Processive Nanostepping of Formin mDia1 Loosely Coupled with Actin Polymerization.

Formins are actin-binding proteins that construct nanoscale machinery with the growing barbed end of actin filaments and serve as key regulators of actin polymerization and depolymerization. To maintain the regulation of actin dynamics, formins have been proposed to processively move at every association or dissociation of a single actin molecule toward newly formed barbed ends. However, the current models for the motile mechanisms were established without direct observation of the elementary processes of this movement.

Biphasic Effect of Profilin Impacts the Formin mDia1 Force-Sensing Mechanism in Actin Polymerization.

Formins are force-sensing proteins that regulate actin polymerization dynamics. Here, we applied stretching tension to individual actin filaments under the regulation of formin mDia1 to investigate the mechanical responses in actin polymerization dynamics. We found that the elongation of an actin filament was accelerated to a greater degree by stretching tension for ADP-G-actin than that for ATP-G-actin.

Perfect chemomechanical coupling of FF-ATP synthase.

FF-ATP synthase (FF) couples H flow in F domain and ATP synthesis/hydrolysis in F domain through rotation of the central rotor shaft, and the H/ATP ratio is crucial to understand the coupling mechanism and energy yield in cells. Although H/ATP ratio of the perfectly coupling enzyme can be predicted from the copy number of catalytic β subunits and that of H binding subunits as /β, the actual H/ATP ratio can vary depending on coupling efficiency. Here, we report actual H/ATP ratio of thermophilic FF, whose /β is 10/3.

Simple mechanism whereby the F1-ATPase motor rotates with near-perfect chemomechanical energy conversion.

F1-ATPase is a motor enzyme in which a central shaft γ subunit rotates 120° per ATP in the cylinder made of α3β3 subunits. During rotation, the chemical energy of ATP hydrolysis (ΔGATP) is converted almost entirely into mechanical work by an elusive mechanism. We measured the force for rotation (torque) under various ΔGATP conditions as a function of rotation angles of the γ subunit with quasi-static, single-molecule manipulation and estimated mechanical work (torque × traveled angle) from the area of the function.

Direct observation of DNA overwinding by reverse gyrase.

Reverse gyrase, found in hyperthermophiles, is the only enzyme known to overwind (introduce positive supercoils into) DNA. The ATP-dependent activity, detected at >70 °C, has so far been studied solely by gel electrophoresis; thus, the reaction dynamics remain obscure. Here, we image the overwinding reaction at 71 °C under a microscope, using DNA containing consecutive 30 mismatched base pairs that serve as a well-defined substrate site.

None of the rotor residues of F1-ATPase are essential for torque generation.

F1-ATPase is a powerful rotary molecular motor that can rotate an object several hundred times as large as the motor itself against the viscous friction of water. Forced reverse rotation has been shown to lead to ATP synthesis, implying that the mechanical work against the motor's high torque can be converted into the chemical energy of ATP. The minimal composition of the motor protein is α3β3γ subunits, where the central rotor subunit γ turns inside a stator cylinder made of alternately arranged α3β3 subunits using the energy derived from ATP hydrolysis.

Controlled rotation of the F₁-ATPase reveals differential and continuous binding changes for ATP synthesis.

F(1)-ATPase is an ATP-driven rotary molecular motor that synthesizes ATP when rotated in reverse. To elucidate the mechanism of ATP synthesis, we imaged binding and release of fluorescently labelled ADP and ATP while rotating the motor in either direction by magnets. Here we report the binding and release rates for each of the three catalytic sites for 360° of the rotary angle.

Direct observation of strand passage by DNA-topoisomerase and its limited processivity.

Type-II DNA topoisomerases resolve DNA entanglements such as supercoils, knots and catenanes by passing one segment of DNA duplex through a transient enzyme-bridged double-stranded break in another segment. The ATP-dependent passage reaction has previously been demonstrated at the single-molecule level, showing apparent processivity at saturating ATP. Here we directly observed the strand passage by human topoisomerase IIα, after winding a pair of fluorescently stained DNA molecules with optical tweezers for 30 turns into an X-shaped braid.

F(1)-ATPase: a prototypical rotary molecular motor.

F(1)-ATPase, the soluble portion of ATP synthase, has been shown to be a rotary molecular motor in which the central γ subunit rotates inside the cylinder made of α(3)β(3) subunits. The rotation is powered by ATP hydrolysis in three catalytic sites, and reverse rotation of the γ subunit by an external force leads to ATP synthesis in the catalytic sites. Here I look back how our lab became involved in the study of this marvelous rotary machine, and discuss some aspects of its rotary mechanism while confessing we are far from understanding.

Kinetic equivalence of transmembrane pH and electrical potential differences in ATP synthesis.

ATP synthase is the key player of Mitchell's chemiosmotic theory, converting the energy of transmembrane proton flow into the high energy bond between ADP and phosphate. The proton motive force that drives this reaction consists of two components, the pH difference (ΔpH) across the membrane and transmembrane electrical potential (Δψ). The two are considered thermodynamically equivalent, but kinetic equivalence in the actual ATP synthesis is not warranted, and previous experimental results vary.

Torque generation and utilization in motor enzyme F0F1-ATP synthase: half-torque F1 with short-sized pushrod helix and reduced ATP Synthesis by half-torque F0F1.

ATP synthase (F(0)F(1)) is made of two motors, a proton-driven motor (F(0)) and an ATP-driven motor (F(1)), connected by a common rotary shaft, and catalyzes proton flow-driven ATP synthesis and ATP-driven proton pumping. In F(1), the central γ subunit rotates inside the α(3)β(3) ring. Here we report structural features of F(1) responsible for torque generation and the catalytic ability of the low-torque F(0)F(1).

Torque generation in F1-ATPase devoid of the entire amino-terminal helix of the rotor that fills half of the stator orifice.

F(1)-ATPase is an ATP-driven rotary molecular motor in which the central γ-subunit rotates inside a cylinder made of α(3)β(3) subunits. The amino and carboxyl termini of the γ rotor form a coiled coil of α-helices that penetrates the stator cylinder to serve as an axle. Crystal structures indicate that the axle is supported by the stator at two positions, at the orifice and by the hydrophobic sleeve surrounding the axle tip.

Efficient ATP synthesis by thermophilic Bacillus FoF1-ATP synthase.

F(o)F(1)-ATP synthase (F(o)F(1)) synthesizes ATP in the F(1) portion when protons flow through F(o) to rotate the shaft common to F(1) and F(o). Rotary synthesis in isolated F(1) alone has been shown by applying external torque to F(1) of thermophilic origin. Proton-driven ATP synthesis by thermophilic Bacillus PS3 F(o)F(1) (TF(o)F(1)), however, has so far been poor in vitro, of the order of 1 s(-1) or less, hampering reliable characterization.

Direct observation of the myosin Va recovery stroke that contributes to unidirectional stepping along actin.

Myosins are ATP-driven linear molecular motors that work as cellular force generators, transporters, and force sensors. These functions are driven by large-scale nucleotide-dependent conformational changes, termed "strokes"; the "power stroke" is the force-generating swinging of the myosin light chain-binding "neck" domain relative to the motor domain "head" while bound to actin; the "recovery stroke" is the necessary initial motion that primes, or "cocks," myosin while detached from actin. Myosin Va is a processive dimer that steps unidirectionally along actin following a "hand over hand" mechanism in which the trailing head detaches and steps forward ∼72 nm.

Resolving stepping rotation in Thermus thermophilus H(+)-ATPase/synthase with an essentially drag-free probe.

Vacuole-type ATPases (V(o)V₁) and F(o)F₁ ATP synthases couple ATP hydrolysis/synthesis in the soluble V(1) or F₁ portion with proton (or Na(+)) flow in the membrane-embedded V(o) or F(o) portion through rotation of one common shaft. Here we show at submillisecond resolutions the ATP-driven rotation of isolated V₁ and the whole V(o)V₁ from Thermus thermophilus, by attaching a 40-nm gold bead for which viscous drag is almost negligible. V₁ made 120° steps, commensurate with the presence of three catalytic sites.

Chemo-mechanical coupling in F(1)-ATPase revealed by catalytic site occupancy during catalysis.

F(1)-ATPase is a rotary molecular motor in which the central gamma subunit rotates inside a cylinder made of alpha(3)beta(3) subunits. To clarify how ATP hydrolysis in three catalytic sites cooperate to drive rotation, we measured the site occupancy, the number of catalytic sites occupied by a nucleotide, while assessing the hydrolysis activity under identical conditions. The results show hitherto unsettled timings of ADP and phosphate releases: starting with ATP binding to a catalytic site at an ATP-waiting gamma angle defined as 0 degrees , phosphate is released at approximately 200 degrees , and ADP is released during quick rotation between 240 degrees and 320 degrees that is initiated by binding of a third ATP.

Activation and stiffness of the inhibited states of F1-ATPase probed by single-molecule manipulation.

F(1)-ATPase (F(1)), a soluble portion of F(o)F(1)-ATP synthase (F(o)F(1)), is an ATP-driven motor in which gammaepsilon subunits rotate in the alpha(3)beta(3) cylinder. Activity of F(1) and F(o)F(1) from Bacillus PS3 is attenuated by the epsilon subunit in an inhibitory extended form. In this study we observed ATP-dependent transition of epsilon in single F(1) molecules from extended form to hairpin form by fluorescence resonance energy transfer.

Stimulation of F(1)-ATPase activity by sodium dodecyl sulfate.

F(1)-ATPase is a rotary molecular motor in which the gamma subunit rotates inside the cylinder made of alpha(3)beta(3) subunits. We have studied the effects of sodium dodecyl sulfate (SDS) on the rotational and ATP hydrolysis activities of F(1)-ATPase. Bulk hydrolysis activity at various SDS concentrations was examined at 2mM ATP.

A giant liposome for single-molecule observation of conformational changes in membrane proteins.

We present an experimental system that allows visualization of conformational changes in membrane proteins at the single-molecule level. The target membrane protein is reconstituted in a giant liposome for independent control of the aqueous environments on the two sides of the membrane. For direct observation of conformational changes, an extra-liposomal site(s) of the target protein is bound to a glass surface, and a probe that is easily visible under a microscope, such as a micron-sized plastic bead, is attached to another site on the intra-liposomal side.

Effect of epsilon subunit on the rotation of thermophilic Bacillus F1-ATPase.

F(1)-ATPase is an ATP-driven motor in which gammaepsilon rotates in the alpha(3)beta(3)-cylinder. It is attenuated by MgADP inhibition and by the epsilon subunit in an inhibitory form. The non-inhibitory form of epsilon subunit of thermophilic Bacillus PS3 F(1)-ATPase is stabilized by ATP-binding with micromolar K(d) at 25 degrees C.

Neither helix in the coiled coil region of the axle of F1-ATPase plays a significant role in torque production.

F(1)-ATPase is an ATP-driven rotary molecular motor in which the central gamma-subunit rotates inside the cylinder made of alpha(3)beta(3) subunits. The amino and carboxy termini of the gamma-subunit form the axle, an alpha-helical coiled coil that deeply penetrates the stator cylinder. We previously truncated the axle step by step, starting with the longer carboxy terminus and then cutting both termini at the same levels, resulting in a slower yet considerably powerful rotation.

Temperature dependence of the rotation and hydrolysis activities of F1-ATPase.

F(1)-ATPase, a water-soluble portion of the enzyme ATP synthase, is a rotary molecular motor driven by ATP hydrolysis. To learn how the kinetics of rotation are regulated, we have investigated the rotational characteristics of a thermophilic F(1)-ATPase over the temperature range 4-50 degrees C by attaching a polystyrene bead (or bead duplex) to the rotor subunit and observing its rotation under a microscope. The apparent rate of ATP binding estimated at low ATP concentrations increased from 1.

Axle-less F1-ATPase rotates in the correct direction.

F1-adenosine triphosphatase (ATPase) is an ATP-driven rotary molecular motor in which the central gamma subunit rotates inside a cylinder made of three alpha and three beta subunits alternately arranged. The rotor shaft, an antiparallel alpha-helical coiled coil of the amino and carboxyl termini of the gamma subunit, deeply penetrates the central cavity of the stator cylinder. We truncated the shaft step by step until the remaining rotor head would be outside the cavity and simply sat on the concave entrance of the stator orifice.

Coupling of rotation and catalysis in F(1)-ATPase revealed by single-molecule imaging and manipulation.

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.