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Bacterial operons for [F.sub.1][F.sub.o]-ATP synthase typically include an uncl gene that encodes a function-unknown small hydrophobic protein. When we expressed a hybrid [F.sub.1][F.sub.o] ([F.sub.1] from thermophilic Bacillus PS3 and [Na.sup.+]-translocating [F.sub.o] from Propionigenium modestum) in Escherchia coli cells, we found that uncl derived from P. modestum was indispensable to produce active enzyme; without uncl, c-subunits in [F.sub.1][F.sub.o] existed as monomers but not as functional [c.sub.11]-ring. When uncl was expressed from another plasmid at the same time, active [F.sub.1][F.sub.o] with [c.sub.11]-ring was produced. A plasmid containing only uncl and c-subunit gene produced [c.sub.11]-ring, but a plasmid containing only c-subunit gene did not. Direct interaction of Uncl protein with c-subunits was suggested from copurification of His-tagged Uncl protein and c-subunits, both in the state of [c.sub.11]-ring and c-monomers. [Na+] induced dissociation of His-tagged Uncl protein from [c.sub.11]-ring but not from c-monomers. These results show that Uncl is a chaperone-like protein that assists [c.sub.11]-ring assembly from c-monomers in the membrane. protein folding | membrane protein | [Na.sup.+] transport

ATP synthesis by V-ATPase from the thermophilic bacterium Thermus thermophilus driven by the acid-base transition was investigated. The rate of ATP synthesis increased in parallel with the increase in proton motive force (PMF) > 110 mY, which is composed of a difference in proton concentration ([DELTA]pH) and the electrical potential differences ([DELTA][PSI]) across membranes. The optimum rate of synthesis reached 85 [s.sup.-1], and the [H.sup.+]/ATP ratio of 4.0 [+ or -] 0.1 was obtained. ATP was synthesized at a considerable rate solely by [DELTA]pH, indicating [DELTA][PSI] was not absolutely required for synthesis. Consistent with the [H.sup.+]/ATP ratio, cryoelectron micrograph images of 2D crystals of the membrane-bound rotor ring of the V-ATPase at 7.0-[Angstrom] resolution showed the presence of 12 [V.sub.o]-c subunits, each composed of two transmembrane helices. These results indicate that symmetry mismatch between the rotor and catalytic domains is not obligatory for rotary ATPases/synthases. ATP synthase | rotary motor | membrane protein | bioenergetics | two-dimensional crystal

The [epsilon] subunit of bacterial and chloroplast [F.sub.0][F.sub.1]-ATP synthases modulates their ATP hydrolysis activity. Here, we report the crystal structure of the ATP-bound [epsilon] subunit from a thermophilic Bacillus PS3 at 1.9-[Angstrom] resolution. The C-terminal two a-helices were folded into a hairpin, sitting on the [beta] sandwich structure, as reported for Escherichia coli. A previously undescribed ATP binding motif, I(L)DXXRA, recognizes ATP together with three arginine and one glutamate residues. The E. coli [epsilon] subunit binds ATP in a similar manner, as judged on NMR. We also determined solution structures of the C-terminal domain of the PS3 [epsilon] subunit and relaxation parameters of the whole molecule by NMR. The two helices fold into a hairpin in the presence of ATP but extend in the absence of ATP. The latter structure has more helical regions and is much more flexible than the former. These results suggest that the [epsilon] C-terminal domain can undergo an arm-like motion in response to an ATP concentration change and thereby contribute to regulation of [F.sub.0][F.sub.1]-ATP synthase. ATP hydrolysis | ATP-binding motif | ATPase regulation | ATP synthase | [F.sub.1] rotation

[V.sub.1], a water-soluble portion of vacuole-type ATPase (V-ATPase), is an ATP-driven rotary motor, similar to [F.sub.1]-ATPase. Hydrolysis of ATP is coupled to unidirectional rotation of the central rotor D and F subunits relative to the [A.sub.3][B.sub.3] cylinder. In this study, we analyzed the rotation kinetics of [V.sub.1] in detail. At low ATP concentrations, the D subunit rotated stepwise, pausing every 120[degrees]. The dwell time between steps revealed that [V.sub.1] consumes one ATP per 120[degrees] step. [V.sub.1] generated torque of [approximately equal to] 35 pN nm, slightly lower than the [approximately equal to] v46 pN nm measured for [F.sub.1]. Noticeably, the angles for both ATP cleavage and binding were apparently the same in [V.sub.1], in sharp contrast to [F.sub.1], which cleaves ATP at 80[degrees] posterior to the binding of ATP. Thus, the mechanochemical cycle of [V.sub.1] has marked differences to that of [F.sub.1]. ATP synthase | molecular motor | single molecular analysis | vacuole-type ATPase

A rotary motor [F.sub.1], a catalytic part of ATP synthase, makes a 120[degrees] step rotation driven by hydrolysis of one ATP, which consists of 80[degrees] and 40[degrees] substeps initiated by ATP binding and probably by ADP and/or [P.sub.i] dissociation, respectively. During active rotations, [F.sub.1] spontaneously fails in ADP release and pauses after a 80[degrees] substep, which is called the ADP-inhibited form. In the present work, we found that, when pushed >+40[degrees] with magnetic tweezers, the pausing [F.sub.1] resumes its active rotation after releasing inhibitory ADP. The rate constant of the mechanical activation exponentially increased with the pushed angle, implying that [F.sub.1] weakens the affinity of its catalytic site for ADP as the angle goes forward. This finding explains not only its unidirectional nature of rotation, but also its physiological function in ATP synthesis; it would readily bind ADP from solution when rotated backward by an [F.sub.o] motor in the ATP synthase. Furthermore, the mechanical work for the forced rotation was efficiently converted into work for expelling ADP from the catalytic site, supporting the tight coupling between the rotation and catalytic event. ADP inhibition | ATP synthase | F1-ATPase | magnetic tweezers | single-molecule observation

[F.sub.o][F.sub.1]-ATP synthase ([F.sub.o][F.sub.1]) is a motor enzyme that couples ATP synthesis/hydrolysis with a transmembrane proton translocation. [F.sub.1], a water-soluble ATPase portion of [F.sub.o][F.sub.1], rotates by repeating ATP-waiting dwell, 80[degrees] substep rotation, catalytic dwell, and 40[degrees]- substep rotation. Compared with [F.sub.1], rotation of [F.sub.o][F.sub.1] has yet been poorly understood, and, here, we analyzed ATP-driven rotations of [F.sub.o][F.sub.1]. Rotation was probed with an 80-nm bead attached to the ring of c subunits in the immobilized [F.sub.o][F.sub.1] and recorded with a submillisecond fast camera. The rotation rates at various ATP concentrations obeyed the curve defined by a Km of [approximately equal to]30 [micro]M and a Vmax of [approximately equal to]350 revolutions per second (at 37[degrees]C). At low ATP, ATP-waiting dwell was seen and the [k.sub.on]-ATP was estimated to be 3.6 x [10.sup.7] [M.sup.-1] x [s.sup.-1]. At high ATP, fast, poorly defined stepwise motions were observed that probably reflect the catalytic dwells. When a slowly hydrolyzable substrate, adenosine 5'-[[gamma]-thio]triphosphate, was used, the catalytic dwells consisting of two events were seen more clearly at the angular position of [approximately equal to]80[degrees]. The rotational behavior of [F.sub.o][F.sub.1] resembles that of [F.sub.1]. This finding indicates that 'friction' in [F.sub.o] motor is negligible during the ATP-driven rotation. Tributyltin chloride, a specific inhibitor of proton translocation, slowed the rotation rate by 96%. However, dwells at clearly defined angular positions were not observed under these conditions, indicating that inhibition by tributyltin chloride is complex. ATP hydrolysis | binding change mechanism | membrane protein | single-molecule imaging

In a rotary motor [F.sub.o][F.sub.1]-ATP synthase that couples [H.sup.+] transport with ATP synthesis/hydrolysis, it is thought that an [F.sub.o]c subunit oligomer ring (c-ring) in the membrane rotates as protons pass through [F.sub.o] and a 120[degrees] rotation produces one ATP at [F.sub.1]. Despite several structural studies, the copy number of [F.sub.o]c subunits in the c-ring has not been determined for any functional [F.sub.o][F.sub.1]. Here, we have generated and isolated thermophilic Bacillus [F.sub.o][F.sub.1], each containing genetically fused 2-mer-14-mer c ([c.sub.2]-[c.sub.14]). Among them, [F.sub.o][F.sub.1] containing [c.sub.2], [c.sub.5], or [c.sub.10] showed ATP-synthesis and other activities. When [F.sub.1] was removed, [F.sub.o] containing [c.sub.10] worked as an [H.sup.+] channel but [F.sub.o]S containing [c.sub.9], [c.sub.11] or [c.sub.12] did not. Thus, the c-ring of functional [F.sub.o][F.sub.1] of this organism is a decamer. The inevitable consequence of this finding is noninteger ratios of rotation step sizes of [F.sub.1]/[F.sub.o] (120[degrees]/36[degrees]) and of [H.sup.+]/ATP (10:3). This step-mismatch necessitates elastic twisting of the rotor shaft (and/or the side stalk) during rotation and permissive coupling between unit rotations by [H.sup.+] transport at [F.sub.o] and elementary events in catalysis at [F.sub.1].

Author(s): Hiroyasu Itoh (corresponding author) [1, 2]; Akira Takahashi [3]; Kengo Adachi [4]; Hiroyuki Noji [5]; Ryohei Yasuda [6]; Masasuke Yoshida [7]; Kazuhiko Kinosita, Jr [4] ATP, the main biological [...]

The vacuole-type ATPases (V-ATPases) exist in various intracellular compartments of eukaryotic cells to regulate physiological processes by controlling the acidic environment. The crystal structure of the subunit C of Thermus thermophilus V-ATPase, homologous to eukaryotic subunit d of V-ATPases, has been determined at 1.95-A resolution and located into the holoenzyme complex structure obtained by single particle analysis as suggested by the results of subunit cross-linking experiments. The result shows that V-ATPase is substantially longer than the related F-type ATPase, due to the insertion of subunit C between the [V.sub.1] (soluble) and the [V.sub.o] (membrane bound) domains. Subunit C, attached to the [V.sub.o] domain, seems to have a socket like function in attaching the central-stalk subunits of the [V.sub.1] domain. This architecture seems essential for the reversible association/dissociation of the [V.sub.1] and the [V.sub.o] domains, unique for V-ATPase activity regulation.

[F.sub.1], a water-soluble portion of [F.sub.0][F.sub.1]-ATP synthase, is an ATP hydrolysis-driven rotary motor. The central [gamma]-subunit rotates in the [alpha]3[beta]3 cylinder by repeating the following four stages of rotation: ATP-binding dwell, rapid 80[degrees] substep rotation, interim dwell, and rapid 40[degrees] substep rotation. At least two 1-ms catalytic events occur in the interim dwell, but it is still unclear which steps in the ATPase cycle, except for ATP binding, correspond to these events. To discover which steps, we analyzed rotations of [F.sub.1] subcomplex ([[alpha].sub.3][[beta].sub.3][gamma]) from thermophilic Bacillus PS3 under conditions where cleavage of ATP at the catalytic site is decelerated: hydrolysis of ATP by the catalytic-site mutant [F.sub.1] and hydrolysis of a slowly hydrolyzable substrate ATP[gamma]S (adenosine 5'-[[gamma],-thio]triphosphate) by wild-type [F.sub.1]. In both cases, interim dwells were extended as expected from bulk phase kinetics, confirming that cleavage of ATP takes place during the interim dwell. Furthermore, the results of ATP[gamma]S hydrolysis by the mutant [F.sub.1] ensure that cleavage of ATP most likely corresponds to one of the two 1-ms events and not some other faster undetected event. Thus, cleavage of ATP on [F.sub.1] occurs in 1 ms during the interim dwell, and we call this interim dwell catalytic dwell.

[V.sub.o][V.sub.1]-ATPase is responsible for acidification of eukaryotic intracellular compartments and ATP synthesis of Archaea and some eubacteria. From the similarity to [F.sub.o][F.sub.1]-ATP synthase, [V.sub.o][V.sub.1]-ATPase has been assumed to be a rotary motor, but to date there are no experimental data to support this. Here we visualized the rotation of single molecules of [V.sub.1]-ATPase, a catalytic subcomplex of [V.sub.o][V.sub.1]-ATPase. [V.sub.1]-ATPase from Thermus thermophilus was immobilized onto a glass surface, and a bead was attached to the D or F subunit through the biotin-streptavidin linkage. In both cases we observed ATP-dependent rotations of beads, the direction of which was always counterclockwise viewed from the membrane side. Given that three ATP molecules are hydrolyzed per one revolution, rates of rotation agree consistently with rates of ATP hydrolysis at saturating ATP concentrations. This study provides experimental evidence that [V.sub.o][V.sub.1]-ATPase is a rotary motor and that both D and F subunits constitute a rotor shaft.

[F.sub.1]-ATPase is a rotary motor enzyme in which a single ATP molecule drives a 120 [degrees] rotation of the central [gamma] subunit relative to the surrounding [[alpha].sub.3][[beta].sub.3] ring. Here, we show that the rotation of [F.sub.1]-ATPase spontaneously lapses into long ([approximately equal to] 30 s) pauses during steady-state catalysis. The effects of ADP-Mg and mutation on the pauses, as well as kinetic comparison with bulk-phase catalysis, strongly indicate that the paused enzyme corresponds to the inactive state of [F.sub.1]-ATPase previously known as the ADP-Mg inhibited form in which [F.sub.1]-ATPase fails to release ADP-Mg from catalytic sites. The pausing position of the [gamma] subunit deviates from the ATP-waiting position and is most likely the recently found intermediate 90 [degrees] position.

Author(s): Ryohei Yasuda [1, 2, 5]; Hiroyuki Noji [1]; Masasuke Yoshida [3, 1]; Kazuhiko Kinosita, Jr (corresponding author) [2, 1]; Hiroyasu Itoh [4, 1] The ATP synthase is an enzyme [...]

Results show that the protein sequence and the N-terminal amino acid sequence of the polypeptides of the bacterium are similar to an F-type ATPase. Data indicate that the enzyme functions as an ATP synthase and participates in the sulfate respiration.

Functional chaperone cooperation between Hsp70 (DnaK) and Hsp104 (ClpB) was demonstrated in vitro. In a eubacterium Thermus thermophilus, DnaK and DnaJ exist as a stable trigonal ring complex (TDnaK*J complex) and the dnaK gene cluster contains a clpB gene. When substrate proteins were heated at high temperature, none of the chaperones protected them from heat inactivation, but the TDnaK*J complex could suppress the aggregation of proteins in an ATP- and TGrpE-dependent manner. Subsequent incubation of these heated preparations at moderate temperature after addition of TClpB resulted in the efficient reactivation of the proteins. Reactivation was also observed, even though the yield was low, if the substrate protein alone was heated and incubated at moderate temperature with the TDnaK*J complex, TGrpE, TClpB, and ATP. Thus, all these components were necessary for the reactivation. Further, we found that TGroEL/ES could not substitute TClpB.

The study investigated the rotation of the gamma-attached actin at low adenosine triphosphate (ATP) concentrations. This was done by fixing a subcomplex of the protruding portion of the ATP synthase onto a surface-bound bead with ten histidines linked to the amino terminus of the beta subunit. Rotation was observed using a fluorescently labeled actin filament attached to the gamma subunit. Results found that rotation of the actin was resolved into discrete 120 degree-steps with occasional back steps. A high thermodynamic efficiency was also noted in the process.

A review was done to describe a single molecule of F1-adenosine triphosphatase (F1-ATPase), a portion of ATP synthase which functions as a rotary motor enzyme. It involves a central rotor which rotates over unlimited angles against a surrounding stator cylinder of a hexamer. ATP synthase consists of a membrane-embedded, proton-conducting portion F0, and a protruding portion F1. Studies on proton flow through F1 showed that ATP synthase comprises an ATP-driven and a proton-driven motor. A comparison of nucleotide-driven molecular motors is discussed.

The rates of inactivation of the wild-type and mutant alpha3beta3gamma subcomplexes of TF1 in the presence of ADP, Mg2+, Al3+ and F- were investigated. The results showed a slow, but complete, inactivation when Al3+ and F- were added to wild type and mutan alpha3beta3gamma subcomplexes containing MgADP bound to a single catalytic site.

A direct observation of the motion of a single molecule of F(sub 1)-adenosine triphosphatase (ATPase) shows that the molecule acts as a rotary motor. Earlier studies have concluded that the gamma-subunit of F(sub 1)-ATPase rotates within the alpha beta-hexamer. In the presence of ATP, the gamma-subunit was observed to rotate for more than 100 revolutions in an anticlockwise direction when seen from the 'membrane' side.

The (alpha-D(sub 261)N)(sub 3)-beta(sub 3)-gamma mutant of the F(sub 1)-ATPase of Bacillus PS3 is unable to dissociate the MgADP bound to its catalytic sites. The lack of dissociation in the mutant causes a delay in the hydrolysis of ATP. The binding of ATP or ADP to the noncatalytic sites in the presence of MgADP is unaffected. The inability to cause the dissociation is due to a defective cross-talk between the catalytic and noncatalytic sites containing ligands.

Segmental labeling is allied by intein splicing reaction to the beta subunit of FoF1-ATP synthase for obtaining the detailed information on the conformational change of the beta subunit monomer. The intrinsic conformational change of the beta subunit monomer induced by nucleotide binding must be one of the essential driving forces for the rotation of F1ATPase.

Research has been conducted on bacterial and eukaryotic proteins which are capable of rescuing stress-damaged proteins from an aggregated states. The structure of bacterial protein ClpB has been investigated via the use of X-ray crystallography and cryo-electron microscopy, and the results have revealed the mechanism by which ClpBi mediates the disaggregation of high molecular weight aggregates.

The [F.sub.1][F.sub.0] ATP synthase is the smallest motor enzyme known. Previous studies had established that the central stalk, made of the [Gamma] and [Epsilon] subunits in the [F.sub.1] part and c subunit ring in the [F.sub.0] part, rotates relative to a stator composed of [[Alpha].sub.3][[Beta].sub.3][Delta]a[b.sub.2] during ATP hydrolysis and synthesis. How this rotation is regulated has been less clear. Here, we show that the [Epsilon] subunit plays a key role by acting as a switch of this motor. Two different arrangements of the r subunit have been visualized recently. The first has been observed in beef heart mitochondrial [F.sub.1]-ATPase where the C-terminal portion is arranged as a two-[Alpha]-helix hairpin structure that extends away from the [[Alpha].sub.3][[Beta].sub.]3 region, and toward the position of the c subunit ring in the intact [F.sub.1][F.sub.0]. The second arrangement was observed in a structure determination of a complex of the [Gamma] and [Epsilon] subunits of the Escherichia coli [F.sub.1]-ATPase. In this, the two C-terminal helices are apart and extend along the 3, to interact with the [Alpha] and [Beta] subunits in the intact complex. We have been able to trap these two arrangements by cross-linking after introducing appropriate Cys residues in E. coli [F.sub.1][F.sub.0], confirming that both conformations of the r subunit exist in the enzyme complex. With the C-terminal domain of [Epsilon] toward the [F.sub.0], ATP hydrolysis is activated, but the enzyme is fully coupled in both ATP hydrolysis and synthesis. With the C-terminal domain toward the [F.sub.1] part, ATP hydrolysis is inhibited and yet the enzyme is fully functional in ATP synthesis; i.e., it works in one direction only. These results help explain the inhibitory action of the e subunit in the [F.sub.1][F.sub.0] complex and argue for a ratchet function of this subunit.

The [F.sub.1][F.sub.o]-type ATP synthase is the smallest motor enzyme known. Previous studies had established that the central [Gamma] and [Epsilon] subunits of the [F.sub.1] part rotate relative to a stator of [[Alpha].sub.3][[Beta].sub.3] and [Delta] subunits during catalysis. We now show that the ring of c subunits in the [F.sub.o] part moves along with the [Gamma] and [Epsilon] subunits. This was demonstrated by linking the three rotor subunits with disulfide bridges between cysteine residues introduced genetically at the interfaces between the [Gamma], [Epsilon], and c subunits. Essentially complete cross-linking of the [Gamma], [Epsilon], and c subunits was achieved by using Cu[Cl.sub.2] to induce oxidation. This fixing of the three subunits together had no significant effect on ATP hydrolysis, proton translocation, or ATP synthesis, and each of these functions retained inhibitor sensitivity. These results unequivocally place the c subunit oligomer in the rotor part of this molecular machine.

Orientation dependence of single-fluorophore intensity was exploited in order to videotape conformational changes in a protein machine in real time. The fluorophore Cy3 attached to the central subunit of [F.sub.1]-ATPase revealed that the subunit rotates in the molecule in discrete 120 [degrees] steps and that each step is driven by the hydrolysis of one ATP molecule. These results, unlike those from the previous study under a frictional load, show that the 120 [degrees] stepping is a genuine property of this molecular motor. The data also show that the rate of ATP binding is insensitive to the load exerted on the rotor subunit.