COMPARATIVE MOLECULAR DYNAMIC SIMULATIONS OF 2 HELICAL- AMPS FOUND IN SNAKES ATRA-1 AND ATRA-2 by Amr Zein Al-Abideen Majul A Thesis Submitted to the Graduate Faculty of George Mason University In Partial fulfillment of The Requirements for the Degree of Master of Science Chemistry Committee: Dr. Barney Bishop, Thesis Director Dr. Amarda Shehu, Committee Member Dr. Paige, Committee Member Dr. John Schreifels, Director, School of Chemistry and Biochemistry Dr. Peggy Agouris, Dean, College of Science Date: Summer Semester 2015 George Mason University Fairfax, VA Comparative Molecular Dynamic Simulations of 2 helical-AMPS found in snakes, ATRA-1 and ATRA-2 A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at George Mason University By Amr Zein Al-Abideen Majul Bachelor of Science George Mason University, 2007 Director: Dr. Barney Bishop, Professor Department of Chemistry and Biochemistry Summer Semester 2015 George Mason University Fairfax, VA Copyright (cid:13)c 2015 by Amr Zein Al-Abideen Majul All Rights Reserved ii Dedication I dedicate this thesis to my mother, Lubna Z. Dabbagh, who single handedly made all things possible, and provided me the best life one could hope for. iii Acknowledgments I would like to give thanks to my adviser Dr. Bishop whose advice kept me going when I was stuck, and has been patient with a process that took entirely too long. I would like to thankDr. Shehuforprovidingtheresourcesandexpertisethatmadeallthispossible, and Dr. Paige for providing me with the last minute insight that filled the holes in this work. I would like to thank all my fellow members at the Shehu Lab and Chemistry Department who have lent me aid. A special thanks to Dan Veltri who provided invaluable simulating discourse and help, Brian Olson who was always available to answer my simple computer science questions, and Corina Miss Math who spent hours working through many an equation with me, especially the matrix algebra of Singular Value Decomposition. iv Table of Contents Page List of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.1 Motivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.2 Problem Statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.3 Thesis outline and structure . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2 Background Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2.1 On Cationic Anti-Microbial Peptdes (CAMPs) and Cathelicidins . . . . . 4 2.2 CAMP modes of action and mechanism . . . . . . . . . . . . . . . . . . . 5 2.2.1 Barrel-Stave and Toroidal Pore Models. . . . . . . . . . . . . . . . 6 2.2.2 Carpet Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 2.2.3 Macropinocytosis, an energy dependent Model . . . . . . . . . . . 8 2.3 Molecular Dynamics Introduction, Methods and Theory. . . . . . . . . . . 9 2.3.1 Basic Principles of Molecular Mechanics . . . . . . . . . . . . . . . 10 2.3.2 On Forcefields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 2.3.3 On Parameterization . . . . . . . . . . . . . . . . . . . . . . . . . . 14 2.4 Secondary Structure Classification . . . . . . . . . . . . . . . . . . . . . . 14 2.4.1 Ramachandran plots . . . . . . . . . . . . . . . . . . . . . . . . . . 16 2.4.2 XTLSSTR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 2.4.3 Salt Bridges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 2.5 Use of SVD in Plane Fitting . . . . . . . . . . . . . . . . . . . . . . . . . . 19 2.6 Lipid Order Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.7 The MMGBSA method to calculate Free Energy of Binding . . . . . . . . 23 2.8 Radius of Gyration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 2.9 Related Work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 3 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 3.1 Simulation Program and Parameters . . . . . . . . . . . . . . . . . . . . . 27 v 3.2 Peptide-lipid systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 4 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 4.1 Peptide Insertion into the Lipid bilayer . . . . . . . . . . . . . . . . . . . . 36 4.1.1 Peptide Insertion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 4.1.2 Electrostatic and hydrophobic interactions of specific amino acids. 39 4.2 Energy Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 4.2.1 Free energy change . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 4.3 Structural Conformations of ATRA-1 and ATRA-2 . . . . . . . . . . . . 49 4.3.1 Xtlsstr Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 4.3.2 phi-psi Angles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 4.3.3 Radius of Gyration . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 4.4 Order Parameter Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 5 Disc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 5.0.1 Further Work and Direction . . . . . . . . . . . . . . . . . . . . . . 63 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 vi List of Figures Figure Page 2.1 Ramachandran definitions example plots . . . . . . . . . . . . . . . . . . . 17 2.2 Thermodynamic Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 3.1 Lipid Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 3.2 Initial peptide-lipid system example . . . . . . . . . . . . . . . . . . . . . 31 3.3 ATRA-1 vs ATRA-2 Initial setup . . . . . . . . . . . . . . . . . . . . . . 32 4.1 ATRA-1 Snapshots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 4.2 ATRA-2 Snapshots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 4.3 ATRA-1 arginine nitrogen atom snapshot illustrating Salt bridge . . . . . 37 4.4 Peptide insertion into the membrane . . . . . . . . . . . . . . . . . . . . . 38 4.5 ATRA-1 N hydrophilic residues . . . . . . . . . . . . . . . . . . . . . . . . 41 4.6 ATRA-1 N hydrophobic residues . . . . . . . . . . . . . . . . . . . . . . . 42 4.7 ATRA-2 hydrophilic residues . . . . . . . . . . . . . . . . . . . . . . . . . 44 4.8 ATRA-2 hydrophobic residues . . . . . . . . . . . . . . . . . . . . . . . . 45 4.9 Electrostatic Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 4.10 VdW Interactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 4.11 Ramachandran plots of phi-psi angles for entire simulation for each system 51 4.12 Ramachandran plots from 0-5ns for each system . . . . . . . . . . . . . . 52 4.13 Radius of gyration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 4.14 Order Parameters for lipids forming salt bridges. . . . . . . . . . . . . . . 54 4.15 Complete Lipid Bilayer Order Parameters . . . . . . . . . . . . . . . . . . 56 4.16 60ns O.P comparison . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 4.17 60-70ns O.P comparison . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 vii Abstract COMPARATIVE MOLECULAR DYNAMIC SIMULATIONS OF 2 HELICAL-AMPS FOUND IN SNAKES, ATRA-1 AND ATRA-2 Amr Zein Al-Abideen Majul, M.S. George Mason University, 2015 Thesis Director: Dr. Barney Bishop This thesis proposes the use of Molecular Dynamic (MD) simulations to study the two synthetic cationic antimicrobial peptides (CAMPs), ATRA-1 and ATRA-2. The pep- tides are based off a natural antimicrobial peptide found in the elapid snake Naja Atra [de Latour et al., 2010]. Natural AMPs can potentially serve as templates for engineer- ing novel antibiotics. MD simulations provide a valuable resource to supplement existing experimental and database-aided prediction data. Specifically the interaction of the two aforementioned peptides with a model lipid bilayer membrane is studied. The antimi- crobial potencies between the peptides differ appreciably, yet their amino acid sequences differ from each other at only 2 positions. This thesis proposes the use of MD to run simulations to extract qualitative and quantitative information on each peptide. Most other similar computational studies on peptides are done on a single type of peptide. Simulations comparing ATRA-1 and ATRA-2 are analysed in this thesis, focusing on on finding various physical and chemical parameters that differentiate the two peptides. The simulations employ all atom explicit models, with a realistic non-anchored membrane. In addition, the feasibility of using MD simulations to predict and rank the effectiveness of proposed rationally designed novel antimicrobial peptides will be evaluated. Chapter 1: Introduction Antimicrobial peptides (AMPs) are naturally occurring peptides reported in virtually all organisms, including bacteria, fungi, plants and animals, and represent an ancient defensive strategy against infection [Juba et al., 2013]. Cationic antimicrobial peptides (CAMPs) are a class of antimicrobial peptides that are an important component of innate immunity in higher organisms. CAMPs are generally small peptides ranging from 10- 50 amino acids that have an overall positive charge. They are further loosely grouped into families based on structural and functional characteristics, for example magainins, β-defensins and cathelicidins [Boman, 2003]. The two peptides used in this study, ATRA-1 and ATRA-2 are synthetic peptides inspired by a CAMP, identified in the venom glands of the Chinese cobra, Naja Atra. This peptide belongs to the cathelicidin family of vertebrate CAMPs. It contains a semi- conserved repeated 11-residue pattern (KR(F/A)KKFFKK(L/P)K), termed the ATRA motif. The ATRA motif present in the Naja Atra peptide serves as a template for the design of the peptides, ATRA-1 and ATRA-2, which are experimentally tested to probe the differences in how their respective sequences impact their performance. ATRA-1 and ATRA-2 are 11 residue long, having amino acids sequences of KRAKKFFKKLK and KRFKKFFKKPK respectively. Differing in only two positions, their bactericidal potencies has been reported to differ significantly [Juba et al., 2013]. This thesis uses Molecular Dynamics to study the two Atra peptides and their interac- tion with a model membrane. Simulations of various configurations of the aforementioned peptideswithlipidsareperformedtostudythem, usingallexplicitatomisticmodels, such as realistic non-coarse grained membranes, to allow a mathematically rigorous calculation 1