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Single-Molecule Spectroscopy and Imaging Studies of Protein Folding-Unfolding Conformational Dynamics: the Multiple-State and Multiple-Channel Energy Landscape

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Single-Molecule Spectroscopy and Imaging Studies of Protein Folding-Unfolding Conformational Dynamics: the Multiple-State and Multiple-Channel Energy Landscape SINGLE-MOLECULE SPECTROSCOPY AND IMAGING STUDIES OF PROTEIN FOLDING-UNFOLDING CONFORMATIONAL DYNAMICS: THE MULTIPLE-STATE AND MULTIPLE-CHANNEL ENERGY LANDSCAPE Zijian Wang A Dissertation Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY May 2016 Committee: H. Peter Lu, Advisor Gabriela Bidart-Bouzat Graduate Faculty Representative Ksenija D. Glusac George Bullerjahn © 2016 Zijian Wang All Rights Reserved iii ABSTRACT H. Peter Lu, Advisor Protein conformational dynamics often plays a critical role in protein functions. We have characterized the spontaneous folding-unfolding conformational fluctuation dynamics of calmodulin (CaM) at thermodynamic equilibrium conditions by using single-molecule fluorescence resonance energy transfer (FRET) spectroscopy. We studied protein folding dynamics under simulated biological conditions to gain a deep, mechanistic understanding of this important biological process. We have identified multiple folding transition pathways and characterized the underlying energy landscape of the single-molecule protein conformational fluctuation trajectories. Our results suggest that the folding dynamics of CaM molecules involves a complex multiple-pathway multiple-state energy landscape, rather than an energy landscape of two-state dynamical process. Our probing single-molecule FRET fluctuation experiments demonstrate a new approach of studying spontaneous protein folding-unfolding conformational dynamics at the equilibrium that features recording long time single-molecule conformational fluctuation trajectories. This technique yields rich statistical and dynamical information far beyond traditional ensemble-averaged measurements. We characterize the conformational dynamics of single CaM interacting with C28W. The single CaM molecules are partially unfolded by GdmCl, and the folded and unfolded CaM molecules are approximately equally populated. Under this condition, the majority of the single protein CaM undergoes spontaneous folding-unfolding conformational fluctuations. Using single molecule FRET spectroscopy, we study each of the single protein’s conformational dynamics in iv the presence of C28W-CaM interactions. The results show an interesting folding-upon-binding dynamic process, and a conformational selection mechanism is further confirmed. The effect of molecular crowding on protein folding process is a key issue in the understanding of protein folding dynamics in living cells. Due to the complexity and interplay between various interactions existing in an equally favored environment of protein folding and unfolding conformational dynamics, such simple reduced entropic enhancement model do not suffice in describing protein folding conformational dynamics. We observe, at higher concentration of crowding reagent Ficoll 70, single protein molecules spontaneously denature into unfolded proteins which involves a combined process of polymer-polymer interaction, entropic effects and solvation thermodynamics and dynamics. Such heterogeneous unfolding process can serve as a first step to a mechanistic understanding of living cell disease as a result of molecular crowding effect, protein aggregates and fibril formation. v To My Mom Whose Insights Influence Me the Most vi ACKNOWLEDGMENTS First and foremost I want to thank my advisor H. Peter Lu. It has been an honor to be one of his graduate students. He has taught me, both consciously and unconsciously, how good experiments are done. I appreciate all his contributions of time, ideas and funding to make my Ph. D. experience productive. I am also grateful for the excellent example he has provided as a successful chemist and professor. I am thankful to all my dissertation committee members: Dr. Ksenija D. Glusac, Dr. George Bullerjahn and Dr. Gabriela Bidart-Bouzat for their precious time. I also want to acknowledge all the group members, both current members and past members from Dr. H. Peter Lu’s group, for setting a competitive, productive and hard-working environment. I also want to thank Dr. Desheng Zheng, Dr. Yufan He, Dr. Yuanmin Wang, Dr. Jin Cao and Dr. Qing Guo for their friendship and help. Especially, I’d like to thank Dr. Takashige Fujiwara for training me on tuning Lasers and technical support. I gratefully acknowledge Delta Electronics Inc. for generously providing me the Delta Electronics Research fellowship. The Delta Electronics Research fellowship supported me for the 2012-2013 academic year. I would also like to thank many faculty and staff members at the Center for Photochemical Sciences and the Department of Chemistry: Nora Cassidy, Alita Frater, Charles Codding, Doug Martin, and Hilda Miranda, for their help. Lastly, I would like to thank my family for all their love and encouragement, for my parents who raised me with a love of science and supported me in all my pursuits. Without their understanding and love, I would never be able to finish this journey. vii TABLE OF CONTENTS Page CHAPTER I. INTRODUCTION……………………… ...................................................... 1 1.1 Introduction to Single-Molecule Fluorescence Spectroscopy. ............................ 1 1.2 Single-Molecule Studies of Protein Conformational Dynamics .......................... 3 1.3 An Overview of Single-Molecule Studies of Protein Folding ............................. 4 1.4 Our Sample Protein Calmodulin (CaM) .............................................................. 7 1.5 Research Objective, Specific Aims and Dissertation Overview .......................... 9 1.6 Reference ............................................................................................................ 11 CHAPTER II. EXPERIMENTAL SECTION ...................................................................... 18 2.1 Principles of Experimental Techniques ............................................................... 18 2.1.1 Principles of Confocal Microscopy ...................................................... 18 2.1.2 Principles of Fluorescence Resonance Energy Transfer ....................... 20 2.1.3 Signal Detection Techniques: Introduction to APD ............................. 25 2.2 Experimental Details ............................................................................................ 26 2.2.1 Experimental Setup of Single-Molecule FRET combined with Confocal Microscope. .................................................................................................... 26 2.2.2 Materials and Sample Preparation……………………………… ........ 29 2.2.3 Statistical Analyses of Single-Molecule Intensity Trajectories ............ 32 2.3 Protein Folding Dynamics in Condensed Phases................................................. 35 2.4 Theory of Polymer Solutions ............................................................................... 37 2.5 References ............................................................................................................ 38 viii CHAPTER III. PROBING SINGLE- MOLECULE PROTEIN FOLDING CONFORMATIONAL DYNAMICS USING SINGLE-MOLECULE FRET SPECTROSCOPY………………………. ............................................................................ 41 3.1 Introduction .......................................................................................................... 41 3.2 Materials and Methods ......................................................................................... 43 3.2.1 Sample Preparation and Characterization…………………………… . 43 3.2.2 Single-Molecule Imaging and FRET Measurements………………… 45 3.3 Results and Discussion ........................................................................................ 47 3.3.1 Single-Molecule FRET Trajectories Monitored Unfolding of Single CaM molecules…………………………… ........................................................... 47 3.3.2 Autocorrelation Analyses of CaM Folding-Unfolding Conformational Fluctuation Dynamics …………………………… ....................................... 50 3.3.3 Non-Exponential Distribution of Folding Waiting Time Indicates Multiple Folding Intermediates …………………………… ....................................... 58 3.3.4 Model Analyses of Conformational Dynamics and Energy Landscape of Single-Molecule CaM Folding …………………………… ......................... 61 3.4 Conclusions .......................................................................................................... 69 3.5 References ............................................................................................................ 70 CHAPTER IV. PROBING SINGLE-MOLECULE PROTEIN FOLDING-UNPON-BINDING CONFORMATIONAL DYNAMICS USING SINGLE-MOLECULE FRET SPECTROSCOPY. ............………………………................................................................................................ 80 4.1 Introduction .......................................................................................................... 80 4.2 Materials and Methods ......................................................................................... 83 ix 4.2.1 Sample Preparation and Characterization…………………………… . 83 4.2.2 Single-Molecule Imaging and FRET Measurement ............................. 84 4.3 Results and Discussion ........................................................................................ 85 4.4 Conclusions .......................................................................................................... 92 4.5 References ...........................................................................................................
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