Georgia State University ScholarWorks @ Georgia State University Chemistry Dissertations Department of Chemistry Spring 5-7-2012 Dissecting Key Determinants for Calcium and Calmodulin Regulation of GAP Junction and Viral Protein Yanyi Chen Follow this and additional works at: https://scholarworks.gsu.edu/chemistry_diss Recommended Citation Chen, Yanyi, "Dissecting Key Determinants for Calcium and Calmodulin Regulation of GAP Junction and Viral Protein." Dissertation, Georgia State University, 2012. https://scholarworks.gsu.edu/chemistry_diss/64 This Dissertation is brought to you for free and open access by the Department of Chemistry at ScholarWorks @ Georgia State University. It has been accepted for inclusion in Chemistry Dissertations by an authorized administrator of ScholarWorks @ Georgia State University. For more information, please contact [email protected]. DISSECTING KEY DETERMINANTS FOR CALCIUM AND CALMODULIN REGULATION OF GAP JUNCTION AND VIRAL PROTEIN by YANYI CHEN Under the Direction of Professor Jenny J. Yang ABSTRACT Calcium and calmodulin are implicated in mediating the Ca2+-dependent regulation of gap junctions that are essential for the intercellular transmission of molecules such as nutrients, metabolites, metal ions and signal messengers (< 1000 Da) through its specialized cell membrane channels and communication to extracellular environment. To understand the key determinants for calcium and calmodulin regulation of gap junction, in this study, we identified a calmodulin binding domain in the second half of the intracellular loop of Cxonnexin50 (the major gap junction protein found in an eye lens) using peptide fragments that encompass predicted CaM binding sites and various biophysical methods. Our study provides the first direct evidence that CaM binds to a specific region of the ubiquitous gap junction protein Cx50 in a Ca2+-dependent manner. Furthermore, two novel CaM binding regions in cytosolic loop and C-termini of Connexin43 (the most ubiquitous connexin) have been shown to interact with CaM with different binding modes in the presence of Ca2+ using high resolution NMR. Our results also elucidate the molecular determinants of regulation of gap junction by multiple CaM targeting regions and provide insight into the molecular basis of gap junction gating mechanism and the binding of CaM to the cytoslic region Cx43-3p as the major regulation site. Upon response to the cytosolic calcium increase, CaM binds to the cytosolic loop to result in the conformational change of gap junction and close the channel. It is possible for CaM to use an adjacent region as an anchor close to the regulation site to allow for fast response. Since a large number of residues in the Cxs mutated in human diseases reside at the highly identified CaM binding sites in Cxs, our studies provide insights into define the critical cellular changes and molecular mechanisms contributing to human disease pathogenesis as part of an integrated molecular model for the calcium regulation of GJs. In addition, we have applied the grafting approach to probe the metal binding capability of predicted EF-hand motifs within the streptococcal hemoprotein receptor (Shr) of Streptococcus pyrogenes as well as the nonstructural protein 1 (nsP1) of Sindbis virus and Poxvirus. This fast and robust method allows us to analyze putative EF-hand proteins at genome-wide scale and to further visualize the evolutionary scenario of the EF-hand protein family. Further, mass spectrometry has also been applied to probe modification of proteins such as CaM labeling by florescence dye and 7E15 by PEG. INDEX WORDS: Calcium, EF-hand, Calcium binding proteins, Calmodulin, CalbindinD9k, CD2, Calmodulin target protein, Protein-protein interaction, Protein grafted approach, Gap junction, Connexin26, Connexin43, Connexin50 DISSECTING KEY DETERMINANTS FOR CALCIUM AND CALMODULIN REGULATION OF GAP JUNCTION AND VIRAL PROTEIN by YANYI CHEN A Dissertation Submitted in Partial Fulfillment of the Requirements of the Degree of Doctor of Philosophy in the College of Arts and Sciences Georgia State University 2012 Copyright by Yanyi Chen 2012 DISSECTING KEY DETERMINANTS FOR CALCIUM AND CALMODULIN REGULATION OF GAP JUNCTION AND VIRAL PROTEIN by YANYI CHEN Committee Chair: Jenny J. Yang Committee: Charles F. Louis Richard D. Veenstra Aimin Liu Siming Wang Electronic Version Approved: Office of Graduate Studies College of Arts and Sciences Georgia State University May 2012 iv ACKNOWLEDGEMENTS All of the work in this dissertation is carried out under the direction of Prof. Jenny J. Yang. I am deeply indebted to Prof. Jenny J. Yang, whose keen help, inspiring suggestions and stimulating encouragement helped me during the course of my research. The completion of my dissertation and subsequent Ph.D. has been a long journey. Her patience, flexibility, genuine caring and concern and faith in me during the dissertation process enabled me to attend to life while also earning my Ph.D. I would like to express my deepest gratitude to Prof. Richard D. Veenstra, Prof. Charles F. Louis, and Prof. Aimin Liu who gave me their full support and guidance in performing this research. I would like to express my sincere gratitude to Dr. Siming Wang for the immeasurable amount of support and guidance she has provided throughout mass spec. study. This dissertation would not have been possible without the expert guidance of these esteemed advisors. Many people on the faculty, staff and colleagues of the Department of Chemistry assisted and encouraged me in various ways during my graduate years. I gladly express my gratitude to them for all their help, support, interest and valuable hints. Especially I am obliged to Dr. Yubin Zhou for initiating the project and teaching me almost everything about science and research, to Dr. Hing-Cheung Wong and Dr. Hsiau-Wei Lee for their hand-to-hand teaching in NMR, to Dr. Shunyi Li for his help in protein purification, to Dr. Jin Zou for his help in CD and fluoremeter, to Dr. Monica Lurtz for the cell-based assays, to Xu Qin for the majority of the gap junction experiments along with Dr.Xianming Lin on the Cx43 and Cx50 projects, to You Zhuo and Juan Zou for working with me in gap junction project. I would also extend my gratitude to Natalie White and Matthew Cameron for carefully proof- reading all the chapters of this dissertation and provided many stylistic suggestions. This work is supported the grants from National Institutes of Health. Finally, I am especially grateful to my parents Wansheng Chen and Mingxiang Liu for giving the opportunity to follow my dreams, to my wife Yangqian Shao and my daughter Amber Chen, for the patient love to make all of these a reality. v TABLE OF CONTENTS ACKNOWLEDGEMENTS ...................................................................................................................... iv LIST OF TABLES .................................................................................................................................... ix LIST OF FIGURES .................................................................................................................................... x LIST OF ABBREVIATIONS ................................................................................................................. xiv 1. INTRODUCTION ............................................................................................................................... 1 1.1. Roles of Calcium (Ca2+) in biological systems ..................................................................................... 1 1.2. The properties of EF-hand motif in Ca2+-binding proteins. .................................................................. 3 1.3. A grafting approach: exploring the metal binding properties of continuous Ca2+ binding loops. ........ 4 1.4. Roles of Ca2+/Calmodulin in regulating intracellular signaling. ........................................................... 6 1.5. Gap junction in cell communication. .................................................................................................... 9 1.6. Calmodulin-mediated regulation of gap junctions. ............................................................................. 11 1.7. The objectives of this dissertation. ...................................................................................................... 11 1.8. Overview of this dissertation. ............................................................................................................. 12 2. MATERIALS AND METHODS ..................................................................................................... 25 2.1. Molecular cloning and plasmid construction ...................................................................................... 25 2.2. Baculovirus vector construction .......................................................................................................... 25 2.3. Expression and purification of proteins .............................................................................................. 26 2.4. Peptide design and synthesize ............................................................................................................. 27 2.5. Nuclear magnetic resonance spectroscopy .......................................................................................... 28 2.6. Fluorescence spectroscopy .................................................................................................................. 30 2.7. Circular dichroism