University of New Hampshire University of New Hampshire Scholars' Repository Doctoral Dissertations Student Scholarship Fall 2013 Regulation of the catalytic and allosteric properties of photoreceptor phosphodiesterase (PDE6) by the glutamic acid- rich protein-2 (GARP2) Wei Yao Follow this and additional works at: https://scholars.unh.edu/dissertation Recommended Citation Yao, Wei, "Regulation of the catalytic and allosteric properties of photoreceptor phosphodiesterase (PDE6) by the glutamic acid-rich protein-2 (GARP2)" (2013). Doctoral Dissertations. 747. https://scholars.unh.edu/dissertation/747 This Dissertation is brought to you for free and open access by the Student Scholarship at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. REGULATION OF THE CATALYTIC AND ALLOSTERIC PROPERTIES OF PHOTORECEPTOR PHOSPHODIESTERASE (PDE6) BY THE GLUTAMIC ACID-RICH PROTEIN-2 (GARP2) BY WEI YAO B.S., Jinan University, 2007 DISSERTATION Submitted to the University of New Hampshire in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biochemistry September, 2013 UMI Number: 3575987 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Di!ss0?t&iori Piiblist’Mlg UMI 3575987 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 This dissertation has been examined and approved. m u Dissertation Director, Rick H. Cote, Professor of Biochemistry and Molecular Biology Thomas M. Laue, Professor of Biochemistry W. Kelley Thomas, Professor of Genetics and Molecular Biology Cooper, Associate Professor of Microbiology andVaughmS Genetics Roy P. Planalp, Associate Professor of Chemistry 0 X l l > j i ' 7 Date DEDICATION This dissertation is dedicated to my wonderful family, especially my loving parents, Yukuan Yao and Guirong Song, as well as my sister, Jing Yao. I will always appreciate their constant love, encouragement and support through every step of my life. I also dedicate this dissertation to all my friends for their support and help. ACKNOWLEDGEMENTS I would like to express my gratitude to my advisor, Dr. Rick Cote, who has not only taught me to strive for excellence as a researcher but who has also inspired me to be a decent person. It is my pleasure and good fortune to have such a wonderful advisor, whose generous support, great patience and sapient guidance led me to achieve the goal of publishing my dissertation. I would like to thank my committee members, Dr. Thomas Laue, Dr. Kelley Thomas, Dr. Vaughn Cooper and Dr. Roy Planalp, for their time, encouragement, and expertise throughout my doctoral research. I would like to acknowledge everyone in Cote lab for their help, support and advice to me whenever I encountered difficulties in my research and in my life: Xiujun Zhang, Sue Matte, Karyn Cahill, Xiongzhuo Gao, Hannah Gitschier, Kevin Schuster, Katie Kozacka and Mary Awad. I would also like to thank Kyle Burton and Rose Hadley from Dr. Planalp’s lab for their cooperation, as well as Melanie Shields for her proof­ reading of my dissertation. This research was funded by a grant from the National Institutes of Health (EY05798), and University of New Hampshire Dissertation Year Fellowship (2012-2013). TABLE OF CONTENTS DEDICATION........................................................................................................................iii ACKNOWLEDGMENTS......................................................................................................iv LIST OF TABLES................................................................................................................viii LIST OF FIGURES................................................................................................................ix ABSTRACT...........................................................................................................................xii CHAPTER PAGE CHAPTER 1. INTRODUCTION................:.......................................................................... 1 1. Physiology of rod and cone photoreceptor cells ............................... 1 2. Cellular biology of rod and cone photoreceptor cells ........................................................5 3. Visual phototransduction pathway in photoreceptor cells ............................................... 6 4. Structure and functions of PDE6 ...................................................................................... 14 5. PDE6 interacting proteins ..................................................................................................20 6. Characteristics of the glutamic acid-rich protein2 (GARP2) .......................................... 23 CHAPTER 2. CHARACTERIZATION OF THE BIOCHEMICAL AND BIOPHYSICAL PROPERTIES OF GARP2....................................................................... 28 Abstract.................................................................................................................................. 28 Introduction ..................................................................................................................;......... 29 Materials and Methods ...........................................................................................................32 Results and Discussion ..........................................................................................................40 Summary ................................................................................................................................ 71 CHAPTER 3. REGULATION OF THE CATALYTIC AND ALLOSTERIC PROPERTIES OF PHOTORECEPTOR PHOSPHODIESTERASE (PDE6) BY THE GLUTAMIC ACID-RICH PROTEIN-2 (GARP2)............................................................. 73 Abstract.................................................................................................................................. 73 Introduction ............................................................................................................................ 74 Materials and Methods ...........................................................................................................77 Results and Discussion ..........................................................................................................83 Summary ................................................................................................................................ 92 CHAPTER 4. THE EFFECTS OF ZINC ON PDE6 CATALYTIC ACTIVITY..............94 Abstract.................... 94 Introduction ............................................................................................................................ 95 Materials and Methods ...........................................................................................................99 Results and Discussion ........................................................................................................ 100 Summary ...............................................................................................................................109 CONCLUSIONS.................................................................................................................. 110 LIST OF REFERENCES..................................................................................................... 113 APPENDIX I. List of vertebrate CNGB1 and GARP sequences ................................... 128 APPENDIX II. Multiple sequence alignment of the N-terminal region of CNGB1 130 LIST OF TABLES TABLE PAGE CHAPTER 2 Table 2-1. Summary of four GARP2 primary antibodies on immunoblot .................... ....46 CHAPTER 4 Table 4-1. The equilibrium constants of individual species in NTA-chelating zinc solutions at pH 7.4 (in absence of Mg2+) ......................................................... 107 Table 4-2. The equilibrium constants of individual species in NTA-chelating zinc solutions at pH 7.4 (in presence of 1 mM Mg2+) ............................................. 107 Table 4-3. The equilibrium constants of individual species in NTA-chelating zinc solutions at pH 7.0 ..............................................................................................108 Table 4-4. The equilibrium constants of individual species in ADA-chelating zinc solutions at pH 7.4 ..............................................................................................108 Table 4-5. The equilibrium constants of individual species in His-chelating zinc solutions at pH 7.4............................................................................................................. 108 viii LIST OF FIGURES FIGURE PAGE CHAPTER 1 Fig. 1-1. Electron microscopic image of rods and cones .......................................................1 Fig. 1-2. Schematic diagram of rod and cone morphology ....................................................5 Fig. 1-3. Visual