil cationic protein antimicrobial activity two human host defense ribonucleases against mycobacteria, the eosinophil cationic protein (rnase ) and rnase the regulatory mechanism that we are reporting will contribute to prevent both nad exhaustion and accumulation of the toxic bal. to the best of our knowledge, this is the first report of a novel reversible covalent modification of an aldh enzyme involving its own substrate anna lewandrowska , aldona jeli nska , agnieszka wi sniewska acknowledgement: this research was supported by the inactivation of the fxr gene reduces aqp expression and impairs urine concentrating ability, which leads to a polyuria or urine dilution phenotype. we have previously found that pon -/-mice exhibit a polyuria phenotype and produce twice as much -h urine as their wild type pon / littermates (borowczyk k et al. metabolism and neurotoxicity of homocysteine thiolactone in mice: evidence for a protective role of paraoxonase development and application of novel non-ewald methods for calculating electrostatic interactions in molecular simulations ikuo fukuda , narutoshi kamiya the most time-consuming part of molecular simulation is the calculation of long-range interactions of the particles. in particular, appropriate treatment of the electrostatic interaction is critical, since the simple truncation cannot be used due to the slow decay of the coulombic function. thus, it is highly demanded to calculate the electrostatic interactions with high accuracy and low computational cost. for this purpose we have developed the zero-multipole (zm) summation method [ ]. in this method the artificial periodic boundary conditions are not necessary and the fourier part evaluations are not needed, in contrast to the conventional ewald-based methods. instead, a pairwise function that is suitably redefined from the coulombic function is used with a cutoff scheme. the underling physical idea is simple: (a) in a biological system, a particle conformation for which the electrostatic interactions are well cancelled is more stable than other conformations [ ]; (b) since such well-cancelled conformations are essentially physical, we should clip a subset of such a conformation out of the conformation within an ad-hoc given cutoff sphere and calculate the interactions only from this subset. this idea is realized by a rigid mathematical consideration that leads to the deformation of the coulombic function. the efficiency of the zm method has been validated in applications to fundamental systems sema a) is a protein originally described as an axonal chemorepellent cue involved in many physiological processes ranging from embryonic development to bone homeostasis or immune responses sema a signal transduction requires the formation of a heteromeric complex with neuropilin- (nrp ) and plexina [ ]. in addition, sema a interaction with nrp is modulated by the furin protease cleavage at its c-terminal basic domain this c-terminal basic domain has also been suggested to mediate the binding to glycosaminoglycans (gags), an association that locates sema a to perineuronal nets and enhances its function in restricting neuronal plasticity and inhibiting axonal regeneration in the central nervous system two peptides corresponding to the highly positively charged regions on the domain were shown to bind to immobilized heparin by surface plasmon resonance (spr) and the affinity dramatically increased when the complete domain was assayed. the binding was confirmed by nuclear magnetic resonance (nmr) and circular dichroism (cd) the conserved cysteine within this motif, necessary for the dimerization of sema a [ ], is also critical for the helix formation. in addition, fluorescence spectroscopy studies showed that the n-terminal region also has a contribution in the binding to gags. we acknowledge the financial support from the european union seventh framework programme (fp / - ) under the project vision semaphorin a: a new player in bone remodeling neuropilins lock secreted semaphorins onto plexins in a ternary signaling complex furin processing of semaphorin f determines its anti-angiogenic activity by regulating direct binding and competition for neuropilin semaphorin a displays a punctate distribution on the surface of neuronal cells and interacts with proteoglycans in the extracellular matrix semaphorin a binds to the perineuronal nets via chondroitin sulfate type e motifs in rodent brains mechanistic basis for the potent anti-angiogenic activity of semaphorin f. biochemistry collapsin- covalently dimerizes, and dimerization is necessary for collapsing activity prior attempts to create functionally relevant groupings of proteins in the crotonase superfamily suggest that this superfamily is difficult to cluster functionally due in part to the functionally diverse nature of the protein superfamily. we have developed two novel procedures to combat this difficulty: tulip (two-level iterative clustering process), a process that utilizes structural information from active sites to cluster protein structures into hypothesized functional groupings, and misst (multi-level iterative sequence searching technique), a process that uses the protein groupings created in tulip as a starting point for iterative genbank searches and further clustering after each search. through these two methods, the total coverage of the crotonase superfamily has increased, and the generated groups contain proteins from subgroups and families that did not have a structural representative. novel hypothesized functional protein groupings have been created, most notably for a large number of proteins that lack annotation data at the subgroup or family level, and for proteins of the enoyl-coa hydratase family fernandes , teresa sorbo , ivan duka , lia christina appold , marianne ilbert , fabian kiessling cnrs, umr , ucibio-requimte, faculdade de ciências e tecnologia e-selectin is a cell-adhesion molecule induced on the surface of endothelial cells in response to cytokines. its upregulation has been reported in many disorders, including inflammatory and cardiovascular diseases, tumor angiogenesis and metastasis [ ]. this profile suggests e-selectin as a promising target to develop molecular imaging probes for the detection of these diseases cyt c with equivalent of fld in media mimicking the cytoplasm. these include % polyacrylamide gel, g/l bovine serum albumin or polyvinylpyrrolidone , and buffer alone for comparison. electrostatic surface representations of the proteins are shown with their in-cell spectrum pl- a search for anti-melioidosis drug candidates targeted to d-glycero-d-manno-heptose- , -bisphosphate phosphatase from burkholderia pseudomallei bpgmhb converts dglycero-d-manno-heptose- b, -bisphosphate to d-glycero-d-manno-heptose- b-phosphate. this is the third step of the biosynthesis pathway of ndp-heptose responsible for a pleiotropic phenotype. therefore, this biosynthesis pathway is the target for inhibitors increasing the membrane permeability of gram-negative pathogens or adjuvants synergistically working with known antibiotics. to find inhibitors of bpgmhb, we performed homology modeling of bpgmhb and in-silico virtual screening with zinc, a free database of commerciallyavailable compounds. tens of thousands of chemical compounds were docked into the active site of bpgmhb. a number of putative bpgmhb binding compounds better than d-glycero-d-manno-heptose- b, -bisphosphate were found using surflex-dock included in the sybyl software package crystal structure of dimeric d-glycero-d-manno-heptose- , -bisphosphate phosphatase from burkholderia thailandensis ewha womans university we have solved the crystal structures of d-glycero-d-manno-heptose- , -bisphosphate phosphatase from burkholderia thailandensis (btgmhb) catalyzing the removal of the phosphate at the position of d-glycero-d-manno-heptose- , -bisphosphate. it belongs to the haloacid dehalogenase (had) superfamily with an a/b rossman fold composed of six parallel b-strands sandwiched between two sets of three a-helices it reveals a conventional rossman-like a-b-a sandwich fold with a novel b-sheet topology. its c-terminus is longer than its closest relatives and forms an additional b-strand whereas the shorter c-terminus is random coils in the relatives. interestingly, its core structure is similar to that of enzyme iib(cellobiose) from e. coli (eciib(cel)) transferring a phosphate moiety. in the active site of the closest eceiib(fruc) homologues, a unique motif cxxgxaht comprising a p-loop like architecture including a histidine residue is found. the conserved cysteine on this loop may be thiolated to act as a nucleophile similar to that of eciib(cel). the conserved histidine residue is presumed to accommodate negatively charged phosphate during enzymatic catalysis leonor morgado , kornelius zeth , , , bj€ orn m. burmann , timm maier bama is a b-barrel membrane protein with five periplasmic n-terminal polypeptide transport associated (potra) domains. the bama structure has been determined recently by x-ray crystallography ( , ), however its functional mechanism is not well understood. this mechanism comprises the insertion of substrates from a dynamic, chaperone-bound state into the bacterial outer membrane, and nmr spectroscopy is thus a method of choice for its elucidation we demonstrated that knocked down autophagy by shrna (shatg , shbecn , and shatg ) and chloroquine (cq) could enhance high dose of uvb induced cell death in odc overexpressing hela and mcf- cells. here, we also observed that knocked down odc in odc overexpressing hela and mcf- cells inhibited autophagy and enhanced high dose of uvb radiation. because of atg can regulate cell apoptosis and utophagy. site directed mutagenesis was used to mutant the amino acid which can regulate cell apoptosis and autophagy on atg , respectively in these two odc overexpressing cells. according to the results fish ß-parvalbumin acquires allergenic properties by amyloid assembly using atlantic cod b-parvalbumin (rgad m ) displaying high ige crossreactivity, we have found that formation of amyloid fibers under simulated gastrointestinal conditions accounts for the resistance to acid and neutral proteases, for the presence of membrane active species at gastrointestinal relevant conditions and for the ige-recognition in allergic patient sera. incorporation of the anti-amyloid compound epigallocathequin gallate prevents rgad m fibrillation, facilitates its protease digestion and impairs its recognition by ige. conclusions: rgad m amyloid formation explains its degradation resistance, its facilitated passage across the intestinal epithelial barrier and the epitope architecture as allergen autophagy could degrade the citrullinated and unfolding protein. herein, padi could enhance autophagy in jurkat t cells and lead to a degradation of p and the accumulation of lc -ii. autophagy and apoptosis are two critical mechanisms which participate against cellular stress, cell activation, survival and homeostasis. pad -overexpressed jurkat t cells caused the activation of th cells to increase mrna expression of cytokines, such as il- , il- , il- and tnfa. cytokines provoked caspase expression and led to caspase-mediated cleavage of beclin- which was an important factor of apoptotic signaling. knockdown of bcen rescued cell survival due to the increase of bcl-xl and the decrease of caspase- . we suggested that padi participated in the activated t cell-induced autonomous death through triggering er stress pathway studies on secondary metabolites production and proteins and enzymes of in vitro cultivated artemisia alba turra and relations with some endogenous phytohormones yuliana raynova , krassimira idakieva , vaclav motyka , petre dobrev , yuliana markovska , milka todorova , antoaneta trendafilova , ljuba evstatieva switzerland aim: artemisia alba turra is an essential oil bearing shrub, characterized with great variability of the essential oil profile of wild grown plants, related to genetic, geographic and environmental factors. it was previously established that inhibition of rooting in vitro caused by cytokinin/auxin treatment affected the essential oil profile of the plant and these changes were also related to bioactive endogenous cytokinin levels in vitro ( , ) cytokinin and auxin effect on the terpenoid profile of the essential oil and morphological characteristics of shoot cultures of artemisia alba terpenoid profile of artemisia alba is related to endogenous cytokinins in vitro salivary hnp - are conventionally measured using an enzyme-linked immunosorbent assay (elisa) which does not discriminate between individual hnps due to their structural similarities. considering the biological importance of salivary human neutrophil a-defensin (hnps), there is therefore, a need to develop an analytical method that will discriminate between the defensins. an lc-ms method has been established for the separation and detection of hnp - . the method has been optimised, validated and applied to examine the relative level of hnp - in participants undertaking a circuit resistance training workout. to date, no studies have systematically investigated the effect of acute (min to hours) and chronic (days to weeks) change in salivary adefensins family before and after exercise by lc-esi-ms systems and models calorimetry showed no difference in dissociation constants at these ph values, while the binding stoichiometry is increased . fold. furthermore, the binding stoichiometry varied fold among the two alginates corresponding to their difference in average molecular weight and in addition fold higher binding affinity was found with the high as compared to the low molecular weight alginate. in conclusion, the binding stoichiometry of b-lactoglobulin with alginate increases by a factor that correlates to the average molecular weight of the alginate and also a much higher affinity was found for the high molecular weight alginate. acknowledgements: this work is supported by the danish council for the presence of mucins and other high molecular weight glycoproteins in saliva makes the direct analysis of defensins difficult. the lc-ms method was linear for concentrations of hnp- between . and ng/ml (r . ) with a lod of . ng/ml. inter and intra assay precision was . - %, respectively. saliva sample were clean up by solid phase extraction (spe) and without-solid phase extraction (wspe) . mm internal diameter column in relation to the method transfer . during lc-ms optimisation genome-wide docking database (gwidd) provides the most extensive data repository of structures and models of ppi on a genomic scale. currently, we are expanding the gwidd dataset to , ppi in , organisms, up from , ppi in organisms in the previous release. the ppi data were imported from intact and biogrid databases and were subjected to in-house modeling pipeline. gwidd current implementation contains , experimentally determined complexes, and , sequence homology and , structure homology models of complexes. the user-friendly interface offers flexible organism-specific search with advanced functions for a refined search for one or both proteins. the new gwidd version includes also a new interactive visualization screen that allows to view search results in different residue representations with the emphasis on the ppi interface refolding and activation of recombinant trypsin i from sardine fish (sardinops sagax caerulea) amyloid is detectable in human dental plaque and is produced by both clinical and laboratory strains of s. mutans, further supporting a functional role. s. mutans lacking p demonstrates residual amyloid forming properties, however, a mutant lacking sortase, the transpeptidase which covalently links p and several other proteins to the peptidoglycan cell wall, is defective in cell-associated amyloid-like properties. the objectives of this study were to identify additional amyloid forming proteins of s. mutans and to evaluate the effects of buffering conditions and ph on the ability of the identified proteins to form amyloids. a p -deficient mutant strain was grown to stationary-phase in defined minimal media, and secreted proteins from spent culture supernatants were fractionated by ion exchange chromatography. partially purified protein fractions were tested for binding of the amyloidophilic dyes congo red (cr) and thioflavin t (tht), and for characteristic birefringent properties following staining with cr and visualization under crossed polarizing filters. proteins from fractions that tested positive for amyloid-like material were separated by sds page, and identified by lc/ms. these included wapa, gbpa, gbpb, smu_ c and smu_ c. recombinant proteins were expressed in escherichia coli, and purified for confirmation and characterization of individual amyloidogenic properties in vitro. recombinant wapa and smu_ c displayed all the biophysical characteristics of amyloid, including visualization of fibrillar aggregates when viewed by transmission electron microscopy. in contrast, gbpa and smu_ c produced amorphous aggregates. wapa and smu_ c form amyloid at different ph, smu_ c under acidic conditions and wapa under neutral to basic conditions. this suggests that the prevailing environmental ph may represent different in vivo triggers for amyloid fibrillization of different s like other small gtpases, the activity of rheb is dictated by its guanine nucleotide binding states: it is active in its guanosine -triphosphate (gtp) bound form and inactive in the guanosine diphosphate (gdp)-bound form. rheb proteins play critical roles in regulating growth and cell cycle, and this effect is due to its role in regulating the insulin/tor/s k signaling pathway rheb interacts directly with fkbp and prevents its association with mtor in a gtp-dependent manner. moreover, fkbp bound to gtp-g-s, a nonhydrolyzable gtp analogon, has a much higher binding affinity for rheb than the gdp-bound form the second study contradicted both studies, since they could not detect any interaction between rheb and fkbp [ ]. to clarify whether there is an interaction and if it is nucleotide dependent, nmr monitored interaction studies were performed employing a c-terminal truncated construct of human rheb ( - rhebdct) that cannot be farnesylated and the biochemically defined binding region on fkpb (fkbp -like fkbp -bd). based on our data rhebdct -gdp does not significantly interact with fkbp -bp. n-fkbp -bd titrated , we observed a weak interaction between rhebdct bound to a gtpanalogon (gppnhp) and fkbp -bd. mapping of the observed spectral changes on the structure of rheb-gtp suggests that fkbp targets the switch region, loop - and the neighboring b-sheet region. we further analyzed the backbone dynamics of rhebdct -gdp and -gppnhp using n relaxtion data (t , t and heteronuclear noe) .based on these data the phosphorylation loop, the switch regions and the loop around residues - show increased backbone dynamics that modulated by the nucleotide binding international centre for genetic engineering and biotechnology tdp- is an rna processing protein that can form inclusions of debatable nature implicated in neurodegenerative diseases. within the putative aggregation domain, repeats of residues - can recruit endogenous tdp- into aggregates inside cells . recently, we showed that a coil to b-hairpin transition in a short peptide corresponding to tdp- residues - enables oligomerization . we have used a broad battery of biophysical experiments, including chromophore and antibody binding, electron microscopy (em), circular dichroism (cd), solution and solid-state nmr, and x-ray to shed light on the nature of these aggregates. based on these findings, structural models for tdp- ( - ) oligomers have been constructed, refined, verified, and analyzed using computational methods, ranging from docking and molecular dynamics simulations to semiempirical quantum mechanics calculations. interestingly, tdp- ( - ) b-hairpins assemble into a novel parallel b-turn configuration showing crossb spine, cooperative h-bonding and tight side chain packing cellular model of tar dna-binding protein (tdp- ) aggregation based on its c-terminal gln/asn-rich region structural characterization of the minimal segment of tdp- competent for aggregation structural evidence of amyloid fibril formation in the putative aggregation domain of tdp- kyungpook national university scolopendin , aglqfpvgrigrllrk, is a -mer peptide derived from the centipede scolopendra subspinipes mutilans. to investigate its property against fungal and bacterial pathogens, antimicrobial tests were performed. we observed that this peptide exhibited antimicrobial activity in a salt-dependent manner and showed no hemolysis. the circular dichroism (cd) analysis observed that a-helical structure properties. we determined the mechanism(s) of action using flow cytometry and investigated the release of potassium. the results showed that the microbial membrane in escherichia coli o and candida albicans was permeabilized with loss of potassium ions. additionally, the bis-( , -dibutylbarbituric acid) trimethine oxonol [dibac ( )] and , '-dipropylthiacarbocyanine iodide [disc ( )] assay showed membrane depolarization. using calcein-encapsulating giant unilamellar vesicles (guvs) and fitc-dextran containing large unilamellar vesicles (luvs), scolopendin disrupted the cell membrane and the damage size is between . to . nm against composition of microbial plasma membrane of e. coli and c. albicans. thus, we demonstrated that a cationic antimicrobial peptide, scolopendin , possesses broad-spectrum antimicrobial effects that formed pore on the cell membrane. structural and functional investigation of the far c-terminal domain (ctd) of the bifunctional enzyme trai using nmr spectroscopy protein structural biology structural and functional investigation of the far c-terminal domain (ctd) of the bifunctional enzyme trai using nmr spectroscopy b.krishna chaitanya, evelyne schrank and klaus zangger institute of chemistry/organic and bioorganic chemistry university of graz, austria corresponding author email id: krishna.bhattiprolu@uni-graz.at bacterial conjugation is a complex process for the horizontal transfer of single stranded dna from one cell to another. this mechanism also leads, for example, to the spread of antibiotic resistance genes and virulence factors among bacterial species. multi-protein complexes formed at the origin of transfer (orit) region of dna and at the cytoplasmic membrane of the bacterial cell, initiate this process. inside the membrane, the relaxosome identifies the single strand for transfer in a plasmid dna, relaxes and unwinds it, whereas the transferosome is involved in pilus formation (type iv secretion system) and transferring the gene through the cytoplasmic membrane. these events take place in the donor bacterial cell along with several other auxiliary proteins [ ] the bifunctional enzyme trai of plasmid r plays a crucial role in the relaxosome activity, as it contains both a relaxase and helicase domain. to exert its functions on dna, trai works in close co-ordination with other relaxosome proteins like tray, tram and the integration host factor. trai is a residual protein and contains major domains: n-terminal relaxase domain, a central helicase domain and a c terminal domain (ctd). the structure of the c-terminal domain until residue has been solved by crystallography, while the structure and function of the remaining residues remained undetermined [ ]. there are saxs models and crystallographic structures for different parts of trai and also for the full length protein. prediction of cleavage specificity in hcv ns / a serine protease and adv cysteine protease systems by biased sequence search threading gonca ozdemir isik , a.nevra ozer department of bioengineering,faculty of engineering,marmara university proteases are enzymes which recognize specific substrate sequences and catalyze the hydrolysis of designated peptide bonds to activate or degrade them. due to the biological importance of proteases, it is particularly important to identify the recognition and binding mechanisms of protease-substrate complex structures in drug development studies. the assessment of substrate specificity in protease systems is crucial, where interpreting the adaptability of substrate residue positions can be useful in understanding how inhibitors might best fit within the substrate binding sites and aid in the design of potent selective inhibitors. substrate specificity is generally determined by the amino acid profile, structural features and distinct molecular interactions. besides experimental methods, computational tools for prediction of natural substrate cleavage sites, such as threading, have emerged as useful alternative approaches to provide valuable insights into complex enzyme-substrate interactions. in this work, the substrate variability and substrate specificity of the hepatitis c virus (hcv) ns / a serine protease and the adenovirus (adv ) cysteine protease was investigated by the biased sequence search threading (bsst) methodology. using available crystal structures of the proteases, the template structures for the substrate-bound proteases were created in silico by performing various peptide building and docking procedures followed by energy minimization and molecular dynamics (md) simulations. bsst was performed starting with known binding, nonbinding and some random peptide sequences that were threaded onto the template complex structures, and low energy sequences were searched using lowresolution knowledge-based potentials. then, target sequences of yet unidentified potential substrates were predicted by statistical probability approaches applied on the low energy sequences generated. the results show that the majority of the predicted substrate positions correspond to the natural substrate sequences with conserved amino acid preferences, while some positions exhibit variability. for ns / a serine protease cleavage, the significant selection for pro at p and cys at p positions is zearalenone is a mycotoxin produced by fusarium graminearum and related fusarium species. f. graminearum is a powerful plant pathogen and infects major crop plants around the world. acute toxicity of zearalenone is low, but due to its structural similarity to b-estradiol it has binding affinity to the estrogen receptor, which results in interference with hormonal balance. typical effects seen in animals include symptoms like hyperestrogenism and reproductive disorders (reduced fertility, reduced litter size or swelling of uterus and vulva). to reduce the risk for human and animal health posed by the ingestion of contaminated food or feed different decontamination strategies have been studied, including biotransformation. today many microorganisms are known to degrade zearalenone, but for most of them the degradation pathway and formed metabolites remained unknown, hence it is unknown if this degradation also means detoxification. only for the fungal strains trichosporon mycotoxinivorans and gliocladium roseum zen degradation has been studied in detail and loss of estrogenicity of reaction products has been confirmed. we screened for, and isolated zearalenone degrading bacteria from soil samples. the most promising new bacterial isolate was taxonomically assigned to the species rhodococcus erythropolis and designated pfa d - . the zearalenone catabolism pathway of pfa d - was found to be identical as known from g. roseum. the primary reaction product, hydrolysed zearalenone, has so far only been postulated in g. roseum. we prepared hydrolysed zearalenone by preparative hplc and showed loss of estrogenicity in assays with the breast cancer cell line mcf and the estrogen reporter yeast strain yzhb . a genomic library was prepared and screened in zearalenone degradation deficient r. erythropolis pr . the gene encoding zearalenone hydrolase was found and named zena. the hydrolase was identified as member of the a/b-hydrolase family and named zena. it was cloned, recombinantly expressed in e. coli and purified by x his-tag mediated immobilised metal affinity chromatography. activity of his-tagged and untagged enzyme zena was compared in cleared lysate and zena was purified for enzyme characterisation. the influence of ph and temperature on enzyme activity and stability was evaluated and kinetic parameters were determined. a new biding site for snake venom c-type lectins?maria cristina nonato costa , ricardo augusto pereira de p adua , marco aurelio sartim , suely vilela sampaio university of são paulo, fcfrp c-type lectins are proteins that bind different glycan molecules by interactions with a calcium atom present in a carbohydrate recognition domain (crd). many organisms (plants, bacteria, virus and animals) use these proteins in various biological events like lymphocyte adhesion, erythrocyte agglutination and extracellular matrix organization. the c-type lectin fold is plastic and possible for about different sequences, what promoted its adaptation to diverse functions, similarly to the observed for the immunoglobulin fold ( - sequences). it is comprised of about - amino acid residues that folds in two fourstranded b sheets sandwiched by two alpha helices. interestingly, c-type lectins present in snake venoms are possible anti-cancer agents since they are toxic to cancer cells and inhibit the adhesion and proliferation of various cancer cell lines. therefore, we have purified a lactose binding c-type lectin from the venom of bothrops jararacussu (bjcul) to study its structure and binding properties to different sugars. bjcul crystals were obtained by vapor diffusion and the structure solved by x-ray crystallography to . Å resolution. bjcul structure is a decamer formed by a pseudo fivefold axis rotation of a dimer hold by a disulfide bond. each monomer binds a calcium atom and possibly another metal at a second and opposed binding site.