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Design and Study of Novel Antimicrobial Peptides with Proline Substitution

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Design and Study of Novel Antimicrobial Peptides with Proline Substitution Design and Study of Novel Antimicrobial Peptides with Proline Substitution A dissertation presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Doctor of Philosophy Jing He November 2009 © 2009 Jing He. All Rights Reserved. 2 This dissertation titled Design and Study of Novel Antimicrobial Peptides with Proline Substitution by JING HE has been approved for the Department of Chemistry and Biochemistry and the College of Arts and Sciences by John F. Blazyk Professor of Biochemistry Benjamin M. Ogles Dean, College of Arts and Sciences 3 ABSTRACT HE, JING, Ph.D., November 2009, Chemistry Design and Study of Novel Antimicrobial Peptides with Proline Substitution (228 pp.) Director of Dissertation: John F. Blazyk Microorganism-related diseases and their resistance to conventional antibiotics are proliferating at an alarming rate and becoming a severe clinical problem. Therefore, it is urgent to develop novel approaches in antimicrobial therapy. Most living organisms produce and utilize at least some small peptides as part of their defensive system in combating infections by virulent pathogens. Research focusing on the structure and function of these antimicrobial peptides from diverse sources has gained a great number of interests in the past three decades. Generally, many naturally existing antimicrobial peptides are positively charged and have the potential to adopt either amphipathic α-helix or β-sheet conformation. In this project, based on preliminary studies of β-sheet-forming peptides developed in our lab, analogs were designed to investigate the effects of introducing a single leucine-to-proline substitution on the structure and function of the peptides. The leucine-to-proline substitution was selected as a structural perturbation that could influence the antimicrobial and the cytolytic activity toward mammalian cells of these peptides. A series of experiments were performed in this project to investigate these potential changes, beginning with the determination of the antimicrobial activity and hemolytic activity of these peptides. Peptide conformation was determined by circular dichroism spectroscopy. Membrane permeability changes in both synthetic lipid bilayers and bacterial membranes were assessed by measuring peptide-induced calcein leakage 4 from large unilamellar vesicles (LUV) and by peptide-induced entry of o-nitrophenyl-β- D-galactopyranoside into E.coli ML-35 cells. The ability of peptides to bind to lipid bilayers of defined composition was measured by tryptophan fluorescence enhancement, acrylamide quenching and 10-doxylnonadecane quenching. The activity of these peptides was further studied by measuring the planktonic bacterial cell killing (the live vs. dead bacterial viability), bacterial biofilm formation inhibition and inhibition of bacterial cells growth within established bacterial biofilms. The results show that the position of proline has a significant influence on the antimicrobial and hemolytic activity of these peptides. Some of these proline-containing analogs show high antimicrobial activity and good selectivity between bacterial vs. mammalian cells. In addition, the peptide binding studies suggest that at least some of the proline-containing peptides may kill bacteria by mechanisms other than simply inducing membrane leakage. In summary, amphipathic β- sheet-forming antimicrobial peptides with proline substitutions appear to be promising models for novel antimicrobial agents. Approved: _____________________________________________________________ John F. Blazyk Professor of Biochemistry 5 ACKNOWLEDGMENTS My sincere gratitude goes to all people who assisted and encouraged me in various ways during the course of my study over the last few years, who generously contributed their time and support to help me make this dissertation possible. First and foremost, I would like to extend my sincerest appreciation to my research advisor, Dr. John F. Blazyk for providing me excellent guidance and consistent encouragement throughout the entire course of my study. I am greatly inspired by him in many ways and also would like to thank him for his great effort and valuable suggestions on revising my manuscript. My thanks also go to my dissertation committee, Drs. Douglas Goetz, Jennifer Hines, Marcia Kieliszewski and Hugh Richardson. I sincerely appreciate their consideration and suggestions in the completion of my dissertation. I would like to give thanks to my group members, Janet Hammer and Michelle Pate, for their support and help, especially at the beginning of my research. To Dr. Kenneth Goodrum, I would like to give thanks for his generous help in using the absorbance plate reader in his lab. I am especially grateful to my family. With their unconditional love, the course of pursuing my degree and the writing of my dissertation has never been a lonely journey. 6 TABLE OF CONTENTS Page Abstract ............................................................................................................................... 3 Acknowledgments............................................................................................................... 5 List of Tables .................................................................................................................... 10 List of Figures ................................................................................................................... 11 Chapter 1: Introduction ..................................................................................................... 16 A. History and Background .......................................................................................... 16 B. Naturally Occurring Antimicrobial Peptides ........................................................... 20 1. Conformation ........................................................................................................ 23 2. Charge ................................................................................................................... 26 3. Amphipathicity ..................................................................................................... 28 4. Hydrophobicity ..................................................................................................... 29 C. Comparison of α-helix- and β-sheet-forming Antimicrobial Peptides and Their Analogs ......................................................................................................................... 31 1. α-Helix-forming Antimicrobial Peptides .............................................................. 32 2. β-sheet-forming Antimicrobial Peptides ............................................................... 36 D. Proposed Mechanisms of Peptide Antimicrobial Action ......................................... 42 1. The Barrel-Stave Model ........................................................................................ 45 2. The Toroidal Pore (Wormhole) Model ................................................................. 46 3. The Carpet Model ................................................................................................. 47 4. Other Possible Action Mechanisms ...................................................................... 49 7 E. Biological Membranes ............................................................................................. 52 1. Composition of Biological Membranes ................................................................ 54 2. Difference between Bacterial and Mammalian Cell Membranes ......................... 59 F. Endotoxin and Biofilm of Bacteria ........................................................................... 66 G. Synthetic Antimicrobial Peptides Designed to Form α-helix or β-sheet ................. 71 1. Comparison of (KIAGKIA)3-NH2 and (KIGAKI)3-NH2 Peptides ....................... 74 2. Synthetic KL-repeat Containing Peptides and Tryptophan Incorporation............ 76 3. Synthetic KL-repeat Containing Peptides with Leucine-to-Proline Substitutions 79 H. Research Goals ......................................................................................................... 88 Chapter 2: Materials and Methods .................................................................................... 90 A. Materials .................................................................................................................. 90 B. Peptide Design ......................................................................................................... 91 C. Determination of Antimicrobial Activity ................................................................. 93 D. Determination of Hemolytic Activity ...................................................................... 94 E. Determination of Phospholipid Concentration ......................................................... 95 F. Preparation of Large Unilamellar Vesicles (LUV) ................................................... 96 G. Measurement of Peptide-induced Calcein Leakage in LUV ................................... 96 H. Determination of Peptide-induced Leakage in E. coli ML-35 ................................. 97 I. Peptide Binding with LUV Measured by Tryptophan Fluorescence Emission ........ 99 J. Peptide Binding with LUV Measured by Acrylamide Quenching ......................... 100 K. Peptide Binding with LUV Measured by 10-Doxylnonadecane Quenching ......... 101 L. Determination of Peptide Secondary Structure by Circular
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