Interacting cytoplasmic loops of subunits a and c of Escherichia coli F1F0 ATP synthase gate H+ transport to the cytoplasm.

H(+)-transporting F1F0 ATP synthase catalyzes the synthesis of ATP via coupled rotary motors within F0 and F1. H(+) transport at the subunit a-c interface in transmembranous F0 drives rotation of a cylindrical c10 oligomer within the membrane, which is coupled to rotation of subunit γ within the α3ß3 sector of F1 to mechanically drive ATP synthesis. F1F0 functions in a reversible manner, with ATP hydrolysis driving H(+) transport. ATP-driven H(+) transport in a select group of cysteine mutants in subunits a and c is inhibited after chelation of Ag(+) and/or Cd(+2) with the substituted sulfhydryl groups. The H(+) transport pathway mapped via these Ag(+)(Cd(+2))-sensitive Cys extends from the transmembrane helices (TMHs) of subunits a and c into cytoplasmic loops connecting the TMHs, suggesting these loop regions could be involved in gating H(+) release to the cytoplasm. Here, using select loop-region Cys from the single cytoplasmic loop of subunit c and multiple cytoplasmic loops of subunit a, we show that Cd(+2) directly inhibits passive H(+) transport mediated by F0 reconstituted in liposomes. Further, in extensions of previous studies, we show that the regions mediating passive H(+) transport can be cross-linked to each other. We conclude that the loop-regions in subunits a and c that are implicated in H(+) transport likely interact in a single structural domain, which then functions in gating H(+) release to the cytoplasm.

Half channels mediating H(+) transport and the mechanism of gating in the Fo sector of Escherichia coli F1Fo ATP synthase.

H(+)-transporting F1Fo ATP synthase catalyzes the synthesis of ATP via coupled rotary motors within Fo and F1. H(+) transport at the subunit a-c interface in trans-membranous Fo drives rotation of the c-ring within the membrane, with subunit c being bound in a complex with the γ and ε subunits extending from the membrane. Finally, the rotation of subunit γ within the α3ß3 sector of F1 mechanically drives ATP synthesis within the catalytic sites. In this review, we propose and provide evidence supporting the route of proton transfer via half channels from one side of the membrane to the other, and the mechanism of gating H(+) binding to and release from Asp61 of subunit c, via conformational movements of Arg210 in subunit a. We propose that protons are gated from the inside of a four-helix bundle at the periplasmic side of subunit a to drive protonation of cAsp61, and that this gating movement is facilitated by the swiveling of trans-membrane helices (TMHs) 4 and 5 at the site of interaction with cAsp61 on the periphery of the c-ring. Proton release to the cytoplasmic half channel is facilitated by the movement of aArg210 as a consequence of this proposed helical swiveling. Finally, release from the cytoplasmic half channel is mediated by residues in a complex of interacting extra-membraneous loops formed between TMHs of both subunits a and c. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

Residues in the polar loop of subunit c in Escherichia coli ATP synthase function in gating proton transport to the cytoplasm.

Rotary catalysis in F1F0 ATP synthase is powered by proton translocation through the membrane-embedded F0 sector. Proton binding and release occur in the middle of the membrane at Asp-61 on the second transmembrane helix (TMH) of subunit c, which folds in a hairpin-like structure with two TMHs. Previously, the aqueous accessibility of Cys substitutions in the transmembrane regions of subunit c was probed by testing the inhibitory effects of Ag(+) or Cd(2+) on function, which revealed extensive aqueous access in the region around Asp-61 and on the half of TMH2 extending to the cytoplasm. In the current study, we surveyed the Ag(+) and Cd(2+) sensitivity of Cys substitutions in the loop of the helical hairpin and used a variety of assays to categorize the mechanisms by which Ag(+) or Cd(2+) chelation with the Cys thiolates caused inhibition. We identified two distinct metal-sensitive regions in the cytoplasmic loop where function was inhibited by different mechanisms. Metal binding to Cys substitutions in the N-terminal half of the loop resulted in an uncoupling of F1 from F0 with release of F1 from the membrane. In contrast, substitutions in the C-terminal half of the loop retained membrane-bound F1 after metal treatment. In several of these cases, inhibition was shown to be due to blockage of passive H(+) translocation through F0 as assayed with F0 reconstituted into liposomes. The results suggest that the C-terminal domain of the cytoplasmic loop may function in gating H(+) translocation to the cytoplasm.

Obstruction of transmembrane helical movements in subunit a blocks proton pumping by F1Fo ATP synthase.

Subunit a plays a key role in promoting H(+) transport-coupled rotary motion of the subunit c ring in F1Fo ATP synthase. H(+) binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of Fo subunit c. H(+) are thought to reach cAsp61 via aqueous half-channels formed by TMHs 2-5 of subunit a. Movements of TMH4 and TMH5 have been proposed to facilitate protonation of cAsp61 from a half channel centered in a four helix bundle at the periplasmic side of subunit a. The possible necessity of these proposed TMH movements was investigated by assaying ATP driven H(+) pumping function before and after cross-linking paired Cys substitutions at the center of TMHs within subunit a. The cross-linking of the Cys pairs aG218C/I248C in TMH4 and TMH5, and aL120C/H245C in TMH2 and TMH5, inhibited H(+) pumping by 85-90%. H(+) pumping function was largely unaffected by modification of the same Cys residues in the absence of cross-link formation. The inhibition is consistent with the proposed requirement for TMH movements during the gating of periplasmic H(+) access to cAsp61. The cytoplasmic loops of subunit a have been implicated in gating H(+) release to the cytoplasm, and previous cross-linking experiments suggest that the chemically reactive regions of the loops may pack as a single domain. Here we show that Cys substitutions in these domains can be cross-linked with retention of function and conclude that these domains need not undergo large conformational changes during enzyme function.

Interactions between subunits a and b in the rotary ATP synthase as determined by cross-linking.

The interaction of the membrane traversing stator subunits a and b of the rotary ATP synthase was probed by substitution of a single Cys into each subunit with subsequent Cu(2+) catalyzed cross-linking. Extensive interaction between the transmembrane (TM) region of one b subunit and TM2 of subunit a was indicated by cross-linking with 6 Cys pairs introduced into these regions. Additional disulfide cross-linking was observed between the N-terminus of subunit b and the periplasmic loop connecting TM4 and TM5 of subunit a. Finally, benzophenone-4-maleimide derivatized Cys in the 2-3 periplasmic loop of subunit a were shown to cross-link with the periplasmic N-terminal region of subunit b. These experiments help to define the juxtaposition of subunits b and a in the ATP synthase.

Cell-free synthesis of membrane subunits of ATP synthase in phospholipid bicelles: NMR shows subunit a fold similar to the protein in the cell membrane.

NMR structure determination of large membrane proteins is hampered by broad spectral lines, overlap, and ambiguity of signal assignment. Chemical shift and NOE assignment can be facilitated by amino acid selective isotope labeling in cell-free protein synthesis system. However, many biological detergents are incompatible with the cell-free synthesis, and membrane proteins often have to be synthesized in an insoluble form. We report cell-free synthesis of subunits a and c of the proton channel of Escherichia coli ATP synthase in a soluble form in a mixture of phosphatidylcholine derivatives. In comparison, subunit a was purified from the cell-free system and from the bacterial cell membranes. NMR spectra of both preparations were similar, indicating that our procedure for cell-free synthesis produces protein structurally similar to that prepared from the cell membranes.

Chemical reactivities of cysteine substitutions in subunit a of ATP synthase define residues gating H+ transport from each side of the membrane.

Subunit a plays a key role in coupling H(+) transport to rotations of the subunit c-ring in F(1)F(o) ATP synthase. In Escherichia coli, H(+) binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of F(o) subunit c. Based upon the Ag(+) sensitivity of Cys substituted into subunit a, H(+) are thought to reach Asp-61 via aqueous pathways mapping to surfaces of TMH 2-5. In this study we have extended characterization of the most Ag(+)-sensitive residues in subunit a with cysteine reactive methanethiosulfonate (MTS) reagents and Cd(2+). The effect of these reagents on ATPase-coupled H(+) transport was measured using inside-out membrane vesicles. Cd(2+) inhibited the activity of all Ag(+)-sensitive Cys on the cytoplasmic side of the TMHs, and three of these substitutions were also sensitive to inhibition by MTS reagents. On the other hand, Cd(2+) did not inhibit the activities of substitutions at residues 119 and 120 on the periplasmic side of TMH2, and residues 214 and 215 in TMH4 and 252 in TMH5 at the center of the membrane. When inside-out membrane vesicles from each of these substitutions were sonicated during Cd(2+) treatment to expose the periplasmic surface, the ATPase-coupled H(+) transport activity was strongly inhibited. The periplasmic access to N214C and Q252C, and their positioning in the protein at the a-c interface, is consistent with previous proposals that these residues may be involved in gating H(+) access from the periplasmic half-channel to Asp-61 during the protonation step.

Aqueous accessibility to the transmembrane regions of subunit c of the Escherichia coli F1F0 ATP synthase.

Rotary catalysis in F(1)F(0) ATP synthase is powered by proton translocation through the membrane-embedded F(0) sector. Proton binding and release occur in the middle of the membrane at Asp-61 on transmembrane helix (TMH) 2 of subunit c. Previously the reactivity of Cys substituted into TMH2 revealed extensive aqueous access at the cytoplasmic side as probed with Ag(+) and other thiolate-directed reagents. The analysis of aqueous accessibility of membrane-embedded regions in subunit c was extended here to TMH1 and the periplasmic side of TMH2. The Ag(+) sensitivity of Cys substitutions was more limited on the periplasmic versus cytoplasmic side of TMH2. In TMH1, Ag(+) sensitivity was restricted to a pocket of four residues lying directly behind Asp-61. Aqueous accessibility was also probed using Cd(2+), a membrane-impermeant soft metal ion with properties similar to Ag(+). Cd(2+) inhibition was restricted to the I28C substitution in TMH1 and residues surrounding Asp-61 in TMH2. The overall pattern of inhibition, by all of the reagents tested, indicates highest accessibility on the cytoplasmic side of TMH2 and in a pocket of residues around Asp-61, including proximal residues in TMH1. Additionally subunit a was shown to mediate access to this region by the membrane-impermeant probe 2-(trimethylammonium)ethyl methanethiosulfonate. Based upon these results and other information, a pocket of aqueous accessible residues, bordered by the peripheral surface of TMH4 of subunit a, is proposed to extend from the cytoplasmic side of cTMH2 to Asp-61 in the center of the membrane.

Structural interactions between transmembrane helices 4 and 5 of subunit a and the subunit c ring of Escherichia coli ATP synthase.

Subunit a plays a key role in promoting H+ transport and the coupled rotary motion of the subunit c ring in F1F0-ATP synthase. H+ binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of F0 subunit c. H+ are thought to reach Asp-61 via aqueous pathways mapping to the surfaces of TMHs 2-5 of subunit a. TMH4 of subunit a is thought to pack close to TMH2 of subunit c based upon disulfide cross-link formation between Cys substitutions in both TMHs. Here we substituted Cys into the fifth TMH of subunit a and the second TMH of subunit c and tested for cross-linking using bis-methanethiosulfonate (bis-MTS) reagents. A total of 62 Cys pairs were tested and 12 positive cross-links were identified with variable alkyl length linkers. Cross-linking was achieved near the middle of the bilayer for the Cys pairs a248C/c62C, a248C/ c63C, a248C/c65C, a251C/c57C, a251C/c59C, a251C/c62C, a252C/c62C, and a252C/c65C. Cross-linking was achieved near the cytoplasmic side of the bilayer for Cys pairs a262C/c53C, a262C/c54C, a262C/c55C, and a263C/c54C. We conclude that both aTMH4 and aTMH5 pack proximately to cTMH2 of the c-ring. In other experiments we demonstrate that aTMH4 and aTMH5 can be simultaneously cross-linked to different subunit c monomers in the c-ring. Five mutants showed pH-dependent cross-linking consistent with aTMH5 changing conformation at lower pH values to facilitate cross-linking. We suggest that the pH-dependent conformational change may be related to the proposed role of aTMH5 in gating H+ access from the periplasm to the cAsp-61 residue in cTMH2.

Subunit a facilitates aqueous access to a membrane-embedded region of subunit c in Escherichia coli F1F0 ATP synthase.

Rotary catalysis in F(1)F(0) ATP synthase is powered by proton translocation through the membrane-embedded F(0) sector. Proton binding and release occurs in the middle of the membrane at Asp-61 on transmembrane helix 2 of subunit c. Previously, the reactivity of cysteines substituted into F(0) subunit a revealed two regions of aqueous access, one extending from the periplasm to the middle of the membrane and a second extending from the middle of the membrane to the cytoplasm. To further characterize aqueous accessibility at the subunit a-c interface, we have substituted Cys for residues on the cytoplasmic side of transmembrane helix 2 of subunit c and probed the accessibility to these substituted positions using thiolate-reactive reagents. The Cys substitutions tested were uniformly inhibited by Ag(+) treatment, which suggested widespread aqueous access to this generally hydrophobic region. Sensitivity to N-ethylmaleimide (NEM) and methanethiosulfonate reagents was localized to a membrane-embedded pocket surrounding Asp-61. The cG58C substitution was profoundly inhibited by all the reagents tested, including membrane impermeant methanethiosulfonate reagents. Further studies of the highly reactive cG58C substitution revealed that NEM modification of a single c subunit in the oligomeric c-ring was sufficient to cause complete inhibition. In addition, NEM modification of subunit c was dependent upon the presence of subunit a. The results described here provide further evidence for an aqueous-accessible region at the interface of subunits a and c extending from the middle of the membrane to the cytoplasm.

The cytoplasmic loops of subunit a of Escherichia coli ATP synthase may participate in the proton translocating mechanism.

Subunit a plays a key role in promoting H(+) transport and the coupled rotary motion of the subunit c ring in F(1)F(0)-ATP synthase. H(+) binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of F(0) subunit c. H(+) are thought to reach Asp-61 via aqueous pathways mapping to the surfaces of TMHs 2-5 of subunit a based upon the chemical reactivity of Cys substituted into these helices. Here we substituted Cys into loops connecting TMHs 1 and 2 (loop 1-2) and TMHs 3 and 4 (loop 3-4). A large segment of loop 3-4 extending from loop residue 192 loop to residue 203 in TMH4 at the lipid bilayer surface proved to be very sensitive to inhibition by Ag(+). Cys-161 and -165 at the other end of the loop bordering TMH3 were also sensitive to inhibition by Ag(+). Further Cys substitutions in residues 86 and 93 in the middle of the 1-2 loop proved to be Ag(+)-sensitive. We next asked whether the regions of Ag(+)-sensitive residues clustered together near the surface of the membrane by combining Cys substitutions from two domains and testing for cross-linking. Cys-161 and -165 in loop 3-4 were found to cross-link with Cys-202, -203, or -205, which extend into TMH4 from the cytoplasm. Further Cys at residues 86 and 93 in loop 1-2 were found to cross-link with Cys-195 in loop 3-4. We conclude that the Ag(+)-sensitive regions of loops 1-2 and 3-4 may pack in a single domain that packs at the ends of TMHs 3 and 4. We suggest that the Ag(+)-sensitive domain may be involved in gating H(+) release at the cytoplasmic side of the aqueous access channel extending through F(0).

Interaction of transmembrane helices in ATP synthase subunit a in solution as revealed by spin label difference NMR.

Subunit a in the membrane traversing F0 sector of Escherichia coli ATP synthase is known to fold with five transmembrane helices (TMHs) with residue 218 in TMH IV packing close to residue 248 in TMH V. In this study, we have introduced a spin label probe at Cys residues substituted at positions 222 or 223 and measured the effects on the Trp epsilon NH indole NMR signals of the seven Trp residues in the protein. The protein was purified and NMR experiments were carried out in a chloroform-methanol-H2O (4:4:1) solvent mixture. The spin label at positions 222 or 223 proved to broaden the signals of W231, W232, W235 and W241 located at the periplasmic ends of TMH IV and TMH V and the connecting loop between these helices. The broadening of W241 would require that the loop residues fold back on themselves in a hairpin-like structure much like it is predicted to fold in the native membrane. Placement of the spin label probe at several other positions also proved to have broadening effects on some of these Trp residues and provided additional constraints on folding of TMH IV and TMH V. The effects of the 223 probes on backbone amide resonances of subunit a were also measured by an HNCO experiment and the results are consistent with the two helices folding back on themselves in this solvent mixture. When Cys and Trp were substituted at residues 206 and 254 at the cytoplasmic ends of TMHs IV and V respectively, the W254 resonance was not broadened by the spin label at position 206. We conclude that the helices fold back on themselves in this solvent system and then pack at an angle such that the cytoplasmic ends of the polypeptide backbone are significantly displaced from each other.

The rigid connecting loop stabilizes hairpin folding of the two helices of the ATP synthase subunit c.

We have tested the role of the polar loop of subunit c of the Escherichia coli ATP synthase in stabilizing the hairpin structure of this protein. The structure of the c(32-52) peptide corresponding to the cytoplasmic region of subunit c bound to the dodecylphosphocholine micelles was solved by high-resolution NMR. The region comprising residues 41-47 forms a well-ordered structure rather similar to the conformation of the polar loop region in the solution structure of the full-length subunit c and is flanked by short alpha-helical segments. This result suggests that the rigidity of the polar loop significantly contributes to the stability of the hairpin formed by the two helices of subunit c. This experimental system may be useful for NMR studies of interactions between subunit c and subunits gamma and epsilon, which together form the rotor of the ATP synthase.

Fluidity of structure and swiveling of helices in the subunit c ring of Escherichia coli ATP synthase as revealed by cysteine-cysteine cross-linking.

Subunit c in the membrane-traversing F(0) sector of Escherichia coli ATP synthase is known to fold with two transmembrane helices and form an oligomeric ring of 10 or more subunits in the membrane. Models for the E. coli ring structure have been proposed based upon NMR solution structures and intersubunit cross-linking of Cys residues in the membrane. The E. coli models differ from the recent x-ray diffraction structure of the isolated Ilyobacter tartaricus c-ring. Furthermore, key cross-linking results supporting the E. coli model prove to be incompatible with the I. tartaricus structure. To test the applicability of the I. tartaricus model to the E. coli c-ring, we compared the cross-linking of a pair of doubly Cys substituted c-subunits, each of which was compatible with one model but not the other. The key finding of this study is that both A21C/M65C and A21C/I66C doubly substituted c-subunits form high yield oligomeric structures, c(2), c(3)... c(10), via intersubunit disulfide bond formation. The results indicate that helical swiveling, with resultant interconversion of the two conformers predicted by the E. coli and I. tartaricus models, must be occurring over the time course of the cross-linking experiment. In the additional experiments reported here, we tried to ascertain the preferred conformation in the membrane to help define the most likely structural model. We conclude that both structures must be able to form in the membrane, but that the helical swiveling that promotes their interconversion may not be necessary during rotary function.

Aqueous access pathways in ATP synthase subunit a. Reactivity of cysteine substituted into transmembrane helices 1, 3, and 5.

Subunit a is thought to play a key role in H+ transport-driven rotation of the subunit c ring in Escherichia coli F1F0 ATP synthase. In the membrane-traversing F0 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 aqueous channels formed at least in part by one or more of the five TMHs of subunit a. Aqueous access to surfaces of TMHs 2, 4, and 5 was previously suggested based upon the chemical reactivity of cysteine residues substituted into these helices. Here we have substituted Cys into TMH1 and TMH3 and extended the substitutions in TMH5 to the cytoplasmic surface. One region of TMH3 proved to be moderately Ag+-sensitive and may connect with the Ag+-sensitive region found previously on the periplasmic side of TMH2. A single Cys substitution in TMH1 proved to be both N-ethylmaleimide (NEM)-sensitive and Ag+-sensitive and suggests a possible packing interaction of TMH1 with TMH2 and TMH3. New Ag+- and NEM-sensitive residues were found at the cytoplasmic end of TMH5 and suggest a possible connection of this region to the NEM- and Ag+-sensitive region of TMH4 described previously. From the now complete pattern of TMH residue reactivity, we conclude that aqueous access from the periplasmic side of F0 to cAsp-61 at the center of the membrane is likely to be mediated by residues of TMHs 2, 3, 4, and 5 at the center of a four-helix bundle. Further, aqueous access between cAsp-61 and the cytoplasmic surface is likely to be mediated by residues in TMH4 and TMH5 at the exterior of the four-helix bundle that are in contact with the c-ring.

Cross-linking between helices within subunit a of Escherichia coli ATP synthase defines the transmembrane packing of a four-helix bundle.

Subunit a of F(1)F(0) ATP synthase is required in the H(+) transport driven rotation of the c-ring of F(0), the rotation of which is coupled to ATP synthesis in F(1). The three-dimensional structure of subunit a is unknown. In this study, Cys substitutions were introduced into two different transmembrane helices (TMHs) of subunit a, and the proximity of the thiol side chains was tested via attempted oxidative cross-linking to form the disulfide bond. Pairs of Cys substitutions were made in TMHs 2/3, 2/4, 2/5, 3/4, 3/5, and 4/5. Cu(+2)-catalyzed oxidation led to cross-link formation between Cys pairs L120C(TMH2) and S144C(TMH3), L120C(TMH2) and G218C(TMH4), L120C(TMH2) and H245C(TMH5), L120C(TMH2) and I246C(TMH5), N148C(TMH3) and E219C(TMH4), N148C(TMH3) and H245C(TMH5), and G218C(TMH4) and I248C(TMH5). Iodine, but not Cu(+2), was found to catalyze cross-link formation between D119C(TMH2) and G218C(TMH4). The results suggest that TMHs 2, 3, 4, and 5 form a four-helix bundle with one set of key functional residues in TMH4 (Ser-206, Arg-210, and Asn-214) located at the periphery facing subunit c. Other key residues in TMHs 2, 4, and 5, which were concluded previously to compose a possible aqueous access pathway from the periplasm, were found to locate to the inside of the four-helix bundle.

Backbone 1H, 15N and 13C assignments for the subunit a of the E. coli ATP synthase.

The structure of the 30 KDa subunit a of the membrane component (F(0)) of E. coli ATP synthase is investigated in a mixture of chloroform, methanol and water, a solvent previously used for solving the structure of another integral membrane protein, subunit c. Near complete backbone chemical shift assignments were made from a set of TROSY experiments including HNCO, HNCA, HN(CA)CB, HN(CO)CACB and 4D HNCOCA and HNCACO. Secondary structure of subunit a was predicted from the backbone chemical shifts using TALOS program. The protein was found to consist of multiple elongated alpha-helical segments. This finding is generally consistent with previous predictions of multiple transmembrane alpha-helices in this polytopic protein.

Insights into the molecular mechanism of rotation in the Fo sector of ATP synthase.

F(1)F(o)-ATP synthase is a ubiquitous membrane protein complex that efficiently converts a cell's transmembrane proton gradient into chemical energy stored as ATP. The protein is made of two molecular motors, F(o) and F(1), which are coupled by a central stalk. The membrane unit, F(o), converts the transmembrane electrochemical potential into mechanical rotation of a rotor in F(o) and the physically connected central stalk. Based on available data of individual components, we have built an all-atom model of F(o) and investigated through molecular dynamics simulations and mathematical modeling the mechanism of torque generation in F(o). The mechanism that emerged generates the torque at the interface of the a- and c-subunits of F(o) through side groups aSer-206, aArg-210, and aAsn-214 of the a-subunit and side groups cAsp-61 of the c-subunits. The mechanism couples protonation/deprotonation of two cAsp-61 side groups, juxtaposed to the a-subunit at any moment in time, to rotations of individual c-subunit helices as well as rotation of the entire c-subunit. The aArg-210 side group orients the cAsp-61 side groups and, thereby, establishes proton transfer via aSer-206 and aAsn-214 to proton half-channels, while preventing direct proton transfer between the half-channels. A mathematical model proves the feasibility of torque generation by the stated mechanism against loads typical during ATP synthesis; the essential model characteristics, e.g., helix and subunit rotation and associated friction constants, have been tested and furnished by steered molecular dynamics simulations.

Subunit A of the E. coli ATP synthase: reconstitution and high resolution NMR with protein purified in a mixed polarity solvent.

Subunit a of the Escherichia coli ATP synthase, a 30 kDa integral membrane protein, was purified to homogeneity by a novel procedure incorporating selective extraction into a monophasic mixture of chloroform, methanol and water, followed by Ni-NTA chromatography in the mixed solvent. Pure subunit a was reconstituted with subunits b and c and phospholipids to form a functional proton-translocating unit. Nuclear magnetic resonance (NMR) spectra of the pure subunit a in the mixed solvent show good chemical shift dispersion and demonstrate the potential of the solvent mixture for NMR studies of the large membrane proteins that are currently intractable in aqueous detergent solutions.

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.