TITLE PAGE STRUCTURAL AND BIOCHEMICAL STUDIES OF RPE65, THE RETINOID ISOMERASE OF THE VISUAL CYCLE by PHILIP DAVID KISER Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Adviser: Dr. Krzysztof Palczewski Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY May, 2010 SIGNATURE PAGE CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of ______________________________________________________ candidate for the ________________________________degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. ii DEDICATION I dedicate this work to: My family for their love and support throughout the years and My teachers for inspiration iii TABLE OF CONTENTS TITLE PAGE.......................................................................................................... i SIGNATURE PAGE...............................................................................................ii DEDICATION .......................................................................................................iii TABLE OF CONTENTS .......................................................................................iv LIST OF TABLES ...............................................................................................viii LIST OF FIGURES ...............................................................................................ix ACKNOWLEDGEMENTS....................................................................................xii LIST OF ABBREVIATIONS ................................................................................xiv ABSTRACT .......................................................................................................... 1 CHAPTER 1: BACKGROUND ON RPE65 AND RETINOID ISOMERIZATION ... 3 1.1. Introduction to retinal structure and function .............................................. 4 1.1.1. Structure and function of the photoreceptor and pigment epithelium layers of the retina......................................................................................... 4 1.1.2. The retinoid (visual) cycle.................................................................... 5 1.2. Prior studies on RPE65 function and membrane binding........................... 6 1.2.1. Functional characterization of RPE65 ................................................. 7 1.2.2. Biochemical studies on RPE65 membrane binding........................... 10 1.3. Chemistry of Retinoid Isomerization ........................................................ 14 1.3.1. Substrates and products.................................................................... 14 1.3.2. Thermodynamic and kinetic considerations....................................... 16 1.3.3. Mechanisms of retinoid isomerization................................................ 17 1.3.4. Substrate specificity........................................................................... 21 iv 1.3.5. The requirement of an iron cofactor................................................... 22 1.4. Experimental outline, goals and rationale ................................................ 24 Table............................................................................................................... 27 Figures............................................................................................................ 28 CHAPTER 2: EXPRESSION, PURIFICATION AND BIOCHEMICAL CHARACTERIZATION OF RPE65..................................................................... 37 2.1. Expression and purification of Zebrafish RPE65...................................... 38 2.1.1. Rationale and Methods...................................................................... 38 2.1.2. Results and Discussion ..................................................................... 40 2.2. Expression and Purification of Human RPE65......................................... 41 2.2.1. Rationale and Methods...................................................................... 41 2.2.2. Results and Discussion ..................................................................... 43 2.3. Generation of an RPE65 monoclonal antibody and mapping of its epitope ........................................................................................................................ 44 2.3.1. Methods............................................................................................. 44 2.3.2. Results and discussion...................................................................... 47 2.4. Extraction and phase separation studies on native RPE65 ..................... 48 2.4.1. Methods............................................................................................. 48 2.4.2. Results and discussion...................................................................... 50 2.5. Purification of Native RPE65 from Bovine RPE ....................................... 52 2.5.1. Rationale and Methods...................................................................... 52 2.5.2. Results and discussion...................................................................... 55 Table............................................................................................................... 58 v Figures............................................................................................................ 59 CHAPTER 3: CRYSTALLIZATION AND STRUCTURE DETERMINATION OF RPE65 ................................................................................................................ 77 3.1. Construction of an RPE65 homology model ............................................ 78 3.2. Attempted crystallization of recombinant RPE65 ..................................... 79 3.3. Crystallization of native bovine RPE65 .................................................... 79 3.4. Diffraction data collection and phasing .................................................... 80 3.5. Model building, refinement and analysis of the model ............................. 86 Table............................................................................................................... 89 Figures............................................................................................................ 90 CHAPTER 4: DESCRIPTION AND INTERPRETATION OF THE RPE65 CRYSTAL STRUCTURE.................................................................................... 98 4.1. A general description of the structure ...................................................... 99 4.2. The membrane-binding surface ............................................................. 100 4.3. The active site cavity and iron-binding site ............................................ 101 4.4. Dimeric structure found in the asymmetric unit and analysis of crystal packing ......................................................................................................... 106 4.5. RPE65 amino acid substitutions associated with Leber congenital amaurosis ..................................................................................................... 109 4.6. Comparison of the RPE65 and apocarotenoid oxygenase structures.... 110 Figures.......................................................................................................... 114 CHAPTER 5: SUMMARY AND FUTURE DIRECTIONS .................................. 142 APPENDIX I: CLASSIFICATION OF MEMBRANE PROTEINS ....................... 152 vi BIBLIOGRAPHY............................................................................................... 155 vii LIST OF TABLES Table 1: RPE65 substrate specificity .................................................................. 27 Table 2: Detergents tested for extraction, purification and crystallization of bovine RPE65 ................................................................................................................ 58 Table 3: Data collection, phasing and refinement statistics. ............................... 89 viii LIST OF FIGURES Figure 1: Role of the RPE in maintaining photoreceptor health and function. .... 28 Figure 2: Schematic representation of the retinoid (visual) cycle........................ 30 Figure 3: Potential mechanisms of RPE65 membrane association. ................... 32 Figure 4: Proposed catalytic mechanisms of retinoid isomerization. .................. 34 Figure 5: Expression of an MBP-His10-zRPE65a fusion protein in E. coli and its purification. ......................................................................................................... 59 Figure 6: Purification of recombinant human RPE65 from insect cells. .............. 61 Figure 7: Immunostaining of a mouse retinal section using the monoclonal RPE65 antibody.................................................................................................. 63 Figure 8: Identification of the epitope-containing RPE65 segment recognized by a RPE65 monoclonal antibody. ............................................................................. 65 Figure 9: SDS-PAGE analysis of bovine RPE microsomal proteins after incubation with reagents