ATP-dependent rotation of mutant ATP synthases defective in proton transport

During ATP hydrolysis, the gammaepsilon c10 complex (gamma and epsilon subunits and a c subunit ring formed from 10 monomers) of F0F1 ATPase (ATP synthase) rotates relative to the alpha3beta3delta ab2 complex, leading to proton transport through the interface between the a subunit and the c subunit ring. In this study, we replaced the two pertinent residues for proton transport, cAsp-61 and aArg-210 of the c and a subunits, respectively. The mutant enzymes exhibited lower ATPase activities than that of the wild type but exhibited ATP-dependent rotation in planar membranes, in which their original assemblies are maintained. The mutant enzymes were defective in proton transport, as shown previously. These results suggest that proton transport can be separated from rotation in ATP hydrolysis, although rotation ensures continuous proton transport by bringing the cAsp-61 and aArg-210 residues into the correct interacting positions.     

Kinetic model of ATP synthase: pH dependence of the rate of ATP synthesis

Recently, a novel molecular mechanism of torque generation in the F(0) portion of ATP synthase was proposed [Rohatgi, Saha and Nath (1998) Curr. Sci. 75, 716-718]. In this mechanism, rotation of the c-subunit was conceived to take place in 12 discrete steps of 30 degrees each due to the binding and unbinding of protons to/from the leading and trailing Asp-61 residues of the c-subunit, respectively. Based on this molecular mechanism, a kinetic scheme has been developed in this work. The scheme considers proton transport driven by a concentration gradient of protons across the proton half-channels, and the rotation of the c-subunit by changes in the electrical potential only. This kinetic scheme has been analyzed mathematically and an expression has been obtained to explain the pH dependence of the rate of ATP synthesis by ATP synthase under steady state operating conditions. For a single set of three enzymological kinetic parameters, this expression predicts the rates of ATP synthesis which agree well with the experimental data over a wide range of pH(in) and pH(out). A logical consequence of our analysis is that DeltapH and Deltapsi are kinetically inequivalent driving forces for ATP synthesis.     

A computational model of the inhibition of Mycobacterium tuberculosis ATPase by a new drug candidate R207910

Diarylquinolines (DARQs) are a new class of potent inhibitors of the ATPase of Mycobacterium tuberculosis. We have created a homology model of a binding site for this class of compounds located on the contact area of the a-subunit (gene atpB) and c-subunits (gene atpE) of Mycobacterium tuberculosis ATPase. The binding pocket that was identified from the analysis of the homology model is formed by 4 helices of three c-subunits and 2 helices of the a-subunit. The lead compound of the DARQ series, R207910, was docked into the pocket using a simulated annealing, multiple conformer, docking algorithm. Different stereoisomers were treated separately. The best docking pose for each stereoisomer was optimized by molecular dynamics simulation on the 5300 atoms of the binding region and ligand. The interaction energies in the computed complexes enable us to rank the different stereoisomers in order of interaction strength with the ATPase binding pockets. We propose that the activity of R207910 against Mycobacterium tuberculosis is based on interference of the compound with the escapement geometry of the proton transfer chain. Upon binding the compound mimics the conserved Arg-186 residue of the a-subunit and interacts in its place with the conserved acidic residue Glu-61 of the c-subunit. This mode of action is corroborated by the good agreement between the computed interaction energies and the observed pattern of stereo-specificity in the model of the binding region.     

Aqueous access channels in subunit a of rotary ATP synthase

The role of subunit a in proton translocation by the Escherichia coli F(1)F(o) ATP synthase is poorly understood. In the membrane-bound F(o) sector of the enzyme, H(+) binding and release occurs at Asp(61) in the middle of the second transmembrane helix (TMH) of subunit c. Protons are thought to reach Asp(61) via an aqueous access pathway formed at least in part by one or more of the five TMHs of subunit a. In this report, we have substituted Cys into a 19-residue span of the fourth TMH of subunit a and used chemical modification to obtain information about the aqueous accessibility of residues along this helix. Residues 206, 210, and 214 are N-ethylmaleimide-accessible from the cytoplasmic side of the membrane and may lie on the H(+) transport route. Residues 215 and 218 on TMH4, as well as residue 245 on TMH5, are Ag(+)-accessible but N-ethylmaleimide-inaccessible and may form part of an aqueous pocket extending from Asp(61) of subunit c to the periplasmic surface.     

Mechanics of coupling proton movements to c-ring rotation in ATP synthase

F1F0 ATP synthases generate ATP by a rotary catalytic mechanism in which H+ transport is coupled to rotation of an oligomeric ring of c subunits extending through the membrane. Protons bind to and then are released from the aspartyl-61 residue of subunit c at the center of the membrane. Subunit a of the F0 sector is thought to provide proton access channels to and from aspartyl-61. Here, we summarize new information on the structural organization of Escherichia coli subunit a and the mapping of aqueous-accessible residues in the second, fourth and fifth transmembrane helices (TMHs). Aqueous-accessible regions of these helices extend to both the cytoplasmic and periplasmic surface. We propose that aTMH4 rotates to alternately expose the periplasmic or cytoplasmic half-channels to aspartyl-61 of subunit c during the proton transport cycle. The concerted rotation of interacting helices in subunit a and subunit c is proposed to be the mechanical force driving rotation of the c-rotor, using a mechanism akin to meshed gears.     

The proton pore of the F0F1-ATPase of Escherichia coli: Ser-206 is not required for proton translocation

A series of experiments was carried out to investigate the role of some polar amino acids in the a-subunit of the ATP synthase of Escherichia coli. Site-directed mutagenesis resulted in the amino acid substitutions Ser-199----Ala, Ser-202----Ala, Ser-206----Ala, Arg-61----Gln or Asp-44----Asn. None of these amino acid substitutions affected the ability of the cells to carry out oxidative phosphorylation. It was concluded therefore that the effect of the substitution of leucine for Ser-206 reported previously (Cain, B.D. and Simoni, R.D. (1986) J. Biol. Chem. 261, 10043-10050) was due to the presence of the leucine rather than the absence of serine. Even though cells carrying the Asp-44----Asn substitution were able to carry out oxidative phosphorylation, membranes from such cells remained proton-impermeable after removal of the F1-ATPase. It appears likely that the proton pore of the F0 of the ATP synthase of E. coli consists of four amino acids, namely Arg-219, Glu-210 and His-245 of the a-subunit and Asp-61 of the c-subunit.     

Resolving the Negative Potential Side (n-side) Water-accessible Proton Pathway of F-type ATP Synthase by Molecular Dynamics Simulations

The rotation of F₁F_o-ATP synthase is powered by the proton motive force across the energy-transducing membrane. The protein complex functions like a turbine; the proton flow drives the rotation of the c-ring of the transmembrane F_o domain, which is coupled to the ATP-producing F₁ domain. The hairpin-structured c-protomers transport the protons by reversible protonation/deprotonation of a conserved Asp/Glu at the outer transmembrane helix (TMH). An open question is the proton transfer pathway through the membrane at atomic resolution. The protons are thought to be transferred via two half-channels to and from the conserved cAsp/Glu in the middle of the membrane. By molecular dynamics simulations of c-ring structures in a lipid bilayer, we mapped a water channel as one of the half-channels. We also analyzed the suppressor mutant cP24D/E61G in which the functional carboxylate is shifted to the inner TMH of the c-protomers. Current models concentrating on the “locked” and “open” conformations of the conserved carboxylate side chain are unable to explain the molecular function of this mutant. Our molecular dynamics simulations revealed an extended water channel with additional water molecules bridging the distance of the outer to the inner TMH. We suggest that the geometry of the water channel is an important feature for the molecular function of the membrane part of F₁F_o-ATP synthase. The inclination of the proton pathway isolates the two half-channels and may contribute to a favorable clockwise rotation in ATP synthesis mode.     

Coupling proton movements to c-ring rotation in F(1)F(o) ATP synthase: aqueous access channels and helix rotations at the a-c interface

F(1)F(o) ATP synthases generate ATP by a rotary catalytic mechanism in which H(+) transport is coupled to rotation of a ring of c subunits within the transmembrane sector of the enzyme. Protons bind to and then are released from the aspartyl-61 residue of subunit c at the center of the membrane. Proton access channels to and from aspartyl-61 are thought to form in subunit a of the F(o) sector. Here, we summarize new information on the structural organization of subunit a and the mapping of aqueous accessible residues in the fourth and fifth transmembrane helices (TMHs). Cysteine substituted residues, lying on opposite faces of aTMH-4, preferentially react with either N-ethyl-maleimide (NEM) or Ag(+). We propose that aTMH-4 rotates to alternately expose each helical face to aspartyl-61 of subunit c during the proton transport cycle. The concerted helical rotation of aTMH-4 and cTMH-2 are proposed to be coupled to the stepwise mechanical movement of the c-rotor.     

Structure of the mitochondrial ATP synthase by electron cryomicroscopy

We have determined the structure of intact ATP synthase from bovine heart mitochondria by electron cryomicroscopy of single particles. Docking of an atomic model of the F1-c10 subcomplex into a major segment of the map has allowed the 32 A resolution density to be interpreted as the F1-ATPase, a central and a peripheral stalk and an FO membrane region that is composed of two domains. One domain of FO corresponds to the ring of c-subunits, and the other probably contains the a-subunit, the transmembrane portion of the b-subunit and the remaining integral membrane proteins of FO. The peripheral stalk wraps around the molecule and connects the apex of F1 to the second domain of FO. The interaction of the peripheral stalk with F1-c10 implies that it binds to a non-catalytic alpha-beta interface in F1 and its inclination where it is not attached to F1 suggests that it has a flexible region that can serve as a stator during both ATP synthesis and ATP hydrolysis.     

Characterization of domain interfaces in monomeric and dimeric ATP synthase

We disassembled monomeric and dimeric yeast ATP synthase under mild conditions to identify labile proteins and transiently stable subcomplexes that had not been observed before. Specific removal of subunits alpha, beta, oligomycin sensitivity conferring protein (OSCP), and h disrupted the ATP synthase at the gamma-alpha(3)beta(3) rotor-stator interface. Loss of two F(1)-parts from dimeric ATP synthase led to the isolation of a dimeric subcomplex containing membrane and peripheral stalk proteins thus identifying the membrane/peripheral stalk sectors immediately as the dimerizing parts of ATP synthase. Almost all subunit a was found associated with a ring of 10 c-subunits in two-dimensional blue native/SDS gels. We therefore postulate that c10a1-complex is a stable structure in resting ATP synthase until the entry of protons induces a breaking of interactions and stepwise rotation of the c-ring relative to the a-subunit in the catalytic mechanism. Dimeric subunit a was identified in SDS gels in association with two c10-rings suggesting that a c10a2c10-complex may constitute an important part of the monomer-monomer interface in dimeric ATP synthase that seems to be further tightened by subunits b, i, e, g, and h. In contrast to the monomer-monomer interface, the interface between dimers in higher oligomeric structures remains largely unknown. However, we could show that the natural inhibitor protein Inh1 is not required for oligomerization.     

Mutations in the uncE gene affecting assembly of the c-subunit of the adenosine triphosphatase of Escherichia coli.

The amino acid substitutions in the mutant c-subunits of Escherichia coli F1F0-ATPase coded for by the uncE429, uncE408 and uncE463 alleles affect the incorporation of these proteins into the cell membrane. The DNA sequence of the uncE429 allele differed from normal in that a G leads to A base change occurred at nucleotide 68 of the uncE gene, resulting in glycine being replaced by aspartic acid at position 23 in the c-subunit. The uncE408 and uncE463 mutant DNA sequences were identical and differed from normal in that a C leads to T base change occurred at nucleotide 91 of the uncE gene, resulting in leucine being replaced by phenylalanine at position 31 in the c-subunit. An increased gene dosage of the uncE408 or uncE463 alleles resulted in the incorporation into the membranes of the mutant c-subunits. The results are discussed in terms of the 'Helical Hairpin Hypothesis' of Engelman & Steitz [(1981) Cell 23,411-422].     

Conservation of Complete Trimethylation of Lysine-43 in the Rotor Ring of c-Subunits of Metazoan Adenosine Triphosphate (ATP) Synthases

The rotors of ATP synthases turn about 100 times every second. One essential component of the rotor is a ring of hydrophobic c-subunits in the membrane domain of the enzyme. The rotation of these c-rings is driven by a transmembrane proton-motive force, and they turn against a surface provided by another membrane protein, known as subunit a. Together, the rotating c-ring and the static subunit a provide a pathway for protons through the membrane in which the c-ring and subunit a are embedded. Vertebrate and invertebrate c-subunits are well conserved. In the structure of the bovine F₁-ATPase-c-ring subcomplex, the 75 amino acid c-subunit is folded into two transmembrane α-helices linked by a short loop. Each bovine rotor-ring consists of eight c-subunits with the N- and C-terminal α-helices forming concentric inner and outer rings, with the loop regions exposed to the phospholipid head-group region on the matrix side of the inner membrane. Lysine-43 is in the loop region and its ε-amino group is completely trimethylated. The role of this modification is unknown. If the trimethylated lysine-43 plays some important role in the functioning, assembly or degradation of the c-ring, it would be expected to persist throughout vertebrates and possibly invertebrates also. Therefore, we have carried out a proteomic analysis of c-subunits across representative species from different classes of vertebrates and from invertebrate phyla. In the twenty-nine metazoan species that have been examined, the complete methylation of lysine-43 is conserved, and it is likely to be conserved throughout the more than two million extant metazoan species. In unicellular eukaryotes and prokaryotes, when the lysine is conserved it is unmethylated, and the stoichiometries of c-subunits vary from 9–15. One possible role for the trimethylated residue is to provide a site for the specific binding of cardiolipin, an essential component of ATP synthases in mitochondria.The ATP synthase (or F-ATPase)¹ embedded in the inner membranes of mitochondria is a multi-protein complex of about thirty polypeptides that couples the transmembrane proton-motive force across the membrane to the synthesis of ATP from ADP and inorganic phosphate in the matrix of the organelle (1). The coupling mechanism involves a mechanical rotation of the enzyme''s rotor at about 100 Hz (2) driven by the proton motive force (3). The rotor itself consists of a hydrophobic ring of c-subunits in the membrane domain of the enzyme plus a central stalk. The central stalk penetrates into the catalytic F₁ domain of the enzyme, which protrudes into the matrix space, and the turning of the rotor brings about conformational changes in the three catalytic sites in each F₁ domain that lead to the binding of substrates, and the formation of ATP and its release into the matrix (4). The c-subunits that constitute the rotor-ring are among the most hydrophobic proteins in nature, and, because their properties are similar to those of lipids, they have been classified as proteolipids (5, 6). In vertebrates, c-subunits are highly conserved and they are well conserved in invertebrates also (4). In the structure of the bovine F₁-c-ring subcomplex, the 75 amino acid c-subunit is folded into two transmembrane α-helices linked by a short loop (4, 7). Each rotor-ring consists of eight c-subunits with the N-and C-terminal α-helices forming concentric inner and outer rings, linked by eight loop regions exposed to the phospholipid head-group region on the matrix side of the inner membrane. Some of the loops are in contact with subunits γ-, δ-, and ε- in the “foot” of the central stalk (4).One striking feature of bovine c-subunits is that glutamate-58 in the C-terminal α-helix is exposed to the lipid bilayer around the mid-point of the membrane, and the protonation and deprotonation of this residue via an arginine residue in an adjacent a-subunit in the membrane domain of the enzyme is an essential feature in the generation of rotation (8). Each complete rotation of the rotor produces three ATP molecules, one from each of the three catalytic sites in the F₁-domain (9), and requires the translocation through the membrane of one proton per c-subunit (10). Thus, the number of translocated protons required to make each ATP is the number of c-subunits comprising the ring divided by three, and this parameter is referred to as the “energy cost” for making each ATP molecule (4). The identity, or near identity, of the sequences of vertebrate c-subunits makes it highly likely that c₈-rings observed in the bovine enzyme will persist throughout vertebrate F-ATPases, and hence the energy cost in their F-ATPases will be 2.7 translocated protons per ATP, the lowest value so far observed (4). The high conservation of the sequences of c-subunits in invertebrates suggests that their F-ATPases will also have c₈-rings, with an associated energy cost of 2.7 protons per ATP (4). The c-rings in fungi, eubacteria, and plant chloroplasts are larger and are made variously from 10–15 subunits depending on the species, implying that the energy cost in these enzymes is 3.3–5.0 protons per ATP (7, 11–16).Another striking feature of the bovine c-subunit, and the topic of this paper, is that the ε-amino group of lysine-43 is completely trimethylated (17). In the structure of the bovine c-ring, these residues are in loop regions of each c-subunit near to the boundary between the lipid bilayer and the aqueous phase of the matrix (4). Their role is not known, but if trimethylated lysine-43 plays some important role in the functioning, assembly or degradation of the c-ring, it would be expected to persist throughout vertebrates and possibly invertebrates also. Therefore, as described here, we have isolated F-ATPases and c-subunits from a wide range of metazoans and have characterized the methylation status of lysine-43 in their c-subunits.     

A point mutation in the ATP synthase of Rhodobacter capsulatus results in differential contributions of Delta(pH) and Delta(phi) in driving the ATP synthesis reaction.

The interface between the c-subunit oligomer and the a subunit in the F0 sector of the ATP synthase is believed to form the core of the rotating motor powered by the protonic flow. Besides the essential cAsp61 and aArg210 residues (Escherichia coli numbering), a few other residues at this interface, although nonessential, show a high degree of conservation, among these aGlu219. The homologous residue aGlu210 in the ATP synthase of the photosynthetic bacterium Rhodobacter capsulatus has been substituted by a lysine. Inner membranes prepared from the mutant strain showed approximately half of the ATP synthesis activity when driven both by light and by acid-base transitions. As estimated with the ACMA assay, proton pumping rates in the inner membranes were also reduced to a similar extent in the mutant. The most striking impairment of ATP synthesis in the mutant, a decrease as low as 12 times as compared to the wild-type, was observed in the absence of a transmembrane electrical membrane potential (Delta(phi)) at low transmembrane pH difference (Delta(pH)). Therefore, the mutation seems to affect both the mechanism responsible for coupling F1 with proton translocation by F0, and the mechanism determining the relative contribution of Delta(pH) and Delta(phi) in driving ATP synthesis.     

Structural interpretations of F(0) rotary function in the Escherichia coli F(1)F(0) ATP synthase

F(1)F(0) ATP synthases are known to synthesize ATP by rotary catalysis in the F(1) sector of the enzyme. Proton translocation through the F(0) membrane sector is now proposed to drive rotation of an oligomer of c subunits, which in turn drives rotation of subunit gamma in F(1). The primary emphasis of this review will be on recent work from our laboratory on the structural organization of F(0), which proves to be consistent with the concept of a c(12) oligomeric rotor. From the NMR structure of subunit c and cross-linking studies, we can now suggest a detailed model for the organization of the c(12) oligomer in F(0) and some of the transmembrane interactions with subunits a and b. The structural model indicates that the H(+)-carrying carboxyl of subunit c is located between subunits of the c(12) oligomer and that two c subunits pack in a front-to-back manner to form the proton (cation) binding site. The proton carrying Asp61 side chain is occluded between subunits and access to it, for protonation and deprotonation via alternate entrance and exit half-channels, requires a swiveled opening of the packed c subunits and stepwise association with different transmembrane helices of subunit a. We suggest how some of the structural information can be incorporated into models of rotary movement of the c(12) oligomer during coupled synthesis of ATP in the F(1) portion of the molecule.     

Structural organization of mitochondrial ATP synthase

Specific modules and subcomplexes like F(1) and F(0)-parts, F(1)-c subcomplexes, peripheral and central stalks, and the rotor part comprising a ring of c-subunits with attached subunits gamma, delta, and epsilon can be identified in yeast and mammalian ATP synthase. Four subunits, alpha(3)beta(3), OSCP, and h, seem to form a structural entity at the extramembranous rotor/stator interface (gamma/alpha(3)beta(3)) to hold and stabilize the rotor in the holo-enzyme. The intramembranous rotor/stator interface (c-ring/a-subunit) must be dynamic to guarantee unhindered rotation. Unexpectedly, a c(10)a-assembly could be isolated with almost quantitive yield suggesting that an intermediate step in the rotating mechanism was frozen under the conditions used. Isolation of dimeric a-subunit and (c(10))(2)a(2)-complex from dimeric ATP synthase suggested that the a-subunit stabilizes the same monomer-monomer interface that had been shown to involve also subunits e, g, b, i, and h. The natural inhibitor protein Inh1 does not favor oligomerization of yeast ATP synthase. Other candidates for the oligomerization of dimeric ATP synthase building blocks are discussed, e.g. the transporters for inorganic phosphate and ADP/ATP that had been identified as constituents of ATP synthasomes. Independent approaches are presented that support previous reports on the existence of ATP synthasomes in the mitochondrial membrane.     

Microscopic rotary mechanism of ion translocation in the F(o) complex of ATP synthases

The microscopic mechanism of coupled c-ring rotation and ion translocation in F(1)F(o)-ATP synthases is unknown. Here we present conclusive evidence supporting the notion that the ability of c-rings to rotate within the F(o) complex derives from the interplay between the ion-binding sites and their nonhomogenous microenvironment. This evidence rests on three atomic structures of the c(15) rotor from crystals grown at low pH, soaked at high pH and, after N,N'-dicyclohexylcarbodiimide (DCCD) modification, resolved at 1.8, 3.0 and 2.2 ?, respectively. Alongside a quantitative DCCD-labeling assay and free-energy molecular dynamics calculations, these data demonstrate how the thermodynamic stability of the so-called proton-locked state is maximized by the lipid membrane. By contrast, a hydrophilic environment at the a-subunit-c-ring interface appears to unlock the binding-site conformation and promotes proton exchange with the surrounding solution. Rotation thus occurs as c-subunits stochastically alternate between these environments, directionally biased by the electrochemical transmembrane gradient.     

Solution structure of subunit F(6) from the peripheral stalk region of ATP synthase from bovine heart mitochondria

The ATP synthase enzyme structure includes two stalk assemblies, the central stalk and the peripheral stalk. Catalysis involves rotation of the central stalk assembly together with the membrane-embedded ring of c-subunits driven by the trans-membrane proton-motive force, while the alpha and beta-subunits of F(1) are prevented from co-rotating by their attachment to the peripheral stalk. In the absence of structures of either the intact peripheral stalk or larger complexes containing it, we are studying its individual components and their interactions to build up an overall picture of its structure. Here, we describe an NMR structural characterisation of F(6), which is a 76-residue protein located in the peripheral stalk of the bovine ATP synthase and is essential for coupling between the proton-motive force and catalysis. Isolated F(6) has a highly flexible structure comprising two helices packed together through a loose hydrophobic core and connected by an unstructured linker. Analysis of chemical shifts, (15)N relaxation and RDC measurements confirm that the F(6) structure is flexible on a wide range of timescales ranging from nanoseconds to seconds. The relationship between this structure for isolated F(6) and its role in the intact peripheral stalk is discussed.     

Proton translocation by the F1F0ATPase of Escherichia coli. Mutagenic analysis of the a subunit

Cassette site-directed mutagenesis was employed to generate mutations in the a subunit (uncB (a) gene) of F1F0ATP synthase. Using sequence homology with similar subunits of other F1F0ATP synthases as a guide, 20 mutations were targeted to a region of the a subunit thought to constitute part of the proton translocation mechanism. ATP-driven proton pumping activity is lost with the substitution of lys, ile, val, or glu for arginine 210. Substitution of val, leu, gln, or glu for asparagine 214 does not completely block proton conduction, however, replacement of asparagine 214 with histidine does reduce enzyme activity below that necessary for significant function. Two or three mutations were constructed in each of four nonpolar amino acids, leucine 207, leucine 211, alanine 217, and glycine 218. Certain specific mutations in these positions result in partial loss of F1F0ATP synthase activity, but only the substitution of arginine for alanine 217 reduces ATP-driven proton pumping activity to undetectable levels. It is concluded that of the six amino acids studied, only arginine 210 is an essential component of the proton translocation mechanism. Fractionation of cell-free extracts of a subunit mutation strains generally reveals normal amounts of F1 specifically bound to the particulate fraction. One possible exception is the arginine 210 to isoleucine mutation which results in somewhat elevated levels of free F1 detectable in the soluble fraction. For nearly all a subunit mutations, F1F0-mediated ATP hydrolysis activity remains sensitive to inhibition by dicyclohexylcarbodiimide in spite of the fact that the mutations block proton translocation.     

The peripheral stalk of the mitochondrial ATP synthase

The peripheral stalk of F-ATPases is an essential component of these enzymes. It extends from the membrane distal point of the F1 catalytic domain along the surface of the F1 domain with subunit a in the membrane domain. Then, it reaches down some 45 A to the membrane surface, and traverses the membrane, where it is associated with the a-subunit. Its role is to act as a stator to hold the catalytic alpha3beta3 subcomplex and the a-subunit static relative to the rotary element of the enzyme, which consists of the c-ring in the membrane and the attached central stalk. The central stalk extends up about 45 A from the membrane surface and then penetrates into the alpha3beta3 subcomplex along its central axis. The mitochondrial peripheral stalk is an assembly of single copies of the oligomycin sensitivity conferral protein (the OSCP) and subunits b, d and F6. In the F-ATPase in Escherichia coli, its composition is simpler, and it consists of a single copy of the delta-subunit with two copies of subunit b. In some bacteria and in chloroplasts, the two copies of subunit b are replaced by single copies of the related proteins b and b' (known as subunits I and II in chloroplasts). As summarized in this review, considerable progress has been made towards establishing the structure and biophysical properties of the peripheral stalk in both the mitochondrial and bacterial enzymes. However, key issues are unresolved, and so our understanding of the role of the peripheral stalk and the mechanism of synthesis of ATP are incomplete.     

Purification, characterization and reconstitution into membranes of the oligomeric c-subunit ring of thermophilic F(o)F(1)-ATP synthase expressed in Escherichia coli

F(o)F(1)-ATP synthase catalyzes ATP synthesis coupled with proton-translocation across the membrane. The membrane-embedded F(o) portion is responsible for the H(+) translocation coupled with rotation of the oligomeric c-subunit ring, which induces rotation of the γ subunit of F(1). For solid-state NMR measurements, F(o)F(1) of thermophilic Bacillus PS3 (TF(o)F(1)) was overexpressed in Escherichia coli and the intact c-subunit ring (TF(o)c-ring) was isolated by new procedures. One of the key improvement in this purification was the introduction of a His residue to each c-subunit that acts as a virtual His(10)-tag of the c-ring. After solubilization from membranes by sodium deoxycholate, the c-ring was purified by Ni-NTA affinity chromatography, followed by anion-exchange chromatography. The intactness of the isolated c-ring was confirmed by high-resolution clear native PAGE, sedimentation analysis, and H(+)-translocation activity. The isotope-labeled intact TF(o)c-ring was successfully purified in such an amount as enough for solid-state NMR measurements. The isolated TF(o)c-rings were reconstituted into lipid membranes. A solid-state NMR spectrum at a high quality was obtained with this membrane sample, revealing that this purification procedure was suitable for the investigation by solid-state NMR. The purification method developed here can also be used for other physicochemical investigations.