Characterization of cytochrome c structure and function by site-directed mutagenesis. Item Type text; Dissertation-Reproduction (electronic) Authors Caffrey, Michael Stephen. Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 07/10/2021 10:29:38 Link to Item http://hdl.handle.net/10150/185346 INFORMATION TO USERS The most advanced technology has been used to photograph and reproduce this manuscript from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. 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Ann Arbor, MI 48106 CHARACTERIZATION OF CYTOCHROME~ STRUCTURE AND FUNCTION BY SITE-DIRECTED MlITAGENESIS by Michael Stephen Caffrey A Dissertation Submitted to the Faculty of the DEPARTMENT OF BIOCHEMISTRY In Partial Fulfillment of the Requirements For the Degree of OOCTOROF PHiLOSOPHY In the Graduate College of THE UNIVERSITY OF ARIZONA 1991 THE UNIVERSITY OF ARIZONA 2 GRADUATE COLLEGE As members of the Final Examination Committee, we certify that we have read the dissertation prepared by Michael Stephen Caffrey entitled Characterization of Cytochrome c Structure and Function by Site-Directed Mutagenesis and recommend that it be accepted as fulfilling the dissertation requirement Doctor of Philosophy for the Degree of Date Date Date Date Date Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation ' requirement. ~~ ~~r_e_c_t~o-r~. --~~----~------- Date 3 STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. ... 4 DEDICATION This work Is dedicated to my wife, Veronique; my parents, Maxine and Steve; and my sister, Mary. 5 ACKNOWLEDGEMENTS First, I would like to acknowledge the psychological and financial support of my parents during the course of my education. It has been a long journey from wanting to be a fireman, archeologist, or oceanographer to becoming a biochemist. I would like to thank Veronique, my wife, for her support during the writing of this thesis and for reminding me that cytochromes were not the prime focus of the universe. I would like to acknowledge my thesis director Michael Cusanovich for his patience, Intellectual Input, and encouragement over the years (and also for Introducing me to my wife). Many thanks go to the "lab", Jo, John and Terry, who made It fun to do science each day. In addition I would like to thank Bob Bartsch for his technical and spiritual guidance. Finally, I would like to acknowledge Fevzl Oaldal for his support in starting this project and Hazel Holden for providing the preliminary coordinates of Bhodobacter capsulatus cytochrome c2' 6 TABlE OF CONTENTS PAGE usr OF IIllJS1RA1l0NS •••.••.•••.••••. "•••.•..••••••••••.....•......•.••••••••••••••••••.......•..•.•.•••...••...•...••.•. 7 IJSTOF'fABIES ...................................................................................................................... 10 usr OF .ABBREVIA1l0NS ................................................................................................... 12 .ABSIRACr ................................................................................................................................ 15 CliAPIER.I. INIRODUCI10N ........................................................................................ 18 18 =~i"d·~·~~·~.. ::·:::::::::::·:::::::::::::·:::::::::::·.:'::::::::::::'::::::::::::':::::::::::.:. 25 RedOK Mutations ......................................................................................................: .•..••• 30 ChaIge Mutatlons ............................................................................................................ 34 St:ruCtural Mutations ..................................................................................................... 36 CHAPTER ll. EXPERIMENTAL MA'lERIALS AND ME1HODS •..••.•...••....•.....•...•.. 39 Mater1a1s .............................................................................................................................. 39 Genetic Procedures ........................................................................................................ 40 Sequencing and Mutagenic PrDners ........................................................................ 43 Mutagenesis ....................................................................................................................... 45 'I'rtparentaI Crosses ........ .................. .................................... .... ......... ......... .... .............. ... 46 Small-Scale Preparations andAml.lys1s of Cell Extracts ................................... 47 ~;~~~g~eroides·Ph~t~~ih~tl~·R{;~Ctl~~·c~~t~~··:·::::::·::::::.::::: §g Spectroscopy ..................................................................................................................... 51 RedOK PoteIltial I>etenn1natlons ......... .... .......... ...... ....... ............ ............ .................... 52 Kinetfc Mea.suren1eIlts .... ...... .......... ............... .......................... ...................................... 52 Protein Denaturations .................................................................................................... 53 Canputer Graphics ......................................................................................................... 55 CHAPI'ER m. 11IE RHODOBACIER CAPSULATUS/ RHODOBACIER SPHAEROIDES GENEIlC SYSIEM ..••.•.•••••...•••.•••••••.•.••.•.•.•.•.• 57 Ba~ ...................................................................................................................... .. 57 Const:Iuctlon of SUe-Directed Mutants ................................................................. .. 64 Product1on of Mutants 1nR ~ ................................................................. .. 66 Grnwth Properties of Merod1p Id Strains ........................................................... 70 Control ExperfInents ..................................................................................................... 71 Discussion of the Dual Genetic System .................................................................. 72 Sfgn1flcance of the RedOK Mutants ......................................................................... .. 78 Sfgn1flcance of the Charge Mutants ........................................................................ .. 80 S~ of the Structural Mutants .................................................................. .. 82 General Observations ..................................................................................................... 85 CHAP'IER IV. CHARACI'ERIZATION OF TIlE REDOX MUI'ANI'S ..................... 86 Introdud:Ion ...................................................................................................................... 86 Spectroscopy ...................................................................................................................