Cells employ a variety of linear motors, such as myosin, kinesin and RNA polymerase, which move along and exert force on a filamentous structure. But only one rotary motor has been investigated in detail, the bacterial flagellum (a complex of about 100 protein molecules). We now show that a single molecule of F1-ATPase acts as a rotary motor, the smallest known, by direct observation of its motion. A central rotor of radius approximately 1 nm, formed by its gamma-subunit, turns in a stator barrel of radius approximately 5nm formed by three alpha- and three beta-subunits. F1-ATPase, together with the membrane-embedded proton-conducting unit F0, forms the H+-ATP synthase that reversibly couples transmembrane proton flow to ATP synthesis/hydrolysis in respiring and photosynthetic cells. It has been suggested that the gamma-subunit of F1-ATPase rotates within the alphabeta-hexamer, a conjecture supported by structural, biochemical and spectroscopic studies. We attached a fluorescent actin filament to the gamma-subunit as a marker, which enabled us to observe this motion directly. In the presence of ATP, the filament rotated for more than 100 revolutions in an anticlockwise direction when viewed from the 'membrane' side. The rotary torque produced reached more than 40 pN nm(-1) under high load.

The enzyme F1-ATPase has been shown to be a rotary motor in which the central gamma-subunit rotates inside the cylinder made of alpha3beta3 subunits. At low ATP concentrations, the motor rotates in discrete 120 degrees steps, consistent with sequential ATP hydrolysis on the three beta-subunits. The mechanism of stepping is unknown. Here we show by high-speed imaging that the 120 degrees step consists of roughly 90 degrees and 30 degrees substeps, each taking only a fraction of a millisecond. ATP binding drives the 90 degrees substep, and the 30 degrees substep is probably driven by release of a hydrolysis product. The two substeps are separated by two reactions of about 1 ms, which together occupy most of the ATP hydrolysis cycle. This scheme probably applies to rotation at full speed ( approximately 130 revolutions per second at saturating ATP) down to occasional stepping at nanomolar ATP concentrations, and supports the binding-change model for ATP synthesis by reverse rotation of F1-ATPase.

A single molecule of F1-ATPase, a portion of ATP synthase, is by itself a rotary motor in which a central gamma subunit rotates against a surrounding cylinder made of alpha3beta3 subunits. Driven by three catalytic betas, each fueled with ATP, gamma makes discrete 120 degree steps, occasionally stepping backward. The work done in each step is constant over a broad range of imposed load and is close to the free energy of hydrolysis of one ATP molecule.

Decay of fluorescence polarization after an impulsive excitation is correlated with wobbling motion of fluorescent molecules in membranes. The motion is characterized by two parameters, a "wobbling diffusion constant" and a "degree of orientational constraint" both of which can be determined directly from experimentally obtained decay. Detailed discussion, including theoretically calculated time-courses of polarization decay, is given for several types of molecules embedded in lipid bilayers; these types cover a large part of fluorescent probes available at present. The theory is useful for the analysis of fluorescence polarization decay in any system where the orientation of fluorophore is restricted by the surrounding structure.

F(1)-ATPase is a rotary molecular motor that proceeds in 120 degrees steps, each driven by ATP hydrolysis. How the chemical reactions that occur in three catalytic sites are coupled to mechanical rotation is the central question. Here, we show by high-speed imaging of rotation in single molecules of F(1) that phosphate release drives the last 40 degrees of the 120 degrees step, and that the 40 degrees rotation accompanies reduction of the affinity for phosphate. We also show, by single-molecule imaging of a fluorescent ATP analog Cy3-ATP while F(1) is forced to rotate slowly, that release of Cy3-ADP occurs at approximately 240 degrees after it is bound as Cy3-ATP at 0 degrees . This and other results suggest that the affinity for ADP also decreases with rotation, and thus ADP release contributes part of energy for rotation. Together with previous results, the coupling scheme is now basically complete.

ATP, the main biological energy currency, is synthesized from ADP and inorganic phosphate by ATP synthase in an energy-requiring reaction. The F1 portion of ATP synthase, also known as F1-ATPase, functions as a rotary molecular motor: in vitro its gamma-subunit rotates against the surrounding alpha3beta3 subunits, hydrolysing ATP in three separate catalytic sites on the beta-subunits. It is widely believed that reverse rotation of the gamma-subunit, driven by proton flow through the associated F(o) portion of ATP synthase, leads to ATP synthesis in biological systems. Here we present direct evidence for the chemical synthesis of ATP driven by mechanical energy. We attached a magnetic bead to the gamma-subunit of isolated F1 on a glass surface, and rotated the bead using electrical magnets. Rotation in the appropriate direction resulted in the appearance of ATP in the medium as detected by the luciferase-luciferin reaction. This shows that a vectorial force (torque) working at one particular point on a protein machine can influence a chemical reaction occurring in physically remote catalytic sites, driving the reaction far from equilibrium.

Unilamellar liposomes with diameters of 25-100 microns were prepared in various physiological salt solutions, e.g., 100 mM KCl plus 1 mM CaCl2. Successful preparation of the giant liposomes at high ionic strengths required the inclusion of 10-20% of a charged lipid, such as phosphatidylglycerol, phosphatidylserine, phosphatidic acid, or cardiolipin, in phosphatidylcholine or phosphatidylethanolamine. Three criteria were employed to identify unilamellar liposomes, yielding consistent results. Under a phase-contrast microscope those liposomes that showed the thinnest contour and had a vigorously undulating membrane were judged unilamellar. When liposomes were stained with the lipophilic fluorescent dye octadecyl rhodamine B, fluorescence intensities of the membrane of individual liposomes were integer multiples (up to four) of the lowest ones, the least fluorescent liposomes being those also judged unilamellar in the phase-contrast image. Micropipette aspiration test showed that the liposomes judged unilamellar in phase and fluorescence images had an area elastic modulus of approximately 160 dyn/cm, in agreement with literature values. The giant liposomes were stable and retained a concentration gradient of K+ across the membrane, as evidenced in fluorescence images of the K(+)-indicator PBFI encapsulated in the liposomes. Ionophore-induced K+ transport and associated volume change were observed in individual liposomes.

F1, a water-soluble portion of FoF1-ATP synthase, is an ATP hydrolysis-driven rotary motor. The central ␥-subunit rotates in the ␣3␤3 cylinder by repeating the following four stages of rotation: ATPbinding dwell, rapid 80°substep rotation, interim dwell, and rapid 40°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 1 subcomplex (␣3␤3␥) 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 1 and hydrolysis of a slowly hydrolyzable substrate ATP␥S (adenosine 5-[␥-thio]triphosphate) by wild-type F 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␥S hydrolysis by the mutant F 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 1 occurs in 1 ms during the interim dwell, and we call this interim dwell catalytic dwell. Fo F 1 -ATP synthase is an enzyme ubiquitous from bacteria to animals and plants. It synthesizes ATP from ADP and inorganic phosphate by using ⌬ H ϩ -driven proton flow through a membrane (1, 2). F 0 F 1 -ATP synthase can easily be separated into two major portions: water-soluble F 1 and membraneembedded F o . The isolated F 1 (␣ 3 ␤ 3 ␥␦) has an ATP hydrolysis activity and is often called F 1 -ATPase (3, 4). The crystal structure of F 1 shows that the rod-shaped ␥-subunit is surrounded by a cylinder made of three ␣-and three ␤-subunits arranged alternatively (5). The catalytic sites are located in ␤-subunits but residues from adjacent ␣-subunits also contribute. It has been thought that F o F 1 -ATP synthase is a complex of F o motor and F 1 motor that share a common rotor: a downhill proton flow through F o drives rotation of the rotor, causing conformational changes in F 1 that result in ATP synthesis. Conversely, ATP hydrolysis in F 1 causes a reverse rotation of the rotor that enforces F o to pump protons in the reverse direction (6). The rotor is made of a c-subunit ring of F o (7-11) and ␥ subunits of F 1 (12-15).We have visualized and analyzed the ATP-driven rotation of the ␥-subunit in the minimum assembly of F 1 motor, ␣ 3 ␤ 3 ␥ subcomplex (hereafter in this article, this subcomplex is called F 1 ) (13,16). To date, the following features have been established. The ␥-subunit makes a 120°step per one ATP consumption (17), which is further divided into 90°and 30°substeps (18). The dwelling time before the 90°substep rotation depends on ATP concentration and disappears beyond the limit of time resolution of the observation methods as ATP concentration ([ATP]) increases. Therefore, the dwell before the 90°substep rotation is a dwell for ATP binding, and the 90°substep rota...