Hydrophobic Burial and Dynamic Conformations of Estrogen Receptor N-Terminus

Monday, February 13, 2017 280 K to 460 K were used to monitor the globule to coil transition for our two peptide models. Our data show a stabilization of the globule form of AK42_PSDP to higher temperatures when monitoring the radius of gyration (Rg) compared to that of AK42. We will also report on the position of chain reversals using clustering algorithms which allow us to see prominent structures at each temperature and thus the influence of the PSDP sequence on the narrowness of the conformational distribution of the alanine-based peptide. 973-Pos Board B41 Degradation of Calponin 2 is Required for Cytokinesis Dipak Maskey, Airong Qian, Jian-Ping Jin. Department of Physiology, Institute of Medicine, Detroit, MI, USA. Calponin 2 is an actin filament-associated protein and plays a role in regulating cytoskeletal functions such as cell adhesion, migration, phagocytosis and cell division. We previously showed that the level of calponin 2 is inversely correlated with the rate of cell proliferation. However, the mechanism by which calponin 2 is down-regulated during cytokinesis is unknown. In the present study, we investigate the levels of endogenous calponin 2 in NIH/3T3 cells and HEK293 cells decreased prior to cell division characterized by an absence in the F-actin-based contractile ring. Nocodazole-synchronized NIH/3T3 cells showed decreased calponin 2, and MG132, a ubiquitin proteasomal inhibitor (UPS), increased the overall calponin 2 level in the cells post nocodazole treatment. The results suggest that M phase cells have a downregulation of calponin 2, which is likely mediated through UPS. Furthermore, GFP-fusion calponin 2 was constructed for dynamic studies in live cells. Control experiments using NIH/3T3 cells and HEK293 cells showed that non-fusion and GFP-fusion calponin 2 had minimal difference in cytoskeleton association and effects on cell proliferation. A smooth muscle-originated cell line SM3 lacking endogenous calponin was transiently transfected with GFP-calponin 2 expression plasmid and imaged using fluorescent microscopy at a series of time points. The results showed that 1) the level of calponin 2 decreased prior to cell division, especially at the contractile ring; 2) the overall level of calponin 2 expression had an inverted correlation to the duration of cytokinesis; and 3) cells had high level ectopic expression of calponin 2 could not complete cytokinesis, which was followed by cell death. The data suggest that degradation of calponin 2 is a factor determining the rate and fate of cytokinesis, which may be targeted to control cell proliferation during development, wound healing, inflammation, pathological remodeling, and tumor growth and metastasis. 974-Pos Board B42 Insulin Structure and Stability Assessed by Intrinsic Fluorescence and Simultaneous UV-vis Absorbance Spectroscopy Coupled with Chemometric Analysis Karen E. Steege Gall1, Marinella Sandros2. 1 Fluorescence Division, HORIBA Scientific, Edison, NJ, USA, 2HORIBA Scientific, Edison, NJ, USA. Stability and aggregation of insulin is studied using simultaneous fluorescence excitation emission matrices (EEMs) and UV-vis absorbance spectroscopy. Insulin is a protein-hormone, produced by the pancreas and is necessary for basic metabolic processes. The different types of insulin therapeutics, used to treat approximately 1.25 million people in the US with Type 1 Diabetes, generally fall into two categories: short-acting and long-acting insulin. The difference between short-acting and long-acting insulin is one residue in the protein sequence. This residue change, along with controlled pH of storage and delivery, is used to either trigger or prevent the formation of insulin dimers and hexamers in the blood stream. The formation of these aggregates lets the body absorb insulin more slowly and the absence of aggregates makes it absorb more quickly. Changes in protein stability and structure, such as those important to the pharmacokinetics of insulin, are often measured using fluorescence emission spectra, UV-vis absorbance spectra or sometimes both, using intrinsically fluorescent amino acids. Furthermore, UV-vis spectrophotometers and fluorometers are typically separate instruments. A novel instrument will measure both techniques simultaneously. Fluorescence EEMs are used in various fields to track changes in complex mixtures including water quality and food science, but the use of EEMs in biotherapeutics is relatively new and still being explored. In this study, the stability and structural differences of two commercially available insulin formulations are shown using fluorescence EEMs and simultaneous UV-visible absorbance spectroscopy. Using a novel instrument, absorbance spectra are simultaneously collected with each EEM to correct for non-linearities in fluorescence intensity, specifically resulting from innerfilter effects at high absorbance solutions. Component analysis from these automatically corrected fluorescence EEMs will be presented for tyrosine and

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phenylalanine residues between the two types of insulin, varying in pH and structure. 975-Pos Board B43 Macromolecular Crowding Effects on Pressure-Induced Protein Folding/ Unfolding Andrei G. Gasic1,2, Dirar Homouz1,3, Margaret S. Cheung1,2. 1 Physics, University of Houston, Houston, TX, USA, 2Center for Theoretical Biological Physics, Rice University, Houston, TX, USA, 3Department of Applied Mathematics and Sciences, Khalifa University, Abu Dhabi, United Arab Emirates. In the interior of a cell, protein folding occurs in a highly crowded environment. It is still unclear how excluded volume from macromolecules affects folding/unfolding transitions. To gather local features of the folding landscape, we use high hydrostatic pressure to unfold the protein in our coarse-grained simulation. Pressure perturbs the protein heterogeneously, which greatly facilitates characterization of the folding mechanisms. Our model uses a mean field approach to account for pressure by adjusting the parameters describing the native stabilizing interactions and the desolvation barrier. Macromolecular crowding agents were modeled as hard spheres to mimic the cell-like environment. We provide theoretical incite into the mechanism of pressure-denaturation in highly crowded conditions, and show water gradually penetrating the hydrophobic core over a wide range of pressures. Furthermore, this study takes us one step closer in understanding the ultimate goal of protein folding in vivo. This research was funded by the National Science Foundation, MCB-1412532, PHY-1427654 and ACI1531814. 976-Pos Board B44 Examining the Stability of b-Sheets using the Charmm Drude Polarizable Force Field Anthony Hazel, James C. Gumbart. Georgia Institute of Technology, Atlanta, GA, USA. b-Sheets are some of the most common secondary structure motifs in proteins, and are important for mediating protein-protein interactions through their association. This association can also lead to the aggregation of misfolded proteins into b-pleated-sheets in neurodegenerative disorders like Alzheimer’s disease. Fixed charge, all-atom molecular dynamics simulations have adequately reproduced the folding free energy landscape of a small b-hairpin, the GB1 domain of protein G, which is a prototype for a larger b-pleated-sheet. Polarizable force fields should, in theory, be able to more accurately reproduce experimental results as they are a more realistic model of the molecular system. The CHARMM Drude model is a relatively efficient polarizable force field that has been shown to perform as well or better than traditional nonpolarizable force fields in reproducing helical content and folding mechanisms of model a-helical peptides. To assess the model’s accuracy for b-sheets, we examined the stability of the GB1 b-hairpin using the CHARMM Drude polarizable force field and two non-polarizable force fields, CHARMM36 and CHARMM22*. Two-dimensional replica exchange umbrella sampling (REUS) simulations show that the b-hairpin is unstable in the Drude system, whereas it is either stable or quasistable in CHARMM36 and CHARMM22*, respectively. The instability in Drude appears to be driven mostly by interactions between the peptide backbone and the surrounding water molecules. By tuning these interactions, we have shiftedthe stability back towards the b-hairpin state. 977-Pos Board B45 Hydrophobic Burial and Dynamic Conformations of Estrogen Receptor N-Terminus Sichun Yang, Yi Peng. Department of Nutrition, Case Western Reserve University, Cleveland, OH, USA. Estrogen receptor (ER) a, a key driver of breast cancer growth, consists of an unstructured transactivation domain located at its N terminus that is critical for hormonal activation. This N-terminal domain (NTD) is viewed as an intrinsically-disordered, dynamic system to fold by a natural osmolyte TMAO, but how TMAO stabilizes the NTD folding and conformation is unclear. We found that TMAO-induced folding of the ERa NTD in response to limited thermolysin digestion results in large-scale structural changes involving in an 18-residue fragment of V141-R158 that is resistant to cleavage. Sequence identification of this NTD fragment reveals four hydrophobic residues (V141, A144, A148 and F149) that are buried and not prone to thermolysin digestion. Molecular dynamics simulations of the NTD-TAMO system show that the NTD adopts a diverse network of conformers, but further generate a best-fit ensemble of structures by maintaining the hydrophobic residues buried. These

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results identify large-scale structural burial of four key hydrophobic residues toward its C-terminal end and provide a molecular view of its dynamic structure-ensemble at the TMAO-induced folded state of this intrinsically disordered transactivation domain of ERa.

Protein Assemblies I 978-Pos Board B46 Studying Peptide Aggregation using Mixed All-Atom/Coarse Grain Molecular Dynamics Simulations John C. Shelley1, Mee Shelley1, Myvizhi Esai Selvan2, Jun Zhao3, Volodymyr Babin1, Chenyi Liao4, Jianing Li5. 1 Development, Schrodinger, Inc., Portland, OR, USA, 2Development, Schrodinger, Inc., New York, NY, USA, 3Computational and Structural Biology Section, Cancer and Inflammation Program, National Cancer Institute, NIH, Frederick, MD, USA, 4Chemistry, University of Vermont, Burlington, VT, USA, 5Chemisstry, University of Vermont, Burlington, VT, USA. Peptide-peptide and peptide-excipient interactions need to be taken into account in biologics formulations. With the desire to formulate at very high peptide concentrations to improve patient experience and compliance, peptide-peptide interactions can lead to very high viscosity, denaturation, aggregation and precipitation. These problematic behaviors can complicate and slow down drug development, sometimes forcing compromises on target characteristics for the drug. We are exploring the use of molecular dynamics simulations to provide a molecular level understanding of peptide association. Our mixed all-atom/coarse grain model represents the protein using an allatom representation with full flexibility while using a simplified coarse grain model for the environment. This approach permits a modest increase in system size and a significant increase in simulation speed relative to all-atom simulations while retaining the ability to study conformational changes of peptides in response to their environment. Such mixed resolution modeling approaches seem ideally suited to studying peptide-peptide association. We present results from a study of the aggregation of melittin, the predominant peptidic component of bee venom, to illustrate the potential for this approach. This approach yields the expected variation of aggregate size with the introduction of salt and provides residue level information. These results suggest that this model should also be useful for studying excipient effects in peptidic solutions. 979-Pos Board B47 Molecular Mechanism of Surface-Assisted Self-Assembly of Amyloid-Like Peptides Seung-gu Kang. IBM Thomas J. Watson Research Center, Yorktown Heights, NY, USA. Peptide self-assembly attracted huge interests due to its clinical importance in protein-aggregation diseases, as well as potential applications for novel material design. Large efforts have been exerted on unraveling factors for the growth dynamics and morphologies. However, an intrinsic delicacy present in molecular systems and environmental elements, such as electrophysical properties of surface and solution, often results in controversy. Recently, a 9-residue long peptide designed from consensus sequences of amyloidogenic proteins was shown to assemble into a highly-ordered epitaxial structure on both hydrophilic mica and hydrophobic highly-oriented pyrolytic graphite (HOPG) surfaces but with very different morphologies: upright conformations on mica and lying-down on HOPG. Furthermore, with raised salt concentrations the peptides formed a highly-ordered multilayered nanofilaments at the mica/water interface in highly controllable fashion, which is unusual compared to uncontrollable, disordered, amorphous aggregates observed in other proteins in the similar condition. In our study, we investigated in molecular level details on how various environmental factors determine the peptide assemblies on mica and HOPG surfaces from their morphologies to the epitaxial growth mechanism, using atomistic molecular dynamics simulations. Firstly, not only surface polarity but also surface structure closely incorporate with individual peptide structure, as synergistically inducing a highly-optimized parallel b-stranded arrays on mica. Next, we showed that hydrophobic sidechain interaction indeed drives the longitudinal length growth of the assembly, while hydrophilic backbone hydrogen bonds rather control the transversal thickness. Finally, in raised salt concentration, we found that double-layered structures of all upright conformation are energetically favorable on mica but with anti-parallel b-stands for the upper layer, while the lower still in the parallel configuration, which again emphasizes the importance of environmental factors like contacting surface.

980-Pos Board B48 Variable Binding of Thioflavin T by Amyloid Fibrils Hiroaki Komatsu1, Claire Meurice1, Giuseppe Grasso1,2, Lisa G. Lippert3, Yale E. Goldman3, Paul H. Axelsen1. 1 Pharmacology, University of Pennsylvania, Philadelphia, PA, USA, 2 Scienze Chimiche, Universita` degli Studi di Catania, Catania, Italy, 3 Physiology, University of Pennsylvania, Philadelphia, PA, USA. Thioflavin T (ThioT) fluorescence is commonly used to quantify amyloid formation in vivo and in vitro, but its binding mode and basis for fibril selectivity is not understood, and there is no consensus on the relationship between fluorescence intensity and fibril mass. To determine whether the rate of fibril formation affected ThioT fluorescence, fibrils of amyloid beta (Ab) proteins were formed at different concentrations of the monomeric protein. The intensity of ThioT fluorescence increased with the monomer concentration during fibril formation. A similar dependence on monomer concentration was observed for insulin fibrils. The affinity of the dye for Ab fibrils formed at different rates was assessed by surface-plasmon resonance (SPR), indicating that fibrils grown from more concentrated monomer solutions bound larger amounts of ThioT per mass of Ab, consistent with the steady-state fluorescence results. However, the dissociation constants and the affinity of individual binding sites were independent of the monomer concentration of Ab proteins. The orientation of ThioT in Ab fibrils was evaluated with polarized fluorescence microscopy, and found to be aligned toward the long axis of the fibril. On the basis of these results, it is proposed that ThioT binds to localized polymorphic sites along the length of a fibril, rather than to well-defined binding sites in a mature fibril with regular structure. 981-Pos Board B49 The Levinthal Problem in Amyloid Aggregation: Identification of Good Coordinates in a Flat Reaction Space Zhiguang Jia1, Jianhan Chen1, Jeremy D. Schmit2. 1 Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS, USA, 2Department of Physics, Kansas State University, Manhattan, KS, USA. Successful protein folding in physiological timescales requires a biased free energy landscape to restrict the otherwise prohibitive search space. The existence of this bias is a necessary outcome of evolution since sequences that fold slowly do not contribute to the fitness of the organism. In contrast, the evolutionary pressure for efficient folding pathways is not present in the formation of pathological aggregates. Accordingly, the measured growth rates for amyloid fibrils are much slower than might be expected for the formation of beta sheets. Analytic theory shows that fibril growth rates are consistent with a random search over the alignments of intermolecular H-bonds and that solution conditions that accelerate this search (i.e. weakening bonds) can increase aggregation rates. This theory identifies two reaction coordinates, the alignment between molecules and the number of formed H-bonds, which we use to devise a novel Markov State Model to simulate fibril growth in atomistic detail. This model is used to simulate the growth of beta amyloid (16-22) and three mutants. The simulations qualitatively capture the non-additive effects of the mutations, but interestingly there is no obvious trend to the mutation effects in the lifetimes of the molecular alignments or individual H-bonds. Instead, the changes in the growth rate emerge from the accumulation of many small perturbations over a large ensemble of trajectories. We conclude with a discussion of theory development with an eye towards the simulation of very slow processes like fibril nucleation. 982-Pos Board B50 Prediction of Protein Aggregation Propensities using GOR Method Maksim Kouza1, Girik Malik1, Eshel Faraggi2, Andrzej Kolinski3, Irina Buhimschi4, Andrzej Kloczkowski1. 1 Battelle Center for Mathematical Medicine, Nationwide Children’s Hopital, Columbus, OH, USA, 2Indiana University School of Medicine, Indianapolis, IN, USA, 3University of Warsaw, Warsaw, Poland, 4Center for Perinatal Research, Nationwide Children’s Hopital, Columbus, OH, USA. The original GOR method published by Garnier, Osguthorpe, and Robson in 1978 was one of the first successful methods to predict protein secondary structure from amino acid sequence [1]. The method is based on information theory. The analysis of frequencies of occurrence of secondary structure for singlets and doublets of residues in a protein database enables prediction of secondary structure for new amino acid sequences. Because of these simple physical assumptions and the ability to predict the probability of formation of betastrands for each residue in protein sequence GOR method has a conceptual advantage over other later developed methods such as PHD, PSIPRED, and others that are based on Machine Learning methods that have a ‘‘black box’’ nature. The GOR method has been continuously improved and modified for