Equilibrium Molecular Dynamics of the Monomer and Dimer Units of Streptococcus Pnuemonae and Corynebacterium Diphtheriae Pili

Sunday, February 12, 2017 thermal response of i.corA differed considerably from that of the outer component o.corA. Analysis of the radius of gyration revealed that the inner TM component underwent a continuous transition from a globular conformation to a random coil structure on raising the temperature. In contrast, the outer transmembrane component exhibited an abrupt (almost discontinuous) thermal response in a narrow range of temperature. Scaling of the structure factor showed a globular structure of i.corA at low temperature with an effective dimension D ~ 3 and random coil at high temperature with D ~ 2. The residue distribution in o.corA is slightly sparser than that of i.corA in a narrow thermosresponsive regime. The difference in thermo-response characteristics of these components (i.corA, o.corA) may reflect their unique transmembrane functions. Attempts are being made to incorporate such realistic features as explicit solvent and membrane; corresponding results may also be presented as data become available. 256-Pos Board B21 The Competition between Electrostatic-Steering and Conformational Dynamics in the Diffusion-Limited Association of Calcineurin and Calmodulin Peter M. Kekenes-Huskey1, Bin Sun1, Eric C. Cook2, Trevor P. Creamer3. 1 Chemistry, UK, Lexington, KY, USA, 2Biochemistry, University of Kentucky, Lexington, KY, USA, 3Chemistry, University of Kentucky, Lexington, KY, USA. Calcineurin (CaN) is a serine/threonine phosphatase that regulates a variety of physiological and pathophysiological processes in most mammalian tissue. In its inactive state, the CaN catalytic domain is inhibited by the auto-inhibitory domain (AID); the active state is obtained upon CaM binding to the CaN regulatory domain (RD). It has been established that the RD is highly disordered when inhibiting CaN, yet it undergoes a disorder-to-order transition upon binding calmodulin (CaM) to activate the phosphatase. Given that the RD is richly populated with polar and charged amino acids, arguably electrostatic interactions may influence the rate of CaM association. However, it is likely that properties of the RD conformational ensemble, such as its ‘effective volume’ and accessibility of its CaM binding motif, influence the CaM/CaN association rate. In present study, we investigated via computational modeling the extent to which electrostatics and structural disorder co-facilitate or hinder CaM/CaN binding kinetics. We examined several peptides containing the CaM binding motif, for which lengths and amino acid charge distributions were varied, to isolate the contributions of electrostatics versus conformational diversity to predicted, diffusion-limited association rates. These rates were predicted using Replica Exchange Molecular Dynamics (REMD) and Brownian Dynamics (BD) simulations. Our results indicate that association rates vary as a function of increasing CaN RD length (beyond the required CaM recognition sequence), thus indicating that RD conformational ensemble properties influence CaM binding. Second, we found that increasing the solvent ionic strength generally depressed CaM/CaN association rates, owing to the attenuation of long-range electrostatic interactions that would normally accelerate protein-protein association. Finally, CaN peptides with positively-charged amino acids substituted at native negatively-charged sites had complex effects on the predicted association rate, owing to ‘off-target’ interactions that in some cases competed with the intending binding site. Our findings detail the interplay between conformational diversity and electrostatically-driven protein-protein association involving CaN, which are likely to extend to wide-ranging processes regulated by intrinsicallydisordered proteins. 257-Pos Board B22 Equilibrium Molecular Dynamics of the Monomer and Dimer Units of Streptococcus Pnuemonae and Corynebacterium Diphtheriae Pili Emmanuel Naziga, Jeff Wereszczynski. Illinios Institute of Technology, Chicago, IL, USA. Pili are elongated protein structures that enhance the adhesive abilities and virulence of bacteria. As a result, it is important to understand their structure and mechanical properties. Pili are constructed by joining monomeric units (or pilins) into polymeric fibers via intermolecular isopeptide bonds. Interestingly, the pilins additionally possess intramolecular isopeptide bonds that are formed between the sidechains of lysine and asparagine residues in the same domain of the protein, which are believed to impart mechanical stability to the pili. In this work, we use all atom molecular dynamics simulations to study the conformational dynamics of monomeric and dimeric forms of the pilins of streptococcus pnuemonae (RrgB) and corynebacterium diphtheriae (spaA) in the presence and absence of intramolecular isopeptide bonds. The results reveal how the energetics and dynamics of these subunits are affected by intramolecular isopeptide bonds, and demonstrate how they affect pili stability.

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258-Pos Board B23 Biophysical and Structural Characterization of Antibody Responses to Malaria Antigens Stephen Scally1, Alexander Bosch1, Brandon McLeod1, Gianna Triller2, Katharina Imkeller2, Rajagopal Murugan2, Sebastian R€amisch3, Rick King4, William Schief3, Hedda Wardemann2, Jean-Philippe Julien1. 1 Molecular Structure & Function, The Hospital for Sick Children Research Institute, Toronto, ON, Canada, 2Max Planck Institute for Infection Biology, Heidelberg, Germany, 3The Scripps Research Institute, San Diego, CA, USA, 4 Malaria Vaccine Inititive, Washington, DC, USA. Malaria is a global health priority: 214 million malaria cases were reported in 2015 alone, predominantly in Africa and resulting in 438,000 deaths - 70% of which were in children below five years of age. Reverse vaccinology holds promise to design effective immunogens for the development of malaria vaccines. This concept is based on interrogating the B cell repertoire of vaccinated or infected subjects to identify protective antibodies that will guide immunogen design. Our efforts have focused on plasmodium falciparum targets associated with the development of pre-erythrocytic and transmission-blocking vaccines. We isolated B cells from individuals naturally exposed to plasmodium falciparum, and from vaccinated animals and humans having undergone controlled infection. We performed extensive binding experiments to characterize affinities and competition of dozens of antibodies by isothermal titration calorimetry and biolayer interferometry. These studies uncovered several epitope bins, which give biophysical insights into immunodominant and protective B cell responses. X-ray crystallography, small-angle X-ray scattering and singleparticle electron microscopy were used and integrated to define antibody recognition at the molecular level. Our structural delineation of protective epitopes provides the blueprints to engineer optimized antigens that can be formulated and tested as pre-erythrocytic and transmission-blocking malaria vaccines. 259-Pos Board B24 Structural Analysis of the Precursor Protein of Atrial Natriuretic Peptide Sumika Futori, Satomi Higashigawa, Shigeru Shimamoto, Yuji Hidaka. Kindai university, Higashi-Osaka, Osaka, Japan. Atrial natriuretic peptide (ANP) is produced in the atrium and functions as a vasodilator. ANP is expressed in the form of a precursor, prepro-ANP, and is then processed into pro-ANP, which consists of the pro-peptide (98 residues) and mature ANP (28 residues, one disulfide bond) regions, in the endoplasmic reticulum. The biological role of ANP has been studied extensively. However, our knowledge of the role of the pro-peptide region of pro-ANP is still unclear. Therefore, to study the processing mechanism of pro-ANP and the role of the pro-peptide region, recombinant pro-ANP was prepared by an E. coli expression system and its conformation examined by means of circular dichroism (CD). The recombinant pro-ANP, which was readily over-expressed as a soluble form in E. coli cells, was purified by ion exchange chromatography, and identified by MALDI-TOF/MS. To obtain structural information regarding pro-ANP, CD measurements were carried out in 20mM Tris/HCl buffer (pH7.4). However, the results suggested that pro-ANP does not possess specific secondary structures similar to other intrinsically disordered proteins. Therefore, to further investigate the tertiary structure of pro-ANP, we employed trifluoroethanol (TFE) as a structural mediator and collected CD spectra of pro-ANP under several sets of conditions. The results indicated that TFE dramatically induced the formation of an a-helical structure in pro-ANP, suggesting that the pro-peptide region of pro-ANP is able to form an a-helical structure under a hydrophobic environment. Therefore, we propose that the tertiary structure of pro-ANP may be stabilized by interacting with hydrophobic compounds, such as serum albumin or membrane lipids, in vivo. 260-Pos Board B25 Inhibition of Aggregation in B-Sheet Model Peptide by PPII Helix Capping Heng Chi1, Min Zhou1, Timothy A. Keiderling2. 1 pharmacy, Jiangsu Food and Pharmaceutical Science College, Huaian, China, 2Chemistry, University of Illinois at Chicago, Chicago, IL, USA. A 12mer peptide (Beta, SWTVEGNKYTYK-NH2) was designed based on the Trpzip model. Beta can form an aggregate with fibril like morphology imaged by TEM. The secondary structure of the fibril was characterized to be antiparallel b-sheets by FT-IR spectra. VCD (Vibrational Circular Dichroism) spectrum demonstrated the supramolecular chirality of the fibril and UV-CD spectrum of this peptide confirmed the tertiary contact between aromatic residues in the sequence. Upon mutation with an added PPII helix inducing combo to the C-terminal of this peptide, a new peptide BP (SWTVEGNKYTYKN GAPPPK-NH2) was synthesized. BP did not enhance ThT fluorescence, its secondary structure was characterized to be unordered by UV-CD and FT-IR spectra. Further FRET analysis with a dansylated version of BP shows that the end to end distance is consistent with the proline mutations disrupting the aromatic contacts and thus stabilized the unordered conformation. In contrast,