PAGE 1 CONTRIBUTIONS OF THE INDIVIDUAL b SUBUNITS TO THE FUNCTION OF THE PERIPHERAL STALK OF F 1 F 0 ATP SYNTHASE By TAMMY WENG BOHANNON GRABAR A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2004 PAGE 2 Copyright 2004 by Tammy Weng Bohannon Grabar PAGE 3 This document is dedicated to my husband, Chuck, and my daughter, Kaia. PAGE 4 ACKNOWLEDGMENTS The work illustrated in this dissertation and my growth as a scientist could not have been accomplished without the guidance, encouragement and support of several people on both the professional and personal levels. The first person I would like to thank is my mentor, Dr. Brian Cain. He allowed me to join his laboratory when I was fresh out of college, even though I had no real experiences in a scientific lab. He exhibited extreme patience while teaching me everything from how to hold and operate a pipette to pouring agarose gels to cloning my own plasmids. His evident passion and excitement about science opened my eyes to a whole new world of opportunity. Prior to joining his laboratory, I had never even dreamed of joining graduate school and pursuing a PhD; therefore, I feel extreme gratitude and consider myself very fortunate to have joined his lab. Once I joined the lab, Dr. Cain allowed me the freedom to make my own initial scientific and experimental decisions, which was an excellent teaching method for me, but he was always there for guidance and support whenever it was needed. I would also like to thank him for being so involved in his lab. On any given day, I knew I could have his undivided attention if I needed to consult with him. Over the years Dr. Cain has spent a tremendous amount of time teaching me to think critically about scientific experiments, how to communicate my data and ideas to others, how to give a professional scientific presentation, and how to write scientific papers, and for those countless hours I thank him. iv PAGE 5 I would also like to thank every person on my committee: Dr. Linda Bloom, Dr. Art Edison and Dr. Dan Purich from my department, and Dr. Julie Maupin-Furlow from the Microbiology and Cell Sciences Department. I have known Dr. Maupin-Furlow the longest. She taught one of the most challenging courses I took as an undergraduate. When an unexpected death occurred in my family, she was kind enough to allow me to postpone an exam without questioning my motive, which was very unusual for most of my professors while I was an undergraduate. Thanks to her, that was the only class in which my grade was not affected that semester. I would also like to thank her for her continual support in helping me get into graduate school and then subsequently taking the time to hike across campus to join my committee meetings. I would like to thank Dr. Purich for teaching me, in the middle of a physical biochemistry class, that sometimes we have to take some time off to go outside and get some fresh air. That was always an important lesson when endless hours in the lab led to careless mistakes. I also enjoyed his sometimes unusual stories and adventures that he had to share with me when I was spending entire days in the biochemistry library studying for exams. I would like to thank Dr. Edison for his constant support and encouragement. He has always been the first person who publicly and very kindly commended me after my journal club presentation. I believe his kind words of support through the years helped me to gain the courage I needed to believe in myself to really deliver a good presentation. I would also like to thank him for his eagerness to understand every aspect about my project. And last, but not least, I would like to thank Dr. Bloom. As a woman in my department and a new mother with a career in academia, she has been a wonderful role model. She has always had kind words and smiles to bestow on me. I would also like to thank her for v PAGE 6 assisting me during my committee meetings when discussions of fluorescence started to go over my head. I would also like to take the time to thank everyone that I had the pleasure of working with in the lab. These are the people I spent countless hours with during the course of the day and held many scientific and personal conversations with, and I am happy to be able to call them my friends. I could not have spent the last five years with a better group of people. Drs. Tammy Otto and Debra Zies were members of the lab when I first joined and were the ones who taught me the ways of the lab. Dr. Michelle Gumz joined the graduate program and subsequently joined Dr. Cains lab the same time as I. I would like to thank Tammy, Debbie and Michelle for their scientific and personal support as well as sharing with me memories of pool barbeques, wedding showers and baby showers. Dr. Deepa Bhatt joined the lab as a postdoc during my graduate career. Her friendship and scientific guidance have been very valuable to me. I would like to thank her for giving me fantastic advice on all of my oral presentations and reviewing all of my papers. And finally, I would like to thank my family for all the encouragement, love and support they have unwaveringly offered over the years. I would like to thank my dad for always finding the positive in everything that was negative and always encouraging me to overcome the many obstacles that graduate school hurled towards me. He never lost faith in me, even when I was ready to give up. I would like to thank my mom for her tremendous support as well. She spent a lot of time and energy stocking my refrigerator and freezer full of meals when I found that I did not have the time to care for myself. She has spent many weeks and weekends at my home since my daughter was born so that I vi PAGE 7 could have extra time to work on my dissertation. And last, but not least, I would like to thank my husband for his constant emotional support and belief in me. He has been with me through the thick and thin of graduate school and never once complained of my emotional torment when things were not going my way. I could not have accomplished this without him. vii PAGE 8 TABLE OF CONTENTS Page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................xii LIST OF FIGURES.........................................................................................................xiii ABBREVIATIONS.........................................................................................................xvi ABSTRACT.......................................................................................................................xx CHAPTER 1 BACKGROUND AND SIGNIFICANCE....................................................................1 Introduction...................................................................................................................1 Structure and Function of F 1 F 0 ATP Synthase.............................................................3 The Catalytic Core.................................................................................................6 The hexamer.............................................................................................8 The subunit................................................................................................12 The Rotor Stalk....................................................................................................12 The subunit................................................................................................14 The subunit................................................................................................16 The ring of c subunits...................................................................................21 The Stator Stalk...................................................................................................25 The a subunit................................................................................................27 The subunit................................................................................................33 The b subunit................................................................................................37 Subunit Equivalence...................................................................................................51 F 1 F 0 ATP Synthase Mechanism..................................................................................56 Proton Translocation: Driving Rotation..............................................................57 Coupling..............................................................................................................60 Catalysis: The Binding Change Mechanism.......................................................62 Genetic Expression and Assembly.............................................................................63 Summary.....................................................................................................................65 viii PAGE 9 2 INTEGRATION OF UNEQUAL LENGTH b SUBUNITS INTO F 1 F 0 ATP SYNTHASE...............................................................................................................67 Introduction.................................................................................................................67 Materials and Methods...............................................................................................69 Materials..............................................................................................................69 Strains and Media................................................................................................70 Recombinant DNA Techniques...........................................................................70 Mutagenesis and Strain Construction..................................................................75 Crude Preparative Procedures.............................................................................78 Determination of Protein Concentration.............................................................80 Ni-Resin Purification...........................................................................................80 Assays of F 1 F 0 ATP Synthase Activity...............................................................82 Immunoblot Analysis..........................................................................................85 Results.........................................................................................................................88 HA-Epitope Tagged b Subunits...........................................................................88 Construction and growth characteristics of mutants....................................88 Effects of epitope tags..................................................................................90 Expression of different b subunits in the same cell......................................91 Ni-Resin Purification....................................................................................92 V5-Epitope Tagged b Subunits...........................................................................97 Construction and growth characteristics of mutants....................................97 Effects of epitope tags................................................................................100 Detections of heterodimers.........................................................................103 Formation of mixed length b subunits in F 1 F 0 ATP synthase...........................107 Discussion.................................................................................................................111 3 GENETIC COMPLEMENTATION BETWEEN MUTANT b SUBUNITS IN F 1 F 0 ATP SYNTHASE.....................................................................................................114 Introduction...............................................................................................................114 Materials and Methods.............................................................................................116 Materials............................................................................................................116 Strains and Media..............................................................................................117 Recombinant DNA Techniques.........................................................................117 Mutagenesis and Strain Construction................................................................118 Preparative Procedures......................................................................................120 Immunoblot Analysis........................................................................................120 Assays of F 1 F 0 ATP Synthase Activity.............................................................121 Results.......................................................................................................................121 Construction and Growth Characteristics of Mutants.......................................121 Heterodimer Formation of b arg36 Defective Subunits with b wt ..........................123 Heterodimer formation of b 153end-his Complemented with b wt-V5 ...................128 Heterodimer Formation of b +124-130-his Complemented with b wt-V5 ....................131 ix PAGE 10 Mutual Complementation..................................................................................135 Discussion.................................................................................................................139 4 DEVELOPMENT OF CYSTEINE CHEMICAL MODIFICATIONS OF ALTERED b SUBUNITS............................................................................................................143 Introduction...............................................................................................................143 Materials and Methods.............................................................................................146 Materials............................................................................................................146 Strains and Media..............................................................................................146 Recombinant DNA Techniques.........................................................................147 Mutagenesis and Strain Construction................................................................148 Crude Preparative Procedures...........................................................................152 Assays of F 1 F 0 ATP Synthase Activity.............................................................152 Immunoblot Analysis........................................................................................153 Results.......................................................................................................................153 Construction and Growth Characteristics of Mutants.......................................153 Effects of Cysteine Mutations...........................................................................157 Discussion.................................................................................................................158 5 MUTAGENISIS OF THE AMINO AND CARBOXYL TERMINI OF THE b SUBUNIT IN F 1 F 0 ATP SYNTHASE.....................................................................161 Introduction...............................................................................................................161 Materials and Methods.............................................................................................165 Materials............................................................................................................165 Strains and Media..............................................................................................165 Recombinant DNA Techniques.........................................................................166 Mutagenesis and Strain Construction................................................................166 Crude Preparative Procedures...........................................................................168 Assays of F 1 F 0 ATP Synthase Activity.............................................................169 Results.......................................................................................................................169 Amino Terminal Mutations...............................................................................169 Construction and growth characteristics of mutants..................................169 Effects of amino terminal mutations..........................................................171 Carboxyl Terminal Mutations...........................................................................173 Construction and growth characteristics of mutants..................................173 Effects of carboxyl terminal mutation........................................................175 Discussion.................................................................................................................176 6 CONCLUSIONS AND FUTURE DIRECTIONS...................................................180 Conclusions...............................................................................................................180 Integration of Unequal Length b Subunits into F 1 F 0 ATP Synthase.................181 x PAGE 11 Genetic Complementation between Mutant b Subunits in F 1 F 0 ATP synthase..........................................................................................................183 Development of Cysteine Chemical Modifications of Altered b Subunits.......185 Mutagenesis of the Amino and Carboxyl Termini of the b subunit in F 1 F 0 ATP Synthase.................................................................................................186 Future Directions......................................................................................................188 Complementing Mutant b Subunits...................................................................189 Function of F 1 F 0 ATP Synthase Incorporated with b Subunit Heterodimers....190 Positions of the Individual b Subunits in F 1 F 0 ATP Synthase...........................190 Length of the Peripheral Stalk in F 1 F 0 ATP Synthase Complexes Incorporated with Shortened and Lengthened b Subunits.............................191 Other Implications....................................................................................................195 APPENDIX A MUTAGENIC OLIGONULCEOTIDES.................................................................202 B DEVELOPING PROTOCOL FOR PURIFYING F 1 F 0 ATP SYNTHASE.............206 Purification of Enzyme Complexes Incorporated with b Subunit Heterodimers.....206 Culture...............................................................................................................206 Disruption of Bacteria.......................................................................................207 Ni-Resin Purification.........................................................................................209 V5-Epitope Iimmunoprecipitation.....................................................................210 Detection of Purified Enzyme Complexes........................................................210 Assays of F 1 F 0 ATP Synthase Activity.............................................................211 LIST OF REFERENCES.................................................................................................212 BIOGRAPHICAL SKETCH...........................................................................................237 xi PAGE 12 LIST OF TABLES Table page 1-1. F 1 F 0 ATP synthase subunit equivalency....................................................................52 2-1. Aerobic growth properties and membrane-associated ATP hydrolysis activity of mutants expressing epitope tagged uncF(b) genes...................................................90 3-1. Aerobic growth properties and membrane-associated ATP hydrolysis activity of mutants expressing epitope tagged uncF(b) genes............................................122 4-1. Description of uncF(b) cysteine mutations.............................................................149 4-2. Description of the unc operon cysteine mutations 1 .................................................154 4-3. Aerobic growth properties and membrane-associated ATP hydrolysis activity of mutants expressing cysteine...............................................................................156 5-1. Aerobic growth properties and membrane-associated ATP hydrolysis activity of mutants expressing uncF(b) mutations at the amino terminus...........................170 5-2. Aerobic growth properties and membrane-associated ATP hydrolysis activity of mutants expressing uncF(b) insertions or deletions throughout the b subunit..175 6-1. Preliminary data of coexpressed mutant b subunits................................................190 A-1. Oligonucleotide sequences.....................................................................................203 A-2. Oligonucleotide description....................................................................................204 xii PAGE 13 LIST OF FIGURES Figure page 1-1. Timeline of developing views of F 1 F 0 ATP synthase...................................................5 1-2. Space-filling structural model of Escherichia coli F 1 F 0 ATP synthase........................7 1-3. Structure of the subunit of E. coli F 1 F 0 ATP synthase.............................................19 1-4. Controversial models of the a subunit topology.........................................................29 1-5. Amino acid sequence of the E. coli F 1 F 0 ATP synthase b subunit.............................38 1-6. Gross structure of the E. coli F 1 F 0 ATP synthase and the domains of the b subunit......................................................................................................................40 1-7. Model for F 1 F 0 ATP synthase peripheral stalk orientation dependent upon the direction of rotation during ATP synthesis or hydrolysis........................................47 1-8. Speculative models for the b-like subunits.................................................................55 1-9. Model of proton translocation and torque generation in F 0 ........................................59 1-10. The binding change mechanism...............................................................................63 2-1. Oligonucleotides for epitope tags and mutagenesis of uncF(b).................................74 2-2. Construction of the single transcript expression system............................................77 2-3. Histidine and HA-epitope-tagged b subunit expression system.................................89 2-4. Western blot analysis of histidine and HA-epitope tagged b subunits.......................92 2-5. Investigation of detergent solubilization of F 1 F 0 ATP synthase complexes..............94 2-6. Ni-resin purification of F 1 F 0 expressing different length b subunits, treated with the cross-linker BS 3 ..................................................................................................95 2-7. Ni-resin purification of histidine and HA-epitope tagged F 1 F 0 treated with the cross-linker BS 3 ........................................................................................................96 2-8. Histidine and V5-epitope-tagged b subunit expression system..................................98 xiii PAGE 14 2-9. ATP-driven energization of membrane vesicles prepared from uncF(b) gene mutants...................................................................................................................101 2-10. NADH-driven acidification of membrane vesicles prepared from uncF(b) mutants...................................................................................................................102 2-11. Ni-resin purification of F 1 F 0 ATP synthase treated with the cross-linker BS 3 .......106 2-12. Ni-resin purification of F 1 F 0 ATP synthase expressing unequal length b subunits...................................................................................................................108 2-13. Quantitation of b subunit heterodimeric F 1 F 0 .........................................................110 2-14. Interactions of b subunits of unequal lengths.........................................................112 3-1. Oligonucleotides for epitope tags and C-terminal truncation of uncF(b)................119 3-2. Ni-resin purification of F 1 F 0 ATP synthase incorporated with b arg36 subunit mutations................................................................................................................124 3-3. ATP-driven energization of membrane vesicles prepared from uncF(b) arg36 gene mutants...........................................................................................................127 3-4. Ni-resin purification of F 1 F 0 ATP synthase containing a b subunit carboxylterminal truncation.................................................................................................129 3-5. ATP-driven energization of membrane vesicles incorporated with F 1 F 0 ATP synthase containing a b subunit carboxyl-terminal truncation...............................130 3-6. Ni-resin purification of membranes incorporated with b +124-130-his subunit mutation..................................................................................................................132 3-7. ATP-driven energization of membrane vesicles incorporated with a defective b +124-130 subunit mutation........................................................................................133 3-8. Ni-resin purification of F 1 F 0 ATP synthase incorporated with complementing defective b subunits................................................................................................136 3-9. ATP-driven energization of membrane vesicles incorporated with F 1 F 0 ATP synthase containing complementing defective b subunits.....................................137 3-10. Interactions of defective b subunit with wild type b subunits found in intact F 1 F 0 ATP synthase complexes...............................................................................140 3-11. Model of F 1 F 0 ATP synthase incorporated with complementing defective b subunits ..................................................................................................................142 xiv PAGE 15 4-1. Model of E. coli F 1 F 0 ATP synthase.........................................................................144 4-2. Oligonucleotides for cysteine mutagenesis of the unc operon.................................150 4-3. Expression plasmid of cysteine mutants...................................................................155 4-4. Western blot analysis of cysteine mutant b subunits of differing length.................158 4-5. Model of F 1 F 0 ATP synthase with cysteine substitutions in the b and subunits..159 5-1 Amino acid sequence and domains of the E. coli b subunit......................................162 5-2. Oligonucleotides for mutagenesis at the amino and carboxyl termini in the unc operon.....................................................................................................................167 5-3. ATP-driven energization of membrane vesicles prepared from b subunit membrane domain mutants....................................................................................172 5-4. Amino acid insertion and deletion analysis of the E. coli b subunit........................174 5-5. Mutations constructed throughout the b subunit......................................................177 6-1. Design of FRET experiments to measure the peripheral stalk.................................193 6-2. Model of rotation inhibition due to a fusion protein on the subunit.....................195 6-3. Sequence alignments of subunits b and b from various species with the b subunit of E. coli....................................................................................................200 B-1. Diagram of purification procedures for homogeneous heterodimeric b V5 /b his F 1 F 0 ATP synthase complexes...............................................................................208 xv PAGE 16 ABBREVIATIONS ACMA, 9-amino-6-chloro-2-methoxyacridine ADP, adenosine-5-diphosphate ala, alanine AO, tegamineoxide WS-35 Ap, ampicillin Ap r ampicillin resistant asn, asparagine ADP, adenosine-5-diphosphate ATP, adenosine-5-triphosphate b +7-his seven amino acid insertion in the b subunit with a 6X histidine epitope tag at the amino terminus b 7-V5 seven amino acid deletion in the b subunit with a V5 epitope tag at the carboxyl terminus b ser84cys substitution of a cysteine for serine at amino acid position 84 in the b subunit -ME, -mercaptoethanol bp, base pair BS 3 bis(3-sulfo-N-hydroxysuccinimide ester) BCA, bicinchoninic acid BSA, bovine serum albumin Cm, chloramphenicol xvi PAGE 17 Cm r chloramphenicol resistance cys, cysteine DACM, N-(7-dimethylamino-4-methyl-coumarinyl)-maleimide DCCD, dicyclohexylcarbodiimide 2 the second -helix in the epsilon () subunit ECD, 1-ethyl-3[3-dimethylamino]propyl carbodiimide ECL, enhanced electrochemiluminescence EDTA, ethylenediaminetetraacetic acid FPLC, fast polynucleotide liquid chromatography FRET, fluorescence resonance energy transfer g, gravitational force gln, glutamine glu, glutamate GFP, green fluorescent protein HA, peptide epitope of hemagglutinin protein of human influenza virus ICBR, Interdisciplinary Center for Biotechnology Research IPTG, isopropyl-1-thio--D-galactopyranoside kb, kilobase kD, kilodalton LB, Luria Bertani medium LBG, Luria Bertani media supplemented with 0.2% glucose LDAO, lauryldimethylamine oxide LSB, Laemmli sample buffer mG, milligram xvii PAGE 18 mL, milliliter MOPS, 3-[N-morpholino]propanesulfonic acid NADH, -nicotineamide adenine dinucleotide, reduced form NFDM, nonfat dry milk Ni-CAM, high capacity nickel chelate affinity matrix NMR, nuclear magnetic resonance spectroscopy PAGE, polyacrylamide gel electrophoresis PBS, phosphate-buffered saline PBST, phosphate-buffered saline supplemented with 0.1% tween20 PCR, polymerase chain reaction P i inorganic phosphate P/O, number of ATPs made per 2 e transferred to oxygen PVDF, polyvinylidene fluoride rms, root mean square ser, serine SDS, sodium dodecyl sulfate (lauryl sulfate) TID, 3-(trifluoromethyl)-3-(m-[ 125 I]iodophenyl)diazirine TBS, tris-buffered saline thr, threonine TTBS, tris-buffered saline supplemented with 0.1% tween20 TD, taurodeoxycholate TE, tris[hydoxymethyl]aminomethane, ethylenediaminetetraacetic acid buffer, pH 8.0 T M tris[hydoxymethyl]aminomethane, magnesium sulfate buffer, pH 7.5 xviii PAGE 19 Tm, melting temperature of double stranded DNA Tris, tris[hydoxymethyl]aminomethane g, microgram L, microliter V5, epitope found in the P and V proteins of the paramyxovirus, SV5 v/v, volume/volume wt, wild type w/v, weight/volume xix PAGE 20 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CONTRIBUTIONS OF THE INDIVIDUAL b SUBUNITS TO THE FUNCTION OF THE PERIPHERAL STALK OF F 1 F 0 ATP SYNTHASE By Tammy Weng Bohannon Grabar August 2004 Chair: Brian D. Cain Major Department: Biochemistry and Molecular Biology The universal molecule of biological energetics is adenosine triphosphate (ATP), and the enzyme involved in providing the majority of cellular ATP is F 1 F 0 ATP synthase. Enzymes in this family utilize the electrochemical gradient of protons across membranes to synthesize ATP from ADP and inorganic phosphate in a coupled reaction. The cytoplasmic F 1 and the membrane-bound F 0 sectors are linked by two stalk structures, the rotor stalk and the peripheral stalk. Proton conduction through the F 0 sector drives the rotation of the rotor stalk within the catalytic core, which is held steadfast by the peripheral stalk. In Escherichia coli, the subunit of F 1 and a parallel homodimer of identical b subunits constitute the peripheral stalk of F 1 F 0 ATP synthase. Work accomplished in this dissertation indicates that the bacterial enzyme does not require two identical b subunits to form the dimer. Two different length b subunits, with a size difference of at least 14 amino acids, were capable of forming the b dimer of an intact F 1 F 0 ATP synthase complex. Also, in work presented in this dissertation, a defective xx PAGE 21 mutation in one region of the b subunit was overcome by dimer formation with a second b subunit that contained defective mutation in a different region but had a wild-type sequence in the region of the former defective b subunit. This mutual complementation between fully defective b subunits indicated that each of the two b subunits makes a unique contribution to the functions of the peripheral stalk, such that one mutant b subunit is making up for what the other is lacking. Interestingly, the equivalent of the bacterial b subunit in plants exists as two genetically different subunits, and the mammal counterpart exists as at least four subunits. This work suggests that the individual functions of the b subunits may be reflected in the fact that higher organisms evolved to encode multiple b-type subunits. xxi PAGE 22 CHAPTER 1 BACKGROUND AND SIGNIFICANCE Introduction The premiere of Peter Mitchells chemiosmotic theory in 1961 eventually resulted in the major breakthrough of the characterization of F 1 F 0 ATP synthases. Basically, his theory stated that protons are pumped across energy transducing membranes, thereby creating an electrochemical gradient of protons (1). This proton gradient, also known as the proton-motive force, consists of two components: i) a chemical component due to the concentration gradient of protons and ii) an electrical component, or membrane potential, due to the positive charge of the protons (H + ). As a result, one side of the membrane is more positive than the other. The potential energy of this gradient can then be transduced to chemical energy or utilized to perform work when the protons diffuse back across the membrane from the higher to the lower potential (2). The protons can diffuse across the membrane through specific transmembrane proton conductors, which can synthesize adenosine 5-triphosphate (ATP) or co-transport solutes, and in the case of bacteria drive flagellar rotation. The ability to consume nutrients and convert them to energy is required of all living organisms, from microscopic bacteria to plants to humans. The universal molecule of biological energetics is ATP, and in almost all organisms, the central enzyme involved in providing the majority of cellular ATP is F 1 F 0 ATP synthase (3-6). F 1 F 0 ATP synthases are responsible for the production of ATP in the final step of processes called oxidative phosphorylation and photophosphorylation. They provide the bulk of cellular energy in 1 PAGE 23 2 the majority of eukaryotes and prokaryotes. The synthesis of ATP occurs at a rate of about 100 s -1 which maintains a concentration of about 3 mM ATP in Escherichia coli and greater concentrations in mitochondria and chloroplasts with no noticeable product inhibition (3). In eukaryotes, they are located in the inner mitochondrial membrane, or in the thylakoid membrane of chloroplasts. In most bacteria, F 1 F 0 ATP synthase is located in the cytoplasmic membrane. Enzymes in this family utilize the electrochemical gradient of protons across these membranes in order to synthesize ATP from ADP and inorganic phosphate (P i ) in a coupled reaction. In bacteria, the reaction of ATP synthases can be reversed if the situation of a dissipated electrochemical proton gradient arises. In this case, ATP derived from glycolysis can be hydrolyzed in order to pump protons across the membrane, creating a membrane potential. The membrane potential can then be utilized to drive other cellular processes such as the extrusion of sodium ions, nutrient uptake and flagellar rotation. An explosion of research concerning F 1 F 0 ATP synthase has occurred during the past few decades. In particular, a great deal of knowledge of the enzyme has been solved only in the past decade. A plethora of relatively recent reviews concerning every aspect of F 1 F 0 ATP synthase can be found in the special editions of Journal of Bioenergetics and Biomembranes (volume 32, 2000) and Biochimica et Biophysica Acta (volume 1458, 2000) as well as reviews authored by Noji and Yoshida (2001), Senior et al. (2002), Capaldi et al. (2002) and Weber and Senior (2004). This chapter will provide detailed explanations of what is currently known about F 1 F 0 ATP synthase including the mechanism of the enzyme as a whole as well as structure and functions of individual subunits, equivalence of the bacterial enzyme to its eukaryotic equivalents and genetic PAGE 24 3 expression and assembly. The research presented in this dissertation primarily concerns the b subunit of the Escherichia coli (E. coli) F 1 F 0 ATP synthase. Hence the b subunit will be discussed extensively later in this chapter. Structure and Function of F 1 F 0 ATP Synthase The structure and function of F 1 F 0 ATP synthases are remarkably similar from bacteria to humans. In E. coli, the simplest form of the enzyme, F 1 F 0 ATP synthase is a complex enzyme composed of twenty-two polypeptides of eight different types with the stoichiometry of 3 3 ab 2 c 10 (Figure 1-2) (6, 7). The deduced molecular size is approximately 530 kDa. The structure of F 1 F 0 ATP synthase in chloroplasts is very similar with the exception that there are two isoforms of the b subunit. On the other hand, the mitochondrial enzyme is more complex, including an extra 7-9 small subunits which are thought to have roles in enzyme regulation (8-10). Discussion of F 1 F 0 ATP synthase is commonly divided into two portions, F 1 and F 0 The F 1 portion of the enzyme is composed of the cytoplasmic subunits, 3 3 and is responsible for the synthesis of ATP. The F 0 portion consists of the membrane-bound subunits, ab 2 c 10 and is responsible for the translocation of protons through the membrane. New insights concerning the functions and the intersubunit contacts have refined the way F 1 F 0 ATP synthase is perceived, dividing discussions of the enzyme into the catalytic core, the rotor (central) stalk and the stator (peripheral) stalk (3, 11-14). The catalytic core consists primarily of the 3 3 subunits, the rotor stalk consists of the c 10 subunits and the stator stalk consists of the b 2 subunits. Over the years, F 1 F 0 ATP synthase has received worldwide recognition as the tiniest rotary motor known to mankind (15, 16). Protons passing through the enzyme PAGE 25 4 complex drive the rotation of the rotor c 10 subunits at about 100 Hz. This rotation, which is absolutely essential for the machinery of the enzyme, transmits energy over a distance greater than 100 by providing the means by which conformational changes in the F 1 catalytic core, 3 3 take place for the synthesis of ATP (3, 4). Structural studies of F 1 F 0 ATP synthase commenced in the early 1960s and persist to this day in pursuit of a complete high-resolution structure. Negative staining procedures in the early 1960s initially revealed the traditional tripartite features of the enzyme complex from sub-mitochondrial particles, consisting of what was referred to as the headpiece, stalk and basepiece (Figure 1-1) (17). Ten years later, the first electron micrograph of a detergent-solubilized F 1 F 0 ATP synthase was published, confirming the existing idea of a tripartite molecule (18). Appreciably, electron microscopy (EM) in combination with other biochemical data of isolated F 1 exposed a hexagonal arrangement of alternating subunits with a seventh mass found in the center of the array (Figure 1-1) (7, 19). Based on this premise it was first suggested that F 1 consisted of an alternating hexagonal array of three and three subunits with the and situated centrally (7). The idea did not gain favorable recognition for some twenty years until verified by x-ray crystallography (20, 21). Continuous improvements in EM technology led to numerous publications of various ATP synthases which defined the average overall dimensions of about 190 from top to bottom and about 37 assigned to the stalk structure (22-25). Using a combination of traditional biochemical, molecular biological and immunological techniques along with EM, many important discoveries were made that led to what we now understand of F 1 F 0 ATP synthase. The first direct evidence for rotation of the stalk appeared in 1990 (24), but was not followed by the visualization of the peripheral stalk, PAGE 26 5 Figure 1-1. Timeline of developing views of F 1 F 0 ATP synthase. Electron microscopy and biochemical analysis from the early 1970s through the 1980s allowed visualization of the classical tripartite features of F 1 F 0 ATP synthase consisting of what was referred to as the headpiece, stalk and basepiece. Furthermore, the arrangement of the F 1 subunits were first proposed in 1974, though it did not gain favor until high resolution structure was obtained twenty years later. The first direct evidence for rotational catalysis appeared in 1990. Improved EM techniques showed the existence of a peripheral stalk, assigned the role of the stator to hold the F 1 sector in place against the proposed rotation, and a cap in the late 1990s. In 1994, the first high-resolution structure (2.8 ) of F 1 appeared, consisting of the 3 3 hexamer with partial structure of the subunit. Currently there is no high resolution structure for the entire F 1 F 0 ATP synthase complex though many of the subunits have been solved individually or in part by model polypeptides. PAGE 27 6 assigned the task of the stator to hold F 1 in place against the rotation of the centrally located stalk, until many years later (26-29). Today there is still no high-resolution structure of the entire F 1 F 0 ATP synthase enzyme complex from any organism. X-ray crystallographic and NMR data of partial complex structures from rat, bovine, yeast and E. coli or model polypeptides deduced from nucleotide sequences have accumulated over the past twenty years to allow for a composite structural model with both highand low-resolution structures (Figure 1-2). Currently, complete high-resolution structures are available for the , and c subunits and partial structures for the and b subunit. There is currently no high-resolution data for the membrane-integral a subunit. The Catalytic Core The first high-resolution structure, resolved to 2.85 consisted of 3 3 of F 1 prepared from bovine heart under inhibited conditions and in the absence of P i and substoichiometric amounts of ADP (20). This major breakthrough was shortly followed by a 2.80 F 1 isolated from rat liver in the absence of the physiological cation, Mg 2+ (21). The arrangement of the subunits in the two structures obtained were exceptionally similar and confirmed Catteral and Pedersens proposal made two decades prior by showing the three and three subunits arranged alternatively with the amino and carboxyl-termini of the subunit, each forming an -helix, extending up through the center of the hexamer (Figure 1-2) The only difference in the structures, which was the occupancy of the three nucleotide binding sites located at the interfaces, was likely due to the difference in preparation conditions (7) or crystal quality. In the former, PAGE 28 7 Figure 1-2. Space-filling structural model of Escherichia coli F 1 F 0 ATP synthase. The model is based on a composite of high and low resolution structures taken from E. coli, yeast and bovine F 1 F 0 ATP synthases. F 1 F 0 subunits included in the model were 3 3 ab 2 c 10 The subunits were color coded as follows: red; green; cyan; orange; yellow; b, blue; a, brighter yellow; c, darker blue. The direction of the arrow indicates the direction of rotation of c 10 during ATP synthesis. The yellow and blue cylinders represent the a subunit and portions of the b subunit that currently have no high-resolution structures from any species. PAGE 29 8 one of the catalytic sites was empty and in the latter, all three active sites were occupied with nucleotides (20, 21). Since the occupancy state of the three catalytic sites has been of considerable debate, a more accurate depiction of the F 1 moiety, which would give some insight about the mechanism of ATP synthesis, must be attained from crystals obtained under physiological conditions. A more accurate depiction of the structures and roles of each individual subunits of the F 1 ATPase follows. The hexamer Homology. The subunit of the E. coli F 1 F 0 ATP synthase, product of the uncA gene, is the largest subunit consisting of 513 amino acids with a deduced molecular weight of 55,313 Da. The subunit, a product of the uncD gene, is a 459 amino acid subunit, with a molecular weight of 50,325 Da. Based on the primary sequences, the and subunits of E. coli F 1 have the most obvious homologies in the chlororplast and mitochondrial enzymes (30). The highest conserved subunit from the E. coli F 1 F 0 ATP synthase is the subunit with approximately 70% homology with the chloroplast and mitochondria equivalents (31). The subunits exhibit roughly 50% homology (31). A total of 6 nucleotide binding sites are housed at the interfaces, three catalytic contributed primarily by the subunit and three noncatalytic housed primarily by the subunit (32, 33). The nucleotide binding regions have sequence homologies with other proteins that bind nucleotide or phosphate, including secA protein, N-ethylmaleimide sensitive fusion protein, herpes simplex virus UL15, Ca 2+ -ATPase, H + /K + ATPase and Na + /K + ATPase (34-37). Furthermore, the nucleotide binding motif, GXXXXGKT/S, known as the Walker A motif, which was first identified in the and sequences of F 1 has been found to be conserved in the high-resolution structures of other proteins PAGE 30 9 including p21 ras adenylate kinase, RecA, elongation factor Tu, and transducin(20, 38-42). Tertiary structure. The first high-resolution structure of bovine F 1 resolved at the atomic level (2.8 ) was solved by Walkers group a decade ago (20). It was found to be a flattened sphere approximately 80 high and 100 wide with the three and three subunits arranged as a hexamer of alternating subunits around a centrally located 90 long -helix formed by the subunit. A dimple 15 deep is located at the top of F 1 The amino-terminal regions of the and subunits were once thought to be in close proximity to the membrane due to labeling experiments (43). Contrary to this early data, the crystallographic data placed the amino-terminal regions on the top of the 3 3 hexamer over 100 away from the lipid bilayer. The folds of the and subunits were found to be nearly identical. They each consisted of a six-stranded -barrel at the amino terminus ( 19-95 9-82 ), a central domain containing the nucleotide-binding site ( 96-379 83-363 ) and a bundle of seven and six helices at the carboxyl termini of the and subunits, respectively ( 380-510 364-474 ) (20). The nucleotide binding domain consisted of a nine stranded -sheet with nine associated -helices, of which the -carbons of the seven -strands and the seven associated helices can be superimposed onto the RecA protein ATP binding site with an rms separation of 1.9 (20). The three catalytic sites were located at the interfaces of the 3 3 hexamer. In the original crystal structure, now commonly referred to as the reference structure, two of the three sites were occupied by nucleotide, containing MgADP ( DP site) and MgAMP-PNP ( TP site). The third site was empty and designated E . The DP and TP PAGE 31 10 subunits were in similar, closed conformations whereas the E adopted an open conformation, differing from the other two by a large hinge motion of the carboxyl terminal domain of greater that 20 Subsequently, several high-resolution structures of crystals obtained under various nucleotide conditions gave the same overall structure of bovine F 1 with two nucleotides bound (two nucleotide structures) (44-49). The TP site was found to occasionally contain a diphosphate nucleotide, establishing that there is no requirement for TP to be occupied by a nucleotide triphoshate to produce a conformational change (48). A more recent structure of bovine F 1 solved by the Walker group at 2.0 showed all three catalytic sites bound by nucleotide (50). Both the TP and DP sites contained MgADP, adopting the closed conformation, whereas the site corresponding to the E site in previous structures contained MgADP+P i and adopted a half-closed conformation. It is thought that the DP site is actually the catalytic site. The structure of rat liver F 1 was solved (2.8 ) in the presence of physiological concentrations of nucleotides but in the absence of the physiological cation, Mg 2+ In this structure, all three nucleotide binding sites adopted strikingly similar conformations, analogous to the DP and TP of the previously reported structure of the bovine F 1 This structure had no indication of the open conformation and showed the presence of nucleotide in all three sites (21). The structure of an 3 3 complex from a thermophilic bacterium was solved in the absence of nucleotides and exhibited all three subunits in the open conformation, suggesting there is a correlation between the open conformation and the absence of nucleotide (51). A low resolution crystal structure (4.4 ) of the E. coli F 1 has been obtained by Capaldis group, in which the catalytic sites are thought to be very similar to that of the bovine structure; however, the occupancy state of the nucleotide binding sites PAGE 32 11 was unclear (52). The frequent reports of two nucleotide structures have bewildered scientists due to the vast body of biochemical data from numerous laboratories, using a variety of techniques, which establish indisputably that all three catalytic sites are readily filled with nucleotide (32, 53). It is possible that the enzyme preferentially crystallizes in a ground state intermediate which may occur after the release of product, leaving one site empty and opened (54). Nevertheless, the accumulating structural data may be indicative of several intermediary steps that may form during the synthesis of ATP. A detailed account of the mechanism of ATP synthesis follows later in this chapter. The crystal structure does offer some insight of the chemical mechanism of ATP synthesis (20). In the subunit, 4.4 from the terminal phosphate of the bound nucleotide triphosphate, there is clearly a density for a water molecule hydrogen bonded to the carboxylate of glu188 This carboxylate is positioned to allow an inline nucleophilic attack of the water molecule on the terminal phosphate. The guanidinium of a neighboring residue, arg373 is thought to help stabilize the negative charge on the terminal phosphate during the transition state (20). This same arrangement can be found in the catalytic site of transducin(42). The crystal structure also provides some insight as to why the nucleotide binding sites in the subunit are noncatalytic. There is no spacial equivalent of the carboxylate of glu188 in the subunit. The spatial equivalence in the subunit is filled by a gln208 with the side chain pointed away from the terminal phosphate (20). The binding of the adenine to the noncatalytic site of the subunit is highly specific, unlike the subunit nucleotide binding site, which can accommodate GTP, ITP as well as ATP (55, 56). This specificity is due to several hydrogen bonds as well as the presence of the tyr368 close to the 2-position of the adenine ring in the PAGE 33 12 subunit, while in the subunit the adenine is in contact with a hydrophobic interface (20). Though the binding sites in the subunits are highly specific, the roles remain obscure. The subunit The subunit plays an important role in the catalytic core. Interactions between the aminoand carboxyl-terminal -helices and the 3 3 subunits are responsible for the conformational changes that result in ATP catalysis. The subunit is a fundamental part of the rotor stalk. The Rotor Stalk Two narrow stalks, a centrally located stalk and a peripheral stalk, have been observed to link the catalytic core of F 1 and the membrane-bound proton translocating F 0 with about of 40-45 in between (27). The central stalk came into view three decades ago via EM and has since become widely referred to as the rotor stalk. The rotor stalk consists of the and subunits. The bottom of the rotor stalk is connected firmly to the F 0 ring of c subunits located in the membrane and the top extends 90 within the 3 3 hexamer of F 1 where it forms crucial interactions with both the and subunits (57-59). F 1 F 0 ATP synthase is an extraordinary enzyme due to its ability to couple potential energy, obtained from proton translocation through F 0 in the membrane, to the synthesis of chemical energy, over 100 away in F 1 by a rapid rotation of subunits. Although predicted by Boyer in the 1970s, evidence of rotation did not appear until the early 1990s. The X-ray crystal structure solved by Walkers group suggested that the subunit was the rotating subunit by suggesting it could distribute itself to all three subunits as opposed to just one (20). Consistent with this idea was inhibition of the F 1 PAGE 34 13 complex by crosslinking the subunit to one of the or subunits (60, 61) and recovery after photobleaching experiments (62, 63). More convincing evidence was provided when Duncan et al. crosslinked the subunit to an unlabeled subunit by disulfide bond and then mixed the complex with 35 S-labeled subunit (along with the and subunits). When the disulfide bond was broken and ATP was added, the subunit was observed to switch from labeled to unlabeled subunit (64). Finally, direct evidence was achieved in single molecule experiments by attachment of a fluorescent actin filament to the subunit and observance of unidirectional rotation of the actin filament upon addition of ATP (15). Direct observation of the rotating subunit was soon followed by observance of the rotation of the and c subunits at the same speed and direction, indicating that these three subunits rotate in synchrony, forming the central rotary machinery of the enzyme complex (65-67). Until very recently, rotation has only been observed in the direction of ATP hydrolysis. Direct evidence for the synthesis of ATP by F 1 has been shown by attaching a magnetic bead to the subunit of F 1 fixed to a glass surface and the rotating the bead, in the appropriate direction, using electrical magnets (68). The first structural information obtained for the subunit of E. coli was accomplished by nuclear magnetic resonance (NMR) studies (69) and is good agreement with the crystal structure solved at 2.3 (70). In all previous crystal structures of the rotor stalk, the portion of the rotor stalks subunit protruding from the F 1 hexamer and the subunit were disordered. Recently, the structure of the bovine homologs of the rotor stalk and subunits has been solved and refined to 2.4 (48). The structures of the E. coli and subunits are remarkably similar with that from bovine F 1 When PAGE 35 14 comparing the structures of the and rotor stalk obtained under different conditions or from different sources, in combination with an overwhelming amount of biochemical and immunological evidence, it is clear that the domains of the two subunits undergo major shifts in position, which reflects its fundamental role in the synthesis of ATP (20, 44, 48, 71-80). The subunit In E. coli, the subunit is the third largest subunit of F 1 F 0 ATP synthase, encoded by the uncG gene as a 286 residue polypeptide with a deduced molecular weight of 31,563 Da. It plays an essential role in coupling proton transport to the synthesis of ATP. The first visualization of a portion of the subunit was a bovine F 1 partial structure solved in combination along with the 3 3 hexamer revealing three -helices (20). The 209-272 (residues 223-286 in the E. coli sequence) carboxyl terminus formed a long (90 ) -helix extending from the stalk structure seen by EM to about 15 from the top of the hexamer. The bottom half of this helix formed a left-handed anti-parallel coiled coil with a shorter -helix composed of the amino-terminal residues 1-45 (20). The two helices protruded about 30 from the bottom of F 1 An approximately 20 kink in the latter helix was produced by pro40 and a similar but less pronounced kink was induced by leu217 in the former (48). A third, much smaller -helix, composed of 73-90 (residues 83-99 in the E. coli sequence) was inclined at about 45 degrees from the larger helices and located directly under the F 1 hexamer. More recently, the complete structure of the bovine rotor stalk has been solved to 2.4 (48). The overall length of the stalk, from the carboxyl terminus of the subunit to the very bottom where it contacts the ring of c subunits, was 114 The portion that PAGE 36 15 protrudes from the 3 3 hexamer, i.e. the part seen in electron micrographs, was 47 long and 54 wide at its largest cross-section. A completely new / domain, consisting of a five-strand -sheet (1-5) and six -helices (a-f), was identified in the complete structure of the subunit. Helices a and f extended into the 3 3 hexamer to form the antiparallel coiled coil discussed previously. Strands 1-3 along with helices b and c formed a Rossman fold that forms extensive interactions with the subunit as well as the ring of c subunits in the membrane (discussed below). This fold was linked to a -hairpin, formed by strands 4 and 5, by helix d. Overall, this / domain had a globular, oval shape with the dimensions 51 wide by 41 high. The positioning of the / domain at the base of the rotor stalk may provide stability to the structure of the rotor stalk during rotational catalysis (48). A low-resolution crystal structure of the E. coli F 1 F 0 ATP synthase subunit was solved to 4.4 (52). Upon comparison with the high-resolution bovine structure obtained one year later, a few differences were observed (48). Helices a and f (see above) were extended by and extra 12 and 20 residues, respectively. Four additional putative -helices, designated B and D-F, were found in the E. coli structure and had little agreement with the bovine subunit structure. E. coli helix B runs parallel with the bovine -strand 5 and may correspond to it. Helix D has no apparent equivalent in the bovine model and helices E and F appear to overlap with regions in the bovine (bacterial see Table 1-1) and (no equivalent in bacteria) subunits. The remainder of the E. coli structure appeared similar to the bovine structure. The crystal structure displays a strikingly asymmetrical F 1 due to differences in the domains of the and subunits and the interactions formed with the single subunit PAGE 37 16 (20). The obvious asymmetric positioning of the coiled coil of the subunit is a key feature to the mechanics of the binding change mechanism of F 1 F 0 ATP synthase. Its large carboxyl terminus -helix passes through a hydrophobic sleeve formed by six proline-rich loops of the and subunits, undoubtingly resulting in the conformational changes occurring in the catalytic sites (20). In the E subunit (see above), several hydrogen bonds are formed with the subunit, which forms a catch, resulting in conformational changes. Specifically, residues arg254 and gln255 in the carboxyl terminal helix form hydrogen bongs with E-asp317 E-thr318 and E-asp319 Also, a second catch is formed between the carboxyl terminal domain of the T subunit and the short helix of the subunit. Hydrogen bonds formed between lys87 lys90 and ala80 with T-asp394 and T-glu398 This sequence of the subunit, DELSEED ( 394 400 ), is a portion of the binding site of amphipathic cationic inhibitors and putatively the ATPase inhibitor protein (81-83). Recently, mutations of residues involved in the catch loops were shown in inhibit ATP hydrolysis activity by the soluble F 1 -ATPase (84). Structural information suggests the two antiparallel coiled coil -helices of the subunit may unwind during rotational catalysis and the subunit rotates around the F 1 axis while undertaking a net translation of about 23 (85). It is likely that these gross changes observed in the structures revealed individual functional states of the enzyme complex during catalysis. The subunit The subunit of E. coli F 1 F 0 ATP synthase consists of 138 amino acids with a molecular weight of 15, 068 Da and is encoded by the uncC gene. The subunit has several putative functions in the F 1 F 0 ATP synthase complex including structural, inhibitory and coupling roles. Structurally, the binding of F 1 to F 0 has long been known PAGE 38 17 to require the presence of the subunit, which had implicated it as part of stalk structure (86). In isolated F 1 and in isolated F 1 F 0 to a lesser extent, it has been shown to inhibit ATP hydrolysis activity (87-89). The removal of from isolated F 1 resulted in up to a 10-fold increase in ATP hydrolysis activity. Furthermore, a truncated version of the subunit lost all inhibitory functions but still promoted binding of F 1 to F 0 hence the inhibitory feature has been assigned to the extreme carboxyl terminus (90). It was speculated that it acts as an inhibitor by reducing the rate at which the product is released from the catalytic site (91). Also, the subunit, as part of the rotor stalk, plays a role in the coupling of proton translocation to the catalytic site. The subunits diversity of functions is supported by the findings that it produces several points of interactions with the (61, 74, 92), (61, 88, 89, 93, 94) and (74, 95-97) subunits of F 1 and the c (98, 99) subunits of F 0 An innovative set of experiments conducted by members of the Dunn laboratory made use of different sized fluorescent proteins, ranging from the 12 kDa cytochrome b 562 protein to the 30 kDa flavodoxin reductase protein with a 20 residue linker (100). The proteins were fused to the carboxyl-terminus of the subunit. Since the subunit is part of the rotor stalk, according to the concept of rotational catalysis, the fusion of a large protein at this site should sterically hinder rotation due to the presence of the peripheral stalk. Cells expressing the smaller cytochrome b 562 protein fused to the subunit grew on minimal media, indicating a functional F 1 F 0 ATP synthase complex. However, cells expressing the larger flavodoxin reductase protein fused to the subunit, though found in an intact enzyme, failed to grow. These results provided the first evidence, in vivo, supporting rotational catalysis. PAGE 39 18 High-resolution structural data of the E. coli subunit was first solved by NMR followed by the X-ray crystal structures of the isolated and complexed subunits (69, 70, 101). The isolated subunit consisted of an 84 residue amino-terminal -sandwich domain and a 48 residue carboxyl-terminal helix-turn-helix domain in which the two-helices formed an antiparallel hairpin (70). The -sandwich consisted of two five-stranded -sheets folded as a rigid, flattened -barrel. The structure of the isolated subunit from E. coli was very similar to the F 1 complex isolated from bovine, which included the 3 3 subunits (48, 70). Superimposition of 127 of the amino acid C resulted in an rms deviation of 1.6 On the other hand, in the E. coli complex, resolved to 2.1 the subunit assumed a strikingly different conformation, in which the two -helices of the antiparallel hairpin at the carboxyl-terminus are wide apart and wrapped around the subunit (Figure 1-3A) (101). Subsequently, both conformations of the subunit have been trapped in E. coli F 1 F 0 ATP synthase by crosslinking experiments, confirming the existence of both in an intact enzyme complex (102). Furthermore, Capaldis group observed that when the carboxyl-terminal helices assume the hairpin conformation, bringing them closest to the F 0 sector, ATP hydrolysis was activated. Still, the enzyme was fully coupled in the direction of either hydrolysis or synthesis. In contrast, when the two helices were open, assuming a position closer to the F 1 sector, ATP hydrolysis was inhibited and the enzyme functioned only in the direction PAGE 40 19 Figure 1-3. Structure of the subunit of E. coli F 1 F 0 ATP synthase. The residue numbers and subunit labels are color coded to match the subunits it represents. The subunit has been suggested to undergo large conformational changes during catalysis from an overwhelming amount of biochemical data. Two different structures have been obtained for the subunit, confirming the previous data. A) Superimposition of the C trace of the structure obtained from isolated subunit (-helices shown in red and the -sandwich shown in blue) with the structure obtained from the complex (yellow). B) Composite structural model. Rotor stalk is based on the crystal structure obtained from the complex. The DELSEED 2 and the -sandwich are indicated to show the close proximity of DELSEED and 2 as well as the relative distance between the 2 and the -sandwich. PAGE 41 20 of ATP synthesis. This conformational switch of the subunit was therefore suggested to play a key role as a selective inhibitor of ATP hydrolysis and directional regulator of rotational catalysis by acting as a ratchet (102). Movement of the two -helices was consistent with other observations. Changes in the subunit conformation due to nucleotide occupancy in the catalytic sites has been observed in tryptic proteolysis experiments (89). Cysteine replacements in the carboxyl-terminal -helix ( 2 ) resulted in crosslinks with the and subunit (61, 103). More importantly, treatment with a zero-length crosslinker, 1-ethyl-3[3-dimethylamino]propyl carbodiimide (EDC), resulted in a high yield of crosslinks between the subunit and the DELSEED ( 380 386 ) region of the subunit ( DELSEED ) following ATP hydrolysis in the catalytic sites, but these interactions are disrupted upon the subsequent binding of ATP. Also, in a composite structure of F 1 F 0 ATP synthase incorporated with the E. coli complex as solved by Rodgers and Wilce, the sandwich was at least 10 away from the DELSEED region (Figure 1-3B) (101). The carboxyl-terminal 2 produces several points of interactions with the and as well as points of interactions with its own -sandwich domain (61, 74, 88, 89, 92-99). In order for the 2 to interact with the DELSEED 1 and the subunit -sandwich domain, it is clear from the structure that the subunit would be required to undergo large movements during the catalytic cycle. In E. coli, F 1 F 0 ATP synthase can act in two functional directions. In the case of a dissipated electrochemical gradient, the F 1 F 0 complex acts primarily as an ATPase in order to pump protons across the membrane to provide a gradient to drive various ion transport activities in the cell. Under severe conditions where cellular ATP levels are exceedingly low the enzyme acts predominantly in the direction of ATP synthesis. PAGE 42 21 Therefore, one can imagine that the ability to selectively turn off ATP hydrolysis, while preserving ATP synthesis function, may be important for E. coli. In mitochondria the ability to control the F 1 F 0 ATP synthase complex is essential. It is believed to act exclusively in the direction of ATP synthesis and is strictly regulated (104). The ring of c subunits The c subunit is one of the three membrane-bound F 0 subunits of F 1 F 0 ATP synthase. Ten copies of the c subunit form a ring in the membrane that plays a crucial role in both proton translocation and rotation of the rotor stalk (105). It is the smallest subunit of the F 1 F 0 ATP synthase enzyme complex with 79 amino acids and a molecular weight of 8,256 Da and it is encoded by the uncE gene. Structure and topology. Early biochemical, genetic and immunological data had suggested the structure of the c subunit to be that of a helical hairpin with two lipophilic -helices (amino acids 1-41 and 50-79) separated by a hydrophilic loop (amino acids 42-49). Both of the putative transmembrane helices in the regions of c leu4-leu19 and c phe53-phe76 were vulnerable to chemical modification by the nonpolar photoreactive reagent 3-(trifluoromethyl)-3-(m-[ 125 I]iodophenyl)diazirine (TID), which is a hydrophobic carbene generator that is believed to react from the nonpolar region of the lipid bilayer, indicating that these regions were in fact in the hydrophobic phase of the bilayer (106). The loop region of the hairpin is substantially more polar and antibodies against it were shown to bind to F 1 -stripped inverted membrane vesicles suggesting that it resides in the cytoplasmic of the cell (107, 108). Both of the transmembrane helices are devoid of charged amino acids with the noteworthy exception of c asp61 in the center of the second helix, which undergoes a protonation and deprotonation cycle during proton translocation PAGE 43 22 (discussed in F 1 F 0 ATP Synthase Mechanism below) Dicyclohexylcarbodiimide (DCCD) reacts specifically with c asp61 blocking proton translocation, and this reaction is blocked by the mutations c ala24ser or c ile28thr suggesting that the c subunit is folded in such a way so that the asp61 of the second helix is in close vicinity to residues 24 and 28 of the first helix (109, 110). This model was further supported by the ability to move the critical aspartate from residue 61 in the second helix to residue 24 in the first helix without disruption of enzyme function (111). Also, only one subunit in the ring of 10 c subunits need be modified by DCCD to inhibit activity, indicating that each one of the c subunits is consecutively involved in proton translocation (112, 113). Furthermore, modification of the c subunit by DCCD trapped the configuration of the subunit (discussed above), providing evidence for a connection between the c and subunits (89). Intersubunit contacts made by the c subunit are evident from mutational data and gives some insight to the topology. Mutations constructed in the polar loop region can disrupt the binding of F 1 to F 0 (114-117). Three conserved amino acids, c arg41 -c gln42 -c pro43 lie at the apex of the polar loop region and are predicted to interact with the F 1 subunit (98, 116). F 1 F 0 ATP synthase complexes with the uncoupling mutation, c gln42glu were found to be recoupled with a second site suppressor mutation in the subunit of F 1 glu31gly, val, or cys (98) and was shortly followed by the observance of disulfide bridge formation between the c subunit and the and subunits (99, 118). Also, switching the essential aspartate from residue 61 in the second helix of the c subunit to residue 24 of the first helix (discussed above) resulted in a functional F 1 F 0 ATP synthase complex though the cells were not as healthy compared to cells containing a wild-type enzyme complex (111). Eighteen third-site suppressor mutants were found that helped to PAGE 44 23 optimize this c ala24asp,asp61gly defect, with only five laying on the c subunit and 13 in the a subunit, all near the a arg210 residue, which is required for proton translocation (further discussed below) (119, 120). Early models of the organization of F 0 suggested that the ring of c subunits were situated on the periphery, surrounding the centrally located a and b subunits, which rotated in the center of the ring (121). This model was proved wrong by high-resolution NMR data (122) and cross-linking experiments (123, 124) which indicated that the oligomer of c subunits are closely packed with a lipid filled core less than 25 wide. The individual c subunits are packed front-to-back such that the second helix of each is situated towards the exterior and the first helix is located on the interior, which renders the c asp61 exposed to the lipid environment. The uncommonly high pKa (7.1) of the c asp61 carboxyl side chain is likely due to this hydrophobic environment (125). Furthermore, scanning force and cryoelectron microscopy demonstrated that F 0 is asymmetrically arranged in the membrane (27, 126, 127). For these reasons, the a and b subunits are thought to be situated to the periphery of the ring of c subunits. High resolution structures of membrane-bound proteins were nonexistent for many decades past structural determination of soluble proteins and still prove difficult to this day due to their highly hydrophobic nature. The membrane intrinsic c subunit of E. coli, which was solved by NMR in an organic solvent (chloroform-methanol-water) in the 1990s, was one of the earliest high-resolution structures of a transmembrane helical protein (122, 128-130). Notable, the c subunit could be reconstituted from the organic solvent mixture with complete preservation of function; therefore, it was clearly not irreversibly denatured (113). As predicted two decades prior, the c subunit folds as a hairpin of two extended -helices with the c asp61 of the second helix packed less than 5 PAGE 45 24 from the c ala24 and c ile28 of the first helix (122). With the exception of c asp61 in the second helix, both helices consist entirely of nonpolar amino acids. The first helix is greatly enriched in glycines and alanines, which led to a smaller diameter. The -helical structure of the second helix is interrupted around c asp61 due to disrupted hydrogen bonds around c pro64 which cause the angle of the helical packing to change direction from there to the carboxyl terminus (122). A recent study, using parallax analysis of fluorescence quenching, the proton binding site c asp61 was found to be deeply embedded in the membrane at about 1.8 from the center of the bilayer (131). Stoichiometry. The stoichiometry of c subunits would be valuable in determining the number of protons transported per ATP synthesized and will directly relate to the P/O ratio of oxidative phosphorylation. However, the number of c subunits in F 0 had been a matter of controversy for many years. The number of c subunits in an F 1 F 0 ATP synthase complex was suggested to be between 9 and 14, but whether this number fluctuated based on the species or environmental conditions or whether it was a fixed number were the two prevailing arguments until just a few years prior. Based on a related family of vacuolar (V-type) ATPases, in which the proposed subunit c had evolved into a fused dimer of four transmembrane helices with a single proton-transporting glutamate in the center of the fourth helix, Fillingame et al. set out to genetically fuse the E. coli c subunit by introducing a flexible loop of similar length (123). The generated c-c dimers and c-c-c trimers resulted in functional enzyme complexes. In combination with crosslinking studies and normalization to the / content of the membranes, the favored stoichiometry was fixed to 12 c subunits per F 1 F 0 ATP synthase complex (132). More recently, the experiment was revised to include only trimers and tetramers of the c subunit (105). PAGE 46 25 Partial activity was observed in complexes incorporated with eight (c 4 + c 4 ) or nine c (c 3 + c 3 ) subunits and crosslinked products of more than 10 c subunits were observed but did not purify in intact enzyme complexes. Crosslinking showed that the preferred stoichiometry of c subunits in intact E. coli F 1 F 0 ATP synthase was c 4 + c 3 + c 3 or 10 c subunits. This number is consistent with the c 10 oligomer found in the yeast crystal structure of a yeast F 1 F 0 ATP synthase consisting of 3 3 c 10 resolved to 3.9 (58). However, the preferred number may still vary in different species. The archaebacteria Methanococcus jannascjii ATP synthase has a natural c subunit trimer and therefore cannot incorporate the E. coli equivalent of c 10 in the membrane (133). With 10 c subunits present in the membrane 3.3 protons are required per ATP synthesized, which was compatible with the early experimentally determined ratio of 3 H + /ATP estimated from E. coli whole cells (134). This value also indicates a P/O value of 2.3 from NADH-linked substrates and 1.4 for succinate, also compatible with the predicted values of 3 and 2, respectively (135). The Stator Stalk The b and subunits were once believed to form the central, rotating stalk of F 1 F 0 ATP synthase. However, high resolution crystallographic data refuted this idea in the mid 1990s (20). The stator stalk did not come into view until improved EM technology observed a peripheral stalk in the late 1990s (26-29) and the visualization of the subunit, as a cap structure atop F 1 F 0 ATP synthase, soon followed (27-29). As its name implies, the role of the stator is to hold the 3 3 hexamer in place against the rotation of the subunit during rotational catalysis. Based on chemical crosslinking data, it is currently believed that the a subunit resides to the periphery of the ring of c subunits with PAGE 47 26 the membrane-spanning domain of the b dimer situated to one side of the a subunit where it is in close proximity to both the a and c subunits. The b dimer extends out of the membrane and in a highly elongated conformation reaches to near the top of F 1 making contacts with theand subunits along the way and the subunit at its extreme carboxyl terminus. The stator stalk consists of the b subunit of F 0 and the subunit of F 1 Although the primary function of the a subunit of F 0 is considered to be a role in proton translocation along with the c 10 subunits, it nevertheless plays the part as a stator and will be discussed in this section. Structurally, the a subunit plays a role in both the formation of a dynamic interface with the ring of c subunits as well as the formation of a secure complex with the b dimer. Pursuit of a high-resolution structure for the a subunit remains a challenge to this day. The high-resolution structure of the region of the a subunit that forms the interface with the c 10 subunits is eagerly anticipated since it appears to be the crucial region for proton translocation. In regard to the stator stalk, partial structural information has been obtained for the E. coli b subunit membrane spanning domain and the subunit amino terminus by NMR studies (136, 137). X-ray crystallography has solved the structure of a model polypeptide based on the dimerization domain of the b subunit (138). The binding of F 1 to the membrane-bound F 0 requires both the and subunits, suggesting that each of the subunits are involved with the stalk structures of F 1 F 0 ATP synthase (87, 139, 140). In fact, the subunit forms an integral part of the peripheral stalk and the subunit functions as part of the central stalk (discussed below). The subunit of F 1 has been visualized seated at the very top of the F 1 3 3 hexamer by EM (11). However, recent PAGE 48 27 evidence has suggested that the subunit may actually be positioned slightly to the side of F 1 in association with only a single subunit (Figure 1-2) (100, 141-143). The b subunit is the primary focus of this dissertation and will be discussed at length later in this chapter. The a subunit The E. coli a subunit is a large, extremely hydrophobic protein encoded by the uncB gene and consists of 271 amino acids with a molecular weight of 30,276 Da. All enzymes of the F 1 F 0 ATP synthase family contain an a subunit homolog with strong primary sequence homology even among evolutionarily diverse species (144). The most highly conserved region resides in the carboxyl-terminal one-third end, amino acid residues a 190-263 Notably, in the region that is involved in proton translocation, there is a remarkable conservation of the amino acid residues a leu207 a arg210 a leu211, a asn214 and a gln252 and an evident conservation of a glu219 and a his245 at the homologous positions in all a subunits from different species (144). The a arg210 is the most strictly conserved among all species and does not tolerate substitution with any other amino acid (further discussed below) (145-147). As mentioned above, there is no high-resolution structural data for the a subunit. Also, contradicting models exist concerning the number of transmembrane helices as well as the orientation in the membrane. Difficulty in studying the structure of the a subunit arises from its extreme hydrophobicity and the necessity to include the denaturant, trichloroacetate, in purification procedures. This is compounded by the fact that it cannot be expressed at high levels in E. coli and is not found in the membrane without the presence of both the b and c subunits (148-150). Furthermore, the a subunit is known to PAGE 49 28 be a substrate of the protease FtsH, which will rapidly degrade the subunit if it is not in its native state (151). It was readily labeled with TID, which is a hydrophobic carbene generator that is believed to react from the nonpolar region of the lipid bilayer, but its solubility properties made it unsuitable for analysis as was done with the c and b subunit (106). Consequently, the amino acids in contact with the lipid phase of the bilayer were not identified. Due to difficulties in obtaining high-resolution structural data, much of what is known of the a subunit arises from mutational studies. Topology. Hydropathy analyses indicated five definite membrane-spanning regions and one putative membrane span (121, 140, 152, 153). Much of what is known of the a subunit structure and has come from the analysis of cysteine mutagenesis. Greater than 50 cysteine substitutions, which resulted in a functional F 1 F 0 ATP synthase, were used in two kinds of experiments (154-158). Various maleimide derivatives were used to search for the surface-accessible regions (154, 155). And double cysteine mutations were used to search for disulfide formation between a-a and a-c (153). The results supported the model in which the a subunit spans the membrane five times and the fourth span, which includes a arg210 is in contact with the second transmembrane -helix of the c subunit (Figure 1-4A). Additionally, residues that were originally thought to be located in the cytoplasm were not labeled, indicating that the six-membrane span model was incorrect (132, 159). The location of the amino-terminus of the a subunit has also been very controversial. A substantial amount of evidence indicates that the carboxyl terminus PAGE 50 29 Figure 1-4. Controversial models of the a subunit topology. There is no high-resolution structural data for the a subunit. Mutagenesis, crosslinking and immunological experiments were used to study the topology. Roman numerals indicate the number of transmembrane helices. Small numbers indicate the relative position of the amino acid residue. Several crosslinking reactions were observed between the fourth helix of the a subunit (IV*) and the second helix of the c subunit in double cysteine mutants. Contradicting models exist for the topology of the a subunit. A) Model with five-transmembrane helices and the amino terminus residing in the periplasm. B) Model with six transmembrane helices and the amino terminus located in the cytoplasm. PAGE 51 30 resides in the cytoplasm (154, 155, 160). This observation, in combination with the five-transmembrane helices, indicates that the amino-terminus should reside in the periplasmic space. Polyclonal antibodies against a peptide model of the extreme carboxyl-terminus as well as antibodies against epitope tags constructed at the carboxyl terminus of the a subunit revealed this region to be located in the cytoplasm (160). Moreover, cysteine substitutions at a 266 or a 277 were highly reactive on the cytoplasmic side of the membrane (154, 155). The orientation of the amino and carboxyl-termini was studied by gene fusion proteins and peptide-directed antibodies, revealing a cytoplasmic location of both termini (161, 162). Insertion of epitope tags at various positions also confirmed the cytoplasmic local of both termini, arguing in favor of the controversial six-transmembrane model of the a subunit (Figure 1-4B) (160). In the five-transmembrane model a stretch of about 37 amino acids at the amino-terminus resides in the periplasm with only one transmembrane helix, approximately up to residue a 66 present (Figure 1-4A) (154-156, 158). In the six-transmembrane model the amino-terminus resides in the cytoplasm with two transmembrane helices present before the first cytoplasmic loop, which range from approximately residues a 33-49 and a 54-70 (Figure 1-4B) (160). A series of a subunit amino-terminal truncations and internal deletions were constructed and the F 1 F 0 ATP synthase function was tested by growth on a succinate minimal media. Assembly of intact complexes was tested by membrane-associated ATPase activity and the presence of the a subunit was analyzed by immunoblot analysis (163). Four sections were found to be particularly interesting. The first 33 residues at the amino terminus were shown to be necessary for the insertion of the a subunit into the membrane. Two internal deletions, from residues a 91-99 and a 163-177 resulted in functional PAGE 52 31 enzyme complexes, indicating that these regions were not important for function. A fourth deletion, from residues a 120-124 was concluded to be important for function, but not assemble because high levels of a subunit were found in the membrane, but the enzyme was not functional. The importance of the carboxyl-terminus was also analyzed by constructing a series of early termination codons (164). Sequence alignment of the a subunit demonstrates that many bacterial homologues contain glutamate and histidine residues at the extreme carboxyl-terminus (glu-glu-his in E. coli). However, truncation of the final four residues had no effect, and truncation of the final nine residues were tolerated at 25C, suggesting that the extreme carboxyl terminus of the a subunit did not significantly contribute to proton conduction or functional interactions with other subunits. Proton translocation. The first indication that the a subunit was directly involved in proton translocation appeared nearly two decades ago when mutations constructed in the a subunit (a ser206leu and a his245-tyr ) were found to affect F 0 -mediated proton pumping without influencing F 1 F 0 ATP synthase assembly (165). Since then, not including the cysteine mutations described above, more than 75 missense mutations have been constructed and analyzed in or near the conserved regions of the a subunit to chart the amino acids involved in proton translocation. In general, mutation of a conserved amino acid residue impaired F 0 -mediated proton translocation, but the severity of the defects varied (166). The only F 1 F 0 ATP synthase a subunit residue that is strictly conserved amongst all species, from bacteria to humans, and cannot endure any amino acid substitution, whether basic, acidic or nonpolar, was a arg210 (145-147). Mutations at this site abolished both PAGE 53 32 ATP-driven proton pumping and passive F 0 -mediated proton translocation. Growth on succinate minimal media indicated no ATP synthesis by the mutants. The observed effects were shown not to be due to failure of F 1 F 0 ATP synthase to assemble because treatment with the detergent lauryldimethylamine oxide (LDAO) released F 1 from the prepared membranes and revealed abundant ATP hydrolysis activity. The presence of assembled F 1 F 0 ATP synthase complexes incorporated with an a arg210 mutant was later directly confirmed by Dr. James Gardner (167). Substitution with an alanine allowed passive F 0 -mediated proton translocation indicating that the proton channel was intact and suggested that the a arg210 is not obligatorily protonated or deprotonated during proton conduction (168). A second site suppressor mutation, a gln252arg which partially compensated for the a arg210gln mutation, was identified, and suggested to be in close proximity to each other with residence on the transmembrane helix 5 and 4, respectively, in the five-transmembrane model (121). The a 210 residue is thought to have a direct role in proton translocation. The orientation of the a subunits fourth transmembrane helix had been determined relative to the orientation of the c subunits second transmembrane -helix by crosslinking double cysteine mutants (157). Crosslinking data has positioned a 214 in close proximity to c 62 and c 65 and a 211 close to c 69 (157). This places the putative fourth helix of the a subunit in contact with the second helix of the c subunit. Models have the a 210 residue positioned near the center of the fourth helix at a level in the lipid bilayer very close to the essential c asp61 residue (14). Whether a 210 is directly protonated/deprotonated or controls protonation of the c asp61 residue remain unanswered (169). Insight from a high resolution structure of an intact F 0 is greatly desired and would provide extremely valuable answers to many of the unsolved questions. PAGE 54 33 Single mutations at residues a 218 a 219 or a 245 were shown to have a considerable impact on F 0 -mediated proton conduction (144, 146, 170). When comparing amino acid sequences of various mitochondria, chloroplast and bacteria, there appears to be an instance of evolutionary covariation with these three amino acids (144). This suggests that when a mutation occurred in one of the three residues, it was accompanied by a second mutation to compensate for any loss in activity. This would cause the two residues to pass through evolution as a hereditary unit. Based on this observation, double mutants were constructed in the E. coli a subunit to imitate other lines of evolution (144, 171). Every double mutant studied resulted in functional F 1 F 0 ATP synthase complexes with considerably more activity than any of the single residue mutants. Due to the functional relationship, it is possible that these three amino acids are in close proximity to each other. A few other strongly conserved amino acid residues located on the fourth and fifth transmembrane helices are worth mentioning. Residues a asp214 and a gln252 were both strongly conserve but found nonessential, with the effects of mutations at these residues varied widely (146, 170, 172, 173). Models of the a subunit have these residues lining a water-filled proton channel. Recently, the aqueous accessibility of residues along transmembrane helices 2 and 5 has been shown to extend to both sides of the membrane (174). Also, a mutation at residue 217, a ala217arg blocked proton conduction and inhibited F 1 F 0 ATP synthase activity (167). The subunit The E. coli subunit is one of the F 1 subunits. It is discussed here because it is an essential part of the F 1 F 0 ATP synthase stator stalk. The simplest stator stalks occur in PAGE 55 34 nonphotosynthetic prokaryotes and consist of a dimer of the F 0 b subunits (discussed in the following section) and a single F 1 subunit. The subunit displays a very low level of conservation across various species. It is a globular protein encoded by the uncH gene that consists of 177 residues with a molecular weight of 19,332 Da. It plays essential roles in both the binding of F 1 to F 0 as well as coupling of the catalytic activities of F 1 and F 0 (139, 175-180). Circular dichroism (CD) spectroscopy and sedimentation analysis studies performed on the subunit suggested a highly helical and elongated conformation (139). Structure of the subunit. A partial high-resolution structure of the subunit has been solved by NMR (137). During the purification procedure, a truncated form of the subunit was produced by a bacterial protease. It was revealed to be the amino-terminal 134 amino acids ( 1-134 ) by mass spectroscopy and N-terminal sequencing. The same sized subunit fragment was often seen in F 1 preparations and could be produced in isolated E. coli F 1 by treatment with trypsin without liberating the 1-134 from the F 1 complex (89). Furthermore, purified 1-134 stably binds to -free F 1 preparations. The high affinity of the 1-134 subunit for F 1 indicated that the conformation of the fragment was preserved during the purification procedure. NMR was performed on both the 1-134 fragment and the intact subunit; however, the quality of data for the intact subunit was not sufficient enough for structural analysis due to its propensity to aggregate at high concentrations. To date, the carboxyl-terminal 43 amino acid residues ( 135-177 ) of the subunit is the only portion of the F 1 sector not known at the atomic level. The amino-terminal 105 residues of the subunit formed a dense globular domain, while the region from residues 106-134 was mostly disordered with the exception of one PAGE 56 35 -helix (137). The amino-terminal domain, 1-105 consisted of a six -helix bundle with the dimensions 45 x 20 x 30 Helices 1 ( 4-20 ) and 2 ( 24-38 ), and helices 5 ( 70-81 ) and 6 ( 88-104 ) organized into V-shapes that intercalated to form a core. Helices 3 ( 41-47 ) and 4 ( 53-64 ) were packed compactly against this four-helix core. Following this globularly packed domain there was a loop region followed by a seventh -helix ( 118-129 ). Comparison of the structural data for the intact subunit against that of the 1-134 fragment illustrated the same structure for residues 1-104, but the spectral shift of residues 105-134 was very different. It was possible that the carboxyl-terminal 42 residues missing from the 1-134 fragment affects this region of the subunit. subunit topology. Taken together with biochemical and immunological data, the structure revealed by NMR revealed that the subunit consists of two domains, an amino terminal domain, 1-104 and a carboxyl-terminal domain, 105-177 Under oxidizing conditions, two native cysteines present in the aminoand carboxyl-terminal domains of the intact subunit, cys64 and cys140 respectively, formed a disulfide bond. Furthermore, NMR data indicated some NOEs between the carboxyl-terminal -helix and the amino-terminal domain. The data indicated that there is probably a close interaction between the aminoand carboxyl-terminal domains of the intact subunit. Proteinase accessibility and immunological analysis were used to examine the topology of the subunit (89). The subunit was susceptible to trypsin digestion at the carboxyl-terminal 20 residues in isolated F 1 but not in intact F 1 F 0 ATP synthase, indicating a protection of the amino-terminal region by F 1 Deletion analysis of the carboxyl-terminal region also implied the importance of the subunit in binding F 1 to F 0 PAGE 57 36 Taken together, these observations suggested that the amino-terminal domain is predominantly involved in the binding of the subunit to F 1 and the carboxyl-terminal domain is involved in binding to F 0 The location of the subunit has had a history of being very controversial. Prior to the high resolution structure obtained by Abrahams et al. (1994), the b and subunits were expected to form part of the central stalk of the F 1 F 0 ATP synthase enzyme, which is now known to consist of the and subunits (20). Due to the dimensions, it seemed unlikely that the b and subunits could fit as part of the central stalk, which implied that they must form a separate connection between F 1 and F 0 Improving EM technology did not allow visualization of the second stalk structure at the periphery of the F 1 F 0 ATP synthase complex until many years later (27, 28). Prior to visualization by EM, several early crosslinking studies had been reported in the quest to find the location of the subunit binding on F 1 finding it to be on the subunit (89, 181-184). Notably, crosslinking the subunit to the subunit did not have a great impact on F 1 F 0 ATP synthase function, as would be expected if the subunit formed part of the stator stalk (185). High-resolution structure of the F 1 3 3 hexamer with a partial structure of the subunit had revealed a dimple in the top of the hexamer approximately 15 deep that was adjacent to the core space where the aminoand carboxyl-terminal -helices of the subunit resided (20). EM studies had revealed a cap structure at the very top of F 1 in both E. coli and mitochondrial complexes (27, 29, 186). It was thus believed that the subunit resided in the dimple of F 1 as the cap seen in the EM structures (187). This possibility was refuted when Prescott et al. demonstrated the ability to stably incorporate the green fluorescent protein (GFP), via varying length peptide linkers (0, 4 or 27 amino PAGE 58 37 acids), to the carboxyl-terminus of the subunit without interrupting function of the enzyme complex. GFP forms a rigid, stable structure with the dimensions 24 wide and 48 high (188). This study indicated that the putative cap structure could not possibly occupy the entire dimple atop F 1 More recent evidence has suggested that the subunit may actually be positioned slightly to the side of F 1 in association with only a single subunit (Figure 1-2) (100, 141, 142, 189). The b subunit The b subunit is required for the normal assembly and function of F 1 F 0 ATP synthase (190). The E. coli F 1 F 0 ATP synthase has two identical b subunits, which form a homodimer, that are the product of a single gene (Figure 1-2). It is an elongated amphipathic polypeptide that crosses the membrane one time at its amino-terminus and has an extensive hydrophilic carboxyl-terminal domain. This pattern is characteristic of b-type subunits of ATP synthases, although the mitochondrial b has two consecutive membrane-spanning segments at the amino-terminus (191). Most ATP synthase b-type subunits consist of between 150 and 170 amino acid residues. The E. coli b subunit, encoded by the uncF gene, consists of 156 amino acid residues and has a deduced molecular weight of 17, 265 Da (Figure 1-5). Domains and Structure. Currently there is no high-resolution structure of the entire b subunit. Several factors probably contribute to the difficulty of structural analysis. The b dimer is a thin, highly extended, mostly -helical structure, its dimerization is comparatively weak and reversible (192), and it displays evidence of flexibility (193-195). This has led to alternative low-resolution approaches to study the structure of the b dimer such as circular dichroism (CD) spectroscopy, deletion analysis, PAGE 59 38 Figure 1-5. Amino acid sequence of the E. coli F 1 F 0 ATP synthase b subunit. The E. coli b subunit is a 156 residue amphipathic polypeptide. The amino acid sequence and the four domains are shown. The transmembrane domain (b 1-22 ), tether domain (b 24-60 ), dimerization domain (b 63-122 ) and the -binding domain (b 123-156 ) are shown in blue, orange, green and red, respectively. The large purple stars indicate residues capable of forming high yields of b-b crosslinks upon cysteine substitution. The smaller purple stars indicate residues found to form low-yields of crosslinks. The arrows indicate positions crosslinked to other subunits of ATP synthase. High-resolution structures based on model polypeptides consisting of b 1-34 and b 62-122 (underlined residues) have been solved by NMR and crystallography, respectively. NMR analysis of residues 1-34 has revealed a -helical structure with a rigid 20 bend at positions 23-26. X-ray crystallography revealed a highly -helical structure with modeled into a right-handed coiled coil. PAGE 60 39 analytical ultracentrifugation, chemical crosslinking, and the analysis of tendencies for disulfide bond formation. CD spectroscopy analysis has predicted the secondary structure of the b subunit to be approximately 80% -helical with about 14% -turn conformation (196). Although there is no high-resolution structure of the intact F 0 sector, an abundance of evidence suggests the necessity of the b subunit to exist in the dimeric state. The hydrophilic region of b, consisting of residues b 24-156 (also known as b sol ), has been expressed and shown to form highly extended dimers capable of binding to F 1 -ATPase in solution (197). Sedimentation equilibrium ultracentrifugation gives a molecular weight value of about 30,000 Da for b sol consistent with a dimer of two 15,000 Da b sol monomers (13). The existence of the dimeric state of the b subunits was confirmed by covalently cross-linking the two b subunits in the complex and verifying the activity of the enzyme (198). Furthermore, the ability of b to bind to F 1 was discovered to be directly proportional to the ability of b to form dimers, suggesting the necessity of the b dimer formation before the binding of F 1 to the complex (199). The dimerization of the b subunit has been shown to be relatively weak and reversible. The monomeric and dimeric forms of b sol were shown to exist in a dynamic equilibrium and the dimer was converted to the monomeric state at 40C (192). This same melting characteristic was observed with CD spectroscopy (200). Furthermore, the similar traits were observed in photosynthetic organisms, which encode two different b-type subunits, b and b (13). When the cytoplasmic regions of the b and b subunits from the cyanobacterium Synechocystis were expressed individually, the polypeptides were found to only exist in the monomeric state. However, when they were mixed together, PAGE 61 40 the formation of the dimers was observed by chemical crosslinking and sedimentation equilibrium ultracentrifugation (13). Also, the dimers were observed to melt at 40C as was the case with the E. coli b subunit. The striking similarity between the E. coli b subunit and the photosynthetic organism b and b subunits indicate that the former is a good model in which to study the b subunit. Cross-linking and deletion analysis has led to the development of a four-domain model of the E. coli F 1 F 0 ATP synthase b subunit (Figures 1-5 and 1-6) (12). Amino acid Figure 1-6. Gross structure of the E. coli F 1 F 0 ATP synthase and the domains of the b subunit. The b subunit domains were described by Dunn et al. (12) The membrane-spanning domain roughly corresponds to amino acid residues 1-22, the tether domain is approximately residues 24-60, the dimerization domain is considered to be residues 60-122, and the F 1 -binding domain is roughly residues 123-156. PAGE 62 41 residues b 1-22 corresponds to the hydrophilic membrane spanning domain. Residues b 24-60 roughly corresponds to the tether domain, which is the portion of the peripheral stalk often observed in electron micrographs. The dimerization domain, approximately b 60-122 is required for the dimerization of the two b subunits. And finally, the F 1 binding domain, roughly amino acid residues b 123-156 is required for the binding of F 1 to F 0 The amino-terminal membrane spanning domain, b 1-22 forms a single transmembrane span while the large remainder is a polar hydrophilic domain which extends above the cytoplasmic leaflet of the lipid bilayer and reaches towards the top of F 1 (Figure 1-6) (13, 191). A wealth of evidence has suggested this proposed topology for the b subunit. The amino terminal region was uniformly vulnerable to chemical modification by the a nonpolar photoreactive reagent, TID, which is a hydrophobic carbene generator that is believed to react from the nonpolar region of the lipid bilayer, indicating that this region was in fact in the hydrophobic phase of the bilayer (106). This observation was consistent with other labeling procedures including the labeling of b cys21 by hydrophobic nitrenes (201) or the hydrophobic maleimide N-(7-dimethylamino-4-methyl-coumarinyl)-maleimide (DACM) (148). Modification of b cys21 interfered with intersubunit interactions within F 0 Furthermore, reconstitution of the F 0 subunits upon labeling the b subunit with DACM resulted in reduced proton translocation as well as F 1 binding affinities (148). TID failed to label the region b asn2gln10 indicating that the first few residues at the amino-terminus protrude into the periplasm (106) Despite attempts by several laboratories, there is presently no high-resolution structure of the entire b subunit. Therefore, model polypeptides have been constructed in order to elucidate the structure of the b subunit by domain. A model polypeptide PAGE 63 42 comparable to residues b 1-34 which contains the membrane-spanning domain, dissolved in a 4:4:1 v/v mixture of chloroform/methanol/water previously used for solving the structure of subunit c, has been solved by NMR (136). The data revealed an -helical monomeric structure with a 20 bend at residues 23-26 (KYVW) (Figure 1-5). The hydrophobic residues, b 4-22 formed an -helix, followed by the 20 bend, and then resumed with -helical structure from residues b 27-34 The bend was proposed to be positioned as the b subunit exits the membrane. A series of cysteine substitutions resulted in a high yield of crosslinks formed at residues b 2 b 6 and b 10 (Figure 1-5). A lower yield of crosslinks were observed to form at residues b 3 b 8 b 9 and b 11 (Figure 1-5). No crosslinks were observed when cysteines were substituted for residues 12-21. The observance of continuous crosslinks between residues b 6-11 were suggested to indicate a dynamic interaction between the contacting faces of the two b subunits in this region (132). These observations led to a dimeric model in which the extreme amino-termini of the b subunits crossed each other in close proximity at an angle of about 35 in the region of residues b 4-11 and then the two b subunits angled apart as they traverse the membrane towards the cytoplasmic side (Figure 1-5) (136). The region of the 20 bend, b 23-26 was suggested to change the direction of the second -helix, b 27-33 such that it would extend into the cytoplasm at an angle perpendicular to the plan of the membrane. This model was confirmed by a systematic mutational analysis of the membrane-spanning domain performed by Hardy et al. (202). The tether domain of the b subunit, roughly b 24-60 is the least defined part of the subunit domains from a structural point of view. It corresponds to the portion of the peripheral stalk often seen in electron micrographs and is called tether simply because PAGE 64 43 it is the section of the b subunit that links the more defined membrane-spanning and dimerization domains Figure 1-6). There is no high resolution structure for this region of the b subunit. The NMR structure of b 1-34 described above extended slightly within this domain, revealing an -helix at least up through residue b 34 .(136) Also, a heptad repeat, extending from just outside of the membrane and continuing without interruption up to residue b ala79 suggests the structure to be a coiled coil (197, 203). Though crosslinking studies showed that the tether domains of the two b subunits are in parallel and in close proximity, this domain contributes little to the stability of the dimerization of the b subunits (192). Deletion constructs analyzed by sedimentation equilibrium experiments suggested that a form of the b subunit truncated in each end, b 53-122 was capable of forming dimers with an efficiency close to the complete cytoplasmic domain, b 24-156 indicating that the most pertinent intersubunit contacts of the b subunit was located within this central region, referred to as the dimerization domain (192). More recently, the dimerization domain has been refined to residues b 63-122 ; however, the amino-terminal boundary is likely to decrease even further due to the observation that deletion of residues b 54-64 or b 65-75 resulted in intact and functional F 1 F 0 ATP synthase complexes (Figure 1-6) (194). A crystal structure of a monomeric dimerization domain, based on a model polypeptide consisting of residues b 62-122 has recently been solved and refined to 1.55 (138). Based on an undecad repeat and crosslinking data, Dunn and coworkers have constructed a model in which the two -helices of the b 62-122 region formed a coiled-coil with a right-handed superhelical twist. A number of previous studies supported this coiled-coil arrangement. First, the shape of the b 53-122 polypeptide was consistent with a PAGE 65 44 coiled-coil of similar length as determined from its frictional coefficient (1.60) in an ultracentrifuge and from NMR relaxation parameters (192). Secondly, small-angle X-ray scattering by b 52-122 in solution specified a maximum length to be about 95 consistent with the expected coiled-coil length (13). Thirdly, CD spectroscopy indicated that this polypeptide was 100% -helical and the similar intensities of the minima suggested the helices to be arranged in a coiled-coil (204). Fourthly, cysteine substitution and crosslinking studies suggested a periodicity consistent with a parallel coiled-coil (Figure 1-5) (204). Finally, b subunit sequence analysis of E. coli and other prokaryotes revealed a conservation of an undecad pattern, which is a distinctive characteristic of a right-handed coiled coil. Mutation of amino acid residue b arg-83 which interrupts the undecad repeat, markedly stabilized the dimer, as expected for the proposed two-stranded, right-handed coiled-coil structure. The carboxyl-terminal F 1 -binding domain, b 124-156 also referred to as the -binding domain, has a more globular conformation and is required for the binding of F 1 to F 0 (205, 206). Work accomplished by Futai and coworkers two decades ago revealed that truncation of the extreme carboxyl-terminus of the F 1 -binding domain by only a few amino acids resulted in assembly defects in F 1 F 0 ATP synthase (206). Subsequently, work performed by Dunn and coworkers demonstrated that the final two to four amino acids of the b subunit were necessary for binding the subunit of F 1 (205). An addition of a cysteine at the carboxyl-terminus was chemically crosslinked to a cysteine introduced at 158 A close association of the two b subunits in the F 1 -binding domain was indicated by crosslinks formed between cysteines individually substituted at positions b 124 b 125 PAGE 66 45 b 126 b 127 b 128 b 129 b 130 b 131 b 132 b 139 b 144 b 146 or b 156 (Figure 1-5) (198, 200, 207). Hydrodynamic evidence favors a folded structure for this domain of the b subunit as opposed to the highly elongated structure of the remainder of the b subunit. Also, several studies have shown that either a b ala128glu mutation, deletion of the last four residues, or cold temperature dramatically decreased the sedimentation coefficient, by 23%, suggesting that the F 1 -binding domain underwent a conformational change from a globular structure to a less folded more extended conformation (192, 205, 208). The mutation, b ala128glu may have caused an electrostatic repulsion that would cause the two b subunits to push apart. The carboxyl-terminal residues may form an amphipathic helix, so the deletion would have disrupted essential interactions. And cold temperatures have been shown to weaken hydrophobic interactions in proteins, suggesting the importance of the hydrophobic amino acids in the folding of this domain (13). Dunn et al. suggested that these observations implied that the carboxyl-terminus of the b subunit has a weakly folded structure in which the hydrophobic amino acids are arranged to impart structural stability and create hydrophobic patches on the surface (13). The folded conformation appears to be required for the exposed hydrophobic patches to interact with F 1 Mutagenesis. Several mutant searches and site-directed mutagenesis studies have been performed in the membrane-spanning domain of the b subunit. However, only a single mutation, b gly9asp located near the periplasmic side of the lipid bilayer, resulted in a defective proton pore in an intact F 1 F 0 ATP synthase complex (209). Second site suppressors of this mutation have been found in the a(a pro240ala or a pro240leu ) and c (c ala62ser ) subunits that partially repaired the defect, indicating an interaction between the b subunit and both the a and c subunits (210, 211). The membrane-spanning domain PAGE 67 46 contains one charged residue, b lys23 but mutations generated at this site did not influence proton translocation, suggesting that this domain of the b subunit did not have a direct function in proton conduction, although it was required for the maintenance of a functional F 0 complex (212). A systematic mutational study of the membrane-spanning domain conducted by Hardy et al. was described above and a triple mutant, b N2A,T6A,Q10A is described in Chapter 5 of this dissertation (202). A couple notable characteristics can be attributed to the tether region of the b subunit. Relatively large deletions and insertions of up to 11 and 14 amino acids, respectively, were accommodated in this region and the altered b subunits still assembled into fully functional F 1 F 0 ATP synthase complexes (193, 194). In fact, decreases observed in enzyme activity paralleled the decrease of b subunit found in the membrane, suggesting that the alterations affected assembly of the enzyme, but not the function. Assuming -helical structure, an 11 amino acid deletion would shorten the b subunit tether region by approximately 16.5 and a duplication of 14 amino acids would increase the length by about 21 This implies that the b subunit is highly flexible and the altered b subunits may compensate for the lost or gained distance via that flexibility. The fact that the peripheral stalk must extend from within the membrane to up near the top of F 1 suggests that some part of the stalk must be flexible enough to stretch or straighten in the shortened b subunits, or in the case of the lengthened b subunits, bend to take up the slack. Prior to these observations, the b subunit was often viewed as a rigid, rod-like structural feature during rotational catalysis. Also, though the b subunit is the least conserved subunit of F 1 F 0 ATP synthase, gaps are rarely found in sequence alignments of numerous organisms (213, 214). Therefore, the ability to manipulate the PAGE 68 47 Figure 1-7. Model for F 1 F 0 ATP synthase peripheral stalk orientation dependent upon the direction of rotation during ATP synthesis or hydrolysis. The subunits are color coded as follows: light blue; grey; dark blue; green; orange; a, yellow; b, red; c, cyan. The panel on the left indicates the orientation of the b 2 dimer if the enzyme is actively synthesizing ATP. The panel on the right represents the position of the b 2 dimer during ATP hydrolysis. The arrows indicate the direction of rotation of the rotor stalk subunits (c 10 ). The red cylinders indicate regions of the b subunits for which there is no high-resolution structure. PAGE 69 48 length of the peripheral stalk was an unexpected surprise to the field. The length of the wild-type b subunit is probably the optimum length for assembly of F 1 F 0 ATP synthase. The apparent flexibility has been proposed to help alleviate torsional strain brought about by rotational catalysis (193). Another hypothesis concerning the flexibility of the tether domain is that this region of the b 2 dimer serves as a hinge, allowing reorientation of the stator depending on the direction of rotation as the enzyme carries out ATP synthesis or hydrolysis (Figure 1-7) (195, 202). Another important feature of the tether domain is the evolutionarily conserved b arg36 that has been implicated in a structural role influencing proton conduction through F 0 Mutational studies at this amino acid residue led to numerous defects from failure to assemble or function to uncoupling phenotypes (215). Amino acid substitutions, b arg26ile or b arg26glu resulted in assembled F 1 F 0 ATP synthase complexes that displayed defects in F 0 -mediated proton translocation or a disruption of coupling activity, respectively. Substitution with a cysteine at this residue led to a crosslink product with the a subunit of F 0 (216, 217). The close proximity of this conserved residue to the a subunit indicates that it may play a role in aligning the proton exit channel. Protein-protein interactions between the two b subunit dimerization domains have been shown to be essential for forming the peripheral stalk (13). Mutations at a conserved residue, b ala79 resulted in major F 1 F 0 ATP synthase assembly defects (203, 218). The b ala79 mutations were modeled in the b sol polypeptide to investigate the affects of the mutations. The model polypeptides were shown to retain -helical structure, but chemical crosslinking and sedimentation experiments suggested that the b ala79 mutants were incapable of forming dimers (199). PAGE 70 49 In the F 1 -binding domain, a mutation was found, b ala128asp that had little effect on the dimerization of the b subunits but led to an assembly defect of F 1 F 0 ATP synthase (208). However, the mutant was found to have a reduced tendency to interact with F 1 and sedimentation equilibrium ultracentrifugation experiments revealed a 12% decrease in the sedimentation coefficient, indicating a structural perturbation (discussed above). The studies suggested that the b ala128 residue was not important in b subunit dimerization, but it had an important structural role in the F 1 -binding domain. Intersubunit interactions. The formation of disulfide bonds between cysteine residues introduced in the membrane-spanning domain of the b subunit and the a subunit as well as second site suppressors of the b gly9asp found in the a subunit (discussed above) strongly suggests an interaction between the two stator subunits (209-211). The b gly9asp mutation was also partially suppressed by a mutation in the c subunit (discussed above), but it is not known whether it is due to a direct interaction between the b and c subunits or if the suppression is mediated through the a subunit. The b subunit has also been shown to interact with the and subunits of F 1 (198, 216). The interaction of the F 0 b subunit with the F 1 subunit has been well documented (200, 205, 219, 220). The interaction is mediated by the carboxyl-termini of both subunits and is essential for the binding of F 1 and F 0 The critical role of the subunit mediating the interaction between the b subunit and the bulk of F 1 has been demonstrated by the inability of -depleted F 1 to bind to F 0 (219). Crosslinking the b and subunits via introduced cysteines did not affect F 1 F 0 ATP synthase activity, which was consistent with the proposed role of the b 2 peripheral stalk as a stator and demonstrated that the binteraction need not be dynamic (221). Though it is believed that the binding PAGE 71 50 of F 1 to F 0 heavily relies on the binteraction, evidence of other subunit contacts may also influence binding. Furthermore, the binding of b to was shown to be relatively weak by analytical ultracentrifugation, indicating that other subunit contacts may contribute to binding (220). Many crosslink formations were found when cysteines were introduced into the b and or subunits (Figure 1-5). A cysteine introduced to the carboxyl-terminus of the b subunit has been shown to crosslink to cys90 (198). Also, cysteines positioned at b 92 or b 109 formed crosslinked products with the subunit or both the and subunits, respectively (216). These results confirm that the b subunit is proximal to the 3 3 hexamer, but a direct interaction has not been confirmed. Stator stalk function. The necessity of a stator in F 1 F 0 ATP synthase was recognized after the realization that rotation was a fundamental feature of ATP catalysis. The current view of the peripheral stalk is primarily that of the stator which forms a connection the a subunit and the 3 3 hexamer, holding these subunits in place against the rotation of the rotor subunits, c 10 The idea of a flexible stator stalk has led to other proposed features of the b subunit. The apparent flexibility of the b subunit has been suggested to transiently store energy during rotational catalysis which could be potentially be expended to force the conformational change that allows the release of ATP(222). Another model describes the flexibility of the tether region as a hinge that could reorient the b subunit when switching between ATP synthesis and hydrolysis (discussed above) (Figure 1-7) (202). However, there is no direct evidence of whether the b subunit is actually acting in a flexible manner. Finally, the b subunit has recently been suggested to influence the nucleotide binding sites in the subunits (223). In these experiments, a spin label was incorporated at residue 331. Upon addition of b sol the PAGE 72 51 spectrum of this spin label was observed to change in a way that implied that the catalytic sites were in a more open conformation. These results indicate that the current view of the stator stalk as a structural feature may soon be revised such that the b subunit has a more direct role during rotational catalysis. Subunit Equivalence The E. coli ATP synthase complex has, by far, the simplest architecture of all the F 1 F 0 ATP synthase enzyme complexes. It is composed of twenty-two polypeptides of eight different types with the stoichiometry 3 3 ab 2 c 10 (Figure 1-2, Table 1-1) (112, 224). The 3 3 subunits comprise the F 1 sector and the ab 2 c 10 subunits comprise the F 0 sector. In the E. coli enzyme, all eight subunits are necessary for the function of F 1 F 0 ATP synthase (225, 226). Chloroplasts also have a relatively simple architecture with the exception that they have nine different subunits (227) due to the fact that the two b subunits are products from two different genes and are not identical (Figure 1-8). In contrast, the F 1 F 0 ATP synthase from mammalian mitochondria is composed of at least thirty-one polypeptides of sixteen different types with the F 1 stoichiometry of 3 3 and a much more complex F 0 consisting of a, b 2 c 10-14 d, e, f, g, (F 6 ) 2 A 6 L, OSCP, IF 1 (7, 228-230). Yeast mitochondria F 1 F 0 ATP synthase has an extra three subunits compared to the mammalian enzyme, stf1p, i, and k. The F 1 sector. In the F 1 sectors, homologues of the E. coli F 1 F 0 ATP synthase have been identified for the and subunits, based on amino acid sequence homology, in the chloroplast and mitochondrial enzymes (30). Based on the primary sequences, the highest conserved subunit from the E. coli F 1 F 0 ATP synthase is the subunit with approximately 70% homology with the chloroplast and mitochondria PAGE 73 52 Table 1-1. F 1 F 0 ATP synthase subunit equivalency Mitochondria Bacteria Chloroplast Yeast Bovine Function catalytic site catalytic site rotor OSCP OSCP stator rotor stabilization? a a (or IV) a (or 6) a proton channel, stator b b (or I) b (or 4) b stator b (or II) 9 stator c c (or III) c (or 8) c proton channel, rotor d d stator? 8 A6L stator? e e ? f f ? g g ? h F6 stator inh1p IF1 inhibitor stf1p ? j ? k ? equivalents (31). The subunits exhibit roughly 50% homology (31). The nucleotide binding regions of these two subunits also have sequence homologies with other proteins that bind nucleotide or phosphate, including the E. coli secA protein, N-ethylmaleimide sensitive fusion protein, herpes simplex virus UL15, Ca 2+ -ATPase, H + /K + ATPase and Na + /K + ATPase (34-37). Furthermore, the nucleotide binding motif, GXXXXGKT/S, which was first identified in the and sequences of F 1 has been found to be conserved in the high-resolution structures of other proteins including p21 ras adenylate kinase, RecA, elongation factor Tu, and transducin(20, 38-42). Interestingly, the subunit in the chloroplast F 1 F 0 ATP synthase complex contains an insert of about 35 amino acids that is not present in the mitochondrial or PAGE 74 53 nonphotosynthetic eubacteria (231). This loop contained two cysteine residues that were found to be reduced in the active enzyme complex during photosynthesis and oxidized to a disulfide bond in the inactive enzyme while in the dark (232). The E. coli subunit is unfortunately known as the subunit in the mitochondrial enzyme (233) and also shares primary sequence homology with the mitochondrial IF 1 inhibitor protein. The E. coli and bovine subunits share 60% sequence identity, which had suggested that they are functionally equivalent (191, 234). The availability of high resolution structures for these subunits has revealed that they are strikingly similar. Superimposition yields a 1.64 rms deviation (48). The bacterial subunit has been suggested to be an inhibitor of ATP hydrolysis, undergoing large, ratchet-like conformational changes to selectively switch off ATP hydrolysis (102). In mitochondria, this inhibitor action of the bacterial subunit is ascribed to the IF 1 protein of the F 0 sector. It was suggested that the bacterial subunit was separated into the two polypeptides in the mitochondrial enzyme complex, and IF 1 Finally, the bacterial and chloroplast subunits of F 1 share a significant sequence homology (234). The subunits equivalent in the mitochondrial F 1 F 0 ATP synthase is known as oligomycin-sensitivity-conferring protein (OSCP) (235-237). The carboxyl-terminal region of the mitochondrial b subunit was been demonstrated to bind to the OSCP subunit (E. coli subunit) through subunit interactions (Collinson et al., 1994) and chemical crosslinking analysis (Soubannier et al., 1999). The F 0 sector. The F 0 sectors of the F 1 F 0 ATP synthase family is by far more diverse than the F 1 sector with an additional eight different subunit types in mammalian and an extra ten different subunits in yeast mitochondria (Table 1-1). The E. coli a and c subunits are respectively equivalent to the chloroplast IV and III subunits and the PAGE 75 54 mitochondrial ATPase-6 and ATPase-8 subunits. In every case, both subunits are hydrophobic proteins required for proton translocation (120, 140). Unlike the a and c subunits, the E. coli b subunit does not have any obvious homologues in chloroplasts or cyanobacteria F 1 F 0 ATP synthases. However, both of them have two distinct subunits with similar hydrophobic and hydrophilic residue distribution (238). These subunits are referred to as subunits b and b in cyanobacteria and subunits I and II in chloroplasts. It is believed that only one of each of these subunits is present in the F 1 F 0 ATP synthase complex, incorporating into the enzyme as a b-b heterodimer as opposed to the b subunit homodimer present in E. coli. No obvious homologue of the E. coli b subunit has been found in the mitochondrial F 1 F 0 ATP synthase, even upon analysis of sequence, function or three-dimensional structure (239). However, hydropathy plot analysis does indicate that the mammalian mitochondrial b subunit that may be analogous to the E. coli b subunit (240, 241). Proteolysis studies and crosslinking data supported the location of the mitochondria b subunit at a position analogous to the E. coli b subunit (242, 243). At least three other subunits may play the role of the E. coli b dimer including subunit 8 (or A6L), d and F6. The mitochondrial b subunit is believed to have two -helical transmembrane spans at its amino-terminus arranged in an antiparallel configuration. The extreme amino-terminus of the mitochondrial b subunit is thought to begin on the cytoplasmic side of the membrane, traverses the membrane to the periplasmic leaflet of the membrane as an -helix, then turn back and traverses the membrane again as it exits the membrane in the cytoplasm and reaches towards the top of F 1 (Figure 1-8). The remainder of what would be equivalent to the E. coli b dimer is highly speculative, though the overall shape and characteristics of the b8dF6 subunits favor this explanation PAGE 76 55 (Figure 1-8). In this model, the mitochondrial subunit 8 contributes to a third membrane spanning region, and a combination of subunit d and subunit F6 forms the hydrophilic domain. The F 0 subunit known as the subunit in mitochondrial F 1 F 0 ATP synthase has no counterpart in the bacterial or chloroplast enzymes. It is a small polypeptide (50 amino acids) folded into a helix-loop-helix. It is believed to play a role in the stabilization of the central stalk and its absence in the bacterial and chloroplast enzymes may explain why Figure 1-8. Speculative models for the b-like subunits. Shown are models for the bacterial, chloroplast and mitochondrial b subunits. The membrane-spanning regions are indicated by the black lines. An abundance of evidence supports the parallel arrangement of the bacterial b 2 homodimer and the chloroplast bb heterodimer. The mitochondrial analogue of the b subunit is believed to consist of up to four polypeptides. The model shown for the mitochondrial b8dF 6 structure is highly speculative. PAGE 77 56 the bacterial subunit of the F 1 sector (equivalent to the subunit in mitochondria) easily dissociates from the F 1 complex whereas this has not been observed for the mitochondrial enzyme. Mitochondrial F 0 consists of several additional subunits not found in the E. coli or chloroplast enzymes including the e, f and g subunits as well as an extra three in yeast, stf1p, j, and k subunits. E. coli F 1 F 0 ATP synthase as a model. Initial studies of the F 1 F 0 ATP synthase complex were achieved with enzymes isolated from mitochondria or chloroplast. Although the bacterial, chloroplast and mitochondrial enzymes differ in oligomeric complexity, the enzymes show acceptable overall structural resemblance and primary sequence homology that it is widely accepted that the mechanism of action is the same in all organisms (240, 244). Therefore, studies using the bacterial model became widely accepted since it was more versatile and offered a large range of research that could not be readily undertaken with the more complex organisms. Other advantages include ease of genetic techniques, the ability of bacteria to grow via glycolysis, which allowed characterization of defective F 1 F 0 ATP synthases, and ease of large-scale purification procedures due to a practically unlimited supply of bacteria. F 1 F 0 ATP Synthase Mechanism The overall function of F 1 F 0 ATP synthase can be divided into three distinct parts: proton conduction, coupling, and catalysis. The a and c subunits of F 0 are responsible for the translocation of protons through the membrane. The F 1 and subunits of the rotor stalk are responsible for coupling the energy acquired from the proton gradient to the F 1 catalytic sites. And the three catalytic sites located at the interfaces of the three and PAGE 78 57 subunits are responsible for the synthesis of ATP or sometimes, as in the case of bacteria, ATP hydrolysis. All three functions must be tightly integrated for the production of ATP. Proton Translocation: Driving Rotation The demonstration that the electrochemical gradient of protons drives the rotation of bacterial flagella (245) in combination with Peter Mitchells chemiosmotic theory (2) began the search for evidence of rotation in F 1 F 0 ATP synthesis. At the same time, a model for proton transport was suggested by Cox et al. (212) and Boyer developed his ideas for the binding change mechanism (discussed below) (246). But an indication of rotational catalysis was not evident until the high-resolution crystal structure of F 1 became available (20). This was followed a few years later by the first direct observation of rotation when Noji et al. fixed the top of F 1 to a glass coverslip and attached a fluorescently labeled actin filament to the subunit (15). Upon addition of ATP, rotation of the actin filament was observed under an optical microscope at 0.2-10 revolutions per second. At low concentrations of ATP (<600 nM), the actin filaments were observed to rotate in a step-wise manner at 120 intervals, which reflects the three catalytic sites in the F 1 3 3 hexamer (247). Experiments, in which two phenylalanine residues in the nucleotide binding pocket were mutated to reduce the binding affinity of ATP, indicated that the binding and hydrolysis of ATP is initially accompanied by a 90 substep followed by a 30 substep attributed to product release (248, 249). These observations were consistent with the two proposed catches observed between the and subunits in the high resolution structure (see The subunit above) (20). The observance of the rotation of the and c subunits at the same speed and direction, indicating that these three subunits rotate in synchrony, forming the central rotary machinery of the enzyme PAGE 79 58 complex (65-67). The concept of rotational catalysis with the rotation of the c 10 subunits relative to the 3 3 hexamer is now well accepted. Several models have been proposed for proton translocation. One of the earliest models suggested a series of side chains spanning the lipid bilayer formed a proton wire, involving amino acid residues c asp61 a arg210 a glu219 and a his245 in which the protons hop from one side chain to another until it passes through the membrane (212, 226, 250). Other models include a water-filled proton channel model, formed by the charged residues of the a and c subunits, in which hydronium ions (H 3 O + ) pass through the lipid bilayer (251) and a proton carrier model, in which a proton binds on the exterior of the membrane followed by a conformational change that brings the complex through the membrane and releases the proton on the outer surface (252, 253). The current prevailing model suggests that protons, in the form of H 3 O + enter a half channel created by the a subunit (Figure 1-9) (254, 255). The H 3 O + is then believed to protonate one of the c 10 subunits at residue c asp61 which is positioned near the center of the lipid bilayer (131). Besides forming the proposed half channel, the a subunit is thought to play another crucial role in the protonation of the c asp61 The pKa of the c asp61 carboxyl side chain is uncommonly high, which is likely due to its lipid environment (125). The essential a arg210 residue is thought to facilitate a pKa shift of the c asp61 carboxyl side chain to a lower pKa form during proton translocation (14, 132). Either the protonation of the carboxylate, or possibly the release of a proton from a previously protonated c asp61 into an exit channel housed by the a subunit, somehow promotes the generation of torque (Figure 1-9). The torque produced by proton translocation is believed to drive the rotation of the ring of c 10 subunits relative to the a and b subunits PAGE 80 59 during rotational catalysis. Translocation of three to four protons generates enough torque energy to rotate the c 10 ring by 120 and results in the synthesis of a single molecule of ATP. Figure 1-9. Model of proton translocation and torque generation in F 0 Subunits included are (green), (orange), a (yellow), b 2 (purple), c 10 (blue). Protons (red) are traveling in the direction of ATP synthesis. Protons are believed to travel through a half channel housed in the a subunit as hydronium, H 3 O + An essential residue located near the center of the lipid bilayer in the c subunit, c asp61 is thought to be protonated as another proton exits through another exit half channel located on the cytoplasmic side of the membrane. The protonation/deprotonation drives the rotation of the c 10 ring. The model was drawn from schemes proposed by Junge (254) and Vik and Antonio (255). PAGE 81 60 Coupling In F 1 F 0 ATP synthase, the mechanism of energy coupling requires both the rotor stalk and the peripheral stalk. Rotation of the ring of c 10 subunits consequently results in the rotation of the entire rotor stalk, which is essential in coupling the energy obtained from proton translocation to the synthesis of ATP in the catalytic sites of the 3 3 hexamer located over 100 away. The role of the peripheral stalk is to hold the hexamer in place while the aminoand carboxyl-terminal-helices of the subunit rotates within. In a fully functional and coupled enzyme, the kinetics of proton translocation and ATP synthesis are linked so that one cannot proceed without the other, and vise versa (256). Mutational studies suggested that the polar loop of the c 10 subunits was involved in the coupling function. In F 1 F 0 ATP synthases incorporated with the c gln42glu subunit mutant, F 1 was found to bind normally to the F 0 mutant, but the passive leakage of protons through this complex was not prevented as in must be in coupled enzyme complexes (257). Also, ATP was hydrolyzed normally by this mutant, but hydrolysis was not coupled to active proton translocation. A similar uncoupled phenotype was found in complexes incorporated with a c arg41lys mutant (258). Another mutation at the same site, c arg41his was found to prevent the binding of F 1 to F 0 Thus, this loop region in the c subunit appears to play essential roles in both the binding of F 1 and the coupling of proton translocation to ATP synthesis. Second site suppressor mutations to the c gln42glu mutation were found in the subunit, specifically, glu31gly,val,lys that recouped proton translocation and ATP synthesis (98). Crosslinking studies of cysteine double mutants found cross-linked products between cys31 and c cys40 c cys42 and c cys43 (99). Moreover, a functional contribution of the subunit in energy coupling was demonstrated PAGE 82 61 by subunit mutants that uncoupled proton transport and ATP hydrolysis (259). Second site suppressor mutations were found in other regions of the subunit (260, 261). Cysteine substitutions formed crosslinks between the subunit, cys205 and c cys40 c cys42 and c cys43 (118). Crosslinking studies also provided evidence for an interaction between and (75, 77). In addition, the subunit has been cross-linked to both the F 1 and F 0 subunits, via introduced cysteines, indicating that it spans the entire length of the stalk with the subunit (60, 99). Combined, these observations had suggested that the coupling mechanism occurred by direct interactions of the c subunit loop regions and the and rotor stalk of F 1 which convey the proton gradient energy to the catalytic sites, probably by direct interactions. The crystal structure later confirmed these interactions (20). The crystal structure displayed a strikingly asymmetrical F 1 due to differences in the domains of the and subunits and the interactions formed with the single subunit (20). The obvious asymmetric positioning of the coiled coil of the subunit is a key feature to the mechanics of the binding change mechanism (discussed below) of F 1 F 0 ATP synthase. Its large carboxyl terminus -helix passes through a hydrophobic sleeve formed by six proline-rich loops of the and subunits, presumably resulting in the conformational changes occurring in the catalytic sites (20). In the E subunit (see above), several hydrogen bonds are formed with the subunit which forms a catch, resulting in conformational changes. Specifically, residues arg254 and gln255 in the carboxyl terminal helix form hydrogen bongs with E-asp317 E-thr318 and E-asp319 Also, a second catch is formed between the carboxyl terminal domain of the T subunit and the short helix of the PAGE 83 62 subunit. Hydrogen bonds form between lys87 lys90 and ala80 within the DELSEED region, T-asp394 and T-glu398 Structural information suggests the two antiparallel coiled coil -helices of the subunit may unwind during rotational catalysis and the subunit rotates around the F 1 axis while undertaking a net translation of about 23 (85). It is likely that these gross changes observed in the structures revealed individual functional states of the enzyme complex during catalysis. Catalysis: The Binding Change Mechanism F 1 F 0 ATP synthases house three catalytic sites, located at the 3 3 interfaces, with the predominate sites positioned in the subunit and some contributions made by the subunits (20). The minimal complex capable of normal ATP hydrolysis activity is the 3 3 complex (262). Boyer predicted that ATP synthesis requires a chronological involvement of the three catalytic sites, each of which changes its binding affinity for the substrates and products as it continues through a cyclical mechanism, referred to as the binding change mechanism (246, 263). The mechanism of ATP synthesis, in terms of the 3 3 and subunits and the substrates, ADP and P i is described here (Figure 1-10). The principles of the binding change mechanism have become the most commonly used model for recounting the means of ATP synthesis by F 1 F 0 ATP synthase. The three distinct catalytic sites were described as the tight (T) site containing ATP, an empty open (O) site, and the loose (L) site illustrated with ADP and P i bound (Figure 1-10). According to Boyer, catalysis starts with the binding of ADP and P i at the open site. The energy input from proton translocation drives the rotation of the rotor stalk, which ultimately results in the conformational changes responsible for ATP synthesis such that the tight site is converted to an open site, the open site assumes a loose site conformation, PAGE 84 63 and the loose sites becomes a tight sight. ATP is formed in the new tight site and the molecule of ATP that was found in the original tight site is released and the binding change mechanism starts fresh (Figure 1-10). Boyers mechanism included three proposals: i) only one site is actively synthesizing ATP at any given moment, ii) the reaction occurs reversibly at this site, and iii) energy input is required to bind the substrates, ADP and P i into the catalytic sites and to release the synthesized ATP, but not for the actual reaction to occur. Strong evidence for this model has come from the crystal structure of F 1 (20) and the observance of rotation via an attached fluorescent actin filament (discussed previously) (15, 65-67). Figure 1-10. The binding change mechanism. This is a simplified model of a more detailed enzymatic mechanism described by Weber and Senior (189) in which all three catalytic sites are transiently filled with nucleotide during ATP synthesis. Subunits included are (red), (green) and (pink). Genetic Expression and Assembly The E. coli F 1 F 0 ATP synthase is encoded in the 7 kb unc operon, which was cloned and sequenced in its entirety (224, 264). The genes encoded are called the uncB, PAGE 85 64 uncE, uncF, uncH, uncA, uncG, uncD and uncC coding for the a, c, b, , and subunits, respectively. A single copy of each gene is transcribed into a single polycysternic mRNA transcript, from which multiple polypeptides can be translated (265-267). The synthesis of the correct number of subunits, resulting in the stoichiometry of 3 3 ab 2 c 10 is thought to occur by translational regulation (264, 268). The efficiency at which the individual subunits are synthesized were observed to be variable and roughly corresponded to the stoichiometry of the intact enzyme complex (269, 270). Far less is understood about the assembly of the complex F 1 F 0 ATP synthase enzyme compared to the structural and mechanistic studies. Some have proposed that no particular pathway is necessary based on the observation that the complex can be dissembled into individual subunits and then reconstituted in vitro (113, 148, 271). On the contrary, some believe that the assembly follows an integrated pathway in vivo (272). The idea of an assembly pathway appealed to many since it would prevent a newly assembled F 1 sectors from freely hydrolyzing cellular ATP in the cytoplasm, and newly assembled F 0 sectors from acting as open proton pores in the membrane. If assembly were a random event, the potential to create isolated F 1 and F 0 sectors would exist. Some evidence supporting the integrated pathway does exist. The subunit is thought to function as an inhibitor of ATP hydrolysis activity, undertaking large conformational changes to allow the enzyme to switch from ATP synthesis to ATP hydrolysis under conditions of low ATP or low proton gradient, respectively (102, 177, 273-276). Its inhibitory action may possibly act to inhibit free ATPase activity in the partially assembled state. Some have speculated that the binding of the F 1 subunits to F 0 may PAGE 86 65 influence the opening of the F 0 proton channel in vivo (176, 277-279). The a subunit was observed to be absent from membranes of cells lacking the b or c subunits (150). Furthermore, work accomplished in our laboratory has showed that the b subunit monomer does not integrate into the membrane has no affinity for F 1 indicating that the formation of the b dimer in the membrane is an early event in F 1 F 0 ATP synthase assembly (199, 203, 218). Summary There is now ample evidence indicating that F 1 F 0 ATP synthases are composed of three functional parts, the catalytic core, the rotor stalk and the stator stalk. In the E. coli enzyme complex, the catalytic core consists of the 3 3 subunits and functions as an ATP synthase or an ATPase. The rotor stalk consists of the c 10 subunits and couples the energy of proton conduction to the synthesis of ATP by rotating within the 3 3 hexamer. Finally, the stator stalk consists of the ab 2 subunits and remains in a fixed position, anchored to the membrane by the a and b 2 subunits and to the 3 3 hexamer via interactions made by the subunit. Much of what was known of F 1 F 0 ATP synthase has been irrefutably confirmed by high resolution structures of partial complexes or model polypeptides. To date, a high resolution structure of the complete enzyme, or at least the complete F 0 is eagerly anticipated. Since the visualization of the peripheral stalk less than a decade ago a plethora of data characterizing the b 2 homodimer has emerged. The observation that the b 2 homodimer was likely not a rigid structure, and possibly more of an elastic structure, created the foundation for the work described in the following chapters. The work illustrated in Chapters 2, 3, 4, 5 and 6 of this dissertation will characterize the role of the PAGE 87 66 peripheral stalks dimer of identical b subunits in the E. coli F 1 F 0 ATP synthase by using a combination of site-directed mutagenesis and biochemical methods. Chapter 2 demonstrates the ability of the E. coli b subunit to form heterodimers and the capability of F 1 F 0 ATP synthase complex to tolerate the incorporation of two different length b subunits with a size difference of at least 14 amino acids (195). Chapter 3 demonstrates the formation of b heterodimers including at least one and up to two defective b subunits and documents indisputable evidence that F 1 F 0 ATP synthases incorporated with b subunit heterodimers are functional (280). Furthermore, the work accomplished in the chapter indicates, for the first time, that each of the two b subunits makes a unique contribution to the functions of the peripheral stalk, such that one mutant b subunit is making up for what the other is lacking. Chapter 4 describes cysteine chemical modifications constructed in the subunit and shortened, lengthened and wild-type length b subunits. The unc operon expression plasmids generated in this study will be used in future fluorescent labeling experiments. Chapter 5 documents mutagenesis experiments conducted on the extreme aminoand carboxyl termini of the b subunit (202) (Bhatt et al., manuscript in preparation, 2004). Finally, Chapter 6 summarizes the conclusions of this study and suggests the future directions that the work described in this dissertation has offered. PAGE 88 CHAPTER 2 INTEGRATION OF UNEQUAL LENGTH b SUBUNITS INTO F 1 F 0 ATP SYNTHASE Introduction F 1 F 0 ATP synthases provide the bulk of cellular energy production in both eukaryotes and prokaryotes (3, 5, 6). Enzymes in this family utilize the electrochemical gradient of protons across membranes in order to synthesize ATP from ADP and inorganic phosphate in a coupled reaction (16). In Escherichia coli, F 1 F 0 ATP synthase is a complex enzyme composed of approximately twenty-two polypeptides with the stoichiometry of 3 3 ab 2 c 10 (6, 7). The F 1 portion is composed of the subunits 3 3 and is responsible for enzymatic catalysis. The F 0 portion of the enzyme consists of the ab 2 c 10 subunits and is responsible for the translocation of protons through the membrane. Electron microscopy has shown that the F 1 and F 0 sectors are linked by two slender stalk structures (11). During ATP synthesis proton translocation drives the rotation of the central stalk, which consists of subunits within the 3 3 hexamer held stationary by the peripheral stalk. This rotation propagates the conformational changes in the active sites located at the interfaces driving catalytic activity (3, 15, 62, 65, 281, 282). The subunit of F 1 and a dimer of two identical b subunits from F 0 comprise the peripheral stalk acting as the stator. The subunit has been visualized seated at the top of the F 1 3 3 hexamer (187). However, recent evidence has suggested that the subunit may be positioned slightly to the side of F 1 in association with a single subunit (100, 141-143). 67 PAGE 89 68 The C-terminal region of the subunit is in direct contact with the extreme C-terminal end of the b dimer (3, 185, 200, 219, 220). The b subunit dimer constitutes the majority of the peripheral stalk stretching from within the membrane to near the top of F 1 (12). Dimerization of the b subunits is required for the normal assembly and function of F 1 F 0 ATP synthase (199). The two b subunits are believed to exist in parallel as an extended structure spanning from the periplasmic side of the membrane to near the top of F 1 Each has a N-terminal transmembrane domain, a tether domain extending from the surface of the membrane to the bottom of F 1 a dimerization domain and a -binding domain (13). The ability of b to bind to F 1 was proportional to the ability of b to form dimers, suggesting the necessity of the b dimer formation before the binding of F 1 to the complex (199). Presently, there is no high-resolution structure of the entire b subunit. A model polypeptide of the first 34 residues of the N-terminus has been solved by NMR, revealing a hydrophobic membrane-spanning -helix (136). A crystal structure of a monomeric dimerization domain, consisting of residues 62-122, has been solved and refined to 1.55 (138). Dunn and coworkers have constructed a model in which the two -helices of the b 62-122 region form a right-handed coiled coil. Much of the structural information on the b dimer has been gleaned from classical biochemical approaches such as CD-spectroscopy, crosslinking and sedimentation experiments (30, 166, 192, 196, 197, 203, 283). These studies revealed that the overall structure of the b subunit dimer is a highly extended conformation with approximately 80% -helix. Previous studies have shown that b subunits with deletions of up to eleven amino acids and insertions of up to fourteen amino acids, corresponding to approximately 16 and 21 respectively, formed functional F 1 F 0 complexes (193, 194). When b subunits PAGE 90 69 with either a seven amino acid deletion or an insertion, b 7 or b +7 respectively, were incorporated into the F 1 F 0 ATP synthase complex, the properties of the enzymes were essentially wild type. These observations suggested that the role of the b dimer is more of a flexible structural feature. However, it was not known whether this flexibility extended to the dimerization of two b subunits of unequal lengths and their incorporation into an enzyme complex. In the present study an experimental system was developed to allow expression of two different b subunit genes and determine whether the differing b subunits were assembled into an F 1 F 0 ATP synthase complex. Here, we demonstrate that the F 1 F 0 ATP synthase complex can tolerate b subunits with a size difference of at least 14 amino acids. Materials and Methods Materials Molecular biology enzymes and mutagenic oligonucleotides were obtained from Invitrogen (Carlsbad, CA), Life Technologies, Inc. (Grand Island, NY), New England Biolabs (Beverly, MA) and Stratagene (La Jolla, CA). Reagents were obtained from Sigma (St. Louis, MO), BioRad Laboratories (Hercules, CA) and Fisher Scientific (Pittsburgh, PA). Plasmid purification kits were acquired from Qiagen Inc. (Valencia, CA). The anti-rabbit immunoglobulin horseradish peroxidase-linked whole antibody (from donkey), anti-mouse immunoglobulin horseradish peroxidase-linked whole antibody (from sheep), Hybond ECL Nitrocellulose membrane, electrochemiluminescence Western blotting reagents and high performance chemiluminescence film were purchased from Amersham Biosciences (Piscataway, NJ). Polyclonal antibodies against SDS-denatured b subunit (284, 285) were generously PAGE 91 70 provided by Dr. Karlheinz Altendorf (Universitt Osnabrck, Osnabrck, Germany). Mouse monoclonal antibodies against the peptide epitope of hemagglutinin protein of human influenza virus (HA epitope tag) were purchased from Roche Molecular Biochemicals (Indianapolis, IN). Monoclonal antibodies against the epitope found in the P and V proteins of the paramyxovirus, SV5 (V5 epitope tag) were purchased from Invitrogen. Strains and Media The bacterial strains used to create the epitope tagged b subunits include the wild type b subunit expression plasmid, pKAM14, and plasmids used to express b subunits shortened or lengthened by 7 amino acids, pAUL3 and pAUL19, respectively, and have been described previously (193, 194, 203). The plasmids encoding the different uncF(b) genes were used to compliment E. coli strain KM2 (b) carrying a chromosomal deletion of the gene (218). All strains were streaked onto plates containing Minimal A media supplemented with succinate (0.2% w/v), to determine enzyme viability. Cells harvested for membrane preparation were grown in Luria Bertani media supplemented with glucose (0.2% w/v) (LBG). Isopropyl-1-thio--D-galactopyranoside (IPTG)(40 g/ml), ampicillin (Ap) (100g/ml), and chloramphenicol (Cm) (25 g/ml) were added to media as needed. All cultures were incubated at 37C for the appropriate duration. Recombinant DNA Techniques Plasmid purification. Plasmid DNA was purified with the Qiagen Mini-Prep and Maxi-Prep kits according to the protocols provided from the manufacturer. Mini-preps required 4 mL (high copy plasmid) or 6 mL (low copy plasmid) of an overnight bacterial culture carrying the desired plasmid. Maxi-preps required a 500 mL culture grown PAGE 92 71 overnight. Final elution volumes for the mini-prep kit was 30 or 50 L, for low copy and high copy plasmids, respectively, and 200-250 L for the maxi-prep kit. Final concentrations of 0.5 and 1.0 g/l plasmid were routinely obtained with the Qiagen mini and maxi-prep kits. Digestions, ligations, and transformations. Restriction endonuclease digestions, ligations, and transformations were performed according to the recommendations of the manufacturers (New England Biolabs, Stratagene and Invitrogen). For analytical purposes, restriction endonuclease digestions were normally prepared in a total volume of 20 L, including plasmid DNA, enzyme, buffer, ddH 2 O and occasionally BSA, and then incubated for an hour at the temperature specified by the manufacturer. Ligations required two purified double-stranded DNA fragments, a vector and an insert, of known length and concentration (ng/L). DNA fragments were routinely separated in a 0.8 % agarose gel by electrophoresis and the appropriate sized fragment was excised and purified using a Qiagen, Inc. QIAquick Gel Extraction kit. The vector fragment contained the antibiotic resistance gene and the origin of replication. The insert typically contained the desired gene or a site specific mutation. Two control reactions and two ligation reactions were set up. The typical reaction was set up in 20-40 L and included vector, insert, ATP, T4 DNA ligase buffer, T4 DNA ligase and ddH 2 O. The first control reaction was a control for uncut plasmids, containing no insert and no ligase, and the second, containing no insert, was a control for the vectors ability to ligate with itself. The femptomolar concentration (fmol/L) was determined from the known size and measured concentration. Two ligation reactions were then set up, the first had 1 part vector to 3 parts insert and the second was 1:10. Typically 5-15 fmol of vector was used. PAGE 93 72 The four reactions were incubated for 5 minutes at room temperature if T4 High Concentration DNA ligase was used or overnight at 16 C if T4 DNA ligase was used and then transformed into competent E. coli. Transformations were performed in one of three different E. coli strains. DH5 competent cells and XL10-Gold Ultracompetent cells were competent bacteria purchased from Invitrogen and Stratagene. These bacteria were used for purposes of plasmid preparations or to transform with a mutagenesis reaction such as Quikchange or ligations. The DH5 and XL10-Gold bacteria were stored at C. Basically, 25 L pre-aliquoted cells were thawed on ice and 1-5 L plasmid DNA was added and mixed by gentle stirring with the pipette tip. The bacteria were incubated on ice for 30 minutes, heat shocked at 42 C for 45 seconds, and then incubated on ice for 2 minutes. 1 mL LBG was added and then incubated at 37 C taped to a roller drum. The cells were harvested by centrifugation for 1 minute, the supernatant was discarded, the bacteria were resuspended in the remaining media, spread onto a LBG plate supplemented with the appropriate antibiotic and incubated at 37 C overnight. XL10-Gold cells required treatment with -mercaptoethanol (-ME) before transformation to increase the efficiency. 2 L of the provided -ME mix was added to 45 L pre-aliquoted cells and incubated on ice for 10 minutes prior to transformation with gentle swirling every 2 minutes. The transformation proceeded as described above. KM2 (b) was a strain of E. coli generated by a previous lab member (218) and maintained in our laboratory. This strain was used for purposes of plasmid expression and the study of the b subunit of F 1 F 0 ATP synthase. Before transformation, KM2 had to be made competent. Sterile technique was important since KM2 cells cannot be selected for by an antibiotic. All reagents and supplies were pre-chilled unless otherwise noted. A culture of KM2 PAGE 94 73 cells was grown overnight at 37 C in 5 mL LBG. The overnight culture (100 L) was inoculated into 10 mL pre-warmed (37 C) LBG and allowed to grow for 2-4 hours. The fresh culture was poured into sterile polypropylene tubes and incubated on ice for 10 minutes. The bacteria were then harvested by centrifugation (6,000 rpm in a ss-34 rotor) for 10 minutes. The supernatant was discarded and the tubes were inverted on a Kimwipe for 1 minute to allow excess LBG to drain. The bacteria pellet was resuspended in 10 mL cold, sterile 50 mM CaCl 2 and incubated on ice for 45-60 minutes. The bacteria were harvested as described above, resuspended in 1 mL of the 50 mM CaCl 2 and stored at 4 C until use. Generally, the competent KM2 cells could be used after two hours on ice but were most efficient for transformation at 24 hours and expired at 72 hours. For transformation, 100-200 L of cells were used as previously described. Site-directed mutagenesis. Site-directed mutagenesis was performed either by means of a Stratagene Quikchange XL kit or by ligation-mediated mutagenesis. Oligonucleotides containing the desired mutation(s) were designed to anneal to the same sequence on opposite strands of the plasmid (sense and antisense primers) (Appendix A) (Figure 2-1). When possible, a silent mutation was encoded to add or delete a restriction endonuclease recognition sequence to allow for easy screening of the mutation. Primers were optimally designed by ensuring the mutation was in the middle of the sequence, a cytosine (C) or guanine (G) flanked both ends of the sequence, and the melting temperature (T m ) was greater than or equal to 78 C. The T m was calculated as T m =81.5+0.41(%GC)-675/N-%mismatch, where N was the primer length (bases). When introducing insertions or deletions, "%mismatch" was dropped from the formula. PAGE 95 74 Figure 2-1. Oligonucleotides for epitope tags and mutagenesis of uncF(b). Shown are the sense strands of the mutagenic primer pairs. Green and red codons specify translation start and stop sites, respectively. A) Mutagenic primers encoding the sequence of the desired epitope tag insertion along with a silent mutation that introduced a new endonuclease recognition sequence. Epitope tags were inserted as described in the Materials and Methods. The oligonucleotides sequences specifying the histidine, HA or V5 epitope (bold blue) tag are labeled with the corresponding amino acid. The SphI, NdeI and SacI restriction sites (underlined) were added along with the histidine, HA and V5 tags, respectively, to facilitate screening. B) Oligonucleotides designed to construct the one-plasmid expression system. PAGE 96 75 Primers were not fast polynucleotide liquid chromatography (FPLC) or polyacrylamide gel electrophoresis (PAGE) purified as called for in the protocol. The reaction mixture consisted of 5 L Stratagenes 10X Pfu buffer, 3 L QuikSolution, 50 ng wild type plasmid, 125 ng each of the sense and antisense oligonucleotides, 1 L of 25 mM dNTPs, and 1 unit Pfu turbo polymerase. The total volume was brought up to 50 L with ddH 2 O and PCR was performed in a Perkin Elmer GeneAmp PCR System 2400 thermocycler according to the following cycling parameters: 95C 1 minute presoak; 18 cycles of 95C 50 seconds, 60C 50 seconds, 68C 1 minute per kb of plasmid length; 4C 7 min. Upon completion of the thermocycling, 1 L DpnI restriction endonuclease was added to the reactions in order to digest the methylated (nonmutated) plasmid DNA. The plasmids carrying the desired mutation were then transformed into competent DH5 cells, purchased from Life Technologies, and grown on LBG plates supplemented with the appropriate antibiotic. Plasmids carrying the desired mutation(s) were screened for by restriction endonuclease analysis and then the nucleotide sequences were directly determined by automated sequencing in the core facility of the University of Florida Interdisciplinary Center for Biotechnology Research (ICBR). Mutagenesis and Strain Construction Plasmids pKAM14 (b, Ap r ) (203), pAUL3 (b 7 Ap r ) (194) or pAUL19 (b +7 Ap r ) (193) were used to construct the epitope tagged b subunits. Epitope tags were inserted into each of the plasmids using the Stratagene Quikchange kit. A histidine epitope tag was inserted at the N-terminus by mutagenesis between the first and second codons of the uncF(b) gene to express b his or b +7-his (Appendix A) (Figure 2-1). Plasmids pTAM37 (b his ) and pTAM35 (b +7-his ) were created by digesting both of the recombinant histidine PAGE 97 76 tagged b subunit plasmids with PstI and NdeI and subsequently ligating the genes into a plasmid conferring the chloramphenicol resistance gene and the pACYC184 origin of replication (Table 2-1). Likewise, an HA epitope tag was inserted at the N-terminus by mutagenesis between the first and second codons of the uncF(b) gene to generate plasmids pTAM36 (b HA ) or pTAM34 (b 7-HA ). A V5 epitope tag was added to the C-terminus by site-directed mutagenesis before the termination codon of the uncF(b) gene to express b V5 or b 7-V5 from plasmids pTAM46 and pTAM47 (Figure 2-1). The recombinant HA-tagged and V5-tagged b subunit plasmids included the ampicillin resistance gene and the pUC18 origin of replication. Unique restriction enzyme sites SphI, NdeI and SacI were constructed near the histidine, HA and V5 epitope tag sequence, respectively, for an initial detection of the insertions, and then the nucleotide sequence was subsequently confirmed by automated sequencing in the ICBR core facility. Additionally, a set of plasmids was designed in order to express two different-tagged b subunits from a single transcript (Figure 2-2). As an example, a unique restriction enzyme site, SphI, was created in conjunction with the histidine tag between the Shine Dalgarno sequence and the first codon of uncF(b) to create pTAM35 (Figure 2-2A). In a separate site-directed mutagenesis reaction, an SphI site was added to the pTAM34 plasmid downstream of the HA-tagged b subunit and behind another favorable Shine Dalgarno sequence (Figure 2-2B). The two plasmids were digested with SphI and BstEII restriction endonucleases. The 3.2 kb vector fragment from pTAM34 and the 623 bp insert fragment from pTAM35 were isolated and then excised from a 0.8 % (w/v) agarose gel and purified with a QIAquick gel extraction kit. The vector and insert fragments were then allowed to ligate overnight at 16 C. It was then crucial to mutate an PAGE 98 77 Figure 2-2. Construction of the single transcript expression system. A) A unique restriction enzyme site, SphI, was created in conjunction with the histidine tag between the Shine Dalgarno sequence and the first codon of uncF(b) to create pTAM3. B) An SphI site was added to pTAM34 downstream of the HA-tagged b subunit and behind another favorable Shine Dalgarno sequence. The two plasmids were digested with SphI and BstEII. The 3.2 kb vector fragment from pTAM34 and the 623 bp insert fragment from pTAM35 were ligated. Additional mutagenesis (see Materials and Methods) resulted in C) a 3.7 kb plasmid, pTAM40, which expressed b 7-HA and b +7-his from the same promoter and included the ampicillin resistance gene and the pUC18 origin of replication. In similar constructions, plasmids pTAM41 (b wt-HA and b +7-his ) and pTAM42 (b wt-HA and b wt-his ) were created. PAGE 99 78 intrinsic added start codon, which is in the SphI recognition sequence, to prevent a missense mutation. The mutagenic oligonucleotide was designed to accomplish three tasks in one reaction: 1) mutate the ATG found in the SphI recognition sequence, 2) add a new Shine Dalgarno sequence in a favorable position from the true start codon, and 3) mutate the GTG start codon to a more favorable ATG start site (Figure 2-1). The resulting 3.7 kb plasmid, pTAM40, expressed b 7-HA and b +7-his from the same promoter and included the ampicillin resistance gene and the pUC18 origin of replication (Figure 2-2C). In similar constructions, plasmids pTAM41 (b wt-HA and b +7-his ) and pTAM42 (b wt-HA and b wt-his) were created (Table 2-1). Throughout this dissertation, the insertion or deletion and the epitope tag are indicated after the plasmid name for clarity, for example plasmid pTAM35 (b +7-his ). Each plasmid and the control plasmids pKAM14 (b) and pBR322 were expressed in the E. coli cell line KM2 (b) for study, so that the only b subunits in the cells were the product of the plasmid genes. The two plasmid expression system successfully allowed expression of various combinations of histidine tagged and V5-tagged b subunits in the same cell (Figure 2-7). Appropriate antibiotics were added to the growth medium, and in the case of the coexpressed plasmids, both ampicillin and chloramphenicol were added to select for cells expressing both plasmids. Crude Preparative Procedures Inverted membrane vesicles from KM2(b) strains expressing the desired b subunit epitope tagged F 1 F 0 ATP synthase complex were prepared for activity assays, Ni-resin purification and Western Blot analysis. Unless otherwise noted, all reagents, rotors and materials were kept at 4 C. The membrane preparations were prepared by inoculating a PAGE 100 79 10 mL starter culture, grown overnight, into a 2 L Erlenmeyer flask containing 500 mL LBG, supplemented with the appropriate antibiotic, ampicillin (Ap) and/or chloramphenicol (Cm). Similarly, 1 mL of a starter culture was inoculated into a nephalo flask containing 50 mL LBG (Ap and/or Cm). The bacteria were grown at 37 C in a New Brunswick Scientific incubator shaker (220 rpm) and the turbidity was monitored using a Klett-Summerson photoelectric colorimeter. IPTG (40 M) was added when the turbidity reached 75 Klett units and the cells were collected when the turbidity reached 150 Klett units. The bacteria were harvested by centrifugation for 10 minutes at 8,000Xg in a Sorvall GSA rotor. The pellets were rinsed once with TM buffer (50 mM tris-HCl, 10 mM MgSO 4 pH 7.5) and then resuspended in a final volume of 10 mL TM buffer. DNaseI (10 mg/mL) was added to a final concentration of 10 g/mL and the bacteria were broken by passing through a French Pressure Cell one time at 14,000 psi. Cellular debris and unbroken cells were removed by centrifuging twice at 10,000Xg for 10 minutes. Membranes were then collected by ultracentrifugation at 150,000Xg in a Beckman 70.1 Ti rotor for 1.5 hours. The membrane pellets were rinsed once with TM buffer and then resuspended in TM buffer to a final volume of 2 mL using a 2 mL Wheaton tissue grinder. For the purposes of Western blot analysis or Ni-resin purification, one ultracentrifugation step sufficed. However, activity analysis required an additional ultracentrifugation step in order to remove nonspecifically bound ATPases. In this case, the membrane pellets were resuspended in a final volume of 10 mL using a 10 mL Wheaton tissue grinder and the ultracentrifugation step was duplicated. PAGE 101 80 Determination of Protein Concentration Total membrane protein concentrations were determined by the bicinchoninic acid (BCA) assay (286). The membrane preparations were diluted 1:10 for this assay. For each sample, triplicates of 10 L, 20 L and sometimes 30 L of the diluted samples were aliquoted for the assay. Bovine serum albumin (BSA) was used to generate a standard curve. A stock of 1 mg/mL BSA was prepared and stored in microcentrifuge tubes at C. The exact BSA concentration was determined from the OD 280 (1.0 mg/mL BSA OD 280 =0.667). The BSA was aliquoted in triplicates ranging from 0 to 100 g (0, 5, 10, 20, 40, 60, 80, and 100 L) of protein. All of the samples were incubated in 3 mL of standard working reagent (SWR), consisting of 50 parts solution A (1% BCA-Na 2 2% Na 2 CO 3 H 2 O, 0.16% sodium potassium tartrate, 0.95% NaHCO 3 pH 11.25 and filtered) to one part solution B (4 % CuSO4H 2 O) plus 1% sodium dodecyl sulfate (SDS) mixed fresh as needed, for 30 minutes at 37 C and then for 10 minutes at room temperature. The absorbance at 562 nm was recorded for each sample with an LKB Biochrom Ultrospec II spectrophotometer. The standard curve was generated from the BSA OD readings, with a typical correlation coefficient of at least 0.997, and then protein concentrations were determined using linear regression. A typical concentration range of the total membrane protein prepared from 500 mL of media was 5-20 g/L. Ni-Resin Purification Ni-resin purification was achieved using the High Capacity Nickel Chelate Affinity Matrix (Ni-CAM) purchased from Sigma. It became necessary to optimize conditions and the ratio of protein to packed Ni-resin volume for each type of experiment. Determining the optimal ratio of protein to Ni-resin was critical because F 1 F 0 ATP PAGE 102 81 synthase complexes with two histidine-tagged b subunits bind with a higher affinity than the complexes containing a b subunit heterodimer, which has only one histidine epitope tag. For the purposes of Western blot analysis, a total of 5 mg of membrane protein was brought up to 1 mL with final concentrations of 0.2% tegamineoxide WS-35, 0.15 M NaCl, and 1 mM imidazole. The purification procedure was accomplished using the trial scale miniprep method essentially described by the manufacturer. The 1 mL of clarified membrane protein was divided up equally into 5 microcentrifuge tubes, containing 100 L of packed Ni-resin that had been washed one time with 500 L equilibration/wash buffer (50 mM NaH 2 PO 4 H 2 0, 0.3 M NaCl, 10 mM imidazole, 0.2% tegamineoxide WS-35, pH8.0), and allowed to mix on a nutator for 5 minutes. The samples were centrifuged for 30 seconds at 5,000Xg and the supernatant was discarded. The resin was washed 8 times with 1 mL wash buffer by gently mixing for 1 minute, centrifuging for 30 seconds at 5,000Xg and discarding the supernatant. The histidine-tagged proteins were eluded by mixing 50 L elution buffer (50 mM NaH 2 PO 4 H 2 0, 0.3 M NaCl, 250 mM imidazole, 0.2% tegamineoxide WS-35, pH8.0) in each microcentrifuge tube for 1 minute, centrifugation, and then the supernatant was extracted. The elution step was repeated one more time to recover more of the target protein. Finally, the target protein was pooled together and then concentrated from 500 L to about 50 L by centrifugation with a Millipore Amicon Bioseparations Microcon YM-10 (14,000Xg for 30 minutes). To control for possible enzyme disruption during the solubilization and purification procedures, the tagged wild-type length b subunits were either mock treated or treated with the homobifunctional crosslinker, bis(3-sulfo-N-hydroxysuccinimide ester) (BS 3 ), after the addition of detergent. Membrane preparations were chemically crosslinked by PAGE 103 82 treating with 1mM BS 3 for 30 minutes at room temperature. The cross-linking reaction was stopped by addition of 100 mM ethanolamine HCl, pH 7.5, for 10 minutes. For the purposes of densitometric analysis, a total of 15 mg membrane protein was solubilized and clarified as described above. The clarified protein was divided into 10 samples and then Ni-resin purified (1 ml packed resin volume divided into 10 microcentrifuge tubes) as described previously, and the final eluate was concentrated with a Microcon YM-10. Attempts to purify the heterodimers with the batch method (15 mg membrane protein to 1 mL packed resin volume in a 15 mL corning conical tube, undivided) proved unsuccessful; therefore division into several microcentrifuge tubes was necessary. Assays of F 1 F 0 ATP Synthase Activity Growth on a minimal succinate medium was used as an initial, in vivo, assay for enzyme viability. ATP hydrolysis activity was assayed by the acid molybdate method (146), which measures the release of P i from ATP, in order to determine the specific activity of the epitope tagged F 1 F 0 ATP synthase enzyme complexes. Membranes were assayed to determine the linearity with respect to time and enzyme concentration. Prior to the assay, all supplies and reagents were distributed, labeled and placed on ice or 37 C as specified below. For each membrane sample, duplicates of 60 g membrane protein were incubated in 4.0 mL of the reaction buffer (50 mM Tris-HCl, 1 mM MgCl 2 pH9.1) at 37 C. Stop buffer, which acts to prevent further hydrolysis of ATP, was prepared as needed and consisted of 1.3 part ddH 2 O to 0.6 part HCl/molybdate solution (2.5% NH 4 Mo 4 O 24 H 2 O, 4.0 N HCl) to 0.4 part 10% SDS. The stop buffer was divided into 2 mL aliquots in 13x100 mm disposable borosilicate glass tubes and kept on ice throughout PAGE 104 83 the assay. Tubes of stop buffer were required for each time point of each sample, phosphate standard curve, and ATP only control. Prior to the beginning of the time course, two measures are taken to subtract background phosphate. First, a zero time point was taken by removing 435 L of the reaction buffer, containing the membranes before the addition of ATP, and adding it to 2 mL of stop buffer. Also, to account for spontaneous hydrolysis, ATP was incubated in 4 mL of reaction buffer with no membrane protein and then 435 L was removed and added to stop buffer. The time course was started by the addition of 80 L ATP (0.15 M in 25 mM Tris-HCl, pH 7.5) to the reaction buffer and additional time points were taken at 2, 5, 7, 10 and 12 minutes. A phosphate standard curve was generated by preparing duplicates of 1 mL reaction buffer containing 0, 0.02, 0.1, 0.2, 0.4 and 0.6 mol phosphate (KH 2 PO 4 ), and then adding 435 L of each to 2 mL stop buffer. Once the time course and the standard curve were completed, all of the samples in stop buffer were removed from the ice and allowed to come to room temperature. The inorganic phosphate concentration was determined by adding 100 L of Eikonogen solution (1 M NaHSO 3 0.1 M Na 2 SO 3 0.01 M 4-amino-3-hydroxy-1-naphthalenesulfonic acid), diluted 1:10, and incubating at room temperature for 30 minutes to allow color development. The absorbance of each time point at 700 nm was recorded with an LKB Biochrom Ultrospec II spectrophotometer and the amount of free phosphate in each sample was determined using linear regression. The rate of ATP hydrolysis was determined and the apparent specific activity was expressed as mol P i /min/mg membrane protein. Membrane energization was detected by the fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine (ACMA) (271). First, F 1 F 0 ATP synthase-mediated ATP PAGE 105 84 driven proton pumping activity in inverted membrane vesicles prepared from the epitope-tagged mutants was used as an indication of coupled activity. Acidification of the inverted membrane vesicles upon addition of ATP was directly examined by fluorescence quenching. Membrane protein (250 g) was suspended in 3 ml of assay buffer (50 mM MOPS, 10 mM MgCl 2 pH 7.3) in a quartz cuvette. The fluorescent dye ACMA was added to a final concentration of 1 M, and fluorescence was monitored with a Perkin-Elmer LS-3B Fluorescence Spectrometer with excitation at 410 nm and emission at 490 nm and directly recorded with a Perkin-Elmer GP-100 Graphics Printer. After several seconds, the fluorescent emission stabilized and was manually set to 85% for the purpose of the scale. The emission was graphically recorded for 1.5 minutes, ATP was added to a final concentration of 0.4mM and the emission was recorded for and additional 10-12 minutes. The level of -nicotinamide adenine dinucleotide, reduced form, (NADH)-driven fluorescence quenching was monitored for all membrane preparations to demonstrate that the vesicles were intact and closed. Membrane protein (250 g) was suspended in 3 ml of assay buffer (50 mM MOPS, 10 mM MgCl 2 pH 7.3). ACMA was added to a final concentration of 1 M, and fluorescence was recorded excitation set at 410 nm and emission set at 490 nm. After several seconds, the fluorescent emission stabilized and was manually set to 95%. The emission was recorded for 1 minute, 5 L NADH (0.1 mM) was added and the emission was continually recorded. Over time, membrane vesicle acidification peaked and the fluorescence quenching reached a maximum. As the fluorescence began to rise, 5 L of 0.3 mM KCN was added and the emission was recorded for a total time of about 10-15 minutes. PAGE 106 85 Immunoblot Analysis Electrophoresis and transfer. Proteins were mixed with 2X Laemmli sample buffer (LSB) (62.5 mM tris-HCl, pH 6.8, 2% w/v SDS, 720 mM -mercaptoethanol, 20% glycerol, and 0.1% Bromophenol blue) and incubated for 5 minutes at 95 C. The proteins were then loaded on either a 15 cm 15% tris-glycine SDS gel or a purchased SDS-PAGE 15% Tris-HCl Ready Gel purchased from BioRad Laboratories and then transferred onto a nitrocellulose (anti-b or anti-V5 antibody incubation) or polyvinylidene fluoride (PVDF) (anti-HA antibody incubation) microporous membrane by electroblot. The proteins were separated in electrode buffer (25 mM tris, 192 mM glycine, and 0.1% SDS) by electrophoresis in a Mini-PROTEAN II cell (small gels) or a PROTEAN II (large gels). When it was necessary to distinguish two different tagged b subunits from the same immunoblot, a maximal separation was required. The proteins were run on a 15 cm 15% polyacrylamide tris-glycine gel at 100 mamp, with current held constant, until the dye reached the stacker and then at 24 mamp for 12 hours. It was necessary to double the amperage when two gels were running at the same time. The protein transfer was accomplished in transfer buffer (25 mM tris, 192 mM glycine, 20% methanol) at 4 C at either 100 V, 0.25 amp limit for 1 hour (small gels) or 100 V, 0.36 amp limit for 4 hours (large gels). Anti-b subunit antibody. The b subunit antibody incubation was performed essentially as described previously by Tamarappoo et al. (287). The current lab stock of anti-b subunit antibodies were diluted 1:40 (long-term stock) and 1:40,000 (short-term stock) for storage on a non-frost-free shelf at C and required and additional 1:40 dilution of the short-term stock for the working concentration. Upon completion of PAGE 107 86 electrophoresis to a nitrocellulose membrane, the protein was visualized, for loading comparison, with fast green stain (50% methanol, 10% glacial acetic acid, 0.01% fast green) by incubation at room temperature with gentle shaking on a Bellco Biotechnology Orbital Shaker for 15 minutes followed by three 2-5 minute (depending on strength of stain) destain washes (50% methanol, 10% glacial acetic acid). The fast green stain was recycled for repetitive use and the destain was discarded. The membrane was washed three times for 5 minutes with tris-buffered saline (TBS) (10 mM tris-HCl, 150 mM NaCl, pH7.2) supplemented with 0.1% polyoxyethylenesorbitan monolaurate (tween 20) (TTBS) and then blocked for 1 hour at room temperature or overnight at 4 C in TBS supplemented with 5% nonfat dry milk (NFDM). The primary antibody incubation was performed with the anti-b subunit antibody (1:40) in TTBS supplemented with 2% BSA for 1 hour followed by three 5 minute washes with TTBS. The anti-b antibody was restored at C and thawed for reuse up to 3-5 times. Finally, secondary antibody incubation was performed with horseradish peroxidase-linked donkey anti-rabbit antibody (1:50,000 in TTBS-BSA), washed 4 times in TTBS (two 5 minute washes, two 10 minute washes) and then the antibody was detected by enhanced chemiluminescence (ECL). Signals were visualized on high performance chemiluminescence film using a Kodak X-Omat. Typical exposure times for the anti-b antibody ranged from 1-5 minutes for strong signals and 30-60 minutes for weaker signals. Anti-HA antibody. The HA-epitope tag antibody was performed essentially as described by the manufacturer followed by a secondary antibody incubation with horseradish peroxidase-linked anti-mouse (from sheep) antibody (1:10,000). Several initial attempts to visualize the HA-epitope tagged b subunit on a nitrocellulose PAGE 108 87 membrane failed to give a clean signal. It was essential to enhance the sensitivity of the antibody by transfer onto a PVDF membrane for a higher signal to noise ratio. Before electrotransfer, it was crucial to prepare the membrane by immersion in 100% methanol for 15 seconds ddH 2 O for 2 minutes and then transfer buffer for 5 minutes. The wash buffer consisted of a phosphate buffered saline (PBS) (1% NaCl, 0.025% KCl, 0.18% Na 2 HPO 4 0.03% KH 2 PO 4 pH 7.4) supplemented with 0.1% tween 20 (PBST), membranes were blocked in PBS with 5% NFDM, and the primary and secondary antibody incubation was performed in PBST supplemented with 2.5% NFDM. Typical exposure times for the anti-HA antibody were about 1 hour. Occasionally, a stronger detection reagent, ECL Plus (Amersham Biosciences), was required for detection. ECL Plus is an extremely sensitive detection system; therefore it was necessary to follow protocol carefully, using optimal primary and secondary antibody conditions. The detection reagents were brought to room temperature before opening and then solutions A and B were mixed in a 40:1 ration. The final volume required was 0.1 mL per cm 2 of membrane. Excess wash buffer was allowed to drip off the membrane and then the detection solution was pipetted onto the membrane and incubated at room temperature for 5 minutes. Finally, it was critical to drain off excess detection reagent by touching the corner of the membrane onto a kimwipe before exposure to film. Anti-V5 antibody. The V5-epitope tag antibody incubation was performed as described by the manufacturer followed by secondary antibody incubation with horseradish peroxidase-linked sheep anti-mouse antibody (1:10,000). The wash buffer consisted of TTBS, membranes were blocked in TBS with 5% NFDM, and the primary and secondary antibody incubation was performed in TBS supplemented with 2.5% PAGE 109 88 NFDM. Typical exposure times for the anti-V5 antibody were about 30 seconds to 1 minute for membrane proteins and about 1 hour to overnight to visualize Ni-resin purified proteins. Results HA-Epitope Tagged b Subunits To investigate whether it is possible for two b subunits of unequal length to dimerize to form the peripheral stalk in a functional enzyme complex, a collection of plasmids expressing two different epitope tagged b subunits were generated by site-directed mutagenesis. Initially a histidine-epitope tag and a HA-epitope tag were employed. Construction and growth characteristics of mutants The first approach developed to express two different b subunits in the same cell was a two plasmid expression system (Figure 2-2). A total of four plasmids were constructed expressing b wt-his (Cm r ), b +7-his (Cm r ), b wt-HA (Ap r ) or b 7-HA (Ap r ) (Table 2-1). The histidine and HA epitope tags were needed to facilitate enzyme purification and subunit detection on Western blot, respectively. Coexpression of both b subunits was selected for by media that contained both ampicillin and chloramphenicol. A previous analysis of F 1 F 0 ATP synthase complexes with b subunits shortened and lengthened by 7 amino acids found the mutants to be essentially wild-type (193, 194). Functional F 1 F 0 ATP synthase complexes have been studied with epitope tags positioned on many of the subunits, however, an epitope tag on the b subunit had not been attempted. It was possible that the epitope tags would affect enzyme assembly or function. The effects of the added epitope tags were studied by the ability of the plasmids to complement the E. coli strain KM2 (b) (218). Growth on succinate minimal PAGE 110 89 medium was used as an initial qualitative gauge of enzyme activity in vivo since E. coli strains lacking F 1 F 0 ATP synthase cannot derive energy from nonfermentable carbon sources. In each case, the strains expressing the histidine and HA-epitope tagged b subunits grew comparably to the wild type strain (Table 2-1). Figure 2-3. Histidine and HA-epitope-tagged b subunit expression system. We employed an epitope-tag system in order to determine whether b subunits of unequal length can interact to form the dimer in an intact and functional enzyme complex. B) Both b 7-HA and b +7-his were expressed together in KM2 (b) cells using a two-plasmid expression system. Plasmid pTAM34 was designed to express high levels of HA-tagged b 7 This plasmid contains the genes conferring ampicillin resistance and the pUC18 origin of replication. Plasmid pTAM35 includes the chloramphenicol resistance gene, the pACYC184 origin of replication and expresses histidine-tagged b +7 In similar constructions, plasmids pTAM37 (b wt-his Cm r ) and pTAM36 (b wt-HA Ap r ), or pTAM35 (b +7-his Cmr) and pTAM46 (b wt-HA Ap r ) were developed for coexpression experiments. PAGE 111 90 Table 2-1. Aerobic growth properties and membrane-associated ATP hydrolysis activity of mutants expressing epitope tagged uncF(b) genes Strains Description Growth 1 Specific activity 2 KM2/pKAM14 (+) b wt Ap r +++ 1.72 0.04 KM2/pBR322 (-) b, Ap r 0.50 0.01 KM2/pTAM37 b wt-his Cm r +++ 1.53 0.08 KM2/pTAM35 b +7-his Cm r +++ 1.35 0.03 KM2/pTAM36 b wt-HA Ap r +++ 1.48 0.03 KM2/pTAM34 b 7-HA Ap r +++ 1.39 0.04 KM2/pTAM37/pTAM36 b wt-his + b wt-HA Cm r & Ap r +++ nd KM2/pTAM35/pTAM36 b +7-his + b wt-HA Cm r & Ap r +++ nd KM2/pTAM35/pTAM34 b +7-his + b 7-HA Cm r & Ap r +++ 1.41 0.04 KM2/pTAM42 b wt-his + b wt-HA Ap r +++ nd KM2/pTAM41 b +7-his + b wt-HA Ap r +++ nd KM2/pTAM40 b +7-his + b 7-HA Ap r +++ nd KM2/pTAM46 b wt-V5 Ap r +++ 1.63 0.02 KM2/pTAM47 b 7-V5 Ap r +++ 1.40 0.05 KM2/pTAM37/pTAM46 b wt-his + b wt-V5 Cm r & Ap r +++ 1.70 0.03 KM2/pTAM35/pTAM46 b +7-his + b wt-V5 Cm r & Ap r +++ 1.58 0.02 KM2/pTAM35/pTAM47 b +7-his + b 7-V5 Cm r & Ap r +++ 1.53 0.03 1 E. coli strains were grown aerobically on succinate minimal medium. Colony size was scored after 72-hr incubation at 37C as: +++, 1.0 mm; ++, 0.3-0.5 mm; +, ~0.1 mm; -, no growth. 2 ATPase activities were measured as described under Materials and Methods. Units of specific activity = mol of PO 4 released per mg of protein/min S.D. Units were calculated from the slope of the line based on three measurements with incubations for 12 minutes. Effects of epitope tags Since F 1 has little affinity for the membrane in the absence of intact F 0 total membrane associated F 1 -ATP hydrolysis activity was used as a test of F 1 F 0 ATP synthase complex assembly. Under conditions of high pH, F 1 can be released from the influence of F 0 (146), so the amount of ATPase activity in the solution was used as a measure of the amount of intact enzyme complex located in the membrane vesicles. The histidine and HA-epitope tags had a slight, but not vitally significant affect on enzyme assembly. Membrane preparations with a b wt-HA or b 7-HA incorporated into the F 1 F 0 ATP synthase complex had specific activities of about 86% and 81% of the wild type strain, respectively (Table 2-1). The latter value was comparable to the effect of the seven PAGE 112 91 amino acid deletion in the absence of the epitope tag. Similar activities were observed in membrane vesicles isolated from cells with a histidine epitope tag was incorporated onto the enzyme. F 1 F 0 ATP synthase complexes with a b wt-his or b 7-his dimer had specific activities of about 90% and 79% of the wild type strain, respectively (Table 2-1). Expression of different b subunits in the same cell The ability to express two different b subunits in the same cell in roughly equal quantities was crucial to the success of the experiment. Therefore it was necessary to establish whether two separate plasmids could be used to direct production of two different b subunits. Two b subunits, expressed from pTAM35 (b 7-his ) and pTAM34 (b 7-HA ), were expressed in the same KM2 (b) cell and their ability to exist in the same cell was determined by Western blot analysis (Figure 2-4A). As expected, immunoblot analysis of crude membrane preparations using anti-b antibodies showed the presence of the b subunit in all strains complemented with an epitope-tagged b subunit (Figure 2-4A, Lanes 3-6). As shown in the first lane, KM2 cells did not express b subunit. After transforming with plasmid pKAM14 (b wt ), the KM2 cells expressed the wild type b subunit (Lane 2). The third and forth lanes represented KM2 cells expressing the b 7-HA and b +7-his subunits, respectively. In lane 5, individual membrane preparations representing lanes 3 and 4 were mixed together. Finally, lane 6 represented the KM2 expressing both the short and long b subunits in the same cell. This figure demonstrated our ability to separate the short and long subunits enough to be sufficiently distinguishable. Notably, the KM2 cells successfully expressed the two different b subunits in roughly equal quantities from the same cell. PAGE 113 92 Figure 2-4. Western blot analysis of histidine and HA-epitope tagged b subunits. A) KM2 (b) cells were transformed with pKAM14 (b wt ), pTAM34 (b 7-HA), pTAM35 (b +7-his ), or both pTAM34 and pTAM35. Crude membrane preparations were made and proteins separated on a 15% polyacrylamide Tris-HCl-SDS BioRad Ready gel. The proteins were transferred to a nitrocellulose membrane and probed with anti-b anitbodies. Lane 5 represents a mixture of membranes in lanes 3 and 4. B) The crude membrane preparations were solubilized with 0.2% LDAO and then subjected to Ni-resin purification under native conditions (see Materials and Methods). The purified proteins were then separated with SDS-PAGE and Western blotted with anti-b antibodies. Ni-Resin Purification Initial experiments utilizing the two plasmid expression system approach indicated that the two different b subunits were incorporated into F 1 F 0 ATP synthase in a segregated manner, suggesting that the b subunit dimer must form in parallel during F 1 F 0 ATP synthase assembly. The two b subunits, expressed from pTAM35 (b 7-his ) and pTAM34 (b 7-HA ), were expressed in the same KM2 (b) cell and their ability to exist in the same F 1 F 0 ATP synthase enzyme complex was determined by Ni-resin purification followed by Western blot analysis (Figure 2-4B). Upon Ni-resin purification, only enzyme containing the histidine epitope tagged b subunit should have been present. As PAGE 114 93 anticipated, KM2 cells did not express b subunit. Western blot analysis confirmed that b subunits without a histidine epitope tag were washed off the resin during the wash steps (Figure 2-4B, Lanes 2 and 3) whereas b +7-his was retained by the Ni-resin (Figure 2-4B, Lane 4). In lane 5, when individual membrane preparations of KM2 individually expressing either the short or long subunit were mixed together, as expected, only the histidine epitope tagged b subunit was purified from the Ni-resin. In the KM2 cells expressing both b +7-his and b 7-HA only the histidine-tagged long b subunit appeared, suggesting the different b subunits were segregated into separate enzyme complexes. However, there were two important caveats: the enzyme complex may have dissociated before coming off the Ni-resin, and a difference of fourteen amino acids may have simply been too much for the stability of the enzyme. Both issues were studied. First, two additional detergents to solublize the membrane were investigated. The capability of the detergents to allow the enzyme to come off the resin intact was investigated by cross-linking the b subunits after solubilization and Ni-resin purification and then visualized by Western blots. Secondly, the size difference of the two b subunits was decreased by testing for the ability of the b +7-his subunit to dimerize with a b wt-HA subunit. Membrane preparations of KM2 (b) expressing b +7-his were solubilized with 0.2% solutions of tegamineoxide WS-35 (AO), taurodeoxycholate (TD), lauryldimethylamine oxide (LDAO) or SDS and allowed to incubate at room temperature for 30 minutes. AO, TD and LDAO were detergents commonly used to solubilize membrane proteins. LDAO was likely to dissociate F 1 from F 0 In theory, however, F 0 should have remained intact through the purification. The solubilized proteins were then mock treated (-) or treated (+) with the homobifuctional crosslinker, BS 3 The cross-linking reaction was stopped by PAGE 115 94 Figure 2-5. Investigation of detergent solubilization of F 1 F 0 ATP synthase complexes. Membrane preparations of KM2 (b) expressing b +7-his were solubilized with 0.2% solutions of detergents: tegamineoxide WS-35 (AO), taurodeoxycholate (TD), lauryldimethylamine oxide (LDAO) or SDS. The solubilized proteins were then mock treated (-) or treated (+) with 1mM bis(3-sulfo-N-hydroxysuccinimide ester) (BS 3 ) for 20 minutes at room temperature. The cross-linking reaction was stopped by addition of 100 mM ethanolamine HCl, pH 7.5 for 10 minutes. The products were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-b antibodies. addition of ethanolamine HCl, pH 7.5 and then the products were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-b antibodies for Western blot analysis (Figure 2-5). The b +7-his dimers were detected in membrane preparations treated with BS 3 after solubilization, therefore, the crosslinked product showed that the b +7-his subunit dimer was stable during solubilization with AO, TD and LDAO (Figure 2-5, Lanes 4,6, and 8), but not with SDS (Figure 2-5, Lane 10), which was expected to denature the proteins. Once the detergent conditions were determined to be safe for the enzyme complex, the ability of the different b subunits, b +7-his and b 7-HA to heterodimerize was reevaluated by a slightly different approach. This time, membrane preparations were treated with the crosslinker, BS 3 prior to Ni-resin purification to ensure no disruption of the dimers (Figure 2-6). Immunoblot analysis of crude membrane PAGE 116 95 preparations using anti-b antibodies showed the presence of the b subunit in all strains complimented with a b or an epitope-tagged b subunit (Figure 2-6A, Lanes 2-5). As expected, only b subunits with a histidine tag were retained by Ni-resin purification (Figure 2-6A, Lanes 6-11). F 1 F 0 complexes with only the HA-tagged b subunits were completely washed off the resin (Figure 2-6A, Lanes 6-7). To detect a heterodimer formation, immunoblot analysis using the anti-HA antibody was performed (Figure 2-6B). When the two plasmids were coexpressed in the same cell the anti-HA antibody did not detect the presence of b 7-HA after Ni-resin purification, indicating the two different Figure 2-6. Ni-resin purification of F 1 F 0 expressing different length b subunits, treated with the cross-linker BS 3 Crude membrane preparations of KM2 expressing the different b subunits were cross-linked (Materials and Methods). The products were then solubilized and subjected to Ni-resin purification before separation by SDS-PAGE and subsequent Western blot analysis. A) Proteins were transferred to a nitrocellulose membrane and probed using a polyclonal anti-b antibody. B) Proteins were transferred to a PVDF membrane and probed using an anti-HA mouse monoclonal antibody. PAGE 117 96 b subunits, b +7-his and b 7-HA did not dimerize. Similar experiments were performed with a seven amino acid size difference as opposed to the fourteen amino acid difference just attempted (data not shown). The immunoblot analysis gave identical results to the previous data, b +7-his and b wt-HA dimerization was not detected, suggesting the two different b subunits had incorporated into F 1 F 0 ATP synthase in a segregated manner. However, further experimentation utilizing this experimental approach indicated that dimerization was not detected between two wild type length b subunits, b wt-his and b wt-HA Figure 2-7. Ni-resin purification of histidine and HA-epitope tagged F 1 F 0 treated with the cross-linker BS 3 Crude membrane preparations of KM2 expressing the different b subunits were cross-linked (Materials and Methods). The products were then solubilized and subjected to Ni-resin purification before separation by SDS-PAGE and subsequent Western blot analysis. A) Proteins were transferred to a nitrocellulose membrane and probed using a polyclonal anti-b antibody. B) Proteins were transferred to a PVDF membrane and probed using an anti-HA mouse monoclonal antibody. PAGE 118 97 rendering the previous data worthless (Figure 2-7). In order to conclude that the different b subunits formed F 1 F 0 ATP synthase complexes in a segregated manner, it was necessary to detect an interaction between two wild type length b subunits. Several factors may have contributed to the failure of the first experimental system: unknown interactions between the HAand histidine-epitope tag may have hindered an interaction between the two b subunits, the anti-HA antibody was not sensitive enough or the signal-to-noise ratio was too small for detection, or expression from two different plasmids may have placed an early, detrimental affect on the experimental system. The latter was considered first. Dimerization of the b subunits is thought to be an early event in enzyme assembly, making the requirement for immediate dimerization of b via co-translation of a single b subunit transcript feasible. Therefore, three plasmids were designed to express two different tagged b subunits from a single transcript (Figure 2-3C, and Table 2-1). All three plasmids, pTAM40, pTAM41 and pTAM42, successfully expressed two different b subunits, nevertheless, an interaction between b wt-his and b wt-HA was not found. V5-Epitope Tagged b Subunits Several attempts to visualize an interaction between two different wild type length b subunits with a histidine and a HA-epitope tag failed. Attention was then focused on the types of epitope tags used and we decided to replace the HA tag with a V5-epitope tag. Construction and growth characteristics of mutants In a third attempt to investigate whether it is possible for two b subunits of unequal length to dimerize to form the peripheral stalk in a functional enzyme complex, a collection of plasmids expressing histidine or V5 epitope-tagged b subunits were PAGE 119 98 Figure 2-8. Histidine and V5-epitope-tagged b subunit expression system. A) Both b 7-V5 and b +7-his were expressed together in KM2 (b) cells using a two-plasmid expression system. Plasmid pTAM47 was designed to express high levels of V5-tagged b 7 This plasmid contains the genes conferring ampicillin resistance and the pUC18 origin of replication. Plasmid pTAM35 includes the chloramphenicol resistance gene, the pACYC184 origin of replication and expresses histidine-tagged b +7 In similar constructions, plasmids pTAM37 (b wt-his Cm r ) and pTAM46 (b wt-V5 Apr), or pTAM35 (b +7-his Cm r ) and pTAM46 (b wt-V5 Apr) were developed for coexpression experiments. B) Line diagram of the different b subunits that were coexpressed in the same cell. Interactions between b wt-his / b wt-V5 b +7-his / b wt-V5 and b +7-his /b 7-V5 were investigated. PAGE 120 99 generated by site-directed mutagenesis. Again, the epitope tags were needed to facilitate enzyme purification and subunit detection on a Western blot, respectively. In order to express two different b subunits in the same cell, we first used the two-plasmid expression system (Figure 2-8A). A total of four plasmids were constructed expressing b wt-his (Cm r ), b +7-his (Cm r ), b wt-V5 (Ap r ) or b 7-V5 (Ap r ) (Table I). The deletion removed the segment from Leu54-Ser60, and the insertion resulted in duplication of the same series of amino acids. Previous work had shown that F 1 F 0 ATP synthase complexes with b subunits shortened and lengthened by 7 amino acids were essentially wild-type (193, 194). However, it was possible that the epitope tags would affect enzyme assembly or function. This was particularly a concern at the C-terminus where small deletions at the extreme C-terminal end of the subunit had been shown to inhibit F 1 F 0 ATP synthase function (206, 288). Addition of the fourteen amino acid V5-epitope tag to the C-terminus of b might have impinged on enzyme assembly. The effects of the added epitope tags were studied by the ability of the plasmids to complement the E. coli strain KM2 (b) (218). Growth on succinate minimal medium was used as an initial qualitative gauge of enzyme activity in vivo since E. coli strains lacking F 1 F 0 ATP synthase cannot derive energy from nonfermentable carbon sources. In each case, the strains expressing the epitope-tagged b subunits grew comparably to the wild type strain (Table I). Hence, even though deletion of as few as two amino acids affected the ability of the b dimer to interact with the F 1 subunit (41,42), addition of fourteen amino acids to the C-terminus did not interfere with the interaction of the b and subunits. PAGE 121 100 Effects of epitope tags Since F 1 has little affinity for the membrane in the absence of intact F 0 total membrane associated F 1 -ATP hydrolysis activity was used as a test of F 1 F 0 ATP synthase complex assembly. Under conditions of high pH, F 1 can be released from the influence of F 0 (146), so the amount of ATPase activity in the solution was used as a measure of the amount of intact enzyme complex located in the membrane vesicles. The V5-epitope tag did not have a significant affect on enzyme assembly. Membrane preparations with a b wt-V5 or b 7-V5 incorporated into the F 1 F 0 ATP synthase complex had specific activities of about 95% and 82% of the wild type strain, respectively (Table I). The latter value was comparable to the effect of the seven amino acid deletion in the absence of the epitope (194). A slightly greater decrease in specific activity was reproducibly observed in membrane vesicles isolated from cells when a histidine epitope tag was incorporated onto the b wt or b +7 subunit, about 90% and 79% of wild type, respectively. Nevertheless, abundant activity was retained, suggesting very limited effects resulting from addition of the epitope tags. Furthermore, comparable activities were observed in samples when the histidine-tagged and V5-tagged b subunits were coexpressed. F 1 F 0 ATP synthase-mediated ATP-driven proton pumping activity in membrane vesicles prepared from the epitope-tagged mutants was used as an indication of coupled activity. Acidification of inverted membrane vesicles was examined by fluorescence of ACMA (Figure 2-9). The level of NADH-driven fluorescence quenching was monitored for all membrane preparations to demonstrate that the vesicles were intact and closed. The levels of NADH-driven fluorescence quenching were strong and directly comparable in every case (Figure 2-10). Membranes isolated from cells with a V5 epitope tag PAGE 122 101 Figure 2-9. ATP-driven energization of membrane vesicles prepared from uncF(b) gene mutants. Cell membrane vesicles were prepared by differential centrifugation (see Materials and Methods). Membrane protein (200 g) was suspended in 3 ml of assay buffer (50 mM MOPS, 10 mM MgCl 2 pH 7.3). The fluorescent dye ACMA was added to a final concentration of 1 M, and fluorescence was recorded with excitation at 410 nm and emission at 490 nm. ATP was added as indicated to a final concentration of 1mM. The samples for each trace have been labeled according to the amino acid insertion or deletion and the epitope tag, so the strains used as the sources of the samples were as follows: b wt KM2/pKAM14; b wt-V5 KM2/pTAM46; b 7-V5 KM2/pTAM47; b wt-his KM2/pTAM37; b +7-his KM2/pTAM35; b wt-his / b wt-V5 KM2/pTAM37/pTAM46; b +7-his /b wt-V5 KM2/pTAM35/pTAM46; b +7his/b 7-V5 KM2/ pTAM35/pTAM47. PAGE 123 102 Figure 2-10. NADH-driven acidification of membrane vesicles prepared from uncF(b) mutants. Membrane protein (250 g) was suspended in 3 ml of assay buffer (50 mM MOPS, 10 mM MgCl 2 pH 7.3). The fluorescent dye ACMA was added to a final concentration of 1 M, and fluorescence was recorded with excitation at 410 nm and emission at 490 nm. 5 mL NADH (0.1 mM) was added and the emission was continually recorded. Over time, membrane vesicle acidification peaked and the fluorescence quenching reached a maximum. As the fluorescence began to rise, 5 mL of 0.3 mM KCN was added and the emission was recorded for a total time of about 10-15 minutes. The samples for each trace have been labeled according to the amino acid insertion or deletion and the epitope tag, so the strains used as the sources of the samples were as follows: b wt KM2/pKAM14; b wt-V5 KM2/pTAM46; b 7-V5 KM2/pTAM47; b wt-his KM2/pTAM37; b +7-his KM2/pTAM35; b wt-his / b wt-V5 KM2/pTAM37/pTAM46; b +7-his /b wt-V5 KM2/pTAM35/pTAM46; b +7his/b 7-V5 KM2/ pTAM35/pTAM47. PAGE 124 103 incorporated onto the b wt or b 7 subunit, KM2/pTAM46 or KM2/pTAM47, respectively, displayed a very slight reduction in coupled activity (Figure 2-9). The reduction in coupled activity correlated very well with the minor reduction in F 1 -ATP hydrolysis activity. A larger reduction in coupled activity, of about 20-25%, was observed in membrane vesicles isolated from cells when a histidine epitope tag was incorporated onto the b wt or b +7 subunit, KM2/pTAM37 or KM2/pTAM35, respectively. The decrease in coupled activity also paralleled with the reduction seen in F 1 -ATP hydrolysis activity. Moreover, the coupled activities observed in membranes isolated from cells coexpressing histidine-tagged and V5-tagged b subunits were, as expected, intermediary between the V5-tagged species and the histidine-tagged species. The larger reduction seen in the cells expressing a histidine epitope tag arose in part from the plasmid vector. The moderate-copy plasmid vector pACYC184 was used to express the histidine-tagged b subunits. When histidine-tagged b subunits were expressed from the pUC18 plasmid vector, both coupled activity and ATP hydrolysis activity were similar to untagged b subunits (data not shown). Hence the reductions in activity observed in membranes from the histidine-tagged strains reflected reduced amounts of intact F 1 F 0 ATP synthase incorporated into the membranes. For the purposes of this study, the important parameters were the presence of an intact and functional F 1 F 0 ATP synthase enzyme complex and the ability to distinguish the two different tagged b subunits via Western blot. Detections of heterodimers Dimerization of the b subunits is thought to occur early during enzyme assembly, making the requirement for immediate dimerization of b via co-translation of a single b subunit transcript, feasible. Therefore, it was necessary to establish whether a two PAGE 125 104 plasmid expression system could be utilized in order to direct production of two different b subunits that will dimerize to form an intact F 1 F 0 enzyme complex. Two wild-type length b subunits, expressed from plasmids pTAM37 (b wt-his Cm r ) and pTAM46 (b wt-V5 Ap r ), were expressed in the same cell and studied for their ability to dimerize. Under the appropriate conditions, the histidine-tagged b subunits allowed for the easy purification of enzyme from a crude membrane preparation using a nickel affinity resin. Via immunoblot analysis, the presence or absence of the V5 epitope tag provided a means to determine whether the two different tagged b subunits are interacting to form a heterodimer. Immunoblot analysis of crude membrane preparations using anti-b antibodies showed the presence of the b subunit in all strains complemented with an epitope-tagged b subunit (Figure 2-11A, Lanes 2-4). As expected, only b subunits with a histidine tag were retained by Ni-resin purification (Figure 2-11A, Lanes 5-12). Importantly, F 1 F 0 complexes with only the V5-tagged b subunits were completely removed from the resin during the wash steps (Figure 2-11A, Lanes 7-8). To detect a heterodimer formation consisting of wild-type length b subunits, b wt-his and b wt-V5 expressed from two plasmids, immunoblot analysis using the anti-V5 antibody was performed (Figure 2-11B). When the two plasmids were coexpressed in the same cell the anti-V5 antibody detected the presence of b wt-V5 after Ni-purification, indicating that the expression system was successful and the tags do not hinder hetero-dimerization (Figure 2-11B, lanes 11-12). To confirm that the F 1 F 0 ATP synthase complex did not rupture during the solubilization and purification procedures, the tagged b subunits were either mock treated or treated with the homobifunctional crosslinker, BS 3 after the addition of detergent. As PAGE 126 105 expected, crosslinked b subunits were readily observed only in samples that had undergone BS 3 treatment and survived Ni-resin purification (Figure 2-11A, Lanes 6, 10, 12). BS 3 chemical crosslinking had also demonstrated that the b +7-his subunit dimer was stable during solubilization with tegamineoxide WS-35, taurodeoxycholate and lauryldimethylamine oxide, but not with sodium dodecyl sulfate (SDS) (Figure 2-5). As an added precaution to ensure there was no nonspecific aggregation of b subunits after membrane solubilization or during Ni-resin purification, two independent membrane preparations were mixed together (Figure 2-11, Lanes 9-10). Membrane vesicles derived from strains KM2/pTAM37 (b wt-his ) and KM2/pTAM46 (b wt-V5 ) were mixed, the membranes were solubilized with 0.2% tegamineoxide and allowed to incubate at room temperature for 30 minutes, and the treated with BS 3 before performing Ni-resin purification. Immunoblot analysis with anti-b antibodies showed normal levels of histidine-tagged b subunit upon Ni-resin purification (Figure 2-11A, Lanes 9-10). No V5-epitope was present after Ni-resin purification indicating nonspecific aggregation was not a factor in the observed results (Figure 2-11B, Lanes 9-10). Therefore, any observed interactions of b subunits must have been due to heterodimers integrating within an intact F 1 F 0 ATP synthase complex in a cell expressing both b subunits. Importantly, immunoblotting using the anti-V5 antibody clearly detected V5 epitope tagged b subunits in Ni-resin purified samples when the two were coexpressed (Figure 2-11B, Lanes 11-12). PAGE 127 106 Figure 2-11. Ni-resin purification of F 1 F 0 ATP synthase treated with the cross-linker BS 3 Crude membrane preparations of KM2 expressing the normal length b subunits with epitope tags were mock treated (-) or treated (+) with 1mM bis(3-sulfo-N-hydroxysuccinimide ester) (BS 3 ) for 20 minutes at room temperature. The cross-linking reaction was stopped by addition of 100 mM ethanolamine HCl, pH 7.5 for 10 minutes. The products were then solubilized with 0.2% tegamineoxide WS-35 and subjected to Ni-resin purification before separation on a 15% polyacrylamide Tris-glycine SDS gel and subsequent Western blot analysis. A) Proteins were transferred to a nitrocellulose membrane and probed using a polyclonal anti-b antibody. B) Proteins were transferred to a nitrocellulose membrane and probed using an anti-V5 mouse monoclonal antibody. PAGE 128 107 Formation of mixed length b subunits in F 1 F 0 ATP synthase The two plasmid expression system was used to study whether unequal length b subunits could form a dimer or, alternatively, whether the b subunits dimerize and incorporate into enzyme complexes in a segregated manner. In order to determine if there is a direct protein-protein interaction between the different length b subunits, we investigated the ability of b +7-his to retain an interaction with a b wt-V5 or a b 7-V5 subunit following Ni-purification (Figure 2-12). All membranes prepared from strains expressing a b subunit were readily detectable and distinguishable by size on an immunoblot (Figure 2-12, Lanes 1-7). Only b subunits with a histidine tag were retained by Ni-resin purification (Figure 2-12A, Lanes 8-12). Immunoblot analysis using an anti-V5 antibody was performed on the membrane preparations and Ni-purified products (Figure 2-12B). The V5-epitope tag was detected only in membrane vesicles derived from KM2 strains expressing either the V5-tagged b subunit or the coexpressed V5-tagged and his-tagged b subunits (Figure 2-12B, Lanes 1-7). As expected, upon Ni-resin purification, the V5 epitope was not detected in samples containing only histidine-tagged b subunit (Figure 2-12B, Lane 8). Likewise, samples containing only the V5-tagged b subunits were not detected upon Ni-resin purification, signifying that they were efficiently removed from the resin during wash steps (Figure 2-12B, Lanes 9-10). Finally, we investigated the ability of b +7-his to dimerize to form intact F 1 F 0 ATP synthase complexes with b wt-V5 or b 7-V5 The b wt-V5 was detected with anti-V5 antibodies and was readily observed to dimerize with the b +7-his indicating a b wt-his /b wt-V5 interaction (Figure 2-12B, Lane 11). To a much lesser extent, the b 7-V5 subunit was also distinguishable with anti-V5 antibodies (Figure 2-12B, Lane 12). The data indicated that the F 1 F 0 ATP synthase enzyme PAGE 129 108 complex could tolerate b subunit heterodimers with a size difference of at least 14 amino acids. Detection of b subunit heterodimers led directly to two additional questions. How many b subunit heterodimer F 1 F 0 ATP synthase complexes were formed? Were they active? We had not yet purified a heterodimeric complex to homogeneity so the direct assay has not been performed. However, an indication of activity could be assessed based on the percentage of homodimeric and heterodimeric F 1 F 0 complexes. Therefore, Figure 2-12. Ni-resin purification of F 1 F 0 ATP synthase expressing unequal length b subunits. IPTG-induced cells were lysed in a French pressure cell, and membrane vesicles were isolated by differential ultra-centrifugation. The crude membrane preparation was solubilized with 0.2% tegamineoxide WS-35 and subjected to Ni-resin purification before separation on a 15% polyacrylamide Tris-glycine SDS gel and subsequent Western blot analysis. A) Proteins were transferred to a nitrocellulose membrane and probed using a polyclonal anti-b antibody. B) Proteins were transferred to a nitrocellulose membrane and probed using an anti-V5 mouse monoclonal antibody. PAGE 130 109 membranes were prepared from strains KM2/pTAM37/pTAM46 (b wt-his /b wt-V5 ), KM2/pTAM35/pTAM46 (b +7-his /b wt-V5 ) and KM2/pTAM35/pTAM47 (b +7-his /b 7-V5 ). All membranes prepared from strains expressing an epitope-tagged b subunit were readily detectable and distinguishable by size on an immunoblot of a 15 cm 15% SDS-PAGE gel (Figure 2-13A). Densitometry was used to determine the relative amounts of histidine-tagged and V5-tagged b subunits in samples coexpressing the different subunits (Figure 2-13B). Relative amounts of the histidineand V5-tagged b subunits were determined from crude membrane preparations that contained the different coexpressed b subunits. In each case, expression of the various length b his and b V5 subunits led to nearly equal incorporation into F 1 F 0 ATP synthase complexes (Figure 2-13B). Relative amounts of the b his and b V5 subunits were also determined from Ni-resin purified samples coexpressing both b subunits. Upon Ni-resin purification, all of the b V5 subunit seen on a Western blot must necessarily be incorporated into the enzyme complex as a heterodimer along with a b his subunit. Hence, when the two different wild-type length b subunits were coexpressed, there was at least 15% heterodimer formation (Figure 2-13B). It is possible that further manipulation of subunit expression and optimization of the purification procedure might result in a higher percentage of heterodimers found in the membranes. Somewhat lower percentages of heterodimer formation were found when b +7-his was coexpressed with b wt-V5 or b 7-V5 (Figure 5B). Based on the percentage of heterodimer formation and the activities observed when the different b subunits were coexpressed in the same cell (Figure 2-13, Table 2-1), it is likely that the heterodimeric F 1 F 0 ATP synthase species are functionally active. PAGE 131 110 Figure 2-13. Quantitation of b subunit heterodimeric F 1 F 0 Membranes were prepared from IPTG-induced cells by differential centrifugation. A total of 15 mg membrane protein was solubilized, Ni-resin purified (1 ml packed resin volume), concentrated with a Microcon YM-10 and separated on a 15 cm 15% polyacrylamide Tris SDS gel to allow maximal separation. A) Proteins were transferred to a nitrocellulose membrane and probed using a polyclonal anti-b antibody. This Western blot was deliberately overexposed to allow easy visualization of the V5-tagged b subunits in the Ni-resin purification lanes. B) Densitometric analysis of lower exposures was used to determine the relative amounts of histidine-tagged and V5-tagged b subunits in samples coexpressing the different subunits. Reletive amounts of b subunits were determined for crude membrane preparations and Ni-resin purified samples coexpressing both b subunits. The percent of heterodimer formation was calculated based on three independent membrane preparations as well as three separate exposure times. PAGE 132 111 Discussion In the present chapter, we have developed an expression system that facilitates production, purification and detection of b subunits of unequal lengths within E. coli. The experiments involved an epitope tag system that allowed us to determine if the different b subunits segregated into homodimers, or alternatively, if a heterodimer of long and short b subunits can be incorporated into an F 1 F 0 ATP synthase complex. The histidine and V5 epitope tags introduced into the b subunits did not appreciably affect enzyme assembly or function. Expression of two different wild-type length b subunits led to three distinct F 1 F 0 ATP synthase complexes in the same cell; 1) a homodimer of histidine-tagged b subunits, 2) a homodimer of V5-tagged b subunits and 3) a heterodimer consisting of a histidine-tagged b and a V5-tagged b subunit. More importantly, three different F 1 F 0 ATP synthase complexes were present even when the b subunits were not of identical length. We observed dimerization of b subunits between b +7-his and both b wt-V5 and b 7-V5 This demonstrates that b subunits that differ in length by at least 14 amino acids can be incorporated into an enzyme complex. Given that the tether domain is likely an helix, the difference in length between the b subunits would be approximately 21 Dimerization could occur in two ways. First, assuming that the transmembrane domains were in parallel, the hydrophilic domains could be out of register. Alternatively, we favor a conformation in which both the transmembrane domains and the dimerization domains as defined by Dunn and coworkers (13) exist in parallel. This would require that a section of the tether domain in the longer b subunit be out of contact with the shorter b subunit (Figure 2-14). It is likely that a parallel alignment of the dimerization domain is required for enzyme assembly. PAGE 133 112 Figure 2-14. Interactions of b subunits of unequal lengths. An epitope-tag system novel to F 1 F 0 ATP synthase studies was established in order to determine whether b subunits of unequal length could interact to form the dimer in an intact and functional enzyme complex. The b 7 subunit was expressed with a V5-epitope tag at its C-terminus (represented by the s shaped red line) and b +7 was expressed with a histidine tag at its N-terminus (represented by the curved oragnge line). The lightning bolt indicates a deletion and the orangte patch represents an insertion of amino acids. The histidine tag allowed for purification on a Nickel affinity column and the V5-tag allowed us to detect the presence of the b 7 subunit after Ni-purification using Western blot analysis. Expression of the two different epitope-tagged b subunits in the same cell leads to three distinct interactions within an F 1 F 0 ATP synthase complex (1) a homodimer of b 7-V5 (2) a homodimer of b +7-his and (3) a heterodimer consisting of both the shortened and lengthened b subunit. PAGE 134 113 Recent electron microscopy and NMR studies have revealed a distinctive 20 bend in the b dimer within the tether domain (27, 136, 196). The research presented here suggests the possibility of straightening or further bending of the two b subunits within the peripheral stalk and lends support to the concept of a flexible peripheral stalk. This raises the question, why should the tether domain be so flexible as to allow insertions, deletions and dimerization of b subunits of unequal lengths? If one views the peripheral stalk to be a rope-like structure linking F 1 to F 0 then its position holding F 1 against the rotation of the central stalk would not be expected to be the same for counterclockwise and clockwise rotation. Flexibility of the tether domain might facilitate reorienting the peripheral stalk to act as a stator for rotation in either direction during ATP synthesis and ATP hydrolysis. The ability to generate and purify F 1 F 0 complexes with two genetically different b subunits provides a potentially useful experimental tool. It is now feasible to specifically label a single b subunit within a purified complex. This will facilitate biochemical modification experiments and the use of physical methods. PAGE 135 CHAPTER 3 GENETIC COMPLEMENTATION BETWEEN MUTANT b SUBUNITS IN F 1 F 0 ATP SYNTHASE Introduction In the F 1 F 0 ATP synthase enzyme complex, the peripheral stalk consists of a parallel dimer of identical b subunits. Dimerization of the b subunits is thought to be an early event necessary for enzyme assembly and function (199). The two b subunits exist in an extended -helical conformation, spanning from the periplasmic side of the membrane to near the top of F 1 However, due to the asymmetric nature of the enzyme complex the two b subunits cannot participate in identical protein-protein interactions with the other subunits. In the amino-terminal membrane spanning region, the peripheral stalk contacts a single a subunit. Similarly, at the carboxyl end of the peripheral stalk, the two b subunits interact with a single subunit. The b dimer has been studied by a variety of traditional biochemical approaches such as CD spectroscopy, cross-linking, and sedimentation experiments (30, 166, 192, 196, 197, 203, 283). Limited structural information is available from studies of polypeptides modeling functionally defined domains of the b subunit. Dmitriev et al. studied the amino-terminal membrane spanning domain by NMR using a 34 amino acid polypeptide, revealing an -helix with a 20 bend at pro-27 and pro-28 (136). A systematic mutagenesis approach supported the model described in the NMR paper suggesting that the extreme amino-termini of the two b subunits were in close contact and then the subunits flare apart as they traverse the membrane (136, 202). X-ray crystallography of a model polypeptide reflecting residues 114 PAGE 136 115 62-122, corresponding to the dimerization domain, showed an extended, highly -helical structure (138). The structures of the tether domain contributing to the segment between the membrane surface and the bottom of F 1 and the F 1 binding domain in the carboxyl terminal region have yet to be determined. Previously we showed that b subunits with deletions and insertions in the tether region of up to eleven and fourteen amino acids, respectively, formed functional F 1 F 0 complexes (193, 194). These observations suggested that flexibility is an inherent characteristic of the peripheral stalk. This apparent flexibility also extended to the dimerization of the b subunits. When two b subunits with unequal length tether domains were expressed together, F 1 F 0 ATP synthase complexes containing heterodimeric peripheral stalks were assembled (195). F 1 F 0 complexes were able to tolerate the incorporation of two different b subunits with a size difference of at least fourteen amino acids. Although activity evidence suggested the heterodimeric F 1 F 0 complexes were likely functional, the earlier study was not design to rigorously demonstrate that these were active. A major problem that plagued all previous mutagenesis studies of the b subunit was that mutations constructed in the uncF(b) gene affected both subunits of the b homodimer. One missense mutation led to two amino acid replacements. Our ability to express two different b subunits in the same cell and detect b subunit heterodimer formation provided an approach to study the individual functional roles of the b subunits. In the present study, three different b subunit mutations were studied. Mutation of an evolutionarily conserved arginine, b arg36 to an isoleucine or glutamate has previously been shown to result in an intact, completely defective F 1 F 0 ATP synthase complex PAGE 137 116 (215). Second, deletion of the last four amino acids at the C-terminus has been shown to affect the ability of the b dimer to form a stable interaction with the subunit F 1 resulting in a major defect in F 1 F 0 ATP synthase assembly (205, 206). Thirdly, insertions or deletions constructed in a cytoplasmic region of the b dimer that contains a stretch of hydrophobic amino acids, b !24-130 (VAILAVA) has been shown to completely affect the ability of the b subunit to form a stable dimer and insert into the membrane. Here, we demonstrate functional activity for F 1 F 0 ATP synthase complexes containing a heterodimeric peripheral stalk, with major defects occurring in the different domains of each b subunit. Materials and Methods A thorough account of many of the procedures used in Chapter 4 can be found in previous chapters. Many of the techniques, including recombinant DNA techniques, site directed mutagenesis, western blotting, as well as assays of protein concentration and F 1 F 0 ATP synthase activity have been described in detail in Chapter 2. A detailed description of purification of F 1 F 0 ATP synthases containing b subunit heterodimers to homogeneity can be found in Chapter 3. Materials Molecular biology enzymes and mutagenic oligonucleotides were obtained from Invitrogen (Carlsbad, CA), Life Technologies, Inc. (Grand Island, NY), New England Biolabs (Beverly, MA) and Stratagene (La Jolla, CA). Reagents were obtained from Sigma (St. Louis, MO), BioRad Laboratories (Hercules, CA) and Fisher Scientific (Pittsburgh, PA). Plasmid purification kits were acquired from Qiagen Inc. (Valencia, CA). The anti-rabbit immunoglobulin horseradish peroxidase-linked whole antibody (from donkey), anti-mouse immunoglobulin horseradish peroxidase-linked whole PAGE 138 117 antibody (from sheep), Hybond ECL Nitrocellulose membrane, electrochemiluminescence Western blotting reagents and high performance chemiluminescence film were purchased from Amersham Biosciences (Piscataway, NJ). Polyclonal antibodies against SDS-denatured b subunit (284, 285) were generously provided by Dr. Karlheinz Altendorf (Universitt Osnabrck, Osnabrck, Germany. Monoclonal antibodies against the epitope found in the P and V proteins of the paramyxovirus, SV5 (V5 epitope tag) were purchased from Invitrogen. The Seize Primary Immunoprecipitation Kit was purchased from Pierce Biotechnology (Rockford, IL). Strains and Media The wild type b subunit expression plasmid, pKAM14, and plasmids used to express arg36 mutatations b subunits have been described previously (203, 215). The plasmids encoding the uncF(b) gene were used to complement E. coli strain KM2 (b) carrying a chromosomal deletion of the gene (218). All strains were streaked onto plates containing minimal A media supplemented with succinate (0.2% w/v), to determine enzyme viability. Cells harvested for membrane preparation were grown in Luria Broth supplemented with glucose (0.2% w/v) (LBG). Isopropyl-1-thio--D-galactopyranoside (IPTG)(40 g/ml), ampicillin (Ap) (100g/ml), and chloramphenicol (Cm) (25 g/ml) were added to media as needed. All cultures were incubated at 37C for the appropriate duration. Recombinant DNA Techniques Plasmid DNA was purified with the Qiagen Mini-Prep and Maxi-Prep kits. Restriction endonuclease digestions, ligations, and transformations were performed PAGE 139 118 according to the recommendations of the manufacturers (New England Biolabs, Stratagene and Life Technologies, Inc.). Site-directed mutagenesis was performed either by means of a Stratagene Quikchange kit or by ligation-mediated mutagenesis. DNA fragments were separated in 0.8 % agarose gel by electrophoresis and purified using a Qiagen, Inc. QIAquick Gel Extraction kit. Plasmid sequences were determined by automated sequencing in the core facility of the University of Florida ICBR. Mutagenesis and Strain Construction Plasmid pKAM14 (b, Ap r ) (203) was used to construct b subunits with a deletion of the last four amino acids. Plasmids pKAM14 (b, Ap r ) (203), pTLC11 (b arg36ile Ap r ), or pTLC15 (b arg36glu Ap r ) (215) were used to construct the epitope tagged b subunits. Epitope tags were inserted into each of the plasmids using the Stratagene Quikchange kit. A four amino acid carboxyl-terminal truncation was accomplished by deletion of the final four codons of the uncF(b) gene to express b 153end (Appendix A) (Figure 3-1A). The restriction endonuclease recognition sequence for HindIII was constructed near the deleted sequence for an initial detection of the truncation. A histidine epitope tag was inserted at the N-terminus by mutagenesis between the first and second codons of the uncF(b) gene to express b his b 153end-his or b +124-130-his (Figure 3-1B). All of the recombinant histidine-tagged b subunit plasmids were then digested with PstI and NdeI and subsequently ligated into a plasmid conferring the chloramphenicol resistance gene and the pACYC184 origin of replication (Table 3-1). A V5 epitope tag was added to the C-terminus by site-directed mutagenesis before the termination codon of the uncF(b) gene to express b V5 b arg36ile-V5 or b arg36glu-V5 (Figure 3-1B). The recombinant V5-tagged b subunit plasmids included the ampicillin resistance gene and the pUC18 origin PAGE 140 119 of replication. Unique restriction enzyme sites SphI and NdeI were constructed near the histidine and V5 epitope tag sequence, respectively, for an initial detection of the insertions, and then the nucleotide sequence was subsequently confirmed by automated sequencing in the ICBR core facility. Throughout the paper, the mutation and the epitope Figure 3-1. Oligonucleotides for epitope tags and C-terminal truncation of uncF(b). Shown are the sense strands of the mutagenic primer pairs. Mutagenesis was accomplished as described in the Materials and Methods. Green and red codons specify translation start and stop sites, respectively. The amino acids encoded in the sequences are labeled above the codons. A) Mutagenic primer encoding a four amino acid deletion (codons shown in purple) along with a silent mutations that introduced a new endonuclease recognition sequence, HindIII (underlined). B) Oligonucleotides designed to insert a histidine or V5-epitope tag (bold blue). The SphI and SacI restriction sites (underlined) were added along with the histidine and V5 epitope tag to facilitate screening. PAGE 141 120 tag are indicated along with the plasmid name for clarity, for example plasmid pTAM51 (b 153end-his ). Each plasmid and the control plasmids pKAM14 (b) and pBR322 were expressed in the E. coli cell line KM2 (b) for study, so that the only b subunits in the cells were the product of the plasmid genes. The two plasmid expression system allowed expression of various combinations of histidine tagged and V5-tagged b subunits in the same cell (Figures 2C, 4C, 6C, 8C). Appropriate antibiotics were added to the growth medium, and in the case of the coexpressed plasmids, both ampicillin and chloramphenicol were added to select for cells expressing both plasmids. Preparative Procedures Inverted membrane vesicles from KM2 (b) strains expressing the desired b subunits were prepared essentially as described previously (194). Protein concentrations were determined by the bicinchoninic acid (BCA) assay (289). Ni-resin purification was achieved using the High Capacity Nickel Chelate Affinity Matrix (Ni-CAM) purchased from Sigma. A total of 5 mg of membrane protein was brought up to 1 mL with final concentrations of 0.2% tegamineoxide WS-35, 0.15 M NaCl, and 1 mM imidazole. The purification procedure was accomplished using the batch method as described by the manufacturer. Immunoblot Analysis Proteins were loaded on a 15% tris-glycine SDS gel and transferred onto nitrocellulose by electroblot. The b subunit antibody incubation was performed essentially as described previously (195), using a 1:25,000 dilution of the anti-b subunit antibodies. Secondary antibody incubation was performed with horseradish peroxidase-linked donkey anti-rabbit antibody (1:50,000), and the antibody was detected by PAGE 142 121 enhanced chemiluminescence. The V5-subunit antibody incubation was performed as described by the manufacturer followed by a secondary antibody incubation with horseradish peroxidase-linked sheep anti-mouse antibody (1:10,000). Signals were visualized on high performance chemiluminescence film using a Kodak X-Omat. Assays of F 1 F 0 ATP Synthase Activity Growth on a minimal succinate medium was used as an initial, in vivo, assay for enzyme viability. ATP hydrolysis activity was assayed by the acid molybdate method (146). Membranes were assayed in buffer (50 mM Tris-HCl, 1 mM MgCl2, pH 9.1) to determine the linearity with respect to time and enzyme concentration. Membrane energization was detected by the fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine (ACMA) (271). Results Construction and Growth Characteristics of Mutants To investigate the function of individual b subunits in a F 1 F 0 ATP synthase enzyme complex, plasmids expressing defective b subunits with either a histidine or a V5 epitope-tag were generated by site-directed mutagenesis. A total of five plasmids were constructed expressing the b wt-his b 153end-his b wt-V5 b arg36ile-V5 or b arg36glu-V5 subunits (Table 3-1). The epitope tags were needed to facilitate enzyme purification and subunit detection on a Western blot, respectively. In order to express two different b subunits in the same cell, we used the two-plasmid expression system described previously in Chapter 2(195). Enzyme complexes incorporated with a histidine or V5 epitope-tagged b subunit homodimer have been studied previously and were shown to result in a functional enzyme complex (195). E. coli strain KM2 (b) was used as the host because the uncF(b) gene has been deleted, eliminating any background b subunit. PAGE 143 122 Table 3-1. Aerobic growth properties and membrane-associated ATP hydrolysis activity of mutants expressing epitope tagged uncF(b) genes Strains Description Growth 1 Specific activity 2,3 b, Ap r KM2/pKAM14 (+) +++ 1.18 0.05 wt b, Ap r KM2/pBR322 (-) 0.24 0.03 b, Cm r KM2/pTAM37 +++ 1.08 0.08 wt-his KM2/pTAM51 b, Cm 153end-his 0.22 0.05 r b, Ap r KM2/pTAM46 +++ 1.22 0.04 wt-V5 KM2/pTAM53 b, Ap arg36ile-V5 r 0.80 0.02 b arg36glu-V5 Ap r KM2/pTAM54 0.75 0.08 b, Cm KM2/pTAM38 +124-130-his 0.21 0.04 r b + b KM2/pTAM37/pTAM46 wt-V5 +++ 1.16 0.03 wt-his KM2/pTAM51/pTAM46 b + b 153end-his wt-V5 +++ 1.00 0.05 KM2/pTAM37/pTAM53 +++ 1.12 0.04 b wt-his + b arg36ile-V5 KM2/pTAM37/pTAM54 b + b wt-his +++ 1.10 0.05 arg36glu-V5 KM2/pTAM38/pTAM54 b +124-130-his + b wt-V5 +++ 1.20 0.03 KM2/pTAM51/pTAM53 b 153end-his + b arg36ile-V5 + 0.51 0.02 KM2/pTAM51/pTAM54 b 153end-his + b arg36glu-V5 0.37 0.07 1 E. coli strains were grown aerobically on succinate minimal medium. Colony size was scored after 72-hr incubation at 37C as: +++, 1.0 mm; ++, 0.3-0.5 mm; +, ~0.1 mm; -, no growth. 2 ATPase activities were measured as described under Materials and Methods. Units of specific activity = mol of PO 4 released per mg of protein/min S.D. Units were calculated from the slope of the line based on three measurements with incubations for 12 minutes. 3 Previously reported ATPase activities resulted from an abbreviated membrane preparation protocol. Activities reported here resulted from an additional wash step to remove nonspecifically bound ATPases. Previous analyses of FF ATP synthase complexes incorporated with the mutant b, b, b or b subunits found the mutants to be completely defective (205, 206, 215). Therefore, our ability to form FF complexes containing b heterodimers offered an approach to study activity in a complex with only a single functional b subunit protein. First, the effects of the b, b, b and b mutations were studied for dimerization with a wild type epitope tagged b subunit in order to complement the E. coli strain KM2 (b) (218). Growth on succinate minimal medium was used as an initial qualitative gauge of enzyme activity in vivo since E. coli strains lacking FF ATP synthase cannot derive energy from nonfermentable carbon sources (Table 3-1). As expected, when any one of the mutants was expressed 1 0 arg36ile arg36glu 153end +124-130-his 1 0 arg36ile-V5 arg36glu-V5 153end-his +124-130-his 1 0 PAGE 144 123 alone in the cells no growth was detected. In each case, the strains coexpressing the mutant epitope-tagged b subunits with a wild-type epitope-tagged b subunit grew comparably to the wild type strain. Heterodimer Formation of b arg36 Defective Subunits with b wt Although two unequal length b subunits formed a heterodimer in an intact F 1 F 0 ATP synthase complex (195), it was possible that a single amino acid substitution could affect dimerization, preventing assembly of a complex. To consider whether a V5 epitope-tagged b arg36 subunit, b arg36ile-V5 or b arg36glu-V5 could form a heterodimer with a wild type histidine epitope-tagged b subunit, b wt-his each pair of subunits were expressed in strains KM2/pTAM37/pTAM53 and KM2/pTAM37/pTAM54 (b) (Figure 3-2A and B). The b subunits were detected in membranes prepared from strains expressing a b subunit by immunoblot analysis (Figure 3-2A, Lanes 1-9). However, only b subunits in complexes with at least one histidine tag were retained by Ni-resin purification (Figure 3-2A, lanes 10-13). Immunoblot analysis using an anti-V5 antibody was performed on the membrane preparations and Ni-purified products (Figure 3-2B). The V5-epitope tag was detected only in membrane vesicles derived from KM2 strains expressing either the V5-tagged b subunit or the coexpressed V5-tagged and his-tagged b subunits (Figure 3-2B, Lanes 1-9). When only V5-tagged b subunits were expressed alone in a cell, no F 1 F 0 complexes were recovered from the Ni-resin purification (Figure 3-2B, Lane 10). Finally, we investigated the ability of b wt-his to dimerize to form intact F 1 F 0 ATP synthase complexes with b arg36ile-V5 or b arg36glu-V5 Both b arg36ile-V5 and b arg36glu-V5 were detected with anti-V5 antibodies after Ni-resin purification (Figure 3-2B, Lanes 12-13). The only mechanism for recovery of defective V5-tagged b subunits was through PAGE 145 124 Figure 3-2. Ni-resin purification of F 1 F 0 ATP synthase incorporated with b arg36 subunit mutations. Cells expressing recombinant b subunits were lysed in a French pressure cell, and membrane vesicles were isolated by differential ultracentrifugation. The crude membrane preparation was solubilized with 0.2% tegamineoxide WS-35 and subjected to Ni-resin purification before separation on a 15% polyacrylamide Tris-glycine SDS gel for subsequent anti-b, or on a mini BioRad 15% Ready Gel for anti-V5 Western blot analysis (see Materials and Methods). A) Proteins were transferred to a nitrocellulose membrane and probed using a polyclonal anti-b antibody. B) Proteins were transferred to a nitrocellulose membrane and probed using an anti-V5 mouse monoclonal antibody. C) Line diagram of the different b dimers found in the cell. Green and red lines represent the histidine and V5 epitope tags, respectively. Orange represents the b subunit and the blue star represents the b arg36 mutation. PAGE 146 125 dimerization with b wt-his indicating formation of heterodimeric F 1 F 0 ATP synthase complexes. Therefore, coexpression of the two different b subunits in the same cell led to three distinct interactions within an intact F 1 F 0 ATP synthase complex (Figure 3-2C): 1) a homodimer of b wt-his 2) a homodimer of b arg36ile-V5 or b arg36glu-V5 and 3) a heterodimer consisting of both the defective and wild type b subunit. Since F 1 displays reduced affinity for the membrane in the absence of intact F 0 total membrane associated F 1 -ATP hydrolysis activity was used as a test of F 1 F 0 ATP synthase complex assembly. Under conditions of high pH, F 1 can be released from the influence of F 0 (146), so the amount of ATPase activity in the solution was used as a measure of the amount of intact enzyme complex located in the membrane vesicles. Previous data indicated minimal affects on specific activity due to the epitope tags (195). Confirming these observations, the V5-epitope tag did not have an apparent affect on enzyme assembly. Membrane preparations with a b wt-V5 incorporated into the F 1 F 0 ATP synthase complex had virtually the same specific activity of the wild type strain (Table 3-1). A small decrease in specific activity was reproducibly observed in membrane vesicles isolated from cells when a histidine epitope tag was incorporated onto the b wt about 89% of the wild type strain. Furthermore, comparable activities were observed in samples when the histidine-tagged and V5-tagged b wt subunits were coexpressed. Also verifying previous data (215), when b arg36ile-V5 or b arg36glu-V5 was expressed alone in strains KM2/pTAM53 and KM2/pTAM54, the membranes retained abundant activity, about 60% and 54%, of the wild type strain, respectively (Table 3-1), indicating considerable amounts of intact F 1 F 0 ATP synthase complexes found in the membranes. When b arg36ile-V5 or b arg36glu-V5 were coexpressed with b wt-his by construction of strains PAGE 147 126 KM2/pTAM37/pTAM53 or KM2/pTAM37/pTAM54 specific activities of about 94% and 91%, respectively, were observed indicating a quantity of intact F 1 F 0 ATP synthase complexes approaching the wild type strain levels. F 1 F 0 ATP synthase-mediated ATP-driven proton pumping activity in membrane vesicles prepared from the epitope-tagged mutants was used as an indication of coupled activity. Acidification of inverted membrane vesicles was examined by fluorescence of ACMA (Figure 3-3). Membranes isolated from cells with a V5 epitope tag incorporated onto the b wt subunit, KM2/pTAM46 (b wt-V5 ), reproducibly displayed a very small reduction in coupled activity, correlating very well with the F 1 -ATP hydrolysis activity. Consistent with previous observations, a larger reduction in coupled activity of about 20% was observed in membrane vesicles isolated from strain KM2/pTAM37 (b wt-his ). The coupled activities observed in membranes isolated from cells coexpressing histidine-tagged and V5-tagged b subunits were intermediary between the V5-tagged species and the histidine-tagged species. Strains expressing either b arg36ile-V5 or b arg36glu-V5 displayed no ATP-dependent proton pumping activity as expected. Significantly, when either one of the b arg-36 mutants was coexpressed with b wt-his KM2/pTAM37/pTAM53 (b wt-his /b arg36ile-V5 ) or KM2/pTAM37/pTAM54 (b wt-his /b arg36glu-V5 ), the coupled activity observed was essentially the same as strain KM2/pTAM37/pTAM46 (b wt-his /b wt-V5 ). When expressed alone, the strains with b arg-36-V5 mutants had no activity and the strains expressing b wt-his displayed less activity than the b wt-his /b arg-36-V5 mutant enzyme. As a consequence, it was most likely that the F 1 F 0 ATP synthase complexes with either the b wt-his /b arg36ile-V5 or b wt-his /b arg36glu-V5 peripheral stalks were active. To demonstrate that the PAGE 148 127 Figure 3-3. ATP-driven energization of membrane vesicles prepared from uncF(b) arg36 gene mutants. Membrane vesicles were prepared by differential centrifugation. Membrane protein (200 g) was suspended in 3 ml of assay buffer (50 mM MOPS, 10 mM MgCl 2 pH 7.3). The fluorescent dye ACMA was added to a final concentration of 1 M, and fluorescence was recorded with excitation at 410 nm and emission at 490 nm. ATP was added as indicated to a final concentration of 1mM. The samples for each trace have been labeled according to the b subunit mutation and the epitope tag, so the strains used as the sources of the samples were as follows: b, KM2/pBR322; b wt KM2/pKAM14; b wt-V5 KM2/pTAM46; b wt-his KM2/pTAM37; b arg36ile-V5 KM2/pTAM53; b arg36glu-V5 KM2/pTAM54; b wt-his /b wt-V5 KM2/pTAM37/pTAM46; b wt-his /b arg36ile-V5 KM2/pTAM37/pTAM53; b wt-his /b arg36glu-V5 KM2/ pTAM37/pTAM54. PAGE 149 128 membrane vesicles were intact and closed, the level of NADH-driven fluorescence quenching was monitored for all membrane preparations. The levels of NADH-driven fluorescence quenching were strong and directly comparable in every case (data not shown, for a representative figure, see Figure 2-10). Heterodimer formation of b 153end-his Complemented with b wt-V5 Deletion of the last four amino acids at the C-terminus has been shown to dramatically affect the ability of the b dimer to form a stable interaction with the subunit F 1 resulting in a major F 1 F 0 ATP synthase assembly defect (205, 206). Immunoblot analysis was performed in order to detect a heterodimer interaction between b 153end and b wt (Figure 3-4A and B). Coexpression of pTAM51 (b 153end-his ) and pTAM46 (b wt-V5 ) in the same cell resulted in the appearance of a weak signal representing b wt-V5 in Ni-resin purified material (Figure 3-4B, Lane 8). Although there was no attempt to be quantitative in this assay, the appearance of the band suggested relatively inefficient assembly of these heterodimers. Nevertheless, when the two different b subunits are expressed in the same cell, three distinct F 1 F 0 complexes were assembled (Figure 3-4C). These included a homodimer of b wt-V5 in an intact F 1 F 0 ATP synthase complex, a homodimer of b 153end-his in a partially assembled defective enzyme, and a heterodimeric F 1 F 0 ATP synthase consisting of both subunits. Apparently, the b wt-V5 subunit stabilized the b 153end-his subunit within an intact F 1 F 0 ATP synthase complex. Total membrane associated F 1 -ATP hydrolysis activity was used as a test of F 1 F 0 ATP synthase complex assembly (Table 3-1). As expected, membranes with enzyme complexes incorporated with b 153end-his displayed a specific activity similar to the negative control, indicating virtually no interaction with F 1 Coexpression of pTAM51 PAGE 150 129 Figure 3-4. Ni-resin purification of F 1 F 0 ATP synthase containing a b subunit carboxyl-terminal truncation. Membrane preparation and Ni-resin purification and Western blot analysis were accomplished as described in Materials and Methods. A) Proteins were transferred to a nitrocellulose membrane and probed using a polyclonal anti-b antibody or B) an anti-V5 mouse monoclonal antibody. C) line diagram of the different b dimers found in the cell. PAGE 151 130 Figure 3-5. ATP-driven energization of membrane vesicles incorporated with F 1 F 0 ATP synthase containing a b subunit carboxyl-terminal truncation. Membrane preparation and ATP-driven proton pumping was accomplished as described in Materials and Methods. The samples for each trace have been labeled according to the b subunit mutation and the epitope tag, so the strains used as the sources of the samples were as follows: b, KM2/pBR322; b wt KM2/pKAM14; b wt-V5 KM2/pTAM46; b wt-his KM2/pTAM37; b 153end-his KM2/pTAM51; b wt-his /b wt-V5 KM2/pTAM37/pTAM46; b wt-V5 /b 153end-his KM2/pTAM46/pTAM51. PAGE 152 131 (b 153end-his ) and pTAM46 (b wt-V5 ) resulted in a specific activity of about 81% of the wild type strain (Table 3-1). An indication of coupled activity was shown by F 1 F 0 ATP synthase-mediated ATP-driven proton pumping activity in membrane vesicles prepared from the epitope-tagged mutants (Figure 3-5). Membranes containing only the b 153end-his subunit exhibited no coupled activity as expected. Membranes isolated from cells coexpressing pTAM51 (b 153end-his ) and pTAM46 (b wt-V5 ) displayed a slight reduction in coupled activity, of about 20%, compared to membranes from cells coexpressing b wt-V5 and b wt-his It is feasible that the observed reductions in enzymatic activities were entirely due to formation of the mutant homodimeric species; this could not be directly demonstrated by these methods due to background activity from b wt-V5 homodimeric F 1 F 0 ATP synthases present in the membranes. Heterodimer Formation of b +124-130-his Complemented with b wt-V5 It has been shown by Dr. Deepa Bhatt that insertions or deletions constructed in a hydrophobic stretch corresponding to amino acids 124-130 (VAILAVA) of the b subunit results in loss of enzyme function (Bhatt et al., manuscript in preparation). Immunoblot analysis was performed in order to detect a heterodimer interaction between b+124-130-his and bwt-V5 (Figure 3-6A and B). The b subunit was detected only in membranes prepared from strains expressing a functional b subunit (Figure 3-6A, Lanes 1-6). No b subunit was present in membranes when KM2 (b) was complemented with plasmid pTAM38 (b+124-130-his). Immunoblot analysis using an anti-V5 antibody was performed on the membrane preparations and Ni-purified products (Figure 3-6B). Interestingly, coexpression of pTAM38 (b+124-130-his) and pTAM46 (bwt-V5) in the same cell resulted in the appearance of a signal representing bwt-V5 in a Ni-resin purified sample (Figure 3-6B, PAGE 153 132 Figure 3-6. Ni-resin purification of membranes incorporated with b +124-130-his subunit mutation. Membrane preparation and Ni-resin purification and Western blot analysis were accomplished as described in Materials and Methods. A) Proteins were transferred to a nitrocellulose membrane and probed using a polyclonal anti-b antibody or B) an anti-V5 mouse monoclonal antibody. C) line diagram of the different b dimers found in the cell. PAGE 154 133 Figure 3-7. ATP-driven energization of membrane vesicles incorporated with a defective b +124-130 subunit mutation. Membrane preparation and ATP-driven proton pumping was accomplished as described in Materials and Methods. The samples for each trace have been labeled according to the b subunit mutation and the epitope tag, so the strains used as the sources of the samples were as follows: b, KM2/pBR322; b wt KM2/pKAM14; b wt-V5 KM2/pTAM46; b wt-his KM2/pTAM37; b +124-130-his KM2/pTAM38; b wt-his /b wt-V5 KM2/pTAM37/pTAM46; b wt-V5 /b +124-130-his KM2/pTAM46/pTAM38. PAGE 155 134 Lane 10). The only mechanism for recovery of b wt-V5 was through dimerization with b +124-130-his indicating that the b wt-his subunit rescued some of the defective b subunit and formed heterodimeric F 1 F 0 ATP synthase complexes. Therefore, coexpression of the two different b subunits in the same cell led to two distinct interactions within an intact F 1 F 0 ATP synthase complex (Figure 3-6C): 1) a homodimer of b wt-V5 and 2) a heterodimer consisting of both the defective and wild type b subunit. F 1 -ATP hydrolysis activity of total membrane protein was used as a test of F 1 F 0 ATP synthase assembly (Table 3-1). As expected, membranes with enzyme complexes incorporated with b +124-130-his displayed a specific activity similar to the negative control, indicating virtually no interaction with F 1 Coexpression of pTAM38 (b 124-130-his ) and pTAM46 (b wt-V5 ) resulted in a specific activity similar to the wild type strain (Table 3-1). It was likely that the F 1 F 0 ATP synthase complexes incorporated with the b 124-130-his /b wt-V5 were intact. An indication of coupled activity was shown by F 1 F 0 ATP synthase-mediated ATP-driven proton pumping activity in membrane vesicles prepared from the epitope-tagged mutants (Figure 3-7). Membranes containing only the b 124-130-his subunit exhibited no coupled activity as expected. Membranes isolated from cells coexpressing pTAM38 (b 124-130-his ) and pTAM46 (b wt-V5 ) displayed virtually the same amount of coupled activity as the membranes from cells coexpressing b wt-V5 and b wt-his Since coexpression of the two different b subunits in the same cell led to only two distinct interactions, it was likely that the F 1 F 0 ATP synthase complexes incorporated with the b 124-130-his /b wt-V5 were functional. PAGE 156 135 Mutual Complementation In order to look for mutual complementation of b subunits with defects in different functional domains, b arg36ile-V5 and b arg36glu-V5 were coexpressed with b 153end-his in the absence of a functional wild type b subunit. Significantly, when b 153end-his and b arg36ile-V5 were coexpressed in the same cell, KM2/pTAM51/pTAM53, small colonies were present after 5 days of growth. Expression of either b arg36ile-V5 or b 153end-his alone in KM2 (b) results in a completely defective enzyme complex, any F 1 F 0 ATP synthase activity must necessarily have come from a heterodimer formed from two defective b subunits. Therefore, the b arg36ile and b 153end mutations qualified as intergenic second site suppressor mutations. To determine whether there was a direct protein-protein interaction between two mutant subunits, we investigated the ability of b 153end-his to form an interaction with b arg36ile-V5 or b arg36glu-V5 by the standard immunoblot (Figure 3-8A and B). All of the controls provided on the first immunoblot were included and displayed similar results. An additional control was included to ensure complete solubilization of membrane vesicles was achieved and nonspecific aggregation of the b subunits was not a concern (Figure 3-8A and B, Lanes 10-11). Two independent membrane preparations were mixed together. Membrane vesicles derived from strains KM2/pTAM51 (b 153end-his ) and KM2/pTAM53 (b arg36ile-V5 ) or KM2/pTAM54 (b arg36glu-V5 ) were mixed and the membranes were solubilized with 0.2% tegamineoxide WS-35 prior to Ni-resin purification. Immunoblot analysis with anti-b antibodies showed normal levels of histidine-tagged b subunit upon Ni-resin purification (Figure 3-8A, Lanes 10-11). No V5-epitope was present after Ni-resin purification, indicating that neither non-specific PAGE 157 136 Figure 3-8. Ni-resin purification of F 1 F 0 ATP synthase incorporated with complementing defective b subunits. Membranes preparation, Ni-resin purification and Western blot analysis was accomplished as described in Materials and Methods. A) Proteins were transferred to a nitrocellulose membrane and probed using a polyclonal anti-b antibody or B) an anti-V5 mouse monoclonal antibody. C) line diagram of the different b dimers found in the cell. Three interactions, b arg36mut-V5 /b arg36mut-V5 and b 153end-his /b 153end-his homodimers as well as b arg36mut-V5 /b 153end-his heterodimers, existed in the cell. PAGE 158 137 Figure 3-9. ATP-driven energization of membrane vesicles incorporated with F 1 F 0 ATP synthase containing complementing defective b subunits. Membrane preparation and ATP-driven proton pumping was accomplished as described in Materials and Methods. The samples for each trace have been labeled according to the b subunit mutation and the epitope tag, so the strains used as the sources of the samples were as follows: b, KM2/pBR322; b wt KM2/pKAM14; b 153end-his KM2/pTAM51; b arg36ile-V5 KM2/pTAM53; b arg36glu-V5 KM2/pTAM54; b wt-his /b wt-V5 KM2/pTAM37/pTAM46; b 153end-his /b arg36ile-V5 KM2/pTAM51/pTAM53; b 153end-his /b arg36glu-V5 KM2/pTAM51/pTAM54. PAGE 159 138 aggregation was nor incomplete solubilization was a factor in the observed results (Figure 3-8B, lanes 10-11). Therefore, in cells expressing two different b subunits, any observed interactions of b subunits must have been due to heterodimers integrating into an intact F 1 F 0 ATP synthase complex. Importantly, immunobotting using the anti-V5 antibody clearly detected V5 epitope-tagged b subunits in Ni-resin purified samples when two different b subunits, b 153end-his and b arg36ile-V5 or b arg36glu-V5 were coexpressed (Figure 3-8B, Lanes 12-13). The data indicated that dimerization between the two defective b subunits could occur and the F 1 F 0 ATP synthase complex accepted incorporation of a b heterodimer with mutations affecting two different domains. Expression of two different defective b subunits yielded three distinct F 1 F 0 ATP synthase complexes including the b arg36ile-V5 or b arg36glu-V5 homodimeric F 1 F 0 a partially assembled b 153end-his homodimer complex, and the heterodimeric F 1 F 0 ATP synthase complexes. Total membrane associated F 1 -ATP hydrolysis activity was used as a test of F 1 F 0 ATP synthase complex assembly (Table 3-1). Membrane vesicles isolated from strains KM2/pTAM51/pTAM53 (b 153end-his /b arg36ile-V5 ) and KM2/pTAM51/pTAM54 (b 153end-his /b arg36glu-V5 ) yielded membrane associated specific activities of about 29% and 14%, respectively, which was intermediate between the specific activities if either of the mutants were expressed alone in the cell. An indication of coupled activity was obtained by F 1 F 0 ATP synthase-mediated ATP-driven proton pumping activity in membrane vesicles prepared from the epitope-tagged mutants (Figure 3-9). As expected, membranes from all three strains expressing defective b subunits by themselves showed no proton pumping activity. Membranes isolated from cells expressing b 153end-his /b arg36ile-V5 or b 153end-his /b arg36glu-V5 KM2/pTAM51/pTAM53 PAGE 160 139 or KM2/pTAM51/pTAM54, respectively, displayed coupled ATP-driven proton pumping of about 20% and 16%. Since either homodimer leads to a completely defective enzyme, any coupled activity observed necessarily came from an intact heterodimeric F 1 F 0 ATP synthase. The data indicated that F 1 F 0 ATP synthase complexes incorporated with heterodimers of two mutant b subunits were indeed functional. Furthermore, the results demonstrated that the roles of the two b subunits in the peripheral stalk were not equivalent. Discussion Historically the b subunit dimer has been viewed as a single functional unit. However, the asymmetric nature of the F 1 F 0 ATP synthase enzyme complex suggested that the functional role of each b subunit should not necessarily be considered equivalent. Protein-protein contacts made by one b subunit cannot be made by the other. In the present work, a unique expression system that facilitates the production, purification and detection of F 1 F 0 ATP synthase complexes incorporated with a b subunit heterodimer was utilize in order to study the functional roles of the two b subunits. The experiments involved a two-plasmid expression system that directed production of two different b subunits in the same cell and an epitope tag system that allowed detection of a b subunit heterodimeric F 1 F 0 ATP synthase species. Three regions of the b subunit were considered. An evolutionarily conserved arginine, b arg36 located near the interface between the membrane and tether domains had been found to be crucial for F 1 F 0 ATP synthase function (215). Enzyme complexes incorporated with a b arg36ile-V5 or b arg36glu-V5 were found to be intact yet functionally defective. Second, the C-terminal last four amino acids had been shown to be essential for the F 1 binding domain (206, 288). PAGE 161 140 Enzyme complexes with a b 153end-his were found to be only partially assembled. Thirdly, insertions and deletions in a hydrophobic stretch of amino acids in the b subunit corresponding to amino acids 124-130 (VAILAVA) resulted in a complete loss of enzyme function. The b dimer was not found in membranes when cells expressed only the b +124-130 subunit. Heterodimerization was detected in cells expressing either arg36 mutation, b arg36ile-V5 or b arg36glu-V5 with b wt-his b 153end-his /b wt-V5 and b +124-130-his /b wt-V5 (Figure 3-10). Figure 3-10. Interactions of defective b subunit with wild type b subunits found in intact F 1 F 0 ATP synthase complexes. An epitope-tag system was used in order to determine if a defective b subunit could form a dimer with a wild type b subunit. A V5 epitope tag was constructed at the C-terminus of the b subunit (shown in red) or a histidine epitope tag was placed at the N-terminus of the b subunit. Four mutant b subunits were studied and all were found to form a dimer with a wild type b subunit (1) b arg36ile-V5 (2) b arg36glu-V5 (3) b 153end-his and (4) b +124-130-his PAGE 162 141 Dimerization was observed in membrane preparations from cells expressing both b 153end-his /b arg36ile-V5 and b 153end-his /b arg36glu-V5 More importantly, enzyme complexes incorporated with the mutant heterodimers were functionally active, suggesting that each of the b subunits were complementing the other to form an intact and functional enzyme complex. This observation demonstrated unambiguously that F 1 F 0 ATP synthase complexes containing b heterodimers were active and provided evidence that each of the individual b subunits provide specialized functions within the peripheral stalk. Clearly, each of the mutant b subunits compensates for what the other is lacking. This raises a question concerning the relative positions of the individual b subunits of the peripheral stalk. In order for F 1 F 0 ATP synthase containing the two different mutant b subunits to be intact and functional, it is likely that the b arg36ile-V5 (or b arg36glu-V5 ) subunit must be positioned such that its extreme C-terminus forms the appropriate contacts with the subunit of F 1 Similarly, the b 153end-his subunit must be positioned so that its b arg36 makes the appropriate contacts with the F 0 subunits (Figure 3-11). Incorrect positioning of the mutant b subunits during assembly might be expected to lead to an inactive or partially assembled enzyme complex. PAGE 163 142 Figure 3-11. Model of F 1 F 0 ATP synthase incorporated with complementing defective b subunits. The b arg36ile subunit was expressed with a V5 epitope tag at its carboxyl terminus (represented by the S-shaped red line) and the b 153end subunit was expressed with a histidine tag at its amino terminus (represented by the orange curved line). Complexes incorporated with a b subunit heterodimer resulted in a functional enzyme complex. The b arg36ile-V5 subunit provides an intact F 1 binding domain for interaction with the subunit, and the b 153end-his subunit carries out the interaction with the a subunit. PAGE 164 CHAPTER 4 DEVELOPMENT OF CYSTEINE CHEMICAL MODIFICATIONS OF ALTERED b SUBUNITS Introduction F 1 F 0 ATP synthases utilize the electrochemical gradient of protons across membranes to synthesize ATP from ADP and P i (3, 290, 291). In E. coli, F 1 F 0 ATP synthase is a multimeric enzyme composed of twenty-two polypeptides of eight different types (Figure 4-1). The F 1 portion is composed of the subunits 3 3 and is responsible for catalysis. The F 0 portion of the enzyme consists of the ab 2 c 10 subunits and conducts proton translocation through the membrane. Two stalk structures link the F 1 and F 0 sectors. Proton translocation drives the rotation of the central stalk, known as the rotor, within the stationary 3 3 hexamer, resulting in conformational changes within the catalytic sites (3, 15, 16, 63, 64). A second stalk structure, known as the peripheral stalk, consists of the and b subunits (Figure 4-1). The role of the peripheral stalk is thought to be that of a stator, holding the hexamer in place against the rotation of the central stalk. The subunit can be chemically fixed to a single subunit without loss of enzyme activity, which would be expected if was part of the peripheral stalk linking F 1 and F 0 supporting the stator concept (185). The carboxyl termini of the and b subunits have been shown to be in direct contact (3, 142, 200, 219, 220). 143 PAGE 165 144 Figure 4-1. Model of E. coli F 1 F 0 ATP synthase. The peripheral stalk of F 1 F 0 ATP synthase is composed of the subunit of F 1 and the homodimer of b subunits of F 0 Shown in orange and red are the and b subunits, respectively. It has been shown previously that insertions and deletions of amino acids in the tether region of the b subunit do not affect enzyme activity. However, it was not known how the insertions or deletions were accommodated in the context of an intact enzyme. An understanding of the structure of the peripheral stalk can be obtained by investigating the apparent length of the peripheral stalk in shortened, lengthened and wild type b subunits. PAGE 166 145 A dimer of two b subunits is the major component of the peripheral stalk of F 1 F 0 ATP synthase and is necessary for normal assembly and function. The b dimer spans the cytoplasmic membrane and reaches towards the topside of F 1 where it meets the subunit (Figure 4-1) (100, 141-143). A substantial amount of evidence suggests the b dimer to be at least 80% -helical, with 14% -turn, and exist in a parallel, elongated conformation, spanning about 45 from the top of the membrane to the bottom of the 3 3 hexamer or about 100 to the top of the hexamer (12, 13, 166, 196, 292). Four domains comprise the b subunits: the amino-terminal membrane spanning, tether, dimerization and the carboxyl terminal -binding domains (Figure 4-5) (13). Despite the previous model of the b subunit dimer, describing it as a rigid structural feature of F 1 F 0 ATP synthase, accumulating evidence suggests a more flexible stalk model (193-195, 202). It has been shown by Dr. Paul Sorgen that F 1 F 0 ATP synthase retains sufficient levels of activity upon relatively large deletions or insertions in the b subunit. An eleven amino acid deletion and a fourteen amino acid insertion in the region of the b subunit spanning the tether domain and the beginning of the dimerization domain was accommodated by the enzyme (Figure 4-5). Assuming -helical structure, this 21 insertion and 16 deletion corresponds to well over a third of the length spanning from the top of the membrane to F 1 or right under a quarter of the length spanning towards the top of F 1 Furthermore, four amino acid insertions and deletions were successfully accommodated throughout most of the dimerization and -binding domains of the b subunit (manuscript in preparation). In contrast to the previously accepted role of the b subunit as a rigid stator, these observations suggest that the role of the b dimer is more of a flexible or elastic structural feature during rotational catalysis. In the present PAGE 167 146 chapter, cysteine chemical modifications were created in the and b subunits to provide reactive thiols groups for future labeling studies. Materials and Methods A more thorough account of many of the procedures used in Chapter 6 can be found in previous chapters. Many of the techniques, including recombinant DNA techniques, site directed mutagenesis, crude membrane preparation procedures, western blotting, as well as assays of protein concentration and F 1 F 0 ATP synthase activity have been described in detail in Chapter 2. Materials Molecular biology enzymes and mutagenic oligonucleotides were obtained from Invitrogen (Carlsbad, CA), Life Technologies, Inc. (Grand Island, NY), New England Biolabs (Beverly, MA) and Stratagene (La Jolla, CA). Reagents were obtained from Sigma (St. Louis, MO), BioRad Laboratories (Hercules, CA) and Fisher Scientific (Pittsburgh, PA). Plasmid purification kits were acquired from Qiagen Inc. (Valencia, CA). The anti-rabbit immunoglobulin horseradish peroxidase-linked whole antibody (from donkey), Hybond ECL Nitrocellulose membrane, electrochemiluminescence Western blotting reagents and high performance chemiluminescence film were purchased from Amersham Biosciences (Piscataway, NJ). Polyclonal antibodies against SDS-denatured b subunit (284, 285) were generously provided by Dr. Karlheinz Altendorf (Universitt Osnabrck, Osnabrck, Germany. Strains and Media The bacterial strains used to create the cysteine chemical modifications in the b subunit include the wild type b subunit expression plasmid, pKAM14 (b, Ap r ), and plasmids used to express b subunits shortened or lengthened by 11 amino acids, pAUL5 PAGE 168 147 (b 11 Ap r ) and pAUL47 (b +11 Ap r ), respectively, and have been described previously (193, 194, 203). The plasmids encoding the uncF(b) gene were used to compliment Escherichia coli (E. coli) strain KM2 (b) carrying a chromosomal deletion of the gene. Plasmid pJLG1 (a, c, b, Cm r ), generated by Dr. James Gardner in our lab, was used as a temporary vector in order to facilitate cloning due to its extra unique restriction enzyme sites. The wild type unc operon expression plasmid, pAES9 (abc, Cm r ), was used to create a cysteine-less F 1 F 0 ATP synthase complex and has been described previously (178). The plasmids encoding the unc operon (abc) were used to compliment E. coli strain 1100BC (abc) carrying a chromosomal deletion for the entire operon. All strains were streaked onto plates containing Minimal A media supplemented with succinate (0.2% w/v), to determine enzyme viability. Cells harvested for membrane preparation were grown in Luria Broth supplemented with glucose (0.2% w/v) (LBG). Isopropyl-1-thio--D-galactopyranoside (IPTG)(40 g/ml), ampicillin (Ap) (100g/ml), and chloramphenicol (Cm) (25 g/ml) were added to media as needed. All cultures were incubated at 37C for the appropriate duration. Recombinant DNA Techniques Plasmid DNA was purified with the Qiagen Mini-Prep and Maxi-Prep kits. Restriction endonuclease digestions, ligations, and transformations were performed according to the recommendations of the manufacturers (New England Biolabs, Stratagene and Life Technologies, Inc.). Site-directed mutagenesis was performed either by means of a Stratagene Quikchange kit or by ligation-mediated mutagenesis. DNA fragments were separated in 0.8 % agarose gel by electrophoresis and purified using a PAGE 169 148 Qiagen, Inc. QIAquick Gel Extraction kit. Plasmid sequences were determined by automated sequencing in the core facility of the University of Florida ICBR. Mutagenesis and Strain Construction Plasmids pKAM14 (b, Ap r ) (203), pAUL5 (b 11 Ap r ) (194) or pAUL47 (b +11 Ap r ) (193) and pJLG1 (a, c, b, Cm r ) (167) were used to construct cysteine mutant b subunits. Mutagenesis was performed in three different subunits; therefore, the ultimate goal was to express the entire unc operon containing the desired cysteine mutations from one plasmid. Plasmid pJLG1 (a, c, b, Cm r ) was used as a temporary vector in order to facilitate cloning due to its extra unique restriction enzyme sites. b subunit mutations. In order to construct the b subunit mutations, plasmids pAUL5 (b 11 Ap r ), pAUL47 (b +11 Ap r ) and pJLG1 (a, c, b, Cm r ) were first digested with restriction enzymes PpuMI and NarI in order to move the b subunit deletion and insertion into the pJLG1 plasmid. The resulting 204 and 270 bp cassettes isolated from pAUL5 (b 11 Ap r ) and pAUL47 (b +11 Ap r ), respectively, were subsequently ligated into the pJLG1 vector, resulting in pTAM1 (b 11 Cm r ) and pTAM3 (b +11 Cm r ) (Table 4-1). In order to site specifically label the b subunit in future experiments, a native amino acid was mutated to a cysteine. Two sites were chosen for replacement, b ser84 and b gly43 located respectively above and below the region of length alteration. Furthermore, the b subunit had a native cysteine, b cys20 located in the membrane-spanning portion of the subunit, which was mutated to a serine. The mutations were created in each of the plasmids using the Stratagene Quikchange kit. Sense and antisense mutagenic oligonucleotides were created for each of the desired b subunit mutations (Appendix A) (Figure 4-2A). First, the native cysteine was abolished by mutagenesis of codon 20 PAGE 170 149 Table 4-1. Description of uncF(b) cysteine mutations Plasmid Description 1, 2 b Length b cys20ser b gly43cys b ser84cys Growth 3 pKAM14 b wt Ap r wt +++ pBR322 b, Ap r NA NA NA pTAM1 b 11 Cm r 11 nd pJLG1 4 b wt Cm r wt nd pTAM3 b +11 Cm r +11 nd pTAM8 b 11-cys20ser, gly43cys 11 +++ pTAM9 b cys20ser, gly43cys wt +++ pTAM10 b +11-cys20ser, gly43cys +11 +++ pTAM11 b 11-cys20ser, ser84cys wt +++ pTAM12 b cys20ser, ser84cys 11 +++ pTAM13 b +11-cys20ser, ser84cys +11 +++ 1 Description as found in text. 2 Plasmids pTAM1-pTAM13 all encode the uncB(a) and c genes, but for the sake of brevity, only the b subunit mutations are described. 3 Plasmids were transformed into E. coli strain KM2 (b) and grown aerobically on succinate minimal medium. Colony size was scored after 72-hr incubation at 37C as: +++, 1.0 mm; ++, 0.3-0.5 mm; +, ~0.1 mm; -, no growth. 4 Plasmid courtesy of Dr. James Gardner. of the uncF(b) gene to express b cys20ser b 11-cys20ser and b +11-cys20ser Then, a cysteine was introduced by mutagenesis of codon 43 (b gly43cys ) or codon 84 (b ser84cys ) of the uncF(b) gene to generate a set of six plasmids: pTAM9 (b cys20ser, gly43cys ), pTAM8 (b 11-cys20ser, gly43cys ), pTAM10 (b +11-cys20ser, gly43cys ), pTAM12 (b cys20ser, ser84cys ), pTAM11 (b 11-cys20ser, ser84cys ) and pTAM13 (b +11-cys20ser, ser84cys ) (Table 4-1). The existing unique restriction enzyme sites recognized by SnaBI, Bsp1285I and XbaI were abolished by silent mutation near the encoded b cys20ser b gly43cys and b ser84cys mutations, respectively, for an initial detection of mutations. Subsequently, the nucleotide sequence was subsequently confirmed by automated sequencing in the ICBR core facility (Figure 4-2A). The recombinant altered b subunits included the chloramphenicol resistance gene and the pACYA184 origin of replication. PAGE 171 150 Figure 4-2. Oligonucleotides for cysteine mutagenesis of the unc operon. Shown are the sense strands of the mutagenic primer pairs. The desired mutations are shown in color. Bold script indicates a change in nucleotide. Restriction enzyme recognition sequences that were silently added or abolished are underlined. Mutations were introduced as described in the Materials and Methods. A) Blue indicates the cysteine mutations constructed in uncF(b). The SnaBI, Bsp1285I and XbaI restriction sequences were knocked out along with the b cys20ser b gly43cys and b ser84cys respectively, to facilitate screening. B) Red indicates the cysteine mutations generated in uncH(). The ClaI sequence was knocked out and the EcoRI site was generated along with cys140ser for screening purposes. C) Green indicates the cysteine mutation made in unc(). The BamHI sequence was added to screen for the cys90ser mutation. PAGE 172 151 and subunit mutations. The subunit has two indigenous cysteines, cys64 and cys140 which have been shown to be highly reactive with maleimide reagents (293). We planned to chemically label both sites specifically, so plasmids were constructed which expressed only one of the two cysteines by mutating one, either cys64ser or cys140ser to a serine. Also, another cysteine located within the subunit of F 1 has been shown to be highly reactive to maleimide reagents. This cysteine was mutated to a serine without loss of enzyme function (198). Plasmid pAES9 (abc, Cm r ) was used to construct the cysteine mutant and subunits. Due to the difficulty of performing Quikchange on a 10.9 kb plasmid, it was necessary temporarily move the genes encoding the and subunits into plasmid pBluescript (pBS) by digesting pAES9 (abc, Cm r ) with the restriction enzymes BamHI and EcoRI, flanking the genes of interest, and then ligating the 3.8 kb cassette into pBS, pTAM2. Sense and antisense mutagenic oligonucleotides were created for each of the desired and subunit mutations (Figure 4-2B and C). First, each of the native cysteines found in the subunit were substituted with serines in two different plasmids by site-directed mutagenesis of codon 64 or 140 of the unc () gene to express cys64ser or cys140ser Then the native cysteine found in the subunit was changed to a serine by mutagenesis of codon 90 of the unc () gene to express cys90ser The ClaI sequence was silently knocked out and the EcoRI site was generated along with cys140ser (Figure 4-2B) and the BamHI sequence was added along with the cys90ser mutation (Figure 4-2C) for initial screening purposes and then the nucleotide sequence was subsequently confirmed by automated sequencing in the ICBR core facility. The genes encoding the and subunits with the generated cysteine PAGE 173 152 mutations were then shuttled back into pAES9 (abc, Cm r ) to create plasmids pTAM20 ( cys90ser cys64ser abc, Cm r ) and pTAM21 ( cys90ser cys140ser abc, Cm r ). The recombinant plasmids included the chloramphenicol resistance gene and the pACYA184 origin of replication. Construction of intact mutant unc operons. The final step of plasmid construction was to move each of the previously generated b subunit cysteine mutants (Table 4-1) into both pTAM20 ( cys90ser cys64ser abc, Cm r ) and pTAM21 ( cys90ser cys140ser abc, Cm r ), generating plasmids pTAM22-pTAM33 (Table 4-2). Each of the plasmids encoded the entire unc operon with the cys90ser and b cys20ser mutations as well as every possible combination of shortened, lengthened or wild type length b subunits, b gly43cys or b ser84cys and cys64ser or cys64ser The final products included 12 plasmids that encoded 12 genetically different F 1 F 0 ATP synthase complexes as well as the chloramphenicol resistance gene and the pACYA184 origin of replication (Table 4-2). Crude Preparative Procedures Inverted membrane vesicles from KM2 (b) or 1100BC (abc) strains expressing the desired cysteine mutations were prepared essentially as described in Chapter 2 (194). Bacteria were grown in 500 mL LBG supplemented with the appropriate antibiotic, harvested, and passed through a French Pressure Cell one time at 14,000 psi. Membranes were then collected by differential centrifugation. Protein concentrations were determined by the bicinchoninic acid (BCA) assay (286). Assays of F 1 F 0 ATP Synthase Activity Growth on a minimal succinate medium was used as an initial, in vivo, assay for enzyme viability. ATP hydrolysis activity was assayed by the acid molybdate method PAGE 174 153 (146). Membranes were assayed in buffer (50 mM Tris-HCl, 1 mM MgCl2, pH9.1) to determine the linearity with respect to time and enzyme concentration. Immunoblot Analysis Proteins were loaded on a 15% tris-glycine SDS gel and transferred onto nitrocellulose by electroblot. The b subunit antibody incubation was performed essentially as described previously, using a 1:25,000 dilution of the anti-b subunit antibodies. Secondary antibody incubation was performed with horseradish peroxidase-linked donkey anti-rabbit antibody (1:50,000), and the antibody was detected by enhanced chemiluminescence. Signals were visualized on high performance chemiluminescence film using a Kodak X-Omat. Results Construction and Growth Characteristics of Mutants Single cysteines, which will provide reactive thiols for site-specific chemical modification, were strategically placed within the F 1 F 0 ATP synthase enzyme complex. Several cysteine mutations were generated in the unc operon in a multi-step construction scheme to ultimately result in 12 plasmids encoding different combinations of six different amino acid substitutions as well as b subunits of altered length (Table 4-2) (Figure 4-3). A previous analysis of F 1 F 0 ATP synthase complexes with b subunits shortened and lengthened by 11 amino acids found the mutants to be essentially wild-type when expressed in the presence of 40 M IPTG (193, 194). Functional F 1 F 0 ATP synthase complexes have been studied with many different cysteine mutations; PAGE 175 154 Table 4-2. Description of the unc operon cysteine mutations 1 Plasmid 2 Length 3 cys90ser b cys20ser b gly43cys b ser84cys cys140ser cys64ser pAES9 wt pBR322 NA NA NA NA NA NA pTAM22 11 pTAM23 wt pTAM24 +11 pTAM25 11 pTAM26 wt pTAM27 +11 pTAM28 11 pTAM29 wt pTAM30 +11 pTAM31 11 pTAM32 wt pTAM33 +11 1 Plasmids were designed to contain various combinations of the cysteine mutations in the b and subunits. Symbols: -, plasmid does not contain the listed mutation; plasmid encodes the mutation listed above. 2 All plasmids, with the exception of pBR322, encoded the entire unc operon and conferred chloramphenicol resistance. 3 Length of the tether domain of the b subunit. Symbols: empty plasmid vector; wt, normal length;11, eleven amino acid deletion; +11, eleven amino acid insertion. PAGE 176 155 Figure 4-3. Expression plasmid of cysteine mutants. F 1 F 0 ATP synthase complexes with site-specific cysteine mutations were designed to be expressed from a single plasmid encoding the entire unc operon. Plasmid pTAM24 is one of twelve of the constructed plasmids. The name of the unc gene is listed followed by the subunit it expresses in parenthesis. The black stars represents codons that expressed a native cysteine that had been substituted with a serine. The red star represents a glycine to cysteine substitution at codon 43 of the uncF(b) gene. The green dot represents an 11 amino acid insertion. Plasmid pTAM24 expresses the entire unc operon with the following mutations: b +11, cys20ser, gly43cys cys140ser and cys90ser The plasmids confer chloramphenicol resistance and the pACYA184 origin of replication. Similar constructions include those listed in Table 4-2. however, it was necessary to determine whether the mutations generated in the present study would have affect activity. The effects of the cysteine mutations were studied by the ability of the plasmids to complement either the E. coli strain KM2 (b) (Table 4-1) or 1100BC (abc) (Table 4-3). Growth on succinate minimal medium was used PAGE 177 156 as an initial qualitative gauge of enzyme activity in vivo since E. coli strains lacking F 1 F 0 ATP synthase cannot derive energy from nonfermentable sources. In each case, the strains expressing the different cysteine mutations grew comparable to the wild type strain (Table 4-3). Table 4-3. Aerobic growth properties and membrane-associated ATP hydrolysis activity of mutants expressing cysteine Strains 1 Description 2 Growth 3 Activity 4 pAES9 a, b, c, Cm r +++ 100% pBR322 empty vector, Ap r 12% pAUL5 5 b 11 Ap r +++ 90% pAUL47 5 b +11 Ap r +++ 87% pTAM22 cys90ser cys140ser b 11, cys20ser gly43cys +++ 89% pTAM23 cys90ser cys140ser b cys20ser gly43cys +++ 95% pTAM24 cys90ser cys140ser b +11, cys20ser gly43cys +++ 87% pTAM25 cys90ser cys140ser b 11, cys20ser ser84cys +++ 88% pTAM26 cys90ser cys140ser b cys20ser ser84cys +++ 101% pTAM27 cys90ser cys140ser b +11, cys20ser ser84cys +++ 89% pTAM28 cys90ser cys64ser b 11, cys20ser gly43cys +++ 92% pTAM29 cys90ser cys64ser b cys20ser gly43cys +++ 96% pTAM30 cys90ser cys64ser b +11, cys20ser gly43cys +++ 90% pTAM31 cys90ser cys64ser b 11, cys20ser ser84cys +++ 93% pTAM32 cys90ser cys64ser b cys20ser ser84cys +++ 99% pTAM33 cys90ser cys64ser b +11, cys20ser ser84cys +++ 91 % 1 With the exception of pAUL5 and pAUL47, all plasmids were expressed in the 1100BC cell line. Plasmids pAUL5 and pAUL47 encode only the b subunit and were expressed in the KM2 cell line. 2 The description for plasmids pTAM22-pTAM33 only list the subunits with cysteine mutations for brevity; however, these plasmids also encode the wild type genes for the , a and c subunits. 3 E. coli strains were grown aerobically on succinate minimal medium supplemented with 40 M IPTG. Colony size was scored after 72-hr incubation at 37C as: +++, 1.0 mm; ++, 0.3-0.5 mm; +, ~0.1 mm; -, no growth. 4 E. coli strains were grown in LBG supplemented with the appropriate antibiotics and 40 M IPTG. ATPase activities were measured as described under Materials and Methods. Activity is listed as a percentage of the wild type strain (pAES9), which was set to 100%. Units were calculated from the slope of the line based on three measurements with incubations for 12 minutes. 5 Plasmids courtesy of Dr. Paul Sorgen. PAGE 178 157 Effects of Cysteine Mutations Membrane associated ATP hydrolysis activity. F 1 has very little affinity for the membrane in the absence of intact F 0 therefore, total membrane associated F 1 -ATP hydrolysis activity was used as a test of F 1 F 0 ATP synthase complex assembly. The cysteine mutations had very little, if any at all, affect on enzyme assembly. In general, membranes with the cysteine mutations incorporated into the F 1 F 0 ATP synthase complex had specific activities ranging from about 88 to 95% of the wild types strain (Table 4-3). These values were comparable to the effect of the eleven amino acid deletion and insertions in the absence of the cysteine mutations. In fact, membranes with F 1 F 0 ATP synthase complexes incorporated with b subunits that were wild type in length, yet carried all the cysteine mutations, ranged from 95 to 100% of the wild type strain. It was likely that the slight decrease in ATP hydrolysis activity was due to the change in length in the b subunits and not due to the cysteine mutations. Western blot analysis. If the b subunit of F 1 F 0 ATP synthase does not dimerize and become incorporated into an intact F 1 F 0 ATP synthase complex, it is generally turned over and therefore absent from membrane preparations. Therefore the presence of the b subunit in the membrane preparations was detected by Western blot analysis. Proteins from membrane preparations generated from E. coli strain 1100BD (abc) complemented with pAES9 (abc), pTAM31 ( cys90ser cys64ser ,, a, b 11, cys20ser ser84cys c), pTAM32 ( cys90ser cys64ser ,, a, b cys20ser ser84cys c) and pTAM33 ( cys90ser cys64ser ,, a, b +11, cys20ser ser84cys c) were separated by SDS-PAGE and detected with anti-b antibodes (Figure 4-4). The b subunit expressed from pTAM32 and pAES9 migrated to the same molecular weight as expected. The size PAGE 179 158 difference in the b subunit is clear between the 11 and +11, confirming the expression of unequal length b subunits. Figure 4-4. Western blot analysis of cysteine mutant b subunits of differing length. 1100DBC cells were transformed with plasmids pAES9 (abc), pTAM31 ( cys90ser cys64ser ,, a, b 11, cys20ser ser84cys c), pTAM32 ( cys90ser cys64ser ,, a, b cys20ser ser84cys c) and pTAM33 ( cys90ser cys64ser ,, a, b+11, cys20ser ser84cys c). Proteins from a crude membrane preparation were separated on a 15% polyacrylamide Tris-HCl-SDS BioRad Ready gel and then transferred to a nitrocellulose membrane in order to probe with anti-b antibodies. Discussion In the current chapter, we have developed a set of twelve unc operon expression plasmids that encode amino acid substitutions to void the F 1 F 0 ATP synthase complex of all known reactive thiols as well as generate strategically placed cysteines. Cysteines were chosen because the thiol side chain is highly reactive and can be modified by maleimide reagents. The cysteine mutations did not affect enzyme assembly or function. Plasmids were designed to express a single cysteine at one of two locations in the subunit as well as one position, either above or below the site of insertion or deletion, in the b subunit (Figure 4-5). The idea for establishing a single reactive cysteine in both F 1 and F 0 is to use them as targets for labeling with fluorescence compounds. F 1 can readily be stripped from F 0 PAGE 180 159 Figure 4-5. Model of F 1 F 0 ATP synthase with cysteine substitutions in the b and subunits. The red stars indicate the approximate location of generated cysteines. The region colored green is the approximate location of the eleven amino acid insertion (b +11 ) or deletion (b 11 ). A set of twelve plasmids was designed to express one cysteine in the and unequally length b subunits. each cysteine labeled separately with a "donor" and "acceptor" fluorescent compound, and then stoichiometrically reconstituted under conditions of high ionic strength (203). With a single fluorescent compound located above and below the region of insertion or deletion in the tether domain of the b subunit, physical measurements can be calculated and compared. Comparing the length of the wild type, shortened and lengthened peripheral stalks will give some insight on the overall possible conformation of the altered F 1 F 0 ATP synthase complex. The F 1 F 0 ATP synthase enzyme complex must, in some way, adapt to the shortened or lengthened b subunits. This can occur in at least one of two ways: 1) distortion of F 1 to accommodate the change in length of the peripheral stalk or 2) distortion of the peripheral stalk itself. If the former was true, F 1 would become distorted by bending of the central stalk or compression of the 3 3 hexamer and the rigid stalk hypothesis would gain favor. Since F 1 F 0 ATP synthase function requires the ability of the central stalk to rotate freely while making specific interactions within the catalytic PAGE 181 160 hexamer, this mechanism is highly unlikely. If the latter situation were true, distortion would be limited to the b dimer and the flexible stalk hypothesis would hold true. This could occur by straightening or increasing the naturally occurring 20 bend in the shortened or lengthened peripheral stalk, respectively, or by stretching or compressing the secondary structure in the b dimer. The use of FRET will provide evidence as to which of the hypotheses is true. One major limitation plagued this line of research. F 1 F 0 ATP synthase is incorporated with a homodimer of b subunits, hence two b subunits, each with a reactive cysteine. Since FRET requires only one b subunit be labeled, a system necessarily had to be developed to allow purification of F 1 F 0 ATP synthase complexes with two genetically different b subunits: one cysteine-less and one with a single cysteine. The system utilizes epitope tags placed on the b subunits and is discussed in detail in Chapter 2. Future FRET studies will use this epitope-tagged scheme in order to express F 1 F 0 ATP synthase complexes with a b dimer containing only one cysteine. PAGE 182 CHAPTER 5 MUTAGENISIS OF THE AMINO AND CARBOXYL TERMINI OF THE b SUBUNIT IN F 1 F 0 ATP SYNTHASE Introduction Two stalk structures have been shown to link the F 1 and F 0 sectors (11, 187). The central, or "rotary" stalk, extends within the 3 3 hexamer. Protons diffuse through the membrane, via the a and c subunits of F 0 resulting in the rotation of the central stalk within the hexamer, ultimately generating ATP (4, 16, 189). The peripheral stalk, or "stator", is positioned to the side of the F 1 F 0 ATP synthase complex and reaches from the periplasmic leaflet of the membrane up to near the top of F 1 in an -helical highly extended conformation (12, 13, 166, 204, 294), where it makes contact with a single subunit (100, 141-143). The primary function of the peripheral stalk is to prevent the rotation of the 3 3 hexamer against the rotation of the central stalk. The peripheral stalk consists of the subunit of F 1 and the b subunit dimer of F 0 (198, 288), which have been shown to be in direct contact with each other at their carboxyl termini (3, 4, 200, 219, 220). The b subunit dimer is the major component of the peripheral stalk, anchoring F 1 to the membrane. Despite attempts by several other laboratories, there is presently no high-resolution structure of the entire b subunit. Therefore, model polypeptides have been constructed in order to elucidate the structure of the b subunit by domain. Four domains comprise the b subunits: the amino terminal membrane-spanning, tether, dimerization and the carboxyl terminal -binding domains (Figure 5-1) (12, 13). A model polypeptide comparable to residues 1-34, which contains 161 PAGE 183 162 the membrane-spanning domain, has been solved by nuclear magnetic resonance (NMR). The data revealed an -helical monomeric structure with a 20 bend at residues 23-26 (136). A series of crosslinking studies led to a dimeric model in which the extreme Figure 5-1 Amino acid sequence and domains of the E. coli b subunit. The b subunit is a 156 residue, 17,264 Dalton amphipathic polypeptide. Dunn and coworkers have defined four domains in the b subunit: the membrane spanning (blue), tether (orange), dimerization (green) and -binding domains (red). However, the boundaries of the dimerization domain are continually being refined. Dr. Paul Sorgen demonstrated that F 1 F 0 ATP synthase could retain sufficient levels of activity upon relatively large deletions or insertions of up to eleven and fourteen amino acids, respectively, in the tether and beginning of the dimerization regions. The black lines indicate the amino acids deleted and the cyan indicates the amino acids duplicated that resulted in a functional F 1 F 0 ATP synthase. Red lines indicate amino acids deleted (below sequence) or inserted (above sequence) that resulted in a loss of enzyme function. PAGE 184 163 amino-termini of the b subunits crossed each other in close proximity, the two b subunits then angled apart as they traverse the membrane towards the cytoplasmic side (136). The tether domain, contained within residues 23 and approximately 60, does not add significant stability to the dimerization of the b subunits and its role is the least distinct portion of the b subunit. This is the region of the b subunit that can be seen in electron micrographs (EM) of intact F 1 F 0 ATP synthase complexes (11, 187). There is currently no high-resolution structure for this domain. The structural conformation of the tether domain is suggested to be that of an -helical coiled-coil based on the heptad repeat which extends from the membrane surface to residue b Ala79 (138, 203). It has been shown by Dr. Paul Sorgen that F 1 F 0 ATP synthase retains sufficient levels of activity upon relatively large deletions or insertions of up to eleven and fourteen amino acids, respectively, within the b subunit tether domain, suggesting that this domain contributes a significant degree of flexibility to the peripheral stalk (Figure 5-1) (193, 194). The dimerization domain is currently defined as residues 63-122 and is required for the b subunit to form a dimer. A crystal structure of a monomeric polypeptide, modeled from residues 62-122, has recently been solved and refined to 1.55 (138). Dunn and coworkers have constructed a model in which the two -helices of the b 62-122 region form a right handed coiled coil. Finally, the carboxyl-terminal region, residues 123-156, forms the -binding domain. The extreme two to four amino acids of the carboxyl terminus are significantly important for the ability of the b subunit to form an interaction with the subunit. Deletion of these amino acids has been known to inhibit F 1 F 0 ATP synthase function (205, 206). PAGE 185 164 This chapter focuses on the extreme amino and carboxyl termini of the b subunit. At the amino end, several mutational studies have been conducted in the membrane-spanning region; however, very few were known to produce a deficient F 1 F 0 ATP synthase complex. A single amino acid mutation, b gly9asp has been suggested to have a strong negative influence proton conduction by the a and c subunits (209). A systematic mutational analysis of the membrane domain, performed by Andrew Hardy in our laboratory, supports the model predicted by Dmitriev et al. It appears the extreme amino termini of the b subunit dimer are in close contact with each other, accounting for most of the important b-b interactions in the membrane domain, and as the dimer traverses the membrane the two b subunits flares apart (202). Three amino acids that have been shown to exhibit the strongest crosslinking efficiency in the membrane-spanning domain were replaced with alanines (b asn2ala, thr6ala, gln10ala ), yielding a defecting F 1 F 0 ATP synthase complex. Here, we demonstrate that mutation of only a single amino acid, at positions 2, 6 or 10, does not significantly affect the function of the enzyme. Several insertions and deletions can be tolerated in the tether region of the b subunit dimer (Figure 5-1) (193, 194). Evidence that the b subunit may form specific interactions with an subunit of F 1 suggested that length alterations may not be tolerated in regions of b running parallel to the F 1 hexamer (198). Other members in our laboratory are currently studying a series of insertions and deletions throughout the dimerization and -binding domains. At the carboxyl terminal end of the b subunit, mutation of the last amino acid to a cysteine, b leu156cys and chemical crosslinking forms an uncoupled F 1 F 0 ATP synthase complex (198). Furthermore, deletion of as few as two to four amino acids yields a completely defective enzyme (205, 206). However, an PAGE 186 165 insertion of amino acids had not been attempted. Here, we demonstrate that a four amino acid insertion at the b subunit carboxyl terminus does not affect the activity of the enzyme. Materials and Methods Many of the procedures utilized in Chapter 7 can be found in detail in the previous chapters. Many of the techniques, including recombinant DNA techniques, site directed mutagenesis, western blotting, as well as assays of protein concentration and F 1 F 0 ATP synthase activity have been described in detail in Chapter 2. Materials Molecular biology enzymes and mutagenic oligonucleotides were obtained from Invitrogen (Carlsbad, CA), Life Technologies, Inc. (Grand Island, NY), New England Biolabs (Beverly, MA) and Stratagene (La Jolla, CA). Reagents were obtained from Sigma (St. Louis, MO), BioRad Laboratories (Hercules, CA) and Fisher Scientific (Pittsburgh, PA). Plasmid purification kits were acquired from Qiagen Inc. (Valencia, CA). Strains and Media The wild type b subunit expression plasmid, pKAM14, has been described previously (193, 194, 203). The plasmids encoding the uncF(b) gene were used to compliment E. coli strain KM2 (b) carrying a chromosomal deletion of the gene (218). All strains were streaked onto plates containing Minimal A media supplemented with succinate (0.2% w/v), to determine enzyme viability. Cells harvested for membrane preparation were grown in Luria Broth supplemented with glucose (0.2% w/v) (LBG). Isopropyl-1-thio--D-galactopyranoside (IPTG)(40 g/ml) and ampicillin (Ap) PAGE 187 166 (100g/ml) were added to the media as needed. All cultures were incubated at 37C for the appropriate duration. Recombinant DNA Techniques Plasmid DNA was purified with the Qiagen Mini-Prep and Maxi-Prep kits. Restriction endonuclease digestions, ligations, and transformations were performed according to the recommendations of the manufacturers (New England Biolabs, Stratagene and Life Technologies, Inc.). Site-directed mutagenesis was performed either by means of a Stratagene Quikchange kit or by ligation-mediated mutagenesis. DNA fragments were separated in 0.8 % agarose gel by electrophoresis and purified using a Qiagen, Inc. QIAquick Gel Extraction kit. Plasmid sequences were determined by automated sequencing in the core facility of the University of Florida Interdisciplinary Center for Biotechnology Research (ICBR). Mutagenesis and Strain Construction Plasmid pKAM14 (b, Ap r ) (203) was used to construct all the b subunit mutants. The mutations were created in each of the plasmids using the Stratagene Quikchange kit. Amino terminal mutations. An alanine scan at the amino terminus of the b subunit, producing b asn2ala, thr6ala, gln10ala was performed by Andrew Hardy, resulting in the expression of a defective F 1 F 0 ATP synthase complex. Here, the individual sites were mutated, one at a time, to determine if the defect was the result of any particular amino acid. Sense and antisense mutagenic oligonucleotides were designed for each of the desired b subunit mutations (Appendix A) (Figure 5-2A). Three separate site-directed mutagenesis reactions were performed at codons 2, 6 and 10 of the uncF(b) gene in order to express mutant b subunits from plasmids pTAM43 (b asn2ala ), pTAM44 (b thr6ala ) and PAGE 188 167 Figure 5-2. Oligonucleotides for mutagenesis at the amino and carboxyl termini in the unc operon. Shown are the sense strands of the mutagenic primer pairs. The amino terminus mutations are depicted in orange and the carboxyl terminus insertion is shown in blue. Bold script indicates a change in nucleotide. Green and red indicate start and stop codons, respectively. Restriction enzyme sequences that were silently added are underlined. Mutations were introduced as described in the Materials and Methods. A) The DraI, StuI and MfeI restriction sequences were added along with the b asn2ala b thr6ala and b asn10ala respectively, to facilitate screening. B) The codons expressing the last four amino acids of the b subunit (VAEL) were duplicated or deleted in order to insert or remove four amino acids at the extreme carboxyl terminus. The SacI or HindIII restriction sequences were added, respectively, along with the mutations for screening purposes. PAGE 189 168 pTAM45 (b gln10ala ) (Table 5-1). The restriction enzyme sites recognized by DraI, StuI and MfeI were silently added near the encoded b asn2ala b thr6ala and b gln10ala mutations (Figure 5-2A), respectively, for an initial detection of mutations, and then the nucleotide sequence was subsequently confirmed by automated sequencing in the ICBR core facility. The recombinant mutant b subunits included the ampicillin resistance gene and the pUC18 origin of replication. Carboxyl terminal mutation. The b subunit dimer can tolerate relatively large insertions and deletions in the tether region (193, 194). Insertions have been generated throughout the dimerization and -binding domains, and the corresponding deletions are currently under construction by other members of the laboratory. Here, a four amino acid carboxyl-terminal truncation was accomplished by deletion of the final four codons of the uncF(b) gene to express b 153end from plasmid pTAM51 (Figure 5-2B). Likewise, a four amino acid insertion was constructed by duplication of the final four codons of the uncF(b) gene to express b +153-156 (Figure 5-2B). The restriction endonuclease recognition sequence for HindIII or SacI, respectively, was constructed near the deleted sequence for an initial detection of the truncation and deletion, and then the nucleotide sequence was later established by automated sequencing in the ICBR core facility (Figure 5-2B). The recombinant lengthened b subunit included the pUC18 origin of replication and conferred ampicillin resistance. Crude Preparative Procedures Inverted membrane vesicles from KM2 (b) or 1100BC (abc) strains expressing the desired cysteine mutations were prepared essentially as described in Chapter 2 (194). Bacteria were grown in 500 mL LBG supplemented with the PAGE 190 169 appropriate antibiotic, harvested, and passed through a French Pressure Cell one time at 14,000 psi. Membranes were then collected by differential centrifugation. Protein concentrations were determined by the bicinchoninic acid (BCA) assay (286). Assays of F 1 F 0 ATP Synthase Activity Growth on a minimal succinate medium was used as an initial, in vivo, assay for enzyme viability. ATP hydrolysis activity was assayed by the acid molybdate method (146). Membranes were assayed in buffer (50 mM Tris-HCl, 1 mM MgCl2, pH9.1) to determine the linearity with respect to time and enzyme concentration. Membrane energization was detected by the fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine (ACMA) (271). Results This chapter focuses on the extreme amino and carboxyl termini of the b subunit. Work accomplished at the amino terminal was an extension of studies accomplished by Andrew Hardy. Work accomplished at the carboxyl terminal end was a contribution to an insertion and deletion analysis, currently in progress, of the b subunit dimerization and -binding domains. Amino Terminal Mutations Mutations simultaneously effecting amino acids b asn2 b thr6 and b gln10 produces a completely deficient F 1 F 0 ATP synthase complex (202). To investigate the mutations individually, the sites were each mutated one by one and then the effects were studied. Construction and growth characteristics of mutants A previous analysis of F 1 F 0 ATP synthase complexes incorporated with b subunits including three mutations at codons 2, 6 and 10 (b asn2ala, thr6ala, gln10ala ) found the PAGE 191 170 Table 5-1. Aerobic growth properties and membrane-associated ATP hydrolysis activity of mutants expressing uncF(b) mutations at the amino terminus Strains Description Growth 1 Activity 2 KM2/pKAM14 (+) b wt Ap r +++ 1.46 0.03 KM2/pBR322 (-) b, Ap r 0.17 0.02 KM2/pAWH24 3 b asn2ala, thr6ala, gln10ala Ap r 0.83 0.09 KM2/pTAM43 b asn2ala Ap r +++ 1.49 0.09 KM2/pTAM44 b thr6ala Ap r +++ 1.13 0.07 KM2/pTAM45 b gln10ala Ap r +++ 1.15 0.04 1 E. coli strains were grown aerobically on succinate minimal medium supplemented with 40 M IPTG. Colony size was scored after 72-hr incubation at 37C as: +++, 1.0 mm; ++, 0.3-0.5 mm; +, ~0.1 mm; -, no growth. 2 E. coli strains were grown in LBG supplemented with the appropriate antibiotics and 40 M IPTG. ATPase activities were measured as described under Materials and Methods. Units were calculated from the slope of the line based on three measurements with incubations for 12 minutes. 3 Work accomplished by Andrew W. Hardy. mutant to be completely defective; however, membrane-associated ATP hydrolysis activity and immunoblot analysis revealed that the triple mutant was forming intact F 1 F 0 ATP synthase complexes, suggesting that the mutant was incapable of performing coupled proton translocation (202). As a result of these observations, the alanine substitutions were studied individually. The amino acids expressed by codons 2, 6 and 10 were individually mutated to express an alanine, resulting in plasmids pTAM43 (b asn2ala ), pTAM44 (b thr6ala ), and pTAM45 (b gln10ala ) (Table 5-1). The effects of the mutations were studied by the ability of the plasmids to compliment E. coli strain KM2 (b) (218). Growth on succinate minimal media was used as an initial qualitative gauge of enzyme activity in vivo since E. coli strains lacking F 1 F 0 ATP synthase cannot derive energy from nonfermentable sources. In each case, the strains expressing the different alanine substitutions grew comparably to the wild type strain (Table 5-1). PAGE 192 171 Effects of amino terminal mutations Since F 1 has little affinity for the membrane in the absence of intact F 0 total membrane associated F 1 -ATP hydrolysis activity was used as a test of F 1 F 0 ATP synthase complex assembly. Under conditions of high pH, F 1 can be released from the influence of F 0 (146), so the amount of ATPase activity in the solution was used as a measure of the amount of intact enzyme complex located in the membrane vesicles. The b asn2ala substitution, expressed from KM2/pTAM43, had no effect on enzyme assembly (Table 5-1). The b thr6ala and b gln10ala expressed from KM2/pTAM44 and KM2/pTAM45, had a slight, but not vitally significant effect on enzyme assembly. Membranes with a b thr6ala or b gln10ala incorporated into the F 1 F 0 ATP synthase complex had specific activities of about 77% of the wild type strain. F 1 F 0 ATP synthase-mediated ATP-driven proton pumping activity in membrane vesicles prepared from the mutants was used as an indication of coupled activity. Acidification of inverted membrane vesicles was examined by fluorescence of ACMA (Figure 5-3). The level of NADH-driven fluorescence quenching was monitored for all membrane preparations to demonstrate that the vesicles were intact and closed. The levels of NADH-driven fluorescence quenching were strong and directly comparable in every case (data not shown, for a representative figure, see Figure 2-10). Membranes isolated from cells expressing b asn2ala KM2/pTAM43, displayed an insignificant reduction, approximately 1.6 %, in coupled activity, correlating with the wild-type-like F 1 -ATP hydrolysis activity (Figure 5-2 and Table 5-1). A larger reduction in coupled activity, of about 11%, was observed in membrane vesicles isolated from cells in which a b the6ala or b gln10ala expressed from KM2/pTAM44 or KM2/pTAM45, respectively, was PAGE 193 172 Figure 5-3. ATP-driven energization of membrane vesicles prepared from b subunit membrane domain mutants. Cell membrane vesicles were prepared by differential centrifugation (see Materials and Methods). Membrane protein (200 g) was suspended in 3 mL of assay buffer (50 mM MOPS, 10 mM MgCl 2 pH 7.3). The fluorescent dye ACMA was added to a final concentration of 1 mM and fluorescence was recorded with excitation at 410 nm and emission at 490 nm. ATP was added as indicated to a final concentration of 1 mM. The samples for each trace have been labeled according to the mutation, so the strains used are as follows: b, membranes from strain KM2/pBR322; b wt KM2/pKAM14; b asn2ala, thr6ala, gln10ala KM2/pAWH24; b asn2ala KM2/pTAM43; b thr6ala KM2/pTAM44; b gln10ala KM2/pTAM45. PAGE 194 173 incorporated into F 1 F 0 ATP synthase (Figure 5-3). The decrease in coupled activity paralleled the reduction seen in F 1 -ATP hydrolysis activity (Table 5-1). Carboxyl Terminal Mutations Since the original insertions and deletions constructed by Dr. Paul Sorgen in the in the tether region, a collaborative effort has been made by other members of the laboratory to study the remainder of the b subunit (Figure 5-4). Insertions and deletions constructed in a hydrophobic region of the b subunit in the F 1 binding domain b 124-130 was accomplished by Dr. Deepa Bhatt. Four amino acid duplications, scattered throughout the dimerization and F 1 binding domains, were constructed by an undergraduate in the laboratory Stephanie Cole and the corresponding deletions are currently underway by another undergraduate, Megan Greenlee. Here, a four amino acid insertion and deletion was constructed at the extreme carboxyl terminus of the b subunit. Construction and growth characteristics of mutants The C-terminal region of the b dimer is in direct contact with the extreme C-terminal end of the subunit (3, 185, 200, 219, 220). Small deletions at the extreme C-terminal end of the b subunit had been shown to inhibit F 1 F 0 ATP synthase function (206, 288). Two plasmids were generated to study the affects of deleting or duplicating the final four amino acids of the b subunit. Plasmids pTAM51 and pTAM52 were expressed in the E. coli KM2 (b) cell line to express b 153end or b +153-156 respectively (Table 5-2). Since E. coli strains lacking functional F 1 F 0 ATP synthase cannot derive energy from nonfermentable sources, growth on succinate minimal medium was used as an initial determination of enzyme activity in vivo. As expected, KM2 cells complemented with PAGE 195 174 Figure 5-4. Amino acid insertion and deletion analysis of the E. coli b subunit. Dunn and coworkers have defined four domains in the b subunit: the membrane spanning (blue), tether (orange), dimerization (green) and -binding domains (pink). Dr. Paul Sorgen demonstrated that F 1 F 0 ATP synthase could retain sufficient levels of activity upon relatively large deletions or insertions in the tether and beginning of the dimerization regions. An analysis of the remainder of the b subunit is currently underway. Lines drawn below the sequence correspond to deletions, lines drawn above the sequence correspond to a duplication (insertion). The purple lines indicate the amino acids deleted and the cyan indicates the amino acids duplicated that resulted in a functional F 1 F 0 ATP synthase. Red lines indicate amino acids deleted (below sequence) or inserted (above sequence) that resulted in a loss of enzyme function. Grey lines indicate deletions that are currently under construction. plasmid pTAM51 (b 153end ) failed to grow on a succinate medium (Table 5-2). In contrast, the strain expressing b +153-156 KM2/pTAM52, grew comparably to the wild type strain. Hence, even though deletion of only four amino acids affected the ability of the b PAGE 196 175 dimer to interact with the F 1 subunit, addition of four amino acids to the carboxyl terminus did not interfere with the interaction of the b and subunits. Effects of carboxyl terminal mutation Total membrane associated F 1 -ATP hydrolysis activity was used as a test of F 1 F 0 ATP synthase complex assembly (Table 5-2). As expected, membranes with enzyme complexes incorporated with b 153end displayed a specific activity similar to the negative control, indicating virtually no interaction with F 1 On the other hand, membranes with Table 5-2. Aerobic growth properties and membrane-associated ATP hydrolysis activity of mutants expressing uncF(b) insertions or deletions throughout the b subunit Strains Description Growth 1 % Activity 2 KM2/pKAM14 (+) b wt Ap r +++ 100 KM2/pBR322 (-) b, Ap r 13 KM2/pSC1 3 b +59-62 Ap r +++ 82 KM2/pSC2 3 b +73-76 Ap r +++ 89 KM2/pSC3 3 b +90-93 Ap r +++ 103 KM2/pSC4 3 b +102-105 Ap r +++ 90 KM2/pMMG2 4 b 102-105 Ap r +++ nd KM2/pDB20 5 b 124-125 Ap r 12 KM2/pDB21 5 b +124-125 Ap r 19 KM2/pDB22 5 b +124-127 Ap r 19 KM2/pDB23 5 b +124-130 Ap r (+) 21 KM2/pDB24 5 b 2x(+124-125) Ap r (+) 16 KM2/ pDB25 5 b 3x(+124-125) Ap r (+) 11 KM2/pSC5 3 b +143-146 Ap r +++ 87 KM2/pMMG1 4 b 143-146 Ap r +++ nd KM2/pTAM52 b +153-156 Ap r +++ 108 KM2/pTAM51 b 153-156-his Ap r 12 1 E. coli strains were grown aerobically on succinate minimal medium supplemented with 40 M IPTG. Colony size was scored after 72-hr incubation at 37C as: +++, 1.0 mm; ++, 0.3-0.5 mm; +, ~0.1 mm; -, no growth; (+), sporadic colonies from reversions. 2 E. coli strains were grown in LBG supplemented with the appropriate antibiotics and 40 M IPTG. ATPase activities were measured as described under Materials and Methods. Units were calculated from the slope of the line based on three measurements with incubations for 12 minutes. Value presented as percentage (%) of the wild type control (KM2/pKAM14). 3 Work accomplished by Stephanie Cole. 4 Work accomplished by Megan Greenlee. 5 Work accomplished by Dr. Deepa Bhatt. PAGE 197 176 enzyme complexes incorporated with b +153-156 displayed a specific activity similar to the wild type strain. An indication of coupled activity was shown by F 1 F 0 ATP synthase-mediated ATP-driven proton pumping activity in membrane vesicles prepared from the mutants (data not shown, work accomplished by Stephanie Cole). Membranes containing the b 153end subunit exhibited no coupled activity as expected. Membranes isolated from cells expressing pTAM52 (b +153-156 ) exhibited coupled activity comparable to the wild type strain. The levels of NADH-driven fluorescence quenching were strong and comparable in every case (data not shown). The coupled activities observed reflected the observations from the F 1 -ATP hydrolysis activities (Table 5-2). Discussion In a collaborative effort, members of the laboratory have conducted an extensive mutational study of the entire length of the b subunit (Figure 5-5). At the amino terminal end of the b subunit, Andrew Hardy conducted a systematic mutational analysis of the membrane-spanning domain. His work established that there were specific sequence requirements in the b subunit membrane spanning domain and supported a model in which the extreme amino-termini of the two b subunits are in close contact with one another, accounting for most of the important b-b interactions in the membrane domain, and then flares apart as they cross the membrane (202). Here, an alanine scan was performed on three amino acids that had been shown to exhibit the strongest crosslinking efficiency in the membrane-spanning domain (b asn2ala, thr6ala, gln10ala ), yielding a defecting yet intact F 1 F 0 ATP synthase complex (202). It appears that there were adequate b-b interactions to support assembly of the enzyme, but the proton channel of F 0 PAGE 198 177 Figure 5-5. Mutations constructed throughout the b subunit. Shown are the approximate locations of the expressed b subunit mutants constructed and studied in our laboratory. Red indicates mutants constructed and studied in the present chapter. Shown in blue is the mutant created and studied in Chapter 3. The purple stars indicate mutants constructed and studied by Stephanie Cole. The blue stars indicate mutants constructed and studied by Megan Greenlee. The orange stars indicate a select few of the mutants constructed and studied by Dr. Deepa Bhatt. The green star designates the region of insertions and deletions studied by Dr. Paul Sorgen. The cyan star shows a mutant constructed and studied by Andrew Hardy. Growth indicates the ability of the strain to compliment the E. coli KM2 (b) cell line and grow on minimal succinate media. Symbols are as follows: +++, colonies 1.5 mm; -, no growth; (+), sporatic colonies from reversions. PAGE 199 178 was not functional, suggesting that the b subunit membrane spanning domain may have a role in allowing the proton channel of the a and c subunits to align correctly (202). Here, we demonstrated that mutation of only a single amino acid, at positions 2, 6 or 10 (b asn2ala, b thr6ala or b gln10ala ), did not significantly affect the function of the enzyme. In fact, the minimal loss of enzyme activity associated from any of the single mutations did not nearly add up to the total defect of all three mutations combined. The results support the idea that although no single amino acid is necessary for the arrangement of a functional proton channel, several sites contribute synergistically to the formation of a functional F 1 F 0 ATP synthase enzyme complex. In the tether region of the b subunit, Dr. Paul Sorgen had constructed a series of insertions and deletions and observed that the altered b subunits were capable of forming the peripheral stalk of a functional F 1 F 0 ATP synthase complex (193, 194). It was not known whether similar insertions or deletions could be accommodated in other regions of the b subunit where crucial interactions with F 1 subunits are believed to occur. Dr. Deepa Bhatt observed that neither insertions nor deletions were tolerated in a small stretch of hydrophobic amino acids found in the F 1 binding domain, b 124130 (Figure 5-5). Four amino acid insertions, as well as the corresponding deletions, scattered throughout the remainder of the soluble portion of the b dimer were constructed by Stephanie Cole and Megan Greenlee and were found to result in a functional F 1 F 0 ATP synthase complex (Figure 5-5). In the present chapter, we confirmed that a four amino acid truncation at the extreme carboxyl terminus of the b subunit (b 153end ) results in a partially assembled defective enzyme complex. However, duplication of the last four amino acids (b +153-156 ) had no appreciable affect on enzyme assembly or function. PAGE 200 179 The research presented here suggests the importance of the extreme amino and caborxyl termini. Although it was believed that the dimerization domain provided the majority of the protein-protein interactions by allowing the formation of the b subunit dimer (12, 13), our studies indicated that other regions of the b subunit are required for normal assembly of a functional F 1 F 0 ATP synthase complex due to contacts made with other subunits in the enzyme. Though the three amino acid substitutions at the amino terminus allowed dimerization and assembly of an intact F 1 F 0 ATP synthase, it appears that its sequence is crucial for the correct alignment of the proton channel formed by the a and c subunits. Likewise, the final four amino acids at the carboxyl terminus is not critical for the dimerization and partial assembly of the enzyme, however it is essential for the binding of F 1 due to its contacts with the subunit. PAGE 201 CHAPTER 6 CONCLUSIONS AND FUTURE DIRECTIONS Conclusions The work presented in the preceding chapters had a considerable impact on the way the b 2 homodimer of the E. coli F 1 F 0 ATP synthase is viewed and may provide some implications concerning the genetically dissimilar b-type subunit heterodimers found in higher organisms. A previous analysis of the b subunit performed in Dr. Cains laboratory had suggested that the b 2 dimer has a flexible characteristic in its tether region (193, 194). A two plasmid expression system novel to the studies of the E. coli b subunit was developed in Chapter 2 in order to determine if the apparent flexibility extended to the dimerization of the b subunit. This work provided interesting information concerning the tether region of the peripheral stalk. The two plasmid expression system was also utilized in Chapter 3 to study the function of individual b subunits in the peripheral stalk by exploiting b subunits with known defective mutations. From this work, a model has been developed concerning the positioning of the two b subunits relative to the F 1 F 0 ATP synthase complex and the intersubunit contacts made by the individual b subunits. On another note, Chapter 4 describes single cysteine substitutions that were generated in different length b subunits with the ultimate goal of studying the apparent flexibility of the b 2 dimer. And finally, Chapter 5 describes mutagenesis work performed on the extreme aminoand carboxyl-termini of the b 2 dimer in contribution to ongoing experiments performed by others in the laboratory. The following sections will summarize and discuss the implications of the results from Chapters 2, 3, 4 and 5 and 180 PAGE 202 181 then discuss the future directions that will be taken as a direct result of the work accomplished in this dissertation. Chapter 2: Integration of Unequal Length b Subunits into F 1 F 0 ATP Synthase The work presented in Chapter 2 stemmed from observations made by Dr. Paul Sorgen in our laboratory that b subunits with deletions of up to eleven amino acids and insertions of up to fourteen amino acids, corresponding to approximately 16 and 21 respectively, formed intact and functional F 1 F 0 ATP synthase complexes (193, 194). This work had suggested that the tether region of the b subunit possesses a certain degree of flexibility. However, it was not known whether this flexibility extended to the dimerization of two b subunits of unequal lengths and their incorporation into an enzyme complex. An experimental system was developed to allow expression of two different b subunit genes and determine whether the differing b subunits were assembled into an F 1 F 0 ATP synthase complex. The experiments involved an epitope tag system that allowed us to determine if the different b subunits segregated into homodimers, or alternatively, if a heterodimer of long and short b subunits can be incorporated into an F 1 F 0 ATP synthase complex. The histidine and V5 epitope tags introduced into the b subunits did not appreciably affect enzyme assembly or function. Expression of two different wild-type length b subunits led to three distinct F 1 F 0 ATP synthase complexes in the same cell; 1) a homodimer of histidine-tagged b subunits, 2) a homodimer of V5-tagged b subunits and 3) a heterodimer consisting of a histidine-tagged b and a V5-tagged b subunit. More importantly, three different F 1 F 0 ATP synthase complexes were present even when the b subunits were not of identical length. We observed dimerization of b PAGE 203 182 subunits between b +7-his and both b wt-V5 and b 7-V5 This demonstrates that b subunits that differ in length by at least 14 amino acids can be incorporated into an enzyme complex. Given that the tether domain is likely an helix, the difference in length between the b subunits would be approximately 21 Dimerization could occur in two ways. First, assuming that the transmembrane domains were in parallel, the hydrophilic domains could be out of register. Alternatively, we favor a conformation in which both the transmembrane domains and the dimerization domains as defined by Dunn and coworkers (13) exist in parallel. This would require that a section of the tether domain in the longer b subunit be out of contact with the shorter b subunit (Figure 2-14). It is likely that a parallel alignment of the dimerization domain is required for enzyme assembly. Recent electron microscopy and NMR studies have revealed a distinctive 20 bend in the b dimer within the tether domain (27, 136, 196). The research presented here suggests the possibility of straightening or further bending of the two b subunits within the peripheral stalk and lends support to the concept of a flexible peripheral stalk. This raises the question, why should the tether domain be so flexible as to allow insertions, deletions and dimerization of b subunits of unequal lengths? If one views the peripheral stalk to be a rope-like structure linking F 1 to F 0 then its position holding F 1 against the rotation of the central stalk would not be expected to be the same for counterclockwise and clockwise rotation. Flexibility of the tether domain might facilitate reorienting the peripheral stalk to act as a stator for rotation in either direction during ATP synthesis and ATP hydrolysis (Figure 1-7). The ability to generate and purify F 1 F 0 complexes with two genetically different b subunits provides a potentially useful experimental tool. It is now feasible to specifically PAGE 204 183 label a single b subunit within a purified complex. This will facilitate biochemical modification experiments and the use of physical methods in future experiments. Chapter 3: Genetic Complementation between Mutant b Subunits in F 1 F 0 ATP synthase The work documented in Chapter 3 directly stemmed from the experimental system developed in Chapter 2. Our ability to genetically express two different b subunits in the same cell and detect heterodimer formation provided us with the unique opportunity to study each of the b subunits in the peripheral stalk as an individual b subunit rather than a dimer forming a single entity. A major problem that had plagued all previous mutagenesis studies of the b subunit was that mutations constructed in the uncF(b) gene affected both subunits of the b homodimer. One missense mutation led to two amino acid replacements. However, the asymmetric nature of the F 1 F 0 ATP synthase enzyme complex suggested that the functional role of each b subunit should not necessarily be considered equivalent. Protein-protein contacts made by one b subunit cannot be made by the other. The work presented Chapter 3 exploited three b subunit mutations that had already been characterized and shown to result in completely defective F 1 F 0 ATP synthase complexes when expressed in both b subunits. An evolutionarily conserved arginine, b arg36 located near the interface between the membrane and tether domains had been found to be crucial for F 1 F 0 ATP synthase function (215). Enzyme complexes incorporated with a b arg36ile-V5 or b arg36glu-V5 were found to be intact yet functionally defective. Second, the C-terminal last four amino acids had been shown to be essential for the F 1 binding domain (206, 288). Enzyme complexes with a b 153end-his were found to be only partially assembled. Thirdly, insertions and deletions in a hydrophobic stretch of amino acids in the b subunit corresponding to residues b 124-130 (VAILAVA) resulted in PAGE 205 184 a complete loss of enzyme function. The b subunit dimer was not found in membranes when cells expressed only the b +124-130 subunit. Heterodimerization was detected in cells expressing either arg36 mutation, b arg36ile-V5 or b arg36glu-V5 with b wt-his b 153end-his /b wt-V5 and b +124-130-his /b wt-V5 (Figure 3-10). Dimerization was also observed in membrane preparations from cells expressing both b 153end-his /b arg36ile-V5 and b 153end-his /b arg36glu-V5 More importantly, enzyme complexes incorporated with the mutant heterodimers were functionally active, suggesting that each of the b subunits were complementing the other to form an intact and functional enzyme complex. This observation demonstrated unambiguously that F 1 F 0 ATP synthase complexes containing b heterodimers were active and provided evidence that each of the individual b subunits provide specialized functions within the peripheral stalk. Clearly, each of the mutant b subunits compensates for what the other is lacking. The work accomplished in this chapter raises a question concerning the relative positions of the individual b subunits of the peripheral stalk. In order for F 1 F 0 ATP synthase containing the two different mutant b subunits to be intact and functional, it is likely that the b arg36ile-V5 (or b arg36glu-V5 ) subunit must be positioned such that its extreme C-terminus forms the appropriate contacts with the subunit of F 1 Similarly, the b 153end-his subunit must be positioned so that its b arg36 makes the appropriate contacts with the F 0 subunits (Figure 3-11). Incorrect positioning of the mutant b subunits during assembly might be expected to lead to an inactive or partially assembled enzyme complex. An answer to this question will be a goal discussed in Future Directions. PAGE 206 185 Chapter 4: Development of Cysteine Chemical Modifications of Altered b Subunits The work described in Chapter 4 was performed in order to examine the nature of the apparent flexibility of the tether region. It has been shown by Dr. Paul Sorgen that an eleven amino acid deletion and a fourteen amino acid insertion in the the b subunit spanning the tether domain and the beginning of the dimerization domain was accommodated by the enzyme (Figure 4-5). Assuming -helical structure, this 21 insertion and 16 deletion corresponds to well over a third of the length spanning from the top of the membrane to F 1 or right under a quarter of the length spanning towards the top of F 1 In contrast to the previously accepted role of the b subunit as a rigid stator, these observations suggested that the role of the b dimer is more of a flexible or elastic structural feature during rotational catalysis. In Chapter 4, cysteine chemical modifications were created in the and b subunits to provide reactive thiols groups for future labeling and measurement studies. A set of twelve unc operon expression plasmids that encode amino acid substitutions were developed to void the F 1 F 0 ATP synthase complex of all known reactive thiols as well as generate strategically placed cysteines. Cysteines were chosen because the thiol side chain is highly reactive and can be modified by maleimide reagents. The cysteine mutations did not affect enzyme assembly or function. Plasmids were designed to express a single cysteine at one of two locations in the subunit, cys64 or cys140 as well as one position, either above or below the site of insertion or deletion in the b subunit, b cys84 or b cys43 (Figure 4-5). The idea for establishing a single reactive cysteine in both F 1 and F 0 is to use them as targets for labeling with fluorescence compounds (see Future Directions). One major limitation plagued this line of research. F 1 F 0 ATP synthase is incorporated with a homodimer of b PAGE 207 186 subunits, hence two b subunits, each with a reactive cysteine. Since only one b subunit was desired to be labeled, a system necessarily had to be developed to allow purification of F 1 F 0 ATP synthase complexes with two genetically different b subunits: one cysteine-less and one with a single cysteine. With the epitope-tag system developed in Chapter 2, this should not be a problem for future studies. Chapter 5: Mutagenesis of the Amino and Carboxyl Termini of the b subunit in F 1 F 0 ATP Synthase In a collaborative effort, members of the laboratory have conducted an extensive mutational study of the entire length of the b subunit (Figure 5-5). The work illustrated in Chapter 5 was achieved in order to contribute to two other projects in the lab. The chapter focuses on the extreme aminoand carboxyl-termini of the b subunit. At the amino end, a systematic mutational analysis of the membrane domain was performed by Andrew Hardy in our laboratory. His work established that there were specific sequence requirements in the b subunit membrane spanning domain and supported a model in which the extreme amino-termini of the two b subunits are in close contact with one another, accounting for most of the important b-b interactions in the membrane domain, and then flares apart as they cross the membrane (202). Three amino acids that have been shown to exhibit the strongest crosslinking efficiency in the membrane-spanning domain were replaced with alanines (b asn2ala, thr6ala, gln10ala ), yielding a defecting F 1 F 0 ATP synthase complex. It appeared that there were adequate b-b interactions to support assembly of the enzyme, but the proton channel of F 0 was not functional, suggesting that the b subunit membrane spanning domain may have a role in allowing the proton channel of the a and c subunits to align correctly (202). Work performed in contribution to this dissertation demonstrated that mutation of only a single amino acid, at positions 2, 6 or PAGE 208 187 10 (b asn2ala, b thr6ala or b gln10ala ), did not significantly affect the function of the enzyme. In fact, the minimal loss of enzyme activity associated from any of the single mutations did not nearly add up to the total defect of all three mutations combined. The results support the idea that although no single amino acid is necessary for the arrangement of a functional proton channel, several sites contribute synergistically to the formation of a functional F 1 F 0 ATP synthase enzyme complex. In the other line of research described in Chapter 5, mutations were constructed at the extreme carboxyl-terminus of the b subunit in contribution to an extensive insertion and deletion analysis. In the tether region of the b subunit, Dr. Paul Sorgen had constructed a series of insertions and deletions and observed that the altered b subunits were capable of forming the peripheral stalk of a functional F 1 F 0 ATP synthase complex (193, 194). It was not known whether similar insertions or deletions could be accommodated in other regions of the b subunit where crucial interactions with F 1 subunits are believed to occur. Dr. Deepa Bhatt observed that neither insertions nor deletions were tolerated in a small stretch of hydrophobic amino acids found in the F 1 binding domain, b 124130 (Figure 5-5). Four amino acid insertions, as well as the corresponding deletions, scattered throughout the remainder of the soluble portion of the b dimer were constructed by Stephanie Cole and Megan Greenlee and were found to result in a functional F 1 F 0 ATP synthase complex (Figure 5-5). Work described in this dissertation confirmed that a four amino acid truncation at the extreme carboxyl terminus of the b subunit (b 153end ) results in a partially assembled defective enzyme complex. However, duplication of the last four amino acids (b +153-156 ) had no appreciable affect on enzyme assembly or function. PAGE 209 188 The research presented here suggests the importance of the extreme amino and carboxyl termini. Although it was believed that the dimerization domain provided the majority of the protein-protein interactions by allowing the formation of the b subunit dimer (12, 13), our studies indicated that other regions of the b subunit are required for normal assembly of a functional F 1 F 0 ATP synthase complex due to contacts made with other subunits in the enzyme. Though the three amino acid substitutions at the amino terminus allowed dimerization and assembly of an intact F 1 F 0 ATP synthase, it appears that its sequence is crucial for the correct alignment of the proton channel formed by the a and c subunits. Likewise, the final four amino acids at the carboxyl terminus is not critical for the dimerization and partial assembly of the enzyme, however it is essential for the binding of F 1 due to its contacts with the subunit. Future Directions One of the major objectives for the field as a whole is to obtain a high-resolution structure of an intact F 1 F 0 ATP synthase complex, or at least a complete F 0 The high resolution structure of F 1 confirmed many existing views and it was also a valuable tool used to assist a plethora of experimental planning that concerned F 1 We believe the same will hold true for F 0 A high-resolution structure will help to unveil many of the mysteries of F 0 such as proton translocation, and the mechanism in which the c 10 ring rotates while the adjacent a and b subunits remain in place. Although the work illustrated in this dissertation has contributed a great deal of novel information concerning the b subunit of the E. coli F 1 F 0 ATP synthase, it also left many open questions for future investigators. The following section discusses some of the future directions that directly resulted from work accomplished in this dissertation. PAGE 210 189 Complementing Mutant b Subunits The data presented in Chapter 3 leads to the immediate question, do additional pairs of mutant b subunits exist that can mutually complement each other? The asymmetric nature of the F 1 F 0 ATP synthase complex suggests that only one b subunit must make the appropriate contact within the enzyme. However, this trait may sometimes be restricted by the quaternary structure of the b subunit dimer. In other words, due to the coiled coil arrangement of the two b subunits, one region of a particular b subunit may make a contact near the amino terminal end and then that same b subunit may also align to make an additional important contact up towards the carboxyl terminal end. Pairs of plasmids encoding a mutant uncF(b) gene, which expresses a defective b subunit, are currently being cotransformed into KM2 (b) cells and studied for their ability to grow on succinate. Since the expression system requires one plasmid to carry the ampicillin resistance gene and the second to carry the chloramphenicol resistance gene, an undergraduate in the laboratory, Stacia Howard, is currently cloning some of the other uncF(b) gene mutants in the laboratorys possession into chloramphenicol resistant plasmids. Table 6-1 describes preliminary evidence of ATP-driven proton pumping of various combinations of mutant b subunits. Indeed it appears that at least one pair of b mutants, b N2A, T6A, Q10A /b arg83pro do not mutually suppress each other (Table 6-1). Furthermore, it appears that some of the complementing pairs have more activity than the other. Whether the apparent reduction in activity is due to a smaller percentage of heterodimeric F 1 F 0 or whether the assembled heterodimeric species are still impaired will require purification of the heterodimeric complexes to homogeneity (described next). PAGE 211 190 Table 6-1. Preliminary data of coexpressed mutant b subunits. b +124-130 b 153end b arg83pro b arg36ile *** *** b arg36glu *** *** *** b N2A, T6A, Q10A * b +124-125 nd nd *** b +124-127 * b 124-127 nd b ala79lys *** *** *** Defective b subunits were coexpressed in the KM2 (b) cell line. Membrane preparation and preliminary ATP-driven proton pumping were accomplished as described in Chapter 2 Materials and Methods. Symbols: ***, proton-pumping activity at least 20% of samples prepared from wild type strains; *, <10% activity; -, no activity; nd, no data. Function of F 1 F 0 ATP Synthase Incorporated with b Subunit Heterodimers Work discussed in Chapters 2 and 3 have provided evidence that F 1 F 0 ATP synthase complexes can be incorporated with b subunit heterodimers. Although it was unambiguously demonstrated that the heterodimeric F 1 F 0 ATP synthase complexes were functional, a direct confirmation of activity will be pursued by performing activity assays on homogeneous enzyme preparations. These assays will also allow us to directly determine the activity of the complementing, double b subunit mutants (Chapter 3) relative to wild type F 1 F 0 complexes. A detailed account of a developing protocol for purifying the F 1 F 0 ATP synthase complexes with b subunit heterodimers can be found in Appendix B. Positions of the Individual b Subunits in F 1 F 0 ATP Synthase The two b subunits of F 1 F 0 ATP synthase have traditionally been viewed as a single functional entity. However, work described in Chapter 3 indicated that this may not be the case. F 1 F 0 ATP synthase complexes incorporated with a heterodimeric b subunit in which each b subunit contains a defective mutation were functional. This observation suggested that the individual b subunits have specialized roles within the enzyme PAGE 212 191 complex. Furthermore, the asymmetric nature of the enzyme suggests that the two b subunits cannot participate in the exact same interactions. For example, the b subunit cannot have the same contacts with the single a in the membrane-spanning region and likewise with the subunit at the carboxyl-terminus. The unique experimental system described in Chapters 2 and 3 will be used to investigate the individual roles and positions of each b subunit. The experiments will heavily rely on the use of defective b subunits along with very efficient and well characterized chemical crosslinking techniques. Length of the Peripheral Stalk in F 1 F 0 ATP Synthase Complexes Incorporated with Shortened and Lengthened b Subunits FRET. Relatively large insertions and deletions in the tether region of the b subunit were accommodated by the F 1 F 0 ATP synthase complex and allowed retention of function (193, 194). Comparing the length of the wild type, shortened and lengthened peripheral stalks will give some insight on the overall possible conformation of the altered F 1 F 0 ATP synthase complex. The F 1 F 0 ATP synthase enzyme complex must, in some way, adapt to the shortened or lengthened b subunits. This can occur in at least one of two ways: i) distortion of F 1 to accommodate the change in length of the peripheral stalk or ii) distortion of the peripheral stalk itself. If the former was true, F 1 could become distorted by bending of the central stalk or compression of the 3 3 hexamer and the rigid stalk hypothesis would gain favor. Since F 1 F 0 ATP synthase function requires the ability of the central stalk to rotate freely while making specific interactions within the catalytic hexamer, this mechanism is highly unlikely. If the latter situation were true, distortion would be limited to the b dimer and the flexible stalk hypothesis would hold true. This could occur by straightening or increasing the naturally occurring PAGE 213 192 20 bend in the shortened or lengthened peripheral stalk, respectively, or by stretching or compressing the secondary structure in the b dimer. The use of fluorescence resonance energy transfer (FRET) will provide evidence as to which of the hypotheses is true. The flexible stalk hypothesis suggests that the overall conformation of the F 1 F 0 ATP synthase complex is maintained regardless of the alterations in the length of the b subunit and the changes in length are accommodated by changes in the tether region. If the flexible stalk hypothesis holds true, two predictions can be made based on the relative measured length of the shortened, lengthened and wild type length b subunits: i) the altered length of the tether region will not effect on the measured distance between a fixed site in F 1 and a site in the dimerization domain of the b subunit and ii) the distance between a fixed site in F 1 and a site below the tether region alteration will vary by only a few due to the bending or straightening of the tether region. On the other hand, if the rigid stalk hypothesis holds true a change in distance from a fixed site in F 1 and a site below the alteration would result in an approximately 16 difference between the wild type length b and b 11 The length of the peripheral stalks with altered length b subunits will be measured by FRET. FRET requires the introduction of single donor and acceptor fluorophores into a purified F 1 F 0 ATP synthase complex. A common approach to performing FRET is to advantageously place a single cysteine at each end of the distance to be measured. Fluorescent maleimide derivatives are highly and specifically reactive with cysteine side chains. In order to perform FRET, single cysteines were strategically placed within the subunit of F 1 and b subunit of F 0 This work has been accomplished and is discussed in Chapter 4. These cysteines will be used to donorand acceptor-label F 1 and F 0 with PAGE 214 193 Figure 6-1. Design of FRET experiments to measure the peripheral stalk. The fluorophores will be added to the thiol side chains of the cysteines generated in Chapter 4. The stars indicate the approximate positions of the d cys140 b cys84 and b cys43 The green shaded area of the b subunit represents the location of the insertions or deletions. maleimide reagents for the FRET studies (Figure 6-1). F 1 can readily be stripped from F 0 each cysteine labeled separately with a "donor" and "acceptor" fluorescent compound, and then stoichiometrically reconstituted under conditions of high ionic strength (203). With a single donor fluorophore positioned in the subunit and a single acceptor fluorescent compound located above or below the region of insertion or deletion in the tether domain of the b subunit, physical measurements can be calculated and compared (Figure 6-1). PAGE 215 194 One major limitation plagued this line of research. F 1 F 0 ATP synthase is incorporated with a homodimer of b subunits, hence two b subunits, each with a reactive cysteine. Since FRET requires only one b subunit be labeled, a system necessarily had to be developed to allow purification of F 1 F 0 ATP synthase complexes with two genetically different b subunits: one cysteine-less and one with a single cysteine. The system utilizes epitope tags placed on the b subunits and was discussed in detail in Chapter 2. Future FRET studies will use this epitope-tagged scheme in order to express F 1 F 0 ATP synthase complexes with a b dimer containing only one cysteine. Alternative approach. Another approach for the investigation of a potentially flexible peripheral stalk may also be attempted in future experiments. An innovative set of experiments conducted by members of the Dunn laboratory made use of different sized fluorescent proteins, ranging from the 12 kDa cytochrome b 562 protein to the 30 kDa flavodoxin reductase protein with a 20 residue linker (100). The proteins were fused to the subunit of the central rotor stalk. If the fusion protein were small enough, one would expect the central stalk to rotate freely. If the fusion protein were too bulky, one could imagine that the protein would run into the peripheral stalk during rotational catalysis. As the fusion proteins became larger, an inhibition of activity resulted due to the bulky fluorescent protein running into the peripheral stalk during rotation. The growth curve of the bacteria was also monitored and revealed that the larger fusion proteins inhibited growth more than the smaller proteins. If this same technique were to be applied to F 1 F 0 ATP synthases incorporated with b 11 and compared to the activity of complexes incorporated with b +14 conclusions could be made concerning the flexibility of the peripheral stalk. For example, if the 20 bend in the b subunit were straightened in PAGE 216 195 Figure 6-2. Model of rotation inhibition due to a fusion protein on the subunit. A natural 20 bend in the b subunit has been observed near the surface of the membrane (136). Shortening the b subunit by 11 amino acids (left panel) or lengthing by 14 amino acids(right panel) may be accompanied by the straightening or further bending, respectively, of this 20 bend. Genetic fusion proteins on the subunit have been described by Cipriano et al. (represented by the yellow oval) (100). The larger the fusion protein, the greater the inhibition of activity due to the inability of the central stalk to rotate during catalysis. We propose a model in which F 1 F 0 ATP synthase complexes incorporated with shorten or lengthened b subunits may accommodate smaller or larger fusion proteins, respectively. the shortened b subunit, inhibition of growth might become apparent with the smaller fusion proteins (Figure 6-2). Conversely, in complexes incorporated with the lengthened b subunit, the tether region may bend further, allowing the larger fusion proteins to pass between the central and peripheral stalks. Comparison of the growth properties of the two extreme tolerable lengths of the b subunit could lend support to the flexible stalk model. Other Implications How does the work presented in this dissertation affect the way in which the b subunit homologues of F 1 F 0 ATP synthase are currently viewed? This work suggests that the importance of the b subunit dimer may be reflected in the fact that higher organisms PAGE 217 196 evolved to encode multiple b subunit equivalents. The b subunit is the least conserved subunit of F 1 F 0 ATP synthase. In E. coli, the peripheral stalk of the F 1 F 0 ATP synthase complex exists as a dimer of identical b subunits expressed from a single gene. However, the equivalent of the bacterial b subunit in photosynthetic bacteria and plants exists as two different subunits, referred to as b and b, and the mammal counterpart exists as at least four subunits, expressed from two and four separate genes, respectively (Figure 1-8). The second non-identical b-type subunit found in photosynthetic organisms has been assumed to appear in order to gain additional functions in connection with photophosphorylation, though there is no direct evidence of this proposal (295, 296). Here, I would like to propose a different view concerning the multiple b-type subunit genes found in higher organisms. Work described in this dissertation suggested that the bacterial enzyme does not require two identical b subunits to form the dimer. Two different length b subunits, with a size difference of at least14 amino acids, were capable of forming the b dimer of an intact F 1 F 0 ATP synthase complex. Interestingly, the b and b subunits of photosynthetic organisms are rarely of equal length, with size differences ranging from about 4 to 40 amino acids due to extensions at either the aminoor carboxyl-termini or sometimes due to gaps found within the center of a b subunit. Also, in work presented in this dissertation, a defective mutation in one region of the b subunit has been shown to be overcome by dimer formation with a second b subunit that contained defective mutation in a different region but had a wild-type sequence in the region of the former defective b subunit. This observation has implications concerning the two individual b subunits in which each b subunit makes individual contacts within the enzyme complex. I believe the presence of the two b subunits are required for the PAGE 218 197 structural stability of the peripheral stalk. However, I would like to take this observation a step further and ask the following question: Why do bacteria encode only one b subunit gene, whereas higher organisms encode multiple b-type subunit genes, if single amino acid mutations are capable of destroying the enzyme function? One could speculate that bacteria do not require a functional F 1 F 0 ATP synthase in order to survive. Though growth would be slow, bacteria can persist via glycolysis until environmental pressures either cause death, a reversion of the uncF(b) back to a wild-type gene, or a second site suppressor mutation. Higher organisms, on the other hand, do not have the luxury of solely surviving from glycolysis due to their higher energy needs. Selective pressures may have caused higher organisms to evolve to encode two different b subunit genes, where important amino acids may occur in only the b subunit or the b subunit. This is reflected in my finding that the b or the b subunits in plants have only about 15-20% identity with the E. coli b subunit, but the amino acids conserved in the chloroplast b and b subunits generally occur at different residues, suggesting that the b dimer, as a whole, actually contains a larger number of conserved residues that are involved in making the functional contacts (Figure 6-3A, C). In fact, when the sequences of the b and b sequences were manually merged together, such that identical or similar amino acids replaced the nonconserved residues, the percent identity and similarity of the fictional merged b subunit nearly doubled (Figure 6-3B, C). It appears the actual number of conserved residues is actually higher than believed to be, the difference being, the conserved residue need only to be in one or the other b subunit. This proposal may account for why the b-type subunits are the least conserved of all the F 1 F 0 ATP synthase. If only one of the b-type subunits is required to make the appropriate contacts, mutations PAGE 219 198 occurring in the other b-type subunits of the organism would not have a dramatic affect on the enzyme function. PAGE 220 199 PAGE 221 200 Figure 6-3. Sequence alignments of subunits b and b from various species with the b subunit of E. coli. The E. coli sequence is entirely in bold script. The blue star indicates residues that are similar across all of the species. The red arrow indicates residues that are identical across all of the species. The hyphen indicates gaps. Sequences were randomly chosen from the National Center for Biotechnology Information (NCBI) database. Residues indicated in red bold script in the b and b subunits of the various species are identical to the corresponding residue in the E. coli b subunit. Residues indicated in blue are similar to the E. coli b subunit. In parenthesis are the percent identity followed by the percent similarity. A) Sequence alignment of subunits b and b of other species with the b subunit of E. coli. B) The b and b subunits of each species were merged together so that the identical or most similar amino acid (relative to E. coli) replaced nonconserved amino acids. C) Graphical representation of sequence alignments. PAGE 222 201 PAGE 223 APPENDIX A MUTAGENIC OLIGONULCEOTIDES Many of the mutations created during the course of this dissertation were via site-directed mutagenesis, performed either by means of a Stratagene Quikchange XL kit or by ligation-mediated mutagenesis. For the Quikchange XL procedure, oligonucleotides containing the desired mutation(s) were designed to anneal to the same sequence on opposite strands of the plasmid (sense and antisense primers). When possible, a silent mutation was encoded to add or delete a restriction endonuclease recognition sequence to allow for easy screening of the mutation. Primers were optimally designed by ensuring the mutation was in the middle of the sequence, a cytosine (C) or guanine (G) flanked both ends of the sequence, and the melting temperature (T m ) was greater than or equal to 78 C. The T m was calculated as T m =81.5+0.41(%GC)-675/N-%mismatch, where N was the primer length (bases). When introducing insertions or deletions, "%mismatch" was dropped from the formula. For a detailed description of the reaction, see Chatper 2 Recombinant DNA Techniques under Materials and Methods. The resulting plasmids that carried the desired mutation were then transformed into competent DH5 cells, purchased from Life Technologies, and grown on LBG plates supplemented with the appropriate antibiotic. Plasmids carrying the desired mutation(s) were screened for by restriction endonuclease analysis and then the nucleotide sequences were directly determined by automated sequencing in the core facility of the University of Florida Interdisciplinary Center for Biotechnology Research (ICBR). A description of the oligonucleotides follows. All oligonucleotides are listed in the 5 to 3 direction. 202 PAGE 224 203 Table A-1. Oligonucleotide sequences. Primer Sequence TB1 5 ctaaatagaagcatgctgctgtgcaccaccaccaccaccacaatcttaacgcaacaatcctcggc 3 TB2 5 gccgaggattgttgcgttaagattgtggtggtggtggtggtgcacagcagcatgcttctatttag 3 TB3 5 ctaaatagaggcattgtgctatgtacccatatgacgtgccggactacgcgaatcttaacgcaacaatcctcggc TB4 5 gccgaggattgttgcgttaagattcgcgtagtccggcacgtcatatgggtacatagcacaatgcctctatttag TB5 5 ctgtaaggagggaggggcatgcgtctgaattttattacg 3 TB6 5 cgtaataaaattcagacgcatgcccctccctccttacag 3 TB7 5 ggagggagggggaggctgctgtgc 3 TB8 5 gcacagcagcctccccctccctcc 3 TB10 5 cgtctgtcacgcaaagttaagctgaattcgaaaattgataagtctgtaatggcagg 3 TB11 5 cctgccattacggacttatcaattttcgaattcagcttaactttgcgtgacagacg 3 TB12 5 ctgttcgttctgttctccatgaagtacgtttggccgcc 3 TB13 5 ggcggccaaacgtacttcatggagaacagaacgaacag 3 TB14 5 gaaattgctgactgccttgcttccgcagaacgagcacataag 3 TB15 5 ccttatgggctcgttctgcggaagcaaggcagtcagcaatttc 3 TB16 5 gcgaacaaacgccgctgccagattctcgacgaagc 3 TB17 5 gcttcgtcgagaatctggcagcggcgtttgttcg 3 TB18 5 gccgagtcgtttatcgcagtttctggtgagcaactggac 3 TB19 5 ccagttgctcaccagaaaccgcgataaacgactc 3 TB20 5 ctgtacgtgatgttcgctgtcgc 3 TB21 5 gctcgcccctacgccaaagcagc 3 TB23 5 ggcatgaaagttaagtctactggccggatcctggaag 3 TB24 5 cttccaggatccggccagtagacttaactttcatgcc 3 TB25 5 ctgctgcgatggaaaaacgtctgtcac 3 TB27 5 gggagggggaggcagatatgcaccaccacc 3 TB28 5 ggtggtggtgcatatctgcctccccctccc 3 TB29 5 gtcctgccagcgttctacactttggtg 3 TB30 5 gaggcattgtgctgtgtggccgccattaatggc 3 TB31 5 gccattaatggcggccacacagcacaatgcctc 3 TB32 5 gaggcattgtgctgtgcaccaccaccaccaccaccaccaccaccactggccgccattaatggc 3 TB33 5 gccattaatggcggccagtggtggtggtggtggtggtggtggtggtgcacagcacaatgcctc 3 TB36 5 ggagggaggggctgatgcaccaccac 3 TB37 5 gtggtgcatcagcccctccctcc 3 TB38 5 cgtggataaacttgtcgctgagctcggtaaaccgatcccgaacccg ctgctgggtctggactctacctaaggagggaggggctgatgtct 3 TB39 5catcagcccctccctccttaggtagagtccagacccagcagcgg gttcgggatcggtttaccgagctcagcgacaagtttatccacg 3 TB405 catcgtggataagctttaaggagggaggggctg 3 TB415 cagcccctccctccttaaagcttatccacgatg 3 TB40+ 5 gataaacttgtcgctgaactggtcgctgagctctaaggagggaggg 3 TB41+ 5 ccctccctccttagagctcagcgaccagttcagcgacaagtttatc 3 PAGE 225 204 Table A-2. Oligonucleotide description. Primer Conc. 1 Sense 2 Description T m 3 TB1 0.892 S TB2 0.606 A addition of histidine-epitope tag (HHHHHH) to the N-terminus of the b subunit; +SphI 85 TB3 0.329 S TB4 0.414 A addition of HA-epitope tag (YPYDVPDYA) to the N-terminus of the b subunit; +NdeI 85 TB5 2.846 S TB6 2.236 A addition of SphI site 3 of the b subunit for cloning purposes; + SphI 78 TB7 1.951 S TB8 1.593 A mutation of SphI site 3 of the b subunit and addition of a favorable ATG prior to next gene; -SphI 78 TB10 0.308 S TB11 0.650 A cys140ser mutation; +EcoRI; -ClaI 80 TB12 0.977 S TB13 1.538 A b cys20ser mutation; -SnaBI 79 TB14 1.151 S TB15 1.446 A b gly43cys mutation; -Bsp1285I 77 TB16 1.100 S TB17 1.290 A b ser84cys mutation; -XbaI 75 TB18 1.366 S TB19 1.242 A cys64ser mutation 82 TB20 2.684 S sequences b in the sense (not a good one) 75 TB21 2.422 S sequences in the sense direction 81 TB23 1.380 S TB24 1.640 A cys90ser mutation; +BamHI 77 TB25 5.016 S sequences in the sense direction 78 TB27 0.886 S TB28 1.235 A mutations to create Shine Delgarno sequence upstream of the 2 nd b gene in mix plasmids 76 TB29 5.279 A sequences b in the inverse complement direction 79 TB30 4.334 S TB31 4.608 A deletion of residues 1-24 of b subunit to create b sol 85 TB32 8.301 S TB33 7.618 A addition of histidine-epitope tag (HHHHHHHHHH) to the N-terminus of the b sol subunit 78 TB36 0.439 S TB37 0.477 A mutations to create a better Shine Delgarno sequence upstream of the 2 nd b gene in mix plasmids 78 TB38 12.33 S TB39 9.310 A addition of V5-epitope tag (GKPIPNPLLGLDST) to the C-terminus of the b subunit; +SacI 79 TB404.522 S TB414.891 A deletion of last 4 amino acids at the C-terminus of the b subunit; +HindIII 76 TB40+ 1.109 S TB41+ 0.983 A duplication of last 4 amino acids at the C-terminus of the b subunit; +SacI 78 1 Concentration of stock oligonucleotide in g/l, stored in the -20C. 2 Direction of the primer: S, sense; A, antisense. 3 Melting temperature in C. PAGE 226 205 PAGE 227 APPENDIX B DEVELOPING PROTOCOL FOR PURIFYING F 1 F 0 ATP SYNTHASE Purification of Enzyme Complexes Incorporated with b Subunit Heterodimers The purification procedure described below has been attempted one time; therefore a detailed account of the current protocol is given here. Once the particulars of the procedure are optimized, the same protocol will be applied to all the F 1 F 0 ATP synthases incorporated with different combinations of heterodimeric mutant b subunits. For the sake of simplicity, the different b subunits are simply referred to as b V5 and b his Culture Inverted membrane vesicles from KM2(b) strains expressing the desired b subunits will be prepared essentially as described previously and optimized for large-scale membrane preparations (199, 297). One of the advantages of studying a bacterial enzyme is the practically unlimited amount of bacteria that may be grown and harvested. The following can be scaled up or down to meet the experimental needs. The bacteria will grown by inoculating 20 mL starter culture, grown overnight, into a 2 L Erlenmeyer flask holding 1 L LBG supplemented with both ampicillin (Ap) (100 g/mL) and chloramphenicol (Cm) (25 g/mL). Similarly, 1 mL of the starter culture will be inoculated into a nephalo flask containing 50 mL LBG (Ap and/or Cm) to monitor growth. For the purposes of the large-scale purification, a total of 10 L LBG-Ap-Cm, contained in 10 flasks, will be grown. The bacteria will be grown at 37 C in a New Brunswick Scientific incubator shaker (220 rpm) and the turbidity monitered using a Klett-Summerson photoelectric colorimeter. IPTG (40 M) will be added when the 206 PAGE 228 207 turbidity reaches 75 Klett units and the cells will subsequently be collected when the turbidity reaches 150 Klett units. The bacteria will be harvested by centrifugation for 10 minutes at 8,000Xg in a Sorvall GSA rotor. The pellets will be rinsed once with TM buffer (50 mM tris-HCl, 10 mM MgSO 4 pH 7.5), weighed, and then stored at C until the following day. Typically, a 10 L culture will yielded approximately 25 g bacteria. Disruption of Bacteria The 25 g frozen bacteria will be allowed to thaw at room temperature and resuspended in a final volume of 200 mL TM buffer. Resuspension requires rigorous vortexing, until the bacterial clumps dislodge from the walls of the bottles, and then continuously pipetted with a 25 mL pipette until no cell clumps are apparent. It is important to sufficiently break all clumps of bacteria in order to efficiently break open the cells. The bacteria will be treated with lysozyme in order to disrupt the bacterial cell wall. Prior to lysozyme treatment, the cells will be prepared by dissolving 0.91 g ethylenediamine tetra acetic acid (EDTA) in the suspension. The 200 mL bacterial suspension should be stirred with a magnetic stir bar at room temperature throughout the lysozyme treatment. 200 mL of 200 mM Tris-HCl, pH 7.5 and 1 M sucrose will be added and stirred for 3 minutes. Lysozyme (0.6 mg per g cells) will be added and stirred for 5 minutes. The bacterial cell walls may then be broken by osmotically shocking with 400 mL ddH 2 O. The suspension will be stirred for an additional 5-10 minutes. Hereafter, all samples, reagents and equipment should be kept at 4 C unless otherwise specified. The bacteria will be harvested by centrifugation and the viscous supernatant may be discarded. The pellet will be resuspended in a final volume of 125 mL TM buffer by pipetting. Notably, in a trial attempt at purification, the bacterial pellet was extremely PAGE 229 208 Figure B-1. Diagram of purification procedures for homogeneous heterodimeric b V5 /b his F 1 F 0 ATP synthase complexes. The b subunit dimers represents intact F 1 F 0 ATP synthase. Basically, membrane preparations will be solubilized in 0.2% tegamineoxide WS-35. The solubilized proteins will then be Ni-resin purified in order to remove the homodimeric b V5 /b V5 F 1 F 0 complexes. This will be followed by a V5 immunoprecipitation step to remove the homodimeric b his /b his F 1 F 0 complexes, leaving only the heterodimeric b V5 /b his complexes in solution. A battery of tests will then be run in order to determine the success of the purification procedures and then ultimately activity assays will be performed. PAGE 230 209 viscous and resuspension required about 30 minutes. DnaseI (10 mg/mL) will be added to help decrease the viscosity during resuspension. The bacteria will be broken by a single pass through the French Pressure Cell at 20,000 psi. Cellular debris and unbroken cells will be removed by centrifuging twice at 10,000Xg for 10 minutes each. Membranes will then be collected by ultracentrifugation at 150,000Xg in a Beckman 70.1 Ti rotor for 1.5 hours. The membranes pellets will be rinsed once with Tm buffer and then resuspended in a final volume of 30 mL Tm buffer using a 30 mL Wheaton tissue grinder. Total membrane protein concentration will be determined by the bicinchoninic acid (BCA) assay (Markwell et al., 1978). Ni-Resin Purification The crude membrane preparation described above will contain all membrane proteins, including homodimeric b V5 /b V5 F 1 F 0 complexes, homodimeric b his /b his F 1 F 0 complexes and heterodimeric b V5 /b his F 1 F 0 complexes (Figure B-1). The membrane preparation will be solubilized in 0.2% tegamineoxide WS-35 and then the homodimeric b his /b his F 1 F 0 complexes and heterodimeric b V5 /b his F 1 F 0 complexes will be isolated using the Ni-CAM resin purification procedure (see Chapter 2 Materials and Methods). The homodimeric b V5 /b V5 F 1 F 0 complexes will be eliminated at this step due to the lack of a histidine epitope tag. Preliminary experiments showed that Ni-CAM resin overloaded with membrane protein only produced the homodimeric b his /b his F 1 F 0 complexes and the heterodimeric complexes were washed away. This can be explained by the fact that the heterodimeric b V5 /b his complexes only contain one histidine epitope tag where the homodimeric b his /b his enzyme complexes are incorporated with two histidine epitope tags. Therefore, an optimization of the ratio of membrane protein to Ni-CAM resin will be PAGE 231 210 necessary. The optimized ratio will lie somewhere around 1.0 mg membrane protein to 0.2 mL packed Ni-CAM resin. V5-Epitope Iimmunoprecipitation The mixture of the homodimeric b his /b his F 1 F 0 ATP synthase complexes and the heterodimeric b V5 /b his F 1 F 0 complexes will then be separated by a second epitope-mediated purification (Figure B-1). Immunoprecipitation of the heterodimeric b V5 /b his F 1 F 0 will be accomplished by immobilizing mouse monoclonal anti-V5 antibody (purchased from Invitrogen) on a resin using the Seize Primary Immunoprecipitation Kit (Purchased from Pierce Biotechnology). This kit has been successfully used several times by Dr. Michelle Gumz in our laboratory and consistently gave high yields of purified proteins. Detection of Purified Enzyme Complexes Once the purified heterodimeric bV5/bhis F1F0 ATP synthase complexes are eluted from the V5-affinity resin, the eluant will be tested for the presence of intact and pure enzyme complexes (Figure B-1). The product will be ran on a 10-18% gradient SDS PAGE gel and then silver stained in order to examine the subunit content of the purified product. All eight subunits should be distinguishable in the stained gel, indicating the presence of intact F1F0 ATP synthase complexes. The homogeneity of the purified product will then be examined by immunoblot analysis. The bV5 and bhis were separable on a 15 cm 15% SDS PAGE gel when ran at 100 mamp, with current held constant, until the dye reached the stacker and then at 24 mamp for 12 hours (see Figure 2-13 for a representative figure). It was necessary to double the amperage when two gels were running at the same time. Upon transfer to a nitrocellulose membrane, immunoblot analysis using anti-b antibodies will be performed in order to determine if the PAGE 232 211 heterodimeric bV5/bhis F1F0 has been purified to homogeneity. Densitometry will be used to determine the relative amounts of the bV5 and bhis subunits. If the product is indeed pure, the bV5 and bhis subunits should be present in a 1:1 ratio. Assays of F1F0 ATP Synthase Activity Once the purified heterodimeric bV5/bhis F1F0 ATP synthase complexes are obtained, the battery of traditional activity assays will be performed. 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PAGE 258 BIOGRAPHICAL SKETCH Tammy Weng Bohannon Grabar, daughter of William and Chu Shia Bohannon, was born in Altus, Oklahoma, on February 6, 1977. She lived in Okinawa, Japan, Patrick Air Force Base, Florida, Selfridge Air National Guard Base, Michigan, and Orange Park, Florida, before settling with her family in Melbourne, Florida. In her youth she participated in a variety of extracurricular activities and sports. She graduated in the top 1% of her class from Eau Gallie High School in June 1995. The following August she attended the University of Florida where she majored in microbiology. She received a Bachelor of Science degree with honors from the College of Agriculture in May 1999. During the summer of 1999 Tammy joined Dr. Brian Cains laboratory in the Department of Biochemistry and Molecular Biology at the University of Florida to gain her first experiences in scientific research. The following Fall semester she was accepted to the University of Florida graduate program in the Department of Biochemistry and Molecular Biology. She joined the laboratory of Dr. Brian Cain in February 2000 where she began the work described in this dissertation. During her graduate career Tammy married her high school sweetheart and boyfriend of 11 years, Charles Raymond Grabar, Jr., on May 11, 2002. Their daughter, Kaia Mae Grabar, was born on August 16, 2003. 237