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Folding and Assembly of Multimeric Proteins: Dimeric HIV-1 Protease and a Trimeric Coiled Coil Component of a Complex Hemoglobin Scaffold: a Dissertation

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Folding and Assembly of Multimeric Proteins: Dimeric HIV-1 Protease and a Trimeric Coiled Coil Component of a Complex Hemoglobin Scaffold: a Dissertation University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2007-08-22 Folding and Assembly of Multimeric Proteins: Dimeric HIV-1 Protease and a Trimeric Coiled Coil Component of a Complex Hemoglobin Scaffold: A Dissertation Amanda Ann Fitzgerald University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Amino Acids, Peptides, and Proteins Commons, Genetic Phenomena Commons, Nucleic Acids, Nucleotides, and Nucleosides Commons, and the Viruses Commons Repository Citation Fitzgerald AA. (2007). Folding and Assembly of Multimeric Proteins: Dimeric HIV-1 Protease and a Trimeric Coiled Coil Component of a Complex Hemoglobin Scaffold: A Dissertation. GSBS Dissertations and Theses. https://doi.org/10.13028/750v-ht77. Retrieved from https://escholarship.umassmed.edu/ gsbs_diss/341 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. A Dissertation Presented By Amanda Ann Fitzgerald Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester in partial fulfillment of the requirements for the degree of Doctor of Philosophy August 22, 2007 Biochemistry and Molecular Pharmacology ii Copyright Page CHAPTER II of this thesis is in preparation as the manuscript “The Folding Free Energy Surface of HIV-1 Protease: Monomer Folding Controls the Formation of the Native Dimeric β-barrel.” A.A.Fitzgerald, O.Bilsel, Y. Wu, A. Kundu, J. A. Zitzewitz., and C.R. Matthews for submission to Journal of Molecular Biology C. Robert Matthews, Ph.D., Thesis Advisor Anthony Carruthers, Ph.D., Member of Committee William Royer, Ph.D., Member of Committee David Lambright, Ph.D., Member of Committee Celia Schiffer, Ph.D., Chair of Committee that the student has met all graduation requirements of the school. Anthony Carruthers, Ph.D., Dean of the Graduate School of Biomedical Sciences iv Acknowledgements My journey to a doctoral degree would never have been possible if I had not had a tremendous amount of support and encouragement from many people. To express my gratitude, I would like to take this opportunity to acknowledge some of the many people who have helped me along the way. I would like to thank Bob Matthews for mentoring my dissertation research. I will always remember our many conversations about chemical kinetics and the intricacies of protein folding. I am also grateful to the members of the Matthews’ lab who were always willing to lend a hand. Jill Zitzewitz guided me every day through my research, helping me to design experiments and analyze my projects in innovative ways. Osman Bilsel was my consulting expert on data analysis, reminding me to consider the physical implications of every possible model. Ying Wu taught me molecular biology, protein purification, and how to work efficiently. Agnita Kundu helped me to persevere through the difficulties of the HIV-1 protease project by sharing with me her own difficulties on a parallel research project. Jessica Vasale taught me peptide synthesis and purification. Karen Welch was a great source of help for everything. Ram Vadrevu, Randy Forsyth, Louise Wallace, Iain Day, Rob Simler, Anna Svensson, Sagar Kathuria, Zhenyu Gu, Xiaoyan Yang, and Can Kayatekin also provided a supportive lab environment. I would like to thank my Thesis Research Advisory Committee. As my committee chair, Celia Schiffer constantly reminded me to remember the biological relevance of the projects I studied and consider all possibilities within the experimental v data. Bill Royer was especially helpful in the coiled coil project and provided structural biology expertise. Dave Lambright gave thoughtful suggestions in data analysis. Tony Carruthers provided helpful suggestions regarding kinetic data and also reminded me of the big picture. I am also thankful to José Argüello for serving as my outside thesis examiner. I am grateful to the people who encouraged me to pursue graduate work to completion. Carol Russell, my advisor at Framingham State College, has been a constant cheerleader in my pursuit of science. Gael Daly has provided a listening ear and prayerful encouragement. Kathryn Bard has reminded me to keep my goals in sight. Moses Prabu has been immensely helpful in editing my thesis and keeping me focused. I would like to thank my fellow graduate students for sharing in the journey. Julie, Tom, and Mandy– thank you for helping me through core course and qualifying. Jared, Jenn, Kristen, Trista, Jeff, Anthony, Steve, Naveen, Nick, Yufeng, Chaitali, and Madhavi– thank you for being supportive colleagues in the BMP department. Mary and Melonnie– I am forever grateful for our friendship that has developed over the past five years. Last, but most definitely not least, I would like to thank my family for their constant love and encouragement. Uncle Reid– thank you for not letting me quit. Michael– thank you for being there. Aunt Maureen– thank you for always reminding me “You go girl!” Mom and Dad– thank you for teaching me to be intellectually curious and dedicated to my passions. And finally, my fiancé Dave– you have been my greatest source of strength, laughter, and love. I could not have done this without you. vi To My Family vii Abstract Knowledge of how a polypeptide folds from a space-filling random coil into a biologically-functional, three-dimensional structure has been the essence of the protein folding problem. Though mechanistic details of DNA transcription and RNA translation are well understood, a specific code by which the primary structure dictates the acquisition of secondary, tertiary, and quarternary structure remains unknown. However, the demonstrated reversibility of in vitro protein folding allows for a thermodynamic analysis of the folding reaction. By probing both the equilibrium and kinetics of protein folding, a protein folding mechanism can be postulated. Over the past 40 years, folding mechanisms have been determined for many proteins; however, a generalized folding code is far from clear. Furthermore, most protein folding studies have focused on monomeric proteins even though a majority of biological processes function via the association of multiple subunits. Consequently, a complete understanding of the acquisition of quarternary protein structure is essential for applying the basic principles of protein folding to biology. The studies presented in this dissertation examined the folding and assembly of two very different multimeric proteins. Underlying both of these investigations is the need for a combined analysis of a repertoire of approaches to dissect the folding mechanism for multimeric proteins. Chapter II elucidates the detailed folding energy landscape of HIV-1 protease, a dimeric protein containing β-barrel subunits. The folding of this viral enzyme exhibited a sequential three-step pathway, involving the rate-limiting viii formation of a monomeric intermediate. The energetics determined from this analysis and their applications to HIV-1 function are discussed. In contrast, Chapter III illustrates the association of a coiled coil component of L. terrestris erythrocruorin. This extracellular hemoglobin consists of a complex scaffold of linker chains with a central ring of interdigitating coiled coils. Allostery is maintained by twelve dodecameric hemoglobin subunits that dock upon this scaffold. Modest association was observed for this coiled coil, and the implications of this fragment to linker assembly are addressed. These studies depict the complexity of multimeric folding reactions. Chapter II demonstrates that a detailed energy landscape of a dimeric protein can be determined by combining traditional equilibrium and kinetic approaches with information from a global analysis of kinetics and a monomer construct. Chapter III indicates that fragmentation of large complexes can show the contributions of separate domains to hierarchical organization. As a whole, this dissertation highlights the importance of pursuing mulitmeric protein folding studies and the implications of these folding mechanisms to biological function. ix Table of Contents Title page i Copyright page ii Approval page iii Acknowledgements iv Abstract vi Table of Contents viii List of Tables xiv List of Figures xv Abbreviations xvii CHAPTER I: INTRODUCTION AND LITERATURE REVIEW 1 Overview of the protein folding problem 2 The central dogma of molecular biology 2 Searching for a folding code 3 Postulated models of protein folding 5 Computational efforts in protein folding 5 Benefits from solving the protein folding problem 8 Protein therapeutics 8 Misfolding and disease 8 Emerging questions in the protein folding field 11 Structure-guided studies 11 Methods of protein engineering 12 Thermodynamic versus kinetic control 13 Chemical kinetics view of folding 13 Technology Development 16 Fluorescence spectroscopy 16 NMR spectroscopy 17 x Mass spectrometry 18 Small angle X-ray scattering 18 Ultra-fast folding detection 19 Mutational analysis of folding reactions 21 Topology and folding 22 Relative contact order 22 Topology modules within a proteins 22 Key protein folding studies on different motifs 24 Folding trends in mainly α-helical proteins 24 Folding trends in α/β proteins 26 β−Protein Folding
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