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Originally published In Press as doi:10.1074/jbc.M700551200 on May 21, 2007

J. Biol. Chem., Vol. 282, Issue 28, 20698-20704, July 13, 2007
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Rotational Catalysis of Escherichia coli ATP Synthase F1 Sector

STOCHASTIC FLUCTUATION AND A KEY DOMAIN OF THE beta SUBUNIT*

Mayumi Nakanishi-Matsui{ddagger}, Sachiko Kashiwagi{ddagger}, Toshiharu Ubukata{ddagger}, Atsuko Iwamoto-Kihara§, Yoh Wada, and Masamitsu Futai{ddagger}1

From the {ddagger}Futai Special Laboratory, Microbial Chemistry Research Center, Microbial Chemistry Research Foundation, Tokyo 141-0021, Japan and Department of Biochemistry, Faculty of Pharmaceutical Sciences, Iwate Medical University, Iwate 028-3694, Japan, §Department of Bioscience, Nagahama Institute of Bioscience and Technology, Nagahama, Shiga 526-0829, Japan, and Department of Biological Sciences, The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0041, Japan

Received for publication, January 19, 2007 , and in revised form, May 16, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A complex of {gamma}, {epsilon}, and c subunits rotates in ATP synthase (FoF1) coupled with proton transport. A gold bead connected to the {gamma} subunit of the Escherichia coli F1 sector exhibited stochastic rotation, confirming a previous study (Nakanishi-Matsui, M., Kashiwagi, S., Hosokawa, H., Cipriano, D. J., Dunn, S. D., Wada, Y., and Futai, M. (2006) J. Biol. Chem. 281, 4126-4131). A similar approach was taken for mutations in the beta subunit key region; consistent with its bulk phase ATPase activities, F1 with the Ser-174 to Phe substitution (betaS174F) exhibited a slower single revolution time (time required for 360 degree revolution) and paused almost 10 times longer than the wild type at one of the three 120° positions during the stepped revolution. The pause positions were probably not at the "ATP waiting" dwell but at the "ATP hydrolysis/product release" dwell, since the ATP concentration used for the assay was ~30-fold higher than the Km value for ATP. A betaGly-149 to Ala substitution in the phosphate binding P-loop suppressed the defect of betaS174F. The revertant (betaG149A/betaS174F) exhibited similar rotation to the wild type, except that it showed long pauses less frequently. Essentially the same results were obtained with the Ser-174 to Leu substitution and the corresponding revertant betaG149A/betaS174L. These results indicate that the domain between beta-sheet 4 (betaSer-174) and P-loop (betaGly-149) is important to drive rotation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A ubiquitous ATP synthase (FoF1) synthesizes ATP coupled with an electrochemical proton gradient formed by a respiratory chain (for reviews, see Refs 1-5). FoF1 consists of a catalytic sector, F1 ({alpha}3beta3{gamma}{delta}{epsilon}), and a membrane-embedded proton path-way, Fo (ab2c10), and can reversibly transport protons coupled with ATP hydrolysis. The {alpha} and beta subunits form a catalytic hexamer ({alpha}3beta3), the central space of which is occupied by the {gamma} subunit {alpha}-helices. ATP is synthesized or hydrolyzed cooperatively at a catalytic site in each beta subunit as the binding change mechanism predicts (2). The {gamma} subunit rotation in {alpha}3beta3 has been supported by biochemical studies (1, 6, 7), a crystal structure of the {alpha}3beta3{gamma} complex (8), and video recorded using an actin filament as a probe (9, 10). Consistent with ATP-dependent proton translocation, a {gamma}{epsilon}c10 complex rotated relative to the {alpha}3beta3{delta}ab2 in the purified FoF1 (11-14) or its membrane-bound form (15, 16). The rotation of FoF1 in liposomes has been revealed by means of single molecule fluorescence resonance energy transfer (17).

Counterclockwise rotation of the {gamma} subunit has been studied more recently with probes giving low viscous drag such as colloidal gold (14, 18, 19). The three 120° steps in one revolution of Bacillus F1 were first observed using an actin filament (20), and later the single 120° step was further subdivided into two substeps (80° and 40°) using gold beads that allow finer observation and analysis (14, 19, 21). The substeps with larger displacement angles (80°) and smaller substeps (40°) are assigned to ATP binding and hydrolysis/product release steps, respectively (19, 21, 22). We have observed that the rotation speed of beads attached to the Escherichia coli {gamma} subunit varied, reflecting stochastic fluctuations (18, 23). Although the average speeds were dependent on the diameter of beads, 40- and 60-nm diameter beads rotated with essentially the same rate (18), suggesting that their rotation speeds were close to that of the {gamma} subunit without a probe attached.

The mechanism underlying the chemistry and energy coupling of FoF1 has been studied by introducing mutations (1, 10, 13, 16, 24). One of the most interesting mutations is the substitution of the betaSer-174 residue (24-27) located in beta-sheet 4, thus being distant from the bound ATP in the beta subunit (8) (see Fig. 1). The size of the residue at this position is pertinent as to the activity (25); the larger the side-chain volume of the residue introduced, the lower the ATPase activity became. The betaS174F (betaSer-174 to Phe substitution) or betaS174L (betaSer-174 to Leu) F1 sector exhibited ~10% of the wild-type activity (25). The defect of betaS174F was suppressed by the replacement of betaGly-149 (betaGly-149 to Ala, Ser, or Cys) in the phosphate binding P-loop near {alpha}-helix B (26, 27). However, rotation of an actin filament connected to {gamma} was not proportional to the ATPase activity; generated torque for betaS174F and betaS174L were 40~100% that of the wild-type level (24). Thus, it became of interest to analyze mechanical revolution of the {gamma} subunit in these mutants using a probe smaller than an actin filament.

In this study we confirmed stochastic fluctuation of the {gamma} subunit rotation by analyzing the single revolution time (time required for 360° revolution). The betaS174F mutant paused at 120° steps longer than the wild type, giving an ~6 times lower revolution time. The rotation of second-site revertant betaG149A/betaS174F was similar to that of the wild type. Essentially the same results were obtained for betaS174L mutant and its second-site revertant betaG149A/betaS174L. We discuss a possible role(s) of the domain including beta-sheet 4 and the phosphate binding P-loop, where betaSer-174 and betaGly-149 are located, respectively (Fig. 1).


Figure 1
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  		FIGURE 1.
Models of the beta-sheet 4/loop/{alpha}-helix B/P-loop domain in E. coli ATP synthase beta subunit. a, the structure of {alpha}3beta3{gamma} is shown with a region discussed in this study (squares): blue, beta subunit; green, {alpha} subunit; gray, {gamma} subunit. b and c, the beta-sheet 4/loop/{alpha}-helix B/P-loop domain structure of E. coli beta subunit was modeled after the bovine structure (8). The sequence identity of the beta subunit between cow and E. coli is 71.7% (24). The nomenclature for {alpha}-helix and beta-sheet is cited from Abrahams et al. (8). The domains of empty (b) and ATP-bound (c) beta subunits are shown together with amino acid residues discussed in under "Results and Discussion." The positions of betaSer-174 and betaGly-149 residue are shown (red line).

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation and Materials—E. coli strain DK8 ({Delta}uncB-C) was used as a host for recombinant plasmids and grown at 37 °C in a synthetic medium containing 0.5% glycerol as a carbon source. A plasmid carrying the unc operon introduced six His residues at the {alpha} subunit amino terminus, and {gamma}S193C and {gamma}K108C substitutions in the {gamma} subunit were described previously (18). Mutations betaS174F and betaS174L and related substitutions of the beta subunit were introduced into F1 engineered for rotation (24). The wild-type and mutant F1 sectors were purified on a glycerol gradient (24). Gel electrophoresis showed the presence of about 1 mol {epsilon}/mol F1 but no {delta} subunit in any of the preparations analyzed. Gold beads (60-nm diameter) were obtained from British Bio Cell International and were coated with biotinylated bovine serum albumin (18). The cover glass used for constructing a cell for observing rotation was soaked in 0.1 N KOH for 3 days followed by extensive washing with high purity water (18).

Assay Procedures—Mutant and wild-type ATPase activities were assayed as described previously (10) under the conditions used for the rotation assay (18). The third (highest) Km values for ATP were obtained with the Mg2+ concentrations that gave the maximal activities. When varying the ATP concentration, the Mg2+:ATP ratio was maintained at 1:1, except that the Mg2+ concentration was 0.5 mM when ATP concentration was lower than 0.5 mM. For the betaG149A mutant, the ratio was maintained at 2:1, except that the Mg2+ concentration was 4 mM for ATP lower than 0.5 mM. Protein concentrations were determined using bovine serum albumin (Sigma, Fraction V) as a standard (28).

Observing {gamma} Subunit Rotation—Rotation was assayed essentially as described previously (18). Briefly, gold beads were connected to the immobilized F1 sector in a flow cell (~30-µm deep) filled with buffer A (10 mg/ml bovine serum albumin, 10 mM MOPS2/KOH, pH 7.0, 50 mM KCl, and 2 mM MgCl2). Immediately after the introduction of buffer A containing 2 mM ATP and its regenerating system, images of the beads illuminated with laser light (JUNO EX, Showa Optronics Co.) were obtained on dark field microscopy (BX51WI-CDEVA-F, Olympus, Tokyo) and recorded with a charge-coupled device camera for data analysis using a Metamorph (Molecular Devices Corp.). The proper camera speeds (1000~4000 frames/s) were selected depending on the mutations of the F1 sectors; wild-type and the revertants, 4000 frames/s; betaS174F, betaS174L, and betaG149A, 1000 frames/s. They were also assayed at a different camera speed when necessary. Other methods, including construction of glass cells for rotation and laser-light illumination, were described previously (18). We occasionally observed apparent clockwise movements of less than two revolutions, although they were not actual rotations. The beads analyzed were those showing such movements amounting to less than 5% of the total counterclockwise revolutions.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Stochastic Rotation of Gold Beads Attached to Wild-type F1—Gold beads connected to F1 rotated with various speeds and often paused for a short period (~ms) as shown previously (18). In the previous paper, we recorded their rotations for 0.25 s, estimated rates every 10 ms, and combined those from different beads (18). Histograms of the rotation rates showed stochastic fluctuation. However, previous analysis may emphasize the frequencies of rates close to 0 rps if beads paused a long time (>10 ms). Furthermore, these histograms may include variation between beads together with fluctuation of the rotation speeds. The previous observation time (18) was not enough to detect long pauses (>0.25 s). Thus, we were prompted to study longer time courses and single rotation events (360° rotations). The present analysis was more appropriate than estimating rates every 10 ms (18), because all revolutions could be included.

In this study the time course of each bead was followed for 2 s. We occasionally observed long pauses (>0.1 s) (Fig. 2a), which were not found in previous time courses (18); a bead rotated about 500 revolutions and paused (yellow curve in Fig. 2a), and others paused after 300 revolutions (green and pink curves). Sometimes we observed beads that started rotating after a long pause (gray curve). On average, about 3 long pauses appeared when we recorded 1000 revolutions. These pauses were possibly because of Mg-ADP inhibition (29), as discussed below. We assumed that the long dwells are Mg-inhibited states of the enzyme and analyzed single rotation events.

When time courses were expanded, apparent smooth rotations (Fig. 2a) exhibited dwells (short pauses, ~ms) and ~120° stepping (Fig. 2b). Therefore, we analyzed the single revolution time, i.e. the time required for 360° of revolution, to evaluate stochastic fluctuation of rotation. This parameter could follow all rotations of a bead in a time course regardless of the length of the pauses. As expected from the various speeds observed in a time course, each bead clearly showed stochastic fluctuation of the single revolution time (for examples, see Fig. 2c). The histograms for the individual beads and those for multiple beads (Fig. 2d) are closely similar, indicating that fluctuation tendency is an intrinsic property of the F1 molecule. The geometric mean of single revolution time was ~2.3 ms (2.0, 2.3, 2.3, and 2.6 ms, for four different beads) (Fig. 2c), and the average rotation rate (reciprocal of single revolution time) was 440 rps, i.e. slightly higher than the reported value, 380 rps (18). A 360° rotation including the long pause was observed with lower frequency and shown in the histograms of single rotation times (see >15, horizontal axis in Fig. 2, c and d).


Figure 2
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  		FIGURE 2.
Stochastic rotations of gold beads attached to F1. a, time courses of 60-nm beads attached to wild-type F1 were followed in the presence of 2 mM ATP. Different colors represent rotations of 10 individual gold beads. b, time courses of apparent smooth rotations were expanded to show dwells and ~120° stepping. c, examples of histograms of single revolution times (time required for 360° revolution) obtained for four single beads are shown. The colors correspond to those in a. d, histograms combined for multiple beads. The single revolution times obtained for 10 randomly selected beads are combined and shown as histograms.

 
ATPase Activity of Mutant F1—It became of interest to analyze the mutant F1 sector to understand the mechanisms underlying rotation and its stochastic fluctuation. betaS174F (Fig. 1b) and related mutations were introduced into the engineered F1 to observe rotation as previously described (18, 24). Steady state ATPase activities were assayed under the same conditions as those for rotation observation. The relative activities (% of wild-type level) were essentially similar to previous results: betaS174F, 8%; betaS174L, 9%; betaG149A/betaS174F, 314%; betaG149A/betaS174L, 200%; betaG149A, 75%. We also estimated the third (highest) Km for ATP to assay rotation in the presence of excess ATP. betaS174F, betaS174L, and the second-site revertant betaG149A/betaS174F exhibited Km values for ATP of 69, 60, and 64 µM, respectively, i.e. essentially the same as that of the wild type (71 µM). betaG149A exhibited a Km value (0.3 mM) of ~4-fold higher than that of the wild type, possibly because of the location of betaGly-149 near bound-Mg-ATP at the catalytic site (Fig. 1c). Based on these biochemical properties of the mutant enzymes, we assayed rotations of betaS174F, betaS174L, and the corresponding second-site revertants in the presence of 2 mM ATP, ~30-fold higher than the Km values to compare steady state ATP hydrolysis and rotation.

Rotation of Mutant F1 with betaSer-174 Replacement—The time course of a gold bead attached to betaS174F was followed for 2 s. The mutant rotated with variable rates and exhibited long pauses similar to the wild type (Fig. 3, a and b). The total revolutions (in 2 s) were much less than those of the wild type (Fig. 3b). Thus, we compared the wild type and mutants by analyzing single rotation events. The mutant apparently exhibited longer single revolution times than the wild type, as shown for the histograms of multiple beads (Fig. 3c). They are similar to those for single beads (data not shown). The peaks of the wild-type and mutant histograms from multiple beads were at 1.75 and 5 ms, respectively, and their geometric means were 2.3 and 14 ms, respectively. Most (~80%) of the mutant single revolution times were longer than 5 ms, and ~50% of them were longer than 10 ms (Fig. 3c), whereas ≥90% of those of the wild type were ≤5 ms. The geometric mean of the betaS174L single revolution times was ~26 ms, i.e. ~11 times longer time than that of the wild type, and >20% of the mutant times were longer than 100 ms, as shown by the histograms (Fig. 3d). These results were consistent with the low ATPase activities of the mutants.

Pausing of the {gamma} Subunit in the Presence of a High Concentration of ATP—The single revolution times of betaS174F and betaS174L were significantly longer than those of the wild type in the presence of 2 mM ATP (Fig. 3, c and d), possibly because the mutants exhibited a high tendency for longer dwell. Thus, we compared dwells of the wild type and mutants. As shown by expanded time courses, the mutant paused longer at 1/3, 2/3, or 3/3 of a 360° revolution compared with the wild type (Fig. 4a, note the time scale). The angular distributions of the centroids showed three peaks at about 120°, 240°, and 360°/0° (Fig. 4b), indicating that beads paused after 120° revolutions, as shown previously with low ATP concentrations (20). However, the pausing observed in the present study may not be at the ATP waiting dwell (time for a catalytic site waiting for ATP binding), since it was not affected by a further increase in the ATP concentration (data not shown), and rotations were assayed in the presence of a ~30-fold higher ATP concentration than the Km.


Figure 3
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  		FIGURE 3.
Observing rotation of beads attached to the {gamma} subunits of betaS174F and betaS174L mutants. a, time courses of 60-nm diameter beads attached to betaS174F mutant F1 are shown. b, the betaS174F mutant time courses (red) together with those of the wild-type (blue) (Fig. 2a) are shown. The frequencies of observing rotating beads were ~2% for both the wild type and mutants, whereas the other beads exhibited Brownian motions. c, betaS174F histograms of single revolution times obtained for 10 randomly selected beads. The frequency of single revolution times longer than 100 ms was 6.6%, distributed randomly from 101 to 1187 ms. d, histograms of betaS174L single revolution times for 32 beads. The frequency of single revolution times longer than 100 ms was 21.6%, distributed randomly from 101 to 1309 ms. A fitted curve for the wild type (blue) obtained from Fig. 2 is also shown (c and d).

 
The pausing dwell contributed significantly to a single revolution time since the stepping (0 -> 120°, 120 -> 240°, or 240° -> 360°/0°) speeds were high (mostly ≤0.25 and ≤1 ms per 120° step for wild-type and mutant, respectively). About 80% of the pausing dwells of the wild type and mutant were ≤0.5 and ≤5ms, respectively (Fig. 4c), indicating that mutant paused at least ~10 times longer than the wild type. Essentially the same results were obtained for the betaS174L mutant. Thus, the longer pausing dwells caused the slow rotation speeds and low ATPase activities of mutants.

Rotation of the Second-site Revertant—As shown previously, a defect of the betaS174F enzyme was suppressed by the second-site mutations, betaGly-149 to Ser, Cys, or Ala (26, 27), giving apparently wild-type ATPase activity. The time course of the revertant betaG149A/betaS174F and betaG149A single mutant was analyzed (Fig. 5, a and b) and shown together with those of the wild type and betaS174F mutant for comparison (Fig. 5c). Beads attached to the revertant F1 rotated similar to those of the wild type, except that they rarely exhibited pauses longer than 0.1 s. The histogram of the single revolution times for the revertant showed a peak at 2.5 ms and a geometric mean of 2.9 ms (Fig. 5d), i.e. similar to the wild-type values (Fig. 2d).

The average rotation rates were estimated as reciprocals of the geometric means for single revolution times, assuming that F1 sectors exhibiting various speeds were present in the assay mixture; those for the wild type, betaS174F, revertant, and betaG149A were 440, 70, 340, and 70 rps, respectively. The results for the two single mutants and the revertant indicate that the two mutations (betaS174F and betaG149A) suppressed each other and gave similar rates as the wild type. Essentially the same results were obtained for betaG149A/betaS174L (Fig. 5e), the second-site revertant of betaS174L.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have previously observed that a gold bead attached to the {gamma} subunit rotated at various rates, indicating stochastic fluctuation of F1 rotation (18). However, the reported results may have included variations between F1 molecules because data for multiple beads were combined to obtain histograms. In this study we analyzed single revolution times that include the pausing dwell and stepping velocity. As expected from the time courses, individual beads exhibited stochastic fluctuations, and their histograms were similar, indicating that the variation among F1 molecule was not significant.

The observed stochastic fluctuation was probably because of the intrinsic properties of the {gamma} subunit driven by catalysis in {alpha}3beta3 hexamer, since they were essentially independent of the bead sizes (40-200-nm diameter) (18, 23), lengths of histidine tag (6 or 10 histidine residues) introduced into the {alpha} subunit,3 and enzyme preparations (F1 or FoF1) (data not shown). The fluctuations were mainly because of the varying pausing dwells (~ms) because stepping was fast. Stochastic fluctuation was also observed in Bacillus F1 with the fluorophore Cy3, attached to the {gamma} subunit (30). Careful definition of wild-type rotation was the basis of further studies on mutant F1.

Replacement of betaSer-174 in beta-sheet 4 lowered the ATPase activity to ~10% of the wild-type level (25). The means of single revolution times of the beads attached to betaS174F and betaS174L were about 6 and 11 times longer than that of the wild type, respectively, consistent with about a 10 times longer pausing dwell of the mutant than that of the wild type. betaS174L took longer single revolution times than betaS174F for an unknown reason, although their steady state ATPase activities were similar (9 and 8% of the wild-type level, respectively). The difference between the results of ATPase activity and single revolution time may be because of the observation times: rotation assay, 2 s; ATPase activity, 3 min. It is possible that betaS174F did not rotate for long time (>2s) more often than betaS174L.


Figure 4
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  		FIGURE 4.
Pausing of the wild-type and mutant rotation. a, rotation of the wild type, betaS174F, and betaS174L. Rotation was followed as described in Fig. 3, and expanded time courses of typical beads are shown: black, wild-type; dark gray, betaS174F; light gray, betaS174L. b, histograms of the angular distribution of the wild-type and mutant beads. c, histograms of the pausing duration of the wild-type, betaS174F, and betaS174L. Rotations of randomly selected beads were followed for 200 revolutions, and histograms of pausing duration are shown. Wild-type histograms are also shown on an expanded time scale (inset).

 
betaS174F and betaS174L exhibited the {epsilon} subunit sensitivity similar to the wild type (data not shown), suggesting that the slow rotations by the mutants were not because of the effect of this subunit. As shown previously, the {epsilon} subunit was dissociated from F1 during its immobilization and washing before rotation assay (18).

The defect of betaS174F mutant activity was suppressed by a series of replacements of the betaGly-149 residue (26, 27). The single revolution times of the revertant betaG149A/betaS174F and betaG149A/betaS174L were similar to that of the wild type. betaGly-149 is the first residue of the P-loop containing catalytic residues such as betaLys-155 and betaThr-156, whereas betaSer-174 is located in beta-sheet 4 (Fig. 1). The conformation of the beta-sheet 4/loop/a-helix B/P-loop domain is strikingly different between the empty (betaE) and nucleotide-bound (betaDP and betaTP) beta subunit (8) (Fig. 1, b, and c). Thus, the conformational changes of the P-loop during catalysis affect beta-sheet 4 through {alpha}-helix B for the rotation. Substitution of betaSer-174 possibly affected the conformational transition (betaD -> betaE or betaE -> betaT) of the entire domain and increased the pausing dwell. The transition became similar to the wild type with the second mutation, betaG149A.

It should be important to discuss which rotation step is related to the conformation transition. 120° revolution was observed initially for an actin filament connected to Bacillus F1, when the ATP concentration was lowered (20). Using 40-nm gold beads, Yasuda et al. (19) further observed 90° and 30° substeps in each 120° step, which were later revised to 80° and 40°, respectively, by the same group (21). The dwell before the 80° revolution was dependent on the ATP concentration, whereas that before the 40° substep was not. These results indicated that the 80° and 40° substeps were driven by ATP binding and hydrolysis/product release, respectively. We assayed rotations with a high ATP concentration and often observed pauses upon 120° revolution possibly at one of the two substeps. As discussed above, they paused not at the ATP waiting dwell but possibly at the ATP hydrolysis/product release dwell. Assuming that the E. coli enzyme has the same two substeps as Bacillus, the mutant F1 paused longer before the 40° stepping. Present results of mutants and revertants indicate that the 40° stepping is driven by the conformation transition of the beta-sheet4/loop/a-helix B/P loop domain. Hydrolysis in or product release from the catalytic site including P-loop apparently originates this transition.

We occasionally observed long pauses (>0.1 s), which apparently lowered bulk-phase ATPase activity. The revertant betaG149A/betaS174F and betaG149A mutant showed long pauses less often than the wild type. These pauses may be because of the Mg-ADP-inhibited form observed previously using duplex beads (440- and 517-nm diameter) (29). The wild-type ATPase activity is sensitive to Mg2+ (31), which stabilizes the Mg-ADP-inhibited form (32, 33). On the other hand, the ATPase activities of betaG149A/betaS174F and betaG149A were less sensitive to Mg2+ than that of the wild type (data not shown), similar to the revertant, betaG149S/betaS174F (26). Thus, the long pauses of the revertant and betaG149A occurred rarely because their Mg-ADP-inhibited forms were unstable. These results were consistent with higher steady state ATPase activities of the revertant and betaG149A. In this regard mutations in the corresponding domain of Bacillus F1 changed the tendency to generate Mg-ADP-inhibited form (34).


Figure 5
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  		FIGURE 5.
Rotation of second-site revertant betaG149A/betaS174F and the betaG149A mutant. a, time courses of 60-nm beads attached to betaG149A/betaS174F. b, time courses of 60-nm beads attached to betaG149A. c, time courses of mutants and revertant. The rotation of mutants and the revertant was compared with wild-type: orange, betaG149A/betaS174F; green, betaG149A; blue, wild-type; red, betaS174F. d, single revolution times of betaG149A/betaS174F (orange) and betaG149A (green). The time courses of 10 beads were followed, and their single revolution times are shown as histograms together with fitted curves for the wild-type (blue) and betaS174F (red) obtained in Figs. 2 and 3, respectively. The fitted curves represent histograms for shorter than 50 ms. e, histograms of single revolution times of betaG149A/betaS174L. Single revolution times were obtained for 10 randomly selected beads. Fitted curves for the wild-type (blue) and betaS174L (purple) are also shown.

 
In conclusion, using gold beads but not actin filaments, present studies clearly exhibited stochastic fluctuation of F1 rotation and its defect in beta subunit mutants. Mutation/suppression studies revealed that the beta-sheet 4/loop/a-helix B/P-loop is an important domain to drive rotation and is at least partially responsible for Mg-ADP inhibition. Further single molecule analysis will provide a new insight in enzyme mechanism (35).


   FOOTNOTES
 
* This study was supported by CREST, Japan Science and Technology Agency, and the Japanese Ministry of Education, Culture, and Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 81-19-651-5111; Fax: 81-19-698-1843; E-mail: futaim{at}iwate-med.ac.jp.

2 The abbreviation used is: MOPS, 3-(N-morpholino) propanesulfonic acid. Back

3 M. Nakanishi-Matsui, S. Kashiwagi, T. Ubukata, A. Iwamoto-Kihara, Y. Wada, and M. Futai, unpublished observation. Back


   ACKNOWLEDGMENTS
 
We are grateful for the support from Daiichi Pharmaceutical Co. and Eisai Co. Ltd.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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