ABSTRACT MARTIN, STANTON LOWELL. Structural Characterization of Red clover necrotic mosaic virus by Small Angle Neutron Scattering and X-ray Crystallography. (Under the direction of Dr. Steven A. Lommel.) Understanding the fundamental structure of viruses has been a key objective in the discipline of virology since its inception. X-ray crystallography, and electron microscopy were the main techniques used for probing viral structures for most of the 20th century. Neutrons were also used in some cases. Over the years the techniques have been incrementally improved to the point where it is now possible to resolve viral structures at atomic or near atomic resolution. This in turn allows investigators to apply computational techniques such as molecular dynamic simulations to predict how changes in viral dynamics may be influenced by the various environment(s) in which viruses exist. To understand function it is essential to investigate changes in viral dynamics. This work began as a purely computational project aimed at understanding the structure of RCNMV using existing data. In the course of the project it became evident that not enough raw structural data was available to make the necessary inferences, so a quest to obtain a higher resolution structure was initiated. Two separate but related techniques were utilized: X-ray crystallography and neutron scattering. Chapter one of this thesis traces the history of the science of virology from its inception, and tells the story of how the discovery of X-rays caused a convergence of the disciplines of crystallography and virology resulting in significant advances in our understanding of viral structural biology. Chapter two reports our own experience with X- rays applied to the study of RCNMV. Chapter three traces the history of the neutron sciences. Ironically, some of the most important advances in the neutron sciences were made possible because of war related research. Chapter four reports our own experience with neutron scattering. Chapter five reports some of our own successes and failures in the quest for better understanding of our virion. Our own experience with its missteps, false starts, and occasional lucky breaks, seems to mimic the experience of thousands of scientists who, despite sometimes adverse circumstances and personal failures, managed collectively to advance the knowledge of structural virology over the last 150 years. Chapter six concludes with a report on the current stage of our research and outlines some of the recent breakthroughs in technology that guide our aspirations for future investigations. Structural Characterization of Red clover necrotic mosaic virus by Small Angle Neutron Scattering and X-Ray Crystallography by Stanton Lowell Martin A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Plant Pathology Raleigh, North Carolina 2012 APPROVED BY: ____________________________ ____________________________ Steven A. Lommel Stefan Franzen Committee Chair Committee Co-Chair ____________________________ ____________________________ James A. Moyer Ignazio Carbone ____________________________ Russell D. Wolfinger ii DEDICATION To my lovely wife and my two curious children iii BIOGRAPHY Stan Martin graduated from North Carolina State University with a Master’s Degree in Bioinformatics in 2002. He worked as a Bioinformatics Computing Consultant for North Carolina State University from 2002-2012. During his tenure as a Senior Bioinformatics Computing Consultant, he enrolled in the Ph.D. program in Plant Pathology at NCSU where he studied structural virology under the direction of Dr. Steven A. Lommel. iv ACKNOWLEDGMENTS Many people contributed to the development of this thesis. Besides the committee members, the author gratefully acknowledges the contribution of members of the NCSU biochemistry department, Dr. Robert Rose, Dr. Paul Swartz, and Dr. Flora Meilleur for their assistance with the X-ray crystallography work. Dr. William Heller and Dr. Lilin He of Oak Ridge National Laboratory assisted in the neutron scattering work. Mr. Richard Guenther, and Ms. Carol George assisted in virus purification and sample preparation for both the X-ray and neutron scattering studies. Dr. Tim Sit provided invaluable advice during both the experimental and reporting phases of this work. v TABLE OF CONTENTS LIST OF TABLES ............................................................................................................. viii LIST OF FIGURES ........................................................................................................... ix CHAPTER ONE: THE DEVELOPMENT OF STRUCTURAL VIROLOGY AND X-RAY CRYSTALLOGRAPHY.................................................................................................... 1 Beginnings of the Science of Virology .................................................................. 1 Crookes Tubes and Energy Rays ........................................................................... 5 The Discovery of X-Rays ...................................................................................... 6 The Genesis of Crystallography............................................................................. 10 The Development of X-Ray Crystallography ........................................................ 12 X-ray Crystallography and Virology ..................................................................... 19 Powder Diffraction of Tobacco mosaic virus ........................................................ 20 Theoretical Advances in Structural Virology ........................................................ 22 Spherical Viruses Studied by Single Crystal Diffraction Techniques ................... 25 Solution of the Tobacco mosaic virus Structure .................................................... 30 The Discovery of Synchrotron Radiation .............................................................. 32 Virus Crystal Structures Solved Using Synchrotron Radiation ............................. 37 Advances in Structural Virology (1990-present) ................................................... 45 References Cited .................................................................................................... 51 CHAPTER TWO: CRYSTALLIZATION AND PRELIMINARY X-RAY DIFFRACTION ANALYSIS OF RED CLOVER NECROTIC MOSAIC VIRUS ......................................... 64 Abstract .............................................................................................................................. 64 Introduction to RCNMV ........................................................................................ 65 Materials and Methods ........................................................................................... 67 Virus Propagation ...................................................................................... 67 Virus Purification ....................................................................................... 67 Virus Purity Assays.................................................................................... 69 Crystallization ............................................................................................ 69 Data Collection and Processing ................................................................. 71 Results and Discussion .......................................................................................... 73 Sample Characterization ............................................................................ 73 RCNMV Crystals ....................................................................................... 75 Conclusions ............................................................................................................ 76 References Cited .................................................................................................... 78 CHAPTER THREE: THE DEVELOPMENT OF NEUTRON SCATTERING AND ITS CONTRIBUTION TO STRUCTURAL VIROLOGY ...................................................... 80 The Discovery of the Neutron................................................................................ 80 The Quest for Neutrons .......................................................................................... 82 Effect of World War II on the Development of Neutron Sciences ........................ 83 Nuclear Reactors Used in Science ......................................................................... 88 Application of Neutron Sciences to Biology ......................................................... 91 Neutron Scattering and Virology ........................................................................... 93 References Cited .................................................................................................... 96 vi CHAPTER FOUR: THE INTERNAL STRUCTURE OF THE RED CLOVER NECROTIC MOSAIC VIRUS AND THE ROLE OF DIVALENT CATIONS IN THE VIRUS LIFECYCLE ...................................................................................................................... 99 Abstract .................................................................................................................. 100 Introduction ............................................................................................................ 101 Results .................................................................................................................... 106 Discussion .............................................................................................................