key: cord-0003479-eoz1l5fi authors: nan title: Award Winners and Abstracts of the 32nd Annual Symposium of The Protein Society; Boston, MA, July 9–12, 2018 date: 2018-11-01 journal: Protein Science DOI: 10.1002/pro.3513 sha: 041e50456e5c62980ff8bb61d83283f58f09ef6d doc_id: 3479 cord_uid: eoz1l5fi nan Full-length immunoglobulins (Igs) are widely considered difficult to crystallize because of their large size, N-linked glycosylation, and flexible hinge region. However, numerous cases of intracellular Ig crystallization are reported in plasma cell dyscrasias. What makes some Ig clones more prone to crystallize during biosynthesis as well as the biochemical and cell biological requirements for this cryptic event remain poorly understood. To investigate the underlying process of intracellular Ig crystallization events we searched for model IgGs that can induce crystalline inclusions during recombinant overexpression. We identified and characterized three distinct human IgG mAb clones that induced crystalline inclusions. In all three cases, crystallization occurred only when the two subunits were co-transfected and when the export-ready IgG species reached a certain threshold concentration in the ER. Some mAbs crystallized spontaneously, while others required a pharmacological blockade of ER-to-Golgi transport. Subtype switching had drastic effects on crystal morphology and crystallization propensity. Elimination of prominent acidic patch on the variable domain surface (when present) also abrogated the crystallization events. The evidence supported that different mAbs call for distinct modes of intermolecular interactions and threshold concentrations before crystallization ensues in the ER lumen. Whether such conditions are met under physiological or abnormal cell growth conditions is an important factor that dictates the prevalence of intracellular Ig crystallization events in a monoclonal population of antibody-expressing cells. Genetic or epigenetic changes in transformed plasma cells may play additional roles in promoting the occurrence of Ig crystallization by modulating the biosynthetic and protein trafficking capacity of afflicted cells. 6 Massachusetts Institute of Technology (Cambridge, United States); 2 Fudan University (Shanghai, United States); 3 Shanghai Jiaotong University (Shanghai, United States) G protein-coupled receptors (GPCRs) are the largest family of cell-surface molecules that detects information outside the cell and transduce to internal signals. Nearly half of all marketed drugs use GPCRs as targets. These membrane proteins also play a critical role in tumor initiation, progression, invasion and metastasis, which renders them a promising target against many cancers. Yet the study of their structure and function are notoriously difficult. GPCR proteins contain 7-transmembrane alpha-helical segments comprised of large numbers of hydrophobic residues. These proteins can only be solubilized in aqueous systems with detergents. Lack of detergents will result in protein aggregation. Here we present a useful tool, the QTY code, to genetically modify the hydrophobic domains of GPCRs to become water-soluble without diminishing their functions. Up to~56% of the transmembrane segments were altered. QTY designed GPCRs were screened via yeast two-hybrid system and produced in several host systems, such as SF9 insect cell and e coli. The proteins can be effectively extracted without any detergents from E.coli inclusion bodies and refolded into functional structure. Final storage buffer of modified proteins is detergent-free and the solubility is regulated by arginine content. The modified GPCRs exhibit affinity to their natural ligands as verified by MircoScale Thermophoresis measurements, even after heat-treatment at elevated temperatures. QTY Code provide a simple method for engineering membrane proteins without the presence of detergents. This approach enables a novel pathway through which difficult GPCR proteins may be studied, modified and utilized for in vivo and in vitro applications. Wei-Ven Tee 1 , Enrico Guarnera 1 , Igor Berezovsky 1 1 Bioinformatics Institute (Singapore, Singapore) The omnipresence of allosteric regulation together with the fundamental role of structural dynamics in this phenomenon have initiated a great interest to the detection of regulatory exosites and design of corresponding effectors. However, despite a general consensus on the key role of dynamics most of the earlier efforts on the prediction of allosteric sites are heavily crippled by the static nature of the underlying methods. Because of the critical role of global protein dynamics in allosteric signaling, we postulate the existence of reversibility in allosteric communication, according to which allosteric sites can be detected from the perturbation of the functional sites. We tested this hypothesis using our structure-based model of allostery, which allows one to analyze the causality and energetics of the allosteric communication. The reverse perturbation hypothesis and its predictive power were validated on the set of classical allosteric proteins, then, on an independent extended benchmark set. We show that, in addition to known allosteric sites, the perturbation of the functional sites unravels rather wide protein regions, which can host latent regulatory exosites. These protein parts that are dynamically coupled with functional sites can be used for inducing and tuning of allosteric communication, and an exhaustive scanning of the perresidue allosteric effects can eventually lead to the desired modulation of protein activity. The siteeffector interactions necessary for a specific mode and level of allosteric communication can be finetuned by both adjusting the sites structure to the available effector molecule or by the design or selection of an appropriate ligand. Despite decades of research on the tumor-suppressor known as breast cancer 1 early onset protein (BRCA1), fundamental understanding of its function and how it prevents breast and ovarian cancer risk is lacking. For example, although it has been known for over 15 years that the BRCA1-associated RING domain protein (BARD1) is required for BRCA1 ubiquitin ligase activity, the role of BARD1 remains enigmatic. Does BARD1 play a direct role in BRCA1 activity or is it only a silent partner that prevents BRCA1 degradation? We have applied biochemistry and cell biology techniques to investigate the role of the BARD1 RING domain in ligase activity. Paradoxically, although heterodimerization with BARD1 is required for BRCA1 ligase activity, we found that the BARD1 RING per se is dispensable in vitro for the ubiquitylation of several substrates. However, three different BARD1 RING domain missense mutations discovered in families with a history of breast cancer all show specific loss of ubiquitylation activity towards histone H2A in nucleosomes suggesting a novel role for BARD1 in substrate selectivity. While ubiquitylation is commonly thought of as a degradation signal, we use cell biology techniques to show that ubiquitylation of H2A by BRCA1/BARD1 leads to regulation of estrogen metabolism gene expression. We find that while BARD1 does protect BRCA1 from degradation, it is not its primary essential function. I will present our biochemical and cellular findings, a working model for the role of BARD1 in BRCA1 ligase function, and its implications for breast cancer risk. that we could introduce the small molecules to the inside of TIP60 without dissociation and reassociation processes. This is advantageous for application to the drug delivery system. Moreover, we found that TIP60 was further assembled to the larger structure by the addition of cationic molecule because TIP60 has the anionic charged surface. Interestingly, when we added the lysozyme as the cationic molecule, TIP60 forms visible precipitations via the 100 nm-scale intermediate particle formation. Therefore, TIP60 can be also used as the chemically modifiable building block. Our understanding of how ubiquitination influences ubiquitin signaling pathways is hindered by the difficulties in preparing distinct ubiquitinated substrates. Inspired by the protease-mediated ligation, our group has utilized a ubiquitin C-terminus hydrolase, Yuh1, to catalyze aminolysis of ubiquitin with small amines. However, this aminolysis can only occur at high pH and high concentration of amines. To address these problems, we engineered a Yuh1 variant, which we named as Programmable Ubiquitin Ligase (PULSE), which can autoubiquitinate with designer ubiquitin chains under physiological conditions. The evolution of Yuh1 into a ubiquitin ligase started from alanine scanning at the primary sphere of Yuh1, where we discovered a Yuh1 mutant that can carry out aminolysis at physiological pH. Kinetic characterization of this Yuh1 mutant suggested it has lower catalytic acitivity compare to WT but the kcat of aminolysis is four times higher than the kcat of hydrolysis. In addition, by introducing a Lys at proximity to the active site of Yuh1 mutant, it can autoubiquitinate at the incorporated Lys with defined ubiquitin chains with short lifetime. Yeast display was used to evolve Yuh1 mutant for efficient autoubiquitination and we have isolated a clone, PULSE, with extended autoubiquitination lifetime. We envision that PULSE will be useful as ubiquitinated substrates with designated ubiquitin chains in various biochemical assay in the ubiquitin field. In the future, we would like to further engineer PULSE as a versatile ubiquitin ligase to catalyze ubiquitination of our desired substrates. The ability to control the location and timing of enzymatic activity using environmental and/or external stimuli is a fundamental design challenge with myriad applications in biology and medicine. We have developed structure-based, computation-guided predictive methods for controlling enzyme activity using (1) covalently attached photoresponsive azobenzene groups for reversible activation, and (2) designed prodomains that enable irreversible activation in response to environment-specific proteolytic activity. Coupling computational design with deep sequencing and growth selections enables massively parallel screening and optimization of designed activity switching. Applying the first (photosensitization) method to the therapeutically useful enzyme yeast cytosine deaminase, we obtained a change in enzyme activity by the photocontrolled modulation of the enzymes active site lid structure, while fully maintaining thermostability. Multiple cycles of switching, controllable in real time, are possible. The predictiveness of the method is demonstrated by the construction of a variant that does not photoswitch as expected from computational modeling. Our design approach opens new avenues for optically controlling enzyme function. The designed photocontrolled cytosine deaminases may also aid in improving chemotherapy approaches that utilize this enzyme. Applying the second (chemosensitization) method to the FDA-approved enzyme carboxypeptidase G2 (CPG2), we have developed matrix metalloprotease (MMP) activity-dependent CPG2 variants. As MMP activity is a hallmark of growing tumor microenvironments, our designs may be useful for devising efficient versions of spatially targeted enzyme-prodrug therapies. Reference Ribonuclease Sa (RNase Sa), from Streptomyces aureofaciens, is one of the oldest enzymes and is involved in RNA substrate binding. RNase Sa was stabilized by 1.3 kcal/mol upon mutating the -turn sequence 48SYGY51 to 48PYGY51 in the protein. We have performed molecular dynamics simulations in explicit water on the wild type and the S48P mutant of RNase Sa, as well as the peptide models of -turns: Ac-SYGY-NMe and Ac-PYGY-NMe. We found an increase in -turn propensity on S48P mutation in Ac-PYGY-NMe and the mutant protein model, S48P. In addition, we found slightly better hydrophobic packing in the S48P mutant protein. An overall reduction in fluctuations was observed for the S48P mutant protein, especially in the loop comprising of the residues 61-67 (60s loop). The 60s loop consists of most of the positively charged residues in the protein and is involved in substrate binding. Network Analysis of the structure shows that the residues involved in the path between the point of mutation, i.e. 48, to the 60s loop involve residues that are conserved in the ribonuclease family. The stability of the mutant protein was also accounted for by an increased conformational entropy in the mutant protein (TSmutant-wild type = 6.8 kcal/mol), due to a loss of correlations, and the presence of more salt-bridges and their enhanced sampling in the mutant protein. The Evolution of Dynamic Amino Acid Interaction Networks Around the Catalytic Cycle of a Tryptophan Synthase molecule force spectroscopy, applying force parallel to the helical axis in the shear geometry. Modifications in the hydrophobic core or the helix propensity both alter the binding potential, but with different outcomes: a less tightly packed hydrophobic core increases the potential width (x), without significantly affecting the barrier height (koff ). In contrast, a reduced helix propensity decreases both potential width and barrier height. Our goal is to use this information for developing a library of mechanically characterized CCs that can be applied as calibrated building blocks for a wide range of applications: from molecular force sensors to mechanosensitive material crosslinks in protein nanostructures and synthetic ECM mimics. Small heat shock proteins (sHsps) comprise a family of molecular chaperones broadly employed across many organisms to prevent aggregation of partially unfolded protein substrates. A defining feature of sHsps is their ability to form dynamic, polydisperse oligomers that exhibit subunit exchange under certain conditions. The physiological relevance of oligomerization and mode(s) of chaperone function remain undetermined. sHsps contain the canonical " alpha-crystallin domain" (ACD). This domain is flanked by N-and C-terminal regions (NTR and CTR, respectively) of varying length and sequence, believed to participate in substrate and quaternary interactions. In order to identify functional regions of sHsps that contain chaperone activity, we utilize a multipronged approach. First, we have purified the relatively unstructured N-terminal sequence from human HspB1. Using model substrates, we have identified an 88-residue sequence that exhibits chaperone activity in solution. To determine the importance of oligomeric organization (multivalent interactions) in chaperone activity we constructed gold nanoparticles (AuNPs) appended with the HspB1 NTR. These sHsp-AuNPs exhibit chaperone activity, with the chaperone capacity varying by substrate. Additionally, to complement these experiments we have utilized mutagenesis of full-length sHsps to identify amino acid residues that contribute to chaperone activity and alter substrate-sHsp interactions. Our combined results indicate the particular importance of NTRs in chaperone activity and demonstrate the therapeutic potential of sHsp-AuNPs. ProxiMAX randomization technology (Colibra) is a defined saturation mutagenesis process that delivers precision control of both identity and relative ratio of amino acids at specified locations within a protein library. The process is non-degenerate, meaning that encoded DNA libraries are as small as is physically possible. Since no constraints are imposed by the genetic code, ProxiMAX can encode all 20 or any desired subsets of amino acids with ease. Moreover, its use of a maximum of 20 codons in saturated positions, without sequence restraints, means that many codons remain available to encode additional, unnatural amino acids (UAAs). Incorporation of UAAs is particularly relevant in expanding engineered protein repertoires. Ultimately, we aim to combine ProxiMAX with an in vitro transcription/translation system to design and express synthetic protein libraries containing multiple UAAs simultaneously. We have employed ProxiMAX randomisation to encode 18 natural amino acids (excluding Cys and Met) in various, putative amino acid-binding locations of an E. coli alanyl-tRNA synthetase. Two variant libraries, each lacking any amino acid editing function, have been created. Our poster will describe the synthesis of those libraries, each encoding >107 novel variant proteins, their composition, quality and our progress towards screening and deconvolution of the libraries to discover novel synthetases, with an initial focus on specificity for D-amino acids. Recent advances in next-generation sequencing and proteogenomics have revealed thousands of microprotein-encoding small open reading frames in prokaryotic and eukaryotic genomes, but we are only beginning to understand their cellular roles. We hope that by studying the regulation and interactions of microproteins, we can generate hypotheses about their functions. First, we describe a quantitative proteomic method that has revealed novel stress-responsive microproteins in bacteria. We describe features of these microproteins that are consistent with function, including predicted structure, proteinprotein interactions, and/or subcellular localization. Second, we describe functional characterization of the human microprotein NoBody, which associates with the mRNA decapping complex and inhibits 5-to-3mRNA decay. We conclude that differential expression analysis and interactomics are powerful approaches to reveal the biological processes in which microproteins participate. Sensing environmental cues and converting them into cellular actions is key for all living organisms. Many complex systems have evolved to sense and react to various stimuli, often through protein conformational changes and protein-protein interactions. One ubiquitous environmental stimulus, which is also easy to apply in a laboratory setting, is light. Sensor proteins often avoid the black-and-white paradigm of wholly inactive and wholly active states to allow for more subtle regulation. Using the blue-lightsensing histidine kinase EL346 from Erythrobacter litoralis as a model for such dynamic signal transduction systems, we sought to determine the conformational changes that occur during light activation. In order to access the dynamic states of this protein, we used double electron-electron resonance (DEER) electron spin resonance (ESR) spectroscopy to measure changes in distances between domains, as well as hydrogen-deuterium exchange mass spectrometry (HDX-MS) to report on conformational changes. We find that the dark-adapted state shows characteristics of both active and inactive states, consistent with previously-determined high background activity in the dark and modest increase upon illumination. The activating mutation V115A, which decouples the sensor and kinase domains, highlights certain conformational changes characteristic activation, in the interface between sensor domain and phosphoacceptor site. Comparison of ADP and ATP bound states suggests that interactions with the gamma phosphate of the nucleotide are essential for efficient activation. These data show that substantial populations of inactive and active conformations coexist in the dark state, and that the coincidence of light and appropriate nucleotide is necessary to activate autophosphorylation activity. Simulating the Dynamics of Gtpases on the Ribosome barriers in this assembly can be modulated by EFs. A common theme that has emerges is that elongation factors can accelerate dynamics through simple excluded volume effects. Specifically, by confining the mobility of tRNA molecules, the associated free-energy barriers can be reduced. This role of EFs is in contrast to many other proteins, where functional dynamics often require power-stroke dynamics. Eyal Arbely 1 1 Ben-Gurion University of the Negev, Department of Chemistry and the National Institute for Biotechnology in the Negev (Beer Sheva, Israel) The reversible posttranslational modification (PTM) of lysine residues by acetylation is mostly associated with the regulation of chromatin remodeling and gene expression. That said, in recent years, thousands of potential lysine acylation sites have been identified, demonstrating that lysine acylationmainly acetylationis a widespread PTM that occurs on proteins involved in diverse cellular processes. However, current studies of the molecular mechanism of acylation-regulated cellular processes are limited to the use of acylated peptides and mutational analyses. In addition, the underlying molecular basis of histone deacetylases (HDACs)-substrate specificity and the regulation of acylation by HDACs, are still elusive. These limitations can be removed by exploiting recent advances in the field of genetic code expansion that enable the genetic encoding and site-specific incorporation of acyl-lysine derivatives into ribosomally synthesized proteins. Utilizing this technology, we reconstituted mammalian HDAC-catalyzed deacylation reactions in live bacteria and monitored the deacylase activity of specific mammalian NAD+-dependent sirtuins and Zn2+-dependent HDACs. By studying site-specifically acylated full-length substrates, as well as short acylated peptides, we found novel catalytic activities and substrates of mammalian HDACs; for example, the deacetylation of the transcription factor STAT3. Together with in vitro measurements and studies in cultured mammalian cells, we also studied the effect of acetylation on the structure and transcriptional activity of STAT3. Our data show that lysine-acetylation increases the transcriptional activity of STAT3 by increasing protein stability. We anticipate that this methodology will advance the field of lysine-acylation and deacylation, by providing information that is inaccessible by current approaches. Translocon Declogger Ste24/Zmpste24 Rescues IAPP-Oligomer Induced Proteotoxicity be Ste24, a protease that is evolutionarily conserved from yeast to humans (ZMPSTE24). In yeast, Ste24 relieved IAPP oligomer induced ER stress and translocation defects. These findings, combined with the recently discovery that Ste24 cleaves polypeptides clogging the translocon, led us to hypothesize that IAPP oligomers might clog the translocon. We tested this hypothesis and extended our discovery beyond yeast. First, we validated the protective function of Ste24 in a rat insulinoma cell line. Next, we functionally replaced yeast Ste24 with human ZMPSTE24 and demonstrated that ZMPSTE24 also rescued IAPP-induced toxicity. Using mutants of ZMPSTE24 with varying levels of de-clogging activity, it became clear that the rescue of IAPP toxicity was proportional to the declogging activity. This relationship allowed the yeast model to become a functional genomics platform with which we characterized the activity of 111 ZMPSTE24 variants uncovered in the exome sequencing of 45,231 subjects for T2D risk genes. Fourteen partial loss-of-function variants were identified and these were apparently enriched among diabetes patients over 2 fold. Thus, we conclude that IAPP oligomers could contribute to beta-cell failure by impairing ER-to-cytoplasm transport. Serum amyloid A (SAA), the protein precursor of AA amyloidosis, is an acute-phase reactant whose action in innate immunity and lipid homeostasis is incompletely understood. Most human SAA circulates bound to plasma high-density lipoproteins (HDL). This study addresses how SAA binds phospholipids, a critical interaction for both normal functions of SAA and its misfolding in amyloid. The structure and dynamics of SAA in model complexes with phospholipids were characterized using circular dichroism and fluorescence spectroscopy, gel electrophoresis, and hydrogen-deuterium exchange mass spectrometry, combined with bioinformatics analyses and the existing x-ray crystal structures. SAA is largely disordered in solution,~80% -helical in the crystals, and~50% -helical when bound to lipoproteins. Additionally, lipid binding increases the SAA thermostability from Tm~18 C to 52 C. SAA helices h1 and h3 exhibit classic amphipathic lipid surfacebinding properties. In the SAA monomer, these helices form an exposed, concave, apolar face with curvature commensurate with HDL. This curvature is defined by a unique, strictly-conserved GPGG motif that facilitates remarkably close interhelical main-chain spacing of 3.6Å. Lipid binding by SAA N-terminal fragments suggests comparable affinity for fragments containing h1-h3. Lastly, hydrogen-deuterium exchange MS reveals increased protection in h1 and h3 upon lipid binding. Together, our results reveal that h1 and h3 form a curved lipid-binding site in SAA that is conserved throughout evolution, explaining preferential binding of SAA to HDL and its ability to solubilize diverse lipids. We propose that this ability reflects the primordial function of SAA in clearing cellular membrane debris from injured sites. Protein structures are key to understanding biomolecular mechanisms and diseases, yet their interpretation is hampered by limited knowledge of their biologically relevant quaternary structure (QS). A critical challenge in inferring QS information from crystallographic data is distinguishing biological interfaces from fortuitous crystal-packing contacts. Here, we tackled this problem by developing strategies for aligning and comparing QS states across both homologs and data repositories. QS conservation across homologs proved remarkably strong at predicting biological relevance and is implemented in two methods, QSalign and anti-QSalign, for annotating homo-oligomers and monomers, respectively. QS conservation across repositories is implemented in QSbio (http://www.QSbio.org), which approaches the accuracy of manual curation and allowed us to predict >100,000 QS states across the Protein Data Bank. Based on this high-quality dataset, we analyzed pairs of structurally conserved interfaces, and this analysis revealed a striking plasticity whereby evolutionary distant interfaces maintain similar interaction geometries through widely divergent chemical properties. Engineering Synthetic Antibodies as Probes to Dissect the Energetic Contributions of Conformational Changes in Proteins Upon Ligand Binding 1 Institute of Nano Science and Technology (Mohali, India); 2 Institute of Nano Science and Technology, Mohali, India (Mohali, India); 3 IISER, Mohali, India (Mohali, India) Bacterial microcompartments (MCPs) are one of the primitive paradigms of compartmentalization and the only ones known in the prokaryotes. These polyhedral organelles contain an enzyme cluster wrapped inside a protein shell and carry out specific metabolic functions in bacteria. Although these organelles have been explored meticulously using genetic and biochemical tools, they have not been explored in biomaterial science. Here we have developed a protocol to uniformly coat the MCPs with a monolayer of gold nanoparticles so that the conjugate material may behave as a hybrid catalyst with an inorganic shell over an active enzyme cluster. The fabricated gold nanoparticles are less than 3nm in size with interparticle distance close to 6nm. This unique assembly can show simultaneously the diol-dehydratase enzyme activity along with the reduction of p-nitrophenol to p-aminophenol in the presence of NaBH4. Under the fluctuating environments of inorganic reactions, the shell proteins of the MCPs firmly maintain a barrier between the core enzyme and the surface inorganic catalysts. The stability and integrity of the shell proteins allow that fabrication of such an intricate hybrid system. The MCP-AuNPs hybrid catalysts inspired a new class of materials which can be used in a chemical reaction condition without perturbing core biological (physiological) environment for enzymatic activity. This system provides a clue for scaffolding uniformly ordered nanoparticles in 3D and the development of new class of hybrid catalytic material based on supramolecular protein assemblies. Resistance to our current antibiotics is reaching crisis levels and there is an urgent need to develop antibacterial agents with novel modes of action. A promising alternative to antibiotics are the naturally occurring endolysin enzymes from bacteriophage. Endolysins cause bacterial lysis by degrading the bacterial peptidoglycan cell wall. Exogenous application of endolysins results in rapid and specific elimination of Gram-positive bacteria making them an excellent alternative and/or adjunct to traditional antibiotics. The streptococcal C1 phage lysin, PlyC, is the most potent endolysin described to date and can rapidly lyse Group A, C and E Streptococci. We intend to engineer the specificity of PlyC using a directed evolution approach, to retarget its bacteriolytic activity to other groups of Streptococci. We have previously determined the X-ray crystal structure of PlyC, revealing a complicated and unique arrangement of two catalytic domains bound to an octameric cell-wall docking assembly. In this assembly, five residues, previously shown to be important for cellwall binding, were targeted for site-saturation mutagenesis. This generated five libraries of several hundred clones, which are currently being screened for lytic activity against different bacterial strains. These results will provide several new PlyC mutants that display lytic activity against previously untargeted bacterial strains. In addition, these results will provide an exhaustive mutational analysis of the known cell-wall binding site in PlyC, delivering valuable insight into the interaction between PlyC and its target bacterial cell wall. The construction of metal-binding sites into designed protein scaffolds is an ultimate test of our understanding of structure-function relationships and it will eventually allow us to construct new proteins for biochemistry and biotechnology applications and biological pathways. The negative effects of cadmium ion (Cd2+) exposure on human health have been recognized for many years. Thus, new technologies that can sequester and remediate cadmium efficiently from dilute environmental sources and sites are highly desirable. Inspired by our previous work on uranyl (UO22+) binding protein design1, we present a strategy here to rational design and develop a cadmium selective binding protein starting from a pre-existing protein scaffold, ribose binding protein (RBP). We developed a computational algorithm for designing Cd2+ binding sites based on the preferred coordination geometries of Cd2+. Several potential binding sites were designed in RBP. The initial experimental screening was performed at bacteria level. Mutants that allow E. coli to grow at high Cd2+ concentration were purified and tested for their binding strength with Cd2+ by using isothermal titration calorimetry. Two of them bind to cadmium at nanomolar range. One of the mutant protein was expressed in the periplasmic region of E coli and we observed some downstream beta-gal gene expression when induced with Cd2+. Thereby, we can regulate the conformational changes by incorporating cadmium binding sites into E. coli RBP, and use the engineered bacterium to sense and scavenge environmental cadmiums. Huabin Zhou 1 , Luhua Lai 2 1 Peking University (Beijing, China); 2 College of Chemistry and Molecular Engineering, Peking University (Beijing, China) Phase separation of proteins/nucleic acids forming non-membrane organelles play an important role in multiple cellular processes. Sequestration of molecules in condensate phase efficiently alter their activity, thus facilitate signal transduction and stress response. We recently discovered that some plant specific transcriptional factors (TFs) undergo liquid-liquid phase separation (LLPS). These liquid droplets undergo fusion and fission in the time scale of seconds. Both DNA binding domains and intrinsic region of TFs are crucial for LLPS. LLPS is driven by electrostatic interaction of intrinsic disorder region and promoted by hydrophobic residues. With increasing ionic strength, protein dissolve from condensed phase and disperse into solvent. Our results suggested that LLPS might be a novel mechanism of epigenetic establishment and transcriptional repression. In addition, TFs LLPS would be an ideal platform for enzyme catalysis and DNA encapsulation. Comparison of Crystal and Cryoem Structures of Hsp104 and ClpB Disaggregases studied in more depth. Thus, the spectrum of Hsp104 molecular architecture, and the extent of protein folding and remodeling activities enabled by it, is inadequately examined. We report two structures of Hsp104 from the thermophilic fungus Calcarisporiella thermophila (CtHsp104). The 2.70 Å crystal structure and 3.8 Å cryo-EM structure reveal left-handed, helical assemblies. We provide the highest resolution and most complete view of Hsp104 hexamers with all domains resolved in the "resting" ADP state to date. We compare these structures with previous models of yeast and fungus Hsp104 and bacterial ClpBs. We also functionally characterize CtHsp104 in vitro and in yeast. While CtHsp104 functionally complements Hsp104 from S. cerevisiae (ScHsp104) in yeast acquired thermotolerance and has many similar biochemical properties to ScHsp104, CtHsp104 is able to antagonize a distinct set of proteotoxic misfolding events under conditions where ScHsp104 is ineffective. Our results are consistent with recent structures of yeast Hsp104. We also establish that naturally occurring Hsp104 orthologues can have therapeutic disaggregase activity against protein substrates involved in neurodegeneration. We suggest that sequence variation among Hsp104s may be a valuable, untapped resource in the engineering of therapeutics for fatal neurodegenerative diseases. Supported by NIH GM094585,GM115586, NIAID HHSN272201200026C, HHSN272201700060C, R01GM099836, and DOE/BER DE-AC02-6CH11357. Karolina Michalska 1 , Grant Gucinski 2 , Fernando Garza-Sánchez 2 , Parker Johnson 3 , Lucy Stols 1 , William Eschenfeldt 1 , Gyorgy Babnigg 1 , David Low 2 , Celia Goulding 3 , Christopher Hayes 2 , Andrzej Joachimiak 1 1 Argonne National Laboratory (Lemont, United States); 2 University of California, Santa Barbara (Santa Barbara, United States); 3 University of California, Irvine (Irvine, United States) Contact Dependent growth Inhibition (CDI) is a mechanism of inter-bacterial competition utilized between closely related strains. This system is based on the exchange of the toxin protein, often a nuclease, between the cells that touch each other. Here we present a novel CDI toxin tRNase that exploits elongation factor EF-Tu to specifically remove the single-stranded 3´-ends from tRNAs that contain guanine discriminator nucleotides. The ternary complex between the toxin, its cognate immunity protein and GDP-bound EF-Tu shows that tRNase interacts only with domain 2 of EF-Tu, partially overlapping the site that anchors the 3´-end of aminoacyl-tRNA. While EF-Tu is necessary for the tRNA cleavage, superposition of the toxin onto the GTPEF-Tuaa-tRNA complex reveals several potential steric clashes between EF-Tu-bound toxin and aa-tRNA. We propose that the toxin remodels the GTPEF-Tuaa-tRNA structure, displacing the 3´-end of aa-tRNA to allow its entry into the nuclease active site. This work was supported by National Institutes of Health (GM094585 and GM115586 to A.J., GM117373 to C.W.G., D.A.L., C.S.H.). The use of Structural Biology Center beamlines was supported by the U. S. Department of Energy, Office of Biological and Environmental Research (DE-AC02-06CH11357 to A.J.). The coral-derived fluorescent protein, Azami Green, is a homotetramer with two distinct binding interfaces. We sought to disrupt each interface independently, in order to quantify the binding affinity of the other. Our strategy was to mutate interface residues to threonine (Thr), which should preserve local structure because it is energetically favored in -sheets, but which is hydrophilic, and so should disrupt an interface and promote soluble dimers. We introduced mutations to Thr at one interface and tracked the oligomeric state as a function of concentration by monitoring the anisotropy of the intrinsic fluorescence of Azami Green fluorophore. Although one or two Thr residues did indeed disrupt binding, a triple mutation to Thr reversed this trend, resulting in tighter binding. In this triple mutant, all twelve central interface residues, six per monomer, are Thr. To determine how these Thr residues form a stable interface, we solved the x-ray crystal structure of Azami Green with the artificial Thr-rich interface. The structural data suggest the interface is stabilized by closepacking of the Thr residues, with only one potential H-bonding interaction between two central Thr residues. This Thr-rich interface provides a special opportunity to study the behavior of Thr in the context of many other Thr residues. We show a hard sphere plus stereochemical constraints model is able to predict the positions of the Thr sidechains. We speculate the size and shape of Thr at a homodimeric interface between beta sheets is ideal for close-packing and other residues would not form such an interface. Calin Plesa 1 , Angus Sidore 1 , Nathan Lubock 1 , Di Zhang 2 , Sriram Kosuri 1 1 UCLA (Los Angeles, United States); 2 University of Pennsylvania (Philadelphia, United States) We demonstrate a new approach to investigate the effect of mutations across large libraries of evolutionarily divergent sequences that share function. DropSynth gene synthesis is used to assemble thousands of homologs of two essential bacterial proteins (phosphopantetheine adenylyltransferase and dihydrofolate reductase). We then characterize the ability of different homologs to complement a knockout E. coli strain in a multiplexed functional assay. Synthetic errors in our assemblies allow us to explore local mutational landscapes around each of the designed homologs. By aligning homolog sequences and collapsing the data onto a reference sequence we observe core functional motifs for complementing homologs. Low-fitness homologs provide gain-of-function mutations, which reveal the reasons underlying homolog incompatibility. Broad mutational scanning using DropSynth is a useful tool to explore protein functional landscapes at large scales. Bispecific antibodies and fusion proteins are novel biologics that combine the specificities of two different molecules and simultaneously target two epitopes. Such biologics with two target functionality can interfere with multiple surface receptors and aid in enhanced bio-therapeutic efficacy. Assembling a bispecific molecule from two different parental molecules expressed in the same producer cell may result in various non-functional forms with respect to bispecificity. [1] Here we look into the active and inactive forms of a bispecific Fc-fusion protein. A mass spectrometric analysis of IdeS subunits revealed the identical amino acid sequence of the two forms, but significant differences in inter-chain hinge region disulfide bonding and some variations in O-linked glycosylation. Majority of the inactive form was found to lack inter-chain hinge region disulfide bonds and therefore present as half-molecules. SE-HPLC analysis was performed on 40 C heating time-course samples, where a rapid formation of half-molecules for the inactive form validates the previous findings. Interestingly, the hinge region cysteines were all in oxidized form as shown by peptide map LC-MS analysis, indicating the occurrence of intrachain disulfide bond shuffling, instead of simple under-disulfide bonding. Disulfide bond mapping is currently underway to resolve the detailed disulfide bonding structures of both active and inactive forms.Intrinsic tryptophan fluorescence study has shown the upper-level structural differences between the two, and further biophysical characterization will be performed to demonstrate the inactive form is a conformer of the active form. [1] Kontermann, R. E.; Brinkmann, U., Bispecific antibodies. Drug Discov Today 2015, 20 (7) Kanosamine is an antibiotic and antifungal, and a component of several different aminoglycosides. Kanosamine is produced by Bacillus cereus UW85, which contains a kab operon required for kanosamine biosynthesis. Here we describe the first characterization of the encoded kab enzymes that catalyze the biosynthesis of kanosamine in three steps from glucose-6-phosphate: KabC, a NAD+-dependent dehydrogenase: KabA, a pyridoxal-5-phosphate (PLP)-dependent glutamate aminotransferase and KabB, a kanosamine-6-phosphate (K6P) phosphatase. We also report the steady-state kinetic parameters of three Kab enzymes along with four crystal structures of KabA: the wild-type enzyme PMP, to PLP, to a covalent adduct of PLP and K6P, and to a ternary complex of PLP and the glutamate analog glutarate. Together, our structures provide consecutive snapshots of the reaction to support the catalytic mechanism for KabA, and reveal the key active-site residues necessary for catalysis. Yonghong Zhang 1 , James Bulllard 1 1 The University of Texas Rio Grande Valley (Edinburg, United States) Pseudomonas aeruginosa is an opportunistic human pathogen and a primary cause of nosocomial infections. The rate of antibiotic resistance in P. aeruginosa is increasing worldwide leading to an unmet need for discovery of new antibiotics with high efficacy. Bacterial protein synthesis is an essential metabolic process and a validated target for antibiotic development. Structural information of protein targets in P. aeruginosa protein synthesis (PAPS) is therefore needed for rational design of inhibitors based on structure-activity relationship. This study aims to structure determination of translation initiation factor 1 (Pa-IF1) and its interaction with the 30S ribosomal subunit to get structural insight into initiation of translation in PAPS. Solution NMR techniques were used to determine Pa-IF1 structure, and to map out the binding interface of Pa-IF1 to the 30S. The key residues of Pa-IF1 involved in the binding were identified by NMR titration. Pa-IF1 functional assay was performed using the established A/T protein synthesis system from P. aeruginosa. Pa-IF1 consists of a five-stranded -sheet with an unusual extended -strand at the C-terminus, and one short -helix. The structure adopts a -barrel fold and contains an oligomerbinding motif. A cluster of basic residues (K39, R41, K42, K64, R66, R70, and R72) on the surface near the short -helix compose the binding interface with the 30S subunit. A structural model of Pa-IF1 and its interaction with the 30S subunit was built based on the titration result and provides structural information for understanding PAPS machinery. Shellfish hypersensitivity has risen in prevalence worldwide and shrimp allergy is one of the main causes of anaphylaxis. Since avoidance is the only passively way in preventing allergic reactions to shrimp, a better understanding of molecular evens in the induction and progression of shrimp allergy is needed. Pentraxin 3 (PTX3) is rapidly produced directly from inflammatory or damaged tissues and involved in acute immunoinflammatory responses. However, the role of PTX3 in the development of immediate IgEmediated shrimp allergy remains unknown. Here we observe that mice with shrimp allergy have increased PTX3 levels in serum and small intestine compare with healthy control mice. We also demonstrate that PTX3 augments degranulation, formation of pro-inflammatory mediators and Fc epsilon receptor I (FcRI) signaling pathways of mast cells in response to pharmaceutical stimulation. In addition, mast cells challenged with shrimp extract have similar effects to PTX3. Furthermore, inhibition of PTX3 by RI37 also rescues exaggerated degranulation and pro-inflammatory mediators expression, suggesting that PTX3 in collaboration with the FcRI downstream pathway contributes to the facilitating of allergic response. Our data provide the first evidence that PTX3 expression is increased in shrimp allergy. These results rise the possibility that PTX3 participate in the cause of shrimp-allergic inflammation and supply further insight into the application of PTX3 inhibitior as a therapeutic reagent. Miao Yu 1 , Zhirong Liu 1 1 Peking University (Beijing, China) In the process of allostery, distal sites correlate with each other to transmit information through the whole protein. As an effective method, correlation analysis employs atom fluctuations to decipher the codes of distal correlations. However, conventional correlation analysis failed to take into account the orthogonal correlations, which are largely populated among the entire correlations. We achieved unbiased assessment of correlation, based on singular value decomposition (SVD). New method based on SVD is employed in coarse-grained G-like model in MD simulations. The potency of the SVD method is confirmed by the experimental results in a model allosteric system, the second PDZ domain in human PTP1E protein. The new method also demonstrated explicit improvement on allosteric site prediction accuracy, examined by a dataset of 23 known allosteric monomer proteins. The proposed method not only provides new insights into correlation analysis, but also suggests a common strategy for investigations other than allostery, such as protein dynamics and drug design. Aggregation is an unresolved problem in biotechnology, where many therapeutic proteins never reach the market because aggregates form during manufacturing/storage. To prevent aggregation, the mechanism needs to be understood. This begins with elucidating the conformational states that lead to aggregation. In this study, we combine: Small-Angle X-ray Scattering (SAXS), Molecular Dynamic (MD) simulations and Single-Molecule FRET, to characterize an aggregation-prone state of the humanized A33 Antibody Fragment (Fab). SAXS showed that A33 Fab adopts a more expanded conformation at acidic pH (5.5, 4.5 and 3.5) compared to neutral pH (7.0 and 9.0), with a 4% increase in the radius of gyration. The same conditions lead to accelerated aggregation, and small amounts of aggregate were also detected by SAXS. SAXS data was combined with structures from MD simulations to reveal that the conformational change at low pH occurred in the constant domains, particularly in the light chain (CL). This same domain displacement was confirmed with smFRET. Further salt bridge analysis and Rosetta mutational study revealed the location of the first residues to get protonated and the domains that could be stabilized further. Online tools were used to predict aggregation prone regions (APR) in the interior of the protein. The SASA of one of these increased due to the low-pH CL displacement. In conclusion, this work elucidated the conformation of an expanded aggregation-prone specie of A33Fab. The findings highlight the importance of identifying local instabilities that might reveal APR regions in the protein. Ultimately, this knowledge can provide a rational basis for protein stabilization. Tilmann Kuenzl 1 , Elisabetta Groaz 2 , Piet Herdewijn 2 , Philippe Marlière 3 , Sven Panke 1 1 ETH Zurich (Basel, Switzerland); 2 KU Leuven (Leuven, Belgium); 3 Génopole (Evry, France) Integration of non-natural compounds into biological processes offers great potential to modify or expand existing cellular functions. Cellular uptake of these compounds, however, is often hindered by the selective permeability of cell membranes. Here, we present versatile solutions to overcome this limitation by covalently attaching natural and non-natural cargo molecules to transport vectors that are efficiently taken up by native transport proteins present in the cell membrane. Most notably, peptide and sulfonate transporters from E. coli are promising entry gates into the cell, as they have exceptionally broad substrate ranges. We demonstrate that attaching different cargo molecules to the carboxyl group of a peptides glutamate side chain or the sulfonate sulfobutanoic acid allows for uptake via these transporters. To release cargo molecules from peptides once they are inside the cell, we exploited the enzyme -glutamyl transferase (GGT), which is known to hydrolyze a wide range of -substituted glutamates. For the sulfonate-based transport system, a GGT variant was rationally engineered to efficiently hydrolyze sulfobutanoic acid-cargo constructs. The versatility of these synthetic transport systems was demonstrated by delivering structurally diverse amino acids and non-natural dyes into the cell. Furthermore, the system was applied to discover a novel synthesis pathway to generate NAD from a non-natural precursor. Given the promiscuity of the transport proteins and GGT, the synthetic transport systems that we have developed offer a highly generalized solution to overcome limitations in cellular uptake and can hence be used for a wide range of biotechnological and synthetic biology applications. The promyelocytic leukemia protein (PML) functions in various cellular pathways by binding and concentrating partner proteins in PML nuclear bodies (NBs). PML-NB formation begins with oxidationdependent PML multimerization, which creates a mesh of covalently cross-linked PML. This mesh becomes the outer shell of a PML-NB that encases a set of partner proteins. PML cross-linking has been proposed to occur through oxidation of cysteine side chains, but which cysteine residues in PML are involved in this process, and how cross-linking leads to formation of the PML mesh, remain poorly understood. We hypothesize that surface-exposed cysteine residues form intermolecular disulfide bonds under oxidizing conditions. Our lab has generated a structural model of the tripartite motif (TRIM) of PML, which comprises the cysteine-rich RING, B-box 1, and B-box 2 domains as well as the cysteine-free coiled-coil domain. This model identifies cysteine residues in the RING and B-box 2 domains that are potentially oxidation-prone. The model further predicts PML to be an antiparallel dimer under nonoxidizing conditions. The arrangement of the monomers precludes intermolecular cross-linking between cysteine-rich domains within the dimer. This facilitates the formation of the PML mesh by allowing each dimer to covalently link to at least two other dimers. Future work will complement this model by using nuclear magnetic resonance (NMR) to compare the structures of PML domains under reducing and oxidizing conditions. Ubiquitin specific proteases (USPs) comprise one major class of deubiquitinating enzymes (DUBs) that contains over 50 proteins. These enzymes counteract ubiquitination in cells by cleaving the isopeptide bond that attaches ubiquitin to its substrate. USP7 is a DUB whose role is to deubiquitinate and stabilize the well-known tumor suppressor, p53. The USP7 catalytic domain structure as an apoenzyme and in complex with ubiquitin aldehyde (Ubal) has been previously determined. Interestingly, three residues of the active site (C223, H464, D481) undergo rearrangement upon Ubal binding. Coined as the catalytic triad, this conformational change switches the enzyme from an inactive to active state. To date, this structural rearrangement has only been reported in USP7. This event may be a potential regulatory mechanism of the enzyme and implies that dynamics may play a role in its activation. However, dynamics of USP7 has never been studied and remains a gap in our knowledge. To address this gap in the structure-function relationship, nuclear magnetic resonance (NMR) 15N CPMG NMR spin relaxation dispersions and T1, T2, NOE spin relaxation studies were used to study protein structure and dynamics of the catalytic domain. Remarkably, the dynamic and flexible residues localize to two distinct regions of the catalytic domain: the ubiquitin binding site and the catalytic groove of the enzyme. These data suggest that conformational dynamics play a role in both enzymatic catalysis and ubiquitin recognition of the catalytic domain. Interrogating the Selectivity of Proteasome Associated Deubiquitinase UCH37 and Developing Inhibitors using High Throughput Screening Jiale Du 1 1 Umass-Amherst (Amherst, United States) Protein ubiquitination is a post-translational modification process which represents a complex signaling system that coordinates essential biological functions. Deubiquitinases (DUBs) regulate key biological pathways by removing or editing ubiquitin chains formed on ubiquitinated substrates. In particular, DUBs play a critical regulatory role within protein degradation through the ubiquitin proteasome system (UPS). In this pathway, misfolded or unwanted proteins are first modified by ubiquitin chains, which targets them for degradation by the 26S proteasome. Once targeted, removal of ubiquitin chains from the modified substrate by DUBs must be achieved prior to substrate unfolding and subsequent degradation. UCH37 is a proteasome associated DUB, which is recruited to the proteasome by its binding partner, RPN13. Our lab discovered that UCH37Rpn13 complex exhibits hydrolytic activity on Lys48 linked branched ubiquitin chains. Investigations aimed at understanding how chain selectivity is achieved led to the finding that UCH37 preferentially binds Lys48 linkage, regardless of chain length and architecture. Our data suggest UCH37 could use Lys48 chains as a conduit in the search for Lys48 branch points. These findings are significant, as the UCH37 plays a major role in Lys48 chain-targeted degradation via the proteasome. Optimization of the BLIP-II Interaction with PBP2a as a Protein-Based Therapeutic Option for MRSA David Boragine 1 , Carolyn Adamski 1 , Shrenik Mehta 1 , Timothy Palzkill 1 1 Baylor College of Medicine (Houston, United States) Antibiotic resistance has manifested into a global health epidemic. One of the most widespread human pathogens, Methicillin-resistant Staphylococcus aureus (MRSA), contains the mecA gene that encodes a novel penicillin-binding-protein, PBP2a. Production of PBP2a by MRSA confers resistance to nearly all -lactam antibiotics by continued peptidoglycan cell wall synthesis, even at high concentrations of antibiotic. The transpeptidase domain of PBP2a shares structural homology with class A -lactamases, bacterial enzymes that inactivate -lactam antibiotics. Class A -lactamases are inhibited by protein-based inhibitors named -lactamase inhibitory proteins (BLIPs) and BLIP-II inhibits class A -lactamases with subnanomolar affinity. It was previously found that BLIP-II inhibits PBP2a in the low micromolar range (KD 1.5M), which is in contrast to BLIP-IIs potent inhibition of class A -lactamases. This primarily is due to a~44,000 times slower association rate of BLIP-II with PBP2a. Alanine scanning mutagenesis of BLIP-II revealed a majority of the mutations exhibited an increased association rate (ka) with PBP2a, while all mutations resulted in the normally fast dissociation rate (kd) to further increase. The alanine scan of BLIP-II identified binding hotspots for binding PBP2a and demonstrated that the binding interface between BLIP-II and PBP2a could be further optimized. In addition, a directed evolution approach with phage display affinity selection was used to identify a BLIP-II double mutation, N50A:Y113H, that enhanced the binding affinity to PBP2a 30-fold to a 50nM KD. These results suggest that BLIP-II can be further optimized and serve as a scaffold for developing potential PBP2a inhibitors. The E. coli -clamp increases the speed and processivity of DNA polymerase III during DNA synthesis. In order for the ring-shaped -clamp dimer to be loaded onto the DNA template, the -clamp and the fivesubunit -clamp loader complex must interact in an ATP-dependent manner. The clamp loader traps or induces an open conformation of the -clamp and loads it onto DNA. Upon ATP hydrolysis, the clamp loader dissociates from the clamp, which enables the recruitment of DNA polymerase III. Although these major steps are well known, the detailed mechanisms of how the subunits of the -clamp and -clamp loader interact in this clamp loading process remain poorly understood. In a previous paper, the N-terminal domain of the clamp loader subunit (called mini-) was crystallized with a -clamp variant I272A/L273A, which binds to mini-50 times stronger than it bins -clamp wild type (WT). The crystal structure of this complex revealed an extended conformation that resembles the open clamp. We hypothesize that the -clamp undergoes transient opening events and becomes trapped by the clamp loader in an open conformation. We designed -clamp variants with dimer breaking mutations and used isothermal titration calorimetry (ITC) to measure binding affinities of mini-to these -clamp variants. Mini-binds to variant I272A/L273A about 20-fold stronger than to the -clamp WT. Similarity, mini-interacts with variant I272A/ L273A/K74E stronger than with -clamp WT. These data suggest that mini-preferentially recognizes the open conformation of the clamp, which is more accessible in the destabilized -clamp variants. Li He 1 , Rachel Strodel 1 , Emily Cohen 1 , Erin Heim 1 , Daniel DiMaio 1 1 Yale University (New Haven, United States) Transmembrane domains (TMDs) usually adopt simple, predictable conformations (e.g. an -helix), but they can have profound effects on protein function and signal transduction. For example, the TMD of the mouse erythropoietin receptor (EPOR), which is essential for erythroid cell proliferation and differentiation, interacts with the TMD of gp55, a transmembrane protein from the spleen focus-forming virus. This interaction activates EPOR-induced signaling, resulting in cell proliferation. Here we developed a genetic system to isolate and characterize artificial transmembrane proteins that are small, simple, and free of post-translational modifications, yet still specifically bind to and activate EPOR. These artificial proteins, termed traptamers (transmembrane protein aptamers), are only 26 residues long and consist of certain sequences of leucine and isoleucine following an initiating methionine. Mutational analysis of one traptamer that activates both the human EPOR and mouse EPOR revealed that the placement of a single methyl group at certain positions guided it to specifically bind to and activate only the human EPOR or only the mouse EPOR. However, the specificity of another traptamer that specifically activates only the human EPOR but not the mouse EPOR cannot be altered by the placement of any single methyl group; rather this traptamer appears to be locked into a structure that can activate only the human EPOR. In addition, a serine at position 238 of the mouse EPOR TMD is important for traptamers to distinguish between human and mouse EPOR. Thus, minimal chemical differences in TMDs can determine the specificity of receptor binding and downstream biological consequences. Nramp family secondary transporters enable uptake of essential divalent transition metals. We use three crystal structures of Deinococcus radiodurans (Dra)Nramp at complementary stages of its transport cycle to illustrate the distinct conformational rearrangements of Nramp transporters and demonstrate that the metal-coordination sphere changes during the transport cycle. While metal transport requires cycling from outward-to inward-open states, efficient proton transport still occurs in outward-locked (but not inward-locked) DraNramp. We propose a model in which metal and proton enter the transporter via the same external aqueous pathway to the binding site, but follow separate routes to the cytoplasm. Additionally, we untangle DraNramps proton-metal coupling into two distinct phenomena: pH stimulation of metal transport and metal stimulation of proton co-transport. Surprisingly, metal elemental identity dictates co-transport stoichiometry, with manganese undergoing symport and cadmium undergoing uniport. We also demonstrate the overarching importance of the membrane potential to the kinetics as well as thermodynamics of Nramp transition metal transport. A conserved salt-bridge network adjacent to the metal-binding site imparts the observed voltage dependence and enables proton co-transport, two properties that allow Nramp to maximize metal uptake and prevent deleterious back-transport of acquired metals. We provide a new mechanistic model for Nramp metal-proton symport in which, in addition to substrate gradients determining directionality as in traditional secondary transport, synergy between protein structure and physiological voltage enforces unidirectional substrate movement. Philipp Schmidpeter 1 , Xiaolong Gao 1 , Jan Rheinberger 1 , Crina Nimigean 1 1 Weill Cornell Medicine (New York, United States) Cyclic nucleotide-modulated ion channels (CNG and HCN) are important in visual and olfactory signaling as well as in pacemaking activity in the heart and brain. So far, these channels mostly were studied in cellular systems and little is known about the purified proteins in defined environments. This, however, is necessary to understand their function and regulation in molecular detail. Here, we present an expression and purification protocol for SthK, a cyclic nucleotide-modulated channel from Spirochaeta thermophila, and establish this protein as a model for investigating gating of eukaryotic cyclic nucleotide-gated ion channels. SthK has high sequence homology with its eukaryotic counterparts and was reported to be activated by cAMP in patch-clamp experiments using Xenopus oocytes. We now are able to obtain large quantities of recombinant SthK channel protein from E. coli. Using negative-stain electron microscopy we demonstrate the homogeneity of our protein preparation. Radioactivity and fluorescence flux assays, as well as single-channel recordings in lipid bilayers, show that the protein is activated by micromolar concentrations of cAMP, which functions a partial agonist, and that channel activity is increased by depolarization. Unlike previous reports, we found that cGMP is also able to activate SthK, although significantly less efficient than cAMP, similar to eukaryotic CNG/HCN channels that show distinct sensitivities to different ligands. Binding assays reveal that cAMP and cGMP bind to SthK with similar apparent affinities, suggesting that the difference in channel activation is due to the efficacy with which each ligand promotes the conformational changes towards the open state. Specific proteins are triggered in response to cellular stress events. For example, when E. coli enter the nutrient-limited conditions of stationary phase growth, cyclopropane fatty acid (CFA) synthase is upregulated and rapidly converts nearly all alkenes found in unsaturated fatty acids of the inner membrane into cyclopropyl groups. It is then degraded by an unknown protease. To gain a better understanding of these events, we solved the crystal structure of E. coli CFA synthase at 2.1 Å. The enzyme is a dimer with each subunit composed of two closely-associating domains. Dimerization and inter-domain linkage were both found to be very important for catalysis. We also discovered that CFA synthase, a folded and thermodynamically stable protein, is degraded in vitro and in E. coli by the AAA+ protease FtsH, commonly thought to be a weak unfoldase. Our data support an avidity-based model whereby one subunit of CFA synthase binds to the inner membrane and allows the other subunit to catalyze cyclopropanation, after which the enzyme is degraded by FtsH. These findings yield new insight into the function and regulation of a soluble protein which acts at the lipid interface to remodel the inner membrane. The novel knob-socket (KS) model provides a construct to interpret and analyze the contributions of amino acid residues to the stability in -helical protein structures. Based on residue preferences derived from a set of protein structures, the KS construct characterizes intra-and inter-helical packing into regular patterns of simple motifs. Intra-helical interactions consist of three residue triangular motifs called sockets, which contribute to stability. For inter-helical interactions, a single amino acid knob from one -helix packs into a three amino acid socket within another -helix. Sockets are defined in three categories: (1) free, unpacked and favoring intra-helical interactions, (2) filled, packed and favoring inter-helical interactions, and (3) non, unpacked and disfavoring -helical structure. The three amino acid socket composition serves as a code that can be used to predict protein packing and by extension, can also be used to understand individual amino acid contributions to helical stability. The KS model was used in the de novo design of an -helical homodimer, KS1.1. Using SDM, KS1.1 point mutants were generated to correlate KS propensities with changes in -helical structure and stability. In the KS -helical model, each point mutation affects six sockets by altering the free/filled propensity values. By analyzing the changes in these propensities, KS based stability predictions were made for each mutant. Predicted values are compared to the experimentally determined stability of each peptide from denaturation studies as measured by circular dichroism spectroscopy. This study serves as a starting point to reveal how residue packing contributes to protein stability. Disulfide bonds are a major factor in protein aggregation, but their exact role in this context is not entirely clear. To address this problem, we have investigated the behavior of RRM2 domain from neuropathological protein TDP-43 under oxidizing conditions. We have found that disulfide-bonded dimers of RRM2 are not unstable per se. However, their ability to spontaneously (re)fold is compromised. Consequently, such dimers become vulnerable to large thermal fluctuations, which ultimately leads to their misfolding and aggregation. As a result, the dynamic equilibrium is established which involves globular monomeric form of RRM2 and the aggregate particles comprised of disordered peptide chains. This equilibrium is mediated by thiol-disulfide exchange reaction. Our in-vitro study of RRM2 relies on a combination of several experimental techniques. In particular, a specially adapted version of the NMR H/D exchange experiment reports on the status of RRM2 peptide chains within the aggregate particles. Furthermore, pulsed-field-gradient NMR experiment that makes use of the spectral signals from the proteins highly dynamic C-terminal tail yields the diffusion coefficient of the aggregate particles. Beyond that, MD modeling has been instrumental in elucidating the mechanism of misfolding. In this context, a new empirical algorithm has been developed which allows one to model formation of disulfide bonds within the framework of conventional MD simulations. Finally, we have also conducted cell culture experiments, demonstrating that cytotoxicity of cysteine-containing peptides can be attributed to their properties of (weak and weakly-specific) disulfide oxidoreductases. This work was supported by the RSF grant 15-14-20038. Conformational Plasticity of Late Embryogenesis Abundant (LEA) Proteins 1 and AfrLEA6 from group 6. We predict that LEA proteins from different groups initiate conformational transitions under contrasting conditions and that this group-specific behavior is relevant to their function(s). We tested this hypothesis by comparing AfLEA1.1 and AfrLEA6 using high-resolution microscopy techniques and measured conformational behavior in defined chemical environments. AfLEA1.1 binds to quaternary ammonium groups at a pH of 8.0 despite a theoretical isoelectric point of 9.37, and the purified protein elutes in three distinct fractions during anion-exchange chromatography. Desiccated AfLEA1.1 forms crystallike aggregates that are stabilized by adding 200 mM trehalose, a sugar that is present in the cysts of A. franciscana. AfrLEA6 does not form crystal-like aggregates during desiccation, but the relatively high hydrophobicity of the polypeptide causes a liquid-liquid phase separation at concentrations above of 2mg/mL (pH 7.2). This reversible phase separation is induced at physiological protein concentrations by the addition of monovalent ions, crowding by Ficoll-400, shifts in pH, temperatures below 5C, and desiccation in a solution mimicking the solute composition of the cytoplasm in embryonic cells of A. franciscana. Our results suggest that different LEA proteins undergo distinct and contrasting conformational transitions in response to desiccation-induced stimuli. This work was funded by NSF IOS-1659970/IOS-145706. Protein self-assemblies modulate protein activities over biological time scales that can exceed the lifetimes of the proteins or even the cells that harbor them. We hypothesized that these time scales relate to kinetic barriers inherent to the nucleation of ordered phases. To investigate nucleation barriers in living cells, we developed Distributed Amphifluoric FRET (DAmFRET). DAmFRET exploits a photoconvertible fluorophore, heterogeneous expression, and large cell numbers to quantify via imaging flow cytometry the extent of a proteins selfassembly as a function of its cellular concentration. We show that kinetic barriers limit the nucleation of ordered self-assemblies, and that the persistence of the barriers with respect to concentration relates to the structure of the assembly. DAmFRET illuminated nucleation mechanisms, distinguishing amyloid from non-amyloid polymers as well as one-step from multi-step nucleation. Supersaturation resulting from sequence-encoded nucleation barriers gave rise to prion behavior, and enabled a prion-forming protein, Sup35 PrD, to partition into dynamic intracellular condensates or to form toxic aggregates. Our results obtained from DAmFRET investigations of over two hundred self-assembling proteins suggest that nucleation barriers commonly exert temporal control over cytoplasmic inheritance, signal transduction, subcellular organization, and proteotoxicity. The tardigrade, a microscopic animal that survives a mystifying array of extreme conditions, synthesizes a unique family of intrinsically disordered proteins (IDPs) that protect its cellular components during desiccation.1 These IDPs are an order of magnitude more effective than trehalose, an FDA approved excipient, at protecting lactate dehydrogenase and the restriction endonuclease DpnI during desiccation and rehydration. Tardigrade IDPs even protect the activity of desiccated lactate dehydrogenase for over an hour at 95 C. We also show that desiccation-inactivated lactate dehydrogenase can be resurrected by resuspension in a solution of the tardigrade IDP, but not osmolyte controls. The evolutionary selection of tardigrade IDPs as protectants against desiccation likely arose from the optimization of this inherent ability to resurrect enzymes combined with the inertness of these proteins with respect to other biochemical pathways. 1) Boothby, Tapia, Brozena, Piszkiewicz, Smith, Giovannini, Rebecchi, Pielak, Koshland, and Goldstein (2017) Tardigrades use intrinsically disordered proteins to survive desiccation. Mol Cell 65 (6) Protein function depends on dynamically transitioning between folded conformational substates. A prime example of this is the homodecameric metabolic enzyme human glutamine synthetase (GS). Disease mutations in this enzyme lead to a rare recessive disease, glutamine deficiency. Specifically, the R341C disease mutation is hypothesized to be due to local unfolding of a helix necessary for a conformational transition during catalysis. We have assessed the GS disease mutants affect on stability, kinetics, and oligomeric assembly using small angle x-ray scattering, negative stain electron microscopy, and differential scanning flourimetry and found distinct mechanisms for individual mutants. Additionally, we have preliminary evidence that human GS may be regulated by the reversible assembly of filaments under conditions of increased manganese concentrations, which has been observed for GS in other species. These experiments will define the often elusive mechanism whereby specific defects can often be rescued by the addition of compensatory mutations. After characterization of the disease states of GS, we will test whether these mutations can be compensated for in a way not sampled by evolution through a deep mutational scan of human GS. Our experimental analysis will reveal how epistasis constrains the conformational ensemble of GS. E. coli guanine deaminse (EcGuaD) catalyzes the conversion of guanine to xanthine. EcGuaD is a member of the aminohydrolase superfamily. Here, we have determined the X-ray crystal structure of EcGuaD. We hope to use information from the structure to modify the enzyme to deaminate other nucleobases. Site directed mutants were generated and biochemically characterized for enzymatic activity. This work describes the in vitro characterization of the full-length form of human Beclin-1 (BECN1) that was prepared as a recombinant protein from Escherichia coli (3-5 mg/L culture). BECN1 is an essential component of macroautophagy, a highly conserved cellular recycling process of damaged cellular components (e.g., proteins, organelles) or pathogens in a bilayer vesicle. BECN1 has been linked to various cancers and neurodegenerative diseases and is also targeted by numerous viruses, including HIV and Herpes Simplex Virus 1 (HSV-1), as a means to evade host defense mechanisms. The inactive BECN1 homodimer is stabilized by interactions with Bcl-2, a pro-survival protein in the apoptotic pathway. Disruption of this complex by an unknown mechanism enables BECN1 to become a component of the phosphatidylinositol-3 kinase complex that initiates autophagy. Consistent with the literature, full-length BECN1 interacts with Bcl-2 (KD = 4.3AE1.2μM) and binds to lipid vesicles. Our analysis also shows that the full-length protein exists a soluble homodimer in solution with a sub-micromolar KD (~0.45μM) that is significantly tighter than published values of the isolated coiled-coil domain (48μM). The coiled-coil region of BECN1 is proposed to control dimerization, but a construct lacking the membrane-binding BARA domain exhibits a 3.5-fold weaker homodimer KD than the full-length protein. This result indicates that both the BARA and coiled-coil domains contribute to formation of the homodimer. Mutagenesis of the interface of the coiled-coil and BARA domains in the full-length protein also weaken the homodimer KD by~5-fold, supporting that these structures interact to stabilize the homodimer. 16 Intracellular fluid-like RNA-and protein-rich granules (RNP granules) are primarily formed by Liquidliquid phase separation (LLPS) of low-complexity intrinsically disordered protein domains (LCDDs). The arginine-rich (R-rich) LCCDs are ubiquitous in eukaryotic RNA binding proteome, act as multi-valent RNA/protein binding modules for homotypic and heterotypic phase transition, and implicated in c9orf72-related Amyotrophic lateral sclerosis disease etiology. What is the mechanism by which the phase transition dynamics of R-rich LCDDs are regulated? Recently, we showed that RNA can modulate their phase behavior by controlling both droplet assembly and dissolution (1) . Monotonically increasing the RNA concentration initially leads to droplet assembly by complex coacervation and subsequently triggers an LCDD charge inversion, which promotes disassembly. Using designed and naturally occurring R-rich LCDD sequences as well as the stress-granule associated protein FUS, here we evaluate the robustness of the RNA-mediated reentrant phase transition. Combining biophysical experiments and polymer physics theories, we show that RNA controls (a) mesoscale droplet dynamics, (b) droplet viscoelasticity, and (c) the condensed phase morphology via charge regulated electrostatics. Importantly, if the homotypic protein-protein and/or RNA-RNA interactions are substantial, further complexities are observed in the phase behavior of the system, including the formation of distinct condensed phases (droplets) displaying orthogonal physical and functional properties. Together, our experiment and freeenergy surface modeling suggest a remarkable degree of plasticity in the RNP-granule phase behavior, manifested by the interplay between homotypic and heterotypic interactions. The transfer of an acetyl group from Ac-CoA to an acceptor amine is catalyzed by Gcn5-related Nacetyltransferases (GNATs). Despite the agreement that this acetylation process proceeds through an ordered sequential mechanism, the role of the acetyl group in driving this mechanism remains elusive. Using NMR spectroscopy, ITC, and kinetic experiment, we show that the position of the acetyl group during catalysis regulates protein conformational dynamics. Part of the substrate binding site switches from a stable-ordered state in the presence of acetyl-CoA to a conformational labile ensemble of structures in the presence of CoA which may allow for the ordered binding of substrates and release of products. HIV reverse transcriptase (RT) is an essential enzyme that is the target of about half of all approved anti-AIDS drugs. An NcRTI (nucleotide-competing RT inhibitor) may block the binding of nucleotides to RT, rather than blocking DNA chain elongation, and could work synergistically with both nucleoside and non-nucleoside RT inhibitors (NRTIs and NNRTIs, respectively). The discovery of indolopyridone-1 (INDOPY-1) and its biochemical characterization provided a path towards the development of NcRTIs as a new potential class of RT drug. However, the lack of structural information regarding INDOPY-1 binding to RT/nucleic acid hindered further progress. Here we report the crystal structure of HIV-1 RT bound to a 38-mer hairpin template-primer DNA aptamer (Apt) and INDOPY-1 at 2.4 Å resolution, obtained by soaking binary RT/Apt crystals with the compound. The structure reveals that INDOPY-1 is able to intercalate between the last nucleotide base-pair in the 3'end of the primer and the adjacent template n+1 nucleotide (5 direction) and displays contacts with RT residues Arg72 and Tyr115. Besides, INDOPY-1 binding forces the latter nucleotide base to shift towards the fingers subdomain. This open conformation would clash with this subdomain in pretranslocated complexes, characterized by its closing upon incoming nucleotide binding, explaining the preference of INDOPY-1 to interact with post-translocated state complexes. The ternary complex also provides a rationale for the distinctive resistance mutation profile of INDOPY-1 with respect to NRTIs. Overall, this structure provides a foundation for designing and developing novel compounds to add to the anti-AIDS drugs toolbox. The dimeric protein, CISD2, belongs to a unique family of [2Fe-2S] cluster containing proteins (CISD proteins), which are implicated in a variety of disorders including type-2 diabetes, Wolfram syndrome 2, and neurodegeneration. Localizing to membranes of the mitochondrion, endoplasmic reticulum, and mitochondrial-associated ER membranes, CISD2 has been linked to autophagy and mitochondrial bioenergetics, but the specific mechanism(s) of action remain ill-defined. Further, CISD2 has been demonstrated to bind the anti-diabetes drug pioglitazone, but the therapeutic impact of binding remains uncharacterized. To investigate the potential function(s) of CISD2 in human hepatocellular carcinoma cells (HepG2) the protein was overexpressed and respirometry was utilized to assess mitochondrial performance. Surprisingly, increased CISD2 levels enhanced cellular oxygen consumption by 86% above control, however no differences were detected during chemically uncoupled respiration. To shift metabolic flux from glycolysis to oxidative phosphorylation, glucose was replaced by galactose as the primary carbon source. Under these conditions, we observed that both control and CISD2 overexpressing cells displayed increased routine respiration, but cells overexpressing CISD2 had 64.5% higher chemically uncoupled respiration than non-transfected controls. Permeabilized cells also demonstrated significantly higher uncoupling rates compared to control regardless of the substrate used. Finally, molecular docking studies were used to investigate the pioglitazone-CISD2 interaction and 30 uM pioglitazone was introduced during respirometric studies and did not negate the enhanced respiratory capacity. Together our results suggest that CISD2 is a critical, yet often overlooked regulator of mitochondrial bioenergetics and may impact the supply of reduced redox equivalents to the electron transport system. Some time ago, we developed a pair of reporter proteins called dual split proteins to monitor membrane fusion. Each DSP (DSP1-7 and DSP8-11) is a chimera of split GFPopt (split between the 7th and 8th betastrands) and split Renilla luciferase (RL). DSP recovers both GFP and RL activities upon self-association mediated by split GFPopt. Recently, superpositively charged GFP (sc(+)GFP) has been described. Here we replaced the GFPopt domain of DSP with sc(+)GFP and examined the outcome. Despite the expected repulsion of positively charged GFP domains, we observed both green fluorescence and RL activities upon co-expression of sc(+)GFP version of DSPs (scDSP1-7 and scDSP8-11) in transfected cells. This suggested that scDSPs were able to self-associate themselves. However, different from DSP, scDSP showed dots-like nuclear localization in the transfected cells, and the RL activity was much lower than that of DSP. The analyses of RL with inserted sc(+)GFP, which mimics the reassociated scDSP, also showed the lower activities and indicated that the lower RL activity of scDSP was not solely due to poor self-association. Since in vitro translated scDSP also showed the low RL activity, the nuclear localization of scDSP was unable to account for the low RL activity. Our data suggest that the presence of superpositive charge near RL may negatively affect the RL activity. Because co-expression of split scGFP1-7 and scGFP8-11 did not recover green fluorescence, the self-association mechanism of scDSP1-7 and scDSP8-11 remains elusive. The induction of potent antibody responses towards key neutralization epitopes is deemed necessary to solve long-standing challenges in vaccinology. The inherent complexity of B-cell responses and poorly understood phenomena, such as immunodominance of distracting epitopes, present major obstacles to focus antibody responses. Thus, synthetic immunogens are seen as a promising strategy to elicit epitope-specific antibodies. To explore their full potential, we developed a computational protocol to extract multi-segment viral epitopes from their native protein and stabilize them in synthetic scaffolds. Our design pipeline consists of an initial step to build secondary structure elements around the epitope, followed by in silico folding, sequence design and assembling of connecting loops. Iterative rounds of design, biophysical characterization and in vitro evolution have yielded a panel of four synthetic proteins presenting major antigenic sites from the Respiratory Syncytial Virus (RSV) fusion protein. These immunogens showed broad reactivity against several human RSV neutralizing antibodies, suggesting they could potentially activate a relevant subset of B-cells in vivo. Mouse immunization studies revealed superior elicitation of epitope-specific antibodies as compared to the native viral protein. Importantly, the in vivo studies showed that single immunogens and cocktail formulations elicited RSV neutralizing activity. Currently, we are elucidating rules and principles of such a synthetic vaccine approach using an indepth immunological analysis. We foresee that our RSV-based studies will be broadly applicable to overcome limitations of other vaccine development efforts. Our methodological pipeline to design novel proteins with embedded complex structural motifs is generally applicable for the design of functional proteins. Tankyrases (TNKS) are members of the poly ADP-ribose polymerase (PARP) enzymes family. Due to their proven involvement in cancer development, and specifically Wnt signalling, TNKS have become attractive therapeutic targets. Unlike conventional small-molecules targeting the catalytic PARP domain, which do not effectively distinguish between TNKS proteins and other PARP family members, we made a series of molecules designed to specifically inhibit the TNKS proteins by targeting their unique substrate-binding domains. To this end we grafted a TNKS-binding sequence onto a repeat-protein scaffold. This TNKS-binding epitope was repeated up to twelve times within this scaffold, generating a series of multivalent molecules, including both monomeric and trimeric display. Taking our cue from nature, the rationale behind this design approach was to enhance the binding affinity of the engineered protein constructs for TNKS, which is itself multivalent. Data will be presented showing the successful suppression of Wnt-dependent transcription by our TNKS-binding molecules, with particularly potent effects achieved by those that are multi-valent. An alternative method, inhibiting TNKS by targeting it for degradation, was also explored and shown to produce similar results. Aflatoxin-oxidase (AFO), a newly discovered oxidase isolated from Armillariella tabescens, was reported to perform aflatoxin B1 (AFB1) detoxification through breaking the bisfuran ring of AFB1. However, based on sequence alignment, we found that AFO shares high sequence identities with dipeptidyl peptidase III (DPP III) family members. To understand the functions of AFO, we determined its crystal structures in the absence and presence of zinc, copper ion, and employed HPLC to test if AFO could cleave the substrates of DPP III. Our structures reveal that AFO contains the classic DPP III activity center and the HPLC results further confirm that AFO possesses the dipeptidyl peptidase activity. Therefore, AFO should belong to DPP III family. Interestingly, unlike reported classic DPP III structure that has a large domain movement upon substrate binding, the AFO structures all adopt the closed conformation, independent of substrate binding. This conformation characteristic of AFO may be related to its enzyme activities. Taken together, our results demonstrate that AFO is a dual activity enzyme with both aflatoxin-oxidase and dipeptidyl peptidase activities and its unique conformation feature expands our understanding on the mode of reaction for this enzyme family. Deep mutational scanning is a widely-used method for multiplex measurement of the functional consequences of protein variants, greatly enhancing our ability to probe sequence-function relationships. Deep mutational scanning has many applications, including understanding protein evolution, exploring protein structure, improving protein function, and comprehensive interpretation of variants found in diseaserelated proteins. To improve upon previous approaches and facilitate comparison between datasets, we have developed a unified statistical framework for analyzing deep mutational scanning data that generates error estimates for each measurement, capturing both common sources of experimental error and consistency between replicates. We applied our model to diverse datasets with different targets and experimental designs comprising many hundreds of thousands of variants, and demonstrated its superiority in removing noisy variants and conducting hypothesis testing. We implemented our model in Enrich2, easy-to-use software that can empower researchers analyzing deep mutational scanning data. Enrich2 is extensible, providing a plugin-based interface for new scoring methods that allows data analysis to keep pace with the rapid advances in experimental methodology. To help make the growing number of deep mutational scanning datasets accessible, we have also created MAVEDB, the Multiplexed Assay of Variant Effect DataBase. MAVEDB allows researchers to upload full deep mutational scanning datasets, including methods and metadata such as keywords and relevant references, and assigns stable accession numbers suitable for publication. MAVEDB also enables users to download these datasets in a standard format, facilitating a new generation of modelling and machine learning applications. Together these tools provide necessary infrastructure for this growing field. E-cadherin is a major component of adherens junctions on cell surface. SNX16 is a unique member of sorting nexins that contains a coiled-coil (CC) domain downstream of the PX domain. We report here that SNX16 regulates the recycling trafficking of E-cadherin. We solved the crystal structure of PX-CC unit of SNX16 and revealed a unique shear shaped homodimer. We identified a novel PI3P binding pocket in SNX16 that consists of both the PX and the CC domain. Surprisingly, we showed that the PPII/2 loop, which is generally regarded as a membrane insertion loop in PX family proteins, is involved in the Ecadherin binding with SNX16. We then proposed a multivalent membrane-binding model for SNX16. Our study postulates a new mechanism for coordinated membrane binding and cargo binding for SNX family proteins in general and provide novel insights into recycling trafficking of E-cadherin. Peptide Grafting into a Repeat Protein Imparts Low Nanomolar Affinity for Keap1 Sarah Madden 1 , Albert Perez-Riba 1 , Laura Itzhaki 1 1 Department of Pharmacology, University of Cambridge (Cambridge, United Kingdom) The grafting of known binding peptides into stable protein scaffolds can impart increased peptide stability as well as constrain the grafted peptide in its bioactive conformation. However, it can be difficult to predict the effect of grafting on the stability and solubility of the protein scaffold and on the affinity of the functionalised protein for its target. Consensus-designed repeat proteins are hyper-stable and can be produced in exceptionally high yields in E. coli, making them ideal for protein engineering projects and for exploitation as biotherapeutics. Here we have grafted a nuclear factor erythroid-2-related factor (Nrf2) peptide onto a loop of the repeat-protein scaffold, enabling binding to Kelch-like ECH-associated protein 1 (Keap1). We then systematically modified the design of the functionalised repeat proteins in order to maximise their stability, solubility and binding affinity for Keap1. This optimisation process yielded highly soluble repeat proteins with melting temperatures above 70oC and Keap1-binding affinities in the low nanomolar range. This study explores the scope of repeat proteins for functionalisation and provides a systematic approach to the optimisation of the stability, solubility and binding affinity of functionalised proteins that could be applied to other scaffolds amenable to peptide grafting. Proteins must be stable in their folded, native state to carry out their function but thorough understanding of the sequence determinants of stability has remained elusive. To understand how stability is encoded in sequence, we surveyed the effects of a disulfide bridge on the energy landscapes of two homologous enzymes involved in antibiotic resistance, TEM-1 and CTX-M-9. The homologs have high structural similarity but differ in stability and in the number of states populated at equilibrium: CTX-M-9 exhibits a two-state denaturation curve, whereas TEM-1 has an additional intermediate state (I). We hypothesized that a disulfide in the alpha domain was a major underlying determinant for these differences. To test this hypothesis, we removed the disulfide bridge from TEM-1 and introduced a disulfide bridge at the same location in CTX-M-9 and then determined the variants stabilities by monitoring denaturation through fluorescence and circular dichroism (CD). As predicted, removing the disulfide from TEM-1 eliminated the intermediate and adding a disulfide bridge to CTX-M-9 introduced an intermediate state. Unexpectedly, however, the TEM-1 and CTX-M-9 intermediates appear structurally different. The TEM-1 I is partially unfolded whereas the CTX-M-9 I shows the same secondary structure as the native state but with different intrinsic fluorescence. We are developing structural models for the two intermediates through strategic mutagenesis of native tryptophan residues and fluorescence measurements. By uncovering the sequence determinants of two-state versus three-state folding in this family of enzymes, we hope to better understand the impact of energy landscape architecture on evolution of antibiotic resistance. Drug resistance is always a hindrance to disease alleviation whether in bacterial or viral infections or even cancers. Resistance has been observed for almost every kind of antibiotic in clinical use and antibiotic resistance has now been labeled as a global threat. Therefore, in order to maintain an upper hand in the drug resistance arms race, it is important to understand the factors that drive resistance. We are conducting a comparative study of mutations in TEM-1 beta-lactamase that underlie broad-spectrum resistance phenotypes in bacteria: M182T occurs naturally in many strains found clinically and M182S is a synthetic variant we have identified and studied in our lab but that has not been documented clinically. M182T is known to increase thermodynamic stability of TEM beta-lactamase but does not have a large effect on activity measured in vitro. The activity and stability parameters of M182S alone and in combination with other clinically relevant mutations were determined using UV-vis spectroscopy, circular dichroism, and intrinsic fluorescence measurements. We found that neither kcat/Km nor thermodynamic stability was significantly different between the enzymes bearing M182S or M182T, regardless of the background mutations. We suspect any fitness difference between the natural and synthetic variants would be very small and are testing this hypothesis through quantitative fitness competition assays. We believe that our findings will lend more insight into the drivers of evolution and drug resistance. As part of the first line of defense against a wide range of microorganisms, antimicrobial peptides (AMPs) are ideal candidates as novel antibacterial agents. AMPs can function via the undisruptive crossing of bacterial cell membranes with the targeting of an intracellular component. For example, buforin II (BF2) is believed to target nucleic acids within the cell. Most assays performed on AMPs focus on peptide activity under dilute buffer conditions, but this does not accurately represent intracellular environments, which are crowded with macromolecules. To this end, the effect of macromolecular crowding on BF2-DNA binding is studied through the use of molecular dynamics simulations and a range of computational models. Crowding is represented as a lowered outer dielectric constant, as spherical blobs, or as explicit protein crowders. These models are applied to frames taken over the course of a molecular dynamics simulation, allowing for better modelling of the dynamics and potential arrangements of crowders in solution. Initial analyses suggest particular buforin II cationic residues exhibit an increased contribution to binding in a crowded environment, independent of the crowding model used. However, the magnitude of this increased contribution appears to be dependent on the particular crowding model. Analyses further suggest that crowder placement is more important to the robustness of calculations than whether the BF2-DNA complex was extracted from a crowded or uncrowded simulation. In fact, random placement of crowding agents post-simulation was not able to reproduce the electrostatic contribution to the free energy as calculated from simulation, electrostatics-driven, placement of macromolecular crowders. Ana Torres-Ocampo 1 , Margaret Stratton 1 1 University of Massachusetts-Amherst (Amherst, United States) Ca2+/calmodulin dependent protein kinase II (CaMKII) is a serine/threonine kinase that organizes into a dodecameric or tetradecameric conformation. CaMKII is the most concentrated enzyme in dendritic spines, where its interaction with the NMDA receptor has been shown to be vital to long-term potentiation (LTP), the underlying cellular mechanism for memory formation. In humans, CaMKII is encoded by 4 genes: CaMKII, CaMKII, CaMKII and CaMKII. CaMKII, and have been shown to be important in a signaling pathway that regulates changes in gene expression during LTP. CaMKII and have been shown to undergo activation-triggered subunit exchange, which leads to a spread of CaMKII activation. Here, we measure subunit exchange in other CaMKII isoforms to investigate its importance in gene expression during LTP. To measure exchange, we label purified CaMKII with red and green dyes and measure FRET over time, comparing activated CaMKII (+ Ca2+/CaM/ATP) to unactivated CaMKII. We also perform a more detailed analysis at single-molecule resolution by attaching biotinylated CaMKII to a coverslip using PEG-biotin and we determine the number of molecules that have both red and green dyes, which we interpret as subunit exchange. We have tested subunit exchange between CaMKII/CaMKII and CaMKII/CaMKII using our FRET approach and we observe subunit exchange in both of these cases. The half-lives show that CaMKII/CaM-KII occurs slower (~70 minutes) compared to CaMKII/CaMKII (~20 minutes). This indicates that exchanging between hetero-oligomers is significantly slowed, potentially due to some minor differences between these isomers, which warrants further study. Background: S. mutans is a causative agent of dental caries and forms recalcitrant biofilms. It produces three amyloidogenic proteins, P1-C123, AgA, and Smu63c. Our objective is to develop tools to differentiate amyloid from monomeric proteins in situ within biofilms to evaluate environmental conditions that trigger amyloidogenesis. Methods: P1-C123 amyloid was induced by mechanical agitation of purified protein. Residual monomer and oligomers were removed with proteinase K and Triton X100, followed by ultracentrifugation to harvest protease-resistant fibrils. Purification of amyloid fibrils was confirmed by TEM. Murine immunizations with purified fibrils to produce amyloid-specific monoclonal antibodies are underway. Three fluorescence dyes, CDy11, BD-oligo, and MK-H4 (2016 JACS: 138(1):402-7) were tested for their ability to differentiate P1-C123, AgA and Smu63c amyloid from monomeric forms. Results: Purified P1-C123 fibrils visualized by TEM were distinct from the mat-like aggregates seen after amyloid induction, but prior to protease digestion. The three dyes tested, CDy11, BD-oligo and MK-H4, were each able to differentiate agitation-induced amyloid from P1-C123, AgA, and Smu63c monomers. CDy11 demonstrated the highest reactivity and specificity against all three amyloid preparations including the purified C1-123 fibrils. CDy11 and BD-Oligo were more effective at discriminating amyloid from monomer than conventional Thioflavin T dye uptake assays. Conclusions: An effective method was developed for purification of P1-C123 fibrils following amyloid induction in vitro. Fluorescent probes, particularly CDy11, were able to differentiate multiple S. mutans amyloid proteins from corresponding monomers and will be utilized in future studies of S. mutans biofilm development. 16 Double-stranded RNA-binding domains (dsRBDs) are commonly found in proteins that interact with dsRNA. Two varieties of dsRBD exist: canonical Type-A dsRBDs interact with dsRNA, while non-canonical Type-B dsRBDs lack essential RNA-binding residues and instead interact with proteins. In higher eukaryotes, the microRNA biogenesis enzyme Dicer interacts with a dsRNA-binding protein (dsRBP) that contains a conserved Type-B dsRBD. In humans, Dicer associates TRBP or PACT, while in Drosophila, Dicer-1 associates with Loquacious (Loqs). The Type-B dsRBD in each of these dsRBPs interacts with the helicase domain of Dicer. We report that the Type-B dsRBDs of Loqs, PACT and TRBP self-associate to form homodimers that have significant structural asymmetry. We have elucidated the 3D structures of the Type-B dsRBDs of TRBP and PACT. These structures reveal an asymmetric self-association mechanism that involves a parallel -strand at the homodimer interface. NMR analysis of the Type-B dsRBDs of Loqs, PACT and TRBP reveal that this asymmetry is conserved from flies to humans. Mutation of a single conserved leucine residue on the homodimer interface abolishes self-association in all three dsRBPs. Moreover, mutations that make TRBP more like PACT enhance self-association, whereas the reciprocal mutations in PACT reduce selfassociation. Lastly, we have determined that the Type-B dsRBDs of TRBP and PACT preferentially heterodimerise. Our data show that dsRBD-dsRBD interactions involving PACT, TRBP and Loqs utilize the same surface that is required for binding Dicer, which suggests that the dissociation of dsRBD-dsRBD interactions may be a key step in the assembly of a functional Dicer complex. CRISPR/Cas9, originally derived from bacterial adaptive immune systems, has recently been harnessed as a versatile and powerful tool for genome editing and gene regulation in many eukaryotic organisms and shows great promise in treating cancer diseases at a genetic level. Interestingly, bacteriophages encode CRISPR-Cas inhibitor proteins-"anti-CRISPRs"-to actively circumvent bacterial CRISPR immunity. Those natural Cas9-specific "anti-CRISPRs" present important tools that can be used to regulate CRISPR/ Cas9-mediated genome editing specificity. My research is to use biochemical and biophysical techniques to provide a better understanding of the fundamental mechanism of CRISPR/Cas9-based specific DNA targeting and editing, as well as to uncover viral anti-CRISPR mechanisms to further support its use for site-specific genetic control and therapeutic applications. To this end, I have solved a series of CRISPR/ Cas9 structures at the different stages of DNA target surveillance and EM structures of CRISPR-Cas9 bound to different anti-CRISPR inhibitor proteins using X-ray crystallography and single-particle cryoelectron microscopy. These structural studies provide not only a fundamental understanding of the molecular mechanisms of RNA-guided DNA targeting and cleavage by CRIPSR-Cas9 and viral anti-CRISPR mechanisms, but also a framework to develop more effective and precise CRISPR-Cas9 tool for biomedical research and therapeutic applications. The post-translational modification or proteins by ubiquitin and ubiquitin chains represents one of the most versatile signals employed by the cell. Ubiquitin (Ub) modifies substrates at lysine residues through a highly regulated enzymatic cascade involving three classes of enzymes. The process is also reversible, and is antagonized by a class of specialized proteases known as deubiquitinases or DUBs. Interestingly, one Ub molecule can be further modified by another Ub molecule resulting in the formation of polymeric Ub chains. These chains can adopt either linear or branched configurations. Recently, it has been demonstrated that branched Ub chains bearing a Lys48 Ub modification can act as potent signals for substrate destruction by the proteasome. The proteasome itself is a large multi-subunit protein complex that acts as the primary protein degradation machine in eukaryotic cells. This large complex contains 3 DUBs (RPN11, USP14 and UCH37) whose primary responsibility is to regulate protein turnover. Of these three DUBs, the role of UCH37 is the least studied. Here we use biochemical techniques and mass-spectrometry to show that UCH37 is a deubiquitinase that selectively hydrolyzes branched Ub chains bearing a Lys48 linkage. We show this in the context of apo-UCH37 as well as the proteasome bound enzyme, demonstrating a potential role for UCH37 regulating proteasomal activity. We further demonstrate that UCH37's catalytic activity is necessary for the proper degradation of substrates tagged with branched Ub chains bearing a Lys48 Ub linkage in cell culture. The unfoldase-protease ClpXP is an important regulator of protein homeostasis. ClpX is a ring-shaped AAA+ homohexamer that unfolds target proteins and translocates them into the ClpP protease for degradation. Previous work has probed the mechanisms of substrate recognition and unfolding by ClpX, but less is understood about how its subunits work together to promote efficient substrate processing. Here, we investigate the conserved linker regions that connect the large and small AAA+ domains and allow ClpX subunits to form a closedring motor. Using targeted disruption of individual linkers, we find that conformational changes triggered by ATP hydrolysis can propagate to neighboring subunits in either an N-to-C terminal or C-to-N terminal direction. By coupling hinge disruption to mutations altering ATP binding and hydrolysis, we determine that the direction in which ATP hydrolysis events propagate is biased by the nucleotide occupancy of each subunit. We also demonstrate that linker length and flexibility are optimized for efficient substrate unfolding. Additionally, by engineering variants of ClpX with missing linkers, we find that ClpX engages and translocates substrates inefficiently when freed from the conformational constraints imposed by the hinges. Together, these results support a model in which the hinges of ClpX facilitate efficient degradation by maintaining proper geometry between neighboring subunits, mediating an efficient mode of degradation wherein multiple subunits hydrolyze ATP in rapid succession. This model informs our understanding of ClpX as well as the larger AAA+ family of motor proteins, which play diverse roles converting chemical into mechanical energy across all domains of life. Recognition of single-stranded DNA (ssDNA) and RNA (ssRNA) is important for many cellular functions and a variety of ssDNA/ssRNA binding proteins (SSB) have evolved for specific binding with ssDNA/ssRNA to form stable complexes. Computational modeling of the specific recognition process and predicting their structure is challenging primarily due to i) inherent flexibility of ssDNA/ssRNA, and ii) heterogeneity in their binding energy landscape. We developed a physical interaction-based coarse-grained model to study SSB-ssDNA/ ssRNA interactions and predict their complex structures. The model uses only the SSB structure and does not require either ssDNA/ssRNA structure or binding site information. Apart from the electrostatic interactions between the positively charged residues and the negatively charged phosphate backbone, the pi-pi stacking interactions between nucleobases and aromatic residues are also included in the force field. Two major factors were tuned that can modulate sequence-specific recognition and binding stability-i) base-aromatic stacking strengths, and ii) flexibility of ssDNA vis-à-vis ssRNA. Applied to different SSB folds, the model could successfully predict their complex structures with respective ssDNA/ssRNA strands. Their native-like simulated conformations correspond to the minima in the binding energy-funnel; this minimum binding energy correlates with corresponding experimental binding affinities. Our results predict that the difference in flexibility of ssDNA and ssRNA plays the major role for the difference in their binding stability and specificity. Coarse-grained simulations are faster, and our findings suggest that this approach is appropriate to study SSB-ssDNA/ssRNA interaction, and can further be used to study complex events such as sliding of SSB along ssDNAs/ssRNAs. Purinergic gliotransmission occurs when astrocytes release ATP to modulate neuronal communication. However, this phenomenon is not well understood, and our goal is to engineer genetically-encoded fluorescent protein biosensors to study purinergic signaling in healthy and pathogenic models. We recently reported the first ratiometric FRET-based sensor that detects extracellular ATP. This first-generation sensor exhibits an affinity for ATP in the micromolar range, which is well suited to studying changes in extracellular ATP levels after stimulated release. We would also like to study fluctuations in basal levels of extracellular ATP, and therefore the goal of this project is to increase the sensors affinity for ATP to nanomolar concentrations. To do this, we mutagenized the sensors ATP binding domain, and we have identified mutants with nanomolar affinity in protein solution studies. We characterized these mutants with fluorescence spectroscopy in protein solutions and with microscopy studies in live cells. Ultimately, we plan to use these sensors to study ATP signaling in primary astrocyte-neuron co-culture and organotypic brain slices to directly visualize neuron activity-dependent release of astrocytic ATP. Deep Mutational Scanning of the Beta-2 Adrenergic Receptor Eric Jones 1 , Nathan Lubock 2 , Daniel Cancilla 2 , Megan Satyadi 2 , Rishi Jajoo 2 , Sriram Kosuri 2 1 UCLA (Los Angeles, United States); 2 UCLA Department of Chemistry and Biochemistry (Los Angeles, United States) G protein-coupled receptors (GPCRs) are principal mediators of cell signaling in humans. GPCRs are widely implicated in disease and targeted by roughly 30% of all FDA approved drugs. How their genetic variation affects ligand binding and signaling remains unknown on a comprehensive scale. The difficulty to interrogate GPCR structure through traditional methods further obfuscates this relationship. Deep mutational scanning presents a way to probe the fitness effect of every missense mutation possible for a protein and understand the contribution of each individual residue to function. We have leveraged advances in DNA synthesis, gene editing, and next-generation sequencing to create a platform able to measure the difference in drug-induced G-protein signaling of all beta-2 adrenergic receptor missense variants (~8000) in multiplex. Our assay enables us to identify broad structural rules for domains of the protein, constitutively active mutants, and how residue tolerance varies for both substitution from different classes of amino acids and the location within the 3D structure of the receptor. Blue light sensing A (blsA) is a blue light using flavin (BLUF) domain isolated from opportunistic pathogen Acinetobacter baumanii. BlsA is vital for A. baumaniis motility, biofilm formation and virulence. When blue light activates blsA [in the N-terminus], the protein interacts with its effector domain(s) [in the C-terminus] with implications in downstream signaling events. Photo absorption modifies the hydrogen bonds and the electrostatic interactions between flavin and the BLUF domain with consequent structural changes that are integral in the signal transduction. Spectroscopic data highlight involvement of residues methionine (94) and tryptophan (92) in the photoactivated signaling state of blsA. My goal is to solve blsAs structure using x-ray crystallography to determine the specific residues involved in the signaling state. Comparison of crystal structures of blsA (~1.8 Å) in the dark state and the photoactivated light state does not show movement of the residues glutamine and tryptophan in the two states. However, we detect a distinct conformational change of an alpha-helix the C-terminus-the effector protein binding terminus-of blsA. 16 Detailed mechanistic understanding of protein complex function can only come from insights into their three-dimensional structure. Traditional methods of protein structure elucidation remain expensive and labor-intensive. Chemical cross-linking coupled with mass spectrometry offers an alternative that has been gaining popularity, especially in combination with other experimental approaches. Here we report advances in method development, combining orthogonal cross-linking chemistries as well as improvements in search algorithms, statistical analysis and computational cost to achieve coverage of one unique cross-linked position pair for every 7 amino acids at 3% false discovery rate. This is achieved without any sample fractionation, enrichment, or isotopic labeling. We use our methods to model the complex between human Carbonic Anhydrase and murine Carbonic Anhydrase Inhibitor that has long resisted attempts of crystallographers. We show that the crosslinks are self-consistent, agree with existing literature, and use Rosetta to define the interaction interface at high resolution. We next apply our pipeline to the yeast 26S proteasome, identifying over 1600 unique cross-linked position pairs from just three mass spectrometer runs. We use integrative modeling to demonstrate that this level of cross-linking density is sufficient to reconstruct the subunit architecture of the regulatory particle into a low-resolution (30 angstrom) cryo-electron microscopy map without any additional structural information for the subunits. High-density constraints derived from cross-linking coupled to mass spectrometry are poised to make impact in unraveling the function of protein complexes which have been resistant to more conventional techniques. In this study solid-state NMR measurements on bacterial chemotaxis receptors in functional complexes are used to test proposed changes in dynamics between signaling states. Chemotaxis receptors, a tractable model for studying membrane protein signal transduction, enable bacteria to sense molecules from outside the cell and direct their swimming towards favorable environments. It is widely accepted that binding of attractant ligands causes a 2 Å piston displacement of a helix that extends through the periplasmic and transmembrane domains. However, it is not clear how the signal propagates 200 Å further to control activity of a kinase bound at the tip of the receptor, but the mechanism is thought to involve changes in dynamics within the cytoplasmic domain. We employed mobility-filtered solid-state NMR experiments to study dynamics of a receptor cytoplasmic fragment (U-13C,15N-CF) in functional complexes with the kinase CheA and coupling protein CheW. NMR of native-like, homogeneous arrays of these complexes (mediated by either vesicle binding or a molecular crowding agent) reveals regions with dynamics on the nanosecond or shorter time scale, which were assigned through a combination of chemical shift prediction and protein truncation approaches. We conclude that the connecting segment between the transmembrane and cytoplasmic domains is highly mobile and its mobility increases in the kinase-off state. Further NMR studies are in progress to identify regions with ms-timescale dynamics and any signaling-related changes in such dynamics, with the goal of understanding the role of dynamics in the signaling mechanism that controls kinase activity. Supported by GM120195. The activity of human UDP-Glucose Dehydrogenase is regulated by the conformation of a buried allosteric switch (T131-loop/6). In the absence of ligand, the allosteric switch is in an inactive E* conformation. Substrate binding induces the slow isomerization of the enzyme to the active E state, which can be observed as hysteresis in progress curves. Inhibitor binding favors adopting the low substrate affinity E state. In order for the allosteric switch to isomerize, the protein core undergoes a remarkable repacking that was hypothesized to be facilitated by large packing defects surrounding the T131-loop that are distinct between the E and E states. Here, we test this hypothesis by using RosettaVIP to design the A104L substitution (hUGDHA104L) that fills a packing defect in the E state. hUGDHA104L had a 13-fold lower affinity for inhibitor which bound non-cooperatively, supporting the hypothesis that packing defects are required for the E to E isomerization. The A104L substitution also abolished hysteresis, which was attributed to the E* to E transition. Crystallographic studies showed hUGDHA104L still adopted the E* state; however, the substitution improved the packing of the protein core. This suggests that hysteresis is not simply caused by the isomerization of the allosteric switch but is instead a result of core repacking. Based on this we propose hysteresis in hUGDH is caused by the high entropy of the E* state protein core and an associated energy barrier that must be overcome to adopt the low entropy active E state. Creatine Kinase [ CK EC 2.7.3.2] is a key enzyme in muscle metabolism, reversibly converting creatine phosphate (PCr) to creatine and ATP. PCr is an energy buffer when ATP levels fall during exercise or when oxidative phosphorylation is compromised. CK has been shown to be post-translationally modified by phosphorylation. We used a model system of hypoxia [severely dehydrated frogs] to study the phosphorylation control of CK during a naturally occurring stress state. Frogs that experience whole body dehydration have impaired blood circulation to the peripheral tissue that hampers oxygen delivery. This creates tissue hypoxia that the animals naturally endure. As follows, CK from the dehydrated muscle showed a significantly higher Vmax for phospho-creatine compared with control CK. Immunoblotting to probe purified CK also demonstrated that CK from the dehydrated muscle has a decreased relative phosphoserine content (25% decrease) compared to CK from control muscle. Isolated CK from dehydrated muscle also had a higher melting temperature (Tm= 49.5 C) than CK from control (Tm= 47.7 C) when thermostability was tested using differential scanning fluorimetry. Drug resistance is a pertinent medical problem associated with drug effectiveness and antibiotic treatment. Bacterial resistance to antibiotics is linked to the function of multidrug efflux transporters, which expel a broad range of toxic substances, including drugs, out of bacteria. AcrB (acriflavine resistance B) is the main representative of the resistance nodulation cell division (RND) family of efflux transporters and plays a major role in the antibiotic resistance phenotype of E. coli. Although AcrB is well studied by traditional structural tools, the impact of mutations, drug, and/or lipid binding on its conformational states and dynamics remains largely unknown. We study both wildtype AcrB and a mutated form responsible for clinically relevant antibiotic resistance and altered substrate specificity. Native MS and IM-MS experiments were used to optimize AcrB purification procedures enabling a completely delipidated, but correctly folded (as judged by IM-MS and circular dichroism), detergent solubilized AcrB to be attained. Moreover, we developed native nanodisc conditions which enabled AcrB to be interrogated within a native lipid environment, permitting lipid and drug binding synergy to be studied. Subsequently, HDX-MS was used to study the AcrB conformational dynamics and ligand binding under these synergistic conditions. Here, we present the ability of advanced structural mass spectrometry (MS) methods to inform on the synergistic effects of drug, antibiotic, and lipid binding on the structure and function of a multidrug efflux transporter involved in bacterial antibiotic resistance. Approximately 44% of human proteins contain intrinsically disordered (ID) peptide segments >30 residues in length, with the majority having no known function. Here we show that an ID segment (ID-tail) in UDP--D-glucose-6-dehydrogenase enhances the affinity of an effector binding site by modifying the dynamics of an allosteric network. The function of the ID-tail does not depend on its sequence or electrostatics. Instead, changes in effector binding affinity can be accurately predicted based on segment length alone. Using a combination of transient state kinetics, hydrogen-deuterium exchange mass spectrometry, thermal denaturation studies, computer simulation and crystal structure analysis, we show that the ID-tail generates a destabilizing force that can alter the energy landscape of a folded protein to favor an allosteric response. Our analysis suggests that any disordered segment attached to folded protein will generate a similar destabilizing force. Thus, the persistence of intrinsic disorder in the proteome may reflect the evolution of low complexity structural elements that can tune a specific protein function. In humans, uridine monophosphate synthase (UMPS) catalyzes the last two reactions in the pyrimidine de novo biosynthetic pathway, with N-terminal OPRT domain and C-terminal OMPDC domain. OMPDC has been reported to dimerize in its active state, whereas the oligomerization and structural characterization of OPRT and UMPS are not known. To understand the regulation of proteins in this pathway, we are investigating the structure of UMPS and its spatio-temporal localization in the cell. Wild type and mutants UMPS were expressed in BL21 E.coli and purified with affinity chromatography and size exclusion chromatography for structural and biochemical studies. UMPS mutants are domain inactive but structurally conserved constructs, E123Q (OPRT inactive) and D312N (OMPDC inactive) to help with crystallization and kinetics measurements. Our results show that WT UMPS and mutants equilibrate in two different oligomeric states, but with different amount in each state, indicating that the active sites might contribute to the different oligomeric states. In cells, UMPS has been reported to be a cytosolic protein, however, its localization has not been observed during increased pyrimidine nucleotide demand. Here we show that FLAG-tagged UMPS does not only localize in the cytosol, but also in the nucleus under both nucleotide rich and depleted conditions in HeLa cells. UMPS is not only an essential protein that can cause devastating diseases when defective, but is also an attractive therapeutic drug target for cancer and autoimmune diseases among others, therefore, structural characterization and cellular localization of UMPS is crucial to determine the regulatory mechanism of the pathway. The extracellular matrix (ECM) protein collagen provides structural integrity to all connective tissues and is extremely biologically active, interacting with numerous receptors and matrix molecules to achieve cellular functions. In the ECM, collagen assembles into highly ordered fibrils, and many of the receptor interaction sites are buried away from the interaction surface. Mechanisms by which collagen receptors recognize and access their binding sites in the collagen fibril are poorly understood, and the extreme size and complexity of collagen fibrils creates a challenge for atomic-level studies. Using an integrative computational and experimental approach, we investigate weak collagen-receptor interactions, with a focus on collagen-integrin interactions, which are critically important for platelet aggregation and hemostasis. First, we present an all-atom molecular dynamics simulation of a solvated collagen fibril model with an explicit interaction surface. Supported by experimental atomic force microscopy imaging, we observe that the fibril surface undergoes nanosecond fluctuations and samples alternate conformations that expose cryptic receptor binding sites. Second, in order to gain detailed atomic-level insight into the interactions between collagen triple helices and integrin, we use collagen mimetic peptides (CMP) that model native and collagen disease states. Using nuclear magnetic resonance spectroscopy, biological assays, and computation to directly probe integrin binding to biologically active CMPs, we show that integrin adhesion depends critically on the local conformation and dynamics of the triple helix at the recognition site. These findings enhance our understanding of collagen as smart fibrils, the dynamics of the surface acting as a gatekeeper to regulate critical protein interactions. 16 The dimeric proteins CISD1 and CISD2 belong to a unique family of iron-sulfur cluster containing proteins and are implicated in a variety of disorders including type-2 diabetes, Wolfram syndrome 2, and neurodegeneration. CISD proteins are directly or indirectly involved in cellular energy homeostasis, but the specific mechanism(s) of regulation remain still ill-defined. Lack of mechanistic explanation is in part due to the fact that interacting proteins and ligands are still mostly uncharacterized. We demonstrated that the soluble portion of CISD1 and CISD2 have one tyrosine and tryptophan residue per monomer. Excitation at ex = 280 nm results in fluorescent emission of both tyrosine and tryptophan, while excitation at ex = 295 nm generates only tryptophan emission. By monitoring the fluorescent emission of CISD1 and CISD2, we observed that certain ligands (e.g. NADH, FMN) quench both ex = 280 nm emission and ex = 295 nm emissions, meanwhile other ligands (e.g. NAD+ and ATP) quench only the ex = 280 nm emission. These observations suggest either one binding site is present and quenching of the ex = 295 nm emission is ligand dependent or two binding sites are present (coupled or uncoupled). Dissociation constants indicate that binding of NAD+ to CISD1 and CISD2 and binding of ATP and ADP to CISD1 is within the physiological concentration range. This demonstrates that CISD1 and CISD2 could be governing or responding to the cellular redox state by binding to a variety of compounds that are directly or indirectly related to the cellular energy state. implicated in breast and ovarian cancer progression due to its immunosuppressive properties. Gal-7 can trigger T-cell apoptosis via a glycan binding site (GBS) that recognizes T-cell glycoreceptors. Previously, drug development favored GBS inhibition to limit Gal-7 binding to immune cells. However, this strategy was met with limited success due to low GBS inhibitor specificity and high GBS similarity among different galectin homologs in the cell. Since the immunosuppressive activity of Gal-7 appears to require a homodimeric form of the protein, perturbing dimer formation could alter its biological function and act as a novel strategy to increase drug specificity and lower off-target binding. We have developed a structural protein-protein interaction strategy to dissect the molecular importance of the homodimeric architecture in Gal-7 function. In this study, we present X-ray crystallography structures of Gal-7 mutants at the homodimeric interface, in absence and presence of -galactoside. The thermal stability of these mutants and their binding affinity towards known Gal-7 ligands were evaluated using circular dichroism and isothermal titration calorimetry, respectively. Finally, the biological activity of these Gal-7 interface mutants was tested on Jurkat T-cells by monitoring their ability to induce apoptosis. These results will be an asset in the design of new galectin inhibitors targeting the homodimeric interface of Gal-7. The major histocompatibility complex (MHC) is a set of genes that play a key role in the vertebrate adaptive immune system in which MHC presents the antigen to be recognized by T cell and activate immune responses. In humans, the corresponding genes encode proteins termed Human Leukocyte Antigens (HLAs). Their expressed cell surface glycoproteins are divided into two classes: class I (MHC I) and II (MHC II). Although MHC I and MHC II have very different chemical compositions, they both adopt a similar three-dimensional structure: two membrane-proximal Ig-like domains supporting an antigen-presenting groove. This groove is composed of a large eight-stranded sheet that forms a platform, topped by two helices that flank the bound peptide. Despite the overall similarity of antigen-presenting architecture, there is a striking distinction in the preferred peptide length loaded onto the two classes of MHC molecules. MHC I tends to present peptides of 8-10 amino acid residues in length, whereas MHC II can accommodate peptides of almost unlimited length. Here, through structural analysis of an immunodominant HIV-1 Gag epitope TW10 (TSTLQEQIGW) and its mutants presented by the HLA-B*5801, a class I molecule derived from patients bearing potential HIV-1 protective HLA class I allele B*5801, we report a surprising observation that the antigenic peptide can be presented in an unconventional fashion which is different from what we know from previous immunology knowledge. These structures also explain the functional differences in T cell response to antigenic peptide mutations. Double-stranded DNA viruses package their genomes into pre-assembled protein capsids, which is remarkable considering the various forces that resist compaction of DNA. Therefore, viruses must encode for highly efficient molecular motors that convert the energy released during ATP hydrolysis into DNA translocation. The packaging motor in bacteriophage phi29 is well-studied. Single molecule experiments show that it is one of the most powerful molecular motors in nature, and that it operates via a highly coordinated mechano-chemical cycle wherein 10-bp DNA translocation bursts are followed by dwells where no translocation occurs. In addition to the ATPase, the phi29 motor also consists of a portal protein, and a virally coded structural RNA (pRNA). The only motor component whose structure is currently unknown is the Cterminal domain of the ATPase (CTD). We thus determined the structure of the CTD by NMR spectroscopy. The structure is reminiscent of the RNase H-nuclease fold. While gp16 does not cleave its genome like other bacteriophages, this structural similarity suggests the CTD retained nuclease binding and translocating functions. Fitting the CTD into cryoEM density of the entire motor complex completes the first atomic structure of a functional DNA packaging motor. The CTD may simultaneously interact with DNA and pRNA via two clusters of basic residues on opposite sides of a central beta-sheet. Together with previous biochemical, biophysical, and single molecule results, this structure provides insight into how the activities of motor subunits are coordinated to translocate DNA, and how the motor transitions between burst and dwell phases. Alpha-galactosidase A is a homodimeric glycoprotein that hydrolyses the terminal alpha-glactosyl moieties from glycolipids and glycoproteins. The primary substrate is ceramide trihexoside. Fabry disease, a lysosomal storage disorder, is caused by mutations in the enzyme that result in an inability to process its substrate. Several companies produce and purify Alpha-galactosidase A at commercial scale to provide the enzyme as a protein replacement therapy for Fabry disease. Advancements in the purification process are actively being developed to facilitate cost-effective and time saving purification schemes. A novel biomimetic small molecule based affinity purification step has been developed, resulting in a significant simplification of the purification process. Experimentation that focused on the biomimetic small molecule binding capacity, stability, reusability, and scalability demonstrated it is suitable for process development toward commercial manufacturing. Utilization of this affinity resin would result in a purification process with fewer chromatography steps, resulting in significant production costs reduction and reduce process times. TDP43 is an RNAbinding protein active in splicing that concentrates into membraneless ribonucleoprotein granules and forms aggregates in amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Although best known for its predominantly disordered Cterminal domain which mediates ALS inclusions, TDP43 has a globular Nterminal domain (NTD). Here, we show that TDP43 NTD assembles into headtotail linear chains and that phosphomimetic substitution at S48 disrupts TDP43 polymeric assembly, discourages liquidliquid phase separation (LLPS) in vitro, fluidizes liquidliquid phase separated nuclear TDP43 reporter constructs in cells, and disrupts RNA splicing activity. We present the solution NMR structure of a headtotail NTD dimer comprised of two engineered variants that allow saturation of the native polymerization interface while disrupting higherorder polymerization. I will also present our latest data on the dynamics of NTD and the role of NTD polymerization in the self-assembly of full length TDP-43. These data provide structural detail for the established mechanistic role of the wellfolded TDP43 NTD in splicing and link this function to LLPS. Parkinsons disease is the worlds second most prominent neurodegenerative disease that affects and depletes basic motor and non-motor sensory mechanisms in humans. The mutations in PARKIN (PARK2 gene), a RING-between-RING E3 ubiquitin ligase, along with PINK1 (PARK6 gene), a protein kinase, are responsible for autosomal-recessive juvenile Parkinsonism [1] . PARKIN is auto-inhibited or inactive in the cytosol; however, phosphorylated-Ub binding to PARKIN or phosphorylation of Ser65 in the ubiquitinlike domain of PARKIN leads to conformational changes, activating PARKIN [1] . The onset of mitophagy occurs when PINK is no longer imported into depolarised or dysfunctional mitochondria for degradation, instead, accumulated on mitochondrial outer membrane where PARKIN is recruited to ubiquitinate mitochondria [1] . Having understood the mechanism, we utilized phage-display technology to design an Ubiquitin (Ub) library and select against full-length PARKIN and PARKIN in the absence of the ubiquitin-like domain (77-464aa). Subsequent rounds of panning with Ub-library against two constructs of PARKIN gave rise to approximately 40 unique binders. In the next steps, we aim to establish a high-throughput screening method based on the turn-over of mitochondria by autophagy that allows us to interrogate the pool of binders with regards to their modulatory effect on PARKIN E3 ligase activity. Loss of protein stability and resulting degradation has been found to underlie pathogenicity for missense variants in individual disease-associated proteins. At the same time, advances in genome sequencing have provided us with an avalanche of variant data across the human proteome, yet especially subtle variants remain hard to interpret. Here we set out to assess whether loss of stability is a general mechanism underlying pathogenicity, and whether biophysical stability predictions can distinguish pathogenic from neutral variation. We selected 21 proteins associated with different classes of disease and inheritance models. Common variants observed in the population were used as a proxy for neutral variation. We found that common variants have near-neutral stability, while some rare variants show loss of stability, and disease-associated variants are significantly destabilized on average. Overall, stability calculations successfully identified 68% of pathogenic variants. We also applied co-variation analysis to capture detrimental variation regardless of molecular mechanisms, identifying 73% of pathogenic variants. The large agreement between stability predictions and co-variation analysis indicates that conservation of stability is a driving factor in evolution. Both methods provide good indication of pathogenicity, and will thus be valuable in the interpretation of novel variants identified in genome sequencing. Indeed, a number of disease-associated variants with neutral scores by both methods turned out to be erroneous database entries. An additional advantage of biophysical stability calculations is that they help identify the molecular mechanism, loss of functional protein levels via degradation, leading also to putative avenues for developing treatments. Upon binding of UDP-xylose, human UDP-glucose dehydrogenase (hUGDH) isomerizes from a catalytically active 32-symmetry hexamer (E) to an allosterically inhibited horseshoe-shaped complex (E). During this transition, the active site residue D280 rotates into the protein core where it forms a hydrogen bond with a backbone carbonyl. The cost of burying and protonating D280 can be seen in the pH dependence of UDP-xylose binding, which shows decreased affinity from pH 7.5 to 8.5. This pH dependency is reflected in the stability of the hexamer, which dissociates at pH 8.5. We hypothesize that in solution hUGDH isomerizes to the E complex in the absence of UDP-xylose, which requires the protonation of D280. To test our hypothesis, we generated a D280N substituted protein. A crystal structure of hUGDHD280N:UDP-xylose shows that the hydrogen bond between the backbone carbonyl and D280N is maintained. Sedimentation velocity studies show that hUGDHD280N is stable at both pH 7.5 and 8.5. The stability of hUGDHD280N suggests it is forming the E complex without binding UDP-xylose. To further test our hypothesis, we are currently determining the affinity of UDP-xylose with fluorescence binding assays at pH 7.5 and 8.5. We expect hUGHDD280N to have higher UDP-xylose affinity that shows no pH dependence. Once the Kds have been determined for both hUGDH and hUGDHD280N, we will be able to calculate the energy cost of burying D280 based on the difference in affinities. Complex responses have been developed by organisms in response to double stranded RNA. RNA silencing pathway is one of the most conserved pathways within organisms. In order to better understand and study this pathway and its ligands, we utilize the viral suppressor of RNA silencing (VSRS) p19 expressed by Tombusviruses. p19 is a viral RNA silencing protein that was first identified in the plant virus, Carnation Italian Ringspot Virus. p19 works on inhibiting RNA silencing by binding and sequestering siRNA. The ability to incorporate biophysical probes in proteins is a powerful tool to interrogate protein-ligand interactions and protein intrinsic dynamics. Herein we report the use of genetic code expansion technology through the introduction of p-Azido phenylalanine (p-AzF) to design a p19 sensor protein for small RNA detection. We provide a strategy to design fluorescence resonance energy transfer (FRET) sensor proteins by incorporation of UAAs at specific sites in the p19 dimer allowing us to change the functional characteristics of the protein. p-AzF incorporation allows subsequent labelling for accurate measurements of FRET. Our results show that we have successfully yielded FRET signals indicating ligand binding. Herein we describe a steady state FRET probe that can be used for small RNA detection. We demonstrate the successful use of a modified version of the probe to track the release of RNA in mammalian cells, utilizing our previously designed p19 fusion protein linked to the cell penetrating TAT peptide. 16 Uranium is necessary for nuclear power. The production of enriched unranium generates thousands of tons of uranyl salts as by-products every year. Its acute toxicity in the environment impels the development of new methods for the detection and the cleanup of UO22+. A few methods have been developed for UO22+ detection, including spectrophotometry, electrochemistry or mass spectrometry. However, all of them present some limitations that prevent the simple, on-field measurement of UO22+ concentration. In this context, we propose the development new UO22+ biosensors based on a recently-described uranyl-binding protein. This small triple-helix protein was engineered to display unique femtomolar affinity for UO22+, and to be highly selective vs. other metal ions. We are investigating several approaches to utilize this protein for the detection of UO22+ and to better understand the selectivity and remarkable affinity of the protein for UO22+. We will present preliminary results demonstrating that SUP protein can be applied to the development of new protein-based UO22+ biosensors, exhibiting high affinity and selectivity. Young Hyun No 1 , Nam Hyeong Kim 1 , Kilho Eom 2 , Yong Ho Kim 1 1 SKKU Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University (Suwon, South Korea); 2 Biomechanics Laboratory, College of Sport Science, Sungkyunkwan University (Suwon, South Korea) Biomolecular assemblies found in nature have received attention due to their biological functions as well as their applications in bionanotechnology as a template unit for designing a functional device and surface. Nevertheless, the design principle and dynamic properties of peptide assemblies on twodimensional solid substrates has been poorly understood. Here firstly, we report nature-inspired twodimensional peptide assembly on graphene, based on both statistical analyses of naturally occurring peptide motifs and equilibrium atomistic simulations of peptide assembly. Two-dimensional peptide self-assembly was designed based on statistical analyses of >104 protein structures existing in nature and atomistic simulation-based structure predictions and optimization of peptide-peptide and peptidegraphene interactions. We characterized the structures and surface properties of the self-assembled peptide formed on pristine graphene. Secondly, we report the effect of secondary structure of peptide into the interaction between peptide and hydrophobic surface. Via SALDI experiment and MD simulation, it is found that random coil structure of peptide is more sensitive to mutation effect than alpha helix structure. Our work may suggest new design strategy that allows for coupling between biological molecules and nanomaterials. Further, such biomolecular design in nano-bio interface may pave a way for establishing programmable material genome toward various biomedical applications. Purinergic signals, such as extracellular adenosine triphosphate (ATP), mediate cell-to-cell communication during fundamental processes such as neural transmission, inflammation, and chemotaxis. Furthermore, the aberrant release of extracellular ATP can contribute to injury and disease, such as in epilepsy, cancer, and infections. Despite its importance in both physiological and pathophysiological conditions, it has remained a challenge to quantitatively measure the spatial and temporal dynamics of extracellular ATP because of the broad ranges of concentrations, distances, and timescales over which it acts. In order to address this challenge, we are developing genetically-encoded biosensors that enable the visualization of the release and clearance of extracellular ATP. When extracellular ATP is released, it can activate P2 receptors, including P2X ligand-gated ion channels and P2Y G-protein coupled receptors, that lead to downstream intracellular signaling via second messengers such as calcium and cyclic adenosine monophosphate (cAMP). Thus, in order to interrogate purinergic signal transduction across the plasma membrane, we are also developing multiplex imaging methods that simultaneously monitor spectrally compatible sensors that correlate the dynamics of extracellular ATP release, purinergic receptor activation, and intracellular second messenger signaling. Using this integrative live-cell imaging approach, we study both activity-dependent and injury-dependent purinergic signaling in neural systems. Reactive oxygen species (ROS), redox enzymes, and redox buffers are central to the propagation of redox signals and oxidative stress between cells in the tissue environment and between organelles in the cellular environment. For example, hydrogen peroxide generated in mitochondria can alter the thiol oxidation states on proteins to alter their function, and subsequently redox active proteins such as glutathione can act as reducing equivalents in the face of oxidative stress. In some cases, it has become apparent that ROS mediate physiologically important signaling across compartments, such as in mitonuclear retrograde signaling. However, it has remained technically challenging to quantitatively correlate compartmentspecific redox dynamics with subcellular resolution within live single cells. In order to address this challenge, we are developing genetically-encoded biosensors that enable the visualization of redox dynamics simultaneously within multiple compartments within the same cell. Our approach utilizes Förster-type resonance energy transfer in a spectral relay strategy to extend the fluorescence emission of the redoxsensitive green fluorescent protein (roGFP) into orange and red emission wavelengths. Furthermore, we are developing multiplexed, multiphoton imaging methods to study redox coupling between organelles and compartment-specific dynamics in thick neural tissues. The M2 protein of Influenza A virus is one of the smallest proton-selective channels found in nature and is the ideal system for studying the use of water when selectively transporting protons across the membrane. Among many interactions, the hydrogen-bonding interaction between membrane proteins and water molecules is of great importance in understanding the role of water molecules for the transport of substrates across membrane. Although methods for obtaining the structural information of transmembrane domains keep advancing, it is still difficult to observe the protein mechanics itself and to measure the interaction with water molecules through hydrogen-bonding. Two-dimensional infrared(2D IR) spectroscopic studies of the pH-dependent structure and hydrogen-bond dynamics of the M2 channel embedded in the supported lipid bilayers are presented here. The M2 channel has a higher tendency of the alpha-helical structure than in the micelle environment and also exhibits a higher content of water molecules in the channel cavity at lower pH(5.5) than at higher pH (7.5) . The presence of more water molecules at low pH was confirmed by the shift of IR absorption frequencies toward the low frequencies and the more pronounced hydrogen-bond exchange dynamics between the M2 proton channel backbone and the surrounding water molecules. Changes in the backbone structure accompanied by the pH change were also observed by polarization-dependent 2D IR experiments. Based on these results, we expect to be able to provide insight into how protons move through the proton channel by identifying a more sophisticated level of hydrogen-bonding network through isotope-labeling of specific residues. Assessing the Ability of Food Protein Nanofibrils to Cross-Seed Amyloid Formation of Related Human Proteins Lida Rahimi Araghi 1 , Tong Zhang 1 , Bingqian Xu 1 , Derek Dee 1 1 University Of Georgia (Athens, United States) Nanofibrils derived from food proteins (e.g., milk, eggs, cereals, and legumes) are gaining interest as nanomaterials and food ingredients owing to their unique functional properties, high aspect ratio and surface chemistry. Food protein nanofibrils resemble amyloid fibrils, some of which are pathological while others are functional. Amyloid fibrils can self-replicate by seeding amyloid formation of identical proteins, or by cross-seeding amyloid formation of different proteins. There is a chance that food protein nanofibrils could cross-seed amyloid formation of proteins endogenous to the human body, which may pose a health risk. This study focused on hen egg-white lysozyme (HEWL) and human lysozyme, characterizing the extent of seeding and cross-seeding reactions under various conditions in vitro. Different HEWL fibril polymorphs were self-assembled under conditions of low and neutral pH, and these were subsequently treated by sonication and proteolysis to replicate processing and digestion conditions to create seeds. Cross-seeding of human lysozyme was measured by ThT fluorescence and microscopy (TEM and AFM). Upon comparison of various conditions of protein concentration, pH, temperature, chemical denaturant, shaking, stirring and the addition of beads, it was found that HEWL nanofibrils cross-seeded human lysozyme fibril formation under certain conditions of both low and neutral pH. These results extend our understanding of amyloid-like fibril cross-seeding and polymorphism of lysozyme, and support the need for further assessment of food-protein nanofibrils before employing them for human consumption. Developing mechanistic models of protein function requires high-resolution experimental information about both structure and dynamics. For example, enzymes often undergo conformational changes that allow them to progress through their catalytic cycles, including substrate binding, chemical transformation, and product release events. Unfortunately, obtaining comprehensive information about macromolecular structure and dynamics can be difficult in practice. Techniques that provide atomic-level structural information (crystallography, cryoEM, SAXS/WAXS) reveal detailed snapshots of the structures present in a conformational ensemble at equilibrium, but yield no insight into the kinetics conformational interconversion. In contrast, spectroscopic techniques (NMR, FRET, etc.) are well suited for probing the kinetics and thermodynamics of conformational changes, but the data from these experiments are generally underdetermined for atomic-level rationalization of the observed signal changes. Breaking this paradox, time-resolved X-ray crystallography and solution scattering experiments offer simultaneous structural and kinetic information at high resolution; however, these experiments have yet to achieve widespread utility. To generalize this powerful technique, we have introduced the use of temperature-jump (Tjump) to initiate conformational dynamics that can be monitored in real time using ultrafast X-ray pulses. This presentation will introduce these new T-jump X-ray experiments, and focus on their application to two enzyme systems, lysozyme and cyclophilin A. In the case of lysozyme, T-jump crystallography revealed time-resolved electron density changes consistent with the well-characterized hinge-bending motion of the enzyme, demonstrating the utility of the method for visualizing atomic-scale protein motions. For cyclophilin A, T-jump SAXS/WAXS experiments elucidated an unexplored coupling between hydration layer dynamics and catalytic protein motions. Amyloids adopt cross-structures composed of long twisted fibrils with -strands running perpendicular to the fibril axis. Recently, a toxic peptide was proposed to form amyloid-like cross-structures in solution, with a planar bilayer-like assembly observed in the crystal structure. Here we design and crystallographically characterize peptides that assemble into spiraling cross-amyloids, which resemble twisted -amyloid fibrils in global geometry. The peptides form helical dimers, stabilized by packing of small and apolar residues, and the dimers further assemble into cross-amyloids with superhelical pitches ranging from 150 Å to 200 Å. Converting a small residue that appeared critical for packing to Leu, resulted in structural rearrangement to a much wider superhelix. Fluorescently tagged versions of the designed peptides form inclusions in mammalian cells, which recover from photobleaching with markedly different kinetics. These designed -amyloids and superhelices can be potentially useful for directing in vivo protein assemblies with predetermined spacings and stabilities. In the last few years, a novel concept emerged where higher-order self-assembly of proteins provides a simple and robust mechanism for signal amplification. This seems to be a universal signalling mechanism in innate immunity, where recognition of pathogens or threats triggers a strong, binary response of the cells (Wu Cell 2013). We have shown recently that the TIR domains of Mal and Myd88 form large ordered assemblies (Ve et al, NSMB 2017). Myd88 actually possesses two domains with self-oligomerization properties, as its Death Domain has been shown to oligomerize with helical symmetry in the Myddosome complex. To visualize the behaviour of full-length Myd88 we used single-molecule fluorescence coupled to eukaryotic cell-free protein expression, shortcutting all purification steps. These experiments show that Myd88 forms prion-like polymers at 100-fold lower concentration than TIR or Death Domain separately. These results pushed us to re-interpret the role of polymerisation in Myd88-related diseases and we studied the impact of mutations L93P, R196C and L252P at the molecular level. We discovered that all mutations completely block the ability of Myd88 to polymerise. Co-expressions between mutants and WT Myd88 explain the homozygous/heterozygous character of the diseases. Interestingly, we found that the cancer-related L252P mutation creates an unexpected gain-of-function: the L252P mutants form extremely stable oligomers, even when expressed at low nanomolar concentrations. Much progress has been made in the field of bioorthogonal chemistry to develop click reactions that enable the coupling of biomolecules, or that with small molecules, under biocompatible conditions. More recent efforts have turned toward developing reversible chemistries that can declick conjugates in a controlled and biocompatible manner for applications such as protein or prodrug activation and drug release from protein therapeutics. Here we examine 2-formylphenylboronic acid (2fPBA)-based chemistry and its utility in both bioorthogonal ligation and cleavage. In particular, we focus on 2fPBA and its conjugation with hydrazide, which produces a boron-nitrogen heterocycle (BNH) as the major product at physiological pH. The stability of the BNH conjugate can be tuned by varying the -substituent to the hydrazide functionality, enabling the formation of a stable or declickable conjugate depending on the substituent. Indeed, we have found that the BNH conjugate with certain hydrazides can be reversed near neutral pH by just using Tris buffer. Tris is a commonly used buffer for biomolecules; this condition for decoupling provides an extremely mild condition for reversing the bioconjugation and allows for the recovery of the biomolecule in its active native state. The results thus show promise of the tunable BNH linker as a valuable tool for generating both stable and cleavable bioconjugates for various protein applications. The Shifting Interactome of Alpha-Synuclein along its Aggregation Pathway Emma Sierecki 1 , James Brown 2 , Andre Leitao 2 , Alexandre Chappard 2 , Yann Gambin 2 1 University of New South Wales (Randwick, Australia); 2 University of New South Wales (Randwick, Australia) The misfolding and aggregation of soluble proteins into insoluble amyloid deposits is associated with a range of neurodegenerative diseases. Amyloid deposits in these diseases are generally primarily composed of a single disease-specific protein, and the main pathological feature of Parkinsons disease is the presence of alpha-synuclein positive cytoplasmic inclusions termed Lewy bodies. The transition from soluble monomeric to fibrillar alpha-synuclein involves the population of multiple intermediate species, including oligomeric and protofibrillar structures. These species have differential toxicities, with certain oligomeric states now appearing to be the major toxic species. While alphasynuclein is the major component of Lewy bodies, a range of other molecules are also associated with these deposits in vivo including molecular chaperones and other proteins involved in cellular quality control processes, as well as many proteins with a high intrinsic aggregation propensity. We couple cell-free expression with single molecule techniques to rapidly screen Lewy body components and alpha-synuclein interactors for their ability to bind to each of these individual species. This approach has allowed the direct comparison of the affinity of on the order of 100 potential alphasynuclein interactors to multiple species along the aggregation pathway. This screen has uncovered a number of potent interactors to specific species of alpha-synuclein, and characterisation of these interactions has revealed insights into how these proteins influence specific microscopic steps in amyloid formation. Carbonylated proteins are a hallmark of diseases such as cancer. Developing tools and technologies for detection of this biomarker can aid in early diagnosis and a better understanding of these debilitating diseases. Classical methods for detection of carbonylated proteins cannot be performed in living systems. Moreover, they require laborious and lengthy downstream processing of samples. To address these existing challenges, we have developed a live cell compatible, real time biomolecule carbonylation monitoring technique. This approach is uncomplicated and can be performed with ease. Here, we will present a series of biocompatible carbonyl reactive fluorescent probes and demonstrate their applicability in the detection of biomolecule carbonyls in isolated systems and in live cancer cells. Unique photochemical properties of these bioorthogonal probes will be discussed and the suitability of each probe for biomarker identification, visualization and quantification will be demonstrated. High density lipoprotein (HDL) is a non-covalent assembly of specific proteins (mainly apoA-I and apoA-II) and lipids, which removes cholesterol from cells via reverse cholesterol transport. During atherogenesis, various cell types in the arterial intima release hydrolytic enzymes that modify HDL proteins and lipids, which can affect HDL structure, stability and functionality. Here we address effects of proteolytic modifications on the structural remodeling of HDL. Human plasma HDL were proteolysed with plasmin, a serine protease active in atherosclerotic lesions. The proteolytic products were analyzed by SDS-PAGE and Western blotting. HDL particle remodeling was monitored under near-physiological conditions. Plasmin action on HDL generated protein fragments in the 10-12 kDa range. Western blotting indicated that these fragments were derived from both apoA-I and apoA-II. Next, intact and plasmin-treated HDL were incubated at 37 oC, pH 7.5 for 6-12 h. Intact HDL showed a release of a fraction of free full-length apoA-I without significant changes in the particle size. In contrast, plasmin-treated HDL underwent fusion with release of full-length and fragmented apoA-I and apoA-II, indicating lipoprotein destabilization. Our results reveal that plasmin can cleave HDL-bound forms of apoA-I and apoA-II and thereby destabilize HDL under near-physiological conditions. We hypothesize that such HDL destabilization and fusion in the arterial intima accompanied by accumulation of free apolipoproteins may contribute to the progression of atherosclerosis. Destabilization of HDL via proteolytic modifications may contribute to excessive accumulation of lipid-free apoA-I in the arterial intima, which probably contributes to the progression of atherosclerosis. Amyloid fibrils play important roles in most neurodegenerative diseases like Parkinson's and Alzheimer's diseases. Understanding the aggregation process and sequence/structure determinants of amyloid fibrils can help understand the molecular mechanisms of their pathogenesis. Almost all known protein fibrils are formed by proteins with or that transform into -sheet structures. Few cases of fibrils constructed by -helices have been found. Recently, Staphylococcus aureus PSM3 was found to form amyloid-like fibrils with a cross-structure. This discovery suggests that -helical proteins also have the potential to aggregate into fibrils and display important biological functions. Here we show a de novo designed 18-residue amphiphilic-helical peptide can aggregate into amyloid-like fibrils as observed in electron microscopy. Circular dichroism spectroscopy gave typical -helical features both before and after aggregation. Molecular dynamic simulations with all-atom or coarse-grained models indicate that the aggregates of this peptide are stable in water. While the lengths of the fibrils differ, their widths were all about 20 nm. We demonstrated that amyloid-like fibrils can be rationally designed using amphiphilic helices as building blocks, which can be helpful to understand the structures of protein fibrils from a new perspective as well as to design new protein aggregates with biological functions. Bilayer fluidity and membrane protein function are both fundamentally dependent on the lipid composition of a biological membrane. To uncover lipid-modulated membrane protein backbone dynamics, timescale-and residue-specific NMR relaxation experiments were recorded which revealed that lipid order, modified either biochemically by acyl-chain saturation and order-inducing cholesterol, or biophysically by temperature variation, not only changes the dynamics of the lipid bilayer, but also the dynamics of the immersed membrane protein in a specific and timescale-dependent manner. Temperaturedependent dynamics analysis of both the membrane protein and the lipids of the bilayer furthermore unveiled a direct coupling between lipid and protein dynamics in the picosecond, microsecond and millisecond timescale, caused by the lipids cis/trans configuration, the rotational diffusion of lipids and reduced fluidity of the lipid phase, respectively. The lipid bilayer has thus the capability to modulate membrane protein function through dynamics in a kinetic range from picoseconds to milliseconds. Protein-based nanostructures have many potential applications in medicine, and materials science. Here we describe a generalizable method that uses short coiled-coil domains to assemble proteins into nanocages of various geometries. The approach requires minimal alteration of the protein and hence should preserve the structural and functional properties of the protein. Our strategy relies on the combination of 2 rotational symmetry elements, one provided by the natural, building block protein (BBP) and the other provided by the coiled coil, to specify a protein cage of the desired geometry. The oligomerization of the coiled-coil brings together the copies of the BBP together, leading to the assembly of protein cages. By employing BBPs and coiled-coils of different rotational symmetries it is possible to design cages with various sizes and geometries. Key to the success of this strategy is optimization of the linker between the BBP and the association strength of coiled-coil strands. Using this strategy we have been able to successfully design an octahedral, tetrahedral and structurally more challenging icosahedral protein cages. Additionally we have elaborated the structure of the previously designed octahedral cage with additional protein domains. Our next focus is on designing protein cages with controlled assembly and here we have made a significant progress in designing a metal dependent protein cage. As the world population ages, the number of people suffering from noncommunicable diseases, such as cancer and neurodegenerative disorders, increases. In 2016, at least 12 million people worldwide were diagnosed with neurodegenerative diseases and this number is estimated to increase by about 20% in 2020. Exposure to environmental toxicants is considered to be a risk factor for developing neurodegenerative diseases. In this study, we aimed to elucidate the toxic effects of fipronil, an N-phenylpyrazole insecticide that can be found as the main ingredient in many insecticide products on human neuronal cells, using the SH-SY5Y cell line. Proteomics analyses of treatments with 43 μM and 78 μM fipronil, which are concentrations giving 75% and 50% cell viabilities at 48 hours, respectively, resulted in differential expression of proteins involved in five functional groups: endoplasmic reticulum (ER) stress and unfolded protein response (UPR) (41% and 38%), neuronal structure and neurite outgrowth projection (23% and 23%), transcription and translation (14% and 15%), oxidative stress (5% and 8%), and others (17% and 16%), compared to untreated control. Immunoblotting of GRP78/BiP, PDI and PGP 9.5 verified the effects of fipronil on ER stress and UPR, whereas ImageJ analysis and 2,7-dihydroethidium (DHE) assay confirmed the effects on neurite outgrowth damage and oxidative stress, respectively. Results from this study indicate that fipronil induces proteostasis stress, a common phenomenon in many neurodegenerative diseases. Further investigations targeting specific proteins with roles in oxidative stress and proteostasis could provide a better understanding of the development of neurodegeneration caused by fipronil. Molecular recognition in protein-protein interactions is commonly understood in terms of the structurefunction paradigm: complementary structural elements interact by forming a defined set of intermolecular interactions. However, interactions between transcriptional activators and the transcriptional machinery eschew this paradigm; a single activator-binding domain (ABD) within a transcriptional coactivator can interact with >10 activators that may share no clear molecular recognition elements. Further one ABD accommodates diverse activator sequences with similar binding affinity. Without clear recognition principles, it has proven nearly impossible to discover small-molecule modulators. Here, we seek to develop a molecular recognition model by characterizing the ABD of Mediator subunit Med25, a structurally unique ABD that is formed from a rigid seven-stranded -barrel core flanked by conformationally dynamic loops and helices. We use transient kinetics and protein NMR to show that the Med25 ABD recognizes activators through a common mechanism of binding followed by a conformational change extending throughout the ABD structure. The equilibrium constant between bound conformations varies among activators, demonstrating that the Med25 ABD uses conformational lability as a mechanism to accommodate diverse sequences with similar affinities. The conformational plasticity of the Med25 ABD results in allosteric communication between its two binding sites, and we use disulfide Tethering to identify a chemical probe that recapitulates allosteric signatures of native ligands. Our data implicate common mechanisms for activator engagement in structurally divergent ABDs. Further, our evidence suggests that small-molecule targeting strategies aimed at conformationally mobile structural elements can take advantage of the plasticity of ABDs to provide chemical probes. Our understanding of protein evolution would greatly benefit from mapping of binding landscapes, i.e. changes in protein-protein binding affinity due to all possible mutations. We studied binding landscapes of four homologous complexes between trypsin-like proteases and their inhibitor BPTI. While structurally very similar, the four complexes span more than nine orders of magnitude in binding affinity. To map the binding landscapes for these interactions, we constructed a library of BPTI mutants containing all possible single and double mutations in the BPTI binding interface. Using the yeast surface display technology, we sorted the BPTI mutants into four affinity groups when interacting with each of the four target proteases. The BPTI populations having high-, WTlike-, lower-and the lowest-affinity for each protease were collected and sequenced with Next Generation Sequencing (NGS). The frequency of each variant in each population was used to create comprehensive binding landscapes of the four homologous PPIs. While in previous studies such an approach was mostly used for qualitative conclusions, several innovations in the experimental methodology have allowed us to achieve quantitative agreement between NGS-derived enrichment values and experimental measurements of binding affinities performed on purified BPTI mutants. The comprehensive data was used to compare the four binding landscapes with each other, to dissect the significance of each binding interface position in binding, and to analyze the additivity/cooperativity effects of single mutations. Our results bring invaluable insights on evolution of proteinprotein interactions and facilitate design of novel protein-based therapeutic molecules. Chaperonin GroEL is a large tetradecameric protein which helps in folding of many cellular proteins under normal and stress conditions. Due to its large size (800 kDa), multimeric nature and irreversibility in unfolding process, the role of critical amino-acid residues in its folding, stability, and assembly process remains still poorly understood. In order to overcome these limitations with the multimeric GroEL molecule, we have used monomeric GroEL, which possesses secondary and tertiary structure and chaperoning function like native GroEL, to study the role of crucial amino-acid residues responsible for stability and assembly of GroEL. The equatorial domain provides most of the intra and inter-subunit interactions during assembly of GroEL. Hence to understand the importance of critical residues in assembly, and stability of the GroEL, we have selectively mutated three residues K3E (N-terminal), D523K (C-terminal) and D473C (Internal residue) and characterised their structural properties, thermodynamic stability, oligomerization state, aggregation prevention property, and refolding activity. Three-state unfolding processes were observed for all the monomeric WT and mutant GroEL versions, where intermediate and equatorial domains unfold first followed by the stable apical domain. Both K3E and D523K mutations causes destabilization to the monomeric and tetradecameric GroEL due to loss of intrasubunit salt-bridge interactions, but K3E causes loss of oligomerization due to loss of both intra and intersubunit interactions. Hence, our study demonstrates that intra-and inter-subunit ionic interactions at the N and C-terminal residues of GroEL are crucial for the thermodynamic stability and multimeric assembly process. We used hydrogen-exchange mass spectrometry (HX MS) to study the working mechanisms of important molecular machines. The high-quality HX MS data presented here reveals detailed information on molecular machines in action that is otherwise inaccessible to other methods.We present two examples to demonstrate the versatility and power of this method: i) assisted folding of maltose binding protein (MBP) by GroEL, a ring-shaped homo-tetradecamer that uses ATP binding and hydrolysis to capture and encapsulate denatured substrate proteins into an enclosed chamber for it to re-gain its native conformation. We've found GroEL rescues the folding defect of an MBP slow folding mutant (V8G) by restoring the stability of an important on-pathway folding intermediate via simple encapsulation. ii) structural dynamics of a AAA+ disaggregase Hsp104 and its importance in functioning as a molecular machine. Its function in solubilizing amorphous aggregates and amyloids hinge upon cooperative effort from the two conserved AAA nucleotide binding domains (NBDs) in response to ATP binding and hydrolysis. HX measurements on Hsp104 performed under native conditions provides not only an independent check on the published static structure but also reveals critical function-related structural dynamics. Binding of different types of nucleotides induce different response all throughout the whole molecule and it indicates the existence of an allosteric pathway that span the entire Hsp104 molecule from the nucleotide binding pocket (Walker A domain) all the way to the business end, i.e. the pore loops and the hexamer interface. Sujata Chakraborty 1 , Ethan Ahler 1 , Emily Dieter 1 , Douglas Fowler 2 , Dustin Maly 2 structure, function and regulatory mechanism is of utmost importance since kinase misregulation leads to numerous diseases including cancer and therefore are also the most sought-after drug targets. Here we have integrated two independent methods, Deep mutational Scanning (DMS) and Cysteine Installation for Modulating Allostery and Targeted Inhibition of Kinases (CystIMATIK), a new chemical genetic approach to allosterically manipulate kinase global conformation to study one of the best studied multidomain protein kinases, Src. Src gets N-terminally myristoylated and together with a short basic SH4 domain, the N-terminal myristic acid helps in Srcs membrane association in a bipartite manner. The involvement of the SH4 domain in any other regulatory mechanism has been unappreciated in literature. Combination of both of these approaches revealed that the SH4 domain acts as an intramolecular regulatory switch in Src that can be enhanced or disrupted resulting in divergent cellular outcomes. Since our integrated approach has uncovered a new layer of regulation in one of the most well characterized protein kinase, we expect them to be broadly applicable throughout the kinome to reveal new regulatory mechanisms to better understand kinase structure, function and regulations in cells. Although few computational methods exist that could potentially be used to model full-length constructs of membrane proteins, none of these methods are perfectly suited for the problem at hand. Existing methods either require an interface or knowledge of the relative orientations of the domains, are not designed for domain assembly, and none of them are developed for membrane proteins. Here we describe the first domain assembly protocol specifically designed for membrane proteins that assembles intra-and extracellular soluble domains and the transmembrane domain into models of the full-length membrane protein. Our protocol does not require an interface between the domains and samples possible domain orientations based on backbone dihedrals in the flexible linker regions which are created via fragment insertion, while keeping the transmembrane domain fixed in the membrane. Our method mp_domain_assembly implemented in RosettaMP samples domain orientations close to the native structure and is best used in conjunction with experimental data to reduce the conformational search space. Matthias Uthoff 1 , Ulrich Baumann 2 1 University of Cologne (Cologne, Germany); 2 Universityof Cologne (Cologne, Germany) AAA+ proteases are hexameric barrel shaped proteases, whose active sites are buried within the so-called proteolytic chamber. The entrance is controlled by six AAA proteins important for substrate recognition, unfolding and translocation. As a homohexamer, FtsH is one of the simplest AAA+ proteases containing one AAA and one protease module, as well as two transmembrane helices and a small periplasmic domain per polypeptide. As the sole membrane-associated AAA+ protease in most bacteria, mitochondria, and chloroplasts, FtsH plays a crucial role in membrane homeostasis. Several substrates are membrane-bound but also soluble often membrane related proteins are processed. Two crystal structures of a transmembrane helix-lacking FtsH construct from Aquifex aeolicus have been determined at 2.9 Å and 3.3 Å resolution in space groups R32 and P312. The typical FtsH hexamer is formed from the two different subunits in the asymmetric unit around the three-fold symmetry axis of the crystals. Similar to other published structures, all subunits are loaded with six ADPs and the two subunits resemble the already known open and the closed conformations. Within the ATPase cycle, while a subunit switches from the opened to the closed state, its pore loop-1 interacts with the substrate and translocates it into the proteolytic chamber. Unique to our models is the inactive conformation of the pore loop, which allows the closed conformation to switch back to the opened state without pushing the substrate out again. Our structures give further insights on how this fold is probably induced and linked to the intersubunit signalling network. Lipases are extensively used for the preparation of optically pure compounds due to their wide-ranging process advantages: they are stable in organic solvents, are chemo-and regioselective, have a broad substrate spectrum and do not need the addition of cofactors. However, they often suffer from a limited range of operating conditions and therefore, different approaches need to be used to enhance enzyme function and stability. B. subtilis lipase LipA is an extremely hydrophobic, alkalophilic microlipase with maximum activity at 35 degrees C and pH 10. The Hepatitis B core protein (HepBc) self-assembles into particles that are stable in the pH range 3-13 and up to 75 degrees C. We have inserted LipA into the surface exposed loop region of His-tagged HepBc. The fusion protein has been characterized using SDS and native PAGE. Zymogram with p-nitrophenyl acetate and lipase assays with p-nitrophenyl palmitate as substrates have been used to demonstrate that the fusion protein is able to retain lipase activity, and to further evaluate activity under various pH and temperature conditions. Under physiological conditions, proteins maintain their native folded structure to perform various biological functions and catalytic activities. However, the protein conformation is affected by changes in cellular and environmental conditions such as temperature, pH, pressure, high salinity and presence of other denaturing chemicals. Osmolytes are known to protect proteins under stress against denaturation. However, the mechanism of stabilization of proteins by osmolytes in the presence of other substances is not completely understood. We have addressed mechanistic aspects of the effect of stabilizing osmolyte Dsorbitol on a model protein (hen egg white lysozyme) in the presence of sodium chloride over a wide range of ionic strength. A combination of high sensitivity isothermal titration calorimetry, spectroscopy, volumetry and sound velocity measurements have been employed to understand the intermolecular interactions operating in these systems both qualitatively and quantitatively. In view of the complexity of protein, the work has also been extended to the building blocks (amino acids). The results suggest that the stabilizing ability of the osmolyte sorbitol is strengthened with an increase in the ionic strength of the solution. A correlation of the nature of the intermolecular interaction of sorbitol, sodium chloride and their mixture with the amino acids and the intact protein hen egg white lysozyme has been attempted. Such studies are important in understanding the mechanism of protein stabilization in the biological systems of the living organisms under a variety of environmental conditions. Amnon Albeck 1 , Ilana Nathan 2 1 Bar Ilan University (Ramat GAn, Israel); 2 Ben Gurion University (Beer Sheba, Israel) Necrosis is one of the most common processes associated with human diseases. It contributes significant pathology to many of the most widespread and lethal diseases, such as myocardial infarction, brain injury, stroke, diabetes, and Alzheimer's disease. Necrosis has long been considered an uncontrolled mode of cell death, and its molecular and cellular details are not clear. We identified an induction of specific proteolytic activities in response to treatment of cells with various necrosis inducers, involving two enzymes. We further demonstrated that inhibition of these enzymes protects from the necrotic process both in-vitro and in-vivo. These studies provide invaluable information on the early biochemical events leading to necrotic cell death. Furthermore, they may also lead to the development of novel anti-necrotic drugs. Taibah Aldakhil 1 , Aftab Ahmed 1 1 Chapman University School of Pharmacy (Irvine, United States) Over many centuries, traditional and complementary medicine has frequently utilized various plant components as a remedy to cure various ailments. The scientific literature is frequently based on small molecules from plants. However, limited reports are available on the biologically active proteins and peptides. There is untapped potential to characterize proteins and peptides that may have a promise to cure various diseases. Fenugreek (Trigonella foenum-graecum) belongs to family Fabaceae, cultivated in southern Europe and Asia. It is frequently used in different cultures as a spice and also as a traditional herbal remedy to treat conditions such as wounds, rashes, diabetes. We present here analysis on proteins/peptides isolated and purified from fenugreek seeds. The ground fenugreek seeds were defatted in hexane. It was then extracted in 1% acetic acid, 20mM Tris/HCl, pH 8.0, and 25mM Sodium phosphate buffer pH6.5, and precipitated in 60% ammonium sulfate. The first-dimensional purification was achieved by gel filtration chromatography on HiLoad Superdex-200 in all three buffer conditions. The fractions were pooled and successfully purified using second-dimensional reverse phase HPLC on Nucleodur C4 column with a gradient of 0-60% acetonitrile. The crude extract and chromatographic fractions were successfully analyzed by Tris-tricine SDS-PAGE gel electrophoresis. The partial characterization of proteins and peptides was achieved through Edman degradation using PPSQ protein sequencer. The homology results revealed partial similarity to late embryogenesis abundant (LEA) and galactosyltransferase protein. Further studies are in progress to completely elucidate the identity of proteins/peptides from fenugreek seeds and further investigate their biological activities. 16 Protein interactions play a key part in most biological processes and understanding their mechanism is a fundamental problem leading to numerous practical applications since proteins represent a major class of therapeutic targets. Docking simulations between two proteins known to interact can be a useful tool for the prediction of likely binding patches on the surface. From the analysis of the protein interfaces generated by massive cross-docking experiments on benchmarks comprising over 150 proteins, where all possible protein pairs, and not only experimental ones, have been docked together, we show that it is possible to predict a proteins binding site without having any prior knowledge regarding its potential interaction partners. In addition, proteins can interact with numerous partners and can present multiple binding sites on their surface, which may alter the binding site prediction quality. For the large majority of the proteins that are associated with very low prediction quality, we show that the predicted alternate binding sites correspond to interaction sites with hidden partners, i.e. partners not included in the original crossdocking dataset. Among those new partners, we find proteins, but also nucleic acid molecules. Finally, a new clustering analysis performed on the binding patches scattered on the protein surface show that their distribution and growth will depend on the proteins functional group. Keywords The spatial-temporal coordination of proteins with their binding targets underlies almost all biological functions in living cells. In order to understand their dynamics, one would rather take account of the binding properties between interacting proteins that are rooted in the structural features at their binding interfaces. Additionally, association and dissociation of a protein complex are often modulated by interactions of other proteins in the PPI networks under variable cellular environments. Computational modeling offers unique advantages to decipher the complexity of this problem. However, current structuralbased simulation methods require enormous resources, thus are only limited to study the dynamics of individual molecular systems. On the other hand, few structural details or energetic features of interacting molecules are included in models of protein reaction networks to study biological functions of cellular processes. Therefore, a feasible and reliable computational model for realistically simulating the complicated dynamics of a protein network is highly demanded. Here we present a new multiscale modeling framework that is integrated with simulations on two different scales. The higher-resolution model incorporates structural information of proteins and energetics of their binding, while the lower-resolution model uses a highly simplified representation of proteins to simulate the long-time-scale dynamics for a system that contains multiple proteins. Through a systematic benchmark test and two practical applications of common network motifs with active cellular functions, we demonstrated that this multiscale framework serves as a powerful approach to understand the molecular mechanisms of dynamic interactions between biomolecules and their functional impacts with high computational efficiency. Autophagy is a highly conserved pathway that mediates the bulk degradation of intracellular components and plays a significant role in the progression and outcome of numerous diseases. A key step in autophagy involves the conjugation of protein LC3 to PE in the membrane. Atg3 catalyzes this reaction, but to date how hAtg3 is activated and regulated for LC3-PE production remains largely unknown. The current Atg3 activation model was proposed based on a study of yeast Atg3 (yAtg3) by Nobel Laureate Dr. Ohsumis group in 2013. Three residues were shown to be critical for its activity. We have examined their corresponding mutations in hAtg3. While hAtg3T244A exhibits an effect similar to yAtg3T214A, surprisingly, hAtg3F296S and hAtg3H266L demonstrate opposite effects as those observed for yAtg3F293S and yAtg3H236L, respectively. In addition, we have found a mutation (hAtg3H262A) that nearly completely abolishes hAtg3s conjugase activity. These results indicate that the molecular mechanism for hAtg3 activation may be different than that of yAtg3. To examine the contributions of a catalytic residue to the conjugation reaction, using ethanolamine (NH2CH2CH2OH) and hydroxylamine (NH2OH) we have developed two single-turnover assays to dissect the role that a catalytic residue plays in activating the amino group for nucleophilic attack or stabilizing the tetrahedral intermediate with a partial negative charge on the carbonyl oxygen (oxyanion hole). We will present our study on understanding the molecular basis of hAtg3 activation and regulation for LC3-PE conjugation using a multidisciplinary approach including structural and biochemical analyses, mutagenesis, and in cell functional assays. The ultimate goal of protein design is to create an amino acid sequence that, when synthesized, will fold into the desired structure as accurately as possible. While computational protein design algorithms are capable of generating a large number of sequences, the practicalities of the currently available methods for testing the designs (cloning, expression, purification) become overwhelming (Johansson et al, 2016) . The aim of this project is to establish a true learning loop by devising an effective method for screening a large and relevant sequence space for folding and function. To achieve this purpose, we have produced a truncated Green Fluorescence Protein (GFP) that lacks strand 10 in its beta-barrel, as described by Boxer et al (2011) . The truncated GFP significantly loses fluorescence and stability compared to the mother protein, but addition of a synthetic beta-strand 10 results in complete recovery of the fluorescence, as well as the stability of the full-length protein. Using this property, we could test how different designs of the beta-strand 10 perform in binding to the truncated GFP and recovery of fluorescence. The peptide-protein interaction is affected by the length of the beta-strand 10, as well as specific amino acid substitutions in the peptide sequence. By extending this work to high-throughput computational design, peptide synthesis and complementation assays, we may explore a large sequence space of GFPs betastrand 10, with the goal of better understanding the relationship between protein sequence, folding and function. Protease (Pr) is an essential enzyme in the life cycle of human immunodeficiency virus (HIV) and hence is one of the most widely studied targets for antiviral drug design. Although there are about ten FDA approved drugs against protease, their long term usage elicits mutations leading to drug resistance. As a result, novel therapeutic approaches are being explored including synthetic antibodies. Recently, a murine monoclonal antibody (mab1696) was reported to inhibit HIV-1 Pr by preventing dimerisation. Crystallographic data revealed only the epitope of protease bound to mab1696. The present study employs computational techniques including knowledge based as well as structure based modeling to generate several possible interacting conformations of Pr-monomer with mab1696. Further, molecular dynamics simulation methods were employed to identify several stable interactions between Protease and mab1696. Results show that mab1696 interacts very strongly with several Pr dimer interface residues, such as Arg8 (N-terminal beta sheet), Cys95 (C-terminal beta-sheet) and Leu24 (near the active site). These observations support the hypothesis that binding of mab1696 prevents the dimerisation of Pr. The interactions and binding conformations identified in this study could form the basis for designing allosteric small molecule inhibitors for preventing the dimerisation of HIV-1 Protease. Soluble expression of recombinant proteins in Escherichia coli is of great importance for biotechnological applications. The most remarkable successes in soluble expression have been attained by genetic fusion of target protein to a solubility enhancer. The maltose-binding protein (MBP) is the most widely used fusion partner, alleviating the formation of inclusion body of targe protein in vivo. The cleavage of fusion tag is required to obtain the sole target protein, however, it may lead to the aggregation of protein that has low solubility in vitro. Even though the passengers can be used without removal of tags, the inherent properties of target protein are largely affected by the bulky fusion partner such as MBP. In this study, we introduce a novel small solubility enhancer 'NEXT tag', a 53 amino acids-long peptide originated from the N-terminal extension of Hydrogenovibrio marinus carbonic anhydrase. When fused to several target proteins including GFPuv, Luciferase and hEGF, the NEXT tag showed highest level of soluble expression among MBP, Glutathione S-transferase (GST) and Fh8 tags. We also fused the tags to an enzyme carbonic anhydrase (taCA) from Thermovibrio ammonificans, a protein exhibiting low solubility in vitro. The purified protein with MBP or NEXT tag showed no visible protein precipitation while the others including wild-type taCA were highly insoluble. More importantly, the NEXT tag allowed the improvement of solubility with minimal effects on the properties of taCA. This novel tag should have general use as an excellent enhancer for solubility and expression of protein. Amanda Peiffer 1 , Matthew Henley 2 , Andrew Henderson 2 , Nicholas Foster 2 , Brian Linhares 2 , Zachary Hill 3 , James Wells 3 , Tomasz Cierpicki 2 , Carol Fierke 2 , Anna Mapp 2 , Charles Brooks III 2 1 University of Michigan (Ann Arbor, United States); 2 University of Michigan (Ann Arbor, United States); 3 University of California at San Francisco (San Francisco, United States) Protein-protein interactions between activators and coactivators mediate transcriptional regulation. Faulty interactions at this level are the origin of many human diseases, yet our limited understanding of these protein complexes has deemed them undruggable, with no current drugs designed to target them. Individual activator binding domains (ABDs) of coactivators interact with a multitude of activators that share minimal sequence similarities, raising interests into the mechanism of molecular recognition between ABDs and respective activators. Most ABDs are helical bundles, forming molecular complexes comprising helix-helix interactions. The ABD Med25 AcID, however, represents a structurally divergent ABD, consisting of a core, seven-stranded beta barrel that is flanked by three alpha helices. Given this structural divergence of Med25 AcID, our work is aimed at identifying the role of the secondary structural elements in Med25 AcID for activator recognition. Using disulfide Tethering and molecular dynamics simulations, we have identified the important molecular recognition elements in Med25 AcID to primarily be the more mobile regions such as unstructured loops and dynamic helices surrounding the stable beta barrel core. Activator recognition and binding ultimately greatly reduces the dynamics of Med25 AcID, particularly in the unstructured regions of the protein. Further, these dynamic regions contribute to the structural plasticity of Med25 AcID, which allows for recognition of a wide array of activators. Future work will focus on exploiting these molecular recognition patterns found in Med25 AcID to develop targeted small molecules, which will allow for us to inhibit specific activatorABD interactions. The Pennsylvania State University (University Park, United States) The TonB system is required for Gram-negative pathogens to actively transport scarce and essential nutrients, such as iron, across their unenergized outer membranes. In a novel form of transport seen only in Gram-negative bacteria, this system uses integral cytoplasmic membrane proteins ExbB, ExbD, and TonB to transduce energy from the proton motive force (pmf ) of the cytoplasmic membrane to outer membrane-embedded TonB-dependent transporters (TBDTs). During the TonB-dependent energy transduction cycle, the periplasmic domains of TonB and ExbD undergo multiple conformational changes and protein-protein interactions. The in vivo evidence suggests that ExbD modulates TonB conformations to allow subsequent proper energization of TBDTs by direct contact with TonB. Having ruled out the participation of TonB and ExbB transmembrane domains (TMDs) in the requirement for pmf, we turned our attention to ExbD, which carries a conserved Aspartate residue in its TMD. In this study, the interactions of the Escherichia coli ExbD TMD and function of its adjacent periplasmic regionpredicted to be disorderedwere explored through in vivo photocrosslinking and mutant analysis. Within the TMD, strong homodimeric ExbD interactions were identified that cast doubt on a recent solved crystal structure. Within the periplasmic predicted disordered region, unique pmf-dependent and pmf-independent interactions with TonB were identified. A comparative analysis identified the conserved ExbD motif (V/I)X(L/I/V)X(L/V)P in many species; alanine scanning of the region from residues 42-63 revealed that V45, V47, L49, and P50 reduce TonB system activity. In addition, ExbD(V45A,47A) alters its protein interactions with itself and with ExbB and TonB. The Pennsylvania State University (University Park, United States) Chorismate mutase catalyzes the conversion of chorismate to prephenate, the first committed step in the biosynthesis of phenylalanine and tyrosine. Saccharomyces cerevisiae chorismate mutase (ScCM) has served as a model system for studying mechanisms of allostery. For this homodimeric enzyme, tyrosine and tryptophan interact with a distant effector binding site, and act as allosteric inhibitor and activator, respectively. The objective of our work is to understand the allosteric communication pathway between the effector binding site and the active site. We used nuclear magnetic resonance (NMR) spectroscopy to gain insight into how enzyme structural dynamics across multiple timescales are altered by effector binding. The 13C-methyl resonance assignments of all Thr and Ile residues were obtained through a nuclear Overhauser effect spectroscopy (NOESY) based strategy, in which we compared the experimental NOE pattern to that expected based on the X-ray crystal structure. Both chemical exchange saturation transfer (CEST) and Carr-Purcell-Meiboom-Gill (CPMG) relaxation-dispersion experiments were performed for the ligand-free, Tyr-bound and Trp-bound enzymes. Trp binding induced s-ms timescale motions in residues that connect the effector-binding site and active site, whereas binding of Tyr tended to repress these motions. Intriguingly, CEST and ligand titration experiments suggest ScCM can bind Tyr and Trp simultaneously, suggesting additional structural states exist in solution that have not yet been observed through X-ray crystallography. These results point to an allosteric model more complex than that given by Monod-Wyman-Changeux and suggest changes in protein structural dynamics might be important for conducting signals between the effector binding site and active site. The nucleotide selection fidelity of the RNA-dependent RNA polymerase (RdRp) is directly linked to the pathogenesis of the virus. Previous studies in our group showed that the Sabin-derived T362I substitution in poliovirus (PV) RdRp decreases fidelity by altering the conformational equilibrium of the motif D active site loop. When correct nucleotide binds, the motif D loop forms a closed conformation in which the highly conserved motif D Lys (Lys359 in PV RdRp) is positioned to act as a general acid to protonate the pyrophosphate leaving group. Binding of incorrect nucleotide make the enzyme remain in an open conformation where the motif D Lys is positioned away from the active site. Based on molecular dynamics simulations, we proposed a model for the motif D conformation equilibrium, where Glu364 interacts with Lys228 in the open state, but in the closed state, Glu364 interacts with Asn370 instead. To test this model, we generated the K228A and N370A variants. Our NMR and kinetic data were consistent with our model. The N370A substitution de-stabilizes the closed state with a higher nucleotide selection fidelity than the wild-type RdRp, and so the protein remains in an open state even with correct nucleotide bound. The K228A variant de-stabilizes the open state with a lower nucleotide selection fidelity than wild-type RdRp, and the variant can more readily access the closed state even with incorrect nucleotide bound. Altogether, our data support the notion that RdRp fidelity can be altered through rational modification of the motif D loop. Swedish Orphan Biovitrum AB (Stockholm, Sweden); 5 Umeå University (Umeå, Sweden); 6 Tel-Aviv University (Tel-Aviv, Israel); 7 Lund University (Lund, Sweden) Ribonucleotide reductases (RNRs) are key enzymes in DNA metabolism, providing the only known de novo pathway for the biosynthesis of deoxyribonucleotides (dNTPs), the immediate precursors for DNA synthesis and repair. Class I RNRs consist of a large, catalytic subunit and a smaller, radical-generating subunit, which together form the active complex. Allosteric mechanisms control substrate specificity and overall activity of these enzymes. In RNRs, the activity master-switch, the ATP-cone, has been found exclusively in the catalytic subunit. In two class I RNR subclasses whose catalytic subunit lacks the ATPcone, we instead discovered ATP-cones in the radical-generating subunit. We employed various biophysical techniques: ITC, DLS, DSC, multi-detection SEC, GEMMA and X-ray crystallography, in combination with activity assays to investigate the allosteric regulation of these unusual RNRs. We explored how binding of nucleotide effectors affected the oligomeric state and as a consequence the activity of the enzyme. We demonstrate that the ATP-cone in the Leeuwenhoekiella blandensis radical-generating subunit regulates activity via quaternary structure induced by binding of nucleotides. ATP induces enzymatically competent dimers, whereas dATP induces non-productive tetramers, resulting in different holoenzymes. The tetramer forms by interactions between ATP-cones, shown by a 2.45 Å crystal structure. To our knowledge, this represents the first observation of transfer of an allosteric domain between components of the same enzyme complex. Protein folding occurs as a set of transitions between structural states within an energy landscape. An oversimplified view of the folding process emerges when transiently populated states are undetected because of limited instrumental resolution. Using force spectroscopy optimized for 1-μs resolution, we reexamined the unfolding of individual bacteriorhodopsin (bR) molecules in native lipid bilayers with a 100-fold improvement in time resolution and a 10-fold improvement in force precision. The resulting data revealed the unfolding pathway in unprecedented detail. Numerous newly detected intermediatesmany separated by as few as 2-3 amino acidsexhibited complex dynamics, including frequent refolding and state occupancies of <10 μs. We next integrated these technical advances with a scheme that covalently coupled bR to a PEG-coated atomic-force-microscopy tip to precisely quantify the initial unfolding of bR. The resulting records revealed rapid near-equilibrium dynamics between three states spaced by a total of 8 amino acids, with the third of these states corresponding to Lys216 where bRs retinal is covalently attached. Dynamic force spectroscopy revealed this retinal-stabilized state was bRs most mechanically robust state and energy landscape analysis showed the energy to extract bRs GF helix pair with the retinal bound was equal to the other five helices combined. We also reconstructed the full 1D freeenergy landscape underlying the unfolding the first two helical turns of bR. Looking forward, this newly developed equilibrium unfolding/refolding assay should provide a platform to precisely quantify the energetics of membrane-protein folding under native-like conditions. Characterizing the Structure of The YG Box Oligomer of the Splicing-Associated SMN Kaylee Mathews 1 , Nicolas L. Fawzi 1 1 Brown University (Providence, United States) Survival of motor neuron (SMN/Gemin1) plays an essential role in small nuclear ribonucleoprotein (snRNP) assembly and localizes to phase-separated bodies in both the nucleus and cytoplasm. Prior to snRNP assembly, SMN must self-assemble via its YG box, and missense mutations in SMN near the YG box cause a severe, neuromuscular degenerative disease called spinal muscular atrophy (SMA). However, the structure of the functional YG box oligomer and the mechanistic effect of the mutations on YG box assembly remains unknown. Here, we present a biophysical and biochemical characterization of the wildtype SMN YG box oligomer. We have successfully purified recombinant, soluble SMN from E. coli. Using circular dichroism (CD) spectroscopy, we have confirmed the presence of -helical character in the Cterminal domain (CTD) of SMN. Using these data in combination with nuclear magnetic resonance (NMR) spectroscopy, we have determined that the CTD of SMN contains one or more disordered regions as well. In addition, we show using gel filtration chromatography that SMN YG box oligomerization has a dissociation constant in the M-range. This work provides new data allowing us to further probe the normal structure SMN and its dysfunction in disease. Anastasia Murthy 1 , Nicolas Fawzi 1 1 Brown University (Providence, United States) Fused in Sarcoma (FUS) is a ribonucleoprotein which functions in transcription, translation, splicing, mRNA transport, and stress responses. FUS is able to undergo the phenomenon of liquid-liquid phase separation, which is the demixing of protein and RNA components from the surrounding cytoplasm or nucleoplasm. The low complexity domain of FUS (FUS LC) is sufficient for phase separation; however, the contribution of the repetitive nature of the low complexity domain to phase separation is not known. Using NMR spectroscopy, we have determined that the interactions that govern the liquid phase involve glutamine, tyrosine, serine, threonine, and glycine, and that these interactions span across the entire polypeptide. This model is consistent with the intrinsically disordered nature of FUS LC. To further parse out how the full-length protein undergoes phase separation, we have begun to investigate the contribution of other domains outside of the LC to phase separation. We have found that other domains in FUS, particularly the RGG domains, interact with the LC to promote phase separation. Interestingly, the RGG domains are sufficient for phase separation alone, indicating a new role for arginine rich sequences in phase separation. In the future, we will investigate which residues in the LC and RGG motifs mediate phase separation of full-length. By understanding how FUS undergoes phase separation we can gain insight into the role of FUS in RNP granules and the mechanism of FUS pathogenesis in neurodegenerative diseases. Intrinsically disordered proteins (IDPs) dynamically regulate cellular activity across all organisms. In Xenopus oocytes, these proteins are found in membrane-less granules in the developing cell, temporally and spatially controlling transcription and localization. Due to their transient nature, it is difficult to examine these proteins without understanding their fundamental properties. Using sophisticated NMR spectroscopy and biophysical imaging methods in vitro, we can isolate individual IDPs for characterization, focusing on phenomenon of inherent liquid-liquid phase separation that can drive the formation, activity, and dissociation of observed complexes. Y-box-binding protein 1 (Ybx1) is a ubiquitous RNA binding protein found in these granules and has diverse roles in the cell from splicing to mRNA transport. Ybx1s exact dynamics in these complexes remains unknown. The protein contains a cold shock domain and is flanked by two predicted disordered domains; the C-terminus region is a unique region of interest as it consists of alternating regions of charged residues, as well as a QYNG enriched prion-like sequence. Here we confirm that the C-terminus region of Ybx1 is intrinsically disordered and show that it is sufficient to induce liquid-liquid phase separation in vitro. Ybx1-NTD is capable of forming structures similar to native frog granules, suggesting that the construct plays a role in inducing and regulating phase separation of granules in the oocyte. These structural discoveries have implications in development biology as well as pathology, as mutations in IDPs have widespread clinical consequence (cancer, ALS, frontotemporal dementia). Veronica Ryan 1 , Anne Hart 1 , Nicolas Fawzi 1 1 Brown University (Providence, United States) hnRNPA2, a component of membraneless organelles, forms inclusions when mutated in a syndrome characterized by the degeneration of neurons, muscle, and bone. Here we provide a unified structural view of hnRNPA2 self-assembly, aggregation, and interaction with other granule components, as well as the distinct effects of small chemical changes including disease mutations and posttranslational modifications on these assemblies. The hnRNPA2 low complexity (LC) domain is compact and intrinsically disordered as a monomer, retaining predominant disorder in a liquid-liquid phase-separated form. Disease mutations D290V and P298L induce aggregation by enhancing and extending, respectively, the aggregation-prone region. Granule components TDP-43 and TOG directly interact with and alter phase separation of hnRNPA2 LC. Arginine methylation reduces hnRNPA2 phase separation, disrupting arginine-mediated contacts. Additionally, generation of a C. elegans model of hnRNPA2-associated neurodegeneration is underway. These results highlight the mechanistic role of specific LC domain interactions and modifications conserved across many hnRNP family members but altered by aggregation-causing pathological mutations. Troy Wymore 1 , Sara Tweedy 2 , Attabey Rodriguez Benitez 3 , Alison Narayan 3 , Charles Brooks III 4 1 University of Michigan (Ann Arbor, United States); 2 University of Michigan, Department of Chemistry (Ann Arbor, United States); 3 University of Michigan, Life Sciences Institute (Ann Arbor, United States); 4 University of Michigan, Departments of Chemistry and Biophysics (Ann Arbor, United States) The synergistic use of molecular phylogenetic analyses, ancestral sequence reconstruction, atomistic simulations, and experimental evaluation was applied to understand the evolution of flavin-dependent hydroxylase (FdH) function and predict strategies for designing new functions. FdHs are ancient enzymes that have evolved several mechanisms to activate O2 and subsequently oxygenate substrates in a site-and stereoselective manner. Thus, these enzymes, if reengineered effectively can replace the need for inefficient, environmentally unfriendly, and extremely costly methods for challenging organic syntheses. A diverse set of FdH sequences was collated and aligned leveraging expectation maximization algorithms and structure as a guide to multiple sequence alignment refinement. Several models of sequence evolution were tested with the highest scoring model (LG +I +G8) used to determine the maximum likelihood phylogenetic tree and infer the sequences of ancestral enzymes. The posterior probability of most positions in the ancestral sequences was high with uncertainties only in variable loop regions and removed from the active site suggesting that a high accuracy reconstruction was obtained. The multiple sequence alignment was then used to construct comparative structural models of extant and ancestral FdHs that served as models for ligand docking of a large variety of aromatic substrates. By comparing the docking results with Quantum Mechanical/Molecular Mechanical (QM/MM) simulations of a reactive state, the reactivity of substrates with extant FdHs and ancestral forms was predicted. The results reveal not only the critical changes to active site residues but also changes to outer-sphere residues that facilitate the emergence of new flavin-dependent hydroxylase functions. Genetically or post-translationally modified Aß proteins can induce endogenous Aß conversation to neurotoxic forms that lead to the formation of amyloid plaques. It has also been proposed that processing by means of specific hydrolysis by arginine endopeptidases such as angiotensin converting enzymes (ACE) generates Aß proteins that form the plaques. In this research the rapid mass-spectrometry based method was used to determine the cleavage sites and activities of N-and C-domains of ACE in Aß hydrolysis, and thereby define the roles of ACE in Alzheimer's disease (AD) pathogenesis. A model system, based on synthetic Aß (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) peptides (that correspond to Aß metal binding domain 1) with free or protected (representing longer forms of Aß 1-40 or 1-42) termini, was used as substrates for N-or C-domain ACE proteolysis. All reaction mixtures were analyzed directly using MALDI-TOF mass-spectrometry in order to identify all products of the hydrolysis. For free-termini Aß peptides, the cleavage sites for the N-and C-domain by ACE are similar, but for protected peptides they are significantly different, and only N-ACE has an effect on the substrate. Experiments with stable isotope labels shows that N-terminus acetylation of Aß decreases the efficiency of hydrolysis. For the rat Aß (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) peptide, even with fullyprotected termini, the digestion pattern is similar to that of human 1-16 peptides with free termini. Asp7 isomerization also accelerates N-ACE cleavage. We conclude that specific ACE hydrolysis of Aß plays a significant role in AD pathogenesis. How Phosphorylation Patterns of tau-441 Modulate its Aggregation Propensity? Iva Ziu 1 1 Oakland University (Rochester, United States) Within the neurons, tau protein binds and stabilizes the microtubules. Post-translational modifications of tau, such as hyperphosphorylation and aggregation, may lead to cell death and cytotoxicity. Phosphorylation of tau protein at specific sites may promote or inhibit its sequential phosphorylation by other protein kinases at different sites (primed and unprimed phosphorylation), but the exact role of this sequential phosphorylation on tau protein biochemistry is unknown. Thus, understanding the mechanisms involved in tau protein biochemistry will provide a better understanding of its role in normal and diseased states. Phosphorylation of tau-441 by GSK-3, MARK4, and/or FYN protein kinases was performed sequentially in vitro. Dot blots and Western blots were used to confirm phosphorylation at specific sites. Aggregation of phosphotau proteins was analyzed by Fluorescence spectroscopy, and aggregate morphologies were evaluated by Transmission Electron Microscopy (TEM). While non-phosphorylated tau is highly disordered and soluble, the phosphorylated tau is prone to aggregation. The phosphotau aggregates, which appeared as amorphous fibril networks, were highly resistant to denaturing conditions. The tau aggregation propensities, aggregate stability and morphologies were dependent on the sequence of phosphorylation. Yuhong Wang 1 , Heng Yin 1 , Shoujun Xu 1 1 Ribosomal translocation is the high fidelity process, but frameshifting is non-error but rare process. We have developed a novel magnetization sensing based force spectroscopy that can measure the ribosome translocase EF-G dynamics and the translocation and frameshifting with single nucleotide precision. We have confirmed a mechanical force associated with the EF-G hydrolysis, in agreement with the active process of ribosome translocation. We also unambiguously observed the highly efficient -1 and -2 frameshiftings on a GA7G slippery mRNA without the downstream secondary structure. The result represents the first experimental evidence of multiple frameshifting steps. It is also one of the rare reports of the -2 frameshifting. Our assay removed the ambiguity of transcriptional slippage involvement in other frameshifting assays. Two significant insights for frameshifting mechanism were revealed. First, EF-G bound with GTP is indispensable to frameshifting. Although EFG bound with GDPCP has been shown to prompt translocation before, we found that it could not induce frameshifting. This implies that the GTP hydrolysis is responsible for the codon-anticodon re-pairing in frameshifting, which corroborates our previous mechanical force measurement of EF-G bound with GTP. Second, translation in all three reading frames of the slippery sequence can be induced by the corresponding in-frame aminoacyl tRNAs. Although Asite tRNA is known to affect the partition between 0 and -1 frameshifting, it has not been reported that all three reading frames can be translated by their corresponding tRNAs. The in vitro results were confirmed by toe-printing assay and protein sequencing. The increasing prevalence of Parkinsons disease (PD) poses a major challenge to aging society in the Western world. Our very limited understanding of the cause of this neurological disorder prevents development of effective therapies. Recent findings suggest that oxidation of the neurotransmitter dopamine is implicated in the pathophysiology of PD. By creating small molecules molecules for bioorthogonal conjugation, we established that oxidation of dopamine is linked to covalent modification of a wide variety of proteins. The application of small synthetic molecules in conjunction with fluorescence microscopy allowed us to monitor the uptake of dopamine and related molecules across the cell membrane, and visualize subsequently the occurrence of intracellular protein modification. We also studied the effect of protein modification mediated by dopamine oxidation on structure and activity of protein substrates in vitro. Our observation link oxidative stress and protein unfolding in PD. In agreement with the finding of wide-spread cell death among dopaminergic neurons in PD patients, our experiments showed the relationship between dopamine oxidation and cytotoxicity in neuroblastoma cells. Our study connects cellular stress and protein unfolding with dopamine oxidation and protein modification. This finding has implications for our understanding of the mechanisms behind neurodegenerative disorders and could help to develop novel therapeutic interventions in the future. Many proteins form quaternary structures stabilized by inter-domain interfaces. We explored how such interfaces change the folding of individual domains by studying superoxide dismutase-1 (SOD-1), a homodimer that can undergo prion-like misfolding in the context of the disease ALS. Force spectroscopy measurements revealed heterogeneous unfolding and refolding via multiple intermediate states for both dimers and isolated monomers, but with distinct differences in the network of transitions observed. Comparing the unfolding pathways for dimers and monomers, the dimer interface was found not only to increase cooperativity by reducing the number of intermediates seen, but also to change the identity and/or order of appearance of the intermediate states, reflecting interface-induced changes in the relative stability of different parts of each domain. Moreover, the dimer misfolded roughly half as much as would be expected from the rate at which monomers misfold, indicating that the interface protects against misfolding, in part by reducing the prevalence of partially folded states that lead to misfolding. These results show how protein-protein interfaces change folding pathways and mechanisms at the level of individual secondary-structure elements. Investigating the Structural Conformations of Elongation Factor G and the Role of GTP Hydrolysis during Translocation. Zeinab Moussa 1 1 Self (HOUSTON, United States) Protein synthesis also known as translation is accomplished by the ribosome. The main stages of translation can be divided into initiation, elongation and termination. The mechanisms of elongation are similar for all ribosomes. During elongation, the coupled movement of mRNA and tRNA is referred to as translocation, and accomplished through a conformational changes catalyzed by the elongation factor G (EF-G). The EF-G is a 5-domain-protein that changes its conformation upon binding to the ribosome. Cryo-EM and crystal structures of EF-G-ribosome complexes revealed that the conserved loops I and II protruding from the tip of domain IV of EF-G insert into the minor groove of the codon-anticodon helix in hybrid and post-translocation complexes. Comparing with the free EF-G structure, the domain IV probably moves a large distance to push the tRNA into the P-site. Its still not clear what is the role of the EF-G compact state, how does the EF-G induce ribosomal and inter-subunit rotation, what is the fate of GTP hydrolysis during translocation. During frameshifting what is roles of multiple EF-G binding? By adopting a FRET pair of (Cy3) and (Cy5) cysteine labeled residues on EF-G we aim at studying the EF-G binding and dissociation dynamics and their relation with GTP hydrolysis in the presence frameshifting signals using single-molecule FRET. We have found through static and realtime measurements that EF-G is flexible when free and bound. These results will help reveal the role for the structural changes of EF-G during translocation and couple that to the importance of GTP hydrolysis Misfolding of the abundant neuronal protein -synuclein is implicated in the etiology of several devastating neurodegenerative diseases, including Parkinsons disease. -Synuclein is known to misfold spontaneously into stable, amyloid-rich conformations; however, the molecular features that underlie its propensity to misfold remain unclear. To reveal these features, we performed a deep mutational scan of -synuclein, screening for the toxicity of each variant in yeast, a well-developed cellular model for -synuclein pathology. Our results identify key residues whose mutations abolish -synuclein toxicity. We also examine the contributions of various physicochemical properties to the toxicity of this poorly structured protein. Finally, we outline the application of this approach to identifying features of -synuclein that mediate specific cellular processes associated with pathology, which we have implemented in a classroom setting. The Initiation of Ubiquitin-Independent Mitophagy by Atg32 Xue Xia 1 , Michael Ragusa 1 , Sarah Katzenell 1 , Erin Reinhart 1 , Katherine Bauer 1 , Maria Pellegrini 1 1 Dartmouth College (Hanover, United States) Autophagy is a cellular process that captures cytosolic material in double membrane vesicles and targets them to lysosomes or the vacuole for degradation. The selective autophagy of mitochondria, termed mitophagy, leads to the degradation of damaged or excess mitochondria. Mitochondria to be degraded are identified by mitophagy receptors. These receptors are divided into two distinct classes. The first set of receptors are cytosolic proteins that recognize ubiquitin moieties on mitochondrial proteins. The second set of mitophagy receptors are outer mitochondrial membrane proteins that initiate mitophagy independent of ubiquitin. Insight into the initiation of ubiquitin-independent mitophagy has been hindered by a lack of structural data on these receptors. In Saccharomyces cerevisiae, ubiquitin-independent mitophagy is initiated by Atg32, a 529 amino acid single pass outer mitochondrial membrane protein. To gain insight into the structure of Atg32 we screened for the location of structured domains. We identified a previously undescribed structured domain residing in the cytosolic region of Atg32. Through a combination of cell biology and yeast genetics we determined that this domain is required for the initiation of mitophagy. We also determined the solution structure of this domain using NMR spectroscopy. To gain insight into the more dynamic regions of Atg32, we used small angle X-ray scattering (SAXS). By combining our SAXS and NMR data we generated a hybrid structural model for the complete cytosolic domain of Atg32. This structural and functional data provides our first insight into the overall architecture of Atg32 and new insight into the mechanisms of mitophagy initiation. Probing Caspase-Nanogel Self-Assembly and Release Francesca Anson 1 , Jeanne Hardy 2 , Sankaran Thayumanavan 2 1 University of Massachusetts, Amherst (Amherst, United States); 2 Corresponding Author (Amherst, United States) Hijacking programmed cell death cascades via systemic delivery is an attractive approach to relieve apoptotic suppression within malignant phenotypes. However, conventional drug administration leads to promiscuous diffusion, depleting healthy and cancerous populations. Alternatively, targeted nanoparticles designed to deliver pro-apoptotic enzymes can activate and direct apoptosis signaling catalytically, with decreased off-target effects. Caspases, the executioners of apoptosis, have been encapsulated and intracellularly delivered using redox-responsive, biocompatible nanogels. We aim to correlate caspase-nanogel composition and potency by probing caspasenanogel self-assembly, cellular uptake and release. This work permits us to elucidate cargo potency, or cargo combinations, for dominating varying levels of pro-survival moieties in mutable environments. As biological macromolecules can occupy up to 40% in volume of the cellular environment, it may be important to account for their effects when studying binding interactions in the cell. System-specific studies have shown that macromolecular crowders indeed have impacts on biomolecular processes. Thus, a further understanding of how to realistically account for these crowding effects when studying biological complexes is integral for experimental and computational models. Instead of approaching the effects of crowding from a system-specific perspective, this computational study aims to bring a general, comprehensive framework to understanding the trends in the relationship between intrinsic, physical properties of binding complexes and the impact of macromolecular crowdingfocusing on solvation properties and electrostatic interactions. Using a continuum electrostatics model, theoretical toy molecules were generated and surrounded by crowders represented as randomly placed, low dielectric cavities. The physical characteristics of these toy molecules could be easily manipulated without biochemical constraint, allowing us to systematically study various intrinsic properties of binding partners, such as the shape of the binding pocket. Preliminary results indicate that physical properties of molecules may predictably impact the strength of the effect of pre-desolvation in which the likelihood of crowders being placed in the unbound state could affect the solvation properties of the binding partners and potentially impact the overall binding free energy. By studying the physical properties of complexes and the effect of crowding through this theoretical toy model, we hope to gain general insights and elucidate crowding trends that may help guide future studies. Merozoite surface protein 2 (MSP2) is an intrinsically disordered protein that is highly abundant on the merozoite surface of malaria parasite P. falciparum. Vaccine trials have shown that MSP2 can confer a protective immune response, albeit in a strain specific manner, suggesting that polymorphic regions of MSP2 are immuno-dominant. One strategy to overcome the hurdle of strain specificity is to bias the immune response towards conserved regions of the protein. The C-terminal region of MSP2 is highly conserved and contains an epitope recognised by two mouse monoclonal antibodies, 4D11 and 9H4. Although both mAbs recognise overlapping epitopes, they have different antigenic properties, with 4D11 able to recognise parasite MSP2 whilst 9H4 cannot, suggesting that the 4D11 epitope is accessible on the parasite surface. In this work, structural biology is combined with vaccine design in the emerging field of structural vaccinology. This strategy was applied to MSP2 using a crystal structure of 4D11 Fv in complex with its cognate minimal binding epitope. Molecular dynamics simulations informed the design of a series of constrained peptides based on the 4D11-bound epitope structure. Subsequently, constrained peptide-KLH conjugates were prepared for immunisation in mice, resulting in high-to-moderate peptidespecific antibody titres in all groups. Furthermore, the specificities of antibody responses revealed that single point mutations can focus the vaccine response towards more favourable epitopes. This rational approach to vaccine design may be useful for not only MSP2-based malaria vaccines but also other intrinsically disordered antigens. The urea-induced unfolding mechanism of the homodimeric detoxification enzyme human glutathione transferase M1a-1a (hGST M1a-1a) was characterised by far-UV circular dichroism, tryptophan fluorescence, 8-anilino-1-naphthalenesulfonate (ANS) binding, size exclusion high-performance liquid chromatography (SE-HPLC), dynamic light scattering and hydrogen-deuterium exchange mass spectrometry (HDX-MS). Additionally, an F56S/R81A mutant monomer was engineered by disrupting both the lock-and-key motif and the mixed charge cluster. The mutant monomer closely resembles the tertiary structure of the WT homodimer subunits, making it a suitable model to study the unfolding mechanism of hGST M1a, in the absence of quaternary interactions. A four-state equilibrium unfolding mechanism is proposed: N22IMonoIOligoD. The disruption of the conserved lock-and-key motif, as well as the structures surrounding the mu loop, results in a destabilisation of domain 1. However, dimer dissociation cannot occur until the mixed charge cluster at the dimer interface has been destabilised. The destabilisation of domain 1 results in destabilisation of 4 and 5 in domain 2, because the domains unfold in a concerted manner. hGST M1a-1a dissociates to form the monomeric intermediate, IMono, which has weak interdomain interactions and compromised short-range contacts. IMono self-associates to form an oligomeric intermediate, IOligo. The destabilisation of 6 and 7 in the hydrophobic core drives the formation of the partially structured denatured state. Further investigation will need to be pursued to determine whether hGST M1a-1a unfolds via transient intermediate states; however, the elucidation of the equilibrium unfolding pathway of a complex homodimeric protein is a valuable addition to the ever-growing knowledge base of protein folding. Structurally-Directed Pyrrolysine-Inspired Cyclization of Therapeutic Proteins Marianne Lee 1 , Michael K Chan 1 , Lin Wang 1 1 The Chinese University of Hong Kong (Shatin, China) Cyclization of linear peptides and proteins can lead to enhanced binding specificity, thermostability, and proteolytic resistance. Hence it is not surprising that cyclic polypeptides are among the most studied and utilized frameworks for the development of new therapeutics. Previous work by our laboratory demonstrated the use of pyrrolysine-inspired cyclization to produce polypeptide lassos. We have further refined this technology to include a structural motif that results in significant improved cyclization efficiencies and yields. Notably, our approach produces a cyclic unit that is inherently stable to reduction in the cytosol, allowing the resulting cyclized lasso to act on intracellular targets, including those implicated in cancer progression and tumorigenesis. As a proof of concept, we have applied this structurally directed pyrrolysine-inspired approach to produce a construct containing a cyclized p16p, a 20-amino acid inhibitory peptide derived from the tumor suppressor protein p16INK4a. We showed by cell cycle arrest assays that this construct is more effective in halting G0/G1 progression than a known linear polyarginine-p16p peptide. PhD student (New Delhi, India); 2 Jawaharlal Nehru University (New Delhi, India); 3 National Institute of Immunology (New Delhi, India) Arginase is a bimetallic Mn2+-enzyme that hydrolyses L-arginine to L-ornithine and urea. The recombinant H. pylori arginase (RocF), a well-studied virulence factor for acid-protection and pathogenesis of the bacterium in the human stomach utilizes either Co2+ or Mn2+ for its catalytic function. However, a higher catalytic activity of the enzyme (~4 fold) was observed with Co2+ ions. This was consistent with higher arginase activity obtained from the extracts of bacteria grown in the presence of Co2+ ions. The difference in the activity could be due to the variation in the metal induced active-site architecture between the Co2+-and Mn2+-proteins. To explore this, we performed a detailed investigation that includes kinetic analysis, biophysical studies and large scale molecular dynamics simulations. The inhibitory mechanism of the two different metalloenzymes with the same inhibitor is found to be different, suggesting the variation of the metal induced conformation of the enzyme primarily at the active-site. The MD simulation studies of the two holo proteins show differences in the distances between the metal ion and its coordinating residues. We also observed a difference in the positioning of the loop that contains the catalytic residues. Time-resolved fluorescence anisotropy measurements also support this observation. All these results suggest the basis for the difference in the catalytic activity between these two holo enzymes. Thus, our study provides a structural insight into the difference of the active-site architecture between the two holo enzymes which may be useful to design small molecule inhibitors with higher efficacy specific to this pathogen. University of the Witwatersrand (Johannesburg, South Africa); 2 University of the Witwatersrand (Johannesburg, South Africa) Secondary polymorphisms can profoundly affect the structure, function, drug susceptibility, and replication capacity of HIV-1 protease. In this research, a South African HIV-1 subtype C Gag-protease variant (W1201i) was investigated. This variant was considered due to the presence of a mutation and insertion (N37TV), located within the hinge region of the protease enzyme. Single-cycle phenotypic assays showed that the mutations present in the N37TV protease conferred a replicative advantage and reduced susceptibility to lopinavir, atazanavir and darunavir. Interestingly, the mutations present in the variant Gag were found to modulate both replication capacity and protease inhibitor susceptibility. In silico studies were performed to understand the physical basis for the observed variations. Molecular dynamics simulations showed that the N37TV protease displayed altered dynamics around the hinge and flap regions. Specifically, the destabilisation of the hinge region allowed a larger and protracted opening of the flap region due to the formation of two key hinge/cantilever salt-bridges, which are absent in the wild-type protease. Furthermore, induced fit docking experiments showed that the mutations affected the thermodynamic landscape of inhibitor binding as there were fewer observable chemical contacts between the N37TV variant protease and the protease inhibitors. Collectively, these data elucidate the biophysical basis for the selection of hinge region mutations and insertions by HIV-1 and adds to the growing knowledge base detailing the mechanisms that govern protease inhibitor drug susceptibility. Davide Tavella 1 , Francesca Massi 1 , Katianna Antkowiak 1 , Reyyan Bulut 1 , Sean Ryder 1 1 University of Massachusetts Medical School (Worcester, United States) RNA-binding proteins (RBPs) control the translation, stability, or localization of maternal mRNAs required for patterning decisions prior to zygotic gene activation in Caenorhabditis elegans. The tandem CCCH zinc finger protein MEX-5 initiates a cascade of RBP localization events that follow fertilization, ensuring correct anterior/posterior axis formation as well as germline/soma segregation. Using NMR spectroscopy, we discovered that the N-terminal zinc finger of MEX-5 is unfolded in the absence of RNA but folds upon RNA-binding. To determine if this disorder-to-order transition contributes to MEX-5 function, we used molecular dynamics simulations to design a variant MEX-5 where both fingers are folded in the absence of RNA. We show that the RNA-binding affinity and specificity is unchanged in this variant. To determine the effect of the increased folded stability of this MEX-5 variant in vivo, we used CRISPR-hr to introduce it into the endogenous mex-5 locus. Homozygotes are sterile and form uterine tumors within a few days of reaching adulthood, while heterozygotes are fertile but form tumors at advanced age. Tumors are derived from embryonic cells wherein nuclei divide, but not the cytoplasm, leading to giant polynucleated embryoid bodies in the uterus. Together, our results show that the unfolded state of MEX-5 is critical to its function in vivo by a mechanism distinct from its RNA-binding activity. Tufts University (Medford, United States) Tandem repeats are a major source of genetic variability in eukaryotes. When found in coding regions, tandem repeats are known to expand and contract, giving rise to phenotypic variation between individuals. With mutation rates up to 100,000 times higher than the genomic average, these regions are often highly unstable, promoting genomic variability and rapid evolution (Gemayel et al., 2010) . We are investigating the mechanisms of tandem repeat instability in the model gene RPB1, which encodes the largest subunit of RNA polymerase II, Rpb1p. Rpb1p has an essential carboxyl-terminal domain (CTD) that is composed of 26 tandem repeats of a seven-amino acid sequence, YSPTSPS. At least 8 repeats are required for survival, but we have shown that yeast with a suboptimal CTD length exhibit spontaneous expansion and contraction of the coding region to improve fitness (Morrill et al., 2016) . In this work, we make use of a tet-off reporter system for RPB1 to monitor expansion and contractions of the repetitive CTD. The mechanisms of CTD instability were investigated using the reporter system and DNA repair mutants. While we have previously determined that repeat expansions require the recombination factor RAD52, our results indicate that CTD contractions occur independently of RAD52 but are dependent on RAD5, suggesting a role of post-replication repair via template switching. Agriculture and Agri-food Canada (London, Canada) Immunoglobulin A (IgA)-nanobody fusions are useful E. coli O157:H7 therapeutics that modularly comprise two highly ordered constant domains, collectively called the fragment crystallisable (Fc), fused to an antigen binding partner known as the variable heavy chain fragment (VHH) derived from camelids. Towards the goal of improving the accumulation of the VHH-Fc fusion, we employed rational design to improve the stability of the Fc. Two rational design strategies, supercharging and disulfide bond introduction, were tested for their effects on accumulation. Since a plant platform offers the prospect of oral delivery, the mutagenized candidates were transiently expressed in leaves of Nicotiana benthamiana and then screened for protein accumulation, thermostability and solubility. We have identified and characterised five supercharging and one disulfide mutant that, in comparison to native, all improve accumulation and enhance thermostability as either Fc alone or when fused to a VHH that binds E. coli O157:H7. We also have found that pyramiding of these mutations result in a complementary increase of accumulation. A binding assay also showed that binding efficacy is retained in the engineered fusion compared to native. A neutralisation assay is currently being performed to determine if the engineered fusion can still prevent binding of the bacteria to mammalian gut cells. The goal of this project is to demonstrate as a proof of concept that a bovine Fc chain can be rationally designed for improved stability and be a viable strategy for improving accumulation of the therapeutic without sacrificing efficacy. Titin is a giant muscle protein coded by 363 exons in human genome. The protein is abundant in striated muscle sarcomere where it plays an important role in sarcomere assembly and force generation. With the complex coding sequence and the number of exons in the gene, titin is capable of expressing a broad spectrum of isoforms. Under different physiological conditions the expression profile of the gene gets altered. These alterations are accounted by both transcript type and variable co-expression ratios of transcripts. Muscular dystrophy with myositis (mdm) is a disease caused by homozygous recessive alleles of the titin gene, which causes 83 amino acids deletion at the N2A/PEVK junction. We have observed alterations in titin exon 11-13 expression in mouse mdm tissues compared to that of wildtype. These exons code for three Z-repeats at the variable N-terminal region of titin, which is important for titin assembly in the sarcomere. We compared expression differences of exon 11-13 between mdm and wildtype tissues using extensor digitorum longus (EDL), psoas and soleus muscle tissues from mice. Predominantly fast twitch muscles EDL and psoas show increased Z-repeats expression under the diseased state while the predominantly slow twitch soleus muscle has no significant difference between the two expression profiles. Therefore we expect titin in predominantly fast twitch muscle to inherit characteristics of slow twitch muscles in mdm mice. The sliding filament model has been the foundation of our understanding of muscle contraction, explaining contraction as the calcium-dependent formation of cross-bridges between actin and myosin filaments. While this has been the prevailing model for muscle contraction for over 50 years, a weakness of the model is its inability to account for all the measurable forces observed in muscle contraction. Recently, it has been suggested that interactions between the titin filament and actin could be the missing component in existing models. The N2A region of titin sits in a unique region of titin, between the Ig-domain region that is elongated under low forces and the PEVK region, which is extended under higher forces. In addition, the muscular dystrophy with myositis (mdm) mice contain a deletion at the junction between the N2A and PEVK regions and muscles from these mice exhibit altered contractile properties. Based on these observations, we hypothesized that the N2A region might be a site of interaction between titin and actin and have used actin co-sedimentation and actin motility assays to investigate this possibility. This work demonstrates that binding between actin and titin occurs in vitro and future work will be focused on demonstrating that this interaction also occurs in vivo to elucidate the role of this interaction in muscle contraction. Stephen Fuchs 1 , Michael Babokhov 1 , Bradley Reinfeld 2 , Kevin Hackbarth 1 , Yotam Bentov 1 1 Tufts University (Medford, United States); 2 Vanderbilt University (Nashville, United States) Copy-number variation in tandem repeat coding regions is more prevalent in eukaryotic genomes than current literature suggests. We have reexamined the genomes of nearly 100 yeast strains looking to map regions of repeat variation. From this analysis we have identified that length variation is highly correlated to intrinsically disordered regions. Furthermore, the majority of length variation is associated with tandem repeats. These repetitive regions are rich in homopolymeric amino acid sequences but nearly half of the variation comes from longer-repeating motifs. Comparisons of repeat copy number and sequence between strains of budding yeast as well as closely related fungi indicate conservation and selection for a particular range of tandem repeat lengths. In some instances, repeat variation has been demonstrated to mediate binding affinity, aggregation, and protein stability. Thus, subsequent analysis has been aimed at identifying proteins for which repeat variation may play conserved and functional roles in modulating protein function. The engineering of microorganisms to produce desirable bioproducts is often reliant on the availability of a suitable biosensor. Here, we report a general strategy for building biosensors in Escherichia coli that act by ligand-dependent stabilization of a transcriptional activator and mediate ligand concentration-dependent expression of a metabolic reporter gene. This strategy allows one to exploit the ligand specificity of natural proteins, such as enzymes or transport proteins, to build new biosensors. We generated such a biosensor by using the lac repressor, LacI, as the ligand-binding domain and fusing it to the Zif268 DNA-binding domain and RNA polymerase omega subunit transcription-activating domain. Using error-prone PCR mutagenesis of lacI and expression-based selection on a reporter gene, we identified a biosensor with multiple mutations, only one of which is essential for biosensor behavior. By tuning parameters of the expression assay, we observed a ligand-dependent response with this biosensor of up to a seven-fold increase in the growth rate of E. coli. The single destabilizing mutation could be combined with a lacI mutation that expands ligand specificity to fucose to generate a biosensor with an improved response both to fucose and to IPTG. However, this destabilizing mutation did not confer ligand-dependent stabilization in either of two periplasmic binding proteins, which are structurally similar to LacI, suggesting that destabilizing mutations may best be identified directly in proteins with the desired ligand-binding property. This general strategy for E. coli biosensors should allow many natural proteins that recognize and bind to ligands to be converted into biosensors. Protein sequence data has been increasing rapidly due to advances in sequencing technology. This sequence data contains rich information about protein structure, function and evolution. Methods that can distill the information from such sequence data are invaluable tools for both understanding and engineering proteins. In this paper, we investigate how probabilistic generative machine-learning models, called variational auto-encoders, can be used to extract protein stability and evolutionary information from multiple sequence alignments (MSA). Utilizing MSA as training data, the variational auto-encoder learns a probability distribution over the protein sequence space, and this probability distribution is shown to be useful in the prediction of protein stability changes upon mutation. In addition, using the variational auto-encoder model, we can project the protein high dimensional sequence data into a low dimensional (2d or 3d) continuous latent space, which provides a facile representation in which to visualize relationships within the sequence space. Through simulated studies, we show that the latent space representation captures evolutionary relationships between sequences and provides an intuitive way to interpolate sequences. Overall, our findings suggest that the variational auto-encoder model is effective at inferring information regarding protein stability and evolutionary relationships from MSA and should be useful as a guide for protein engineering efforts. Acknowledgements: This work is supported through the grants from the National Science Foundation (CHEM1506273). Infection of humans by opportunistic pathogens of the Candida genus, or invasive candidiasis, has a particularly deadly effect on immunocompromised patients. Invasive candidiasis by Candida glabrata, the second most common source of such infections in the US, is dependent upon a variety of cell surface proteins in the adhesin superfamily that promote binding to host tissue and the formation of a biofilm, essential for fungal propagation. Many of these proteins include linker regions containing repetitive amino acid sequences that are hypothesized to be involved in positioning their binding domains in extracellular space. We screened a number of clinical isolates for repeat length variation in a panel of adhesion genes and found that the repetitive regions have a great deal of variability between isolates from clinical sources. Using a biotic adhesion assay, we observed that these clinical strains exhibit a spectrum of binding capacities. To study the contribution of variants in one particular adhesin, Epithelial Adhesin 1 (Epa1p), we heterologously expressed Epa1p in the related yeast, S. cerevisiae, and measured the ability of these transgenic yeast to bind to human epithelial cells. We observe that Epa1p with repeat copy numbers naturally occurring in clinical isolates (3-5 repeats) all confer similar levels of yeast binding to epithelial cells. Mutant EPA1 constructs have been made to express protein with linker region variants not seen in natural populations to identify the role of repeats and the linker region in C. glabrata adhesion and infection. Tau protein is a neuronal microtubule associated protein, however hyperphosphorylation of tau has implications in Alzheimers Disease (AD), leading to microtubule destabilization and contributing to neurofibrillary tangle (NFT) formation. The mechanism by which hyperphosphorylated Tau destabilizes microtubules is still unknown. The specific phosphorylation of Tau may modulate tubulin binding and microtubule polymerization. Towards this goal, the role of phosphorylated tau on microtubule polymerization was evaluated as a function of phosphorylation patterns. The hyperphosphorylated tau441 (2N4R) proteins were prepared in vitro in the presence of Glycogen Synthase Kinase 3ß (GSK-3ß) and/or Fyn Tyrosine kinase. GSK-3ß phosphorylates tau at Ser and Thr residues, while Fyn Tyrosine kinase phosphorylates Tyr residues. Dot blots confirmed the phosphorylation of tau by protein kinases. The polymerization of microtubules, in the presence of phosphorylated tau, was monitored by fluorescence spectroscopy. The non-phosphorylated tau served as a control. The experimental polymerization kinetics data were fitted to determine the rate constants, kapp, and the lag-times, ti, values for microtubule formation in the presence of various phosphorylated tau forms. The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is expressed in members of all three kingdoms of life. Its most well-known role is in the reductive biosynthesis of mevalonate from HMG-CoA as part of the mevalonate pathway for the biosynthesis of isopentenyl diphosphate (IPP) and subsequent isoprenoid derivatives. However, a number of bacteria have been shown to utilize this enzyme in the oxidation of mevalonate to HMG-CoA, presumably as a source of acetyl-CoA. One of these bacteria, Burkholderia cenocepacia, has been shown to express an oxidative HMG-CoA reductase but appears to utilize the nonmevalonate pathway for the biosynthesis of isoprenoids. As such, the physiological role of B. cenocepacia HMG-CoA reductase (BcHMGR) is not entirely clear. Current evidence from a number of kinetic, spectroscopic and chromatographic techniques strongly suggest that BcHMGR is regulated via the morpheein model of allostery. In this model, nonadditive quaternary forms of different levels of activity interconvert in response to changes in substrate concentration, pH, and enzyme concentration. Evidence of BcHMGRs morpheein characteristics will be presented, including unusual kinetic behavior, the presence of multiple oligomeric states of differing activities, a possible alternate function for the enzyme involving GTP hydrolysis, and reversible aggregation in solution. Emerging crystallographic and molecular dynamics simulation studies will also be presented, suggesting possible mechanisms by which this dynamic shapeshifting enzyme responds to changes in ligand and substrate concentrations. In Euryarchaeota, only the Halobacteria and Methanosarcinales groups have cultivable halophilic organisms and it is the proteins from Halobacteria that have been most widely studied. Here, we addressed the halophilic adaptation of the ADP-dependent phosphofructokinase/glucokinase family (ADP-PFK/GK) from Methanosarcinales organisms. Molecular modeling of proteins from non-halophilic and halophilic Methanosarcinales shows a very similar composition and distribution of amino acids but different from those observed in proteins from Halobacteria or Eukarya. Methanosarcinales proteins present a huge increase in the Lys content surface and no hydrophobic core reduction, features that have been suggested to be the main factors responsible for the halophilic character of Halobacteria´s proteins. Biochemical characterization of the Methanosarcinales ADP-PFK/GK from M. evestigatum (halophilic), and M. mazei (non-halophilic) shows that the activity of both of these extant enzymes is only moderately inhibited by salt, but at the same time, they are significantly stabilized by it. Betaine has a protective effect on KCL inhibition and in the thermal stability of both enzymes. The resurrection of the last common ancestor of ADP-PFK/GK from Methanosarcinales shows that the ancestral enzyme displays an extremely high salt tolerance and thermal stability. Structure determination of the ancestral protein reveals unique traits such as an increase in the Lys and Glu content and yet no reduction in the hydrophobic protein core. We demonstrated that the halophilic character is an ancient trait in the evolution of this protein family and that proteins adapt to halophilic environments by non-canonical strategy, different from the proposed models for Halobacteria proteins (Fondecyt1150460). Anabaena Sensory Rhodopsin, ASR and its transducer, ASRT interaction is hypothesized to involve downstream signaling partner during light activated signal cascade. Besides the atomic resolution structures of ASR, ASRT, the signaling mechanism is poorly understood. Atomic structures of ASRT has reveled that this soluble beta stranded rich protein exhibit a helical face at carboxyl terminus. Our recent study has demonstrated that helical face of tetramer is involved in unusual tetrameric stability and destabilize upon phosphorylation. Initial data with efficient donor, acetyl phosphate, acP using fluorescence quenching of lone Trp-9 suggests that ASRT is involved in phosphorylation. We noticed two notable consensus phosphor transfer residue motif in ASRT, University of Massachusetts Amherst (Amherst, United States) Ca2+/calmodulin dependent protein kinase II (CaMKII) has been shown to play an important role in processes crucial to learning and memory. Each subunit of CaMKII is comprised of a kinase domain, hub domain, variable linker region and regulatory segment. CaMKII is expressed from 4 different genes (alpha,beta,delta,gamma) and, to add complexity, there are many splice variants (isoforms) of each. All variants are highly conserved except for the variable linker region. We are trying to understand if and why all of these variants are necessary for proper function. Previously, it has been shown with a small subset of CaMKIIs that there is a relationship between the variable linker length and kinase activity. Here, we measure the activities of a wide range of CaMKII isoforms expressed primarily in the human hippocampus to better understand the role of these variants in memory. Our measurements are performed on human CaMKII (expressed recombinantly) using a coupled-kinase assay. Preliminary data for CaMKII alpha and beta isoforms tested (between 30 and 217 residue linkers) shows that the longer linker has a lower EC50 for Ca2+/CaM relative to the shorter linker. This result is in line with previous data that showed shorter linker variants require more Ca2+/CaM for activation. It will be crucial to compare linker length between genes, which will provide us with some understanding of the contribution from linker composition. Further understanding of the behavior of these isoforms will allow us to dissect the physiological implications of the role of CaMKII variants in different cell types. Anabaena sensory rhodopsin transducer, ASRT a tetrameric soluble protein indicated to function as downstream signaling partner to sensory rhodopsin in Anabaena PCC 7120. The ASRT is indicated to function as downstream signaling partner to sensory rhodopsin. Both solution and crystal structures has reveled that this beta stranded protein exhibit a helical face at carboxyl terminus. Our ASR crystal structure does not include the putative cytoplasmic domain encompassing 230-261 aa residues. Binding data alongwith CABS docking have outlined the significance of this region in ASRT binding, as binding constant of truncated-ASR decreased 5fold to full length- ASR. We have demonstrated that carboxyl helical face of tetramer is involved in unusual tetrameric stability. A blast search matches the ASRT to others of unknown function, with 84% similarity and 74% identity to a protein in the myxobacterium Polyangium cellulosum and with 72% similarity and 58% identity to a protein in the legume symbiont Sinorhizobium meliloti. Often the homologues of this family are termed as DUF, Domain of Unknown Function member. The gene neighbor hood, along with structural fold analysis termed this family as novel small carbohydrate binding protein. We have observed specific xylan binding to ASRT via electrostatic interaction. The xylan binding to ASRT impairs its interaction ASR carboxyl-terminus. Subsequent analysis has revealed a phosphor transfer along with unique structural fold that may transform it as a unique carbohydrate binding module. Xylan and ASR binding to ASRT with putative signaling state will be outlined. Why do organisms use protein hormones as intercellular molecular messengers? Protein hormones, which seem too complex and energetically expensive to use as single-message molecules, usually act via surface receptors linked to intracellular transducers. Earlier studies suggest proteolytic fragments of protein hormones may have additional functions. Thus, if secondary functions are telescoped and released during proteolytic processing in synthetic cells, in circulation, or in target cells, organisms might gain efficiencies by using proteins rather than smaller signal molecules. To probe for active proteolytic peptides, proteolysis prediction software (PROSPER; covers 24 proteases) was applied to 2011 known soluble human protein hormone transcripts or transcript products. Residual peptides left after digestion were identified and compared to other proteins in the human proteome using NCBI BLASTp to identify peptide-matched proteins (PMPs) and, via 3-dimensional structures (PDBs), to see if the matched motifs were on PMP surfaces; these imply the peptides might modulate protein-protein interactions of PMPs via non-canonical signal paths. Of protein translation products examined,~40% have residual peptides of >10-15 residues, many lysine or glutamic acid rich. Sequence alignments with other proteins show~60% of fragment matches are for non-family proteins. Seeding the central node of the network neighborhood program STRING with PMPs indicated~30% of network neighbors have PDBs allowing mapping of the matched peptide relative to protein-protein interaction surfaces. Success of this search algorithm is exemplified by the co-crystal of BMP7 and Noggin that shows a BMP7 surface sequence, matching a TGF1 proteolytic peptide, directly involved in Noggin contact. University of Kansas (Lawrence, United States); 2 Tel Aviv University and University of Haifa (Tel Aviv, Israel); 3 Tel Aviv University (Tel Aviv, Israel); 4 University of Haifa (Haifa, Israel) Outer membrane beta barrels (OMBBs) are the proteins on the surface of Gram negative bacteria. These proteins have diverse functions but only a single topology, the beta barrel. It has been suggested that this common fold is a repeat protein with the repeating unit of a beta hairpin. By grouping structurally solved OMBBs by sequence, a detailed evolutionary story unfolds. A strand-number based pathway manifests with progression from a primordial 8-stranded barrel to 16-stranded and then to 18-stranded barrels. The transitions from 16-to 18-stranded barrels show mechanisms of strand number variation without domain duplication, such as a loop to hairpin transition. This indicates that repeat protein topology can be perpetuated without genetic duplication likely because the topology is being enforced by the membrane environment. Moreover, we find the evolutionary trace is particularly prominent in the C-terminal half of OMBBs which may be relevant to understanding OMBB folding pathways. Conformational change of protein, especially the transition between functional states, is usually critical for biological processes. Therefore, understanding this dynamic process is critical for understanding protein function. However, conformational transitions in proteins are usually not easy to be well characterized by experimental protocols, mainly because of their inadequate temporal and spatial resolution. By providing time-dependent structural information at atomic level, molecular dynamics (MD) simulations serve as a potential tool to dissect the dynamic process of conformational transition of protein, though the sampling of configuration space is still a great challenge. In this work, we proposed a robust and unbiased enhanced conformational sampling protocol with combined MD simulations and principal component analysis, and applied it to explore the conformational transition of adenylate kinase (ADK), a model system with well characterized open and closed state structures. This protocol drives the switching between the open and the closed states of ADK within tens of ns. By analyzing the ensemble of hundreds of one-direction transition in ADK, we reproduced different mechanisms and the associated multiple pathways for domain motion of ADK proposed in literature, and further identified the correlation between the probability of transition pathway and the free energy. Therefore, our work provides a strong evidence for the funnel landscape model, which predicts that protein folding proceeds through multiple kinetically distinct pathways. This reliable and efficient enhanced conformational sampling protocol could be employed to study the dynamics between different functional states of a broad spectrum of proteins and biomolecular machines. Most chemotherapy targets dividing cells, rather than just tumor cells. This causes the failure of chemotherapy, as dosing limited by side-effects leads to drug resistance and relapse. One strategy to overcome this, referred to as Directed Enzyme Prodrug Therapy (DEPT), involves administration of an inactive prodrug that is activated by a foreign enzyme at the tumor site. Enzyme targeting can be achieved using mechanisms that include tumor-directed antibodies or viruses. Clinical trials have demonstrated the effectiveness of DEPT, but were hampered by the complex timing needed to ensure that the enzymeantibody complex was localized exclusively to the tumor before systemic prodrug administered. Otherwise, the circulating enzyme would activate the circulating prodrug, resulting in systemic toxicity. We are employing a massively-parallel computationally-guided design approach using Rosetta, validated with a novel selection system and deep sequencing, to overcome this shortcoming in a model DEPT system, in which the bacterial enzyme, carboxypeptidase G2 (CPG2), is used to activate nitrogen mustard prodrugs. We have designed a pro-enzyme form of CPG2 that is activated by tumor-overexpressed proteases at the tumor site through removal of the designed pro-domain. The pro-enzyme will be dormant in circulation, thereby avoiding activation of the prodrug by circulating enzyme. Proteolytic processing at the tumor site removes the inhibitory pro-domain, restoring the activity exclusively in the tumor microenvironment. In addition to the design approach, the structural and biochemical characterization of our designed enzyme, in comparison to the native form, will be discussed. We have developed a novel approach that allows for the direct production of a highly active immobilized lipase within the bacterium Bacillus thuringiensis. The catalyst was generated by genetically-fusing lipase A from Bacillus subtlis to Cry3Aa, a protein that naturally forms crystals in the bacteria. The Cry3Aa framework significantly stabilized the lipase against denaturation in organic solvents and high temperatures, resulting in an efficient catalyst that could catalyze the production of biodiesel to near-completion over 10 cycles. We believe the simplicity and robustness of our Cry-fusion immobilization system could make it an attractive platform for generating improved industrial biocatalysts for different bioprocesses. Selectively modulating the activity of a desired enzyme in vivo is a major goal in protein design and can aid in the development of methods for understanding and rewiring cell-signaling pathways. Protein kinases and phosphatases are complementary enzymes that catalyze the addition and removal of phosphate groups upon substrates. The high structural homology of kinases and phosphatases presents a challenge in designing selective inhibitors for understanding their cellular roles. Though powerful genetic knockdown and knockout tools exist, they are susceptible to compensatory cellular mechanisms and not easily titratable. We have addressed this problem by designing a potentially general allosteric approach for gating kinase and phosphatase activity utilizing the well-studied protein-protein interactions between Bcl-2 family proteins and their small molecule inhibitors. We have designed a system where specific BH3-only domains, 20 to 25-residues, are inserted into an enzyme, at predetermined non-homologous positions. BH3-only domains, such as Bad, are unstructured but adopt a rigid, -helical conformation upon the addition of a protein binding partner, such as, Bcl-xL. Thus, in our system Bcl-xL acts as a poison and allosterically inhibits the function of the Bad-inserted-enzyme. Subsequently, the addition of a small molecule inhibitor, ABT-737, binds to and displaces Bcl-xL, acting as an antidote, thus restoring enzymatic activity. We have shown that this method allows for controlling both kinases and phosphatases with a small molecule in a dose dependent fashion both in vitro and in cellulo. We are currently optimizing this systems in order to study cell signaling and to redesign new pathways. Spiders spin different types of silk to perform a variety of biological functions. Silk fiber formation involves rapid conversion of soluble silk proteins (spidroins) into tough, insoluble fibers via a complex interplay of pH and ionic gradients, shear forces, and dehydration. It has been a longstanding goal to produce native-like artificial spider silks, but such efforts have largely failed, likely due to the use of oversimplified protein sequences and denaturing conditions during polymerization. It has however been a major challenge to clone highly repetitive DNA sequences characteristic of spidroin coding regions as well maintaining the concentrated precursors in a native state. Here we describe an efficient method for creating full-featured multi-domain constructs of MaSp2, a major component of dragline silk, bearing numerous tandem repetitive elements, and show results of biochemical analyses, toward the biomimetic self-assembly of mesoscale fibers. The design strategy involves sequence optimization to generate a short initial chimeric MaSp2 construct, followed by tandem repeat expansion via iterative cycles of DNA amplification and semi-conservative restriction site ligation, to build a large number of identical poly-alanine/glycine-rich tandem repeats typical of native coding sequences. The recombinant MaSp2 is highly expressed and extremely soluble at high concentrations, and displays native-like secondary structure, oligomer formation, and response to pH changes. We also report liquid-liquid phase separation events in recombinant MaSp2, in common with other protein systems bearing low-complexity, intrinsically disordered regions (IDRs). In response to low pH conditions, soluble MaSp2 rapidly self-assembles into insoluble fibers displaying a high aspect ratio. Chu-Harn Chiang 1 , Yi-Hsuan Fu 1 1 National Tsing-Hua University (Yilan, Taiwan) Collagen is the most predominant component of the extracellular matrix. Exploring the forces to assemble synthetic collagen mimetic peptides (CMPs) into trimers has been an attractive topic in preparing collagen-related biomaterials. Many natural collagens are either AAB-or ABC-type heterotrimers, making heterotrimeric helices better mimics for studying collagen structures in nature. We prepared the CMPs (CR, CK, CF) containing C-terminal cationic (Arg, Lys) or aromatic (Phe) residues to explore collagen heterotrimer folding via cation-interactions. Experimental data indicated that AAB-type heterotrimers could form in both Arg (CR)-Phe (CF) and Lys (CK)-Phe (CF) mixtures, suggesting that the C-terminal cationinteractions between cationic and aromatic residues could serve as a nucleation force and substantially promote the folding of heterotrimers. By controlling the mixing molar ratios of cationic and aromatic peptides in solution, we could obtain the heterotrimers with various compositions. In addition, we prepared CR-Sar and CK-Sar in which a Gly residue near the center of CR and CK was substituted to sarcosine (Sar). Our results revealed that CR-Sar could form homotrimers and AAB-type heterotrimers with CF. CK-Sar, by contrast, could not form either homotrimers or AAB-type heterotrimers with CF. From CR-Sar homotrimers and CR-Sar/CF heterotrimers, each intermolecular hydrogen bond was shown to contribute approximately 10 oC in the melting temperature of a triple helix. This work demonstrates that cation-interactions are an effective force to assist the formation of AAB-type collagen heterotrimers, and such a heterotrimeric system can be used to evaluate the thermodynamic consequences of mutation on collagen heterotrimers. Wei Wang 1 1 Nanjing University (Nanjing, China) The three-dimensional folded structures of proteins, known as native states, make proteins capable of performing related biological functions. To achieve such performance, the structure of the native state of a protein must be susceptible enough to sense the signal and switch to another structure, but also be stable enough to warrant functional specificity and structural robustness. This means a coexistence of high susceptibility and stability for the protein around its native state, which is apparently competing since high susceptibility implies large fluctuations and thus small stability in general, and vice versa. Does the balance of such competition result in a certain kind of critical behavior in proteins? Based on protein structural ensembles determined by NMR, we study the position fluctuations of residues by calculating distance-dependent correlations and conducting finite-size scaling analysis. The fluctuations exhibit high susceptibility and long-range correlations up to the protein sizes. The scaling relations between the correlations or susceptibility and protein sizes resemble those in other physical and biological systems near their critical points. These results indicate that, at the native states, motions of each residue are felt by every other one in the protein. We also find that proteins with larger susceptibility are more frequently observed in nature. Overall, our results suggest that the proteins native state is critical. Loops are widely known to be important for conveying a variety of functions to proteins, especially antibody recognition sites and enzymatic activity. As a result, they have been extensively characterized in soluble proteins. In outer membrane beta-barrels (OMBBs) loops are responsible for enzymatic activity, cell recognition and substrate binding but they have not been well characterized, in part because of the limited crystal structures previously available. We used the loop structures in 110 OMBBs to cluster the turns and loops of 4-6 residues and to characterize position-and cluster-dependent amino acid preferences. As has been previously noted, the extracellular loops tend to be much longer than the periplasmic turns, but we also find the amino acid content is different between loops and turns of the same length. We find a strong preference for aspartate, asparagine, glycine and proline in all lengths of turns and loops which allows for access to unusual regions of the Ramachandran plot. Hydrogen bonding between the sidechain and backbone of residues two to four positions away also plays an important role. Compared to surveys of loops in soluble proteins, we identify very different clusters in the OMBBs, indicating that successful design of OMBB loops and turns will need to rely on these newly described characteristics. The objective of this work was to identify important interacting proteins (IIPs) as a novel potential drug target using protein-protein interaction network (PPIN) and pathway analysis. The PPIN was generated from Leishmania donovani proteome with high confidence score using Cytoscape program. Networks were validated using the Barabasi-Albert (BA) preferential attachment algorithm. In the next step hubs that have higher connectivity than others in network, were identified by normalized degrees. Functional analysis of protein(s) involved in central hub was furthrer carried out by pathway analysis using KOBAS 2.0 and KEGGscape tool. Finally, crucial nodes for overall integrity of PPIN were identified as IIPs. Total 9 IIPs were identifed as central hub proteins, out of which succinyl-CoA synthetase subunit, hsp70, ribosomal L3 and 60S ribosomal L22 protein were found to be involved in metabolic pathways of L. Donovani. Among these central hub proteins, Succinyl-CoA synthetase subunit was selected as most potential drug target in the pathogen on the basis of similarity search. Since the 3D structure of Succinyl-CoA synthetase subunit is not available in the PDB, it was modelled by multiple homology modelling methods. The stability of modelled 3D structure was further validated by molecular dynamics simulation (MDS) at 100 ns time scale. The MDS data revealed, a single structure with consensus having maximum stability in biological system. Our research serves to establish a potential drug target which could be exploited for inhibitor designing using structure-based drug design strategy against L. Donovani. Michael Holliday 1 , Axel Witt 2 , Alberto Estevez 2 , Alexis Rohou 2 , Erin Dueber 2 , Wayne Fairbrother 2 1 Genentech (South San Francisco, United States); 2 Genetech (South San Francisco, United States) CARD9 and CARD11 are closely related signaling proteins that act in myeloid and lymphoid cells, respectively. Both proteins contain an N-terminal CARD that meditates a critical CARD-CARD interaction with Bcl10, leading to Bcl10 oligomerization and downstream activation of NFB. The CARD9 signaling cascade effects an innate immune response to fungal infection, while CARD11 signaling induces T-and B-cell proliferation. Accordingly, a large number of hyperactivating oncogenic mutations in CARD11 have been identified in diffuse large B-cell lymphoma (DLBCL). There has thus far been no detailed structural characterization of mechanism through which these mutations function or of the means by which CARD9 and CARD11 are maintained in an inhibited state. We have therefore determined the NMR solution structures of the CARD9 CARD alone and of a dimeric construct comprising the N-terminal CARD9 CARD with the following linker and 7-helix coiled-coil (CARD-CC). We show that the CARD alone forms filaments in vitro capable of nucleating Bcl10 oligomerization though direct templating of the Bcl10 helical assembly. In the larger construct, however, the CARD forms extensive interactions with the coiled-coil, blocking filament formation and Bcl10 nucleation. All known hyperactivating DLBCL mutations in this region map directly to the CARD-coiled-coil interface. Introduction of these and other CARD-coiled-coil disrupting mutants into an in vivo activity assay also induces hyperactivation of CARD9 signaling. In vitro, disrupting the interface can restore the ability of the CARD-CC construct to nucleate Bcl10 oligomerization. Together, these structural, biochemical, and cellular data demonstrate a novel autoinhibition mechanism utilized by CARD9 and CARD11. Recent observations have shown that a large proportion of the human genome encodes intrinsically unstructured/disordered protein regions (IDRs) that do not adopt a stable structure in isolation. IDRs can carry out their function in a variety of ways that all rely on their flexible state instead of a well-defined conformation. Disordered regions can serve as linkers between ordered domains, exhibit entropic functions, aid the assembly of large macromolecular complexes or drive formation of membrane-less organelles through phase separation. IDRs are also abundant in short binding regions and linear motifs that are involved in protein-protein interactions and post-translational modifications. Through these compact functional modules, IDRs are critical functional elements of many proteins involved in regulation and signaling processes. Proteins with disordered regions have been also associated with various diseases, most notably, with cancer. However, our current understanding of the exact role of IDPs in these diseases is still limited due to biases present in not only structural but in disease-association studies as well. Largescale cancer genome projects now offer an opportunity to move away from the structure centric view and to explore the role of IDPs in diseases in an unbiased way. Through a systematic analysis of the cancer associated mutations collected in the COSMIC database, we could obtain a better understanding of how the distinct biophysical properties of disordered protein regions are reflected in their involvement in cancer. Efrosini Artikis 1 , Charles L. Brooks III 2 1 Biophysics Program (Ann Arbor, United States); 2 Department of Chemistry, Biophysics Program (Ann Arbor, United States) In studying pH-mediated biological processes, NMR is an optimal experimental approach, as the sensitivity of NMR chemical shifts to both molecular composition and local environment allows for a comprehensive description of pH-dependent effects. Currently, empirical and semi-empirical chemical shift predictors are largely insensitive to changes in environmental pH and compute absolute shifts only at, or near physiological pH. Aiming to better understand the manifestation of pH in these NMR observables, we utilize a combination of MD and DFT calculations for the ab initio computation of NMR chemical shift perturbations (CSP). By combining principles observed in model tri-peptides, as well as classical descriptions of electrostatics, we have devised a methodology that recapitulates pH-dependent CSPs in proteins. We anticipate that this predictor will enable greater understanding of electrostatically driven dynamics in proteins involved in pH-mediated processes, and provide a self-consistent way to compare experimental and theoretical models. Small chemical modifications can have significant effects on ligand efficacy and receptor activity, but the underlying structural mechanisms can be difficult to predict from static crystal structures alone. Here we show how a simple phenyl-to-pyridyl substitution between two common covalent orthosteric ligands targeting peroxisome proliferator-activated receptor gamma (PPAR) converts a transcriptionally neutral antagonist (GW9662) into an inverse agonist (T0070907). X-ray crystallography, molecular dynamics simulations, and mutagenesis coupled to activity assays reveal a water-mediated hydrogen bond network linking the T0070907 pyridyl group to Arg288 that is essential for inverse agonism. NMR spectroscopy reveals that PPAR exchanges between two long-lived conformations when bound to T0070907 but not GW9662, including a conformation that prepopulates a corepressor-bound state, priming PPAR for high affinity corepressor binding. Our findings demonstrate that ligand engagement of Arg288 may provide new routes for developing PPAR inverse agonists. This is an important finding because although the structural mechanisms affording activation of PPAR are well understood, it remains poorly understood how to design transcriptionally repressive PPAR inverse agonists. The role of signal transduction domains in modulating the energetics of signaling states of histidine kinases (HKs) is poorly understood. We propose an equilibrium thermodynamic model of signal transduction in HKs, in which sensor, signal transduction and catalytic domains are allowed to sample two-state, kinase-phosphatase equilibria that are allosterically coupled with various efficiencies. The parameter are fit using a large set of transcriptional activity and disulfide cross-linking efficiency assays for wild type and single-point mutants of a Gram negative Histidine kinase, PhoQ. Results and validation of the fit demonstrate that the HAMP signal transduction domain in PhoQ serves to rebalance the energy landscape of the kinase by strongly coupling anti-cooperatively with both the sensor and catalytic domains, priming the sensor to be maximally responsive to ligand binding. Furthermore, we show that it is facile to drastically alter and fine tune the activity of a HK by modulating the stability and allosteric coupling of the HAMP domain, suggesting a robust path to the evolution of new functionalities. Finally, we offer a hypothesis for the structural basis of signal transduction based on this thermodynamic scheme. Solar ultraviolet radiation damages DNA in our cells on a daily basis. UV damage to DNA in skin cells (melanocytes) can lead to melanoma, a type of skin cancer. While we are continually exposed to UV radiation, damaged DNA can go through multiple repair pathways to preserve genomic stability. Specialized enzymes are responsible for fixing various types of damage and ensure the integrity of the DNA. Specifically, DNA polymerases extend double stranded DNA by single nucleotide additions after the damaged is removed. One such polymerase is Human DNA Polymerase Theta (Pol, POLQ), which participates in alternative double strand break repair. Several somatic POLQ variants have been identified in melanoma patients. This research study highlights V2551D, L2538R, and Q2537H, three variants located in the active site of POLQ, which we hypothesize to display aberrant polymerase activity that could affect genomic stability and potentially be a driver for cancer. All Pol mutations, generated by site-directed mutagenesis, were purified via affinity chromatography and assayed for polymerase activity, including nucleotide incorporation and DNA binding. Preliminary results show palm domain variants with altered polymerase behavior compared to wildtype (WT), suggesting that the variants may follow alternate polymerization mechanisms, impacting DNA repair pathways, which could potentially serve as a biomarker of melanoma. Recent advances in gene synthesis, microfluidics, deep sequencing, and microarray techniques have made it possible to construct and assay large libraries of variant protein sequences. This rapid generation of large sets of mutational data has significantly enhanced researchers' ability to study how proteins function and to engineer proteins with new and improved properties. However, there is no standardized format to report this data and no simple mechanism for groups to share the data that they generate. We present ProtaBank, a repository for storing, querying, analyzing, and sharing protein design and engineering data in an actively maintained and updated database. ProtaBank provides a format to describe and compare all types of protein mutational data, spanning a wide range of properties and techniques. It features a user-friendly web interface and programming layer that streamlines data deposition and allows for batch input and queries. A suite of analysis and visualization tools are provided to facilitate discovery, to guide future designs, and to benchmark and train new predictive tools and algorithms. ProtaBank will provide a valuable resource to the protein engineering community by storing and safeguarding newly generated data, allowing for fast searching and identification of relevant data from the existing literature, and exploring correlations between disparate data sets. We demonstrate the importance of a standardized format for reporting protein engineering data that allows for accurate comparisons between different data sets and enables meta-analysis of protein sequence and function as well as future data mining and machine learning approaches. ProtaBank is available at https://protabank.org. Cells use enzymes such as DNA polymerases to maintain genomic stability during DNA repair. DNA Polymerase Theta is the predominant polymerase involved in alternative double-stranded break repair. Despite this, it is often error-prone as it does not accurately match the nucleotide on a DNA template with the correct complementary base. Studies have also found that high expression of Theta leads to increased rate of mutation in cancer cells, suggesting that Theta could be a potential cancer driver. This polymerization mechanism begins once DNA binds to Theta in the region of the thumb domain. This aligns the active site in the palm domain to incorporate the correct nucleotide opposite the template strand with the final rate-limiting step being the release of the extended DNA strand. If this mechanism is flawed, it could lead to mutations, genomic instability, and a pathway for cancer cells to avoid apoptosis. This study aims to elucidate the mechanism of Theta during DNA repair using Fluorescence Resonance Energy Transfer (FRET) in which we internally fluorescently labeled fingers domain of Theta to observe its interaction with DNA and a nucleotide. Preliminary results suggest that only in the presence of correct nucleotide does Theta experience a global conformational change during polymerization to ensure correct matching, suggesting nucleotide selection by Theta is monitored in open conformation and only when the correct pair is formed does polymerization proceed. Our FRET system can be used to further understand mutagenic incorporation pathway of Theta and how it can lead to disease. Botulinum neurotoxins abilities to locally block neurotransmission have made them very useful in treating a wide array of neuromuscular disorders. Conversely, Tetanus neurotoxin (TeNT) selectively targets central nervous system (CNS) neurons after trafficking upstream of the entry site at motoneurons to block central inhibitory signals. Recent molecular and animal studies questioned the assumed receptormediated transport principle of CNTs. In this study, we sought to delineate the essential molecular modules in TeNT that facilitate its rapid retrograde transport to the spinal cord. We generated hybrid clostridial toxins to examine several functional modules within TeNT using biochemical and biophysical in vitro studies, cell-based assays, and in vivo approaches. We find that TeNTs receptor binding subdomain (HCC) is not essential to the toxins entry into its target neurons and subsequent retrograde trafficking selection. Our cell-based and in vivo data further suggests that the N-terminal module of the receptor binding domain (HCN) is the minimal substitution that abolished TeNTs ability to escape entry motoneurons towards retrograde axonal transport. These results provide a clearer view of TeNT/BoNT receptor interaction and intracellular trafficking, and may aid in the design of therapeutic biologics with high specificity to the CNS and other target cells. Heterochromatin protein 1 (HP1) is a reader protein that binds trimethyllysine at position nine of histone H3 (H3K9me3) via cation-interactions to two Tyr and one Trp in the aromatic cage of the HP1 chromodomain. We have observed a linear free energy relationship that exists between the free energy of binding and calculated cation-binding energies of the two Tyr aromatic rings in the binding pocket, and can use this model to predict mutations that will strengthen the cation-interaction between HP1 and H3K9me3. We incorporated a Trp mutation due its increased electron density relative to Tyr. Structure and binding of the two single Tyr-to-Trp mutants as well as the double Trp mutant were determined by circular dichroism, X-ray crystallography, and isothermal titration calorimetry. Binding was not improved as predicted, however, interesting thermodynamic differences were observed relative to wild type HP1. Binding studies show the interaction is more enthalpically favorable and qualitatively our Y24W crystal structure suggests that noncovalent interactions between HP1 and K9me3 have been improved. These results have also been corroborated computationally. The entropic penalty was investigated using 1H-15N HSQC NMR experiments that indicate that in the unbound state, the chomodomains occupy a minor state, and the entropic cost may be the result of Trp mutants paying a larger conformational freedom loss to bind K9me3. The recognition mode of methyllysine reader proteins is deliberately dynamic, and although our mutations did not improve binding, we are interested in further investigating trends in aromatic cage composition in mammalian homologs. Fractal topologies, which are statistically self-similar over multiple length scales, are pervasive in nature. The recurrence of patterns at increasing length scales in fractal-shaped branched objects, e.g., trees, lungs, and sponges, results in high effective surface areas, and provides key functional advantages, e.g., for molecular trapping and exchange. Mimicking these topologies in designed protein-based assemblies will provide access to novel classes of functional biomaterials for wide ranging applications. Here, we describe a modular, multiscale computational design method for the reversible self-assembly of proteins into tunable supramolecular fractal-like topologies in response to phosphorylation. Computationally-guided atomic-resolution modeling of fusions of symmetric, oligomeric proteins with Src homology 2 (SH2) binding domain and its phosphorylatable ligand peptide was used to design iterative branching leading to fractal-like assembly formation by enzymes of the atrazine degradation pathway. Structural characterization using various microscopy techniques and Cryo-electron tomography revealed a variety of dendritic, hyperbranched, and sponge-like topologies which are self-similar over three decades (~10nm-10m) of length scale, in agreement with models from multiscale computational simulations. We demonstrate control over mesoscale topology (by linker design), formation dynamics, and functional enhancements due to dynamic multi-component assemblies constructed with three atrazine degradation pathway enzymes. The described design method should enable the construction of a variety of novel, spatiotemporally responsive catalytic biomaterials featuring fractal topologies. Filament or run-on oligomer (ROO) formation by enzymes is now recognized as a widespread phenomenon with potentially unique enzyme regulatory properties and biological roles. SgrAI is an allosteric type II restriction endonuclease that forms ROO filaments with enhanced DNA cleavage activity and altered DNA sequence specificity. We have characterized the three-dimensional structure of SgrAI in the ROO and non-ROO forms, using x-ray crystallography and cryo-electron microscopy, and show that it forms a left-handed helix with 4 SgrAI/DNA complexes per turn, and that could in principle extend indefinitely. The enzyme also undergoes a conformational change upon oligomerizing into the ROO, which serves to activate the enzyme and alter its DNA sequence specificity. We have also performed extensive thermodynamic and kinetic analyses and have used those to develop models for the full reaction pathway including ROO assembly and disassembly. Global data fitting to these models have allowed us to derive microscopic rate constants for each individual step in the pathway, and to address specific questions of cooperativity, filament growth mechanisms, sequestration of enzyme activity, and advantages over non-ROO mechanisms. We show that the derived rate constants are consistent with ROO sizes determined from electron microscopy and are most consistent with noncooperative growth of the ROO filament. We also show how this mechanism allows for sequestration of DNA cleavage activity on invading DNA, and away from the host genome, and most importantly, how it is advantageous over non-ROO enzyme mechanisms. The flavin dependent hydroxylase TropB catalyzes the site-and stereoselective hydroxylation of phenols. However, the use of this catalyst in organic synthesis is limited by its narrow substrate scope; determination of the substrate binding pose and the mechanism of hydroxylation could be leveraged to rationally redesign TropB and related enzymes to improve activity towards unnatural substrates. These developments would, in turn, reduce the need for costly and difficult organic syntheses of various drug-like molecules. The native substrate, 3-methyl-orcinaldehyde, was computationally docked to the TropB crystal structure, and molecular dynamics (MD) simulations of the ternary complex were employed to predict the structure of the Michaelis complex. Subsequent Quantum Mechanical/Molecular Mechanical (QM/MM) calculations were then utilized to explore the mechanism of substrate hydroxylation and origins of substrate selectivity. Molecular orbital and spin density plots are exploited to provide unprecedented insight into the nature of the catalytic mechanism. The roles of important residues identified by these calculations are consistent with mutational studies, and these results will be applied to expand the substrate scope of TropB. Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3 or A3) proteins act in the innate immune response to viral infections. A3s are a family of cytidine deaminases that hypermutate deoxycytdines to deoxyuridines, resulting in non-viable viruses. Each A3 recognizes a unique motif in a ssDNA substrate -A3A specifically recognizes a CTCA motif, where the second deoxycytidine is deaminated. A3A has been implicated in the restriction of human papillomavirus (HPV), which occurs in the nucleus of the cell. Because A3A cannot differentiate between viral and genomic ssDNA, it can potentially also hypermutate the human genome. This activity may be correlated with HPV-related cancers such as cervical cancer. Thus, it is important to understand how A3A selects and deaminates its substrate. Using recently published structures of A3A bound to different substrate ssDNAs, we have engineered A3A mutants in a key DNA-binding region to assess the binding and deamination of A3A. We have found that this region, while previously not suspected to affect substrate preference, does play a role in the selection of the nucleotide 5 to the deaminated deoxycytidine. However, the catalytic rate of the protein is unaffected by these mutations. These results provide a more detailed understanding of the interplay between the regions that recognize and select ssDNA hotspots in A3A. Phosphorylation is a major regulator of protein interactions; however, the mechanisms by which regulation occurs are not well understood. Here we identify a salt-bridge competition or theft mechanism that enables a phospho-triggered swap of protein partners by Raf Kinase Inhibitory Protein (RKIP). RKIP transitions from inhibiting Raf-1 to inhibiting G-proteincoupled receptor ki-nase 2 upon phosphorylation, thereby bridging MAP kinase and G-ProteinCoupled Receptor signaling. NMR and crystallography indicate that a phosphoserine, but not a phosphomimetic, com-petes for a lysine from a preexisting salt bridge, initiating a partial unfolding event and promoting new protein interactions. Struc-tural elements underlying the theft occurred early in evolution and are found in 10% of homo-oligomers and 30% of hetero-oligomers including Bax, Troponin C, and Early Endosome Antigen 1. In contrast to a direct recognition of phosphorylated residues by binding part-ners, the salt-bridge theft mechanism represents a facile strategy for promoting or disrupting protein interactions using solvent-accessible residues, and it can provide additional specificity at protein interfaces through local unfolding or conformational change. Addressing the urgent need to develop for novel drugs against drug resistant Mycobacterium tuberculosis (Mtb) strains, we are studying ecumicin (ECU) and rufomycin (RUF) as promising new leads that target cellular proteostasis via the peptide ClpC1. ClpC1 Mtb mutants resistant to ECU and RUF show reduced binding affinity by surface plasmon resonance (SPR). Despite certain structural similarities, the macrocyclic peptides, ECU and RUF, generate unique resistance mutants that do not carry cross-resistance. Our SPR competition experiments show that the presence of ECU prevents RUF binding, whereas RUF inhibits ECU binding only partially. The refined X-ray structure (1.4 Å resolution) of the ClpC1-NTD-RUF I complex reveals a binding mode similar to the one observed previously for the ClpC1-NTD-cyclomarin A structure: Phe2 and Phe80 create a hydrophobic ridge, over which the cyclic peptides bind, forming a V-shaped bridge. In addition, the complexed structure revealed that the methyl-epoxide moiety in the Trp of RUF I was opened and covalently bound to Met1 of ClpC1-NTD with a previously unreported thioether linkage. Mass spectrometry after overnight co-incubation confirmed the presence of the covalent ClpC1-NTD-RUF I adduct. The discovered details of the binding topology and the chemical mode of (inter-) action will help drive the further development of potency optimized and bioavailable cyclopeptides as anti-Mtb drugs. Membrane proteins play many critical roles in living cells, including signaling, transport and recognition. Membrane proteins are the most prevalent targets for drug therapies against a wide range of conditions. The interactions between membrane proteins and their binding partners in solution are therefore of central interest to both fundamental research questions and for applications in medicine. Aquaporins (AQPs) are key membrane protein in the regulation of water homeostasis in cells. Here we demonstrate a rapid and quantitative microfluidic approach for studying membrane protein interactions with the calciumdependent signalling, calmodulin (CaM). By measuring the electrophoretic mobilities and diffusion coefficients of the individual components and the resulting complex in the solution phase, we can characterize the binding equilibrium, as well as detect the selective binding of CaM to aquaporin-0 (AQP0) rather than aquaporin-2 (AQP2). While most proteins are susceptible to denaturants, detergents, and proteases, hyperstable or kinetically stable proteins (KSPs) are highly resistant to degradation and unfolding. Of particular interest, KSPs are resistant to denaturation induced by the detergent sodium dodecyl sulfate (SDS). The gel electrophoresis method diagonal two-dimensional (D2D) SDS-PAGE exploits this SDS-resistance and is coupled with MALDI-TOF MS to explore the presence and roles of KSPs in complex biological lysates. This study applies the D2D SDS-PAGE method to the pathogenic model systems Pseudomonas aeruginosa and Staphylococcus aureus to further elucidate structural and functional trends common to hyperstable proteins. Separately changing growth phase, co-culturing the two systems, or altering the active respiration pathways can modulate transcription and ultimately the protein and KSP expression in the systems. These specific environmental changes mimic naturally occurring growth environments that P aeruginosa and S. aureus encounter thus allowing for additional KSP identifications not seen under standard laboratory growth conditions. Analysis of all of the KSPs identified will advance the understanding of protein kinetic stability and may provide information applicable to science and engineering endeavors with medical and biotechnological relevance. Due to the poor efficacy of chemotherapeutics for colorectal cancer, there is a critical need to develop new strategies that are effective against all aspects of disease progression e.g. tumor growth, invasion, angiogenesis, and most importantly, metastasis. Towards this goal, we propose a new therapeutic strategy targeting a key molecular chaperone known as HSP60. In healthy cells, HSP60 is found only in the mitochondria, where its job is to fold other proteins into their proper, functional forms. However, accumulating evidence indicates that cancer cells abnormally retain HSP60 in their cytoplasm, which stabilizes a different set of proteins and helps tumors to grow and metastasize. Through our recent studies, we have identified several hundred small molecules that inhibit the biochemical functioning of HSP60 in vitro. Notably, we found numerous HSP60 inhibitors that are selectively cytotoxic to colon cancer cells with little-to-no toxic effects to non-cancerous cells in cell culture. Furthermore, these HSP60 inhibitors also block colon cancer cells from undergoing anchorage-independent cell growth in cell culture, which is an indicator of tumorgenicity and invasiveness of cancer cells in humans. These promising in vitro results encourage the continued optimization of novel HSP60-targeting chemotherapeutic candidates for efficacy testing in mouse models of colorectal cancer. Despite the therapeutic potential of targeting the abnormal cytosolic HSP60 in cancer cells, no drugs currently function through this mechanism, and thus this strategy gives hope for developing new chemotherapeutic agents that more effectively prevent tumor metastasis, resistance, and recurrence. Protein aggregates, which can result in cellular toxicity, are found in the brains of people afflicted with neurodegenerative diseases. One of the proteins found to aggregate is Tau, a protein that stabilizes microtubules in neuronal cells. Based on its amino acid sequence, Tau is likely to be N-terminally acetylated by the complex NatA, which is responsible for the acetylation of 85% of human proteins and 50% of yeast proteins with this post-translational modification. Loss of the NatA complex results in a variety of pleiotropic detrimental phenotypes, showing that acetylation is a crucial modification for many proteins. Without the presence of NatA and therefore without acetylation Tau may be more prone to aggregation and toxicity. The objective of this study is to determine the effect of N-terminal acetylation on the formation and toxicity of Tau aggregates. Saccharomyces cerevisiae is a useful model organism for these experiments because it is easily genetically modified, and as a eukaryote, it already performs posttranslational modifications such as acetylation. By expressing Tau in yeast strains with and without the presence of NatA, the possible effects of acetylation or lack thereof can be determined by examining protein expression and aggregation within the cells by SDS-PAGE and SDD-AGE. Our results demonstrate that once integrated into the yeast cells, Tau aggregates within both wild-type cells and cells lacking NatA. Future experiments aim to determine whether or not a lack of acetylation leads to increased toxicity within aggregates. Directed Enzyme Prodrug Therapy (DEPT) is a strategy for increasing the therapeutic ratio of chemotherapy, relying on the targeted activation of a relatively non-toxic prodrug in the vicinity of a chosen site, e.g. tumor tissue, by an exogenously delivered enzyme. However, DEPT can be limited by low selectivity for the tumor localization of the enzyme, leading to off-target toxicity and/or low therapeutic efficacy. The development of stimulus-responsive enzymes would allow for precise regulation of prodrug activation, and increased therapeutic ratios. Here, we describe the design of a photoactivatable yeast cytosine deaminase (yCD), a prototypical DEPT enzyme capable of converting the prodrug 5-fluorocytosine to the potent chemotherapeutic 5-fluorouracil, by attaching azobenzene dyes that undergo reversible cis-trans isomerization triggered by light to modulate the enzyme active site structure. We developed a computational protocol for (1) the selection of appropriate azobenzene covalent attachment sites to the active enzyme conformation, and (2) screening the identified sites for their ability to reversibly disrupt the enzyme active site by light-triggered azobenzene isomerization. We show that for an attachment site chosen by our method, the stereochemical changes in the azobenzene dyes, triggered by light of different wavelengths, allow for the reversible modulation of yCD activity. Similarly, for a site where cis-trans isomerization was not predicted to alter the active site configuration, irradiation-mediated switching was not detectable. Our computational design strategy, developed in the context of the Rosetta macromolecular modeling suite, offers a generalizable platform for the photosensitization of protein active sites using attached azobenzene derivatives. The 26S proteasome is the primary protease responsible for regulated protein degradation in eukaryotic cells. This highly complex ATP-dependent protease consists of more than 35 different subunits and has been found to adopt several distinct conformations, yet their functional relevance, the inter-subunit interactions that stabilize these states, and the mechanisms for their interconversion remained elusive. Here, we used a combination of biochemical and EM structural studies on partially recombinant proteasomes to elucidate an allosteric network that controls this conformational switching. Our studies revealed how the distinct proteasome states specifically facilitate and thereby coordinate the individual steps of substrate processing, and how a premature switching of conformations interferes with proteasomal degradation. Elongation factor 2 (EF2) is a GTPase found in eukaryotes and archaea that is required during translation for the movement of ribosomes along mRNA. EF2 contains a unique post-translational modification, called diphthamide, not found in any other protein, including its bacterial homolog EFG. The diphthamide modification is highly conserved, occurs only at a specific histidine in EF2 (His715 in mammals), and requires a complex three-step synthesis pathway involving at least seven different proteins. It is also the target of several bacterial toxins (diphtheria toxin, Pseudomonas exotoxin A, and cholix toxin), which block its function to inhibit protein synthesis. Circumstantial evidence surrounding the diphthamide suggests that it plays an important role in translation, but the function of diphthamide remains elusive. We have systematically mutated the diphthamide histidine to all nineteen other amino acids in human EF2 (hEF2) and stably expressed them in HEK293 cells. Expression was confirmed by gene sequencing and western blot. All transgenic lines grow normally except the H715K mutant, which could not be generated. None of the lines were resistant to Pseudomonas exotoxin A. Work is underway to evaluate the H715K mutation in hEF2 and remove endogenous wild-type hEF2 from cell lines that stably express mutant hEF2. HERC6 is a HECT E3 ubiquitin ligase involved in the innate immune response to viral infections as well as spermatogenesis. HERC6 can facilitate the posttranslational attachment of ubiquitin and ISG15 to various proteins in the cell. This dual ability to ubiquitylate and ISGylate its substrates makes HERC6 a unique HECT E3 ligase family member as all of the other HECT E3 ligases characterized to date work exclusively with either ubiquitin or ISG15. A previous study demonstrated that ubiquitin-linkage specificity is localized in the C-terminal lobe of the HECT domain that contains the conserved cysteine required for catalysis. To determine the unique biochemical and structural basis for HERC6-dependent ubiquitylation and/or ISGylation, we set about solving the 3D structure of HERC6 by x-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. We demonstrate that the C-terminal lobe of HERC6 forms a novel dimeric structure via a 3D domain swapping mechanism, where the protein monomers exchange structural elements to fold into a structurally similar conformation to what would be expected in the monomer. Our in vitro fluorescent ubiquitination and ISGylation assays suggest that the dimeric HERC6 C-lobe is autoinhibited by occluding the catalytic cysteine and we demonstrate that dimer disrupting substitutions of HERC6 relieve this autoinhibited state. This is the first example of a structural domain swapping mechanism for a HECT E3 ubiquitin ligase and demonstrates a novel mode of autoregulation by a unique HECT family member. Post-translational modifications (PTMs) on histone proteins play a central role in epigenetic regulation, and profiling the dynamic readout of these discrete modifications is imperative for therapeutic efforts. Antibodies designed for detecting PTMs on chromatin are an instrumental reagent in epigenetics research, however challenges such as the lack of PTM specificity and reproducibility of detection persist in the use of antibodies. Inspired to improve the tools available for histone PTM detection, we have engineered a "super" reader protein to have a stronger binding affinity than its native counterpart. With a sequence-selective and PTM-specific scaffold as a foundation for directed evolution, we have focused on improving the affinity of heterochromatin protein 1 (HP1) for trimethyl lysine at position 9 of histone 3. Reader:histone interactions typically have dissociation constants in the mid to low micromolar range, so increasing the binding affinity of readers into the nanomolar regime would improve utility in biochemical assays. Targeting highly conserved motifs of the chromodomain, we identified rational mutation sites at which we are screening the incorporation of natural and unnatural amino acids in a combinatorial fashion. We generated a mutant HP1 chromodomain with a 20-fold tighter binding affinity than wildtype and are in the process of evaluating the application of this "super" reader in peptide arrays and dot blot assays. Thus, super readers have the potential to supplement the use of primary antibodies for PTM detection, and engineered reader proteins would both address the limitations of antibodies and be a more cost-effective tool. Many dsDNA viruses use a multi-component ATPase motor to package their genomes into immature capsids during viral maturation. These motors are among the most powerful known molecular machines, and how they couple ATP hydrolysis to DNA translocation is still poorly understood. Using a multifaceted approach combining x-ray crystallography, cryo-electron microscopy, small angle x-ray scattering, protein docking, and biochemical assays, we elucidate the mechanism of ATP hydrolysis-mediated DNA translocation. We present a novel genome packaging system from a thermophilic virus that provides new insight into motor structure and mechanism. We show that interfacial contacts mediate ATP hydrolysis, identify essential DNA binding and cleavage motifs, and reveal ATP-dependent conformational changes of the packaging motor. Additionally, we showed that the motors ATPase domain is both necessary and sufficient for DNA binding, an attribute previously ascribed to the nuclease domain. Finally, we report a cryoEM reconstruction of the virus capsid, revealing mechanisms for capsid expansion and stability, as well as a surprising shared evolutionary history between capsid decoration proteins and the Anti-CRISPR protein AcrIIC1. Overall, our findings lead to a mechanism of translocation with a long leverarm to generate high force, and a novel mechanism for capsid expansion without alteration of underlying icosahedral symmetry. Molecular chaperones TAPBPR (TAP-binding protein related) and tapasin associate with class-I major histocompatibility complex (MHC-I) molecules to promote optimization of peptide cargo. Here, we use solution NMR to investigate the molecular mechanism of peptide exchange. We identify TAPBPR-induced conformational changes on conserved MHC-I surfaces, consistent with our independently determined Xray structure. Conformational dynamics present in the empty MHC-I are stabilized by TAPBPR in a peptide-deficient complex, and become progressively dampened with increasing peptide occupancy. Incoming peptides are recognized by the chaperoned groove according to the global stability of the final pMHC-I product, and anneal in a native-like conformation. Our results demonstrate an inverse relationship between MHC-I occupancy by peptide and the affinity of TAPBPR for such pMHC-I molecules, where the lifetime of transiently bound peptides controls the dynamic regulation of a conformational switch, located near the TAPBPR binding site, which triggers TAPBPR release. These results suggest a similar mechanism for the editing function of tapasin in the peptide-loading complex. Determining the three dimensional structures of macromolecules is a major goal of biological research because of the close relationship between structure and function. Structure determination usually relies on physical techniques including x-ray crystallography, NMR spectroscopy and cryo-electron microscopy. Here we present an approach that allows the high resolution three dimensional structure of a biological macromolecule to be determined only from measurements of the activity of mutant variants of the molecule. Mutations within a protein can have non-independent effects on folding, stability and activity. We show that quantifying how often residue pairs interact non-independently and how such interaction patterns correlate across the protein can robustly identify individual tertiary structure contacts as well as periodic secondary structure elements and their interactions. Moreover, we show that these data can be used to accurately predict the structure of a protein domain at 1.9 Ångström root mean squared error compared to a reference crystal structure. This genetic approach to structure determination should be widely applicable, including to macromolecules whose structures are difficult to determine by physical techniques. Under native conditions, proteins exhibit a range of dynamics from local fluctuations to complete global unfolding and refolding. Investigating these native-state dynamics is important for understanding how proteins function normally, and how they can lose function through misfolding and aggregation. Common experiments to measure folding and unfolding kinetics involve destabilizing a protein by the addition of denaturants to measure kinetic constants. This harsh approach requires significant extrapolation from experimental to physiological conditions and fails for many biologically interesting proteins that tend to aggregate. Hydrogen Exchange Mass Spectrometry (HXMS) is a promising approach that is sensitive to a broad range of folding and unfolding dynamics in the native state, but direct analysis of spectra to determine rate constants is limited to certain experimental regimes. In this work, we present a Markov model to quantitatively analyze native state HXMS across all regimes and extract rate constants directly from measurements on proteins under native conditions. In addition, we develop a Bayesian approach to estimating the rate constants, enabling rigorous approximation of the error in our fit values. Finally, we apply our newly developed tools to native-state HXMS data for Beta-2 Microglobulin, a protein which has been implicated in dialysis related amyloidosis. We find evidence for an on-pathway folding intermediate which may be involved in amyloid formation. In our research, we have encountered multiple structures with misidentified, wrongly modeled, or missing ligands in protein structures deposited to the PDB. We took the liberty of generating the electron density maps using the structure factors deposited and re-refined several crystal structures. When raw diffraction images were available, their re-processing resulted in most remarkable improvements. The set of re-refined structures includes protein complexes with anti-cancer drugs cisplatin and carboplatin, complexes of beta-metallo-lactamases (enzymes on antibiotic resistance) with potential inhibitors, a formate dehydrogenase (biotechnological enzyme), and a LacI family transcriptional regulator. In most cases, the primary ligands were re-modeled, replaced (with a buffer component or a product of ligands conversion), or completely removed. The correctness of protein structures deposited to the PDB is often taken for granted by the users of the structural data. If mistakes are present, they easily propagate into the literature, bioinformatics-based research, and datasets used for training structure-modeling and docking software. Therefore, it is vital for the crystallographic community to safeguard a better quality of the deposited structures and for the users to scrutinize the validity of the structural models. Sicai Zhang 1 1 Boston Children's hospital (Boston, United States) Botulinum neurotoxins (BoNTs) are a family of the most potent bacterial toxins and potential bioterrorism agents. They are produced by various Clostridium strains. Here we report that an Enterococcus faecium strain contains a novel member of the BoNT family, tentatively designated BoNT/En. It cleaves VAMP1/2/3, the same substrate of BoNT/B, D, F, G and X, at a distinct site (A67-D68 in VAMP2). In addition, BoNT/En cleaves multiple other SNARE proteins including SNAP-25, SNAP-23, syntaxin 1B, and syntaxin 4 with low efficiency in vitro, and cleaves SNAP-25 efficiently within neurons. The protease and translocation domains of BoNT/En are functional on rat/mouse neurons, however full-length BoNT/En generated by sortase-mediated ligation do not exhibit neuronal toxicity in mice in vivo, suggesting that the receptor-binding domain of BoNT/En may not target mammalian neurons. Comparative genomic analysis determines that the E. faecium strain containing BoNT/En is a commensal-type, and the BoNT/En gene cluster is located on a 206-kb conjugative plasmid. BoNT/En is the first neurotoxin-like toxin identified in the Enterococcus genus. These findings establish a new member of BoNTs and demonstrate the capability of E. faecium, a commensal organism ubiquitous in humans and animals and a leading cause of hospital-acquired multidrug resistant infections, to horizontally acquire, and possibly disseminate, a BoNT gene cluster. The insecticidal protein, Cry51Aa2.834_16, has been shown to provide effective protection of cotton plants against hemipteran and thysanopteran pest species, including Lygus hesperus and Lygus lineolaris. Cry51Aa2.834_16 has sequence homology to a superfamily of beta pore forming proteins that share structural homology in the pore-forming domain but show significant sequence and structural diversity in the receptor binding or head domain. Previous mechanistic studies revealed that proteolytic activation of dimeric Cry51Aa2.834_16 by Lygus saliva enabled dose dependent binding of monomeric Cry51Aa2.834_16 to a specific protein receptor in the Lygus midgut epithelium with evidence for a membrane inserted protein complex. We have identified the specific protein receptor for Cry51Aa2.834_16 in Lygus hesperus. Both lectin competition analysis and periodate modification implicate glycan modifications on the receptor as contributing elements to the interaction with Cry51Aa2.834_16. Additional structural and mutagenic analyses reveal surface exposed aromatic amino acids in the head domain of Cry51Aa2.834_16 as critical determinants of receptor recognition and insecticidal activity. Together these results expand our understanding of the relationship between structure-function and target specificity of this superfamily of proteins, and provide a model for the mechanism of specificity of Cry51Aa2.834_16 against Lygus species. Wei He 1 , Matthew Coleman 1 1 Lawrence LIvermore National Laborotary (Livermore, United States) Chlamydia is the most common bacterial sexually transmitted infection (STI), affecting over 130 million people every year. Because the chlamydia infection is often without any symptoms, it can go untreated for years and can result in long-term consequences, such as ectopic pregnancy and infertility. The chlamydia major outer membrane protein (MOMP) has been regarded as a promising vaccine candidate. However, isolation of MOMP from infectious chlamydia bacteria is a strenuous and risky job. In addition, MOMP is a highly hydrophobic protein, and it needs to assemble into a trimer to be an effective vaccine. Our lab has developed a cell-free method to de novo synthesize MOMP without using live bacteria. To solubilize and stabilize MOMP in its trimer form, we developed a type of nano-carrier called nanolipoprotein particle (NLP) to incorporate MOMP. NLPs are made of protein scaffold and a lipid bilayer. The lipid bilayer carries the membrane protein like MOMP in its physiological condition. When MOMP is coexpressed with NLP in the presence of an amphiphilic polymer called telodendromer, we were able to achieve high yield of MOMP incorporated telodendrimer NLP (tNLP). We characterized the MOMP functionally as well as structurally and confirmed that MOMP is in a functional trimeric form. We have shown in animal models that our MOMP-tNLP vaccine can induce MOMP specific antibody response and protect animals against chlamydia infection. Our work represents a major breakthrough achieved by combining synthetic biology approaches and cell-free co-expression of MOMP to achieve a highly protective vaccine against Chlamydia infections. Modulation of enzyme structure and flexibility by cofactor/substrate binding provides an important source of enzyme function regulation, yet our understanding of the fundamental mechanisms coupling protein dynamics to biological function is still largely incomplete, therefore limiting our ability to harness protein conformational dynamics to regulate enzymatic activity. Here, we use NMR relaxation dispersion and enzyme kinetic data measured at variable temperature to investigate the role played by conformational dynamics in the phosphoenolpyruvate hydrolysis reaction catalyzed by the C-terminal domain of bacterial enzyme I (EIC). The data, measured for mesophilic (mEIC, physiological temperature = 37 C) and thermophilic (tEIC, physiological temperature = 65 C) homologs of the enzyme, show that the catalytic turnover (kcat) is linearly correlated to the exchange rate constant (kex), which describes s-ms timescale conformational dynamics in EIC. The fact that our measured kcat << kex (i.e. the conformational dynamics are not the rate limiting step in catalysis, Fig. 1 ) suggests that the observed relationship between s-ms dynamics and turnover number may be correlated to the degree of conformational plasticity of the enzyme active site. Guided by our NMR data, we design a hybrid tEIC/mEIC construct that has a thermal stability similar to the one of the thermophilic enzyme, and active site conformational dynamics similar to the ones of the mesophilic protein. The designed hybrid enzyme (hybEIC) has increased high-temperature activity if compared to tEIC (Fig. 1) . We expect our NMR-based design strategy to be applicable to other thermophilic/mesophilic enzyme pairs, and to become a valuable tool in protein engineering. The HIV capsid plays a crucial role in protecting the viral genome from cellular antiviral factors and empowering the virus to take advantage of cofactors. The capsid is composed of capsid protein (CA) monomers, arranged into a lattice of~250 CA hexamers and 12 CA pentamers to assemble a fullerene cone architecture. Many host protein factors recognize features of the higher order capsid lattice, so that capsid assemblies of defined CA oligomers are needed for biochemical and structural studies of capsid-host factor interactions. However, it has been challenging thus far to overcome the natural polymerization tendency of CA to capture discrete oligomers. Recently, a novel llama nanobody (llamabody) has been developed to bind and block the CA polymerization interface with high affinity. To further establish llamabody as a tool to control CA assembly, we sought to understand the biochemical and biophysical details of the CA-llamabody interaction and characterize mutations that allow us to manipulate this interaction. We used size exclusion chromatography binding assays, isothermal titration calorimetry, and X-ray crystallography to determine that llamabody can bind a variety of CA assemblies, that mutating interface residues on CA allows us to tune llamabodys affinity, and that the CA-llamabody interface is not rigid and can adjust to environment. Our findings suggest that llamabody can be used to shield polymerization interfaces during the formation of CA oligomeric assembliespaving the way for future biochemical and structural studies of host factor-capsid interactions. Herein are described three examples where proteins were computationally and experimentally engineered to facilitate their study or to generate novel tools to enable scientific discovery. In the first example, the polycomb repressive complex (PRC2) maintains regions of chromatin in a repressed state, helping dictate cell developmental fates. Using the Rosetta software suite, a protein-based PRC2 disruptor was engineered that abolishes chromatin repression and enables new types of experiments in cancer and stem cells. In a second example, p53 is a transcription factor that, in response to cancer, DNA damage, or cell stress, halts the cell cycle or triggers apoptosis. Cancer cells overexpress the mouse double-minute (Mdm) proteins Mdm2 and Mdm4, which work together to inhibit p53 activity and allow cancer cell survival. A family of highly specific protein-based inhibitors were engineered that allow elucidation of the individual contributions of Mdm2 and Mdm4 on cell cycle regulation. These inhibitor proteins revealed that Mdm2-bound p53 blocks entry into S-phase while Mdm4-bound p53 blocks entry into both S and M phases of the cell cycle. In a final example, the [4Fe4S] cluster of radical S-adenosylmethionine (radical SAM) enzyme pyruvate formate-lyase (PFL-AE) exhibits a rare electronic state in vivo, termed valence localization. To understand how this state occurs, a variant of PFL-AE was engineered that readily forms diffraction-quality crystals. This allowed determination of atomic level structures of PFL-AE bound to various small molecules, revealing that valance localization may be caused by ordering water molecules around the [4Fe4S] cluster. Titin is the largest known molecule in the human body, which in some regards acts as a molecular spring connecting the thin filament in the Z-disk to the thick filament in the M-line. Titin is comprised of regions with distinct physical characteristics, including the N2A region which exists between the proximal immunoglobulin (Ig) region and the PEVK region. The N2A region is of interest as it demonstrates multiple binding interactions and plays a potential signaling role in the sarcomere. Within the N2A region, adjacent to the insertion sequence, are three sequential Ig domains: I81, I82, and I83. These Ig domains demonstrate unique chemical stabilities as compared to one another when unfolded by urea denaturation. It is observed that I83 consistently unfolds at the lowest concentrations of urea and has lowest G, indicating that it is the least stable of the three domains. I82 demonstrates a higher stability, and I81 the highest. The presence of calcium (pCa 4.3) induces a stabilizing effect, most notably in the I83 domain. This was not mimicked as strongly with the presence of another divalent cation (Mg++). Due to the correlation between an influx of Ca++ and the stiffening of titin, this stability may contribute to theories connecting eccentric contractions and changes to the N2A region. 16 Single-stranded DNA, present in numerous aspects of chromosome biology, is managed by a suite of proteins with tailored activities. The majority of these ssDNA-interaction proteins exhibit little sequence preference. Notable exceptions include proteins vital for telomere maintenance, including S. pombe Pot1 and S. cerevisiae Cdc13. These are two of the tightest known binders of ssDNA and are both specific for G-rich telomeric sequences. The C-terminal domain of Pot1, Pot1pC, exhibits non-specific ssDNA recognition, binding a wide range of ssDNA sequences with thermodynamic equivalence. To address how Pot1pC binds ssDNA with no specificity, but discriminates against RNA, multiple structures of Pot1pC bound to non-cognate ssDNA ligands were solved. These structures reveal that specificity is implemented through new binding modes that thermodynamically compensate for base-substitutions through alternate stacking interactions and new H-bonding networks. To investigate how affinity and specificity contribute to function, we created an unbiased panel of alanine mutations across the Cdc13 DNA-binding interface. A subset of mutant proteins exhibited significant loss in affinity in vitro that, as expected, conferred a profound loss of viability in vivo. Unexpectedly, a second category of mutant proteins displayed an increase in specificity, manifest as an inability to accommodate changes in ssDNA sequence. Yeast strains with specificity-enhanced mutations displayed a gradient of viability that paralleled the loss in sequence tolerance in vitro, arguing that binding specificity is fine-tuned to ensure optimal function. These findings suggest that intermolecular interfaces are remarkably sophisticated in their tuning of specificity towards flexible ligands. To effectively develop biotherapeutic molecules and bring them to market, it is critical that researchers thoroughly understand the relationships between specific process parameters and product quality. Design of Experiment studies, which can most effectively define these relationships through full examination of the range of potential variables affecting protein quality, are dependent on the analysis of large numbers of samples. Traditionally, SDS-PAGE, HPLC, and CE based separation techniques have been used to monitor glycosylation, purity, and homogeneity of samples during process development. Unfortunately, these methods limit throughput and limit multifactorial studies. The LabChip ® GXII Touch protein characterization system offers rapid analysis of up to seven different protein quality attributes, thereby enabling significant expansion of process development studies. The same system is available in a lower throughput version suitable for continued use of the same assays as products are transferred to production environments. Here we demonstrate the sensitivity, resolution, and reproducibility of the LabChip ® GXII Touch system using Adalimumab as a reference IgG sample for three different types of standardized CE assays with a maximum separation time of 90 seconds/sample. Data characterizing the glycosylation profile and protein charge heterogeneity were generated with assay kits and protocols for covalent labeling and subsequent separation. Analysis of IgG percent purity, percent non-glycosylated, and percent fragmentation was determined with a CE-SDS assay kit and protocol utilizing on-chip staining and de-staining of proteins and fragments. Kaixian Liu 1 , Christian Kaiser 1 , Kevin Maciuba 1 1 Johns Hopkins University (Baltimore, United States) Multi-domain proteins constituting a large group in all proteomes often require help from molecular chaperones to fold productively, even before the ribosome has finished their synthesis. The mechanisms underlying chaperone function remain poorly understood. We have used optical tweezers to study the folding of elongation factor G (EF-G), a model multi-domain protein, as it emerges from the ribosome. We find that the Nterminal G-domain in nascent EF-G polypeptides folds robustly. The following domain II, in contrast, fails to fold efficiently. Strikingly, interactions with the unfolded domain II convert the natively folded G domain to a non-native state. This non-native state readily unfolds, and the two unfolded domains subsequently form misfolded states, preventing productive folding. Both the conversion of natively folded domains and nonproductive interactions among unfolded domains are efficiently prevented by the ribosome-binding chaperone trigger factor. Thus, our single-molecule measurements of multi-domain protein folding reveal an unexpected role for the chaperone: It protects already folded domains against denaturation resulting from interactions with parts of the nascent polypeptide that are not folded yet. Previous studies had implicated trigger factor in guiding the folding of individual domains, and interactions among domains had been neglected. Avoiding early folding defects is crucial, since they can propagate and result in misfolding of the entire protein. Our experiments define the folding pathway for a complex multi-domain protein and shed light on the molecular mechanism employed by molecular chaperones to ensure productive folding and proteome maintenance. In dividing cells, cytoplasmic dilution is the dominant route of clearance for long-lived proteins whose inherent degradation is slower than the cellular growth rate. Thus, as cells transition from a dividing to a non-dividing state, there is a propensity for long-lived proteins to become stabilized relative to short-lived proteins, leading to alterations in the abundance distribution of the proteome. However, it is not known if cells mount a compensatory response to counter this potentially deleterious proteostatic disruption. We used a dynamic proteomic approach to demonstrate that fibroblasts selectively increase degradation rates of long-lived proteins as they transition from a proliferating to a quiescent state. The selective degradation of long-lived proteins occurs by the concurrent activation of lysosomal biogenesis and upregulation of macroautophagy. Through this mechanism, quiescent cells avoid the accumulation of aged long-lived proteins that would otherwise result from the absence of cytoplasmic dilution by cell division. We have tried to decipher certain consistent patterns present in the residues constituting the allosteric communication sub-system (ACSS). We considered all the PDB annotated proteins from the Allosteric Database (ASD) [1, 2] , categorized into four different classes (kinases, nuclear receptors, peptidases, transcription factors). Thermal fluctuations of hydrophobic residues in ACSSs showed significantly higher values than their non-ACSS counterparts; polar residues showed opposite trends. We found the basic and hydroxyl residues to be slightly more predominant than the acidic and amide residues in ACSSs. Hydrophobic residues were found in abundance in kinase ACSSs; despite varying sequences and lengths of ACSSs, they were found to be structurally similar to each other, suggesting a preferred structural template for communication. Low RMSD and high Akaike Information Criterion (AIC) scores were recorded for ACSS structures, in addition to low degree and closeness centrality, while betweenness centrality followed a non-uniform behavior. ACSSs in general did not demonstrate small world behavior, except for a few sub-graphs and 1XNX. Our current work aims to supplement our previous work [3] of deciphering the general mechanism of allostery using a novel graph-theoretic and complex network approach. The current therapeutic progress for Alzheimers disease (AD) and type-II diabetes (T2D), based on the development of inhibitors of beta-amyloid (A) and human islet-amyloid polypeptide (hIAPP) aggregation, have produced a variety of novel findings but has not been successful in producing treatment for these devastating diseases. In this study, we demonstrate a novel strategy, for the first time, to develop AD and T2D therapy using copolymers and polymers-encased-lipid-nanodiscs. We show the synergistic mechanism of action imposed by solvent exposed charged copolymers and encapsulated-phospholipids on A40/hIAPP self-assembling and amyloid fiber remodeling. Using several biophysical and biochemical assays, we demonstrate that two of bioavailable nanodisc-forming copolymers ( SMAEA-and PMAQA+) inhibit amyloid aggregation, isolate A/hIAPP intermediates and also remodel fibers to small off-pathway oligomers that exhibit variable cell-toxicity (A:SH5Y; hIAPP:RIN5F). Distinct pathological phenotype (A40/hIAPP) intermediates were achieved by varying the nanodiscs molar lipid compositions. We also show the potential anti-amyloidgenic activity of SMAEA-and PMAQA+ copolymers on hIAPP and A40, respectively, at sub-micromolar concentration. SMAEA-and PMAQA+ promote a very quick structural transition (unfolded to -rich) of IAPP and A40, respectively, through electrostatic interactions and inhibit protein aggregation over 5 days. Electron microscopy combined with NMR and molecular dynamics simulations characterize the intermediates of A40/hIAPP trapped by nanodiscs and remodeled from fibers at molecular level. Our study reports a valuable approach to integrate anti-amyloidogenic polymers to design lipid-nanodiscs for strategic anti-amyloid therapy for potential AD and T2D treatments and are exciting as they open avenues for further developments of potent amyloid inhibitors. The channel of matrix-2 (M2) protein from influenza A virus (AM2) has a vital role in transferring protons across viral membranes. A histidine tetrad in the core of the AM2 channel allows proton flux into the virus in response to the low pH of the endosome. AM2 plays an important role in the infective process of this virus; therefore, a deeper understanding of the mechanism, function and structure of the AM2 protein has significant public health relevance. AM2 has been the target of many experimental and computational studies that led to significant findings regarding its structure and function under differing conditions. The central question now facing the field is to understand the mechanism of the proton transfer under different pH conditions. In addition, one important question not fully answered is how other residues in both transmembrane and amphipathic helices in AM2 can accelerate/decelerate the proton flux. To this end, we have applied explicit solvent constant pH molecular dynamics using the multi-site dynamics approach to answer these prevailing questions. We have calculated the pKa values of buried residues in the hydrophobic environment of the transmembrane and amphipathic helices to understand the microenvironment modulates on the charged and neutral states, pH-mediated conformational fluctuations and biological function of membrane proteins. We elucidate their effect on the orientation and position of the helices in the membrane. Our results offer a qualitative image of the mechanism of the proton transfer in AM2 and provide guides for future experimental and theoretical research on this protein. Molecular chaperones, also known as heat shock proteins (HSPs), are a diverse and highly conserved class of enzymes that maintain proteostasis through the mediation of protein structure in vivo via their up-regulation, allowing for recovery from various cellular stressors. Several families of molecular chaperones are known to exist and are classified by molecular weight (kDa). Ssa1 is HSP70 class molecular chaperone found in Saccharomyces cerevisiae that works in tandem with its co-chaperone Sis1 to maintain cellular proteostasis through restoring native conformations of misfolded proteins. Endogenous studies of this protein often prove limiting due to its transient mechanism of activity and the resource-intensive nature of commonly used purification methods. To address this, we developed a highly efficient, one-step purification of Ssa1 utilizing a previously constructed Protein-A transformant strain. Our results indicate that our method is able to natively isolate high yields of pure Ssa1 from small amounts of starting material in a short period of time. Furthermore, our method allows for the native elution of Ssa1 without compromising enzymatic activity, allowing the protein to be utilized immediately after elution for further assays to better understand interactions between Ssa1 and its co-chaperones as well as the effects of post-translational modifications on chaperone function Protein and peptide aggregation leads to the development of several debilitative disorders, such as Alzheimers and Parkinsons diseases or preeclampsia. Misfolded proteins can aggregate forming amyloids that lead to the formation of fibrils and then plaques that disrupt the normal functioning of cells. The development of accurate methods to predict the aggregation of proteins and peptides is of the utmost importance. There are several known methods for prediction of aggregation propensity of a protein from its sequence, such as AGGRESCAN, FoldAmyloid, FISH, AMYLPRED, and others. Recently we used GOR method originally developed for prediction of protein secondary structure from sequence to predict protein aggregation propensities [1] . In the present work we applied SPINE-X method [2] to prediction of aggregation propensity by using a multistep neural-network algorithm. Our preliminary studies obtained using proteins from Curated Protein Aggregation Database (CPAD) [3] show that this method is highly promising and efficient alternative to other known aggregation predicting tools, and leads to improved accuracy of prediction of protein aggregation propensity from sequence. Karl Schmitz 1 , Christopher Presloid 1 , Thomas Swayne 1 1 University of Delaware (Newark, United States) N-end-rule proteolytic pathways couples the stability of cytosolic proteins to the identity of their Nterminal amino acid. In proteobacteria, including E. coli, proteins bearing N-terminal Leu, Phe, Tyr, or Trp residues are recognized by the ClpS adaptor and delivered to the ATP-dependent ClpAP protease for degradation. No equivalent N-end-rule pathway has been described in actinobacteria, yet many members of this phylum, including the globally important human pathogen Mycobacterium tuberculosis, possess a ClpS ortholog. We have solved the X-ray crystal structure of mycobacterial ClpS, revealing overall structural similarity to its proteobacterial counterpart, including a well-defined binding pocket. Fluorescence anisotropy and co-crystallization experiments show that myocbacterial ClpS can bind peptides bearing canonical N-end-rule amino acids, albeit with weaker affinity than E. coli ClpS. Moreover, we find that mycobacterial ClpS interacts with and modulates the activity of the ATP-dependent protease ClpC1P1P2, which bears sequence and structural homology to E. coli ClpAP. Our findings suggest that ClpS functions as an N-end-rule pathway adaptor for the ClpC1P1P2 protease in mycobacteria, and may provide novel avenues for disrupting essential proteolytic processes in Mycobacterium tuberculosis. William Holmes 1 , Anna Lally 1 1 The microtubule-associated protein Tau is associated with neurodegenerative diseases such as Alzheimers Disease and Chronic Traumatic Encephalopathy (CTE). Tau endogenously stabilizes microtubules in neurons, and its function is regulated by phosphorylation. However, hyper-phosphorylation of Tau increases its propensity to aggregate and form toxic oligomers. Tau is also predicted to be a target for the most common mammalian post-translational modification, N-terminal acetylation, which neutralizes the positive charge of the N-terminus by adding an acetyl group. N-terminal truncations of Tau increase aggregation rates over 20 fold, suggesting that the N-terminus affects aggregation. Our understanding of Tau aggregate dynamics primarily relies on Tau expressed and purified in vitro, however these studies utilize E. coli expression systems, which means that our understanding of physiologically relevant Tau is incomplete. Our goal is to determine the effects of N-terminal acetylation on Tau by assessing the differences between acetylated and unacetylated tau. We utilized a co-expression system wherein Tau was coexpressed with the NAT complex in E. coli, and initially determined that Tau was N-terminally acetylated and could be N-terminally acetylated in vitro. Our protocol allows us to express and purify Tau with around 98% purity, and acetylation is determined by SDS-PAGE, IEF, and mass spectrometry. Further analysis by thermal melt and size exclusion chromatography determine any significant structural changes to Tau. Our expression and purification of Tau will produce a more physiological relevant version of Tau that can be used in future studies to more accurately determine changes to structure, aggregation kinetics, and toxicity. Graduate School of Engineering, Nagasaki University (Nagasaki, Japan) C-type lectins play important roles in self-defense systems in marine invertebrates. C-type carbohydrate-recognition domains (CRDs) commonly recognize monosaccharide moiety in oligosaccharide chains through coordinate bonds with Ca2+ ion along with hydrogen bond networks with nearby amino acid residues. Recently, we have found that C-type lectins SPL-1 (heterodimer of A-and B-chains) and SPL-2 (homodimer of B-chains) from the bivalve S. purpuratus bind both GlcNAc and GalNAc without Ca2+ ion through interaction with their acetamido group, which is a novel recognition mode for C-type CRDs. In this study, recombinant A-and B-chains were prepared using Escherichia coli to examine their dimerization properties and individual binding specificities. The proteins were refolded from inclusion bodies in combination of either single or mixed chains. Particle size measurement using dynamic light scattering of the resulting proteins indicated that heterodimer of A/B-chains and homodimer of B/Bchains were correctly formed, whereas refolding of only A-chain resulted in a multimeric form. This suggests that proper interactions for stable homodimer could not be formed between A-chains, which also explains why A/A homodimer has not been found in the natural organism. Detailed carbohydratebinding specificities of A-and B-chains for various oligosaccharides were examined by the glycan array analysis. Both proteins exclusively bound to the oligosaccharides containing GlcNAc or GalNAc at the non-reducing ends, but not to those inside the oligosaccharides. When A-and B-chains were compared, minor differences in specificity were also found, reflecting the structural differences in their binding sites. Thrombospondin-1 (TSP-1), an~450 kDa homotrimeric protein, was discovered as the first endogenous inhibitor of angiogenesis. TSP-1 contains multiple domains and is implicated in calcium homeostasis and cell attachment. Some responses require binding to cell surface receptor CD47, an~50 kDa plasma membrane protein with five transmembrane helices and an extracellular immunoglobulin-like domain. CD47 is central to immune evasion and is overexpressed in many cancers where binding to signal regulatory protein alpha (SIRPalpha) on macrophages leads to immune escape. In endothelial cells, TSP-1 binding to CD47 leads to inhibition of nitric oxide (NO) signaling at multiple steps, including through inhibition of the NO receptor, soluble guanylyl cyclase (sGC). We showed that CD47 is necessary, but insufficient for TSP-1 binding, suggesting that TSP-1 binds a CD47 complex. We seek to discover key functional CD47 complexes in CD47 signal transduction involving TSP-1 and SIRPalpha. To accomplish this, we are developing selective proteomic proximity labeling to identify CD47 binding partners, and developing a CD47 nanodisc assembly for evaluating complex formation and identifying binding surfaces. Here, we will describe expression and purification of full-length CD47, the C-terminal domain of TSP-1 and the N-terminal domain of SIRPalpha, as well as progress with binding analyses using CD47-containing nanodiscs and surface plasmon resonance (SPR). Progress with cellular binding examined by flow cytometry will also be included. Our results will lay the foundation for understanding signal transduction through CD47 and developing new therapeutic strategies for cardiovascular disease and cancer. L-lactate oxidase (LOx) is a flavin mononucleotide (FMN) dependent enzyme that catalyzes the oxidation of L-lactate to pyruvate by using molecular oxygen as a natural electron acceptor with high specificity and stability. Many L-lactate biosensors use artificial electron mediator to facilitate the electron transfer from LOx to the electrode, obtaining current signals that reflect L-lactate concentration. However, dissolved oxygen competes with artificial electron mediator and decrease current signal. Therefore, minimization of the oxygen interference effect is required. To achieve so, using mutagenesis studies, we turned the LOx from Aerococcus viridans (AvLOx) into dehydrogenase. Oxygen accessible path in the AvLOx structure was predicted by software CAVER 3.0.1, revealing a pathway from the enzyme surface to the FMN, and an alteration of Ala96 seemed to block the oxygen pathway. Saturated mutagenesis showed that Ala96Leu mutant decreased oxidase activity (ability to use oxygen as electron acceptor) drastically and maintained the dehydrogenase activity by using the artificial electron mediator (K. Hiraka et al., (2018) Biosens. Bioelectron.). Similarly, we attempted to turn another LOx from Enterococcus (EnLOx) into dehydrogenase. Unfortunately, EnLOx structure was not revealed. Based on the AvLOx structure, EnLOx model structures were constructed, and the oxygen accessible pathway re-analyzed. Interestingly, we found a open pathway that relies on the direction of an amino acid side chain. Saturated mutagenesis study showed that this residue has a critical role for oxygen reactivity. The detailed characteristics of EnLOx mutant will be presented. The e4 allele of apolipoprotein E (APOE4) is strong genetic risk factor for many neurological and cardiovascular conditions. In the case of Alzheimer's disease, the single amino acid that distinguishes the wildtype protein, APOE3, from the risk variant, APOE4, results in a profound (3-4 fold) increase in disease risk. Despite these strong associations with disease, the cellular mechanisms by which APOE4 increases disease susceptibility remain poorly understood. In order to understand the perturbations to basic biology that contribute to APOE4-associated risk, we constructed a yeast model where only APOE4 exhibits a dose-dependent toxicity. In this model, other APOE isoforms, including APOE3, are benign. Through genome-wide screens and functional studies, we identified and characterized two central cellular processes that are perturbed by APOE4 expression-endocytosis and lipid homeostasis. We found that both endocytosis and lipid homeostasis are disrupted not only in yeast but also in human induced pluripotent stem cell (iPSC)-derived astrocytes endogenously expressing APOE4 (and importantly, not in those expressing APOE3). Further characterization allowed us to identify the molecular underpinnings for these disruptions as well as both chemical and genetic means to rescue the phenotypes in yeast and iPSCderived human astrocytes. Our findings demonstrate that we can modulate genetic factors or rewire the metabolic state of the cell to mitigate the widespread consequences of a single point mutation in APOE. With its yeast-to-human cell approach, our work presents a powerful paradigm for studying the influence of both genetics and environment on a common and potent disease risk factor. Fibril formation resulting from protein aggregation is a hallmark of several neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, preeclampsia, a pregnancy-specific disorder, has been shown to share typical pathophysiological features with neurodegenerative diseases (1, 2) . Despite much progress in understanding the protein aggregation process(3), the factors governing fibril formation rates and fibril stability have not been fully understood. Using lattice models we have shown that fibril formation time is controlled by the kinetic stability of the fibril state but not by its energy. Having performed all-atom explicit solvent molecular dynamics simulations with the GROMOS43a1 force field for full-length amyloid beta peptides A40 and A42 and truncated peptides, we demonstrated that kinetic stability can be accessed via mechanical stability in such a way that the higher mechanical stability or kinetic stability the faster fibril formation(4). Pyrazinamide is an essential first-line antitubercular drug which plays pivotal role in tuberculosis treatment. It is a prodrug that requires amide hydrolysis by mycobacterial pyrazinamidase enzyme for conversion into pyrazinoic acid (POA). POA is known to target Ribosomal protein S1 (RpsA), Aspartate decarboxylase (PanD) and some other mycobacterial proteins. Spontaneous chromosomal mutations in RpsA have been reported for phenotypic resistance against Pyrazinamide. Native and mutant RpsA models were constructed and validated through a large set of quality and constraint analysis programs. Then, native and mutant proteins were docked to Pyrazinoic acid and long term molecular dynamics (MD) simulations coupled with per residue binding free energy calculations, free energy landscape analysis and essential dynamics analysis, were performed to outline the mechanism underlying the high-level PZA resistance conferred by the most frequently occurring deletion mutant of RpsA, namely 438A. Our study revealed the secondary structure distortions in mutant responsible for modulation of POA binding site which causes difference in conformational flexibility and collective modes of motions between native and mutant forms. MMPBSA calculations and interaction pattern analysis revealed the difference of energetically favourable binding site in the wild type protein in comparison to the mutant. Size and shape of minimal energy area depicted the higher stability of wild type complex than the mutant forms. Our study reports new insights towards mechanistic details for Pyrazinamide resistance in 438A RpsA mutant that would certainly aid the experimental pipeline for design of new and effective inhibitors targeting the resistant mutants of Mycobacterium tuberculosis. NADPH-cytochrome P450 reductase (CPR) is the key protein that regulates the electron transfer (ET) flow from NADPH to various heme-containing monooxygenases such as cytochrome P450 and heme oxygenase. CPR is composed of two flavin-containing domains that are connected by a hinge region: one contains flavin adenine dinucleotide (FAD), called FAD domain, and the other contains flavin mononucleotide (FMN), called FMN domain. ET is supposed to occur sequentially from NADPH to FAD, FAD to FMN, and FMN to monooxygenase, which is regulated by interdomain open-closed-like motion of CPR that affects the intermolecular interaction with monooxygenase. Therefore the coupling of the redox state and the structural state in CPR plays the key role in understanding the mechanism of ET regulation by CPR, which still remains unsettled. In this study, we examined the dependence of the structural state of CPR on its redox state by molecular dynamics (MD) simulation. We found that the closed state is intrinsically stable and the redox state dependence is only a little as far as the interdomain distance is concerned. However, the distance between the two flavins becomes larger in the reduced state, and furthermore, as the FAD-FMN distance is increased, CPR tends to expose the FMN cofactor to the place where monooxygenase is supposed to be positioned in the complex structure. Thus, our MD simulation clearly indicates that the structural state of CPR is coupled with the redox state, further providing some new insights for the coupling. Neisseria meningitidis is a Gram-negative bacterium that causes most cases of bacterial meningitis. A portion of the population are carriers of N. meningitidis in their nasal cavities. There are six serogroups that cause disease and they differ by the sugars found in the capsule surrounding the bacteria. Capsular sugars are critical components of glycoconjugate vaccines that help prevent meningococcal meningitis. Glycosyltransferase enzymes are responsible for creating these capsular sugars. The bifunctional N. meningitidis serogroup W capsule polymerase synthesizes the sugar coat for this serogroup. It creates a galactose-sialic acid containing heteropolymer. However, the underlying mechanism of this enzyme is still not completely understood. Improved knowledge of how this capsule polymerase synthesizes its sugars can help us harness its power for use as a new biotechnological tool for rational vaccine design. Our aim in this study was to determine the binding affinities of nucleotide sugar donors CMP-sialic acid and UDP-galactose using a coupled transferase assay which were previously unknown. Through this assay we were able to determine a Km value for UDP-galactose (50.7 μM) in the range of other reported values for other retaining galactosyltransferases. However, we were unable to determine a Km value for CMPsialic acid. These experiments suggest that, in future work, an alternate strategy is needed to assess kinetic parameters for this type of enzyme. Jordana Thibado 1 , Joshua Levitz 1 1 Weill Cornell Medicine (New York, United States) As a complement to structural and biochemical approaches, dynamic measurements in the physiological environment of living cells are required for a full, mechanistic understanding of proteins. This is particularly important for membrane receptors, which are tuned to the native spatiotemporal properties of the ligands they sense and are highly sensitive to the local membrane environment in which they reside. To facilitate these types of measurements, we have developed techniques for the optical detection and manipulation of G protein-coupled receptor (GPCRs) with a focus on metabotropic glutamate receptors. Using FRET sensors of receptor conformation at the ensemble level in living cells and at the single molecule level in cell lysates, we have begun to unravel the structural dynamics that initiate receptor activation at the extracellular ligand binding domain and the intersubunit rearrangements that drive G protein activation at the transmembrane domain. These measurements have revealed surprising complexity in the mechanisms of treatment-relevant orthosteric and allosteric drugs and deep heterogeneity between receptor subtypes. To more closely probe the process of ligand binding and activation cooperativity, we have employed photoswitchable tethered ligands that allow extremely rapid control of receptor activation and de-activation. These measurements allow us to begin to reconcile crystal structures and conformational models with binding occupancy and downstream receptor activation measurements. Finally, we have begun to applied y these tools in neurons, brain slices and in vivo to observe and manipulate native receptor function with greatly improved precision compared to classical techniques. Jun Ohnuki 1 , Yasuhiro Arimura 2 , Tomonori Kono 3 , Kuniki Kino 3 , Hitoshi Kurumizaka 2 , Mitsunori Takano 1 1 Department of Pure and Applied Physics, Graduate School of Advanced Science and Engineering, Waseda University (Shinjuku-Ku, Japan); 2 Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University (Shinjuku-Ku, Japan); 3 Department of Applied Chemistry, Graduate School of Advanced Science and Engineering, Waseda University (Shinjuku-Ku, Japan) RimK, a member of the ATP-dependent carboxylate-amine/thiol ligase superfamily, catalyzes not only the polyglutamylation of the C-terminus of ribosomal protein S6 (RpsF) in vivo but also the poly--glutamate synthesis in vitro. Recently, the structure of RimK has been solved, where two different forms (open and closed forms) were observed. In this study, to understand the physical mechanism of polyglutamylation, we predict the substrate-binding sites and ligation mechanism of RimK by molecular dynamics simulation. By statistically analyzing the observed dynamics of the bound and unbound glutamates, we found multiple glutamate-binding sites in RimK near the ATP-binding groove, where glutamate formed electrostatic bonds with positively-charged residues. We also found a large fluctuation in the ATP-binding groove. Interestingly, the glutamate-binding propensity depended on whether RimK is in the open form or in the closed form. To further investigate the substrate-binding site, we determined the crystal structure of RimK in the presence of RpsF. Although the position of RpsF was unclear, we found electron density near one of the predicted glutamate-binding sites, which is likely to be due to the C-terminal glutamic acid residues of RpsF. It is noteworthy that a similar substrate-binding mode can be seen in other proteins in the superfamily to which RimK belongs. On the basis of the electrostatic interactions coupled with the structural polymorphism, we propose a physical mechanism by which RimK processively synthesizes poly--glutamate. Christine Xue 1 , Zhefeng Guo 1 , Joyce Tran 1 , Hongsu Wang 1 , Frederick Hsu 1 1 Deposition of A protein in the form of amyloid plaques is a pathological hallmark of Alzheimers disease. Recent structural studies on patient-derived A amyloids suggest that different sub-type of Alzheimers disease may have different amyloid structures. This correlates with a well-known phenomenon in the in vitro A aggregation studies, called amyloid polymorphism. The questions about the origins and consequences of amyloid polymorphism are of critical importance in terms of developing therapeutic strategies to prevent, diagnose, and treat Alzheimers disease. Here we show that A42 spontaneously forms oligomers with a continuum of sizes ranging from monomers to thousands of kilodaltons. The A42 oligomers quickly form fibrils upon incubation at 37 C. When fractionated using centrifugal filters, the smaller oligomers form fibrils at a faster rate than larger oligomers. Oligomers of different sizes may convert to each other, but the rate of oligomer conversion is slower than the rate of fibrillization. The fibrils formed by oligomers of different sizes show distinct structural features. These results suggest that fibril nucleation is via conformational conversion from oligomers rather than de novo nucleation from monomers, and furthermore, A42 oligomer heterogeneity leads to amyloid fibril polymorphism. Jun Kuwahara 1 , Chisana Uwatoko 1 1 Doshisha Women's University (Kyotanabe, Japan) Zinc finger proteins are among the most abundant proteins in eukaryotic genomes and extraordinarily diverse functions including DNA recognition. In particular, the classical C2H2 zinc fingers possess the characteristics of tandem repeats of zinc-binding module and vary in function, which ranges from DNA to RNA binding. We are interested in versatility of this type of motif and have been searching novel role of the motif. Sp1 is a human ubiquitous transcription factor involved in the early development of an organism and comprises three tandem repeats of C2H2 zinc finger motif and binds directly to a GC-rich upstream element DNA (GC box). Bidirectional traffic between the nucleus and the cytoplasm is routed through the nuclear pore complex (NPC) embedded in the nuclear envelope. Nuclear import of globular proteins of greater than 60 kDa in size is an active process that requires the presence of a suitable nuclear localization signal (NLS) and is mediated by a related family of shuttling transport factors, importins, which can recognize NLS. Although Sp1 is supposed to be actively transported into the nucleus due to its molecular mass (95/105 kDa), none of NLS for Sp1 has been reported. Subcellular localization of Sp1 was examined. We found that C2H2 zinc fingers of Sp1 serve as NLS and can recognize nuclear transport factors, importins. Possible protein interaction of the zinc finger domain will be discussed. [ In recent years, hydroxyl radical-based footprinting (HRBF) coupled with mass spectrometry has been used to identify protein interaction sites and regions of conformational change by using hydroxyl radicals to oxidatively modify the side chains of solvent accessible amino acids. By comparing the oxidation pattern of a protein modified in two states (i.e. ligand-bound and ligand-free), HRBF can identify protein-ligand and proteinprotein interactions sites as well as regions of conformational change. One HRBF method, fast photochemical oxidation of proteins (FPOP), generates hydroxyl radicals via excimer laser photolysis of hydrogen peroxide. To date, HRBF methods have been used in vitro on relatively pure protein systems. We have further extended the FPOP method for in-cell analysis of proteins which will allow for the study of proteins in their native environment. For in-cell studies, we have optimized FPOP conditions for several cell types including the commonly used cell lines HEK and CHO as well as both gram-negative and gram-positive bacteria. Using actin as a model system, we have demonstrated the utility of in-cell FPOP (IC-FPOP) for probing protein structure in the native cellular environment. HEK cells were subjected to FPOP in two conditions, when the monomeric form of actin was predominant and when the polymerized form of actin was predominant. IC-FPOP was able to identify interacting regions in the polymeric form of the protein. These results indicate the efficacy of using in-cell FPOP to study proteins in their native environment. Roman Sloutsky 1 , Christine Battle 1 , Margaret Stratton 1 1 Ca2+-calmodulin dependent protein kinase II (CaMKII) plays a central role in Ca2+ signaling in multiple tissues. In the brain, specifically in the hippocampus, CaMKII is a central player in the formation of long-term memories. CaMKII is comprised of a kinase domain, regulatory segment, variable linker region, and hub domain that is responsible for holoenzyme oligomerization. In vertebrates, CaMKII is encoded by four highly conserved genes: alpha, beta, gamma, and delta. There are alternative splice sites that are all located in the variable linker region. Linker length and composition have been shown to affect the amount of Ca2+/CaM required for activation and the specific frequencies of Ca2+ pulses to which CaMKII responds. Expression of splice variants differs by cell type, likely in accordance with the cells requirements for regulating CaMKII activity. However, although CaMKII is known to be activated by very different Ca2+-pulse frequencies in different cell types, expression of CaMKII splice variants remains poorly understood. We used Illumina sequencing for high-sensitivity identification of CaMKII transcripts in RNA samples isolated from human hippocampus and several mouse tissues. The high degree of sensitivity was achieved by custom construction of sequencing libraries containing only CaMKII inserts. We found that tissues express diverse collections of CaMKII mRNAs, including both previously annotated and numerous novel splice variants. We are now combining the results of this comprehensive transcript detection assay with mass spectrometry data to quantify protein-level abundance of each CaMKII splice variant expressed in human neurons and characterizing their Ca2+/CaM and Ca2+ pulse response profiles. The Escherichia coli Min system, consisting of the proteins MinC, MinD, and MinE, restricts the position of the cell division machinery to the cell center by preventing FtsZ-ring formation at the cell poles. MinD is a Par-like ATPase that associates with the membrane in the ATP-bound state; membrane dissociation occurs after ATP hydrolysis, which is stimulated by a direct interaction with MinE. In vivo, MinD oscillates between the cell poles and oscillation is regulated by MinE-stimulated cycles of MinD membrane association and dissociation. MinC is recruited to the membrane by MinD and therefore also oscillates in vivo. MinC directly engages FtsZ polymers and promotes disassembly. To determine how MinC interacts with FtsZ and MinD, we screened a library of strains containing random mutations in the chromosomal copy of minC for cell division defects in vivo. We identified mutations in the MinC N-and C-domains that impair the ability of purified MinC to inhibit GTPdependent FtsZ polymerization in sedimentation assays and form stable complexes with FtsZ and phospholipid-associated MinD in vitro. Several MinC mutant proteins also exhibit slow oscillation in vivo, suggesting that protein interactions modulate intracellular localization. Together, we identified two distinct sites on the surface of MinC that are important for the FtsZ interaction: one on a cleft in the N-domain, and another site on the C-domain, near the putative MinD interaction site, which is important for the interaction with the FtsZ C-terminus of stable FtsZ polymers but dispensable for the interaction with dynamic FtsZ polymers. Chang Chen 1 , Constance Jeffery 1 1 University of Illinois at Chicago (Chicago, United States) Inflammatory bowel diseases (IBD), including Crohns disease (CD) and ulcerative colitis (UC), comprise a group of immune disorders with symptoms that can have significant negative impact on a patients quality of life. Over one million people are affected in the USA, with similar rates in other Western nations. Non-Westernized nations have a lower prevalence, but it is increasing worldwide. The causes are not understood, but both genetic and environmental factors are involved. The results of studies employing NGS (Next Generation Sequencing) and GWAS (Genome-Wide Association Studies), published by several groups, have identified over 100 alleles that vary between patients and healthy individuals and that might be involved in genetic predisposition to IBDs. We conducted a systematic in silico analysis to predict the impact of the corresponding single amino-acid polymorphisms (SAPs) on the sequences and structures of the proteins. The results of the analysis identified SAPs that are likely to affect protein function, structure and/or stability, and therefore might be involved in genetic predisposition to IBDs. The results suggest which SAPs may be of high priority for in vitro analysis of the purified mutant proteins to aid in further understanding of the molecular mechanisms of disease and for potential future development of therapeutics to alleviate the symptoms of IBD. Engineering protein translation machinery to incorporate noncanonical amino acids (ncAAs) has led to applications ranging from proteomics to bioconjugation and single-molecule studies. As applications of ncAAs in the study and engineering of biological systems continue to emerge, highly efficient encoding of ncAAs is crucial to exploiting their unique chemistries. We have established a quantitative reporter platform to evaluate ncAA incorporation in response to the UAG (amber) codon. Our yeast display-based reporter utilizes an antibody fragment containing an amber codon at which a ncAA is incorporated when the appropriate orthogonal translation system (OTS) is present. Epitope tags at both termini of the antibody allow for flow cytometrybased endpoint readouts of ncAA incorporation efficiency and fidelity. This format supports evaluations of factors that influence amber suppression, including the amber codon position in the reporter, different aminoacyl-tRNA synthetase/tRNA (aaRS/tRNA) pairs, and the vector copy number used to express the OTS. Interestingly, previously described aaRSs evolved from different parent enzymes to incorporate O-methyl-Ltyrosine exhibit different efficiencies and fidelities. E. coli leucyl-tRNA synthetase variants generally exhibit efficient incorporation of a range of ncAAs, and we have discovered activities of variants that have not previously been reported. When compared to plate reader-based assays that utilize fluorescent protein reporters, our assay yields more precise measurements at the bulk level while also supporting single-cell readouts compatible with cell sorting. We expect that this platform will allow us to quantitatively elucidate principles dictating efficient stop codon suppression and evolve OTS components to further enhance genetic code manipulation. Emma Carroll 1 , Andreas Martin 1 , Susan Marqusee 1 Ubiquitination is a common protein posttranslational modification in which the protein ubiquitin is attached to the primary amine of lysine residues on the target protein. Ubiquitination is canonically associated with targeting proteins to the proteasome for degradation; however, it is also involved in many other cellular processes, and the cell must carefully regulate which ubiquitinated proteins should be degraded. While proteasomal degradation is dependent on ubiquitin chain length and topology, it has also been shown that the conformational features of the tagged protein can play a role in proteasomal engagement. However, the biophysical factors driving the in vivo proteasomal degradation code remain experimentally unexplored. These types of studies are hampered by the difficulty of characterizing the energetics and dynamics associated with probing macromolecular complexes such as ubiquitinated proteins. I will present results from a newly developed approach for characterizing the stability and dynamics of proteins with and without defined ubiquitin modifications. We are using both ensemble and single molecule approaches to characterize the effect of ubiquitin modification in structured regions on the thermodynamics and kinetics of protein variants. A repeated insertion mutation in the chromosome 9 open reading frame 72 (c9orf72) gene is linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) pathogenesis. Some of the DiPeptide Repeat proteins (DPRs) created in the G4C2 repeated insertion mutation of the c9orf72 gene are toxic to neurons, but the mechanism of their toxicity is not well understood. An understanding of their biochemical properties and interactions with cellular components could enhance our understanding of toxicity research in vivo. One of their principal potential interactors are G-quadruplex secondary structures in nucleotides, which are implicated in expression control mechanisms. Peptides have been synthesized representing four of the DPR species: (GR) 5, (PR)5, (GA)5, and (AP)5. The peptides were synthesized with a tryptophan near the C-terminal end and a dansyl group attached to the N-terminus for FRET measurements, to determine the degree of collapse in these intrinsically disordered peptides. The FRET distances between the fluorophores correlate most closely with the presence or absence of prolines in the sequences. Analytical ultracentrifugation and circular dichroism experiments have been carried out to determine secondary structure and oligomerization of these peptides, with and without binding to sense and antisense G4C2 DNA oligonucleotides. The quadruplex structure in the sense G4C2 DNA was disrupted, suggesting one possible mode of toxicity. The results of these studies could provide unique insight into the mechanism of DPR interactions with cellular targets in ALS and FTD patients. Hexanucleotide repeat expansions located in the c9orf72 gene are the most common mutations associated with familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) cases. Dipeptide repeat proteins (DPRs) are synthesized from these expansions through repeat-associated non-ATG (RAN) translation and are hypothesized to contribute to the toxicity of these diseases. Our research investigates one of these DPRs, polyGA, and its possible interactions with Hsp104, a protein disaggregase found in yeast. Hsp104 has already shown promise as a possible therapeutic agent to treat amyloid-associated diseases like Parkinsons Disease and Spinocerebellar Ataxia Type 3. However, its effects on C9orf72 DPRs have not been studied. Therefore, this study aims to determine if Hsp104 alters the aggregation of polyGA in Drosophila melanogaster larvae. Confocal microscopy results, when co-expressing Hsp104, revealed a diffuse GFP-(GA)50 fluorescence pattern atypical of normal polyGA expression. Hsp104 antibody staining co-localized with GFP fluorescence, indicating the presence of the protein in the larva. Overall, this outcome suggests that Hsp104 disaggregates polyGA inclusions, producing a novel finding that warrants future investigation into Hsp104 as a potential therapeutic for familial ALS and FTD. The study of protein-based nanomaterials has been an emerging endeavor in the fields of nanotechnology and biomedical engineering. Designing photoresponsive materials that are made of protein is of particular interest, as they are biocompatible, self-assembled tools with nanoscale precision of electron transfer and a promising form of green technology. Originally found in gram-negative bacterium Geobacter sulfurreducens, PilA is a desired protein for the basis for these materials, as there are claims that the protein can engage in electron transfer. PilA protein was heterologously expressed in inclusion bodies in Escherichia coli. The recovered denatured PilA protein was polymerized to its native, fibrillar conformation through a stepwise dialysis as a function of denaturant concentration. The resulting material was studied to determine the intra-and inter-monomeric interactions that are responsible for the resulting PilA nanowire. Atomic force microscopy images as well as Fouriertransform infrared spectra demonstrate that the resulting PilA is linearly organized in polymer form; its morphology is comparable to PilA extracted directly from G. sulfurreducens. Furthermore, the PilA polymers appear to successfully engage in electron transfer, as they produce a significant amount of current once voltage is applied. Tugba Kizilboga Akgun 1 , Nisan Denizce Can 1 , Baran Dingiloglu 1 , Ezgi Basturk 1 , Can Ozden 1 , Evren Onay Ucar 2 , Gizem Dinler Doganay 1 1 Istanbul Technical University (Istanbul, Turkey); 2 Istanbul University (Istanbul, Turkey) BAG-1 is an adaptor protein expressed as four different isoforms that modulates cell survival and apoptosis pathways. By knowing the details of interaction networks of BAG-1 and other chaperone proteins, it is possible to understand how protein homeostasis is regulated in cancer. In this study, the interactions between HER-2 and HSF-1 will be first evaluated and then checked if they play a role in selection between cell survival and apoptosis mechanisms. For the purpose of showing any potential effect on HSF-1, some proteins on this pathway will be checked in BAG-1 upregulated and downregulated breast cancer cells. BAG-1 and Hsp70 (also Hsp27 and HSF-1) interactions were demonstrated by immunoprecipitation. In order to demonstrate the different interactions of each BAG-1 isoform, protein purification was carried out by TAP tag purification. Purified proteins from the BAG-1L, BAG-1M and BAG-1S isoforms were analyzed by LC-MS/MS (Waters Synapt G2-Si HDMS). The obtained findings were evaluated in PRISM protein-protein interaction program and interaction surfaces were tried to be determined. As a result of our study, it was observed that cell survival was increased in breast cancer due to BAG-1 expression. Especially when the HER-2 expression is examined, it has been shown that the expression of BAG-1 directly or indirectly increases the expression of heat shock proteins by affecting the HSF-1 transcription factor on the downstream pathway, thereby protecting the cell against stress. By using advanced techniques (HDX-MS method) of previously undefined surfaces, it will be possible to develop new target molecules and explain the structural mechanisms. Denise Okafor 1 , Jennifer Colucci 1 , Eric Ortlund 1 1 Emory University (Atlanta, United States) Understanding the genetic and biophysical mechanisms by which proteins evolve ligand specificity is crucial for continued progress in evolutionary biology and biochemistry. Androgen receptor is an essential hormonecontrolled transcription factor, a member of the steroid receptor subfamily. AR modulates expression of essential genes by relaying allosteric signals to coregulator proteins, specifically in response to binding of androgenic hormones (e.g. dihydrotestosterone, DHT) in its ligand binding pocket. This preference was fine-tuned over evolution, as ARs immediate phylogenetic ancestor was able to bind and respond to both progestogens and androgens. Here, we seek to understand the evolution of ligand specificity in this receptor using ancAR1, the ancestor of extant androgen receptors. Three historical substitutions have been identified that restore progesterone activation to AR1 in luciferase reporter assays. Our lab has used x-ray crystallography and biophysical methods to characterize the effects of the substitutions on AR1 interactions with both progesterone and DHT. Furthermore, microsecond-long molecular dynamics simulations and network analyses were employed to probe the effects of the replacements on the allosteric networks governing ligand activation within the receptor. This work reveals that allosteric communication between the AR1 ligand binding pocket and activation function helix, a key regulatory region of the receptor, is severely weakened in the non-activating AR1-progesterone complex. The historical substitutions reverse this effect, potentially illuminating the allosteric mechanism that drove the evolution of ligand-specificity in AR1. This work has application for understanding the allosteric network that governs ligand activation of modern-day steroid receptors. Joseph Rehfus 1 , Vincent Hilser 1 1 Johns Hopkins University (Baltimore, United States) Protein molecules are dynamic and conformationally heterogeneous, and these properties contribute to their functions. Ensemble-based equilibrium assays, however, are not always sensitive to low-population conformations or the kinetics that govern their fluctuations. We have applied single-molecule force spectroscopy to study the free energy landscape of adenylate kinase (AK), a three domain protein known to visit excited states defined by local unfolding of its peripheral domains (LID and AMPbd). These states have previously been shown to modulate both the binding affinity and catalytic rate of this essential enzyme. Using entropyenhancing mutations, we selectively lower the free energy of locally unfolded states and find that LID stability is highly correlated with AKs mechanical stability, revealing tight coupling between the LID and CORE domains. Whats more, the coupling is LID domain specific, as destabilizing the AMPbd results in no detectable effect. Our results further suggest that AK exists in at least two states, with very different mechanical stabilities, interconverting with very slow kinetics. The heterogeneity in AKs conformational landscape is greater than currently appreciated and may play additional uncharacterized roles in the proteins function and regulation. Whats more, these adaptations are likely not restricted to AK, and might be leveraged in new strategies for the tuning of protein function via novel therapeutics or through de novo design. Toshiharu Suzuki 1 , Kunio Hirata 2 , Eiki Yamashita 3 , Seiki Baba 4 , Naoya Iida 5 , Takashi Kumasaka 4 , Toshiya Endo 6 , Toru Hisabori 7 , Masasuke Yoshida 6 , Hiroyuki Noji 1 1 Dept of Applied Chem, School of Eng, The University of Tokyo (Tokyo, Japan); 2 SPring8-center, Riken (Tokyo, Japan); 3 Inst of Protein Res, Osaka Univ (Osaka, Japan); 4 Japan Synchrotron Radiation Research Institute (JASRI) (Hyogo, Japan); 5 Waseda Univ (Tokyo, Japan); 6 Dept of Mol Bioscience, Kyoto-Sangyo Univ (Kyoto, Japan); 7 CLS, Inst of Innovative Res, Tokyo Inst of Tech (Yakohama, Japan) Molecular motors are unique proteins because they generate physical power by energy conversion of chemical potential of ATP. The actuator-like function has attracted many researchers, but the detailed molecular mechanism to produce the motion is still obscure because of insufficient structural information. F1-ATPase (F1) is an ATPase domain of FoF1-ATP synthase and functions as rotary molecular motor. Approximately 50kDa rotor rotates relative to 330kDa stator by ATP-hydrolysis. To reveal the mechanism, we have established analytical systems for recombinant human and bovine F1s. Remarkably, the systems have provided valuable clues. Microscopic single-molecule analysis of human F1 had revealed a unique rotation scheme different from bacterial F1. Recent crystallographic study of bovine F1 provided several molecular structures at up to 1.66 Å resolution, which included eight rotation interim snapshots for the release of product phosphate or ADP. The former structures identified stepwise changes in atomic coordinate of the whole structures during rotation, which enabled us to explain the chemo-mechanical coupling mechanism between phosphate-triggered conformation changes in Arginine finger and p-loop lysine at catalytic site and global subunits rearrangement which lead to inducing 20deg-rotation of the rotor part. Furthermore, sequence of the structural transition was confirmed by dynamic time-divided X-ray crystallographic study. Additionally, the ADP-releasing intermediate structures unveiled conformational rearrangement in catalytic site (especially in p-loop) for ADP-releasing. All of the rotation intermediates supported the rotation scheme of human F1 and revealed detailed chemomechanical coupling mechanism to generate the physical power by the elementary steps of ATP-hydrolysis reaction for the first time. Several proteins associate into dimers by exchanging specific, yet identical, secondary structure elements between monomeric subunits, thus leading to an intertwined complex. The functional relevance of this mechanism, known as domain swapping (3D-DS), has been extensively described in other proteins, suggesting that stepping into the unfolded state is required for reaching a monomer-dimer equilibrium. Recent biophysical data have shown that the forkhead domain of human FoxP1 also dimerizes via 3D-DS but its dissociation constant is~3 orders of magnitude lower than most 3D-DS proteins, mainly due to a highly structured monomeric intermediate rather than requiring complete protein unfolding. To obtain a detailed and structural description of the dimerization pathway of FoxP1, we used anisotropy and singlemolecule FRET (smFRET) to study the local effects and heterogeneity in chemical denaturing conditions on three pseudo-heterodimers, in which different fluorophores were attached to each monomer to map both flexible and stable regions. Anisotropy data of unfolding showed that flexible elements located in N and C terminal regions present a low stability (GU 4 kcal/mol), whereas stable and conserved regions present similar stability to global folding parameters (GU~8 kcal/mol). Moreover, smFRET indicated that all flexible regions are unstructured in a previously characterized expanded dimeric intermediate and do not participate in the dissociation pathway, whereas flexibility changes located in the stable regions of FoxP1 promote the expanded dimeric conformation. These findings allow us to have a detailed dimerization mechanism via 3D-DS of FoxP1. The 1918 influenza A virus (IAV) caused the worst flu pandemic (a. k. a. Spanish flu) in human history. Nonstructural protein 1 (NS1) is a multifunctional virulence factor of IAVs, and it is critical to understand the molecular mechanism underlying the unusually high virulence of 1918 IAV. NS1 of 1918 IAV (1918 NS1) hijacks diverse host proteins including CT10-regulator of kinase II (CrkII) and phosphoinositide-3-kinase (PI3K), resulting in enhanced viral replication. To elucidate the molecular mechanisms whereby 1918 NS1 interacts with the host proteins, we investigate the structure and dynamics of 1918 NS1 using NMR spectroscopy and small-angle Xray scattering. Moreover, we study the interaction between 1918 NS1 with human CrkII using NMR and thermodynamic studies. Our study suggests that 1918 NS1 forms a fuzzy complex with CrkII. These results also provide insights into the development of an inhibitor preventing the hijacking of CrkII by NS1 of future pandemic IAVs. The lack of specificity of standard chemotherapies to identify and kill cancer cells has challenged researchers to engineer targeted therapies to replace conventional treatments. One strategy that is currently being optimized to treat cancer is the use of recombinant immunotoxins (RITs). RITs are chimeric proteins that are engineered to contain an antibody conjugated to a toxin. The antibody allows for specific targeting of cancer cells while the toxin domain is internalized by the targeted cell and subsequently induces cell death. These therapies suffer from several problems, including non-specific toxicities, low anti-tumor activity, and immunogenicity. An immune response generated in response to the bacterial domain of the RITs eliminates the possibility of repeated treatment with the targeted protein. Without the possibility of retreatment, the therapeutic effect of RITs is compromised. Deimmunized, humanized, and fully human immunoconjugates have been engineered, but often lack efficacy. We are using specific sequences known to assist cellular intoxication by Pseudomonas exotoxin A (PE) to enhance the activity of human-derived immunotoxins. We are developing a recombinant immunotoxin based on actinresistant human DNase I, using a furin-cleavable linker and KDEL endoplasmic reticulum (ER) retention sequence adapted from the PE intoxication strategy. We expect this construct to have low immunogenicity, high specificity for dividing cells, and enhanced toxicity relative to the unmodified DNase I construct. Terpenes comprise the largest and most structurally diverse class of naturally occurring chemicals. Class I terpene synthases catalyze the cyclization of diphosphorylated acyclic terpene precursors. Limonene is one of the simplest cyclized terpenes, and yet the mechanism behind its formation by the enzyme limonene synthase is still not well defined. To better understand the chemical mechanism of monoterpene (C10) cyclization in atomic detail, we crystallized and solved the structure of (+)-limonene synthase in the apoprotein form. The substrate analog 8,9-difluorogeranyl diphosphate (DFGPP), designed to inhibit the cyclization reaction, was synthesized and soaked it into the limonene synthase crystals. The X-ray crystal structure of the complex solved to 2.7 Å resolution, revealing strong electron density in the active site for three divalent cations, the diphosphate moiety, and the carbon chain of the substrate analog including terminal fluorine atoms. Significantly, DFGPP could not be well modeled in the electron density, with the terminal fluorine atoms barely enclosed, and carbons 3, 4, and 10 completely outside of the 1.0 contour. By contrast, the difluoro-version of the proposed intermediate linalyl diphosphate (DFLPP) could be modeled into the electron density suggesting that DFGPP isomerized to the DFLPP intermediate, which was then trapped in the crystal because the subsequent cyclization step to produce the -terpinyl cation was blocked by the electronegative fluorine atoms at the 8 and 9 positions. This in crystallo formation of DFLPP is the first direct demonstration of the LPP intermediate for any monoterpene cyclase reaction. Nisan Denizce Can 1 , Tu gba Kızılbo ga Akgün 1 , Baran Dingilo glu 1 , Serena Muratçıo glu 2 , Efe Elbeyli 2 , Özlem Keskin 2 , Gizem Dinler Do ganay 1 1 Istanbul Technical University (Istanbul, Turkey); 2 Koç University (Istanbul, Turkey) BAG-1 is an anti-apoptotic adaptor protein which is involved in the regulation of various cellular signaling pathways. BAG-1 has three major isoforms as BAG-1S, BAG-1M and BAG-1L, all of which have different cellular localizations and may also have different interaction partners. In this study, we aimed to detect BAG-1S interacting partners to determine its role in breast cancer and non-tumorigenic breast cells. TAP-tagged BAG-1S containing expression vector were used to transfect MCF-7 and MCF-12A cells. After overexpression, complexes of BAG-1S with its interacting proteins was purified and further identified by tryptic digestion followed by LC-MS/MS. We observed BAG-1S complexes to participate mainly in endoplasmic reticulum-associated degradation (ERAD). We determined Hsp70, Hsp90, BiP, E3 ligase CHIP, AAA ATPase VCP and RAD23B, Calreticulin and Calnexin as BAG-1S interacting partners whose roles are well known for protein homeostasis. To map the interaction surfaces of proteins in complexes, we used a protein-protein interaction prediction tool, PRISM. It is revealed that CHIP, VCP, Hsp70 and Hsp90 can interact with the BAG domain and simultaneously in the same complex RAD23B can interact with the UBL (ubiquitin-like) domain of BAG-1, suggesting a large complex located near endoplasmic reticulum to function in the degradation of unfolded proteins. Functional studies focusing on the delineation of this determined complex in ERAD mechanism are currently under investigation. This work is supported through TUBITAK 115Z169 grant and ITU internal funds. Taylor Cole 1 , Taylor Cole 1 , Dhanusree Viswanathan 1 , Tatyana Igumenova 1 1 Texas A&M University (College Station, United States) Protein kinase C (PKC) family of isoenzymes are Ser/Thr kinases that participate in signal transduction at the membrane surface. Dysregulation of PKC activity has been implicated in cancer progression and heavy metal ion toxicity. The hallmark of PKC activation is the membrane insertion of its C1 and C2 regulatory domains upon sensing their ligands, diacylglycerol and Ca2+/anionic phospholipids, respectively. Our objective was to gain insight into this process by probing how C1 and C2 domains coordinate bivalent interactions with membranes. Using protein-to-membrane FRET experiments, we investigated the influence of C1 ligands on the Ca2+ sensitivity of the membrane-binding process. We found that C1 ligands, the endogenous activator diacylglycerol and tumor-promoting phorbol ester PDBu, reduce the Ca2+ requirement for membrane recruitment of the regulatory region. By varying the properties of the interdomain linker, we found that the Ca2+ requirements for membrane association in the presence of C1 ligands were sensitive to linker length, composition, and rigidity. Increasing rigidity enhanced the Ca2+ sensitivity of membrane association while increasing length and flexibility had the opposite effect. Incorporating low-abundance signaling lipid PtdIns (4,5) bisphosphate in the membrane mimics enhanced Ca2+ sensitivity, bringing the Ca2+ requirement for membrane insertion into the physiologically relevant range irrespective of the linker properties. In aggregate, our data indicate that the properties of the linker region and the presence of the low-abundance signaling lipid in the membranes are essential for the regulatory domains to achieve the maximum thermodynamic benefits of bivalency in membrane interactions. Bag-1 is a multifunctional protein that interacts with diverse array of cellular targets and modulates a wide range of cellular processes, including proliferation, cell survival, transcription, apoptosis, metastasis and motility. In human cells Bag-1 exists as three major isoforms (Bag-1S, -1M, -1L) derived by alternative translation initiation from a single mRNA. It is important to assign the exact interaction partner of each Bag-1 isoform to a specific cell event, and determine the role of each functional Bag-1 isoform in these pathways. To fulfill that gap, we aimed to isolate isoforms with their interacting partners from breast cancer cells as complexes using affinity purification and further determine the interactomes by tryptic digestion followed by LC MS/MS. We focused first the common interactomes of all the isoforms. 46 proteins are found to be common interacting partners of all Bag-1 isoforms, of which are involved in protein processing in ER, stress response, carbon metabolism and cytoskeleton. Bag-1S and Bag-1L have 44 additional common proteins, suggesting redundant roles for these two isoforms. Bag-1M has the least number of interacting partners among the entire set. Results of this work revealed the entire complexes of Bag-1 isoforms and our proteinprotein interaction predictions further showed a fitting model of the determined interacting partners without any structural overlaps, confirming experimental findings. In conclusion, our work determined isoform specific interactomes of Bag-1 and shed light on the roles of these isoforms in breast cancer. This work is supported by TUBITAK 115Z169 project. The cellular interior is crowded, with macromolecules occupying from 10% to 40% of the volume. Under these conditions, proteins experience hard-core repulsions and chemical interactions with cytoplasmic components. Hard-core repulsions stabilize globular proteins, whereas chemical interactions can be either repulsive and stabilizing, or attractive and destabilizing. Several studies have considered crowding effects on globular protein stability, but there are few such studies on proteinprotein interactions. We used 19F NMR to quantify the effects (298 K, pH 7.5) of macromolecular cosolutes on a variant of the B1 domain of protein G (GB1) that forms a domain-swapped homodimer. At a concentration of 200 g/L, the monomer of the synthetic polymer polyethylene glycol (PEG) destabilizes the dimer by 0.30 kcal/mol, while at the same concentration, 3.3 kDa-, 8 kDa-and 20 kDa-PEG stabilize the dimer by 0.08, 0.39 and 0.4 kcal/mol, respectively. These data indicate a stabilizing, but saturable, macromolecular effect. We also showed that the physiologically-relevant cosolutes bovine serum albumin (BSA) and lysozyme have opposite effects; the former (at 100 g/L) stabilizes the dimer by 0.51 kcal/mol, and the latter (at 50 g/L) destabilizes the dimer by 0.12 kcal/ mol. These results can be explained by the differences in charge. BSA has the same charge as GB1, resulting in stabilizing repulsions. Lysozyme and GB1 have complementary charges, resulting in destabilizing attractions. The differing effects of PEG and the protein cosolutes indicate that synthetic polymers are poor mimics of the cellular interior because they do not account for chemical interactions found in cells. Ellen White 1 , Ellen White 1 , Katya Heldwein 1 1 Tufts University (Boston, United States) Human cytomegalovirus (HCMV) causes lifelong latent infections in the majority of the population worldwide. However, HCMV can cause serious illness in individuals with compromised or immature immune systems. Congenital HCMV infections can cause severe developmental problems and will continue to cause problems in children until there is a vaccine. During cell entry, HCMV must fuse its lipid envelope with the host cell membrane. HCMV uses several glycoproteins, including glycoprotein B (gB), expressed on the viral envelope to undergo the membrane fusion process. During the fusion process, gB undergoes a conformational rearrangement from a metastable prefusion form to the stable postfusion form, drawing the viral envelope and the host cell membrane together. The structure of the stable yet inactive postfusion form has been determined but the structure of the active prefusion form remains elusive. Knowing the prefusion form is essential for clarifying the conformational rearrangement gB undergoes during the membrane fusion process. Moreover, prefusion gB is an attractive subunit vaccine candidate. To stabilize the prefusion form, we engineered additional glycosylation sites within gB with the goal of blocking the rearrangement of the prefusion form to the postfusion form. Mass spectrometry was used to confirm glycosylation at the engineered sites. Negative-stain electron microscopy (EM) and small angle x-ray scattering (SAXS) were used to characterize the conformations of the engineered constructs. Our results show that despite the presence of additional glycans, the engineered constructs adopt the postfusion conformation. We hypothesize that gB rapidly folds into the postfusion conformation during protein synthesis. Mutations in UBQLN2 precipitate the protein into intraneuronal inclusions found in rare familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) cases. Normally, UBQLN2 is involved in the regulation of different protein clearance pathways mediating the targeting of accumulated/aggregated proteins for degradation. Intriguingly, aggregated UBQLN2 co-localizes with the pathological inclusions in several common agerelated neurodegenerative disorders. UBQLN2 is a complex protein of four domains: the ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains reside at the N-and C-termini, flanking a central domain that contains multiple STI1 motifs predicted to bind chaperones and the PXX domain of unknown function. Using in vitro protein analysis, longitudinal fluorescence imaging of neuronal cells, and transgenic mouse models, we establish that UBQLN2 is prone to aggregation. In transgenic mouse models overexpressing UBQLN2, we observed that both wild-type protein and the pathogenic P506T mutant form dense aggregates/puncta using immunohistochemistry. However, puncta formed by wild-type protein are smaller and typically spherical, whereas the aggregates formed by P506T mutant are larger and irregular in shape. Furthermore, using longitudinal fluorescence imaging of primary neurons, we find that although both wild-type and P506T UBQLN2 aggregate, the mutant protein displays increased aggregation, which correlates to an increased neurotoxicity. Additionally, aggregation studies of the individual domains conducted in vitro with recombinant proteins, suggest that UBA domain drives the aggregation, and UBL domain is protective. These data were also supported by the longitudinal fluorescence imaging of neurons providing molecular insights in UBQLN2 aggregation and its role in neurotoxicity. The transient receptor potential (TRP) melastatin 4 (TRPM4) cation channel is widely expressed and an important regulator of cellular calcium levels. TRPM4 is activated by increased intracellular calcium in a voltagedependent manner and is, unlike most other TRP cation channels, exclusively permeable to monovalent cations. We determined two structures of the full-length human TRPM4 (hTRPM4) embedded in lipid nanodiscs with added EDTA and CaCl2 at~3 Å resolution using single-particle cryoelectron microscopy. The structures correspond to two distinct closed states, with and without calcium bound, and reveal the general architecture of the TRPM subfamily. Comparison of the two structures allowed de novo identification of a hitherto unassigned, well-defined calcium-binding site within the intracellular side of the transmembrane S1-S4 domain. The binding of calcium induces conformational changes that likely prime the channel for voltage-dependent opening and binding of co-factors such as calmodulin and lipids. The binding of calcium induces conformational changes and provides new leads for the elucidation of how hTRPM4 and other calcium modulated TRPM channels opens in response to calcium. Mutations of the hTRPM4 gene are associated with a variety of cardiovascular disorders while hTRPM4 overexpression is associated with certain types of cancers. The structure of hTRPM4 will improve our current understanding of hTRPM4 function and aid in the development of new pharmacological leads in treatment of cardiovascular diseases and novel tissue biomarkers for detection of various cancers. Dapeng Zhang 1 , Lakshminarayan Iyer 2 , Max Burroughs 2 , Jun Zheng 3 , L Aravind 2 1 Saint Louis University (Clayton, United States); 2 NCBI, NLM, NIH (Bethesda, United States); 3 University of Macau (Macau, China) Protein toxins are the main players mediating bacterial interactions with other bacterial strains/species and their eukaryotic hosts, including plants and humans. However, identification and characterization of novel toxins remain challenging as they are usually fast-evolving driven by evolutionary arms race. Methods: A domain-centric computational pipeline has been developed to screen all sequenced bacterial genomes to identify novel toxins, dissect domain architectures, predict functions and to uncover operonic structures of toxins and associated components involved in immunity, secretion and release. Biochemical experiments were used to test the hypotheses generated by computational analyses. Results: A vast class of polymorphic toxin systems have been identified in all bacterial lineages. We defined two underlying principles of these systems in domain architectures and genomic organizations. We uncovered over 50,000 previously-unknown toxin components and annotated over 150 novel toxin domains, 90 novel immunity families and many other domains associated with different secretion pathways such as T2SS, T5SS, T6SS, T7SS, Photorhabdus virulence cassettes (PVC), PrsW-dependent and MuF phage-capsid-like pathway. We have provided a most comprehensive functional prediction for many toxin domains, including highly divergent versions of peptidases, nucleases, deaminases, ADP ribosyltransferases and ribosylcyclases, nucleotidyltransferases, glycosyltranferases and other lipid and carbohydrate-modifying enzymes. In the end, predicted functions of several toxin families have been verified, including novel T6SS secreted toxins in Vibrio, new ribotoxins targeting rRNA, and novel toxins involved in Citrus Greening, one of the most serious plant diseases. Our discoveries have served as the underlying framework for studying the molecular mechanism of bacterial conflicts. Oshini Ekanayake 1 , Samuel Scinto 2 , Joseph Fox 3 , Sharon Rozovsky 4 1 University of Delaware (Newark, United States); 2 Collaborating graduate student (Newark, United States); 3 Co-Principle Investigator (Newark, United States); 4 Co-Principle investigator; adviser (Newark, United States) Cellular oxidative stress is a result of elevated levels of reactive oxygen species (ROS). In the cell, ROS are converted to less harmful forms such as hydrogen peroxide (H2O2), which subsequently lead to the formation of cysteine sulfenic acids (protein sulfenylation). Additionally, an increase in ROS is also implicated in a number of diseases including cancer, cardiovascular diseases and neurodegenerative diseases. As a result, there is great interest in studying protein sulfenylation stemming from its role in redox regulation and the role of redox modifications in human disease. A number of compounds targeting sulfenic acids have been identified over the years including 5,5-dimethylcyclohexane-1,3-dione or dimedone and more recently 9-hydroxymethylbicyclo[6.1.0]nonyne (BCN). Unfortunately, current reagents suffer from sluggish reaction rates, lack of specificity towards sulfenic acids and poor cell permeability. Here we describe the use of a transcyclooctene (SAM-TCO) as a sulfenic acid reactive molecule, which renders a stable and irreversible thioether adduct with reactive sulfenic acids. We have illustrated its ability to enter live cells through western analysis. More importantly, we have used the rapid TCO-tetrazine reaction to bio-orthogonally quench excess probe inside cells. This will prevent detection of any artefactual sulfenylation that may occur due to unnatural stress introduced during sample processing. SAM-TCO will be used to site specifically modify sulfenic acids in cells and through chemoproteomics, to identify protein targets of cysteine sulfenylation. This work will help improve our understanding of redox biology and identify therapeutic targets for diseases implicated with oxidative stress. Fluorescent biosensors can be used to detect analytes, measure concentration changes, and identify their locations in cells. Here we describe a design for a protein-based biosensor scaffold that can be rationally modified to recognize a target molecule of interest. Our biosensor is based on a stabilized, consensusdesigned variant of the fibronectin3 monobody (cFN3) and utilizes the alternate frame folding (AFF) mechanism to link binding to an engineered conformational change, which is detected by the strategic placement of fluorophores. To create the sensors we: modify the cFN3 active site to bind an antigen of choice (cAbl SH2 domain, hSUMO-1, and hRAS); create a circular permutant (cpFN3) that retains the stability and substrate specificity of cFN3; prime the switch by approximately matching the cFN3 and cpFN3 stabilities; join cFN3 and cpFN3 such that their sequences partially overlap. Binding-knockout point mutations, introduced into either the cFN3 (N)-or cpFN3 (N)-fold, ensure exclusivity of binding, and the shared sequence guarantees exclusivity of folding. Fluorescent donors/acceptors are placed at sites that are proximal to each other in the N-fold and distal in the N-fold, allowing binding/unbinding to be detected by changes in FRET efficiency or static quenching. We demonstrate that the sensors bind their cognate ligands with high affinity, half-times of less than an hour, and with a readily quantifiable fluorescent output. The AFF mechanism, coupled with the FN3 binding domain, establishes a template into which monobodies with different substrate specificities can be substituted to create new biosensors with little additional modification. Kendra Frederick 1 For an organism to survive, its proteins must adopt a diversity of conformations in a challenging environment where macromolecular crowding can derail even robust biological pathways. This situation becomes critical when considering proteins with energetic folding landscapes permit many conformational states. In these cases, the environment can clearly influence the conformation by favoring one pathway over another. Such decisions can have long-lasting biological consequences, as is the case for prions, proteins with the ability to exist stably in distinct functional states with divergent structures: one soluble, the other a highly ordered, self-templating aggregate. Because the aggregating proteins have identical sequences, differences in cellular environment are responsible for the conformational switch. Despite the importance of the environment for protein folding, structural investigations of biomolecules are typically confined to in vitro systems, which cannot capture important structural features imposed by biological environments. Solid-state NMR spectroscopy is currently undergoing a sensitivity renaissance with the development of dynamic nuclear polarization. An experiment that would require decades of experimental time with traditional ssNMR methods can be collected in a day. Applying this technique to the yeast prion protein, Sup35, we found that the native context can have a dramatic influence on protein structure. Enzyme design and engineering seek to develop enzymes that have no natural counterparts, but attempts generally result in low activity. The rational design of enzymes with robust activity is highly desired, particularly in light of increased interest in green chemistry, but is limited by current understanding how to capture and design necessary catalytic properties. Thus reasonable activity levels are only achieved after many rounds of directed evolution. To better understand the residue features needed for catalysis, we are applying our electrostatic method THEMATICS, which identifies functional residues through the perturbed titration behavior of ionizable residues. Directed evolution has been used extensively on the retro-aldolases, and in the case of RA95.5-8F, has resulted in an enzyme with over 200,000 times the catalytic activity of the original designed sequence. We have analyzed the evolutionary history with our electrostatic methodology to understand and rationalize the increases in activity. In silico residue scanning is utilized to generate variants across the entire sequence of the protein, which when combined with the THEMATICS method allows identification of coupling partners of residues with perturbed titration behavior. Applying this to intermediaries along the evolutionary trajectory of RA95, we can identify the functional residues, including the evolutionary catalytic lysine. We show how specific interacting partners help to enhance the catalytic activity, using our computed metrics and reported experimental data. Designing enzymes with these properties in mind may lead to hits with higher activity and ultimately establish design principles for protein engineering. Supported by NSF MCB-1517290. Colin Smith 1 , Adam Mazur 2 , Ashok Rout 2 , Christian Griesinger 2 , Donghan Lee 2 , Bert de Groot 2 1 Wesleyan University (Middletown, United States); 2 Max Planck Institute for Biophysical Chemistry (Goettingen, Germany) One key advantage of structure determination by nuclear magnetic resonance (NMR) is that protein characterization is performed in solution. In this environment, proteins remain in constant motion and often move on timescales from picoseconds to milliseconds. Such motions have been shown to be critical for enzyme catalysis, allosteric regulation, and molecular recognition. With NMR data being particularly attuned to these timescales, a great opportunity is presented to capture both structural and kinetic information. However, nearly all methods of NMR-based structure determination neglect the kinetics, which introduces a rather large approximation to the underlying physics, limiting structural resolution. Here we present a new method called Kinetic Ensemble Refinement (KER) that directly calculates a series of NMR spectra from a set of structures, and then iteratively improves those predicted structures to match experimental data. In our KER method, a hierarchy of temporal interconversion rates between ensemble members are fit to the experimental data simultaneously with the atomic coordinates. Using KER, we were able to fit experimental NOE buildup data significantly better than existing structural ensembles. Furthermore, though NMR derived structures are often considered lower in quality than those determined using x-ray crystallography, analysis of a large set of crystal structures suggests that NOE data contains a surprising amount of high resolution information that is better modeled using our Kinetic Ensemble approach. The KER method enables the integration of experiments that probe both fast and slow timescales, offering the ability to more fully characterize and exploit kinetically isolated protein states. XFELs unique capabilities are utilized to characterize interactions between lipids and apolipoproteins with an eventual goal to study membrane protein dynamics. We present first results from X-ray diffraction studies performed at the LCLS on microcrystals of nanolipoprotein (NLP) particles, or nanodiscs. Cell-free expression of apolipoprotein A1 (ApoA1) in the presence of lipids leads to the formation of discs consisting of a 5 nm thick lipid bi-layer surrounded by a 10-20 nm diameter belt of apolipoprotein. These represent both a stable intermediate state in LDL formation and a membrane-model system into which membrane proteins can be inserted and thus solubilized. Crystals of ApoA1 NLPs were loaded in a humidified chamber onto patterned Si chips. SFX experiments took place at the MFX instrument at LCLS. Bragg peaks at low resolution are apparent within the fiber-like diffraction of the ApoA1 hits. The average layer-line distances correspond to the known height of the A1 disc as characterized by electron microscopy, dynamic light scattering, SAXS and SANS, about 60 Å, while the distances between Bragg peaks within layer lines correspond to the disc width, about 100 Å. This is consistent with NLPs stacking in solution as has been previously documented by TEM. This study represents critical initial steps in both obtaining a structural picture of ApoA1 NLPs and developing this system as a scaffold for future structural characterization of inserted membrane proteins and was enabled by recent advances in fixed stages. The goal of this work is to understand the intrinsic properties that give natural enzymes their catalytic power and to learn how to build these properties into in silico enzyme design. Partial Order Optimum Likelihood (POOL) is a machine learning method developed by us to predict residues important for function, using the 3D structure of the query protein. The input features to POOL are based on computed electrostatic and chemical properties from THEMATICS. These input features are measures of the strength of coupling between protonation events, as catalytic sites in proteins are characterized by networks of strongly coupled protonation states. These networks impart the necessary electrostatic, protontransfer, and ligand binding properties to the active residues in the first layer around the reacting substrate molecule(s). Typically these networks include first-, second-, and sometimes third-layer residues. POOL-predicted, multi-layer active sites with significant participation by distal residues have been verified experimentally by site-directed mutagenesis and kinetics assays for Ps. putida nitrile hydratase, human phosphoglucose isomerase, E. coli replicative DNA polymerase Pol III, E. coli Y family DNA polymerase DinB, and E. coli ornithine transcarbamoylase. In designed enzymes, such as retroaldolases, the residuespecific input features to POOL measures of the strength of coupling between protonation equilibria rise as the enzymes evolve to higher rates of catalytic turnover. We show that high values for these measures in the catalytic residues constitute one necessary feature for catalytic activity. An approach to build these properties into the initial designs is proposed. Acknowledgment: NSF MCB-1517290. Human phosphoglucose isomerase (hPGI) catalyzes the reversible isomerization of glucose 6-phosphate to fructose 6-phosphate, an important step in glycolysis. It serves also as a biomarker for certain types of cancer. Electrostatics-based computational methods THEMATICS and POOL, developed by us, predicted that several residues in the second and third layers beyond the reacting substrate molecule were active in catalysis. These predictions were confirmed by direct biochemical assay, showing that the hPGI second-shell variants K362A, Q388A, E495Q, and D511N, and the third-shell variants H396L and H100L, all show significant loss in catalytic efficiency compared to wild type. K362 is located 11 Å from the ligand; the K362A variant shows no detectable catalytic activity. Observed reductions in kcat/KM for Q388A, E495Q, and D511N in the second shell are 19-, 250-, and 230-fold, respectively; corresponding reductions for H396L and H100L in the third shell are 24-and 630-fold, respectively. Using protein electrostatics and molecular dynamics simulations, we explore the roles that these distal residues play in the catalytic process. We show that electrostatic interactions are partially responsible for these longer-range effects, in which the distal residues influence the electric field within the active site and the proton transfer properties of the first-shell catalytic residues. We also identify conformational dynamical processes that participate in the catalytic mechanism. Supported by NSF MCB-1517290 and an NSF Postdoctoral Fellowship in Biology (SCB). Yujia Xu 1 , Fang Fang Chen 1 , Parminder Jeet Kaven 1 , Faizunnahar Dewan 1 , Carl Guenst 1 , Jui Shivaji Chaugule 1 1 Department of Chemistry, Hunter College of CUNY (New York, United States) Our recent work presented the self-assembly of several designed peptides forming collagen-like protein min-fibrils having an axial repeating structure reminiscent of that of the D-period of native collagens. In this work we characterize the self-assembly of another two designed peptides: peptide Col877 and peptide Col108r. The triple helix domain of Col877 consists of three pseudo-identical units of amino acid sequence arranged in tandem, while that of Col108r consists of three sequence units having the same amino acid composition but very different residue placement. Upon folding into collagen triple helix, Col877 further self-associated laterally to form mini-fibrils having the well-defined alternating gapoverlap zones characteristic of collagen fibrils when examined by electron microscope, except the striated-banding pattern has a size of 35 nm; this banding pattern is subsequently designated as the dperiod of the mini-fibrils. In contrast, the self-assembly Co108r only formed none-specific aggregates with no identifiable shape or other structural features. These results further accentuated the critical involvement of the repeating sequence units in the self-assembly of collagen mini-fibrils. In connection with the unit-staggering self-assembly model of the d-periodic mini-fibrils suggested in our previous studies, we proposed a set of design rules for generating collagen-like protein fibrils with desired stability and specific size of the d-period. Collagen fibrils are essential for the tissue development and function, and are also one of the most sought after biomaterials. The novel design rules will find a wide range of applications in research and in a wide range of biomedical applications. Cytochrome P450s (CYP450) are a ubiquitous superfamily of membrane bound enzymes that are responsible for the catalysis of many substrates including >70% of drugs in the market. Towards this, each turn of the catalytic cycle requires two electrons that are donated by either Cytochrome P450 reductase (CYPOR) or the 2nd electron by cytochrome b5 (cyt-b5). Structural interactions that enable the electron transfer to CYP450 from its redox partner CYPOR is a vital prerequisite for the metabolism. In this study, we used peptide based lipid nanodiscs to successfully reconstitute the CYP450 and REDOX-partners, which was characterized by size exclusion chromatography (SEC) and dynamic light scattering (DLS). Additionally, the size, shape and localization of proteins in nanodiscs were determined using SAXS. However, the correct orientation of two proteins in membrane essential for its catalytic function was determined by SEC-SAXS. Our results also suggest anchorage of the CYP450 proteins in a lipid bilayer to be a minimal requirement for CYP450 catalytic function. Furthermore, high resolution probing of the binding interface of CYP450 with the minimal domain of CYPOR (FMN binding domain (FBD)) was carried out using NMR experiments. The functional ability of the membrane anchored redox complex was successfully evaluated using stopped-flow kinetics. Thus, combining both low and high-resolution data, we report the first structural model of the phospholipid-bilayer-anchored~80-kDa CYP450 and FBD complex. Linking structural details to CYP450's enzymatic mechanism will help unravel the xenobiotic metabolism of diverse microsomal CYPs in their native environment and facilitate the design of new drug entities. Xue Fei 1 1 MIT (cambridge, United States) ClpXP, one of the best characterized AAA+ proteases, consists of ClpX the unfoldase and ClpP the peptidase. A single ClpX ring is hexameric while ClpP is heptameric, raising the question how ClpP and ClpX form a stable complex during ATPase cycle. Although it was known that flexible loops of ClpX stabilize the ClpXP complex by docking into cleft of ClpP ring, it remained unclear what structure rearrangement is necessary to accommodate this inherent symmetry mismatch. Using CryoEM and innovative computational algorithms, we solved structures of ClpXP complex at an average resolution of 5A. The hexameric ClpX adopts a lock washer conformation that lacks six fold symmetry. This conformation allows two distinct types of ClpX-ClpP interaction, to maintain a dynamic but stable interface. The structural information contained in solution scattering data from empty lipid nanodiscs is examined in the context of a multi-component geometric model. X-ray scattering data were collected on nanodiscs of different compositions at scattering vector magnitudes up to 2.0 A-1. Through the calculation of the partial form factor for each of the nanodisc components before the isotropic average, structural parameters in the model were correlated to the features observed in the X-ray scattering data and to the corresponding distance distribution function. It is shown that, in general, the features at~0.30.6 A -1 in the scattering data correlate to the bilayer structure. The data also support the argument that the elliptical shape of nanodiscs found in model fitting is physical, rather than an artifact due to the nanodisc size distribution. The lipid chain packing peak at~1.5 A -1 is visible in the data and reflects the lipid bilayer phase transition. The shape change in the distance distribution function across the phase transition suggests that the nanodiscs are more circular in the fluid phase. The implication of these findings for model fitting of empty and protein-loaded nanodiscs is discussed 16 The cell division protein FtsA is an actin-like ATPase that is highly conserved among prokaryotes and essential in Escherichia coli. FtsA contains a C-terminal membrane targeting sequence (MTS) and directly recruits FtsZ polymers to the inner membrane to establish the Z-ring and coordinate constriction. FtsA directly reorganizes phospholipid (PL) architecture and remodels dynamic FtsZ polymers. To determine if PL engagement by FtsA modifies the interaction with FtsZ, we tested if purified FtsA and a mutant protein lacking the MTS (FtsAMTS) bind to PLs and recruit FtsZ. In sedimentation assays, we observed that FtsA binds PLs with and without ATP. FtsAMTS is unable to bind PLs when ATP is omitted; however, it is recruited in the presence of ATP suggesting that binding PL is ATP-dependent. We observed that FtsAMTS recruits FtsZ to PLs, indicating that interaction between FtsZ and FtsAMTS is maintained. FtsZ polymerizes with GTP, addition of FtsA stoichiometrically inhibits FtsZ polymerization. Interestingly, FtsAMTS is defective for disassembly of FtsZ polymers, suggesting that the MTS of FtsA may regulate FtsA activity and conformation. To determine if there are large conformational differences between FtsA and FtsAMTS, we performed transmission electron microscopy of FtsA and FtsAMTS. We observed FtsAMTS forms long protein polymers, greater than 100 nm, in the presence of ATP. Together, these results suggest that FtsA has a secondary, ATP-dependent, membrane interaction and that the MTS may regulate FtsZ remodeling by FtsA. These studies provide biochemical insight into a highly conserved interaction during early prokaryotic cell division. Predicting the effects of protein mutation on protein folding and binding free energies has a wide variety of applications in protein design, evolution, biology, and medicine. Alchemical free energy methods enable prediction of free energy from molecular dynamics simulations, but have not often been applied to protein mutations. Multisite dynamics (MSD) is a particularly efficient and scalable alchemical free energy method that has developed extensively in recent years. We applied MSD to predict folding free energies in T4 lysozyme for 33 point mutants at 7 sites, and obtain Pearson correlations with experiment between 0.81 and 0.91 depending on the electrostatic treatment used. Encouraged by this success, MSD was used where it is strongest, in exploring concurrent mutations at multiple sites. Combinatorial three site, four site, and five site systems with 8, 24, and 240 sequences respectively were explored and sampled efficiently. Results compare well with experiment, and enable the prediction of epistatic coefficients. Exploring such a large region of sequence space with only a few simulations is unprecedented among alchemical free energy methods. We are hopeful that the ability to search large protein sequence spaces will make MSD broadly useful in years to come. Hsp90 is a dimeric molecular chaperone that undergoes an essential and highly regulated opentoclosedtoopen conformational cycle upon ATP binding and hydrolysis. Although it has been established that a large energy barrier to closure is responsible for Hsp90's low ATP hydrolysis rate, the specific molecular contacts that create this energy barrier are not known. Here we discover that bacterial Hsp90 (HtpG) has a pHdependent ATPase activity that is unique among other Hsp90 homologs. The underlying mechanism is a conformationspecific electrostatic interaction between a single histidine, H255, and bound ATP. H255 stabilizes ATP only while HtpG adopts a catalytically inactive open configuration, resulting in a striking anticorrelation between nucleotide binding affinity and chaperone activity over a wide range of pH. Linkage analysis reveals that the H255ATP salt bridge contributes 1.5 kcal/mol to the energy barrier of closure. This energetic contribution is structurally asymmetric, whereby only one H255ATP saltbridge per dimer of HtpG controls ATPase activation. We find that a similar electrostatic mechanism regulates the ATPase of the endoplasmic reticulum Hsp90, and that pHdependent activity can be engineered into eukaryotic cytosolic Hsp90. These results reveal sitespecific energetic information about an evolutionarily conserved conformational landscape that controls Hsp90 ATPase activity. Ho Leung Ng 1 1 We describe two new computational approaches for predicting drug binding sites on proteins, ConDock and FragSite. ConDock is the first hybrid scoring function to use information from protein surface conservation and ligand docking to predict ligand binding sites. ConDock uses a simple product function of sequence conservation and binding energy scores. We describe the application of ConDock to predict ligand binding sites to two GPCRs with crystal structures, the beta2 adrenergic receptor and A2A adenosine receptor, as well as an uncrystallized GPCR, G-protein coupled estrogen receptor (GPER). We compare the sites predicted by ConDock and traditional methods analyzing surface geometry, surface conservation, and ligand chemical interactions. Incorporating sequence conservation information in Con-Dock avoids errors resulting from physics-based scoring functions and modeling. We also describe Frag-Site, which predicts drug binding sites by docking a virtual library of fragments. FragSite uses a docking score optimized for low molecular weight fragments as opposed to drug-like molecules. We describe how FragSite identifies known and new drug binding sites in K-Ras and Stat3. The chemical properties of the bound fragments provide clues on how to expand to larger hit molecules. Yunsun Nam 1 1 UT Southwestern Med Ctr (Dallas, United States) N6-methyladenosine (m6A) is an abundant, reversible chemical modification that regulates function and stability of many types of RNAs. We use biochemical and structural methods to elucidate how m6A marks are generated by RNA methyltransferases. We show that Mettl3 and Mettl14 cooperate to recognize and catalyze modification of target RNAs. Mettl3 is the catalytically active enzyme, while Mettl14 has a structural role to support the necessary conformation and to bind the substrate RNA. We also show that Mettl16, another m6A writer, uses a distinct mechanism to recognize and bind the RNA substrates. We determine the molecular basis for why the RNA methyltransferases manifest distinct substrate specificities. Moreover, we identify a regulatory mechanism where a conformational change acts as an allosteric switch to tune the enzymatic efficiency of the RNA methyltransferase. Moreover, we reveal how disease mutations of m6A writers lead to aberrant methyltransferase activity, by disrupting the allosteric switch. Together, our study shows that each m6A writer enzyme has evolved to modify a specific set of RNAs with controlled efficiency. Quantitative understanding of the methyltransferases also provides insight into how SAM metabolism and gene regulation are linked. Missense mutations in numerous RNA-binding proteins have been associated with the neurodegenerative disease amyotrophic lateral sclerosis (ALS). These proteins contain at least one RNA-recognition motif (RRM) that may contribute to a common mechanism in neurodegeneration. A molecular dissection approach was used to study the equilibrium unfolding of the individual and tethered RRM domains of TDP-43, and the results revealed a populated intermediate state in the RRM2 domain. NMR studies coupled with simulations indicate that the nuclear export sequence and a predicted aggregation prone stretch are both exposed in this intermediate state, suggesting it contributes to both normal function and aggregation of TDP-43. The equilibrium unfolding of the RRM domains of FUS/TLS, matrin-3 and hnRNPA1 were also investigated to test for the commonality of the RRM intermediate state across a set of RNA-binding proteins linked to ALS. The RRM domains that contained a detectable intermediate state were all adjacent to a disordered prion-like domain with ALS mutation sites. These intermediates are stabilized by a large network of branched aliphatic side chains, which provides a stability core for the proteins. By contrast, they are not necessarily the domains with the highest affinity for RNA. Stable cell lines containing RRM intermediate-enhancing and/or ALS mutations in TDP-43 have been developed to further study the interplay between order and disorder and to test the roles of the intermediate state for normal protein function and for aberrant interactions between the stability core and the disordered prion-like domains that may propagate misfolding in disease pathogenesis. While both archaeal and eukaryotic transcription initiation systems utilize TBP (TATA box-binding protein) and TFIIB (transcription factor IIB), eukaryotic systems include larger numbers of initiation factors. It remains uncertain how eukaryotic transcription initiation systems have evolved. Here, we investigate the evolutionary development of TBP and TFIIB, each of which has an intramolecular direct repeat, using two evolutionary indicators. Inter-repeat sequence dissimilarity (dDR, distance between direct repeats) indicates that the asymmetry of two repeats in TBP and TFIIB has gradually increased during evolution. Interspecies sequence diversity (PD, phylogenetic diversity) indicates that the resultant asymmetric structure, which is related to the ability to interact with multiple factors, diverged in archaeal TBP and archaeal/ eukaryotic TFIIB during evolution. Our findings suggest that eukaryotic TBP initially acquired multiple Eukarya-specific interactors through asymmetric evolution of the two repeats. After the asymmetric TBP generated the complexity of the eukaryotic transcription initiation systems, its diversification halted and its asymmetric structure spread throughout eukaryotic species. To Bud or Not to Bud: Inhibition of the HSV-1 Nuclear Egress Complex Elizabeth Draganova 1 , Ekaterina Heldwein 1 1 Egress is an essential step in replication of Herpes simplex virus-1 (HSV-1) whereby progeny virions are released from the cell, which results in spread to uninfected tissues and hosts. During egress, herpesviruses first bud at the inner nuclear membrane where viral capsids form nascent buds that pinch off into the perinuclear space. This nuclear budding requires the conserved nuclear egress complex (NEC), a heterodimer composed of two viral proteins, UL31 and UL34. Previously, we have shown that the NEC buds synthetic membranes in the absence of other components or energy, which established the NEC as a novel self-contained budding machine. We have also shown that the NEC forms hexagonal honeycomb coats on the inner surface of budded vesicles formed in vitro. While NEC-mediated budding is a rapid process in vitro, it is strictly regulated in infected cells to prevent unproductive budding by an unclear mechanism. Using surface plasmon resonance, confocal microscopy, and cryo-electron microscopy, we show here that another HSV-1 protein binds NEC in vitro and blocks budding. We have identified the regulatory element of this protein responsible for NEC budding inhibition. In the absence of this element, the inhibitory protein can still bind the NEC but no longer inhibits budding. Our work has yielded an indepth biophysical characterization of this inhibitory mechanism of NEC budding. We hypothesize that this inhibitory mechanism is utilized by the virus to negatively regulate NEC-mediated budding during infection. Research on proteolytic mechanisms regulating reproduction was carried out in the teleost to increase our knowledge concerning not only fish, but also vertebrates. Unfertilized eggs obtained from wild pink salmon, Oncorhynchus gorbuscha, were fertilized and maintained during early development in artificial nests within the river Varzuga watercourse (the White Sea basin). Eggs before fertilization and at subsequent stages of embryogenesis and early larvae were sampled. Enzymatic activities of the main proteolytic pathways including proteasome, calpains, and lysosomal autophagy were assessed. Proteases are localized in different cellular compartments and regulated by specific endogenous regulators and substrate availability. Besides, functional specialization of the proteases associated with various stages of embryogenesis and early development was demonstrated. Since ubiquitin-proteasome system primarily mediates protein quality control, the level of chymotrypsin-like proteasome activity in unfertilized eggs and during embryogenesis was similar with substantial increase (up to ten-fold) after hatching corresponding with elevated level of de novo synthesis. Cathepsins sequestrated within multivesicular bodies mostly contribute to routine protein degradation, particularly vitellogenin processing, throughout embryogenesis and in early larvae. Cathepsin hydrolytic capacity raised at eye pigmentation and hatching stages corresponding with organogenesis and egg membrane disintegration, respectively. Calciumdependent proteases (calpains) contribute to highly selective development-associated processes such as cell differentiation or myofibril formation, so gradual increase in calpain activity was shown since fertilization to hatching. Though various proteolytic pathways act cooperatively, they obviously possess particular functions contributing to proteolytic regulation of the early development. Sean Devenish 1 , Chris Thorne 1 , Tom Scheidt 2 , Jackie Carozza 2 , Paolo Arosio 3 , Yingbo Zhang 2 , Alexander Buell 4 , Thomas Müller 1 , Andrew Lynn 1 , Jonathan Faherty 1 , Maya Wright 1 , Maren Butz 1 , Tuomas Knowles 2 1 Fluidic Analytics (Cambridge, United Kingdom); 2 University of Cambridge (Cambridge, United Kingdom); 3 ETH Zurich (Zurich, Switzerland); 4 University of Dusseldorf (Dusseldorf, Germany) The characterisation of biomolecular interactions is a field with applications in health research and diagnostics, yet few current methods are applicable to studies taking place in physiologically-relevant conditions or where sample volumes are limited. Here we discuss a powerful approach for probing the sizes of proteins and their complexes in solution -microfluidic diffusional sizing (MDS). Importantly, with MDS small volumes of protein are studied with high sensitivity in their native state without the use of a matrix or surface. The system is maintained at steady state laminar flow, which enables the diffusion of protein species as they pass through a microfluidic channel to be measured, and consequently their hydrodynamic radius (Rh) to be determined. Preliminary experiments show that this approach has potential applications ranging from basic biophysical analyses to in-solution characterisation of protein-protein interactions (PPIs) and the formation of protein aggregates. We are developing therefore a range of products that will make MDS available to any lab wishing to use it. Knowing structures and dynamics of biomolecular systems is extremely helpful for deciphering their functional mechanisms. The classical normal mode analysis and the elastic network models have been useful computational tools for connecting structures with dynamics and then with functional mechanisms. The recent breakthroughs in experimental technology for structure determination, especially in cryo-EM, have helped unveil the structures of many extremely large assemblies at near atomic resolution, such as HIV-1 capsid that has nearly 5 million atoms. Of these systems, since performing even coarsegrained normal mode studies is challenging, more aggressive course-graining approaches such as the rotation translation block (RTB) were applied. However, they lose a great amount of accuracy in dynamics, especially at higher frequency. In this work, we present a new projection-based method called block of selected electivity (BOSE) that allows the intrinsic flexibility of each structural component of the assembly to be properly maintained when applying projection and thus is able to preserve the accuracy in dynamics. Our results shows that BOSE modes have significantly higher quality than RTB modes though the computational cost of the two are comparable. Using BOSE, we successfully carried out a normal mode analysis of the entire HIV-1 capsid in all-atom details. Our results reveals the role of pentamers dynamics: how they suppress the vibrations of the capsid at both hemispherical ends and thereby stabilize the whole capsid structure. Our results are in agreement with experimental findings that suggest the disassembly and uncoating of HIV-1 capsid start when the pentamers becomes destabilized. Antibodies have at least two binding sites which can cooperate to bind the multivalent antigen stronger than an individual site. This enhancement of binding affinity is called avidity. Despite its importance, avidity is poorly understood in quantitative terms, thus we cannot predict how much this phenomenon will strengthen the interactions between an antibody and a given target. This prevents avidity from being used rationally in antibody engineering. In this project we aim to examine how the monovalent interaction and the structure of the target determine the avidity enhancement of binding to a bivalent target. Our experimental setup involves anti-His-tag antibody for which affinity of the monovalent interaction can be tuned by pH change and a large library of bivalent antigens with epitopes separated by flexible or rigid linkers of various length. Size and structure of the targets will be confirmed by small angle x-ray scattering and formation of cyclic 1:1 antibody-antigen complex will be visualized by negative stain electron microscopy. Finally, we will determine binding affinities of an antibody to each antigen variant by surface plasmon resonance. We aim to use produced experimental dataset to test current theoretical models for how avidity binding occurs and to develop an antibody avidity predictor web applet, capable of predicting the avidity binding constants for binding to multimeric targets of known structure. In this way the project will provide both the fundamental knowledge and practical tools needed to rationally exploit avidity in antibodies. Rizwan Khan 1 , Rizwan Khan 1 , Parvez Alam 2 1 Aligarh Muslim University, India (Aligarh, India); 2 Interdisciplinary Biotechnology Unit, Aligarh Muslim University, India (ALIGARH, India) Protein misfolding and aggregation have been associated with several human diseases such as Alzheimers, Parkinsons and familial amyloid polyneuropathy etc. In this study, anti-fibrillation activity of vitamin k3 and its effect on the kinetics of amyloid formation of hen egg white lysozyme (HEWL) and A-42 peptide were investigated. Here, in combination with Thioflavin T (ThT) fluorescence assay, circular dichroism (CD), transmission electron microscopy and cell cytotoxicity assay, we demonstrated that vitamin k3 significantly inhibits fibril formation as well as the inhibitory effect is dose dependent manner. Our experimental studies inferred that vitamin k3 exert its neuro protective effect against amyloid induced cytotoxicity through concerted pathway, modifying the aggregation formation towards formation of nontoxic aggregates. Molecular docking demonstrated that vitamin k3 mediated inhibition of HEWL and A-42 fibrillogenesis may be initiated by interacting with proteolytic resistant and aggregation prone regions respectively. This work would provide an insight into the mechanism of protein aggregation inhibition by vitamin k3; pave the way for discovery of other small molecules that may exert similar effect against amyloid formation and its associated neurodegenerative diseases. Human astrovirus (HAstV) is a non-enveloped virus with a positive-sense RNA genome and causes gastroenteritis in infants, the elderly and immunocompromised individuals. The virus has no vaccine, a high mutation rate, and emerging clinical symptoms like lethal encephalitis. In order to better understand this virus, we use a structural and biophysical approach to investigate the capsid protein that allows the virus to transport its genome. The HAstV capsid is coded by the viral genome as a 90 kD polyprotein labeled as VP90, which forms a non-infectious particle. The capsid undergoes post-translational modification through proteolytic cleavage to form the mature infectious state, but there is little understanding of the necessary domains, cleavage sites, or structural dynamics. This process can be investigated by expressing the virus particle, simulating its maturation in vitro, then investigating these intermediate particles with structural studies and cell culture testing. We have overexpressed an immature capsid construct in E. coli and purified using nickel-NTA beads and size exclusion chromatography. Constructs with short Nterminal truncations had better expression and solubility when compared to full length constructs. Viruslike particle formation has been confirmed using transmission electron microscopy. Future steps involve optimizing the expression and purification of the viral particles from bacterial and insect cell culture expression systems. These particles will be used for in vitro maturation assays using isolated protease treatments, capsid reconstructions using cryo-electron microscopy and liposome infiltration assays. This research is funded by the Welch Foundation (C-1565 to YJT) and (HAMBP T32GM008280 to MY) The prime cause of homocysteine (Hcy) toxicity towards proteins has been attributed to the modification of protein by homocysteine thiolactone (HTL). But the effects of Hcy (the parent molecule) on proteins are scarce. Hcy also has the ability to modify proteins (cysteine residues, process termed as S-homocysteinylation), which could in turn affect their functionality. However, structural and functional consequences of such modification are not yet clearly understood. Our study aims at investigating these possible effects of Hcy. Using RNase-A, Lyz and horseradish peroxidise (HRP) as models, we studied the effects of Hcy on enzyme activity and conformational status. Enzyme activity measurements after overnight incubation with Hcy led to gradual decrease in enzyme function of HRP in a concentration dependent manner but not in Lyz and RNase-A. Conformational analyses revealed no gross structural alterations in all proteins studied. However, certain alteration in the heme-Trp distance was observed in case of HRP as evident from Trp fluorescence measurements. Loss of function in HRP was due to disruption of heme redox state in presence of Hcy. Similar findings were obtained for three other common heme metalloprotein (Cytochrome c, Cyt c; Hemoglobin, Hb; and Myoglobin, Myo), wherein Hcy induced certain alterations in heme environment rendering it reduced with further disruption of the heme-Trp distance. Hcy was found to alter redox states without direct interaction with proteins (Cyt c and Myo). The findings suggest that heme and heme containing proteins could be potential targets for Hcy modifications and involved in Hcy-induced cytotoxicity. University of Maryland (Rockville, United States); 2 National Institute of Science and Technology (Rockville, United States); 3 University of Maryland, National Institute of Science and Technology (Rockville, United States) A high-throughput method of peptide sequencing would be of great benefit to proteomics, personalized medicine, and peptide drug discovery. A major hurdle in oligopeptide sequencing is discriminating positional information when identifying amino acids. Doing so requires analytical techniques that are not compatible with high-throughput methodology. However, there are proteins found in nature capable of discerning amino acids with a c-terminal bond and a free n-terminal amine. We have adapted two such bacterial proteins, the A. tumefaciens ClpS chaperone and the A. pernix ProX tRNA-editing domain, to a yeast surface display selection scheme, with the intent of selecting mutants able to tightly bind peptides in an n-terminus selective manner. We use a combination of a benchtop yeast pull-down assay and surface plasmon resonance to demonstrate our success. Protein-G is broadly used reagent for antibody purification and detection. C2 domain of Protein-G from Streptococcus has a high affinity for the Fc region (KD~10nM) of the IgG, but much lower affinity (few μM) for the fragment antibody binding (Fab) of the antibodies. To improve its application we engineered new Protein-G variants with 8 point mutations and an approximately 100-fold improved affinity to human Kappa, Lambda, 4D5 and LRT Fab LC scaffold. Initially generated Protein-G-A1 (Bailey et al., 2014) have 24nM binding affinity to 4D5 but do not recognize kappa or lambda scaffolds. Using phage display new LRT fab scaffold with low nM affinity to Protein-G-A1 was generated, but there was still a need for binding to human kappa and lambda. 6 Proten-G libraries randomized in a position of interaction with Fab LC were produced and used in phage display which effect in 12 new Protein-G variants which recognize both kappa and lambda scaffolds. Binding of all of them was characterized using SPR and the affinity of the best from them Protein-G-F is approximately: 3 nM for kappa, 50 nM for lambda, 60 nM for 4D5 and 6 nM for LRT. What is more, selected Protein-G-D variant recognize kappa, lambda and 4D5 slightly weaker, but do not recognize LRT scaffold, which gives opportunity to use it as a unique orthogonal pair with Protein-G-A1. Newly engineered Protein-G variants can be used in number of application in the use of Fab-based affinity reagents, mostly as a purification reagent. Shelly Goomber 1 , Jagdeep Kaur 2 1 National Institute of Malaria Research (New Delhi, India); 2 Department of Biotechnology, Panjab University (Chandigarh, India) Bacillus subtilis lipase LipJ (1.4 subfamily) is one of the smallest, low molecular weight (19kDa) functional enzyme that unfold reversibly as it lacks Cysteine residue and hence disulfide linkage. Therefore it is ideal for structure-function analysis. LipJ mutants were generated by laboratory evolution by error prone PCR and site directed mutagenesis. Three tier high throughput screening was done to select point mutants active and stable at extreme temperature conditions. Extensive biochemical and biophysical assay shortlisted promising point mutants with significant stability and activity at extreme temperature conditions compared to wild type. Thermal assay determines point mutants Gln121Arg, Leu114Pro, Asn166Tyr to be thermostable and active at high temperature, whereas Phe19Tyr, Ile137Met mutants were psychrophilic with significant deviation in temperature optima to cold. Homolog modeling predicted the mutations to be present within loop near surface. Hence the findings lead to idea that random loop, coil and terminal ends of protein may determine the protein physiology, properties whereas non random secondary structures maintain integrity of protein. antagonists, and inverse agonists. To discriminate the distinct efficacy profiles of the ligands, we carried out molecular dynamics (MD) simulations to identify the dynamic behaviors of inactive and active conformations of CB1 with the ligands. The key finding is that -hairpin structure in intracellular loop 3 (ICL3) is formed when agonist, THC, is bound to the active conformation and inverse agonist, taranabant, is bound to the inactive conformation, respectively. On the other hand, -hairpin structure is not formed in both conformations of CB1 when antagonist, THCV, is bound. In addition, the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method was applied to analyze the binding free energy decompositions of the CB1-ligand complexes. The results show that THC and THCV are favorably bound to the active conformation, while taranabant is favorably bound to the inactive conformation. All these observations suggest the possibility that the three classes of ligands can be discriminated. Our findings shed light on the understanding of different efficacy profiles of ligands by analyzing the structural behaviors of CB1 and the binding energies of ligands and yield insights useful for the design of new potent CB1 drugs. Navaneethakrishnan Krishnamoorthy 1 , Hesham Ismail 2 , Gheyath Nasrallah 2 1 Systems Biology, Sidra medical and Research Center (Doha, Qatar); 2 Department of Biomedical Sciences, Qatar University (Doha, Qatar) Homocystinuria is a metabolic disorder that leads to multiple disorders of the central nervous system and cardiovascular system etc. A missense mutation R336C in the protein cystathionine -synthase (CBS) is causing homocystinuria. In particular, it is a founder mutation causes severe effect in Qatari population and the most prevalent inherited monogenic disease in the country. The molecular mechanism of the disease is unclear. Here, we used molecular modelling and experimental studies to characterize the effect of R336C mutation on the CBS to understand the structure-functional relationships. Molecular modeling of human CBS structure with the mutation R336C showed that it is adjacent to the catalytic core. The molecular dynamics simulation suggests that the mutation induces conformational changes, reduces structural stability, impacts the protein surface, and thus, could have an influence on the CBS binding sites of substrates and activity. In addition, we have identified a few potential channels available for the substrates entry and/or exit to/from the catalytic core. Furthermore, we used experimental models of yeast and cell culture created by knocking in the mutant CBS in cell lines (HEK293T and HepG2) to assess the effect of the mutant proteins expression, stability and activity. Altogether, the results show deleterious effect of the mutant and its impact on the structure-functional relationships. This study can provide a basis for the development of novel therapies to treat homocystinuria. Brian Gibbons 1 1 Corteva Agriscience, Agriculture Division of DowDuPont (Hayward, United States) An insecticidal protein, IPD072Aa, from Pseudomonas chlororaphis is highly effective in protecting corn plants from corn rootworm damage when expressed in planta (Schellenberger et. al, Science, 354, (634) (635) (636) (637) . A solution NMR structure of IPD072Aa was resolved and an antiparallel dimer structure was revealed. Strong hydrophobic interactions among several highly conserved residues are responsible for the dimer formation. Biochemical studies showed that dimer dissociation concurs with protein denaturation further supporting a strong dimer interface. Functional evaluation of the active and inactive heterodimer against the target insect indicates that the specific activity is only proportional to the active component suggesting the monomer is likely the active form once ingested into the insect midgut. From the current observations, the data supports a hypothesis that IPD072Aa likely employs a strategy of dimerization to protect its integrity in solution and the highly hydrophobic interface might be the active center interacting with the insect midgut once ingested. Translesion synthesis (TLS), a DNA damage tolerance (DDT) pathway, is an error prone process that recruits specialized polymerases to copy over lesioned areas of DNA. Polymerase Zeta (Pol ), a TLS polymerase capable of transversing over abasic sites, is commonly studied in S. cerevisiae. Although Pol of S. cerevisiae and H. sapiens share a small sequence homology, the binding partners and mechanisms of the two species are surprisingly similar. For example, both human and yeast Pol bind to Rev1, another TLS polymerase, which acts as a scaffold for TLS polymerases. Much of our knowledge of Pol and TLS have been discovered in yeast. However, we have yet to solve a structure of S. cerevisiae Pol. We have begun to optimize expression and purification of the regulatory subunit of yeast Pol, Rev7. Rev7 is unstable in its apo form, however, we were able to stabilize Rev7 by co-expressing it with a fragment of the Pol catalytic subunit, Rev3. This interaction has been verified by Mass Spec of a Rev7/Rev3 complex that has undergone a multi-stage purification process. Furthermore, we were able to co-elute Rev1-C terminal with the Rev7/Rev3 complex during gel filtration. By preparing these constructs, we can study S. cerevisiae Pol through a structural perspective to validate key protein-protein interactions needed for yeast TLS. Othman RECHICHE 1 , Jeff Plowman 2 , Duane Harland 2 , Verne Lee 1 , Shaun Lott 1 1 UNIVERSITY OF AUCKLAND (Auckland, New Zealand); 2 AgResearch Limited (Lincoln, New Zealand) Keratin-associated proteins (KAPs) are encoded by several multi-gene families and are classified into three different groups: ultra-high sulfur (UHS), high sulfur (HS) and high glycine-tyrosine (HGT). KAPs are the major constituent of the matrix between the hair keratin intermediate filaments (IFs), and stabilise hair structure by extensive disulfide bonding. In human, 100 different KAPs are expressed by the hair follicle. These proteins have several distinctive primary structure features, including low sequence complexity and the presence of repeated motifs. The expression and purification of KAPs is challenging because they are cysteine-rich. Therefore, we designed a method that enables a high yield production of pure and soluble KAPs. In order to gain detailed molecular information on HS KAP11.1 and HGT KAP6.1 we carried out biophysical and structural characterization by Circular Dichroism, SEC-MALLS and SAXS. SAXS experiments showed that KAP11.1 adopts an extended shape in solution and is highly flexible, whereas KAP6.1 is more compact and present secondary structure features. SEC-MALLS experiments confirmed that KAP11.1 is monomeric in solution, but KAP6.1 shows a concentration-dependent transition from monomer to dimer. Another interesting feature is that both KAP11.1 and KAP6.1 can form hydrogels. This phenomenon depends on different factors: incubation time, protein concentration and temperature. Small volume, highly concentrated sample tend to gelify more readily. Examples of hair protein and peptide-based hydrogels and their application in severe wound healing cases have been reported in the literature, as well as their commercial derivatives. However, no hydrogel made of a pure KAP has been reported to date. Plantarum. Josef Bober 1 , Nikhil Nair 1 1 Tufts University (Medford, United States) Food-safe probiotic Lactobacillus plantarum is a promising non-model platform bacterium for production of nutraceuticals using metabolic engineering. Biosynthesis of D-tagatose, a low-caloric sugar-substitute with anti-glycemic properties, has been reported using various engineered bacteria expressing Larabinose isomerase. However, low productivity remains a barrier to economical production of this sweetener. Strategies to improve productivity have relied on enzyme engineering to improve kinetic properties toward substrate D-galactose. However, the primary limitation to productivity is not kinetics, but thermodynamics since isomerization of D-galactose to D-tagatose is only mildly favorable. Resultantly, whole cell biocatalysts that disproportionately partition substrate and product across a membrane can circumvent this thermodynamic limitation. Unfortunately, this thermodynamic advantage results in a kinetic penalty due to transport limitations. In this work, we use the mesophilic and acid tolerant L-arabinose isomerase from Lactobacillus sakei in Lactobacillus plantarum as a model system to study D-tagatose production. We confirmed that Dtagatose production was thermodynamically limited in cell-free lysates and transport-limited during whole-cell catalysis. Next, we focused on improving productivity through an investigation and subsequent mitigation of membrane transport barriers. We explored, in detail, cellular engineering strategies including surface display, overexpression of native and non-native sugar transporters, and cell permeabilization techniques to achieve a superior whole-cell biocatalyst for D-tagatose production compared to those reported in literature. Through this investigation, were able to circumvent the thermodynamic conversion barrier while maintaining high reaction rates. This work provides novel insights and demonstrates new tools to guide engineering efforts in probiotic Lactobacillus plantarum as well as other gram-positive bacteria. Protein lysine malonylation, succinylation, crotonylation, and 2-hydroxyisobutyrylation have been recognized as new post-translational modifications (PTMs) in recent years. However, the proteins harboring these modifications and their corresponding functions of the modifications remain largely unknown in cereal plants. Using antibody-based affinity enrichment of modified peptides followed by nano-HPLC/ MS/MS analyses, we identified from a few hundreds to over nine thousands modification sites for these four modifications together with acetylation in developing rice (Oryza sativa) seeds, respectively. Distinct sequence motifs at the modification sites were identified for each of the modifications, respectively. Proteins with different sequence motifs were shown to be favorably associated with unique metabolic pathways or protein function domains. Many of the modified proteins and the corresponding modification sites were conserved from E. coli, human, to plants and many of the modification sites can be modified by multiple different acyl groups, especially in the key enzymes of essential metabolic pathways. Rice proteins with co-modifications of succinylation, malonylation, crotonylation, 2-hydroxyisobutyrylation, acetylation, ubiquitination, and phosphorylation were studied through a comprehensive analysis. The potential cross-talks among different modifications in the regulation of cellular metabolic processes are examined. In addition, the impact of heavy lysine modifications on lysine bioavailability in rice storage proteins has been investigated. Our study delivers a platform for expansive investigation of the molecular networks administrating cereal seed development via post-translational modifications. Emma Livingstone 1 , Andrew Whitten 2 , Saroja Weeratunga 1 , Jennifer Martin 3 , Brett Collins 1 1 Institute for Molecular Bioscience, The University of Queensland (Brisbane, Australia); 2 -Australian Centre for Neutron Scattering, Australian Nuclear Science and Technology Organisation (Sydney, Australia); 3 3Griffith Institute for Drug Discovery, Griffith University (Brisbane, Australia) Vesicle membrane fusion is essential for neurotransmission and blood glucose control, dysfunction of which is associated with neurological disorders and diabetes. The membrane fusion machinery, the topic of the 2013 Nobel Prize in Physiology or Medicine, comprises the soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs), and the Sec1/Munc18 (SM) proteins. Cognate SNAREs from vesicle and target membranes associate to form a trans-SNARE complex. SNARE complex formation provides energy to drive membrane fusion and confers the necessary specificity for regulated delivery of cellular cargo. The SM protein family has been implicated in the regulation of SNARE complex assembly, although their precise regulatory role is controversial. Recent work on an SM protein involved in yeast homotypic fusion suggests that SM proteins may act as templates, orchestrating the alignment of SNAREs from the vesicle and target membranes, promoting membrane fusion. Here we describe recent progress towards the translation of these findings to the mammalian Munc18a and Munc18c proteins, which are involved in neurotransmission and blood glucose control respectively. The healing response of bone to implant insertion is similar to its response to fracture. Therefore, biomaterials are desired to support the healing process of injured bone tissue. A promising approach to improve implant integration into surrounded tissue is the coating of implant surfaces with bio-functional proteins. In this work, a titanium binding peptide was coupled to the chemokine CXCL12, which is a chemoattractant for e.g. progenitor cells (EPC and MSC). Using the IMPACT-system, the N-terminal part of CXCL12 was expressed in E. coli ER2566 as a fusion protein containing a modified intein and a chitin binding domain, such that a C-terminal thioester was produced. The C-terminal part of CXCL12 elongated by the titanium binding peptide was synthesized by solid phase peptide synthesis. Both segments were ligated by native chemical ligation, generating a native peptide bond. The identity and purity of the products were verified by MALDI-ToF mass spectrometry and reversed-phase HPLC. The functionality of the modified chemokines was assessed by inositol phosphate accumulation assay, which displayed a slightly reduced potency compared to native, unmodified CXCL12. Further, the immobilization of the modified chemokines on titanium surfaces indicated a titanium saturation of approx. 40 nM of the CXCL12 variants. Imitating physiologic wound healing, adhesion of SaOs-2 cells on titanium was investigated and cells spread significantly more on coated titanium plates, indicating better cell attachment to the implant surface. Therefore, protein coated surfaces are a promising technique to gain a better integration of implants into the surrounding tissue and enhance wound healing. The activity of Heat shock factor 1 (Hsf1) in regulating the heat shock response is conserved among all eukaryotes, and can play a role in such biological processes as longevity, climate adaptation, and oncogenic transformation Upon sensing an increase in temperature, Hsf1 trimerizes and binds DNA to activate heat shock genes. Here, we use high-throughput fitness measurements of >500,000 variants in the trimerization domain of the Saccharomyces cerevisiae Hsf1 to investigate mechanisms of temperaturespecific Hsf1 function. We find that cell division rate under heat-shock can be modulated by mutations at particular helical positions in the coiled-coil trimerization domain, and find similar phenotypes are conferred in S. cerevisiae by natural trimerization domain variants from other species. Furthermore, exceptional Hsf1 trimerization variants increase the fitness of yeast cells under heat-shock conditions beyond that of wild-type Hsf1. Hsf1 trimerization variants have altered DNA-binding patterns in vivo, and altered transcriptional programs, suggesting that trimerization modulates target specificity. We suggest that Hsf1 variants with altered trimerization can modulate cell division in response to changing temperatures. In a type II Clustered-Regularly-Interspersed-Short-Palindromic-Repeats (CRISPR) system, the CRISPRassociated (Cas) protein Cas9 and RNAs derived from the CRISPR locus form an RNA-guided nuclease to cleave double-stranded DNAs at specific sites. In recent years, the CRISPR-Cas9 system has been successfully adapted for genome editing in a wide range of organisms, leading to a revolution in genome engineering that is still rapidly unfolding. Studies have indicated that a series of conformational changes in Cas9, coordinated by the RNAs and the target DNA, direct the protein into its active conformation. However, details on these conformational changes and their roles in Cas9 function remain to be elucidated. Building on our recent work demonstrating the use of site-directed spin labeling to monitor nucleic-acid dependent conformational changes in Streptococcus pyogenes Cas9, we reported here direct detection of Cas9-mediated DNA unwinding, which is a crucial step in Cas9 target recognition, by a combination of site-directed spin labeling and Molecular Dynamics simulations. Our results support a model in which the unwound non-target strand is stabilized by a positively-charged patch located between the two nuclease domains of Cas9, and reveal uneven increases in flexibility along the unwound non-target strand upon scissions of the DNA backbone. This study establishes the synergistic combination of spin-labeling and Molecular Dynamics to directly monitor Cas9-mediated DNA conformational changes, and yields information on the target DNA in different stages of Cas9 function, thus advancing mechanistic understanding of CRISPR-Cas9 and aiding future technological development. Polymerized hemoglobin (poly-Hb) molecules have been shown to have reduced toxicity compared to cell-free hemoglobins when transfused intravenously. Poly-Hbs are typically generated with non-specific crosslinking agents that yield a product that is polydisperse in molecular weight. We propose a chemoenzymatic approach to generate poly-Hb of defined molecular weight. The proposed method employs the site-specific ligation reaction of the sortase A enzyme from S. aureus. An Hb mutant previously developed in our lab has been modified by adding either the sortase recognition sequence (LPXTG) to the Cterminus of the -subunit or a tetraglycine motif (GGGG) to the N-terminus. We show here that Hb subunits can be ligated directly by sortase A, using a mixture of substrate tagged (LPXTG) and nucleophile tagged (poly-G) Hbs. Additionally, sortase A can be used to modify Hb with a strained cycloalkyne. We show that cycloalkyne-modified Hb molecules can be covalently linked to an azide decorated scaffold, using the well-established method of Huisgen cycloaddition. The work presented here aims to establish the feasibility of generating a monodisperse poly-Hb. Green fluorescent protein (GFP) fusion tags are commonly used to study protein expression and cellular localization in vivo. However, GFP must undergo chromophore maturation to become fluorescent, a process that can have a half-time >30 min inside research model animals. The timescale of chromophore maturation in GFP is thus slower than many key biological processes, preventing their measurement with high spatiotemporal accuracy in model organisms. To address this issue, we have developed a biosensor based on a fully matured but dim GFP. Upon specific binding of a small (15.5 kDa) protein to the sensor, full fluorescence is restored. Thus, by genetically fusing this small protein to a protein of interest, it can be detected as soon as folding is complete, without requiring post-translational modification. Our sensor has been validated in vitro and by flow cytometry in E. coli cells, and in our latest iteration, we have found that the fluorescence signal increases six-fold upon binding, with a dissociation constant of 140 AE 75 nM and a kon of roughly 0.3 μM-1s-1, allowing detection of the protein of interest on the sub-second timescale. Our biosensor opens the door to the study of short-timescale processes in research model animals, such as Drosophila embryogenesis, which we are currently investigating. Methods: A knowledge-based four-body statistical potential, derived by analyzing Delaunay tessellations of coarse-grained protein structures, forms the basis of a computational mutagenesis technique implemented in this study. The approach globally quantifies relative change to protein sequence-structure compatibility upon one or more residue substitutions, referred to as the mutant residual score. Additionally, local environmental perturbations (EP scores) are obtained at each mutated residue position and all residues within 12Å of any mutated position, collectively forming a mutant residual profile. The methodology was used to characterize structural effects for over 46,000 single and multiple residue mutants of green fluorescent protein (GFP) having experimentally known function (fluorescence). Results: A significant (p < 0.0001) GFP structure-function relationship was observed by classifying function of GFP mutants (on/off ) based on fluorescence levels and quantifying structural change using their residual scores. Also, residual scores for over 1,000 single residue GFP variants distinguished residue positions based on structural (buried hydrophobic, exposed surface) and functional (catalytic) roles. Next, residual profiles for all GFP mutants were used to train predictive models of fluorescence by implementing the random forest machine learning algorithm, with on/off classification cross-validation performance reaching 0.93 for sensitivity, 0.91 for precision, and 0.90 for balanced accuracy. Lastly, a regression tree algorithm led to experimental and predicted GFP mutant fluorescence levels that displayed correlation of r = 0.83. Conclusion: An in silico mutagenesis accurately represented GFP structural changes upon mutation. Efficient models were trained for predicting fluorescence of new GFP mutants based on residual profiles, potentially reducing time and cost burdens to researchers. Nicholas Newell 1 1 Newell (Reading, United States) Beta turns constitute more than 20% of all residues in proteins and play crucial roles in structure and function. They are commonly classified by dihedral angles into a small set of types that provides only a low-resolution picture of turn backbone geometries, and more than a quarter of turns remain unclassified. Furthermore, the systematic treatment of side-chain motifs in turns has been limited to the tabulation of single-position amino acid propensities supplemented by visual surveys and counts, and the interactions between turns and the structure in their N-and C-terminal neighborhoods have not been systematically characterized. In this work, a two-stage, least-squares, Cartesian-space clustering algorithm is applied, first to generate a fine-scale partitioning and 3D conformational mapping of the backbone distribution of all beta turns, and then to map, for each backbone geometry identified in the first stage, the distributions of side-chain and rotamer structures for all overrepresented motifs involving one, two, or three amino acids in the turns and in their immediate N-and C-terminal neighborhoods. This work demonstrates that the combination of backbone and then side-chain clustering, followed by statistical motif detection and 3D conformational mapping, is a powerful tool for structural analyses. The results expand the existing picture of beta turns by providing a comprehensive, unified, and highresolution treatment of the backbone and side-chain structure of all beta turns, including associated interactions, and it should prove useful in protein design, structure prediction, and in assessing the structural consequences of disease-associated mutations. Madushi Raththagala 1 , Jordan Alvarez 2 , Craig Vander Kooi 3 , Matthew Gentry 3 1 Skdimore College (Saratoga Springs, United States); 2 Skidmore College (Saratoga Springs, United States); 3 University of Kentucky (Lexington, United States) Reversible phosphorylation of glucose moieties is an integral part of starch synthesis and degradation. Plant glucan phosphatase Starch Excess4 (SEX4) binds to starch granular surface via an extended CBM and DSP domain interface and removes phosphate groups attached to C6 glucose moieties. However, it is still not clear how SEX4 recognizes phosphate groups within the heterogeneous macromolecular entity of starch granular surface and its microenvironment. We report the crystal structure of SEX4 bound to branched meltoheptaose with -1,4 and -1,6 linkages. We identified residues that specifically interact with branched -1,6 linkages of glucans. Invitro enzymatic assays and starch binding assays performed for structure guidede mutants of SEX4 proreins revealed important mechanistic information on how different binding platforms of SEX4 employs to recognize starch. Furthur, Hydrogen-Dueterium Mass Spectroscopy (DXMS) and analytical ultracentrifugation data combined with biophysical analysis of SEX4 suggests conformational dynamics of SEX4 upon starch binding. While of clear biological importance, little is known about how glucan phosphatases recognize and interact with the complex starch granule and integrate to degrade starch. This knowledge is important for designing effective phosphatases to harness starch in industrial settings and future biofuel research. Therefore, the objective of this study is to present a novel sedimentation based assay to measure and define the basis for substrate specificity of glucan phosphatase Starch Excess4(SEX4). This study will expand our understanding glucan phosphatases interact within the starch granule. Here we report binding specificities of SEX4 with different components of the starch granule (linear, branched, phophoologisacharides, etc) . We also compare the values obtained from this novel asasy to the values obtained by currently avaiable methods to detect cabohydrate-protein interactions. The dynamic ensemble of structures of proteins accessible under native conditions plays a key role in defining protein function. In the scope of mass spectrometry (MS), ion mobility (IM) MS has gained its popularity in structural biology because it enables to extract collision cross section (CCS) that can be used for modeling proteins and protein complexes. However, extracting information on the size of the ensemble of structures still remains challenging, which is especially true for traveling wave IM (TWIM) devices as such technology currently lacks a theoretical construct that enables the interpretation of IM arrival time distributions (ATDs) that links, quantitatively, to native protein structural ensembles. In this presentation, we present new developments in TWIM theory that enable utilizing ATD peak width to assess the size of structural ensemble by determining conformational broadening parameter () in conjunction with molecular dynamics (MD) simulation to estimate structural ensembles. For the model peptide, Ac-Ala(n)-Lys peptide, with n ranging from 6 to 19, value increases with increasing n with a sharp transition at n=12, which is in agreement with CCS analysis and MD simulation results. Furthermore, by clustering the structures from MD we were able to infer the possible origin of dynamics in our peptide models. Lastly, using unmodified and cross-linked Avidin, we have also observed decrease in for cross-linked systems. Utilizing TWIM ATD peak width we were able to define conformational heterogeneity of biomolecules and we plan to extract such information on intrinsically disordered proteins. High quality recombinant antigen or therapeutic proteins are essential for the drug discovery process. Low expression levels can be a significant bottleneck and delay project progression. This can often be overcome by either scaling up, designing different expression constructs, or switching expression systems, all of which can put a restraint on resources and timelines. Even more problematic are those proteins that are hard to express in a functional form using mammalian, bacterial, or insect expression systems. As an alternative, we looked at cell free expression to quickly produce small quantities of recombinant protein, which can be sufficient for initial target evaluation and in vitro proof of concept (POC) experiments. We tested commercial E.coli derived cell free expression kits due to their reported robustness, high yields, and low cost. As a POC, we expressed and purified the cytokine IL1 and demonstrated comparable functional and biophysical properties to protein derived from E.coli cells. Encouraged by these results, we expressed more challenging proteins and these efforts will be presented as well. Overall, our data suggest that cell free expression can be a valuable tool for recombinant protein production but its use needs to be considered on a case to case basis. The RNA-binding protein TDP-43 is associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia and is found in stress granules, cytoplasmic membraneless organelles that form in response to stress. While the C-terminal domain (CTD) of TDP-43 is predominately low-complexity, it contains a conserved transient helix that is required for phase separation of the CTD in vitro. In this study we investigate the effects of mutations within this region on the secondary structure and self-assembly of the CTD using NMR spectroscopy and in vitro phase separation assays. Specifically, we introduce mutations at or near G335, a potential helix-disrupting glycine, including the ALS-associated mutation G335D, to determine the correlation between helicity and assembly. We find that increased helicity generally correlates with increased self-assembly propensity and phase separation. These data support a helix-helix interaction process with contributions from conformational selection in which intrinsically low helical structure in the region of G335 limits assembly. SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) that depletes cellular dNTPs in non-cycling cells to promote genome stability and to inhibit retroviral and herpes viral replication. In addition to being substrates, cellular nucleotides also allosterically regulate SAMHD1 activity. Recently, it was shown that high expression levels of SAMHD1 are also correlated with significantly worse patient responses to nucleotide analogue drugs important for treating a variety of cancers, including Acute Myeloid Leukemia (AML). In this study, we used biochemical, structural, and cellular methods to examine the interactions of various cancer drugs with SAMHD1. We found that both the catalytic and the allosteric sites of SAMHD1 are sensitive to sugar modifications of the nucleotide analogs, with the allosteric site being significantly more restrictive. We crystallized cladribine-TP, clofarabine-TP, fludarabine-TP, vidarabine-TP, cytarabine-TP, and gemcitabine-TP in the catalytic pocket of SAMHD1. We find that all of these drugs are substrates of SAMHD1 and that the efficacy of most of these drugs is affected by SAMHD1 activity. Of the nucleotide analogues tested, only cladribine-TP with a deoxyribose sugar efficiently induced the catalytically active SAMHD1 tetramer. Together, these results establish a detailed framework for understanding the substrate specificity and allosteric activation of SAMHD1 with regards to nucleotide analogues, which can be used to improve current cancer and antiviral therapies. Isothermal DNA assembly methods like Gibson assembly have become powerful and flexible tools for DNA cloning and engineering applications with several benefits compared to traditional restriction enzyme-based cloning. Simultaneous, seamless joining of multiple DNA fragments over a broad range of fragment length regardless of the presence or absence of restriction sites facilitates rapid construction of single genes, entire plasmids, and more complex genetic pathways. We describe several methods that apply this technology for the introduction of sequence diversity into proteins. These include the generation of single and multiple simultaneous mutations for rational design and other directed engineering approaches, the preparation of gene shuffling libraries from overlapping PCR fragments from a family of related genes, and the construction of multiple site-saturation or semi-random mutant libraries using inverse PCR and bridging degenerate oligonucleotides for directed evolution approaches. Performance data is provided from a study in which these diversitygeneration methods resulted in a significant increase in activity of a thermostable reverse transcriptase enzyme. Finally, we describe a method for rapidly constructing linear integrating DNA fragments for recombinant strain construction using a combination of isothermal assembly of DNA fragments and in vitro rolling circle amplification (RCA). This method requires no E. coli genomic or plasmid sequences and eliminates propagation in E. coli, which facilitates introduction of large sequences or sequences toxic to the intermediate host and can eliminate several days of strain construction time. We demonstrate application of this method for direct construction of a recombinant protein-secreting Pichia pastoris strain using PCR-generated DNA fragments. Sean Cascarina 1 , Eric Ross 1 1 Colorado State University (Fort Collins, United States) While proteins with low-complexity domains (LCDs) or compositionally biased domains (CBDs) continue to emerge as key players in both normal and pathological cellular processes, a unified, in-depth view of the effects of these domains on protein regulation and function is lacking. Therefore, we have developed a pragmatic bioinformatic approach to understand relationships between sequence composition, protein regulation, and protein function. We find that compositional enrichment affects the translation efficiency, abundance, half-life, and cellular functions of proteins on a proteome-wide scale. However, these effects depend upon the type of amino acid enriched in a given sequence, highlighting the importance of distinguishing between different types of LCDs/CBDs. Furthermore, many of these effects are discernible at amino acid compositions below those required for classification as LCDs or CBDs by traditional methods and in the absence of homopolymeric amino acid repeats, indicating that classical LCD/CBD thresholds may not reflect biologically relevant criteria. Application of our analyses to composition-driven processes, such as the formation of membraneless organelles, reveals distinct composition profiles even for closely related organelles. Collectively, these results provide a unique perspective and detailed insights into relationships between amino acid composition, protein metabolism, and protein functions. Molecular motors are unique proteins because they can generate physical power by a chemical catalytic reaction, hydrolysis of ATP. The actuator-like function has attracted many researchers, but the detailed molecular mechanism to produce the power is still obscure because of insufficient structural information. F1-ATPase (F1) is an ATPase domain of FoF1-ATP synthase and functions as rotary molecular motor. Thẽ 50kDa rotor rotates relative to 330kDa stator by ATP-hydrolysis. To reveal the mechanism, we have established analytical systems for recombinant human F1 and bovine F1. Our recent X-ray crystallographic studies of bovine F1 provided several molecular structures at up to 1.66 Å resolution, which included eight rotation interim snapshots for the release of product phosphate (Pi) or ADP. The former interim structures identified stepwise rotors rotation and widening of the Pi-binding pocket during Pirelease. The stepwise displacements in Pi-mimicking waters, arginine finger, and p-loop Lys residues at the pocket induced a global rearrangement of subunits, which led to driving stepwise rotation of the rotor shaft (~20 deg). The sequence of the structural transition was further confirmed by our dynamic time-divided X-ray crystallographic study. In addition, the ADP-releasing intermediate structures unveiled a conformational rearrangement in the catalytic site (especially in p-loop) for ADP-releasing. These results provide the power-generating chemo-mechanical coupling mechanism of F1 motor that the accumulated elastic force in the protein molecule, originally provided by ATP-binding to the protein, is released by the trigger of the Pi-release and converted into the physical power to drive rotation of the rotor shaft. Most Structural Genomics (SG) protein structures deposited in the PDB have unknown or uncertain function annotations. This accumulated structural information represents a tremendous contribution to structural biology and genomics. Still, the addition of accurate functional annotations for SG proteins would add substantial value to this information. Our approach to functional annotation and validation incorporates predicting functional assignments through structure-based computed chemical properties and local structure matching followed by biochemical validation. This research focuses on four superfamilies: Crotonase, Ribulose Phosphate Binding Barrel, 6-Hairpin Glycosidase, and Concanavlin A-like Lectins and Glucanases. We compare two methods using these superfamilies. Our previously published method Structurally Aligned Local Sites of Activity (SALSA) uses Cartesian alignments to develop spatiallylocalized consensus signatures for proteins of known function in each functional family within each superfamily. In contrast, our new method Graph Representation of Active Sites for Prediction of Function (GRASP-Func) uses sets of tetrahedra generated through Delaunay triangulation for each protein structure and groups proteins with matched tetrahedra. Both methods utilize Partial Order Optimum Likelihood (POOL), a machine learning method that predicts the catalytically important residues in each protein structure. While both methods make similar predictions of function for SG proteins within these superfamilies, GRASP-Func performs faster and enables large-scale comparisons and functional assignments within and across superfamilies. Finally, we are able to test these predictions biochemically to confirm function. The goal of this project is to provide a validated approach to functional annotation to enable applications from drug target identification to green chemistry and biofuel production. The specificity and stability of protein/protein interactions is of vital interest to the pharmaceutical and biotechnological industries. Incorporation of metal-binding sites at target protein interfaces may be one approach to improve the specificity and affinity of naturally occurring and designed protein-protein interactions. The main goal for this research was to generate a metal-mediated protein interface and study the effects of interfacial modification on protein-protein affinity. Our approach recently resulted in the creation of a high-affinity, zinc-mediated protein homodimer. Using a metal-templated approach we engineered novel metal binding sites to generate a high affinity interaction using the 1 domain of Streptoccocal Protein G (G1). Metal coordination enables focused surface redesign, allowing for the preservation of naturally occurring or designed interfaces. Incorporation of metal binding sites generates concerted interactions allowing for enhanced affinity and specificity for protein interactions. Our protein complex is driven by metal coordination through histidine sidechains, allowing for a metal-dependent interaction. Complex formation and assembly was evaluating using size exclusion chromatography and x-ray crystallography. To accurately measure the molecular weight of the protein variants we have used size-exclusion chromatography with multi-angle light scattering (SEC-MALS). The G1 monomer and metal-mediated dimers had molecular weight of 6.2 and 12.2-13.1 kDa, respectively. We also report two high-resolution crystal structures of the designed complexes; both in complex with zinc ion at the designed metal coordination sites. Human protein TGIF1 is a transcription factor with essential roles in cell fate determination, and has been implicated in many human diseases including holoprosencephaly (HPE) and cancers. The function of TGIF1 in transcriptional regulation depends on its homeodomain (HD) that binds DNA in a sequence-specific manner. Before this work, the structure and the DNA-binding mechanism of TGIF1 HD were unclear. In this study, the solution NMR structure of TGIF1 HD was determined, and based on this structure, the underlying causes for two HPE cases that contained P192A and R219C mutation in TGIF1 HD respectively were revealed. P192 and R219 were found to play roles in packing 1 and 2 helix to 3 helix together with A190 and F215 through side-chain interactions. P192A and R219C mutations led to structure disturbances of TGIF1 HD, which subsequently markedly reduced the DNA-binding affinity of TGIF1 HD. Further, the 3 helix of TGIF1 HD was determined as the specific DNA-binding interface using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and chemical exchange saturation transfer (CEST) spectroscopy. The results also suggested that the interface underwent a slow exchange between free and specific DNA-bound states at a rate of 130.2 AE 3.6 s-1. Two residues R220 and R221 located in the interface are crucial for the DNA binding and may account for the DNA binding specificity. Our study provides structural insights of the probable pathogenesis mechanism of two TGIF1-related HPE cases, and the structural and dynamic insights into the specific DNA-binding mechanisms of TGIF1 HD. ADP-dependent glucokinase (ADPGK) is an alternative glucose phosphorylating enzyme. In contrast to classical ATP-dependent hexokinases, ADPGK utilizes ADP as a phosphoryl group donor. ADPGKs involvement in modified bioenergetics of activated T cells has been postulated and elevated ADPGK expression has been reported in various cancer tissues. However, the physiological role of ADPGK is still poorly understood and effective ADPGK inhibitors still await discovery. In the first part of our work we show that 8-bromo substituted adenosine nucleotide inhibits human ADPGK. By solving the crystal structure of archaeal ADPGK in complex with 8-bromoadenosine phosphate (8-Br-AMP) at 1.81 Å resolution we identified the mechanism of inhibition. We observed that 8-Br-AMP is a competitive inhibitor of ADPGK and that the bromine substitution induces marked structural changes within the proteins active site by engaging crucial catalytic residues. In the second part, we present a crystal structure of archaeal ADPGK from Methanocaldococcus jannaschii in complex with an inhibitor, 5-iodotubercidin, d-glucose, inorganic phosphate, and a magnesium ion. The crystal structure shows how the phosphate ion, while mimicking a -phosphate group, is positioned in the proximity of the glucose moiety. In addition, we demonstrate that 5-iodotubercidin inhibits human ADPGK-dependent T cell activation-induced reactive oxygen species (ROS) release and downstream gene expression. The structural insights presented herein provide a critical basis for rational development of novel ADPGK inhibitors. The Nucleotide Excision Repair (NER) pathway requires the coordination of a myriad of proteins in order to recognize and excise the bulky lesions from damaged DNA. XPA, a central scaffolding protein in NER, is recruited to the damage site after the DNA duplex is unwound, where it strongly binds ss/dsDNA junctions. In the NER complex, this DNA binding occurs in the context of additional contacts with other core NER factors, including, but not limited to, the eukaryotic single-stranded binding protein, RPA, which binds and protects the undamaged strand. The interaction of RPA and XPA is essential for NER activity, however, structural data defining the nature of these interactions is currently lacking. To map specific interactions between these two proteins, Small-angle X-ray scattering (SAXS) and NMR spectroscopy were used to build a computational model of the RPA-XPA complex within the Rosetta protein modeling suite. A homology model of the DNA binding domain of XPA was first built from the published crystal structure of the yeast XPA homologue Rad14 bound to DNA. This XPA model was then docked using the Rosetta docking protocol with the crystal structure of the tandem high affinity ssDNA binding domains RPA70AB bound to dC8, employing scattering data as a constraint during the simulations. The final model reveals a well-packed complex with the significant binding surface stabilized by a combination of polar and non-polar interactions. Mutations in the interface are being designed to validate the model and test the functional role of this XPA-RPA contact in NER. Effective management of weedy species in agricultural fields is essential for maintaining crop yields. The introduction of genetically engineered (GE) crops containing herbicide tolerance traits has been a successful tool available to control weeds. FT_T (FT = FOPS/2,4-D tolerant) is an optimized form of RdpA which when over-expressed in crops can oxidize and provide protection from herbicides such as 2,4-D and Quizalofop. Development of this herbicide tolerance trait employed an enzyme engineered with specific enzymatic activity for these two herbicide families. This engineering effort utilized a microbialsourced dioxygenase scaffold to generate variants with improved enzymatic parameters. Additional optimization to enhance in-plant stability of the enzyme enabled an efficacious trait that can withstand the temperature conditions often found in field environments. To address the substrate specificity of engineered enzyme, a list of plant based compounds was generated from a public database and supplemented by compounds tested in the literature as well as ten known herbicidal substrates of RdpA. These compounds (approximately 1200) were computationally screened for structural similarity to 2, 4-D. We derived a similarity threshold from the structures of the known herbicidal inhibitors. The list was further refined using a docking model based on shape and binding energy and the resulting compounds were tested in two FT_T enzymatic assays (LC-MS and coupled) for activity. SERINC3 is one of the newly discovered SERINC proteins that potently inhibits viral infectivity and is antagonized by HIV-1 Nef. Antagonism of SERINC3 by HIV-1 Nef depends on clathrin adaptor protein 2 (AP-2), suggesting that Nef hijacks AP-2 and promotes the internalization of SERINC3 via clathrinmediated endocytosis. While recent studies have identified determinants in SERINC5 required for Nef antagonism, little is known about how SERINC3 interacts with AP-2 and/or Nef. Our previous data show that a loop region of SERINC3, when phosphorylated, binds tightly to the C-terminal domain of the subunit of AP-2 (2CTD) even in the absence of Nef. We have further demonstrated that two serines in the loop are important for tight binding to 2CTD. To probe the binary interaction between SERINC3 and 2CTD, we crystallized and solved the complex structure to 2.1 A resolution. We observed electron density, likely from part of the SERINC3 loop, in the tyrosine-binding pocket of 2CTD, which typically only binds peptide motifs of the form Yxx (where represents a bulky hydrophobic residue). Intriguingly, the side chain density of the peptide in this pocket is considerably smaller than would be expected for a bulky, aromatic tyrosine. This suggests a potential non-tyrosine-based SERINC3 peptide was captured in the tyrosine-binding pocket, and that this peptide may represent a non-canonical motif that binds to 2CTD. These results broaden our understanding of the functional repertoire and structural plasticity in clathrin-mediated endocytosis. Expanding the catalytic capabilities of enzymes beyond native function is of considerable interest to chemists and biologists alike. An efficient and powerful strategy to design novel abiotic activity in enzymes is through the use of artificial cofactors. Enzymes bearing artificial cofactors often possess activity for abiotic reactions that can be optimized through mutation of the protein host. Towards the goal of expanding non-natural enzymatic activity, a cytochrome P450 was engineered to selectively incorporate the artificial metallocofactor, Ir(Me)deuteroporphyrin IX, in lieu of heme, in bacterial cells. Cofactor selectivity was altered by introducing mutations within the hemebinding pocket to discriminate the deuteroporphyrin macrocycle, in combination with mutations to the P450 axial cysteine to accommodate a pendant methyl group on the Ir(Me) center. This artificial metalloenzyme was investigated for activity in nonnative metallocarbenoidmediated olefin cyclopropanation reactions and showed enhanced activity for aliphatic and electrondeficient olefins when compared to the native heme enzyme. This work provides a general strategy to augment the chemical functionality of heme enzymes in cells with application towards abiotic catalysis. Andreia Serra 1 , Andreia Serra 1 , Andreia Monica Serra 2 , Christopher Delgado 2 , Patrick Rodriguez 2 , Emanuele Gabellieri 2 , Francesca Capotosti 2 , Heiko Kroth 2 , Chary Nampally 2 , David Hickman 2 , Andreas Muhs 2 , Andrea Pfeifer 2 1 AC Immune (Lausanne, Switzerland); 2 AC Immune S.A. (Lausanne, Switzerland) Background: Brain deposits of aggregated Tau protein are a common pathological feature of several neurodegenerative disorders including Alzheimer's disease (AD). As the occurrence of such abnormal deposits has been shown to correlate well with cognitive decline in AD, the identification of compounds which bind to, or interfere with the Tau aggregation process are sought for use as PET tracers or as therapeutic agents. We have developed a label-free assay using Surface Plasmon Resonance (SPR) for the characterization of small molecules binding to Tau aggregates. Methods: Using Biacore 8K, several SPR assay conditions were tested including multiple sensor surface matrix (CM3, CM5 and CM7), different ligand densities and buffers (PBS, PBS-P and HBS), ligand capture strategies (with or without antibody), as well as a variety of capture antibodies (Tau13, Tau46 and MC1). Once the assay was optimized, a total of seven compounds (Cpd1-Cpd7) were evaluated for their binding affinities (KD) to full length Tau aggregates (flTau). Results: The optimized assay set-up included a CM3 sensor surface, PBS buffer and use of MC1 antibody for the capture of the Tau aggregates. From the SPR binding affinity assay, four compounds were found to show a KD below 25 nM. Conclusions: Overall a straight-forward label-free SPR assay was developed which enables the identification and selection of compounds capable of binding to flTau aggregates. Such assay can be essential in the development of diagnostic or therapeutic compounds for Tauopathies such as AD. Thioesterase superfamily member 2 (Them2) is a member of the acyl-CoA thioesterase protein family and it can hydrolyze various fatty acyl-CoAs into free acids plus CoA. Them2 is enriched in liver and oxidative tissues, such as brown adipose tissue (BAT). It functions to control adaptive thermogenesis in BAT. In the setting of over-nutrition, Them2 can promote the gluconeogenesis, whereas knockout of Them2 can protect liver against steatosis. Therefore, Them2 is a valuable target for the management of hepatic steatosis and insulin resistance. Here we show the 2.25 Å high resolution crystal structure of Them2 in apo state. It forms a homotetramer, consistent with results obtained from solution studies. Overall, Them2 folded as the classical hotdog domain, comprised of a six antiparallel -strands that wraps around hydrophobic -helixes. High throughput screening was utilized to discovery small molecule inhibitors from diverse libraries targeting Them2. Several hits were identified and currently being examined by differential scanning fluorimetry and isothermal titration calorimetry to validate direct binding. We have also developed a NanoLuc-based protein-fragment complementation assay (NanoPCA) to identify potential interaction partners of Them2. Several lipid shuffling proteins, including PC-TP, have been identified and will be employed to dissect the roles of Them2 in lipid metabolism under distinct nutrition conditions. Production of Subunit Vaccine Candidates Against Bovine Respiratory Disease Pathogen Mannheimia Haemolytica as an Alternative to Antimicrobials inhibit human islet amyloid polypeptide (hIAPP) aggregation in the presence and absence of lipid membranes. In this study we show the effects of CurDAc on three different amyloidogenic peptides: hIAPP, amyloid beta (A) and human calcitonin (hCT). Using commonly employed biophysical techniques; we show that CurDAc has preferential activity on hIAPP over A and hCT in both inhibition of growth and disaggregation of fibers. NMR spectroscopy was used to characterize hIAPP monomers and disaggregated fibers in the presence of CurDAc. Cell toxicity experiments show that CurDAc is a non-toxic compound but that both monomers and fibers treated with CurDAc are more toxic than hIAPP monomers and fibers alone, suggesting that the small molecule stabilizes an increased toxic species. This work shows the potential of CurDAc to be used as a chemical probe to study hIAPP and suggests the mechanism of action is sequence specific. Cytochrome P450s (cytP450s) are a ubiquitous superfamily of enzymes that are responsible for the catalysis of many different substrates including over 75% of the drugs on the market. In order to do this, each turn of the catalytic cycle requires two electrons which are donated by either Cytochrome P450 Reductase (CPR) or cytochrome b¬5 (cytb5). We aim to understand how CPR and cytb5 compete for binding to cytP450 and to unravel the structural and dynamic basis for this competition and how the presence of a lipid membrane environment and various substrates impacts these interactions. Here we use NMR spectroscopy and other biophysical tools to characterize the interplay between this ternary complex of cytP450, CPR, and cytb5. Our data reveals how substrates differently modulate the interaction between the two redox partners and cytP450 by enhancing the preference of cytP450 for one redox counterpart over the other. This work starts to define a complete picture of how this ternary complex works together to metabolize various substrates. Papain-like protease (PLpro) is a multifunctional enzyme encoded in the RNA genome of the infectious Middle East respiratory syndrome (MERS) coronavirus. PLpro acts as a protease to cleave the polyprotein for viral replication and as a deubiquitinating (DUB) and deISGylating (deISG) enzyme for suppression of the innate immune response. For these latter two activities, it is unclear if either or both DUB/deISG activities are necessary for this function. In this study, we determine the structure of MERS PLpro in complex with full-length ISG15 to 2.3 Å resolution. The structure reveals the PLpro-ISG15 interaction interface where PLpro only contacts the C-terminal domain of ISG15. Using this structure as a molecular guide, we designed different PLpro variants with altered substrate specificities, disrupting either or both DUB/deISG activities by targeting residues far away from the active site. Using fluorescence-based assays, we assess each PLpro mutant for protease, DUB, and deISG activity relative to the wild-type. Results show that inserting a positive charge into the hydrophobic pocket of ISG15 or near an Arg residue in Ub completely impairs both DUB/deISG activities. By manipulating an Arg-rich region, we are also able to selectively impair or enhance DUB activity of PLpro. Overall, our study provides functional tools to delineate the importance of DUB versus deISG activity in virus-infected cells as well as potential candidates for attenuating the MERS virus in vivo for vaccine design. Obstructive sleep apnea (OSA) affects up to 24% of the adult population and is associated with several atrial diseases. About 20% of adults have mild OSA and 7% have moderate to severe OSA, with 85% of patients remaining undiagnosed. Although clinical evidence linking OSA to proarrhythmaic atrial changes is well known, the molecular mechanisms by which OSA causes atrial disease remain elusive. To study the OSA-induced cardiac changes, we have implemented a recently developed rat model which closely recapitulates the characteristics of OSA. Rats, aged 50-70 days, received surgically implanted tracheal balloons which were inflated to cause transient airway obstructions. Apnea groups experienced 60 apneas per hour of either 13 seconds (moderate) or 23 seconds (severe) for 2 weeks. Control rats received surgeries but no inflations. Proteomics analysis was done on the rat atria homogenates to identify dysregulated proteins in moderate and severe apnea when compared to control. Both 1-dimentional (1-D) and 2-dimentional (2-D) electrophoresis was performed to separate the proteins and the peptide mixtures. The trypsin digested spots were analyzed by a Nano-Acquity UPLC coupled with Xevo G2 Mass Spectrometer. The proteomics analysis revealed that 3 of the 9 enzymes in glycolysis and 2 proteins related to oxidative phosphorylation were down-regulated in the severe apnea group. In contrast, several structural and pro-hypertrophic proteins were up-regulated with chronic OSA. The data suggests the chronic OSA causes proteins changes which lead to cessation of glycolysis, a diminished capacity to generate reducing equivalents (i.e. NADH) as well as promotion of cardiac hypertrophy. High-throughput experimental techniques have made possible the systematic sampling of the single and double mutation landscape for many proteins. The previous observation that epistatic residue pairs often are in contact in the 3D structure led us to the hypothesis that a systematic double mutant screen may contain sufficient information to discover the 3D fold ab initio. Using the epistasis scores from a mutational scan performed on GB1 by Olson et al., we predict contacts with an 86% true positive rate. Folded models generated from top-ranked epistatic pairs were accurate within 2.4 Å. We also show that only a fraction of double mutation effects is needed to achieve a similar accuracy as the full dataset, suggesting the approach could be used to determine larger proteins that would otherwise be prohibitive experimentally. These results suggest a new experimental approach for determining protein structure. Phenazine M (PhzM) is a class I methyltransferase essential for the biosynthesis of pyocyanin, a virulence factor from Pseudomonas aeruginosa. It is currently an unexplored drug target for treatment of resistant bacteria that cause a large number hospital infections leading to death. Although a crystal structure of the apo protein is available (PDB 2IP2), drug design efforts towards this target have been hampered by the lack of structural information on how the cofactor (SAM) binds in the active site. Here we report the x-ray crystal structure of phzM in complex with SAM solved to a resolution of 2.2Å. Comparison of the apo and holo forms of phzM shows a small RMSD of 0.275 between the two crystal structures. The holo structure shows minor hinge movement in domain 1. SAM interacts with Asp 198, Asp 225, Met 226, Ser240, Arg241 and a crystallographic water molecule. The Arg 241 side chain, which takes on two conformations in the apo structure, is found in a single conformation in the holo protein, where it forms a salt bridge with the carboxylic acid group of the SAM molecule. Furthermore, Asp198 is rotated by approximately 90 degrees about 1 in the holo relative to the apo structure, optimizing H-bonding interaction with the SAM ribose ring. Given the location to which the methyl group must be transferred and the positioning of specific functional groups within the active site, the Phenazine-1-carboxylate (PCA) substrate location can be predicted by docking due to its relatively few degrees of freedom. With the rapid growth of computer power, long-time molecular dynamics simulations are reachable. How to effectively analyze the simulation data to extract thermodynamics and kinetics properties becomes a bottleneck nowadays. We have developed a reaction-path algorithm to obtain path information from simulations. Our method combines the MaxFlux algorithm with the graph theoretic approach to locate the shortest folding pathways. We demonstrated this approach by analyzing the long trajectories of Trp-cage provided by the D. E. Shaw group. Paths were extracted without projecting the conformations onto the reduced dimensions. Two typical folding pathways have been identified from the paths, characterized by the order of formation of -helix and hydrophobic core. The changes of radius of gyration, secondary structure, solvent accessible surface area, and salt bridge formation along the paths were also investigated to reveal the folding mechanism. This promising method can be generally applied to analyze other biomolecular simulations. Monoclonal antibodies (mAbs) are the predominant biopharmaceuticals to treat various diseases. During recombinant mAb manufacturing and storage, some degradative modifications seem to be inevitable, including methionine oxidation, asparagine deamidation and aspartic acid isomerization. These degradations may affect the efficacy and stability of the drug and cause adverse effects such as immunogenicity. Therefore monitoring these modifications are crucial to ensure the quality of therapeutic antibodies. Here an oxidation study of an IgG2 molecule will be presented. A weak anion exchange chromatography (WAX-HPLC) method was developed to analyze the charge heterogeneity of an IgG2 antibody and resolved four peaks. The peaks were separately collected, concentrated, and characterized by subunit and peptide map LC-MS analysis. Upon heat stress and hydrogen peroxide (H2O2) treatment, an increase of the basic peak that elutes earlier than the main peak was observed and confirmed to be oxidized species. Peptide map LC-MS of the H2O2 treated materials also determined the oxidation sites in this IgG2. As this WAX-HPLC method is capable of separating oxidized species from the native form, its a good alternative for the lengthy subunit and peptide mapping methods to monitor the oxidation. Protein sequence space is vast; nature uses only an infinitesimal fraction of possible sequences to sustain life. Are there answers to biological problems other than those provided by nature? Can we create artificial proteins that sustain life? To address this question, the Hecht lab has created combinatorial collections, or libraries, of novel genes with no homology to those found in living organisms. These libraries were subjected to screens and selections, leading to the identification of sequences with roles in catalysis, modulating gene regulation, and metal homeostasis. However, the functional proteins formed dynamic, rather than well-ordered structures. This impeded structural characterization, making it difficult to ascertain a mechanism of action. To overcome this issue, the current work presents a new strategy for library design based on the de novo protein S-824, a fourhelix bundle with a very stable three-dimensional structure. Unlike previous libraries, this one varies a region at the top of the bundle to create a cavity and potential active site. Characterization of S-824 mutants revealed that the structure tolerates amino acid substitutions and is likely a suitable template for library design. A DNA library with an expected diversity of 10^6 was assembled from degenerate oligonucleotides and is being screened for a range of activities. Altogether, this approach represents a step towards creating artificial proteomes capable of carrying out essential biological roles. Conformational transitions are fundamental to the function of many proteins, such as signaling proteins that convert between an enzymatically active and downregulated forms, membrane proteins that transport molecules via open/closed forms, and molecular machines that couple chemical energy to molecular motion. Many conformational transitions are between states with disparate functionality and often tightly regulated for proper control of cellular processes, which highlights the importance of studying the transition processes. Computational methods are valuable for elucidating such transitions in atomistic detail not achievable by experimental observation. The timescales of the transitions are typically longer than can be adequately sampled with current unbiased molecular dynamics (MD) simulations. Enhanced sampling methods are therefore required to overcome the freeenergy barriers that separate different protein conformational states. Adaptively biased path optimization (ABPO) is an approach to optimize conformational transition pathways by constructing the adaptive biasing potential in terms of a one-dimensional path in a reduced-variable space. ABPO allows free sampling of each replica along the path, distinguishing it from other enhanced sampling methods. Here, the application of ABPO is extended to an all-atom description of the protein systems. Our results suggest that ABPO works efficiently to converge an optimized transition pathway using an all-atom description of the protein systems. The reduced variable evolvement during the optimization cycles are analyzed, and the structures on the transition path are extracted to visualize the conformational activation mechanisms. Narutoshi Kamiya 1 , Benson Ma 2 , Gert-Jan Bekker 3 1 University of Hyogo (Kobe, Japan); 2 Georgia Institute of Technology (Atlanta, United States); 3 Osaka University (Osaka, Japan) Antibodies, which consist of heavy and light chains, bind to antigens with high specificity and affinity. Each chain has complementarity-determining region (CDR) loops, which play an important role for antigen binding. Single domain antibodies, sdAbs, function like regular antibodies, however consist of only one chain. Because of their low molecular weight, sdAbs have advantages with respect to production and delivery to their targets. For applications such as antibody drugs and biosensors, an sdAb with a high thermal stability is required. In this work, we chose two sdAbs and two engineered antibodies consisting of only the heavy chain, which have a wide range of melting temperature (Tm) values and have known structures. We then applied molecular dynamics (MD) simulations to these four antibodies to estimate their relative stability and compare them with the experimental data. High temperature MD simulations at 400 K and 500 K were executed with simulations at 300 K as control. Ten 100-ns simulations were carried out for each antibody and temperature to increase statistics. Root-mean-square-deviations against the initial crystal structures did not exhibit a good correlation with the experimental Tm. However, the fraction of native atomic contacts, Q, showed a fair correlation, and so the Q values classified by hydrophobicity and size were subsequently analyzed. Interestingly, the Q value between hydrophilic residues, which are not always located at the CDR loops but also at other regions, exhibited good correlation with the experimental data, suggesting that interactions between hydrophilic residues affect the thermal stability. Streptococcus pneumoniae is a major infectious agent responsible for pneumonia, otitis media, sepsis and meningitis. Pneumococcal surface protein A (PspA) is a well-characterized virulence factor localized on the surface and a target for vaccine development. Pep27, a pneumococcal secreted peptide, initiates pneumococcal autolysis, thereby constituting a major virulence factor. Although a few antisera recognizing PspA and Pep27 have been reported, no monoclonal, well-characterized antibodies for PspA and Pep27 have been developed. In this study, we screened three single-chain antibody variable fragments (scFvs) using phage display from a human synthetic library to select clones 2B11 for PspA and E2 and F9 for Pep27. Dissociation constants (Kd) of 2B11, E2 and F9 were measured to be 5 nM, 1.1 M and 0.5 M, respectively. All three clones showed no cross-reactivity towards other pneumococcal and unrelated proteins. The epitope on PspA and Pep27 were localized to residues 231-242 for PspA and to residues 24, 26 and 27 for Pep27 by mutational analysis. Molecular docking analysis supported the experimentally investigated epitopes on both PspA and Pep27. Comparison of 2B11 with a commercial PspA polyclonal antibody revealed that 2B11 exhibited a better specificity towards recombinant PspA antigen. Whereas 2B11 was capable of detecting endogenous PspA from pneumococcal lysates with affinity similar to that of the commercial antibody, E2 and F9 specifically detected Pep27 in an environment mimicking in vivo conditions, demonstrated in human serum. The scFv clones characterized here represent molecular tools for the detection of pneumococcal diseases with potential for further improvement in affinity. Among the bacteria associated with the human gut microbiome (HGM) the sulfate reducing bacteria (SBR) respire anaerobically using sulfite as a terminal electron acceptor, releasing sulfide. SRB have garnered recent interest for their implication in Irritable Bowel Syndrome (IBS), as the sulfide they release can damage the colonic mucus lining. To better understand the etiology of IBS we must better understand how SRB harness sulfite from the HGM. Sources of sulfite comprise several organosulfonates, including isethionate that is believed to be produced by the HGM through deamination of the abundant metabolite taurine. Although isethionate-sulfite lyase activity was anticipated based on in vivo studies, the corresponding enzymes had been unknown, hampering efforts to inhibit pathological overgrowth of SRBs. Transcriptomic analysis and biochemical characterization performed in the Schleheck and Balskus labs, respectively, have identified isethionate-sulfite lyase enzymes of the glycyl radical enzyme (GRE) superfamily. To better understand the molecular mechanism of this enzyme we solved a crystal structure of the Bilophila wadsworthia enzyme. Surprisingly the active site differs markedly from what is predicted by sequence alignments to other structurally characterized GREs, but maintains essential catalytic features expected of GREs including a cysteine residue, a glycine residue, and a glutamate residue. This structure also reveals two unanticipated arginine residues in the active site that appear to be in a suitable conformation to coordinate the sulfate group of the substrate isethionate. Structural characterization of the enzyme substrate complex is being pursued to elucidate how SRB harness sulfite from a metabolite in the HGM. The nucleolus is an active liquid-like membrane-less organelle that functions as the site for ribosome biogenesis, and a signaling hub for cell cycle regulation and cellular stress responses. The nucleolus contains over 700 ribosomal and non-ribosomal proteins, and several types of RNA. While functions of ribosomal proteins and some non-ribosomal proteins (e.g. Fibrillarin and NPM1) are known, the roles of many other nucleolar proteins are poorly understood. SURF6, an essential non-ribosomal nucleolar protein, promotes ribosome biogenesis and cell proliferation when overexpressed; however, its functional mechanism is unknown. Human SURF6 is intrinsically disordered and contains multiple arginine-rich motifs (R-motifs). SURF6 directly interacts and co-localizes with NPM1 in the nucleolus. NPM1 is a pentameric protein composed of an oligomerization domain, an intrinsically disordered region with two acidic tracts and two basic tracts, and a C-terminal DNA/RNA binding domain. Multivalent R-motifs of SURF6 interact with acidic tracts of NPM1, mediating liquid-liquid phase separation. Interestingly, aside from heterotypic scaffolding NPM1:S6N interactions, competing homotypic scaffolding NPM1:NPM1 interactions are also active in these droplets. Self-association of NPM1 occurs through interactions between the acidic and basic tracts. Here, we show that the composition and physical properties of NPM1:S6N droplets are modulated by competition between the two scaffolding mechanisms, which dynamically and seamlessly adapt to changes in molecular crowding and fluctuations in protein concentrations. We propose modulation of NPM1-dependent nucleolar scaffold by R-motif containing proteins, such as SURF6, as a mechanism for controlling the directionality of the ribosomal biogenesis process within the liquid-like nucleolar microenvironment. Oculocutaneous albinism (OCA), an autosomal recessive disorder, is caused by mutations in human tyrosinase (Tyr) and tyrosinase-related protein 1 (Tyrp1) leading to OCA1 and OCA3, respectively. Currently, the intra-melanosomal domains of both enzymes were successfully purified and characterized. Here, we purified recombinant full-length Tyr and Tyrp1 and used them to investigate this intricate system of various chemical reactions coupled to enzymatic reactions. Recombinant Tyr and Tyrp1 were expressed in Trichoplusia ni larvae and purified using Immobilized Metal Affinity Chromatography (IMAC) and Size-Exclusion Chromatography (SEC) in the presence of 1% Triton X-100. Protein identity was confirmed using specific antibodies and mass spectrometry. The oligomeric and conformational states of both proteins were accessed using sedimentation equilibrium and tryptophan fluorescence, respectively. The SEC profiles and sedimentation equilibrium, confirmed a monomeric state for both Tyr and Tyrp1, at approximately 62 kDa and 64 kDa, respectively. Both proteins show oxidase activity yielding indole-2-carboxylic acid-5,6-quinone (IDCA) from dihydroxyindole-2-carboxylic acid (DHICA) in the presence of 3-Methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH). While only Tyr shows hydroxylase activity of Ltyrosine and oxidase activity of L-3,4-dihydroxyphenylalanine (L-DOPA) yielding dopaquinone. Although they share over 40% of sequence homology, both proteins fail to exhibit the same behavior and may be attributed to developmental functions within the melanosome. Moreover, pure protein remains pivotal for the search of suitable activators of mutant variants and enzyme replacement therapy in a treatment of genetic disorders, such as oculocutaneous albinism. Amyloid light chain (AL) amyloidosis, caused by the aggregation of antibody light chains (LCs), is the most commonly diagnosed form of systemic amyloidosis. Progressive organ damage is caused by amyloid deposition and proteotoxicity. Only some LCs cause amyloidosis, while others are efficiently excreted even when produced at high levels. Finding the variables that determine whether a particular LC will cause amyloidosis could lead to improved diagnosis and therapy. LCs consist of two immunoglobulin domains, an N-terminal variable domain and C-terminal constant domain. We observed that full-length LC dimers, the dominant secreted species, are extremely resistant to aggregation under conditions where their constituent variable domains readily form amyloid. Specific proteolysis between the two domains can release the variable domain which can go on to form amyloid. The rate of this proteolysis depends on a LCs kinetic stability, and we showed that amyloidosis-associated LCs are less kinetically stable than other LCs. This suggests that pharmacological stabilization of amyloidogenic LCs could reduce proteolysis and amyloidogenesis, thus potentially benefitting patients. However, LCs lack an obvious ligand binding site that could be targeted for rational drug design. We therefore developed a high throughput assay for LC stabilization based on proteolytic release of fluorescent dye from specifically labeled LC. Screening identified small molecules that protect LCs from proteolysis and unfolding by urea. These molecules bind with micromolar affinity to LC dimers at the interface between the variable domains. Our work provides proof-of-principle for the use of stabilizing small molecules to treat AL amyloidosis. Dyneins, ATP fueled cytoskeleton motor proteins, play essential role in many biological processes, including ciliary beating, cell division and intracellular transport. They interact with microtubules, made of -and -tubulins, via microtubule binding domains (MTBDs). Here we computationally investigate the role of Ehooks, which are intrinsically disordered regions (IDRs) at the C-terminus of both -and -tubulins, on the MTBD binding. We analyzed the E-hook-MTBD contacts as a function of MTBD-microtubule distance. Several observations were made: (a) the E-hooks constantly grab and release MTBD instead to be permanently bound to it; (b) the E-hooks binding to MTBD is similar to induced-fit mechanism, and E-hooks shift the populations of bound-unbound conformations according to the number of contacts; (c) at different distances, different E-hooks residues bind to MTBD; and (d) the intrinsic conformational changes on MTBD depend of the distance to microtubule and number of contacts with E-hooks, and the MTBD becomes more rigid as the number of contacts increases and distance to microtubule decreases. Complex of phenolic compounds and proteins lead to changes in their structure; which is related to function and nutritional properties. To study those changes in structure, the aim of this work was to measure modifications of a complex of Trypsin from bovine pancreas (Try) with epigallocatechin gallate (EGCG). The successful synthesis of Try-EGCG conjugate was confirmed by spectroscopic and thermal analyses; using ultraviolet (UV), Fourier transform infrared spectroscopy (FT-IR), proton nuclear magnetic resonance (H-NMR), electron spectroscopy for chemical analysis (XPS) and calorimetry (DSC) respectively. The covalent modifications of Try with (EGCG) by a free radical induced as a green grafting method, which was carried out by a redox pair (ascorbic acid/hydrogen peroxide) as the radical initiator in aqueous media. The EGCG concentration covalent binding into the Try molecule, was quantified by Folin-Ciocalteu method. Additionally, the antioxidant and antibacterial of pure EGCG, Try and Try-EGCG conjugate were evaluated. The spectroscopic results indicated the EGCG-Try conjugate obtaining and DSC data indicate an increase in thermal stability of Try in conjugate (p<0.05). Results showed that the covalent binding EGCG amount into the Try molecule was 387.11AE83.01 nmol/mg. In addition, the antibacterial and antioxidant activity of EGCG-Try conjugate were higher than the control Try (p<0.05). According with the last, we can demonstrate the antioxidant function of a protein-antioxidant complex can lead to make further characterization of the complex itself. Producing recombinant proteins can generate misfolded proteins that accumulate in intracellular aggregates, known as inclusion bodies (IB´s). The expression of recombinant trypsinogens in Escherichia coli typically leads to the formation of IB´s. In our work the generation of recombinant trypsin I from Monterey sardine (Sardinops sagax caerulea) (TMSI) was held by overexpression as a fusion protein of trypsinogen with thioredoxin, were the 45 kDa band, corresponding to the fusion protein, was obtained as IB´s, according with this, the aim of this work was to increase the yield of TMSI using N-Lauroylsarcosine sodium salt (sarkosyl). Three percentages of sarkosyl were tested including 0.5,1 and 2% (p/v) once the inclusion bodies were solubilized and obtained from 250mL of cell culture (0.4 g of pellet). Were using 2% (p/v) of sarkosyl showed a remarkable band in the soluble fraction in comparison with the other percentages used and as with the absence of the sarkosyl in the procedure; giving a recovery yield of the soluble form of TMS I of 45%. The biochemical and biophysical characterization of TSMI could have many applications as it can be use to improve functional properties such as solubility, emulsification and gelling properties of food proteins, produce protein hydrolysates and bioactive peptides used in infant formulas and for people with health problems such as hypertension, reduce the concentration of allergens in foods, among others. There are over 2 million cases of bacterial resistant infections per year in the United States resulting in 23,000 deaths. Most antibiotics, like penicillin, function by targeting elements of bacteria that are not present in higher organisms, such as the cross-linking of the peptidoglycan cell wall. Most antibiotics have been overprescribed and lost their effectiveness, so new targets need to be identified that can inhibit or prevent the spread of infection. Our lab has identified the transcription factor, Sigma-28 (28), as a novel potential target for antibiotic development. Sigma-28s primary role is to initiate transcription of the flagella tail proteins in most bacterial species. We hypothesized compounds that inhibit Sigma-28 will impair flagella formation in the bacteria. A high throughput cell-based assay was developed to screen for potential inhibitors. A reporter system containing the Sigma-28 promoter followed by a fluorescent protein was developed to measure promoter activity. Increased Sigma-28 function stimulates production of the fluorescent protein while compounds that inhibit Sigma-28 result in decreased fluorescence. Over 100,000 potential inhibitors were computationally screened and 420 were identified as potential candidate inhibitors. We have screened 320 compounds to date and have identified 20 compounds that inhibit Sigma-28 activity. Further testing is ongoing with these compounds to determine their effective concentrations. 16 Nucleoside diphosphate kinase (NDK) is an enzyme that has a key role in DNA replication as catalyzes the phosphorylation of nucleotides diphosphates to generate nucleotides triphosphates (NTP´s). NDK has been described in the white leg pacific shrimp as a housekeeping enzyme expressed during the infection with the while spot syndrome virus (WSSV). In this study, NDK was obtained as a recombinat protein and its specific activity was measured towards deoxynucleotides diphosphates dGDP, dADP, dTDP and dCDP. NDK activity was 19, 344 U/mg of protein using dTDP as substrate while the lower activity was detected with dGDP. The affinity toward each substrate was determined by isothermal titration calorimetry (ITC) where the data were in the micro-molar range, except for dCTP which had no detectable interaction by this method. The quaternary structure, obtained by size-exclusion chromatography, of the L. vannamei NDK corresponds to a trimer. Crystals of NDK as a binary complex with dGDP were obteined using the macrobatch method, and these crystals are essential for the determination of three dimensional structure of the protein in further studies. Further work may be directed to test if NDK could phosphorylate nucleosides analogs with the aim of blocking the WSSV. Protein transduction domains (PTDs) or cell penetrating peptides (CPPs) have been shown to promote the delivery of therapeutic proteins or peptides into living cells. In previous study, we showed that the double mutant of TCTP-PTD 13 designated TCTP-PTD13M2 was more effective for the delivery of insulin through the nasal membrane than wild TCTP-PTD 13. In this study, we investigated whether modified TCTP-PTDs such as TCTP-PTD M1, M2, and M3, can be also applied to the nasal delivery of exendin-4. Nasal co-administration of TCTP-PTD 13M2 with exendin-4 showed the highest increase of exendin-4 uptake among three TCTP-PTD 13 analogues in normal rats and also decreased more blood glucose levels in diabetic mice without damaging nasal membrane. In conclusion, nasal co-administration of TCTP-PTD 13M2 and exendin-4 showed the best hypoglycemic effect of exendin-4. These results suggest that the modified TCTP-PTD might be universally used for nasal peptide drug delivery. Blood coagulation factor VIII (FVIII) is a non-enzymatic protein cofactor which plays a crucial role in the formation of a stable blood clot. Absence or deficiency of FVIII results in the bleeding disorder hemophilia A. Unfortunately, approximately 30% of patients receiving replacement FVIII generate pathologic anti-FVIII inhibitory antibodies. These inhibitors both reduce the efficacy of the FVIII therapeutic and increase the severity of hemophilia A symptoms. We report the determination of the molecular structure for Et3i, a next-generation human/porcine chimeric FVIII protein for hemophilia A therapy. At 3.2 Å resolution with a Rwork of 0.2146 and Rfree of 0.2879, this will be the highest resolution structure of FVIII to date and will be of substantial interest to the hematological community. Furthermore, an improved model of human FVIII with a Rwork of 0.2655, and Rfree of 0.2895 based on previous 3.7 Å data has been constructed. These models feature more robust geometry and improved amino acid register assignment. Lastly, progress has been made towards the structural determination of the inhibitory antibodies M6143, 2A9, and B136 in complex with the C1 domain of human FVIII. Details of these interactions could inform the future development of less immunogenic hemophilia A protein therapeutics. Blood coagulation factor VIII functions as a cofactor in the blood coagulation cascade for proteolytic activation of factor X. During coagulation, factor VIII is proteolytically activated by thrombin and binds to phosphatidylserine of activated platelet surfaces in coordination with factor IXa to form the intrinsic Xase complex. The C1 and C2 domains of factor VIII are responsible for binding to activated platelet membranes. During replacement therapy for hemophilia A patients, approximately 30% of severe hemophiliacs develop an inhibitory immune response to the therapeutic factor VIII. These inhibitory antibodies inhibit coagulation activity of the therapeutic protein and basal factor VIII. Several of the inhibitory antibodies recognize epitopes on the C1 and C2 domains of factor VIII. Previous structural and phospholipid binding studies indicate the classical monoclonal anti-C2 antibody disrupts factor VIII C2 domain phospholipid binding while the nonclassical monoclonal anti-C2 antibody disrupts the dissociation of factor VIII from von Willebrand factor. No structural or mechanistic data have been collected for the C1 domain. Here, we report a novel protein expression protocol for isolated human factor VIII C1 domain and a fused C1/C2 domain construct. Kinetic data observed between a group A anti-C1 monoclonal antibody (2A9) and fused C1/C2 domain construct through means of biolayer interferometry resemble previously reported with an association constant of 3.0 X 105 1/Ms and a dissociation constant of 2.2 X 10-4 (1/s). Our future studies entail structural characterization of the isolated C1 or C1/C2 domain constructs bound to anti-C1 domain inhibitory antibodies to categorize the immunogenic epitopes. When cytoplasmic extracts of fresh water mussels (Unionoida) are analyzed for superoxide dismutase activity by When cytoplasmic extracts of fresh water mussels (Unionoida) are analyzed for superoxide dismutase activity by SDS-PAGE, instead of the expected dimer of Cu/Zn SOD ie ca. 30 kDa band, an array of multimeric bands ranging down from 250 kDa (ca. 8-30 kDa units) is obtained which have SOD activity when the gel is stained (Beauchamp & Fridovich,1971) . Immunodetection with a specific antibody on a Western blot of a duplicate gel confirm the presence of Cu/Zn SOD. SDS must be removed from the gel by soaking in a mixed bed ion exchange resin before staining for enzyme activity. If the samples are heated or have disulfide bonds reduced by dithiothreitol, a single monomer 15 kDa band that has no enzyme activity is present. Resistance to SDS is illustrative of kinetic stability as defined by Manning & Colon (2004) and attributed to proteins with -sheet structure such as that of Cu/Zn SOD, but does not explain the formation of multimers. That a similar phenomenon also occurs when pure Cu/Zn SOD of bovine erythrocytes or liver are analyzed should eliminate the effect of some other factor in the crude mussel extracts. Osmaan Shahid 1 , Paul Whitford 2 1 Northeastern University (Highland, United States); 2 Department of Physics, Northeastern University (Boston, United States) Elongation factor G (EF-G) uses guanidine triphosphate (GTP) to catalyze the translocation of transfer (tRNA) and messenger RNA (mRNA) through the ribosome during translation, which is accompanied by a rotation between the two ribosomal subunits. EF-G binding to the ribosome is necessary for stabilizing the rotated state, where structures of EF-G before and after translocation have been obtained from x-ray crystallography and cryo-EM experiments. However, characterizing intermediate conformations has remained elusive. To better understand the motion of EF-G during translocation, we use molecular dynamics simulations with an all-atom structure-based model. In this study, we observe a dominant transition path that domain IV of EF-G can take during its large-scale (>100 Å) conformational change, which leads to complete binding of the ribosomal A site. We also observe the extent to which ribosomal subunit rotation can influence conformational rearrangement of EF-G, where there is a balance between rotation and A-site binding. Overall, our results implicate structural and mechanistic aspects of EF-G motion during mRNA-tRNA translocation on the ribosome. Ribosome profiling (Ribo-seq) is a high-throughput sequencing technique in which the ribosomeprotected fragments of mRNA transcripts are isolated and sequenced, providing information about ribosome position at single-codon resolution. Due to the small size of ribosome-protected fragments (~30 bp), a sizable portion of reads generated in Ribo-seq experiments align equally well to multiple locations on a transcriptome (multi-map), making it impossible to unambiguously assign these fragments to their transcripts of origin. In this study, we investigate the extent of multi-mapping in human and yeast Riboseq datasets and find that a significant portion of experimental reads map to multiple positions on the transcriptome. Surprisingly, the identity of the transcripts contributing the most to the total amount of multi-mapping changes upon heat shock, indicating that changes in gene expression cause changes in the way that multi-mapping errors are manifested. We focus on a gene pair, Hsc82 and Hsp82, to show how changes in relative expression can lead to confounded interpretations of ribosome footprints and artifacts that are difficult to predict. Using a k-mer analysis of the transcriptome, we construct a multimapping network describing which transcripts multi-map and where their k-mers multi-map to. This allows us to identify which transcripts are most susceptible to multi-mapping artifacts and identify pathways that are enriched in multi-mapping transcripts. The determinants of protein-protein interactions (PPIs) remain poorly understood despite their necessity in all cellular systems. The study of PPIs is bottlenecked by the low throughput of standard assays and an inability to synthesize many different variants of an interaction. To overcome these problems we built the Next-Generation Bacterial Two-Hybrid (NGB2H), which couples a traditional bacterial two-hybrid with gene synthesis and a quantitative next-generation sequencing (NGS) read out. By uniquely mapping two proteins to a DNA barcode in the 3 UTR of the reporter gene, we can use gene expression as a proxy for interaction strength and are able to simultaneously measure thousands of PPIs with NGS. For proof-ofconcept, we investigated coiled-coilssmall, alpha-helical protein domains. Synthetic coiled-coils have some well-understood design rules and can function as a building blocks for protein nanostructures. However, creating nanostructures requires the constituent coiled-coils to interact with only their intended partner (orthogonality) which must be empirically tested. We use the NGB2H system to assay 8149 pairs of designed coiled-coils for orthogonality. From this we isolate fifty-four strongly orthogonal sets of up to twelve coiled-coils. Furthermore, we find that differences in the coiled-coil distal to the interaction interface strongly effect binding strength and that dipole formation is inhibitory to binding strength. Protein translation via ribosomal activity provides an enticing target for therapeutic inhibition, with applications in antibiotics or tumor suppression. The prokaryotic GTPase translation factor, elongation factor G (EF-G) is a crucial facilitator of ribosomal translocation and recycling of 70S ribosomes. EF-G has previously been demonstrated as the conserved target of the small molecule antibiotic, argyrin B. Recent structural findings indicate that binding of argyrin B occurs between domain III and domain V of Pseudomonas aeruginosa EF-G with an exhibited affinity of 170 nM. There are two proposed effects for the action of argyrin B: an induced conformational change that elongates EF-G and prevents the compact conformation required for proper ribosomal interaction; or the binding of argyrin B prohibits the conformational changes in EF-G required for translocation. Here, we report that argyrin B binds to E. coli EF-G as well as a point mutant engineered to be structurally homologous to EF-G found in P. aeruginosa. The resultant activity of these protein complexes in the context of ribosomal activation are: a 200% increase in relative binding of EF-G to the ribosome with little modulation in GTPase activity; appreciable GTPase activity in the absence of 70S ribosomes; and no appreciable increase in binding affinity for a nonhydrolyzable GTP analog. By examining the binding and structural effects of argyrin B on E. coli derived EF-G we hope to elucidate a potentially novel EF-G conformation that is GTPase-active in the presence of 70S ribosomes and the mechanism of ribosome-dependent GTPase activity. Proteins are attractive as building blocks to assemble supramolecular structures. The capability to design structures with precise topologies at the nanoscale provides great promise to develop next-generation materials for nanotechnology. A rational design approach is to combine protein motifs with known structural and interaction properties into self-assembling building blocks in a modular way. Coiled coils are versatile protein motifs that have been widely used as self-assembling modules. They form supercoiled complexes of two or more alpha helices and have well-understood sequence-to-structure relations that have led to successful design of many sets of non-natural coiled-coil sequences with controlled specificity, orientation and oligomeric state. The synthetic leucine zippers (SYNZIPs) are a set of computationally designed heterodimeric coiled coils. Pairs of SYNZIPs are orthogonally interacting with high affinity at the nanomolar level, while the affinities between non-interacting pairs are lower by more than an order of magnitude. In this study, we designed a protein nanostructure with a triangular topology by modularly arranging three orthogonally interacting SYNZIP pairs into recombinant fusion proteins. The linked-SYNZIP proteins were mixed and assembled into a hetero-trimeric nanostructure. Assembly of a monodisperse, stable, helical, and hetero-trimeric protein triangle was characterized by dynamic light scattering, circular dichroism, analytical ultracentrifugation, atomic force microscopy, and small-angle X-ray scattering. We further organized the protein triangles into two-dimensional arrays on silicon substrates, which directed deposition of gold nanoparticles with controlled inter-particle distances. This method enables 10-nm-scale patterning of biomolecules and inorganic nanomaterials, which provides a promising platform to realize applications in protein-based electronics. Exposure to a virus activates the immune system, which takes rapid measure to protect the body from a spreading infection. Our understanding of the signaling cascade that leads to immuno-activation in response to viral infections is crucial for designing efficient therapeutic drugs that regulate this process. Viral stimulation triggers a signal transduction cascade that activates the interferon regulator factors (IRFs), a group of helix-turn-helix transcription factors that regulate Type I interferon response (i.e. IFN, IFN). All nine human IRFs have a conserved DNA binding domain (DBD) and a less conserved transactivation domain (TAD) that contains an interferon association domain (IAD). IRF3 has a 70 amino acid residue linker linking these two domains. Besides joining the TAD and DBD of IRF3, how the 70-residue linker affects the structure and function of the protein is unknown. NMR data have shown that in isolation, the linker is highly flexible, while when tethered to the TAD the linker is more ordered and probably acts as binding sites for other co-activators. To determine the possible biological role of the IRF3 linker, we investigated the effect of varying the linker length and amino acid composition on inducing IFN response. Here, we show that, the natural of IRF3 linker is not required for robust IFN expression. Although truncation of the natural linker does not affect the transcriptional level of IFN, the linker might still play a structural role in regulation IRF3s function in the cell. Protein dynamics have a crucial role in biomolecular recognition and it has been studied intensively by different techniques for decades, such as NMR, smFRET, AFM and optical tweezer. Recently, nanopore was developed as a biosensor to investigate the protein dynamics and protein-ligand interactions in the physiological condition. The confined space of nanopore allows studying proteins in single molecular level. Here, we use the electrophysiology techniques to film the footage of the direct interactions between this single captured protein molecule and ligands. Taking advantage of the continuous recording of the process in real-time, we can detect not only the different protein conformations but also the transitions between the conformations. Besides, the capture of single molecular was controlled mostly by the electro-osmotic force so the nanopore can trap different target proteins by flipping the voltage and be used as a screening platform to draw a protein population map and find the desired ones. In the future, this nanopore sensor is promising to be developed as a universal sensor to investigate more functional proteins. Mechanistic models for ERAP1 enzymatic activity include alteration between these forms during the catalytic cycle. We have identified four small molecule modulators (A-D) of ERAP1 that differentially affect its activity towards dipeptides and longer peptide substrates. All compounds inhibit peptide hydrolysis but compounds B and C activate Leu-pNA (dipeptide) hydrolysis in a minimal substrate assay. Small-angle Xray scattering (SAXS) studies show that compounds A and D induce opening of ERAP1 whereas compounds B and C induce closure. All the small molecule inhibitors presumably alter the conformation of ERAP1 by binding to either the active site or a regulatory site, hence affecting its activity. To gain insight into the inhibitor binding sites and for a better understanding of the mechanism of catalysis of ERAP1 for both short and long substrates, we elucidated the mode of inhibition of these small molecule inhibitors using Morrison analysis. All inhibitors were competitive for long substrates while compounds B and C were found to exhibit uncompetitive inhibition for short substrates. We hypothesize that compounds B and C bind to a regulatory site within the overall substrate-binding envelope, which remains unoccupied in presence of short substrates, and that the conformational dynamics that play a key role in the catalytic cycle are governed by the occupancy of the regulatory site. The study was designed to evaluate the activities of enzymes of different metabolic pathways, such as aerobic and anaerobic metabolism, pentose phosphate pathway, and protein degradation, in the organs of threespine stickleback spawners from three locations (Kandalaksha Bay, White Sea) that differ in temperature regime, feeding resources, and underwater vegetation. The activities of cytochrome c oxidase (COX), lactate dehydrogenase (LDH), aldolase, glucose-6-phosphate dehydrogenase (G6PDH), and intracellular calcium-dependent proteases (calpains) were measured. The activities of LDH, aldolase and calpains significantly differed in fish from these locations. Moreover, the patterns of enzyme activities were more similar in fish inhabiting biotopes with more similar conditions. It allows us to conclude that the individuals of stickleback from different spawning biotopes differ mainly by the levels of anaerobic metabolism, utilization of carbohydrates and protein degradation. These data point to mechanisms for the adaptive alteration of metabolic pathways of energy supply and protein turnover in dependence on the local environment. The results obtained can be explained by both the initial genetic distance of fish from different spawning grounds and the response of initially homogeneous groups of stickleback to different environmental conditions. The study was carried out under state order (project No 0221-2017-0050). The dengue virus protease (NS2B-NS3pro) plays a critical role in the dengue viral lifecycle, making it an attractive drug target for dengue-related pathologies, including dengue hemorrhagic fever. Crystallographic studies indicate that NS2B-NS3pro transitions between two widely different conformational states: an open (inactive) conformation and a closed (active) conformation. To date, the equilibrium between these states remains in question and the resting conformation of NS2B-NS3pro is also debated. To investigate the importance of such conformational states, we developed versions of NS2B-NS3pro that allow us to trap the enzyme in various distinct conformations. Our data from these variants suggests that the resting state of the enzyme depends largely on the construct used to express the NS2B-NS3pro complex. In an unlinked construct, the enzyme rests predominantly in a closed, active conformation, whereas in a linked construct, NS2B-NS3pro adopts a more relaxed, alternative conformation. Additionally, enzymatic activity appears to be dependent not only on the movement of NS2B, but also on the flexibility of the protease core. The results from these studies provide a more detailed description of the various poses of the dengue virus NS2B-NS3 protease and should help guide future drug discovery efforts toward this enzyme. The Rearranged during Transfection (RET) receptor tyrosine kinase is a multicomponent growth factor receptor involved in kidney development, spermatogenesis, and the development and maintenance of the nervous system1. Glial cell-line derived Neurotrophic Factor (GDNF) and artemin (ART) are two of five GDNF Family Ligands (GFL) that activate RET, each acting via a GPI-linked co-receptor GFR1 for GDNF, and GFR3 for ART2,3. The formation of the RET/GFR/GFL complex is necessary to activate RET signaling, but the mechanism by which binding of the GFLs to GFR brings about activation of RET is not well understood. We have designed scaffolded peptide dimers derived from GDNF and ART to determine if GFR dimerization is sufficient to recruit RET and promote downstream signaling. If successful, these peptide dimers will serve as proof-of-concept for the development of small molecule RET agonists. Conversely, if the dimeric peptides antagonize RET by inhibiting GFL/GFR/RET complex formation, our efforts may enable the discovery of bivalent small molecules that inhibit RET by binding to GFR. This work will broaden our current knowledge of RETs mechanism of activation, while also potentially leading to new strategies for the discovery of receptor tyrosine kinase modulators. Northeastern University (Brighton, India) Nanopore translocation is a promising label-free single molecule technique to distinguish between biomolecules on the basis of their structural or dynamic properties. The confined nature of the nanopore restricts the allowed conformations of the molecules, and in some cases, necessitates a large conformational change to translocate through the nanopore. These effects are reflected in the observed current traces and thus help us in measuring the flexibility of these biomolecules. Recently, we applied molecular dynamics simulations using a structure-based model to observe a correlation between the maximum RMSF of the protein and the width of the experimental current blockade distribution. This suggests that protein translocation can be utilized as a high-throughput method to distinguish between functional conformers in proteins. Applying this technique to translocation of tRNA offers an interesting challenge since the tRNA is expected to undergo a conformational change due to the constricted size of the nanopore. To interpret the structural aspects of the conformation rearrangements associated with nanopore translocation, we apply MD simulations using a simplified structure-based energetics model of tRNA. In our forcefield, interactions between the nanopore and the molecule account for the steric effect of the pore. The mean first passage time (MFPT) for crossing the rate-limiting free-energy barrier is calculated for multiple tRNA species using energy landscape techniques. We find agreement between the MFPT values and the experimental translocation times. Further, these calculations suggest that the experiments specifically observe transient partial unfolding of tRNA. These results provide a structural/energetic interpretation of current nanopore experiments. Ki Hoon Lee 1 , Jungsoo Kim 1 1 Seoul National University (Seoul, South Korea) Silk is an outstanding natural fiber having high toughness. The highly repetitive primary sequence of [GAGAGS]n and [GAGAGY]n allows the close packing of the peptide chain which eventually forms a -sheet crystalline structure in silk. The degree of crystallinity of silk fiber is higher than 60%. If the noncrystalline regions are removed, we can obtain nanocrystals of silk fibroin. In this study, the prepared silk fibroin nanocrystals (SNC) were characterized by various analytic methods such as FT-IR, XRD, and FE-SEM in order to find the nature of SNC. The SNC was further incorporated into alginate hydrogel matrix to improve the mechanical properties of the hydrogel. The interaction between SNC and alginate was analyzed using a rheometer. Finally, the reinforcement effect of SNC on the compressive modulus of hydrogel will be reported. When the silk fiber was treated with NaOH and Na2CO3 at 80 degrees of Celcius, we could obtain SNC as a fine powder. The typical beta-sheet structure was maintained after the treatment. The XRD spectrum indicates the increase in crystallinity due to the removal of the amorphous region. When the SNC was added to the alginate solution, the viscosity was increased proportionally with the amount of SNC added. The SNC reinforced alginate gel had better compressive modulus compared to the original alginate gel. -6) . Insect insulin-like polypeptides binding proteins (IBPs) have been considered as IGFBP-like structural and functional homologues. Previously, the drosophila IBP, the embryo lethal dmIMP-L2, has been shown to increase life span when overexpressed. The first insect IBP structures are reported here; Drosophila Imp-L2 protein, in free-and complex-forms with Drosophila insulin-like peptide 5 (DILP5) and human IGF-1. They reveal a new, Ig-fold rich molecular architecture unrelated to IGFBPs which, provides a novel strategy for the regulation of bioavailability of insulin-like hormones. Highly conserved IBPs sequences suggest similar hormone binding modes in insect vectors such as mosquito, which, together with emerging evidences of the interplay of human insulin/IGF-1 from insect blood meal with both vector and parasite insulin-signalling systems, opens new research routes towards a rational interference of transmission of widespread diseases such as malaria, dengue and yellow fevers. The action of D-serine as a neurotransmitter involved in synaptic plasticity has attracted considerable interest in recent years. Our ability to study D-serine dynamically in brain tissue is limited by the lack of non-invasive methods with high spatial and temporal resolution. Förster Resonance Energy Transfer (FRET)-based optical biosensors have the potential to overcome this, however, require specific and robust binding protein scaffolds that translate ligand-induced conformational changes to optical outputs produced by FRET. The central issue that has prevented the construction of a D-serine-specific FRET sensor is the absence of any naturally occurring and specific D-serine binding proteins that display appropriate conformational changes for this purpose. Here, we have used computational protein design to guide the engineering of a naturally occurring D-alanine/glycine binding protein (DalS) towards increased D-serine specificity. The use of this engineered binding domain as a scaffold for a FRET-sensor created the first FRET-based optical biosensor specific for D-serine. Several iterations of computational design and experimental characterisation yielded two extremely thermostable biosensors, the first of which bound D-serine with~40-fold higher specificity than the competing ligand (glycine), and the second of which bound Dserine with significantly high affinity (Kd = 7 uM). Together, these sensors highlight the power of computational design and represent the first sensors of this class. It is hoped that they will now become widely used experimental tools that could yield new insight into the role of D-serine in the brain. Alpha-ketoglutarate (KG) dependent oxygenases comprise a large superfamily of enzymes that activate O2 for varied reactions. While most of these enzymes contain a non-heme Fe bound by a His2Asp facial triad, a small number of KG-dependent halogenases require only the His2 ligands to bind Fe and activate O2. The enzyme factor inhibiting HIF (FIH) contains a His2Asp facial triad and selectively hydroxylates polypeptides, however removal of the aspartate ligand in the D201G variant leads to a highly active enzyme, seemingly without a complete facial triad. Variants to the facial triad carboxylate are uncoupled as O2 is consumed much faster than primary substrate is hydroxylated. EPR studies established that mechanistic branching occurs following the formation of the Fe(IV)=O intermediate as the metal centers of WT FIH and D201G are sensitive to substrate whereas the metal centers of D201E and D201A are unaffected. Herein, we also report on the formation of an Fe-Cl cofactor structure for the D201G FIH variant using x-ray absorption spectroscopy (XAS), which provides insight into the structure of the His2Cl facial triad found in halogenases. Our results indicated that exogenous ligand binding to form a complete His2X facial triad was essential for coupling O2 activation with hydroxylation, and provide a structural model for the His2Cl-bound nonheme Fe found in halogenases. Investigating the Importance of Distal Residues in Parkin Jenifer Winters 1 , Penny J. Beuning 2 , Lee Makowski 2 , Mary Jo Ondrechen 2 1 Northeastern University (Allston, United States); 2 Northeastern University (Boston, United States) Partial Order Optimum Likelihood (POOL) is a machine learning method that predicts catalytically important residues based on the tertiary structure of the protein, with computed chemical and electrostatic properties of the residues as input features. POOL has predicted spatially extended active sites, where residues that are not in direct contact with the substrate still contribute to catalysis for many enzymes, including for parkin. Parkin is an E3 ubiquitin ligase that mediates the targeting of proteins for degradation in the ubiquitin proteasome pathway. Certain mutations in parkin lead to the accumulation of toxic substrates that damage dopaminergic neurons, causing the autosomal recessive form of Parkinsons disease (PD). POOL has predicted residues in parkin that are important for catalytic activity. Enzyme variants were constructed by single-site directed mutagenesis to test these predictions. Small-angle x-ray solution scattering (SAXS) was used to probe the structural impact of these amino acid replacements. Threedimensional reconstructions calculated from solution scattering data for wild-type parkin and variants were done using the programs GNOM and GASBOR. The resulting shape reconstructions indicate that some distal mutations result in significant domain rearrangements. Currently, the functional impact of these variants are being tested in biochemical assays. Rainbow trout is a typically cold water species highly susceptible to hypoxia and environmentally induced oxidative stress followed by high lethality and fish growth depression under rearing conditions. The aim of this study was to investigate the effects of dietary dihydroquercetin, a powerful antioxidant, on protein oxidation, calpain and proteasome activity levels as well as on growth performance and mortality in farmed rainbow trout. Rainbow trout fingerlings were fed basal diet (control) and diet mixed with dihydroquercetin. In general, dietary inclusion of dihydroquercetin increased final weight and specific growth rate compared to control. Survival rates in experimental group increased significantly compared with the control group. Protein reactive carbonyl products concentration as a marker of oxidative damage and cellular stress in fish has been measured using 2,4 dinitrophenylhydrazine based assay. The proteasome is mainly responsible for the degradation of short-lived and oxidatively modified proteins and has been recently identified as a key participant in protein degradation processes in fish. In the control group, an increase in the protein carbonyl concentration in the muscles associated with proteasome activation has been noted. In the presence of dihydroquercetin, partial decrease in protein oxidation without stimulation of proteasome system has been detected. It should be noted that calpain and proteasome activities levels changed simultaneously. Consequently, dihydroquercetin supplement of the diets could be suggested to increase a resistance to stress inducing factors as well as accelerate growth of rainbow trout. This work was supported by the Russian Science Foundation, project no. 17-74-20098. Nicole Thadani 1 , Kiara Reyes Gamas 1 , Susan Butler 1 , Peter Wolynes 1 , Junghae Suh 1 1 Rice University (Houston, United States) Viruses are complex nanomachines that facilitate the delivery of genetic material into cells and promote their own replication through the use of multifunctional proteins that undergo a series of conformational shifts. Adeno-associated virus in particular is a small, relatively simple ssDNA virus utilized in gene therapy that exhibits dynamic structural changes throughout its life cycle, but the capsid domains required for these transitions are unknown. Using the frustratometer, a computational tool that analyzes protein structures using thermodynamic modeling to identify key regions facilitating binding and structural transformation, we sought to predict residues of AAV proteins promoting capsid assembly, disassembly, and thermal stability. We identified candidate residues favoring assembled and disassembled states using the frustratometer, then conducted capsid mutagenesis to quantify the impact of these sites on virus functionality. We show that residues identified by the frustratometer play essential roles in virus assembly, thermal stability, and transduction. We also demonstrate the relationship between residue properties, capsid position, and virus assembly. This work demonstrates a potential application of the frustratometer (and other tools derived from coarse-grained models) in predicting virus formation and function in silico, elucidating the complex thermodynamics underlying virus metastability and accelerating the process of viral nanotherapeutic design. Marine invertebrates, including bivalve Mytilus edulis, belong to osmoconformers adjusting osmolality of their extracellular fluid to that of surrounding water. They developed diverse adaptive responses to osmotic stress including amino acid release through excessive hydrolysis of cellular proteins. Unlike invertebrates, fish maintains cellular osmolality at constant level and their osmoregulatory system is associated with Na+/K+-ATPase activity. We demonstrated that physiological activities of main proteindegrading enzymes, including cathepsins, calpain-like enzymes, and proteasome, in invertebrates far exceed those in marine fish, such as Atlantic salmon and brown trout. Intracellular proteases are conserved among eukaryote since the similarity of routine protein turnover. Despite this, invertebrate homologues of known proteases have diverged enzymatic characteristics when compared with vertebrate counterparts. Thus, higher protein-degrading capacity and lower substrate selectivity are inherent to invertebrates enzymes. Total protein degradation in mussel organs occurs to be regulated by ambient salinity. Comparing physiological protease activities in mussels exposed to seawater of natural salinity (25 ppm for White Sea), the excessive protease activation was found in mussels at acute rise in water osmolality to 35 or 45 ppm, while substantial decrease in mussels subjected by diluted seawater (15 or 5 ppm). Calpains and cathepsin D more readily responded to salinity variations. High rate of protein hydrolysis provides additional pool of amino acids enable to increase intra-and intercellular osmolality. Despite advanced osmoregulatory system developed in fish, they also exploit protease-mediated adaptive strategy to survive at drastic salinity fluctuations. The research was supported by the state budgetary theme (0221-2017-0050). Valerie Ivancic 1 , Valerie Ivancic 1 , Libo Wang 1 , Eugene Ma 1 , Noel Lazo 2 1 RedShift BioAnalytics (Burlington, United States); 2 Clark Univeristy (Worcester, United States) Insulin-degrading enzyme (IDE) is a ubiquitously expressed Zn2+ metalloprotease that digests several key substrates including insulin, glucagon, amyloid-beta, and amylin. The structure of IDE under conditions of proteolysis is not known. Here, we used a new bioanalytical technique called Microfluidic Modulation Spectroscopy to directly probe the backbone structure of IDE in the absence and presence of ATP and insulin. In the presence of ATP, the backbone structure of IDE does not change. In contrast, the structure of IDE is altered in the presence of insulin such that the percentage of random coil is increased at the expense of beta-sheet. Together, our results show that the interaction of ATP with IDE is localized to sidechains but the interaction of insulin with IDE leads to a perturbation in the backbone structure of the enzyme. Fitness landscapes attempt to describe the relationship between sequence changes and protein function. They are experimentally recorded by combining high-throughput functional selections with Next-Generation sequencing approaches, to estimate effect of mutations. Substitutions, insertions and deletions (InDels) account for the majority of evolutionary changes in Nature, yet understanding the structural and functional effects of InDels (unlike substitutions) remains a challenge. InDels are assumed to be highly deleterious mutations because they are likely to disrupt the structural integrity of proteins. On the other hand, they may induce significant structural changes that substitutions alone cannot cause and thus are believed to be key players in many natural evolutionary processes, such as the modification of active site loops to generate new enzyme functions or the emergence of new protein structures. While the local fitness landscape for substitutions has been recorded for several proteins, such data is absent for InDels. Here we use the green fluorescent protein (GFP) as a model system to record the effect of one-, twoand three-codon deletions on fluorescence using deep mutational scanning, and compare the effect of InDels with substitutions. We sort libraries of random GFP variants by FACS and count the number of times each mutation occurs in each fluorescence fraction. Mutation counts are used to derive the effect of thousands of mutations in eGFP and a stabilized variant. We show that while deletions are better tolerated in loops, multiple mutations retain medium fluorescence in the core of the GFP -barrel, including some that cause a registry shift. Ubiquitin interacting motifs (UIMs) are short -helices embedded in a variety of eukaryotic proteins regulating many critical pathways. UIMs specifically interact with ubiquitinated proteins decrypting and translating the highly complex ubiquitin code into specific molecular events. Since faulty ubiquitination has been observed in several diseases, UIM-containing proteins are emerging as therapeutic targets. To develop selective and potent inhibitors of UIMs we used phage display to engineer ubiquitin variants (UbVs) with unprecedented affinity for all the UIMs within the human proteome. Although UIMs are short -helices of about 20 residues in length, the developed UbVs showed remarkable binding affinity and specificity, with several UbVs displaying absolute specificity for their target UIM. In addition to inhibiting the activity of enzymes in the ubiquitin-proteasome system, the isolated UbVs were able to modulate the proliferation of cancer cell lines in vivo, potentially providing drug candidates for cancer therapy. Our work demonstrates the versatility of UbVs for modulating the activity of UIM-containing proteins, provides a toolkit for probing the function of UIMs, and establishes a general approach for the systematic development of inhibitors targeting short peptide motifs. Recombinant immunotoxins (RITs) are chimeric proteins that join the catalytic portion of a protein toxin to an antibody or receptor ligand for targeted cell killing. RITs utilizing the toxin Pseudomonas exotoxin A (PE) are currently in clinical trials for the treatment of cancer, with one variant under priority review by the FDA for the treatment of hairy cell leukemia. PE-based RITs are constructed by joining the variable fragment of a monoclonal antibody to the catalytic domain of PE using a polypeptide linker that is cleaved by the proprotein convertase furin (PCSK3). Intracellular cleavage of native PE and PE-based RIT by furin is important for cytotoxicity, yet the PE cleavage site is a poor furin substrate. Using rational design to develop more efficiently cleaved furin linkers in PE-based RITs we found that cleavage and cytotoxicity could be enhanced, but the two parameters were not directly correlated. We concluded that the role of furin in the intoxication pathway of PE-based RITs is more complex than a simple cleavage-activation step and hypothesize that furin may also act as an intracellular chaperon of PE and its derivatives. To investigate this hypothesis, we utilized CRISPR-Cas9 to develop a furin deficient HEK293 cell line to which wild type and mutated forms of furin with altered function were stably reintroduced. Here we report our initial findings using this system to investigate the role of furin in the intoxication pathway of PE-based RITs. Membrane proteins represent an essential and diverse component of the proteome. Our understanding of how integral membrane proteins are folded and inserted into the membrane continues to evolve with the development of more sophisticated structural, biochemical, and computational analytical tools. Monotopic integral membrane proteins integrate into the lipid bilayer via one or more reentrant hydrophobic domains that enter and exit on a single face of the membrane. Whereas many membranespanning proteins have been structurally characterized and transmembrane topologies can be predicted computationally, relatively little is known about the determinants of membrane topology in monotopic proteins. In the presented work we have identified two conserved motifs that drive formation of a monotopic membrane topology for PglC, a phosphoglycosyl transferase from Campylobacter jejuni. Whereas many of the structurally characterized monotopic membrane proteins associate with the membrane through hydrophobic loops or amphipathic helices, the recently reported structure of PglC shows that it integrates into the membrane via a highly-ordered, reentrant helix-break-helix motif that significantly penetrates the hydrophobic membrane core. A positively-charged Lys-Arg motif N-terminal to the membrane-inserted domain of PglC and a helix-breaking Ser-Pro motif within the domain act in tandem to enforce a reentrant topology early in the folding process of PglC. Both motifs additionally contribute to the stability of the final folded structure and are important for enzyme activity. These two motifs exemplify principles that are likely to determine topology among diverse monotopic proteins of interest and could inform efforts to predict reentrant topologies more broadly among membrane proteins. Derion Reid 1 , Carla Mattos 1 1 Northeastern University (Boston, United States) Ras GTPases play a central role in a number of signal transduction pathways that regulate cellular proliferation, survival, migration, and apoptosis. The crystal structure of HRas has been used as a representative for all Ras isoforms, however biochemical and structural differences between the Ras isoforms are emerging. Ras functions as a molecular switch where GEFs exchange GDP for GTP to turn its signal on. Ras signaling is turned off either through intrinsic hydrolysis or GAPs. Oncogenic mutants maintain Ras in a constitutively active state, and appear in nearly 30% of human cancers. Yet, currently there are no approved inhibitors of Ras. While KRas has received a lot of attention due to its prominent mutations in pancreatic and colorectal cancers, the structural biology of NRas, which is mutated frequently in melanomas, has been overlooked. We present the crystal structure of NRas and several of its oncogenic mutants accompanied by intrinsic hydrolysis rates, for comparison with those of K-Ras and H-Ras. The NRas hydrolysis rate constant in the presence of the effector Raf-RBD is slower than those of KRas and HRas. This correlates with the presence of Y166 at the C-terminal end of the G-domain, with diminished access to the catalytic conformation for GTP hydrolysis. Our biochemical and structural understanding of NRas mutants will add insight to the development of novel therapeutics for NRas mutant melanomas. Earlier studies by Chow and Skolnick suggest that the diffusion of intranuclear protein through the matrix of bacterial DNA appear to be governed in part by the internal motions of the DNA itself. However, the effect of a proteins size on its diffusion through densely packed DNA is not well understood. For this study, we built and used a program called BDT for Brownian dynamics (BD) simulations of protein-DNA systems using the free-draining approximation. Protein beads were placed into a simulation box containing a fractal DNA chain prepared by arranging DNA beads along a Hilbert space-filling curve where straight segments were at least twice the persistence length of DNA. The box was slowly reduced in volume until in-vivo DNA and protein volume fractions of~13% and~6%, respectively, were reached. BD simulations using this initial configuration were carried out using this preparation method with protein radii from 1.6nm to 6.9nm, and protein diffusion was measured. The diffusion constant for a system with protein radius similar to that of LacI (4.4nm) was found to be comparable to the value obtained from in-vivo studies, suggesting confidence in our methods. The results suggest that, for the range of radii tested, intranuclear protein diffusion increases an order of magnitude linearly to a limit with decrease in protein radii down to the radii of the DNA beads. Blue-light sensing Light-Oxygen-Voltage (LOV) domains represent a widespread way for organisms to detect and respond to light cues in the environment. By harnessing photochemical reactions within an internally-bound flavin chromophore and turning them into protein conformational changes, these photosensors become powerful machines for modulating highly complex physiological pathways. We are trying to understand the molecular mechanisms of this regulation, with the goal of better understanding both these important photobiological pathways and broader issues of coupling of protein structure and function. However, key structural questions about these processes remain unanswered despite extensive interest in LOV photochemistry in both natural and engineered optogenetic-type systems. Here we investigate members of a newly-discovered class of fungal RGS-LOV proteins which contain both RGS (Regulator of G-protein Signaling) and LOV domains, implicating their control of physiologically-important signal transduction pathways. Recent bioinformatic and biochemical analyses suggest that an additionally conserved, but poorly characterized, C-terminal domain plays a role in controlling these proteins by mediating light-dependent membrane recruitment activity. To confirm this, we have adopted a reductionist approach whereby we characterize structure and investigate lipid interactions in this Domain of Unknown Function (DUF). Deciphering the novel photosensory signal transmission mode via an RGS-LOV-DUF combination holds promise to both understanding aspects of biological regulation in this system and in seeding the development of new single-component optogenetic tools with rapid membrane localization and regulated G-protein signaling in response to blue light. Many of the over 6,700 human membrane proteins undergo varying degrees of conformational change, from large changes seen in solute carrier (SLC) transporters to more subtle changes seen in G-protein coupled receptors (GPCRs). These conformational changes are involved in mechanisms such as cellular signaling, substrate transport, and ligand-binding. Although a large number of membrane proteins are thought to have alternative conformations, only a handful of proteins have been successfully crystallized in multiple conformations. In fact, most alternative conformations remain unsolved. By leveraging the rich evolutionary information contained in genomic sequences, it is possible to identify sites that have been under strong coevolution. These evolutionary couplings have been used to accurately predict the three-dimensional structures of ordered and disordered proteins, RNA, and complexes. However, evolutionary couplings have been inefficient in predicting separate conformational states, instead predicting an intermediate structure of the multiple conformations. Here we further develop evolutionary couplings prediction to identify sets of crucial contacts that inform the three-dimensional folds of distinct alternative conformations. For proteins with a known crystal structure for a single conformation, we compare our predicted contacts from evolutionary couplings to determine potential contacts that are most likely to belong to a different conformational state. These predicted contacts are then used to re-fold the protein to predict alternative conformations. With this improved method, it is possible to deconvolve sets of predicted contacts from evolutionary couplings to predict the alternative conformations for a set of human membrane proteins including SLC transporters and GPCRs. HIV-1 protease is a primary target in anti-retroviral therapy. The virus evolves rapidly and thus provides a unique challenge to the design of robust viral therapeutics including protease inhibitors. Over the past decades, a vast number of structures of HIV-1 protease in complex with small molecule inhibitors have been determined making the enzyme an ideal model system for structure-based bioinformatics analysis toward the development of new drug design strategies. In this work we aim to utilize publicly available experimental data to train a machine learning algorithm that can infer inhibitor potency from the 3D structure of an enzyme-inhibitor complex. Over 250 high resolution crystal structures of HIV-1 protease in complex with chemically diverse small molecule inhibitors have been curated from the Protein Data Bank. Inhibition constants of the dataset spanned 9 orders of magnitude. Enzyme-inhibitor interactions were calculated for all structures and compressed into a structure-interaction fingerprint, a binary vector amenable for machine learning. The Interaction fingerprints were used to train a random forest based machine learning algorithm. The algorithm was chosen due to the relatively high transparency and interpretability which allows for the reconstruction of key interactions that determine potency. The trained algorithm was capable of accurately distinguishing low, medium and high affinity binders from one another. Using combinations of interactions as input, we show that both contact-based information and non-bonded-electrostatic descriptors are required for good predictive accuracy. Our results highlight both the potential and limitations of combining traditional structural biology with machine learning to obtain novel insights for drug design. Quantitative real-time PCR (qPCR) is a fluorescence-based molecular biology technique that is currently the gold standard for DNA or RNA detection and quantification in a given sample. Reagents used for nucleic acid amplification often influence the minimum quantity of template that can be reliably detected, and establishing the lower limits of detection and quantification is critical for qPCR-based molecular diagnostic assays. The important parameters that define assay performance at low input are limit of detection (LoD) and limit of quantification (LoQ). We set out to evaluate low input DNA detection for a set of commercially available qPCR master mixes, establishing estimates for the LoD and LoQ under ideal conditions. We used commercially available qPCR primers that target a locus in human genomic DNA present as a single copy per haploid genome and produce a negligible amount of primer dimer. The Luna ® Universal qPCR Master Mixes achieved the theoretical LoD based on the Poisson distribution of 3 molecules in this assay, while the LoQ varied from 32 to 16 molecules for the dye and probe-based qPCR mix, respectively. The data highlights that the master mixes are sensitive enough to amplify a single copy of target DNA. Accuracy during DNA replication is essential for maintaining genetic integrity and viability in all living organisms. DNA polymerases achieve this via several mechanisms, including selective incorporation of correct nucleotides (nucleotide discrimination), delayed synthesis following incorporation of incorrect nucleotides (low rate of mismatch extension), and removal of misincorporated nucleotides via 3-5 exonuclease activity (proofreading). These activities act synergistically to ensure faithful replication, both in vivo and for purified DNA polymerases in in vitro methods such as PCR. High fidelity is critical in many PCRbased technologies, including molecular cloning, library amplification for Next Generation Sequencing (NGS), and molecular diagnostics for cancer and other diseases. Understanding polymerase fidelity mechanisms thus carries significance for these and other applications in biotechnology and medicine. Q5 DNA Polymerase is a commercially available, engineered polymerase belonging to the B family of DNA polymerases. It has robust 3-5 exonuclease (proof¬reading) activity and single-molecule sequencing studies indicate that it has 280-fold higher fidelity than Taq DNA polymerase. While widely used as an ultra-high fidelity DNA polymerase, however, how Q5 achieves this high fidelity has not been previously described. In this work, we report both steady state and pre-steady state kinetic studies of Q5 including rates of correct and incorrect nucleotide incorporation, 3-5 exonuclease activity, and mismatch extension. These studies provide insights into how these factors contribute to the high fidelity of Q5 DNA Polymerase. Enzymes in the Haloacid Dehalogenase superfamily (HADSF) possess a wide range of activities, including phosphatases important in cell membrane biosynthesis and dehalogenases that can degrade halogenated compounds. Unfortunately, the majority of the members of this superfamily, along with most of the 14,000+ Structural Genomics (SG) protein structures in the Protein Data Bank (PDB), have unknown, incorrect, or uncertain biochemical functions. In order to transform SG data into more useful form for benefits for humankind, reliable methods must be developed to functionally annotate these proteins. Functional annotation enables identification of potential applications, such as new pharmaceutical targets or catalysts for bioremediation. Computational methods developed at Northeastern University will be used to predict biochemical function for SG proteins in the HADSF. These methods are Partial Order Optimum Likelihood (POOL) and Structurally Aligned Local Sites of Activity (SALSA). POOL is a machinelearning method that uses the inputs from THEMATICS, ConCavity, and INTREPID to make the functional residue predictions for a given protein. SALSA uses the functional residue predictions from POOL and assigns function to Structural Genomics proteins according to the local spatial arrangement of predicted residues at the active site. So far for the HADSF, using SALSA we have made predictions for 20 SG proteins. These predictions will be experimentally validated by biochemical assays to establish the function of each protein and to verify our computational approach to protein function prediction. The ability to predict computationally the biochemical function of protein structures of unknown or uncertain function adds tremendous value to genomics data. Lauren Viarengo 1 , Adrian Whitty 1 1 Boston University, Department of Chemistry (Boston, United States) There are multiple precedents for macrocyclic compounds (MCs) with structures that violate classical criteria for druglikeness, such as Lipinskis Rule of Five and Vebers Rules, and yet nonetheless are successful oral drugs. These successes, albeit relatively few in number, suggest that to understand what combination of molecular features will confer good pharmaceutical properties on MCs requires consideration of properties beyond the classic metrics for druglikeness. We have devised a set of novel, macrocycle-specific molecular descriptors, all of which can be calculated from 2D chemical structure, which quantify different aspects of MC size, shape and connectivity, atom and group composition, polarity, and molecular flexibility. We additionally use Machine Learning techniques, including Hierarchical Clustering and Principal Component Analysis (PCA), to test how these MC-specific molecular descriptors aid in analyzing and comparing the structures and properties of synthetic MCs intended for use in drug discovery. DNA methylation is a prevalent epigenetic modification in both eukaryotes and prokaryotes, playing important roles in gene regulation, detection of foreign DNA and other cell processes. In prokaryotes, restriction modification systems employ methyltransferases to protect host DNA from methyl-sensitive endonucleases. For molecular biology applications, DNA methylation can be recapitulated in vitro using purified methyltransferases or by directly incorporating methylated nucleotides during DNA amplification by PCR. Here, we characterize the efficacy of 5-methyl-cytosine (5mC) incorporation for a set of commercially available DNA polymerases, examining yield and specificity across a variety of PCR targets. Targets with high GC content proved most challenging for 5mC incorporation, and polymerases that amplified well at high GC content while incorporating standard dNTPs were also most proficient at 5mC incorporation. For Q5 High Fidelity DNA Polymerase, which incorporated 5mC successfully for the broadest range of targets, we further quantitated the effect of dNTP pool composition on 5mC incorporation efficiency. While full substitution of 5m-dCTP for dCTP yielded fully C-methylated PCR products, tuning the dCTP:5m-dCTP ratio allowed us to modulate % 5mC incorporation, yielding PCR products with a range of methylation states. PCR products with different levels of methylation in turn exhibited differential sensitivity to restriction endonucleases, with implications for downstream applications. The D48N/K116Q mutant of lysozyme was expressed in E. coli using a designed and commercially ordered plasmid. It has a substantially changed dipol moment in comparison to the wild type protein. The pure sample of the lysozyme mutant was obtained by solubilization of the inclusion bodies in buffer with 8 M Urea, purification in two steps -ion exchange and gel chromatography, and refolding of the purified protein. Its properties were checked using different biophysical methods: circular dichroism, steady-state fluorescence spectroscopy and stopped-flow spectrofluorimetry. According to the CD data we can assume that, despite small differences, the introduced mutation does not change the secondary and tertiary structure of the protein. Binding of the N-acetylchitotriose decreases the fluorescence band of the lysozyme. This fluorescence change was used to follow kinetics of N-acetylchitotriose binding both to the mutated and wild type lysozyme in a stopped-flow spectrofluorimeter. Experiments were done at 20oC with a 320 nm cutoff filter using an LED light source excitation of 295 nm. The registered progress curves were analyzed with the DynaFit program. It was shown that in both cases (the mutant and wild type lysozyme) binding of N-acetylchitotriose is a two-step process with corresponding rate constants of comparable values. MitoNEET is a recently discovered mitochondrial [2Fe-2S] protein that is a binding partner of the antidiabetic drug pioglitazone. MitoNEET contains a unique ligation, three cysteines and one histidine, of the metal cluster. However, the cellular function of mitoNEET is currently unknown. Several functions have been proposed including a role in cellular respiration, as an iron-sulfur cluster transfer protein, and as an electron-transport protein. Putative protein-binding partners of mitoNEET were collected by a protein pulldown experiment. One result of the pull-down assay, glutamate dehydrogenase 1 (GDH1), is an allosteric enzyme that plays a role in several metabolic cycles and is known to regulate insulin. MitoNEET binds to GDH1 through a disulfide bond and activates the enzyme. Additionally, mammalian GDH1 is allosterically controlled by a number of small molecules. It is activated by ADP and leucine and inhibited by GTP and palmitoyl-CoA. Enzyme kinetics were used to study how mitoNEET binding affects the allosteric control of GDH1. These results have significance in that all of the allosteric regulators are physiologically relevant. Introduction: Systemic sclerosis (SSc) is a multisystem connective tissue disease, characterized by fibrosis and ECM deposition in skin and internal organs, abnormalities of the immune system, and vasculopathy, but molecular mechanisms underlying these abnormalities remain obscure contributing to limited efficacy of treatment. MiRNAs are small non-coding RNAs that play a role in post-transcriptional gene regulation. Because profile of miRNAs in biofluids have been found to be different in pathological states, we predict that circulating miRNAs would be a great blood-based marker for molecular diagnostics. Material and Methods: The study enrolled 10 SSc patients and 6 controls (aged 43-85, 63.10AE12.53 yo). In this study, we compared expression pattern of multiple miRNAs in serum of 10 SSc patients to 6 healthy controls using microarray analysis and RT-qPCR to confirm the obtained results. In addition, bioinformatics analysis was performed to explore miRNAs target genes and the signaling pathways potentially involved in SSc pathogenesis. Results and Conclusion: Microarray profiling demonstrated that 7 miRNAs were markedly down-or up-regulated in SSc patients, compared to the control group. The 18-fold change of miR-4484 level might serve as a potential biomarker for SSc detection, although it has not been reported in previous studies. The most interested target genes for miR-4484 are MMP-21 and ADAM12, which may play a role in fibrosis. MMPs disrupt the basement membrane, allowing inflammatory cells to be easily recruited to the site of injury. These findings demonstrate that gene expression might be regulated by miRNA in SSc patients. The alpha subunits (G) of heterotrimeric G proteins are activated by guanine nucleotide exchange factors (GEFs) that catalyze the release of GDP from the G nucleotide binding site and subsequent loading of GTP into that site. Plasma membrane-bound, agonist-stimulated G protein-coupled receptors (GPCRs) are the best characterized heterotrimeric G protein GEFs, but cytoplasmic GEFs have also been discovered. Of these, Ric-8A has been identified as both a GEF and a folding chaperone for the i, q, and 12/13 classes of G. Previous studies in our laboratory have shown that Ric-8A catalyzes nucleotide exchange by inducing a structurally dynamic and heterogeneous state of G in which the Ras-like and helical domains that enfold the nucleotide are rotated apart, and the beta-sheet scaffold of the Ras domain that supports the nucleotide is structurally dynamic. We have now determined the 2.2Å resolution crystal structure of the stable Ric-8A G-binding domain comprising residues 1-452 of the 530-residue intact protein. This fragment (C78Ric-8A) adopts a continuous armadillo fold, with an architecture similar to -catenin and the Ran-binding nuclear import proteins. Using information derived from small-angle X-ray scattering, chemical crosslinking, hydrogen-deuterium exchange and singlemolecule fluorescence spectroscopy, we are able to deduce the structure of the G:Ric-8A complex, and derive insight into the mechanism by which Ric-8A serves as a G chaperone and GEF. The self-assembly of lysozyme to form amyloid fibrils is associated with systemic amyloidosis, a disease characterized by the presence of amyloid deposits in various organs of the body. The early events associated in the self-assembly of lysozyme are not well understood. In this work, we used Microfluidic Modulation Spectroscopy (MMS) to characterize the early events in the self-assembly of human lysozyme. Through MMS, we were able to probe the mid-IR absorption band of the protein which is sensitive to both -helix and -structure. With heating at 60 C, the -turn content of the protein increases while its -helical content decreases. This result suggests that the first structural transition in the self-assembly of human lysozyme is an -helix to -turn conformational rearrangement. The high stability of the long-lived crystallin proteins is essential for maintaining lens transparency. Aggregation of partially unfolded intermediates of these proteins results in cataract, the leading cause of blindness in the world. Moreover, approximately fifty-percent of juvenile cataracts is due to genetic mutations in crystallin genes. However, there are limited studies on how these mutations lead to protein aggregation. Human D crystallin is a 20 kDa, anti-parallel -sheet Greek Key structural protein and the third most abundant crystallin in the lens. This projects main goal was to optimize a high throughput method for analyzing the relative stability of multiple human D crystallin mutants under varying conditions. The high throughput thermal stability shift assay uses real-time PCR equipment and a fluorescent dye that binds to hydrophobic regions of the protein as it unfolds with increasing temperature. Assay conditions (e.g. ramp speed & protein concentration), buffers, salt concentration, and pH were all tested on human D crystallin. The conditions that were the most comparable to the published Tm value was 5 g protein, 10mM Tris buffer, 10mM NaCl, pH 7.0. Additionally, the absence or presence of varying concentrations (2mM-10mM) of copper sulfate was tested to determine if the presence of copper ions reduced stability. All concentrations above 2mM of copper sulfate reduced the Tm by approximately 20 C. This assay provides a high throughput method to determine regions important for human D crystallins overall stability and analyze mutations that have been identified in juvenile-onset cataract. IGF2 is a major fetal growth hormone, playing a vital role in regulating fetal development and tissue differentiation. In the ER, the IGF2 pro-protein, proIGF2, is composed of a mature region and an E-peptide. The E-peptide is cleaved off in the Golgi, leaving the mature IGF2 hormone. A software that predicts BiP-binding sites (BiPPred) indicates potential BiP-interaction sites on the E-peptide region of proIGF2, which may be intrinsically disordered. Indeed, fluorescent experiments here show that BiP directly interacts with the E-peptide. The next research steps include finding the BiP-binding site(s) on the E-peptide and determining if and where Grp94 interacts with proIGF2. Additionally, it will be determined how Grp94 and BiP may transfer a shared client protein, such as proIGF2, from one chaperone to the other. This exciting work will uncover how Grp94 and BiP collaborate to help fold a shared misfolded client protein, such as proIGF2. Multisubunit tethering complexes are a class of essential regulators of vesicle fusion found in all eukaryotic cells. Although all multisubunit tethering complexes share the ability to bind to at least one SNARE protein, it remains unclear whether these complexes actively regulate SNARE assembly and, if so, what the mechanism of this regulation might be. Here, we report the structure of a subunit of the ER-localized Dsl1 tethering complex bound to one of its cognate SNAREs. This structure reveals that the Dsl1 complex promotes SNARE assembly by orienting the SNARE motifs of two ER SNAREs toward one another. Structural conservation between Dsl1 and other multisubunit tethering complexes suggests that this mode of binding may be generalizable to other members of this family. The syntax for conversion of messenger RNA (mRNA) to protein is provided by the genetic code, the set of universal instructions by which 61 mRNA codons translate to twenty proteinogenic amino acids. This process of information transfer is in part mediated by elongation factor Tu (EF-Tu), a three-domain GTPase found in all domains of life, which chaperones amino-acylated tRNA to the ribosome in complex with GTP during the elongation phase of translation. Here we sought to directly measure the interactions between EF-Tu, tRNA and the ribosome simultaneously using three color single molecule fluorescence resonance energy transfer. Multiple EF-Tu mutants were engineered bearing a single point substitution with a non-canonical amino acid and then modified with an organic fluorophore. The kinetics of EF-Tu dissociation and tRNA accommodation where then determined for pre-steady state tRNA selection at fast time resolution (10-25 ms). Our findings revealed a transient interaction between EF-Tu and the elongating bacterial ribosome during cognate tRNA selection. Surprisingly, our data also uncovered a previously unknown interaction between EF-Tu:GTP and stalled pre-translocation ribosomal complexes that could increase the fidelity of protein synthesis. Together, these findings refine our understanding of the tRNA selection mechanism and highlight the utility of three-color smFRET for interrogating complex biological systems. Biochemical and Biophysical Examination of the Human Hect E3 Ubiquitin Ligase Wwp1 Emilie Ogisu 1 , Donald Spratt 1 1 Clark University (Worcester, United States) WW domain containing Protein 1 (WWP1) is Homologous to E6AP C-Terminus (HECT) E3 Ubiquitin ligase involved in the ubiquitylation-signaling pathway that attaches ubiquitin to substrate molecules for proteosomal degradation. Through this enzymatic cascade, WWP1 can regulate the expression and activity levels of a variety of proteins including Smad proteins involved in the transforming growth factor-(TGF-) signaling pathway, tumor suppressor gene p53, and Ebola matrix protein eVP40. By regulating these substrate molecules, WWP1 is suggested to be involved in different cancers as well as Ebola egression. The C-terminal lobe of WWP1s HECT domain (residues 803-922), the region of the enzyme responsible for ubiquitylation activity, was overexpressed and purified for biochemical and biophysical characterization by circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy, ubiquitin activity assays, pH titrations, and analytical gel filtration. These experiments were performed to further examine WWP1s secondary structure, thermal stability, folding, activity, 3D structure and specific residues in the active site that are required for its function. Our cumulative results demonstrate that the WWP1 C-lobe is a stable, monomeric protein and may form di-ubiquitin chains. Using NMR spectroscopy, we observed the highly conserved residues H888 and F891, both of which are located adjacent to catalytic cysteine C890, showed significant chemical shift perturbations at different pH, suggesting these residues may play a major role in the ubiquitin transfer step. These studies will help us determine the catalytic mechanism of WWP1 at the atomic level and better understand how WWP1 is linked to various human diseases. Apoptosis Resistant E3 Ligase (Arel1) is a member of the Homologous to E6AP Carboxyl Terminus (HECT) E3 Ubiquitin ligase family. All HECT E3 ligases contain a conserved HECT domain comprised of an N-lobe that binds to E2 ubiquitin conjugating enzymes, and a C-lobe that contains the conserved catalytic cysteine required for ubiquitylation. Arel1 regulates apoptosis by attaching ubiquitin to inhibitors of apoptosis proteins (IAPs), second mitochondria-derived activator of caspases (Smac), serine protease HTRA2, and ARTS. Previous studies have found that <80% of the ubiquitin chains formed on these substrates are Lys33, making Arel1 the only HECT E3 ubiquitin ligase known to form Lys33 polyubiquitin chains. To better understand the formation of these atypical ubiquitin chains, nuclear magnetic resonance (NMR) spectroscopy and in vitro ubiquitination activity assays were employed to study the structure of the Arel1 HECT C-lobe (residues 706-823) as well as the function of conserved residues adjacent to the catalytic cysteine (C790). The full backbone resonance assignment of Arel1 C-lobe was completed and the assay found that of the mutants purified (H788A, T789S, T789V, C790A), only the C790A substituted protein was unable to form a thioester bond with ubiquitin. We demonstrate that the wild type C-lobe is able to be charged with ubiquitin in the presence of the E1 and ATP and does not require an E2 enzyme. This study provides initial insight into the unique mechanism and structure of Arel1s C-lobe and its ability to build Lys33-linked polyubiquitin chains. Coagulation Factor XIIIA (FXIIIA) is a transglutaminase that cross-links an array of intra-and extracellular protein substrates via an isopeptide bond. FXIIIA is expressed as an inactive zymogen. During blood coagulation, it is activated in plasma by removal of an N-terminal activation peptide by thrombin. No such proteolytic FXIIIA activation is known in other tissues (i.e. bone) or the intracellular form of FXIIIA. For those locations, FXIIIA is assumed instead to undergo activation by Ca2+ ions. In addition, FXIIIA can be rapidly activated by nonphysiologically high (>50 mM) Ca2+ concentrations. We employed analytical ultracentrifugation to qualitatively and quantitatively characterize intersubunit interactions in FXIIIA. Results revealed dissociation of the dimeric zymogen into monomers upon activation by thrombin and by low (4 mM) or high (100 mM) Ca2+. Compared to 100 mM Ca2+-activated FXIIIA, proteolytically activated FXIIIA showed higher conformational flexibility and lower solubility. A more in depth enzymatic evaluation revealed a lower apparent Km for the glutamine substrate of thrombin-activated FXIIIA. Intriguingly, monomeric active FXIIIA demonstrated a non-Michaelis-Menten kinetic response characteristic of negative cooperativity. Such kinetic behavior may be relevant for regulation of the extent of FXIIIA-catalyzed crosslinking reactions in vivo. The presented research further expands the understanding of FXIIIA function. While the dimeric state stabilizes FXIIIA zymogen in a physiological setting, a dissociation is required for activation. Slow nonproteolytic activation in nonhemostatic environments provides relatively low but constitutive FXIIIA activity. The proteolytic pathway is more effective and promotes fast buildup of the FXIIIA activity during the emergency of blood coagulation. Ubiquitylation is a post-translational modification that involves the covalent attachment of the small signaling protein ubiquitin to a target substrate to control protein turnover and intracellular localization. HECT and RCC-1 domain containing protein 2 (HERC2) is a member of the Homologous to E6AP Carboxyl Terminus (HECT) E3 ubiquitin ligase family, and it is responsible for recruiting proteins for DNA repair in cancer. The HECT domain of HERC2 is required for facilitating the direct transfer of ubiquitin to a substrate through its conserved catalytic cysteine (C4762), however the structural basis for this mechanism is currently unknown. Here we report the first structural and biochemical examination of the HERC2 HECT C-terminal lobe (residues 4776-4834). Using bacterial overexpression and affinity purification in combination with site-directed mutagenesis, in vitro ubiquitylation activity assays, ubiquitin-HERC2 disulfide complex formation, and 2D/3D NMR spectroscopy, a new framework for studying dysfunctional HERC2 in human disease has been established. We demonstrate that the unique acidic C-terminal tail of the HERC2 C-lobe is disordered and is required for HERC2-dependent polyubiquitin chain assembly. We hypothesize that the acidic C-terminal tail is dynamic in the presence of ubiquitin and that HERC2 employs a novel mechanism that differs from other HECT E3 ubiquitin ligases. This work provides the first structural and mechanistic insight into how HERC2 assembles polyubiquitin chains. Due to its ability to survive solely on wood, the shipworm species Lyrodus pedicellatus is an ideal candidate to study the conversion of biomass. Our focus is on biochemical conversion methods that use enzymes to degrade biomass into its components, which can then be used for industrial applications, including biofuel.The purpose of this research is to characterize proteins found in the shipworm and its symbionts that are involved in the breakdown of wood. The shipworm species Lyrodus pedicellatus is an ideal system for this work because it has the ability to reproduce and live on wood as its sole food source. Additionally, the shipworm houses a colony of endosymbiotic bacteria specialized for the degradation of wood, which are localized in the gill. Only a subset of the symbiont metaproteome is transferred to the digestive tract of the host. This provides a working list of important enzymes a small fraction of the metaproteome likely to be directly involved in wood digestion. The proteins encoded by the symbionts of the shipworm have been analyzed using Partial Order Optimum Likelihood (POOL) and Structurally Aligned Local Sites of Activity (SALSA), which are computational techniques developed in the Ondrechen lab. These two computational methods have been used to predict the catalytic residues and functional families of the proteins produced by the shipworm symbionts. Additionally, enzymatic assays, using xylan and various substrates that are components of wood, are being performed with the proteins to confirm experimentally their predicted biochemical functions The perceived capabilities of three-dimensional (3D) single-particle cryo-electron microscopy (cryo-EM) are increasingly expanding in both achievable resolution as well as the size of the target protein structure. Recent technical breakthroughs have not only expanded the technique into the near atomic resolution regime but have also allowed for the structure determination of proteins less than 200 kDa in molecular weight. Here, we report the use of conventional underfocus methodologies in the determination of the structures of bovine alcohol dehydrogenase (~82 kDa) to~2.9 Å resolution and human methemoglobin (~64 kDa) at~2.9 Å and~3.2 Å resolution using an electron microscope operating at 200 kV. Our results demonstrate not only that high-resolution single-particle cryo-EM reconstructions of macromolecular complexes amassing less than 100 kDa can be obtained using conventional defocus-based methodologies but also prove that conformational heterogeneity can be sorted computationally, even for proteins as small as 64 kDa. We further provide evidence that the successful determination of structures of asymmetric molecules less than 45 kDa by cryo-EM is on the horizon. Herpesviruses, such as herpes simplex-1, are double-stranded DNA viruses that infect almost all vertebrates, establishing lifelong latent infections. During reactivation, progeny virions are generated and must exit the cell to spread infection. Herpesvirus egress begins in the nucleus where the fully assembled capsid, too large to pass through the nuclear pores, buds from the inner nuclear membrane (INM) to the perinuclear space. This process relies on the virally-encoded nuclear egress complex (NEC), a heterodimer made of two conserved proteins, UL31 and UL34. The NEC forms a hexagonal coat on the surface of budded vesicles and disruption of this coat formation prevents budding, but it is unclear why oligomerization is required. Additionally, regions of the NEC closest to the INM, membrane proximal regions (MPRs), have been identified and are required for NEC binding and budding, yet the specific segments and residues responsible are unknown. Elucidating how oligomerization and the MPRs enable NEC function will allow us to better understand herpesvirus egress. We have made NEC constructs with truncations and point mutations designed to inhibit oligomerization and MPR functionality. in vitro budding assays and neutron reflectometry were used to determine budding efficiency and membrane interactions for each construct. Our results show that oligomerization changes the NEC-membrane interaction and point mutations within a mini-MPR can impact budding efficiency. Investigating the Functionality of Procaspase-6 and Caspase-6 by Various Nucleotides Ishankumar Soni 1 1 Graduate Student at UMass-Amherst (Sunderland, United States) Caspase-6, a dimeric cysteine aspartate protease, is involved in multiple biological pathways including apoptosis and neurodegeneration. As a result, various researches have been conducted in understanding caspase-6s regulation by biological as well as chemically synthesized molecules. Our research focuses on the direct interactions between nucleotides and (1) procaspase-6: full-length zymogen form as well as (2) caspase-6: active cleaved form. Our preliminary results ATP and GTP acyl probes bind to procaspase-6 (but not caspase-6) motivate us to investigate in (1) where does ATP bind to procaspase-6, (2) how does ATP modulate the functionality of procaspase-6 and caspase-6, and (3) how do other nucleotides (or their derivatives) modulate the functionality of procaspase-6 and caspase-6. Using site directed mutagenesis, we demonstrate that Tyr-198 residue present at the dimer interface of procaspase-6 is a putative binding site for ATP acyl probe as well as ATP. From our gel-based in vitro activity assays, we establish that exogenous ATP can attenuate the self-proteolytic activity of procasase-6. At last, using gel-based in vitro activity assays, we have derived the functionality effects of procaspase-6 by dATP, ADP, AMP, GTP and diadenosine tetraphoshate (Ap4A). There is an urgent public health need to improve the efficacy of the influenza vaccine. The predominant surface antigen on influenza viruses is hemagglutinin (HA), the protein responsible for receptor binding and membrane fusion. The STRucture-guided Influenza Vaccine initiativE (or STRIVE) is a collaboration between NIAID-funded structural genomics centers SSGCID and CSGID and a leading producer of influenza vaccines. The goal of the STRIVE collaboration is to expand structural knowledge of structurally uncharacterized strains of influenza HA to improve pan-strain influenza vaccines. We have established a pipeline for HA expression, purification, and crystallization. HA proteins from twenty-five structurally underrepresented strains entered the pipeline, and twenty-one passed initial expression testing. Of the eight proteins produced in large scale to date, one structure has been solved and deposited in the pdb, and we have crystals of varying quality for three additional HA proteins. These structures will assist in modeling efforts to improve seasonal vaccine efficacy, as well as inform work toward a universal flu vaccine. Collagen is the most prevalent component of the extracellular matrix and provides critical chemical and mechanical cues that control cell fate and tissue formation. The~1000-residue strands of natural collagen are, however, inaccessible through synthetic chemistry or recombinant DNA technology. Self-assembly of short collagen-mimetic peptides (CMPs;~3040 residues), especially strategies that drive triple-helical growth at terminal sticky-ends, provides an alternative route to long, synthetic collagen nanofibers. We theorized that CMP building blocks designed to ensure symmetry in the targeted assembly would also enhance sticky-ended association. Symmetric-assembly design simplifies the placement of attractive and repulsive interstrand interactions on each strand (e.g., using positive and negative charges), eliminating competing associations while maximizing stability. We recently demonstrated the success of this strategy by employing 42-residue CMPs to produce highly-stable triple-helices that match or exceed the length of natural collagens. Here, we establish the general utility of symmetric design by successfully applying it to CMPs of various sizes. We identify the shortest building blocks to date that produce synthetic collagen nanofibers, as well as hydrogels. The assembly and stability of these new building blocks parallel our earlier results, opening the door to mechanistic studies. Symmetric design is a generally applicable and reliable method for the production of synthetic collagen nanofibers and provides an exciting platform for the development of functional biomaterials. Acute myeloid leukemia (AML) is dependent on the interaction between native Myb activator and CREBbinding protein (CBP). The CBP coactivator is capable of directly binding a variety of different activators and transcription factors through its flexible KIX domain, allowing CBP to control transcription of many genes. Unfortunately, the conformational plasticity and the presence of multiple binding sites on KIX make it challenging to target using small molecule inhibitors. The transactivation domains of Myb and MLL bind to two different sites on KIX, and fusing these two domains via a flexible linker produces a peptide (MybLL-tide) that has a greater affinity for KIX than any previously reported compounds while also showing incredible specificity for the KIX domain over other similar coactivator domains. Further modification of the MybLL-tide with a nuclear localization signal and a cell penetrating peptide moiety yield a modified MybLL-tide capable of much greater cellular activity that potently modulates downstream gene expression and decreases the viability of AML cells. These promising results show that MybLL-tide can be an effective, modifiable tool to specifically target the KIX domain and assess transcriptional effects in both AML cells and potentially other cancer cells dependent on aberrant Myb or MLL behavior. Multivalency plays an important role in enhancing specific macromolecular interactions in the immune system of living organisms. Despite its physiological relevance, the accompanying kinetic and thermodynamic effects underpinning multivalent binding interactions are still somewhat unclear. Here, we aim to probe the avidity effects of Fab multimerization using a synthetic polymeric platform of an engineered protein-G variant (pGA1) orthogonal to an artificial Fab scaffold (LRT). pGA1 was obtained previously via phage display selection against a 4D5 Fab scaffold, while LRT resulted from the affinity maturation of the 4D5 Fab light chain using pGA1 as a target. We determined via Surface Plasmon Resonance (SPR) spectroscopy that multivalency improves the analyte-ligand lifespan by decreasing the dissociation rate constant of the complex, Koff, while leaving the association rate constant, Kon, unaffected. We observed a marked six-fold decrease in the KD value for dimeric pGA1 compared to monomeric, but only a two-fold further decrease for trimeric. Additionally, we observed that pGA1 polymer linker length (17 170 Å) does not exert a significant effect on binding kinetics. Together, these data suggest that valency, but not linker length, centrally impacts avidity. The potential applications are widespread for the high-affinity synthetic coupling of pGA1 and LRT. Our pGA1 polymer-based plug-and-play platform may be utilized to effectively increase the valency of LRT Fabs, thereby providing a straightforward strategy of avidity enhancement. Discovery of Allosteric Chaperones to Correct G6Pd (Glucose-6-Phosphate Dehydrogenase) Deficiency SUNHEE HWANG 1 1 STANFORD UNIVERSITY (STANFORD, United States) Glucose-6-phosphate dehydrogenase (G6PD) deficiency, one of the most common human genetic disorders, is caused by over 160 different point mutations and results in various health problems, including hemolytic anemia and neurological damage. As G6PD is a major source of protective anti-oxidants through NADPH production, G6PD deficiency likely contributes to the severity of many acute and chronic diseases associated with oxidative stress. As no medications are available to treat G6PD deficiency, we sought to identify a small molecule that corrects it. Through crystallographic study and mutagenesis analysis, we first identified the functional defect of one common mutant (Canton, R459L). Using high-throughput screening, we identified AG1, a small molecule that increases the activity of the wild-type, the Canton mutant and several other common human G6PD mutants. We confirmed that AG1 reduces oxidative stress and increases NADPH levels in vivo, in zebrafish, and decreases chloroquine-or diamide-induced oxidative stress in human erythrocytes. Our study suggests that a pharmacological agent, of which AG1 may be a lead, will likely alleviate the challenges associated with multiple variants of G6PD deficiency. Bruno Motta Nascimento 1 , Nikhil Nair 1 1 Tufts University (Medford, United States) Poly amino acid synthetases are a small group of enzymes capable of catalyzing the linkage of amino acids independently from ribosomes. Among these, the membrane-associated poly-gamma-glutamic acid (PGA) synthetase found in many Bacillus species can simultaneously polymerize and secrete long polymers of glutamic acid (1000 kDa) by linking alpha amine to the adjacent gamma carboxylate. Our work aims to change the specificity of this enzyme and catalyze the polymerization of different organic compounds, creating a microbial strain that can convert renewable substrates into polymeric materials that are currently produced only by chemical synthesis. First, we have heterologously expressed the PGA synthase subunits (B, C, A and E) in Escherichia coli and developed a semi-quantitative assay for fast detection of the polymer. Instead of randomly changing the substrate specificity of the enzyme, we also tried to elucidate some more information about its cell localization, structure, and reaction to foster our future work in building a mutational library. Sequence alignments show promising similarities with other peptide ligases such as murE and folC. This will help us identify and characterize residues that are relevant for the enzymatic activity. Tagged fusions of the enzyme with fluorescent proteins also indicate its presence at the inner membrane of E. coli. Succeeding, we would like to use some of the discovered putative sites for mutagenesis and asses for activity with the substrate. If successful, this research project will develop in the end a new microbial production platform for polymers with promising industrial applications. The Ohio State University (C, United States) Allostery pervades macromolecular function and drives cooperative binding of ligands to macromolecules. To decipher the mechanisms of cooperative ligand binding it is necessary to define at a microscopic level the structural and thermodynamic consequences of binding of each ligand to its allosterically coupled site(s). However, dynamic sampling of alternative conformations (micro-states) in allosteric molecules complicates interpretation of both structural and thermodynamic data. We study the RNA and TRp binding, un-decameric gene-regulatory protein TRAP from B. stearothermophilus, as a model for understanding how changes in protein structure and dynamics enable both homotropic and heterotropic allostery. We globally fit Trp-TRAP binding data (ITC, native MS, NMR) using a set of related nearest-neighbor statistical thermodynamic models over a range of temperatures and Wiseman c values (c = Affinity * titrand concentration). Fitting the ITC data to nearest neighbor statistical thermodynamic models yield good fits, allowed us to distinguish alternative nearest-neighbor interaction models, and quantified the thermodynamic contribution of neighboring ligands to individual binding sites. These results showed positive cooperativity for binding if one neighboring site was occupied, but no benefit to having both neighboring sites occupied. We also explore the resolving power of native mass spectrometry to better quantified the populations of TRAP with 0-11 bound Trp. Global fitting of mechanistically constrained models to experimental binding data has the potential to yield the microscopic thermodynamic parameters essential for detailing structural and dynamic basis of thermodynamic cooperativity in a wide range of ligand-regulated homo-oligomeric assemblies. Ras GTPases are involved in signal transduction and other cellular processes, including cell proliferation and survival. Ras functions through an on/off switch mechanism. The active GTP-bound state features an allosteric site that interacts with the active site via a water-mediated hydrogen-bonding network. Regulation of active Ras is achieved through its ability to access subtle, distinct conformational states. GTP-bound Ras samples two major conformational states: state 1, characterized by an open active site, and state 2 by a closed active site poised for effector interactions. This delicate balance differs among the isoforms and is affected by point mutations, leading to varying outcomes despite high sequence similarity. Notably, K-Ras oncogenic mutants are associated with colon and pancreatic cancer, while H-Ras is less frequent and seen in bladder and lung cancers. Previously, H-Ras was viewed as a good model for the other isoforms, and thus is represented prominently in the literature. To gain a greater understanding of K-Ras compared to H-Ras, we obtained 1-dimensional NMR signatures associated with switch I and II conformational states. Comparison of GDP and GTP-analog bound H-and K-Ras allow us to distinguish between the on/off states and examine exchange efficiency. Our data show that H-Ras prominently occupies state 2, while K-Ras is more dynamic with a conformational balance shifted toward state 1. However, the oncogenic mutant K-Ras G12D leans towards the state 2 conformation, favoring activation. Understanding the conformational ensemble of Ras and its differences among isoform and mutation is essential for targeted therapies of specific cancers. Much previous work in protein folding has centered on characterizing the difference in free energy between the functional, fully folded native state (N) and unfolded states (I, U). Recent work has highlighted the importance of kinetic barriers as well. The primary transition state barrier to unfolding (G) is important for keeping the protein in its functional form as well as protecting it from states vulnerable to degradation or aggregation. Still, for many proteins, there are other smaller barriers corresponding to motions of the native state that are critical to their normal function and regulation. Small-scale nativestate dynamics are typically measured using techniques that require high protein concentrations far from physiological conditions (NMR), or or require coupling to small molecules (FRET), potentially jeopardizing native state integrity. Here, we demonstrate the observation of such states using hydrogen exchange mass spectrometry (HXMS) under native conditions. In addition, we extract kinetic and thermodynamic parameters for these conformational excusions, in some cases simultaneously. This technique requires only picomoles of sample per acquisition and allows a range of sub-global dynamics to be observed in real time with high temporal resolution on the trypsin precursor, trypsinogen. Furthermore, we can extend the range of dynamics observed using the MS-compatible denaturant acetonitrile. Our nativestate HXMS results provide complementary insight to a suite of HXMS applications performed on a set of functional trypsin variants, attesting to the versatility and utility of the technique. Affinity reagents are invaluable tools for the modulation and detection of proteins. Ubiquitin has been developed as a robust scaffold for generating combinatorial protein libraries capable of yielding highly specific intracellular affinity reagents. Building on our previous successes with biased and naïve ubiquitin libraries, in which a large, contiguous surface of ubiquitin was diversified, we have developed a next-generation naïve ubiquitin library where further diversity is incorporated through the extension and randomization of two loops. Using this library, we are conducting binding selections against a diverse panel of human proteins to identify ubiquitin variants capable of interrogating and disrupting therapeutically relevant protein-protein interactions. Reiya Taniguchi 1 , Asuka Inoue 2 , Misa Sayama 3 , Keitaro Yamashita 4 , Kunio Hirata 4 , Masahito Yoshida 5 , Yoshiko Nakada-Nakura 6 , Yuko Otani 3 , Hideaki Kato 7 , Tomohiro Nishizawa 7 , Takayuki Doi 5 , Tomohiko Ohwada 3 , Ryuichiro Ishitani 7 , Junken Aoki 2 , Osamu Nureki 7 1 the university of Tokyo (Bunky-ku, Japan); 2 Laboratory of Molecular and Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University (Sendai, Miyagi-ken, Japan); 3 Laboratory of Organic and Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo (Tokyo-to, Japan); 4 RIKEN SPring-8 Center (Hyogo-ken, Japan); 5 Laboratory of Heterocyclic Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University (Miyagi-ken, Japan); 6 Department of Cell Biology, Graduate School of Medicine, Kyoto University (Kyoto-fu, Japan); 7 Department of Biological Sciences, Graduate School of Science, The University of Tokyo (Tokyo-to, Japan) Lysophosphatidic acid (LPA) is a bioactive lipid which consists of a phosphate group, a glycerol backbone, and a single acyl chain. LPA activates six class A G-protein coupled receptors (GPCRs) to provoke various cellular reactions. These LPA receptors are subdivided into the Edg (endothelial cell differentiation gene) family (LPA1LPA3) and the phylogenetically distant non-Edg family (LPA4LPA6). The previously reported LPA1 structure enhanced our understanding of the Edg family LPA receptors. In contrast, the functional and pharmacological characteristics of the non-Edg family LPA receptors have remained elusive, due to the lack of structural information. We recently reported the 3.2 Å crystal structure of LPA6, and clarified its ligand recognition mechanism. Surprisingly, its ligand binding pocket is laterally open toward the membrane, and the acyl chain of the lipid used for the crystallization is bound within this pocket, indicating the binding mode of the LPA acyl chain. Docking and mutagenesis analyses also indicated that the conserved positively-charged residues within the central cavity recognize the phosphate head group of LPA. Structural comparison with other related GPCR structures provided further insights into the structural similarity and diversity of GPCRs. In the poster, we would like to also introduce our recent efforts to further understand the ligand recognition mechanism of LPA6. Acute Hepatopancreatic Necrosis Disease (AHPND) is an emerging disease caused by a specific strain of Vibrio parahaemolyticus (Vp AHPND+), which contains PirA and PirB toxins encoded in the pVA1 plasmid. These toxins have been shown to cause the typical histological symptoms of AHPND in infected shrimp, but how this intoxication occurs and how these toxins participate in causing this disease are unresolved questions. Given the scarcity of information on this issue, it is important to study the interaction between PirA and PirB toxins and the epithelial cells of the hepatopancreas. Elucidating this process will make it possible to seek options for controlling outbreaks of intoxication in shrimp. We investigated the macromolecular interactions between these two toxins and once the complex is formed, we tested their effect on membrane component of the Hepatopancreas cells of Litopeneaus vannamei. We found that these two toxins can organize themselves in different oligomerization states, which can suggest that the mechanism of infection could be mediated through the way these toxins oligomerized. The ability to alter genes in mammalian cells enables elucidation of gene function and can highlight new therapeutic targets. To help resolve the genetic dependencies on the extracellular matrix (ECM), we conducted genome-wide CRISPR/Cas9 genetic screens in a human haploid cell line. These fitness screens identified genes Many applications in biotechnology require optimizing protein or peptide binding affinity and specificity for one target among many similar, evolutionarily related family members. For example, this is necessary to make specific detection reagents, or to inhibit one paralog that is implicated in disease without affecting others that are important for the maintenance of healthy cells. The problem is typically treated by screening large libraries of candidate solutions and addressing each specificity design challenge with a new set of experiments. A knowledge of the underlying protein landscape could open new avenues for design and ways to access functional regions in sequence space that are otherwise difficult to discover. We developed a protocol for using thousands of protein-peptide binding affinities, measured with SORTCERY, to parameterize models in a landscape of 3 members of the Bcl-2 family of apoptosis regulating proteins. We showed that models trained on experimental data have predictive ability on unobserved peptide sequences, and that optimization on a landscape defined by these models generates new peptides that are distinct from any previously known binders and have highly optimized interaction affinities and specificities. We generated 36 peptides that bind with high affinity and specificity to just one of Bcl-xL, Mcl-1 or Bfl-1, and additional peptides that can bind selectively to two out of three of these proteins. The successful designs demonstrate the power of this landscape-based design approach, and the resulting peptides have potential for use as diagnostics or therapeutic leads. Lariat-type macrocycles i.e. macrocycles containing a single major substituent are proposed to be a privileged class of compounds for targeting certain protein-protein interaction (PPI) sites, due to their large, structurally complex ring structure that complements a large protein surface, and the large substituent that occupies a nearby cleft or small pocket. Recently significant effort has been directed toward developing large synthetic macrocycle libraries, including some of lariat-type. Computational tools to design libraries for specific protein targets are therefore urgently needed. Unfortunately, computational efforts to dock large, complex macrocycles to protein targets have not yet been successful, presenting an obstacle to structure-based drug design using macrocycles. We describe the development of a computational approach for the virtual screening of lariat-type macrocycle libraries, and its validation using a set of such macrocycles for which co-crystal structures with their protein targets exist. In the method, the largest substituent of the macrocycle is isolated, the ring junction is capped, and the substituent fragment is docked into the target binding site. Analysis of the results for the test set of lariat-type macrocycle substituents found the correct substituent among the top docked poses for many of the complexes. We show that this method is particularly successful for complexes in which the substituent occupies the energetically most important hot spots in the binding site. We propose that this approach can be used to computationally filter macrocycle libraries, to select which substituents should be prioritized for inclusion to bind a particular protein target of interest. Cooperativity is a potent mechanism for fine-tuning the activity of regulatory molecules. However, understanding of cooperativity is still limited and it remains difficult to predict, based on structural information, the magnitude, or sign of cooperative ligand binding. The regulatory protein TRAP (trp RNA-binding Attenuation Protein) which assembles into a homo-undecameric (11-mer) ring can be liganded by up to 11 tryptophan molecules (Trp). The affinity of subsequent Trp ligations can be affected by previous bound Trp, which is the hallmark of cooperativity. These Trp binding events activate TRAP to bind the second set of ligands. If the binding state of adjacent Trp-binding pockets dominates cooperativity, a nearest-neighbor (NN) statistical thermodynamic model can be established and parameterized by fitting the temperaturedependent binding data with a binding polynomial that considers the 2^11 possible Trp-TRAP configurations, the intrinsic binding affinity, and coefficients corresponding to the NN interaction energies. The resulting microscopic parameters quantify the sign and magnitude of cooperativity. To validate the NN model obtained by data fitting, TRAP needs to be engineered into defined configurations in which NN effects cannot occur. Genetically linking two TRAP monomers together coerces TRAP to be assembled into cyclic 12-mer. By installing one mutant monomer that cannot bind Trp on each tandemly-linked dimer, the NN effect can be eliminated. Thus, only intrinsic binding affinity can be observed if NN effect dominates cooperativity. This work is expected to quantify the cooperativity of Trp binding accurately and provide a mechanistic modeling approach to understand cooperativity quantitatively in other oligomeric proteins. Rutgers University (Piscataway, United States) of varying characteristics. However, in the case of positive peptide recognition sequences, significant protein engineering of the OmpG nanopore was required to optimize intrapore electrostatic interactions. Here we show OmpG nanopore sensors containing the positively charged peptide recognition sequence LKKRG that have been developed for detecting West Nile Virus encoded NS2B-NS3 protease. By mutating 23 charged aspartate, glutamate, and arginine residues to neutral serine, threonine, and glycines, as well as adjusting the length of the OmpG peptide recognition loop, we were able to engineer a functional sensor with a positive peptide recognition sequence. Glucan phosphatases are a relatively a newly discovered family of enzymes central to the regulation of starch degradation in plants and glycogen metabolism in humans. Plants comprise three glucan phosphatases, Starch EXcess4 (SEX4), Like Sex Four2 (LSF2), and Like Sex Four1 (LSF1), which dephosphorylate starch. All three plant glucan phosphatases belong to the protein tyrosine phosphatase (PTP) superfamily and are classified in the subgroup of dual-specificity phosphatases (DSPs). However, little is known about the kinetics and regulatory mechanisms of phosphate removal of starch by SEX4, LSF2 and LSF1. Therefore, the focus of this project is to define the enzymology of SEX4 and LSF2. Dephosphorylating kinetic parameters will be obtained using the physiological substrate starch and the generic phosphatase substrate para-nitro phenyl phosphate (pNPP). Ras proteins are important in cell proliferation, inhibition of cell death, and cell transformation. Abnormal expression and function of these proteins are observed in many diseases including several forms of cancer. Cdc42 is a Ras-related protein involved in cell adhesion and cell cycle regulation. Ras protein abnormalities have proven difficult to target and inhibit but recent studies have highlighted potential inhibitors for Cdc42. Studies have found small molecules and miRNAs that can reverse the effects of Cdc42 in cells. However, much is still not understood about the effects of these inhibitors on the protein structure, stability, and function. In this study, a novel Cdc42 inhibitor called ZCL278 was studied. The binding affinity of ZCL278 to Cdc42 was calculated using molecular dynamics simulations, fluorescence titration, and isothermal titration. Changes in conformation and stability of the protein were observed using circular dichroism and fluorescence while exposing the protein to temperature, chemical, and proteolytic digestion. A Cdc42 mutant in the region thought to bind the inhibitor was also studied for comparison. using different methods, a binding affinity of ZCL278 to Cdc42 consistent with the literature was found. The studies also show that ZCL278 stabilizes the protein structure and makes it more resistant to denaturation. Molecular dynamics simulations also showed a number of residues that might be involved in the binding of the inhibitor. These studies further highlight ZCL278 as a potential Cdc42 inhibitor to be used to block Cdc42 abnormal activity and the effects it brings in the cell. Center for Computational Sciences, University of Tsukuba (Tsukuba, Japan) Translation elongation factor-1 alpha (eEF1A) or its paralog Elongation factor-like (EFL) interacts with an aminoacyl-transfer RNA (aa-tRNA) to play its essential role in elongation of amino acid residues during protein synthesis. Some species have been found to have both eEF1A and EFL (dual-EF-containing species). In the dual-EF-containing species, either eEF1A or EFL appeared to be highly divergent in the sequence. While the EFL or eEF1A proteins participate in the translation, the eEF1A or EFL homologues were predicted to play moonlighting functions. Homology modelling and surface analysis of eEF1A and EFL were performed to examine the hypothesis that divergent eEF1A does not strongly interact with aa-tRNA compared to the ordinary eEF1A. The subsequent molecular dynamics simulations were carried out to confirm the validity of modelled structures and to analyse the stability of them. It was found that the molecular surfaces of the both eEF1A in the dual-containing species were negatively charged at the docking site of aa-tRNA, and thus they might not interact with negatively charged aa-tRNA ( Fig. 1 (B) ) (Compare Fig. 1 (E) with (F) and (G) with (H)). The results obtained by molecular docking simulations between eEF1A or EFL and aa-tRNA also support the hypothesis. The present study will contribute to solving the mystery of proteins involved in translation. On top of academic impact, solving the molecular mechanism of translation will also contribute to drug discovery for the fact that many antibiotics have been targeting the translation machinery. Targeting the Beclin 1 Coiled Coil Domain with Stapled Peptides to Enhance Autophagy and Endolysosomal Trafficking Shuai Wu 1 , Yanxiang Zhao 1 , Yunjiao He 1 , Xianxiu Qiu 1 , Wenchao Yang 2 1 The Hong Kong Polytechnic University (Hung Hom, China); 2 Central China Normal University (Wuhan, China) Beclin 1 is a scaffolding molecule within the Beclin 1-Vps34 complex. Vps34 is the only Class III Phosphatidylinositol-3-kinase (PI3KC3) in mammals and a major producer of phosphatidylinositol 3-phosphate (PI3P) on membrane vesicles through phosphorylation of phosphatidylinositol (PI). The Beclin 1-Vps34 complex is indispensable for PI3P-related processes such as autophagy, phagocytosis and endolysosomal trafficking. Beclin 1 coiled coil domain plays a critical role in regulating the activity of the Beclin 1-Vps34 complex. It recruits modulators like Atg14L and UVRAG to form Atg14L-or UVRAG-containing Beclin 1-Vps34 complexes with enhanced activity. Our previous studies have shown that the Beclin 1 coiled coil domain forms an anti-parallel metastable homodimer with "imperfect" coiled coil interface. Beclin 1 readily transits from its metastable homodimeric state to form heterdodimeric complex with Atg14L or UVRAG. Guided with this knowledge we designed all-hydrocarbon stapled peptides that mimics the Nterminal part of the Beclin 1 coiled coil so that they can specifically interact with the C-terminal part to interfere with Beclin 1 homodimerization but does not block the interaction between Beclin 1 and Atg14L/UVRAG. Our in vitro and in vivo data confirm that these designed peptides can reduce Beclin 1 self-association, promote the Beclin 1Atg14L/UVRAG interaction, increase autophagy and enhance endolysosomal degradation of EGFR. These results testify to the strategy of enhancing Vps34-mediated autophagy and endolysosomal trafficking by targeting the Beclin 1 coiled coil domain to redistribute it from the self-associated functionally inactive form to Atg14L/UVRAG-containing complexes with enhanced Vps34 activity. Matthew Spence 1 , Joe Kaczmarski 1 , Nobuhiko Tokuriki 2 , Colin Jackson 1 1 Australian National University (Canberra, Australia); 2 University of British Columbia (Vancouver, Canada) Transcription factors regulate gene expression and are among the most complex and powerful proteins in nature. They have been studied extensively as model systems for a variety of biochemical phenomena and are valuable tools for regulating synthetic gene networks and metabolic pathways. Despite recent interest, the rational design of novel transcription factors for use in biotechnology has remained elusive due to the challenges of computational protein design. The study of molecular evolution can provide insight on the emergence of complex biophysical properties and aid in guiding protein engineering efforts. Here, we have investigated the evolution of the LacI/GalR Family of sugar-binding transcription factors by characterising the in vitro and in vivo functional properties of the Familys putative last common ancestor (AncTF), which was revived using ancestral sequence reconstruction. in vitro binding assays demonstrated that Anc3 was selective for disaccharides cellobiose, a-melibiose and sucrose, emphasizing the importance of promiscuity and selectivity in the evolution of novel protein function. We assayed the in vivo function of AncTF to reveal that it is functionally co-repressible, a result concordant with in vitro assays and the constructed phylogeny. Of equal importance to the evolutionary insights gained on the LacI/GalR Family, the development of an accurate phylogenetic tree, robust biophysical assays and novel reporter/selection methods as part of this work will form the platform for a range of future experiments to investigate the evolution of allostery and potentially produce novel bio-orthogonal transcription factors. PDZ domains play important rolls in anchoring receptor proteins and forming signal transduction complexes in a wide range of organisms. There are roughly 260 PDZ domains in humans alone, and these typically recognize their partner proteins by binding to their C-termini peptides. Despite much prior work, a general method for predicting whether and with what affinity a given peptide would associate with a given PDZ domain is lacking. Yet, an accurate and predictive computational model of PDZ-peptide association specificity would be of great utility towards understanding native signal transduction networks and rewiring them in a targeted fashion with peptides designed to inhibit individual PDZ domains. We propose a method that uses Molecular Dynamics (MD) simulations to compute an estimate of the free energy of PDZ-peptide binding. Each PDZ-peptide pair is initially docked based on similarity to known PDZ-peptide binding positions, and a number of parallel MD simulations thoroughly sample the bound state. As the interaction energy between domain and peptide is a first order approximation of its binding affinity, we use it to rank relative binding strengths to a given domain between tested peptides. The method is still in development as we consider several potential improvements, but initial results suggest that a general physics-based model of PDZ-peptide binding affinity and specificity may be within reach. Computationally Predicting Dynamics and Allostery using Tertiary Motifs Jack Holland 1 , Gevorg Grigoryan 1 1 Dartmouth College (Hanover, United States) Allostery in protein structures underpins many important biological interactions and processes. However, allostery often operates via subtle backbone adjustments and/or minor shifts in the distribution of conformations that a protein occupies, which makes experimentally measuring it difficult and expensive. We have recently shown that the universe of known protein structures is well described, at sub-Angstrom resolution, by a limited set of local structural motifs. These motifs, which we have dubbed TERMs, encompass secondary, tertiary, and quaternary structure and recur broadly across unrelated proteins. This project explores the question of whether allosteric conformational transitions can be explained and/or anticipated by considering how local structural variations, described by the natively observed variability within TERMs, can aggregate towards consorted rearrangements of the entire structure. We describe a procedure, which decomposes an experimental structure of a protein into its constituent TERMs, and generates a structural ensemble for the protein by recombining variant instances of these motifs. Here we present preliminary findings from examining such ensembles and comparing them to experimentally determined dynamics and allostery information. Chitin is a pervasive polymer used by a variety of organisms: it helps to form the exoskeleton of cockroaches, the cell wall of mushrooms, and the beak and pen of squid, among others. These sources mean that it is a consistent environmental element, accounting for up to 1% of indoor dust. Once inhaled, chitin triggers a specific inflammatory immune response, and chitinases (chitin-degrading enzymes) are upregulated to break down and clear the chitin. The inflammation caused by chitin has been associated with lung disorders including allergic asthma and fibrosis. Chitins insolubility, crystalline packing, and water repulsion features pose significant challenges for enzymatic hydrolysis. The primary overexpressed chitinase in the lungs, Acidic Mammalian Chitinase (AMCase), in part solves this problem via processivity. The mechanism underlying this processivity is unclear. Several different mutations of AMCase have been found in human populations that affect susceptibility to allergic asthma: some mutations make the enzyme more active and protect against asthma, while others decrease enzyme activity and increase asthma susceptibility. We are combining structural biology, single molecule microscopy, and activity measurement with simple and complex substrates to understand mechanically how AMCase degrades chitin and how mutations in human populations modulate that activity. In parallel, we are using a directed evolution approach to engineer hyperactive chitinases to investigate their efficacy in treating a variety of inflammatory lung disorders. Neisseria gonorrhoeae is an obligate human pathogen which is responsible for gonococcal infections, and which is a leading cause of transmissible infectious diseases worldwide. Gonococcal infections are currently treated using combination antibiotic therapy, yet the rate of their successful curing is decreasing. This is a result of the rapid development of antibiotic resistance by the pathogen, and this trend necessitates the development of alternative drugs or novel drug targets to combat these infections. Bacteria use lysine or meso-diaminopimelate to crosslink the peptidoglycan monomers in the bacterial cell wall. This pathway, while essential for the survival of most bacteria, is not found in mammals, which may allow antibiotics targeting it to be more selective to the pathogen. 4-hydroxy tetrahydrodipicolinate reductase (DapB), an important enzyme in the meso-diaminopimilate (lysine) biosynthetic pathway, is a promising target for the development of new antibiotics. Thus, we hypothesize that blocking the activity of DapB will induce defects in the bacterial wall similar to those caused by -lactam antibiotics [1] . This study describes the structural and functional characteristics of DapB from Neisseria gonorrhoeae as determined by protein crystallization and enzymatic function studies. [1] Pote, S., Pye, S., Sheahan, T., Majorek, K, & Chruszcz, M. (2018) . 4-hydroxy-tetrahydrodipicolinate reductase from Neisseria gonorrhoeae -structure and interactions with coenzymes. Manuscript in preparation. Estimating free energy of protein conformational changes play an essential role in many areas of biophysics such as enzyme activation, inhibitor specific binding, and allosteric effects. We introduce a novel method called Restrain -Free Energy Perturbation -Release (R-FEP-R) to estimate free energy of protein conformational changes via an alchemical pathway. The R-FEP-R method was developed based on the dual topology FEP scheme which is widely applied to ligand transformation in relative binding free energy calculation of two inhibitors binding to the protein target. In R-FEP-R, the free energy change between two conformational states is calculated by removing atoms of initial conformation and growing atoms of final conformation back simultaneously. Compared with other advanced sampling algorithms such as Umbrella Sampling and Metadynamics, the R-FEP-R method does not require pre-determined transition pathways or reaction coordinates that connect the two conformational states. As a first illustration, the R-FEP-R method was applied to calculate the free energy change between conformational states for alanine dipeptide in solution and for Val111 sidechain in the binding pocket of T4 lysozyme. The results of R-FEP-R agree with the benchmarks very well. Obstructive Sleep Apnea (OSA) is a common sleep disorder that causes partial or complete obstruction of the upper airway during sleep. The diagnosis involves a complete sleep evaluation and the most common treatment is a CPAP mask given to the patients to be worn while they sleep. The mask supplies positive air pressure preventing the blockage of their upper airway. The diagnosis and treatment are highly uncomfortable for the patients and about 85% of the people suffering from OSA remain undiagnosed. Previously, data obtained from rat atria showed that OSA leads to downregulation of proteins involved in aerobic respiration such as Malate dehydrogenase and upregulation of proteins in the cardiac muscle such as Filamin-C. Since insufficient oxygen is passed through the lungs, the proteins involved in aerobic respiration were downregulated, which in turn led to stress in the cardiac muscles. To compensate for the stress, there was an upregulation of proteins involved in cardiac function. Here, we performed a proteomic investigation using mass spectrometry to identify similar dysregulation patterns in the rat brain samples, which would further help us identify a protein biomarker signature for the better diagnosis and treatment of OSA. The proteomic analysis showed downregulation of proteins involved in aerobic metabolism as well as upregulation of those involved in the functioning of the brain such as Myelin basic protein, which functions in the insulation of nerves. Isaline Castan 3 , Rohith Nagari 4 United States); 3 Center for the Development of Therapeutics We recently reported a selective CK1 inhibitor that was discovered using the Connectivity Map. Initially, KINOMEscan ® was used to assess inhibitor selectivity, which was followed by in vitro testing with microfluidics assays to assess potency. To further improve on inhibitory properties, we attempted to characterize the binding mechanism of current lead compounds using X-ray crystallography. Large scale protein production and crystallization required extensive development of protein constructs, and optimization of expression and crystallization conditions. Following optimization, we have obtained crystal structures of CK1 and its homolog, CK1d. Both structures can be used as tools in rational drug design to inform on inhibitor selectivity The list of candidate genes includes novel regulators of ECM-cell interaction with a single-pass transmembrane protein, namely, immunoglobulin superfamily member 8 (IGSF8) being a major hit on several ECM components. The development towards an antibody panel against IGSF8 is currently underway in an effort to study IGSF8 function and ECM relevance. A naïve phage-displayed antibody library was used to conduct affinity-based selections on recombinant IGSF8 protein. As a result of these selections, unique, specific and potent binders for individual domains within IGSF8 have been developed in vitro Peptide Design by Optimization on a High-Dimensional, Data-Parameterized Protein Interaction Landscape Justin Jenson 1 , Vincent Xue 2 , Lindsey Stretz 1 United States); 2 MIT Computational and Systems Biology Program Nuclear magnetic resonance-based relaxation experiments, isothermal titration calorimetry, mutagenesis and adhesion assays reveal that Mg2+ functions as both a structural anchor and dynamic switch of the 11 integrin I domain (1I). Specifically, Mg2+ binding activates micro-to millisecond timescale motions of residues distal to the binding site, particularly those surrounding the salt-bridge at helix-7 and near the metal ion-dependent adhesion site. Mutagenesis of these residues impacts 1I functional activity, thereby suggesting that Mg-bound 1I dynamics are important for collagen binding and consequent allosteric rearrangement of the low-affinity closed to high-affinity open conformation. We propose a multistep recognition mechanism for 1I-Mg-collagen interactions involving both conformational selection and induced fit processes (1) Magnesium Activates Microsecond Dynamics to Regulate Integrin-Collagen Recognition Raf Promotes Ras Dimerization in a Time and Concentration Dependent Manner United States); 2 Department of Chemistry Through in vitro analysis we have identified the Ras binding domain (RBD) of Raf and phospholipid head group mimics, or supported membranes, as necessary for robust Ras dimerization. In solution we observe less than 10% of the Ras population forming dimers, but the majority of Ras forms dimers in the presence of Raf RBD and phospholipid headgroup mimics. Furthermore, we show using supported lipid bilayers that in the context of the membrane the dimerization of the Ras/Raf RBD complex is robust at low concentrations, indicative of a high dimerization Kd Protein nanopore sensors are protein molecules or oligomers that form pores in natural or artificial lipid bilayers, and can be examined using electrophysiology. Nanopore sensors are increasingly relevant in many fields of scientific study, including both basic and applied research, and are useful tools for sensing and studying protein analytes at the single molecule level. E. coli Outer membrane protein G (OmpG) nanopore sensors have been engineered to include chemical labels and peptide recognition sequences Angelo Kaldis 1 , Trevor Alexander 1 , Rima Menassa 1 1 Agriculture and Agri-Food Canada (London, Canada) Bovine Respiratory Disease (BRD), or Shipping Fever, is a multifactorial disease which results in high economic loss of feedlot cattle in North America. The goal of this project is to produce an oral vaccine in plants which provides cattle with protection against infection with Mannheimia haemolytica, the predominant bacterial agent causing BRD, through mucosal immunity. An injectable vaccine is available, but only provides systemic immunity. Constructs containing chimeric M. haemolytica antigens for nuclear (Nicotiana benthamiana) and chloroplast (Nicotiana tabacum) expression were produced and expressed. Each construct contains an N-terminal Cholera Toxin B element for mucosal immunogenicity fused to a modified virulence factor and an antigen of interest (AOI) from M. haemolytica. AOIs were engineered to consist of important epitopes and immunogenic sites of proteins from M. haemolytica. Constructs were transiently expressed and targeted to various subcellular compartments in N. benthamiana. Chimeric AOI-1 peaked in the endoplasmic reticulum at 2.9% of total soluble protein (TSP) or 400 mg/kg of fresh weight. Chimeric AOI-2 accumulation reached 0.02% of TSP in the vacuole or 0.7 mg/kg of fresh weight. Both AOI chimeras have been stably transformed into the Nicotiana tabacum chloroplast genome, accumulating to 2.6 mg/kg and 0.3 mg/kg of fresh weight, respectively. AOI-2 was also overexpressed in E. coli. While most of the protein produced was insoluble, a small portion could be purified from the soluble fraction. The immunogenicity these will be tested in a mouse model of BRD. University of Michigan (Ann Arbor, United States); 2 Technische Universität München (Garching, Germany); 3 Osaka University (Osaka, Japan) Small molecules have largely been studied for their ability to modulate disease related amyloidogenic peptides through inhibition of amyloid fibril growth, promotion of aggregation, or disaggregation of fibers. CurDAc, a previously described water-soluble stable curcumin derivative, has been shown to Cognitive Sciences, Daegu Gyeongbuk Institute of Science and Technology (DGIST) (Daegu, South Korea)The ABL 1 is proto-oncogene encoding non-receptor tyrosine kinase that is one of protein cause chronic myeloid leukemia (CML). Many Abl tyrosine kinase inhibitors was developed and marketed like imatinib, dasatinib, and so on. However, the issue of their side effect still is discussed. Our previous study reported that derivatives of 2-pyrazolinyl-1-carbothioamide (2PC) synthesized from chalcones showed inhibitory effects to Abl tyrosine kinase and their structure and activity relationships (SAR) were also analyzed by QSAR. In this study, we selected 11 derivatives of 2PC and predicted their molecular binding mode between 2PC and Abl kinase using in silico docking. And we carried out molecular dynamics (MD) simulations to identify the dynamic behaviors of Abl kinase upon the binding of 2PC. In addition, the binding free energy between Abl kinase and 2PC was calculated by various method including molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) and 3D-RISM. This study is expected to develop novel Abl tyrosine kinase inhibitors with low side-effects. Multidrug transcriptional repressors (MDTRs) serve as high affinity sensors of multidrug resistance systems, which control the expression of multidrug resistance (MDR) genes, such as multidrug transporters. However, the molecular mechanism by which MDTRs concomitantly achieve promiscuous yet high affinity compound binding to regulate the MDR system remains largely unknown. Here, we structurally and dynamically characterized a MDTR, LmrR, by solution NMR. We found that the compound ligation shifts the preexisting and energetically degenerated conformational equilibrium to varying extents to achieve multidrug recognition and transcriptional regulation. Meanwhile, the compound binding enhances psns dynamics in allosteric sites, which entropically contribute for the high-affinity binding of the compounds. We further found that the transcription regulation of LmrR is achieved through an equilibrium between the operator-bound and the non-specific DNA-adsorption states in the L. lactis genome. Together with the 2:1 binding stoichiometry between LmrR dimer and the operator, the effective operator with the non-specific DNA-adsorption is close to the endogenous LmrR concentration, which allows a substantial reduction of the LmrR occupancy at the operator site upon ligation to the compound. These NMR based quantitative dynamic analysis using site-specific isotope labeling revealed that dynamics is the key structural feature for MDTRs to express their function. The insulin/insulin-like growth factor signalling (IIS) axis is an evolutionary ancient and highly conserved hormonal system involved in the regulation of metabolism, growth, development and longevity in animals. Human insulin is stored in the pancreas, while insulin-like growth factor-1 (IGF-1) is maintained in During the Hsp90-mediated chaperoning of protein kinases, the core components of the machinery, Hsp90 and the cochaperone Cdc37, recycle between different phosphorylation states that regulate progression of the chaperone cycle. We show that Cdc37 phosphorylation at Y298 results in partial unfolding of the C-terminal domain and the population of folding intermediates. Unfolding facilitates Hsp90 phosphorylation at Y197 by unmasking a phosphopeptide sequence, which serves as a docking site to recruit non-receptor tyrosine kinases to the chaperone complex via their SH2 domains. In turn, Hsp90 phosphorylation at Y197 specifically regulates its interaction with Cdc37 and thus affects the chaperoning of only protein kinase clients. In summary, we find that by providing client class specificity, Hsp90 cochaperones such as Cdc37 do not merely assist in client recruitment but also shape the post-translational modification landscape of Hsp90 in a client class-specific manner. Transcriptional coactivators have emerged as exciting opportunities for the development of new therapeutics. For example, the interaction between the transcription factor Myb and the KIX domain of the master coactivator CREB binding protein (CBP) is required for the progression of acute myeloid leukemia, making this protein-protein interaction (PPI) a promising target for therapeutic development. However, the conformational lability of the KIX domain and the lack of well-defined pockets pose major challenges for the development of small molecule inhibitors. Here, we have used a series of screens against homologous KIX motifs to identify the natural product garcinolic acid which is a potent inhibitor of the CBP KIX-Myb PPI. Treatment of leukemia cell lines with low micromolar doses of garcinolic acid results in downregulation of Myb-dependent genes and decreased cell viability. These results demonstrate the utility of our multiple screening approach for identifying modulators of conformationally dynamic targets, and garcinolic acid will be a valuable tool for evaluating the role of critical KIX-activator interactions in cancers.