Multi-Color Single Molecule FRET Study of Intrinsically Disordered Protein Binding

Wednesday, March 2, 2016 of nontoxic amylin monomers into cytotoxic oligomers are not very clear. Studies based on Amyloid-b (Ab), a peptide with similar size, have suggested that a conformational transition along the aggregation pathway makes the oligomers of Ab membrane-binding competent (1). We have explored the possibility of amylin undergoing a similar transition during aggregation as it could shed light on potential commonalities in the conformation and function of different toxic amyloid species. Using fluorescence correlation spectroscopy, we identify two distinct oligomers of amylin along the aggregation pathway having hydrodynamic radius of 0.90 nm and 1.6 nm respectively. The membrane affinity of amylin increases remarkably from ~15 % in smaller species to ~ 85% in the larger one as assessed by an in vitro membrane-binding assay developed in our lab (2). We observe similar difference in the cell membrane attachment ability of these two species in RIN5mf cell lines using confocal microscopy. A preliminary conformational study in artificial lipid bilayers using a SERS based methodology (3) suggests a temporal conformational reorganization in the peptide backbone. Our data suggest that amylin might acquire toxic function by a mechanism which depends on similar conformational features as Ab in presence of membranes. Further studies aimed at obtaining highresolution structural details of amylin oligomers in solution and in membranes are currently in progress. References: 1. Nag S, et al. (2013) Phys. Chem. Chem. Phys. 15:19129-33. 2. Bhowmik D, et al. (2015) Langmuir. 31(14):4049-53. 3. Bhowmik D, et al. (2015) ACS Nano. 9(9):9070-7. 2736-Pos Board B113 Amyloid-b(1-42)Oligomer Models Developed using Combined Solid State NMR and Sequence Specific Hydroxyl Radical Footprinting Data Alexandra Klinger1, Cong Guo2, Huan-Xiang Zhou2, Anant Paravastu3, Janna Kiselar4, Andrew J. Nix5, Terrone L. Rosenberry5. 1 DecipherBio, Wyndmoor, PA, USA, 2Florida State University, Tallahassee, FL, USA, 3FAMU & FSU College of Engineering, Florida State University, Tallahassee, FL, USA, 4Center for Proteomics, Case Western Reserve University, Cleveland, OH, USA, 5Mayo Clinic, Jacksonville, FL, USA. Increasing evidence suggests that soluble aggregates of amyloid-b (Ab) are the pathogenic species in Alzheimer’s disease (AD). However, detailed structural information on these species remains scarce due to low levels of endogenous Ab oligomers and uncertainties surrounding current in vitro model systems. Herein, we describe a hydroxyl radical footprinting (HRF) study of Ab42 monomers, dimers, and specially prepared stable and homogeneous oligomers. Specific side chain solvent accessibilities of individual residues in the folded and aggregated forms of Ab42 are measured with respect to the same residues of Ab42 in a fully exposed reference state. These data provide residue specific side chain solvent accessibility protection factors and are used in complement with biophysical characterizations and ss-NMR analyses of the same systems. Results are discussed in the context of proposed NMR models of Ab oligomers with implications towards further development of therapeutic and diagnostic strategies.

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folding changes the RD structure, which could ultimately lead to a better understanding of how CaM activates CaN. 2738-Pos Board B115 Protein Design for Decreased Disorder: SHERP as an Exemplar Protein Elliot Drew1, David T. Jones2, B.A. Wallace1. 1 Institute of Structural and Molecular Biology, Birkbeck College, University of London, London, United Kingdom, 2Computer Science, University College London, London, United Kingdom. Disorder-to-order transitions are the basis for the promiscuity and diversity of many interactions seen in intrinsically disordered proteins (IDPs), leading to the ubiquity of intrinsic disorder in signalling and regulatory proteins - and thus their important role in many diseases. However, the complexity of IDP dynamics presents a unique challenge to the structural characterisation of these proteins and huge hurdles still exist when designing drugs to affect IDPs. Using the Small Hydrophilic Endoplasmic Reticulum Associated Protein (SHERP) from the parasite L. major, protein design principles have been applied to explore the protein’s disorder-to-order transitions when in the presence of anionic lipids and detergents both computationally and in vitro. A number of mutations were identified which were predicted to significantly decrease protein disorder by both sequence-based methods and molecular dynamics. This has allowed for the design, expression and characterisation of mutant proteins by Synchrotron Radiation Circular Dichroism (SRCD) which decrease disorder while preserving key features of the wild-type ordered structure. Supported by: MRC Fellowship, BBSRC project grant, beamtime grants from ISA, ANKA.

Investigating the Properties of Intrinsically Disordered Proteins

2739-Pos Board B116 Structural Models of an Intrinsically Disordered Protein Adapted for Bacterial Secretion Darragh Patrick O’Brien1, Belen Hernandez2, Dominique Durand3, Veronique Hourdel1, Ana Cristina SotomayorPe´rez1, Patrice Vachette3, Mahmoud Ghomi2, Julia Chamot-Rooke1, Daniel Ladant1, Se´bastien Brier1, Alexandre Chenal1. 1 Structural Biology and Chemistry, Institut Pasteur, Paris, France, 2 Universite´ Paris XIII, Paris, France, 3Universite´ Paris-Sud, Orsay, France. Many Gram-negative bacteria use Type I secretion systems, T1SS, to secrete virulence factors that contain calcium-binding Repeat-in-ToXin (RTX) motifs. Here, we present structural models of an RTX protein, RD, in both its intrinsically disordered calcium-free Apo-state and its folded calcium-bound Holostate. Apo-RD behaves as a disordered polymer chain comprising several statistical elements that exhibit local rigidity with residual secondary structure. Holo-RD is a folded multi-domain protein with an anisometric shape. RTX motifs thus appear remarkably adapted to the structural and mechanistic constraints of the secretion process. In the low calcium environment of the bacterial cytosol, Apo-RD is an elongated disordered coil appropriately sized for transport through the narrow secretion machinery. The progressive folding of Holo-RD in the extracellular calcium-rich environment as it emerges form the TISS may then favor its unidirectional export through the secretory channel. This process is relevant for hundreds of bacterial species producing virulent RTX proteins.

2737-Pos Board B114 NMR Investigation of Calmodulin Induced Folding in the Regulatory Domain of Calcineurin Dinesh K. Yadav. Chemistry, Mississippi State University, Mississippi State, MS, USA. Calcineurin (CaN) plays an important role in the T-cell activation, cardiac system development and nervous system function. Previous studies have suggested that the 97-residue regulatory domain (RD) of CaN binds Calmodulin (CaM) towards the N-terminal end. Calcium/Calmodulin activates the serine/ threonine phosphatase activity of CaN by binding to the regulatory domain, although the mechanistic details of this interaction remain unclear. It is thought that CaM binding at the RD displaces the auto inhibitory domain from the active site of CaN, activating phosphatase activity. In the absence of calcium-loaded CaM, the RD is at least partially disordered, and binding of CaM induces folding in the RD. Previous studies have shown that an a-helical structure forms in the N-terminal half of the RD, but organization may occur in the C-terminal half as well. Here we are interested in the structural transition of the full length RD as it binds to CaM. Using nuclear magnetic resonance (NMR) spectroscopy, we have successfully assigned >85% of the 15N, 13Ca, 13 b C and HN chemical shifts of the unbound, full-length regulatory domain of CaN. While the protein is unstructured, secondary chemical shifts indicate some regions with a-helical propensity, even in the free state. At present, we are studying how the spectrum changes as calcium-loaded CaM is added to the solution. In the long term, this work will identify how binding-induced

2740-Pos Board B117 Multi-Color Single Molecule FRET Study of Intrinsically Disordered Protein Binding Hoi Sung Chung, Fanjie Meng, Jae-Yeol Kim, John M. Louis. Laboratory of Chemical Physics, NIDDK/NIH, Bethesda, MD, USA. Intrinsically disordered proteins (IDPs) are unstructured at the native condition and fold when attaching to their binding partners. Understanding the mechanism of this process requires probing conformational changes of IDPs during binding processes. Since multiple binding pathways should exist as protein folding, single-molecule spectroscopy is expected to provide unique information such as the distribution of binding pathways. In order to probe conformational changes of IDPs and their interactions with binding targets simultaneously, it is necessary to obtain the distance information between more than two fluorophores. In this work, we performed three-color FRET spectroscopy to study the oligomerization of the tetramerization domain (TD) of the tumor suppressor protein p53. Two monomers of TD form a dimer at low nM concentration and subsequently two dimers form a tetramer at higher concentration. In the dimerization experiment, one monomer TD construct was labeled with Alexa 488 and Alexa 647 and immobilized on a PEG-coated glass coverslip via a biotin-streptavidin linkage. Another TD construct was labeled with Alexa 750 as a binding partner in solution. Using the alternating excitation of two picosecond-pulsed lasers (485 and 640 nm) at 40 MHz, it was possible to detect all three FRET efficiencies between three fluorophores. In addition, from the average delay times between photon arrivals and laser excitation, the

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fluorescence lifetime of each state was measured. We determined the dissociation constant and the dissociation rate of the dimer, which are consistent with the previous measurements by fluorescence correlation spectroscopy. More importantly, we observed TD monomer conformations in the dimer that are different from that in the tetramer. The faster formation of the tetramer than the dissociation of the dimer at high TD concentration suggests that these dimers with different conformations should be on pathway intermediates during the tetramerization. 2741-Pos Board B118 Characterization of an Intrinsic Disorder Domain and Functional Activity of ChiZ Membrane Protein from Mycobacterium tuberculosis Cristian A. Escobar1,2, Riqiang Fu2, Timothy A. Cross1,2. 1 Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, USA, 2National High Magnetic Field Laboratory, Tallahassee, FL, USA. ChiZ is a membrane protein from Mycobacterium tuberculosis (MTB) that is involved in cell division. This protein has an intracellular domain that is known to be intrinsically disordered. Interestingly, this cytoplasmic domain is able to hydrolyze peptidoglican (PG) in in vitro assays. ChiZ also contains a C-terminal LysM domain for PG binding in the periplasm. ChiZ is an interesting target for structural characterization and drug development because it is involved in cell division. This work mainly focuses on the nascent structural characterization of the intrinsically disorder region of ChiZ (ChiZ1-64) using solution NMR in addition to biochemical assays for understanding its function. Biochemical assays indicate that ChiZ1-64 is able to hydrolyze PG at pH 4.0 and 7.0. As expected, solution NMR experiments of ChiZ1-64 show a typical spectrum of an unfolded protein. However, HSQC spectra at these two pHs indicate the presence of two distinct regions with different backbone dynamics and signal intensity. Sequence alignment of ChiZ homologs found in other species of Mycobacterium also shows two different regions, one of variable length (residues 1 to 37 in MTB), and another conserved region (residues 38 to 64 in MTB). Both conserved and variable regions match to the regions identified with solution NMR experiments. Removal of the variable region in ChiZ produces an active protein (ChiZ38-165) at low pH. This suggests that the conserved region is enough to hydrolyze PG. However, the variable region appears to be important for full activity at neutral pH. Additional binding assays of ChiZ1-64 show that this region is able to bind PG. This is demonstrated by the depletion of the protein from solution and the detection of protein using solid state NMR of PG and ChiZ1-64 in complex. 2742-Pos Board B119 Experimental Polyproline II Propensities Describe Sequence-Dependent Variability in the Hydrodynamic Size of Intrinsically Disordered Proteins Steven T. Whitten. Chemistry and Biochemistry, Texas State University, San Marcos, TX, USA. The properties of disordered proteins are thought to depend on intrinsic conformational propensities for polyproline II (PPII) structure. While intrinsic PPII propensities have been measured for the common biological amino acids in short peptides, the ability of these experimentally determined propensities to quantitatively reproduce structural behavior in intrinsically disordered proteins (IDPs) has not been established. Presented here are results from molecular simulations of disordered proteins showing that the hydrodynamic radius (Rh) can be predicted from experimental PPII propensities with good agreement. The simulations demonstrate that Rh and chain propensity for PPII structure are linked via a simple power-law scaling relationship, which was tested using the experimental Rh of 22 IDPs covering a wide range of peptide lengths, net charge, and sequence composition. The relationship between Rh and PPII structure was also tested by glycine substitutions applied to a prototypical IDP to demonstrate a method for measuring PPII propensities in disordered proteins. Overall, the results from these studies indicate that the hydrodynamic dimensions of IDPs are evidence of considerable sequence-dependent backbone propensities for PPII structure that qualitatively, if not quantitatively, match conformational propensities measured in peptides. 2743-Pos Board B120 Rescuing the Over-Collapse of Intrinsically Disordered Proteins using a Force Field Derived by a New Paradigm Davide Mercadante1, Sigrid Milles2, Gustavo Fuertes3, Dmitri Svergun3, Edward A. Lemke4, Frauke Gra¨ter1. 1 Heidelberg Institute for Theoretical Studies, Heidelberg, Germany, 2 IBS – Institut de Biologie Structurale, Grenoble, France, 3European Molecular Biology Laboratory, Hamburg, Germany, 4European Molecular Biology Laboratory, Heidelberg, Germany. The sampling of the correct conformational dynamics of intrinsically disordered proteins is key to understand their function. Nevertheless, the

comparison between simulated and experimental ensembles still reveals a considerable gap, as simulations commonly yield overly compact conformers. Recently, attempts to overcome such an artificial collapse have been oriented towards the re-scaling of water dispersion forces to favor protein-water over protein-protein interactions and thus, to aid the occurrence of more extended conformers. However, rather than scaling interactions within an existing force field, a fully re-parameterized force field able to simulate the correct ensembles of intrinsically disordered as well as folded proteins should be preferred. We show that a force field developed on the basis of the Kirkwood-Buff theory of solutions (KBFF) is effective in sampling the dimensions of the ensemble for an intrinsically disordered, particularly compaction-prone Nucleoporin fragment. We compared the ensembles simulated using a wide range of force fields and water models to those obtained using smFRET and SAXS experiments and report on the efficiency of KBFF to reproduce experimental observations. Strikingly, the analysis of specifically designed mutants revealed that KBFF, oppositely to what observed for dispersionrescaled force fields, is able to reproduce the effects that intended mutations are expected to have on the ensemble’s dimensions. Overall, the efficiency of the KBFF in reproducing the correct conformational dynamics of IDPs lies in its viewpoint to retrieve parameters from the quantitative comparison of experimentally measured physico-chemical observables, which are directly related to the solvation of proteins. This solves, as a matter of fact, the problem of over-compaction in the simulations of IDPs and promises to be a valuable route towards a force field adequate for folded as well as disordered proteins. 2744-Pos Board B121 Proteomic and Biophysical Analysis of Polar Tracts Kiersten M. Ruff1, Alex S. Holehouse2, Mary G.O. Richardson3, Rohit V. Pappu4. 1 Computational and Systems Biology, Washington University in St. Louis, St. Louis, MO, USA, 2Computational and Molecular Biophysics, Washington University in St. Louis, St. Louis, MO, USA, 3Biomedical Engineering, Washington University in St. Louis, St. Louis, MO, USA, 4Biomedical Engineering, Center for Biological Systems Engineering, Washington University in St. Louis, St. Louis, MO, USA. Polar tracts are enriched in amino acids such as glutamine, asparagine, serine, and glycine. There is increasing evidence that polar tracts are involved in driving the formation of liquid-like micron-sized membraneless organelles. These membraneless organelles are thought to be important for a range of cellular functions including signal integration, cytoplasmic branching, and transcriptional control at synapses. However, polar tracts are also implicated in the formation of toxic aggregates that are the pathological hallmarks of several neurodegenerative diseases, including Huntington’s disease. Recent results suggest that the formation of functional assemblies enriched in polar tracts may leave the cell susceptible to pathological aggregation if left uncontrolled. The aggregation and phase separation of polar tracts can be modulated by specific amino acid interruptions within the polar tract, as well as specific sequences flanking the polar tract that help to regulate the directionality of self-associations, modulate the overall solubility of polar tracts, or contribute to the fluidity of assemblies formed by polar tracts. Our goal is to uncover the selection of specific patterns of interruptions within polar tracts and / or preferences for specific sequences that flank polar tracts. We present results from a proteomic analysis of polar tracts from 18 different organisms. Distinct organisms are enriched in different types of polar tracts. Additionally, we present results on the enrichment of specific amino acid interruptions within polar tracts, compositional properties of sequences flanking polar tracts, and functional domains within proteins housing polar tracts. The biophysical implications of how interruption and flanking sequence patterns influence the conformational properties and phase behavior of polar tracts will be discussed. 2745-Pos Board B122 Single-Molecule Dissection of the Conformations, Dynamics and Binding of the Disordered 4E-BP2 Protein Zhenfu Zhang1, Alaji Bah2, Hamda Sajjad1, Julie D. Forman-Kay2, Claudiu C. Gradinaru1. 1 Physics, University of Toronto, Mississauga, ON, Canada, 2Molecular Structure Function, The Hospital for Sick Children, Toronto, ON, Canada. Intrinsically disordered proteins (IDPs) play critical roles in regulatory protein interactions. Cap-dependent translation initiation is regulated by the interaction of eukaryotic initiation factor 4E (eIF4E) with disordered eIF4E binding proteins (4E-BPs) in a phosphorylation dependent manner. Single molecule fluorescence resonance energy transfer (smFRET), fluorescence correlation spectroscopy (FCS), time-resolved fluorescence anisotropy (TRFA) and nuclear magnetic resonance (NMR) were used to detect and assess the structural