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1 CONSTRUCTION and DISULFIDE CROSSLINKING of CHIMERIC B CONSTRUCTION AND DISULFIDE CROSSLINKING OF CHIMERIC b SUBUNITS IN THE PERIPHERAL STALK OF F1FO ATP SYNTHASE FROM Escherichia coli By SHANE B. CLAGGETT A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008 1 © 2008 Shane B. Claggett 2 To the highest benefit of all beings. 3 ACKNOWLEDGMENTS I thank my parents for the gift of life and the desire to learn and grow, and I thank all of my teachers for the knowledge they have so diligently acquired and patiently passed on. I especially thank my professor, Dr. Brian Cain, and the members of my committee for their support and guidance. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES...........................................................................................................................8 LIST OF FIGURES .........................................................................................................................9 ABSTRACT...................................................................................................................................13 CHAPTER 1 INTRODUCTION ..................................................................................................................15 Overview of F1FO ATP Synthase............................................................................................15 Crosslinking as a Probe for Molecular Structure....................................................................17 Mechanism of Action of ATP Synthase .................................................................................19 Escherichia coli as a Model System of F1FO ATP Synthase..................................................24 Proton Translocating Subunits in FO ...............................................................................24 Subunit a ..................................................................................................................24 Subunit c...................................................................................................................33 Rotor Stalk Subunits........................................................................................................41 Subunit ...................................................................................................................41 Subunit ...................................................................................................................49 Catalytic Subunits of F1...................................................................................................56 Peripheral Stalk Subunits ................................................................................................63 Subunit ..................................................................................................................63 Subunit b ..................................................................................................................67 Comparison of The Peripheral Stalks of Different Organisms...............................................82 Chloroplast and Chloroplast-like Peripheral Stalks ........................................................83 Mitochondrial Peripheral Stalks......................................................................................84 2 MATERIALS AND METHODS .........................................................................................109 Bacterial Strains and Growth on Succinate ..........................................................................109 Preparation of Membranes....................................................................................................109 Determination of Protein Concentration...............................................................................110 Proton Pumping Assay Driven by ATP................................................................................111 Measuring the Rate of ATP Hydrolysis................................................................................112 Assay Using Acid Molybdate........................................................................................112 Assay Using MESG.......................................................................................................114 Crosslinking Using Cu2+.......................................................................................................115 Formation of the b-b Crosslink......................................................................................115 Formation of the b- Crosslink......................................................................................116 Nickel Resin Purification......................................................................................................116 5 Trypsin Digestion .................................................................................................................118 Western Analysis..................................................................................................................118 Electrophoresis of Proteins and Transfer to Membrane................................................119 Antibody Against the b Subunit ....................................................................................119 Antibody Against the V5 Epitope Tag ..........................................................................120 Densitometry Analysis of Western Blots ......................................................................120 3 FUNCTIONAL INCORPORATION OF CHIMERIC b SUBUNITS INTO F1FO ATP SYNTHASE .........................................................................................................................122 Introduction...........................................................................................................................122 Results...................................................................................................................................123 Plasmid Construction.....................................................................................................123 Complementation Analysis ...........................................................................................124 Stability of F1FO Complexes..........................................................................................125 Coupled F1FO Activity...................................................................................................127 Detection of Heterodimers ............................................................................................128 Discussion.............................................................................................................................130 4 DISULFIDE CROSSLINK FORMATION WITHIN CHIMERIC PERIPHERAL STALKS OF E. coli F1FO ATP SYNTHASE.......................................................................143 Introduction...........................................................................................................................143 Results...................................................................................................................................144 Functional Characterization of Cysteine Mutants .........................................................144 Development of the Crosslinking Assay .......................................................................146 Disulfide Crosslink Formation Between Chimeric Subunits ........................................147 Effects of ATP on Crosslink Formation........................................................................149 Discussion.............................................................................................................................150 5 DISULFIDE CROSSLINK FORMATION WITHIN THE WILD-TYPE PERIPHERAL STALK OF E. coli F1FO ATP SYNTHASE.........................................................................173 Introduction...........................................................................................................................173 Results...................................................................................................................................174 Functional Characterization of Mutants ........................................................................174 Crosslink Formation ......................................................................................................175 Effects of ATP on Crosslink Formation........................................................................176 Discussion.............................................................................................................................176 6 SPECIFIC INTERACTIONS BETWEEN AND THE INDIVIDUAL b SUBUNITS OF ATP SYNTHASE...........................................................................................................185 Introduction...........................................................................................................................185 Results...................................................................................................................................186 Functional Characterization of Mutants ........................................................................186 Development of Crosslinking Assay.............................................................................187 6 Crosslink Formation
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