Structural Models of an Intrinsically Disordered Protein Adapted for Bacterial Secretion

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