Assembling Flagellin Proteins Fromsalmonella Typhimurium and Aquifex Pyrophilus
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Western Michigan University ScholarWorks at WMU Dissertations Graduate College 6-2007 Structure-Function Studies of Self- Assembling Flagellin Proteins fromSalmonella typhimurium and Aquifex pyrophilus Venkata Raghu Ram Malapaka Western Michigan University Follow this and additional works at: https://scholarworks.wmich.edu/dissertations Part of the Biochemistry, Biophysics, and Structural Biology Commons, and the Biology Commons Recommended Citation Malapaka, Venkata Raghu Ram, "Structure-Function Studies of Self- Assembling Flagellin Proteins fromSalmonella typhimurium and Aquifex pyrophilus" (2007). Dissertations. 893. https://scholarworks.wmich.edu/dissertations/893 This Dissertation-Open Access is brought to you for free and open access by the Graduate College at ScholarWorks at WMU. It has been accepted for inclusion in Dissertations by an authorized administrator of ScholarWorks at WMU. For more information, please contact [email protected]. STRUCTURE-FUNCTION STUDIES OF SELF-ASSEMBLING FLAGELLIN PROTEINS FROM SALMONELLA TYPHIMURIUM AND AQUIFEX PY ROPHILUS by Venkata Raghu Ram Malapaka A Dissertation Submitted to the Faculty of The Graduate College in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Department of Biological Sciences Dr. Brian C. Tripp, Advisor Western Michigan University Kalamazoo, Michigan June 2007 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 3265919 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. ® UMI UMI Microform 3265919 Copyright 2007 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Copyright by Venkata Raghu Ram Malapaka 2007 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS I would like to acknowledge Dr. Brian C. Tripp, a wonderful mentor I had during my tenure at Western Michigan University. Dr. Tripp always valued my creative spirit and had a sense of faith in me. His guidance in terms of designing a research project and writing manuscripts have been invaluable. He is always willing to try novel ideas and never afraid of going in a new direction. His critical analysis of my thoughts gave me tremendous confidence in pursuing various ideas during my graduate study. I sincerely believe the various skills that I have learned under him will help me launch my scientific career in a successful way. I would also like to thank my committee; Dr. Bruce Bejcek, Dr. John Geiser, and Dr. Yirong Mo. Their commitment towards their students and colleagues is exemplary. Their suggestions and thoughtful insights have given me an opportunity to critically think about my work and approach it in a rational way. I would like to especially thank Dr. Subra Muralidharan for his financial and intellectual support during my study at WMU. I would like to thank all my lab members for maintaining a healthy work environment in the lab. I would like to thank my father, Mr. Subrahmanyam, for providing me with the best possible means to fullfil my dreams. He has successfully played the role of both the parents after my mother’s demise and made numerous sacrifices for us. I would like to thank my wife Sandhya, for being a wonderful and patient companion. She has been a pillar of strength for me during my study. I would like to acknowledge ii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Acknowledgements - Continued my beloved sisters Vandana and Shilpa, with out whom my life wouldn’t be complete. Finally, I would like to dedicate this work to my mother Suseela and my grandmother Mahalakshmi, who are not here today to share my happiness, but will always be there in our thoughts. Venkata Raghu Ram Malapaka 111 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS ACKNOWLEDGEMENTS....................................................... ...... ii LIST OF TABLES................................ ............. x LIST OF FIGURES ................................................. xi CHAPTER I. INTRODUCTION TO THE DISSERTATION.......................................... 1 References.... ........................................................................ 13 II. INVESTIGATION OF THE HYPERVARIABLE DOMAIN REGION OF THE SALMONELLA TYPHIMURIUM FLAGELLIN PROTEIN IN MOTILITY ............................................................................. 20 Abstract................................................................................ 20 Introduction ......................................................................................... 21 Materials and Methods ....................................................................... 25 Bacterial strains .............................. 25 Plasmids and construction of flagellin internal deletion variant library .............................................................. 26 Motility assays .......................................................................... 31 Colony PCR ..................................... 32 DNA sequence determination and analysis.................. 33 Flagellin protein expression* flagella fiber purification and SDS-PAGE ............................................. 33 Electron microscopy ................................................................. 34 Light microscopy and swimming velocity analysis .................. 35 iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table of Contents - Continued CHAPTER Measurement of power required to stall cells using optical tweezer ................................................................. 35 Structure analysis and homology modeling .............................. 38 Results ......... 38 Description of flagellin hypervariable domain structural features...................................................................... 38 Identification of functional fliC internal deletion variants 39 Analysis of functional deletion variant polypeptide sequences ................................................................................. 42 Purification and transmission electron microscopy analysis of flagella ......................... 70 Swarming agar motility assay ................................................... 74 Video analysis of swimming motility in dilute aqueous media ................................................................. 77 Measurement of stall force in viscous aqueous media by optical trapping .......................................................... 78 Comparison of motility results determined by different methods ................... 80 Discussion .......................................................................................... 81 Effects of internal deletions on cellular motility and stability of flagella .............................................................. 81 Implications for folding, export and assembly ......................... 83 Implications for design of flagellin fusion proteins .................. 85 v Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table of Contents - Continued CHAPTER GenBank accession numbers ....................................... ........... 85 References........................................................................................ 88 Appendices ........................................................................................ 94 A. DNA sequences of the fliC gene deletion variants 94 B. A multiple sequence alignment of all the 39 unique internal deletion variants of FliC ........................................ 132 III. A THEORETICAL MODEL OF AQUIFEX PYROPHILUS FLAGELLIN: IMPLICATIONS FOR ITS THERMOSTABILITY................................................ 133 Abstract.......................................... 133 Introduction ............... ............................... 134 Computational Methods ............................................................. 138 Results and Discussion ....................................................................... 141 Sequence comparison .................................................................. 142 Structure comparison .................................................................. 154 Conclusions .................................................................................. 169 References............................................................ 170 IV. INVESTIGATION OF INSERTION OF GREEN FLUORESCENT PROTEIN IN SALMONELLA TYPHIMURIUM FLAGELLIN PROTEIN TO CREATE A FUNCTIONAL BIONANOTUBE................. 177 Introduction ........................................................................................