Tight Chemomechanical Coupling of the F Motor Relies on Structural Stability.

The F motor is a rotating molecular motor that ensures a tight chemomechanical coupling between ATP hydrolysis/synthesis reactions and rotation steps. However, the mechanism underlying this tight coupling remains to be elucidated. In this study, we used electrorotation in single-molecule experiments using an FβE190D mutant to demonstrate that the stall torque was significantly smaller than the wild-type F, indicating a loose coupling of this mutant, despite showing similar stepping torque as the wild-type.

Characterization of Conformational Ensembles of Protonated N-glycans in the Gas-Phase.

Ion mobility mass spectrometry (IM-MS) is a technique capable of investigating structural changes of biomolecules based on their collision cross section (CCS). Recent advances in IM-MS allow us to separate carbohydrate isomers with subtle conformational differences, but the relationship between CCS and atomic structure remains elusive. Here, we characterize conformational ensembles of gas-phase N-glycans under the electrospray ionization condition using molecular dynamics simulations with enhanced sampling.

Simultaneous Formation and Spatial Patterning of ZnO on ITO Surfaces by Local Laser-Induced Generation of Microbubbles in Aqueous Solutions of [Zn(NH)].

We demonstrate the simultaneous formation and spatial patterning of ZnO nanocrystals on an indium-tin oxide (ITO) surface upon local heating using a laser (1064 nm) and subsequent formation of microbubbles. Laser irradiation of an ITO surface in aqueous [Zn(NH)] solution (1.0 × 10 M at pH 12.

Crystal structures of the ATP-binding and ADP-release dwells of the V rotary motor.

V-ATPases are highly conserved ATP-driven rotary molecular motors found in various membrane systems. We recently reported the crystal structures for the Enterococcus hirae ABDF (V) complex, corresponding to the catalytic dwell state waiting for ATP hydrolysis. Here we present the crystal structures for two other dwell states obtained by soaking nucleotide-free V crystals in ADP.

Formation of M-Like Intermediates in Proteorhodopsin in Alkali Solutions (pH ≥ ∼8.5) Where the Proton Release Occurs First in Contrast to the Sequence at Lower pH.

Proteorhodopsin (PR) is an outward light-driven proton pump observed in marine eubacteria. Despite many structural and functional similarities to bacteriorhodopsin (BR) in archaea, which also acts as an outward proton pump, the mechanism of the photoinduced proton release and uptake is different between two H(+)-pumps. In this study, we investigated the pH dependence of the photocycle and proton transfer in PR reconstituted with the phospholipid membrane under alkaline conditions.

Torque generation of Enterococcus hirae V-ATPase.

V-ATPase (V(o)V1) converts the chemical free energy of ATP into an ion-motive force across the cell membrane via mechanical rotation. This energy conversion requires proper interactions between the rotor and stator in V(o)V1 for tight coupling among chemical reaction, torque generation, and ion transport. We developed an Escherichia coli expression system for Enterococcus hirae V(o)V1 (EhV(o)V1) and established a single-molecule rotation assay to measure the torque generated.

CHARMM Force-Fields with Modified Polyphosphate Parameters Allow Stable Simulation of the ATP-Bound Structure of Ca(2+)-ATPase.

Adenosine triphosphate (ATP) is an indispensable energy source in cells. In a wide variety of biological phenomena like glycolysis, muscle contraction/relaxation, and active ion transport, chemical energy released from ATP hydrolysis is converted to mechanical forces to bring about large-scale conformational changes in proteins. Investigation of structure-function relationships in these proteins by molecular dynamics (MD) simulations requires modeling of ATP in solution and ATP bound to proteins with accurate force-field parameters.

Thermodynamic analyses of nucleotide binding to an isolated monomeric β subunit and the α3β3γ subcomplex of F1-ATPase.

Rotation of the γ subunit of the F1-ATPase plays an essential role in energy transduction by F1-ATPase. Hydrolysis of an ATP molecule induces a 120° step rotation that consists of an 80° substep and 40° substep. ATP binding together with ADP release causes the first 80° step rotation.

Basic properties of rotary dynamics of the molecular motor Enterococcus hirae V1-ATPase.

V-ATPases are rotary molecular motors that generally function as proton pumps. We recently solved the crystal structures of the V1 moiety of Enterococcus hirae V-ATPase (EhV1) and proposed a model for its rotation mechanism. Here, we characterized the rotary dynamics of EhV1 using single-molecule analysis employing a load-free probe.

Energetics of the presequence-binding poses in mitochondrial protein import through Tom20.

Tom20 is located at the outer membrane of mitochondria and functions as a receptor for the N-terminal presequence of mitochondrial-precursor proteins. Recently, three atomic structures of the Tom20-presequence complex were determined using X-ray crystallography and classified into A-, M-, and Y-poses in terms of their presequence-binding modes. Combined with biochemical and NMR data, a dynamic-equilibrium model between the three poses has been proposed.

Information-to-free-energy conversion: Utilizing thermal fluctuations.

Maxwell's demon is a hypothetical creature that can convert information to free energy. A debate that has lasted for more than 100 years has revealed that the demon's operation does not contradict the laws of thermodynamics; hence, the demon can be realized physically. We briefly review the first experimental demonstration of Maxwell's demon of Szilard's engine type that converts information to free energy.

Experimental thermodynamics of single molecular motor.

Molecular motor is a nano-sized chemical engine that converts chemical free energy to mechanical motions. Hence, the energetics is as important as kinetics in order to understand its operation principle. We review experiments to evaluate the thermodynamic properties of a rotational F1-ATPase motor (F1-motor) at a single-molecule level.

Properties of the electrogenic activity of bacteriorhodopsin.

In this study, we analyzed the photoelectric current generated by bacteriorhodopsin adsorbed on a polymer film, "Lumirror" (Muneyuki et al. in FEBS Lett 427:109-114, 1998). We could examine the photoelectric current over a wide range of light intensity and pH values using the same membrane owing to the mechanical and chemical stability of the thin polymer film.

Thermodynamic efficiency and mechanochemical coupling of F1-ATPase.

F(1)-ATPase is a nanosized biological energy transducer working as part of F(o)F(1)-ATP synthase. Its rotary machinery transduces energy between chemical free energy and mechanical work and plays a central role in the cellular energy transduction by synthesizing most ATP in virtually all organisms. However, information about its energetics is limited compared to that of the reaction scheme.

Fabrication and placement of a ring structure of nanoparticles by a laser-induced micronanobubble on a gold surface.

We have developed a new fabrication method for a ring structure of assembled nanoparticles on a gold surface by the use of continuous Nd:YAG laser light. A micronanobubble on a gold surface, created by laser local heating, acts as a template for the formation of the ring structure. Both Marangoni convection flow and capillary flow around the micronanobubble are responsible for the driving force to assemble nanoparticles such as CdSe Q-dots into the ring structure from the solution.

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.

Nonequilibrium energetics of a single F1-ATPase molecule.

Molecular motors drive mechanical motions utilizing the free energy liberated from chemical reactions such as ATP hydrolysis. Although it is essential to know the efficiency of this free energy transduction, it has been a challenge due to the system's microscopic scale. Here, we evaluate the single-molecule energetics of a rotary molecular motor, F1-ATPase, by applying a recently derived nonequilibrium equality together with an electrorotation method.

Observation of DNA pinning at laser focal point on Au surface and its application to single DNA nanowire and cross-wire formation.

We report a new technique for fabricating a single DNA nanowire at a desired position in a sequential manner using the micronanobubble generated by laser local heating at the Au/water interface. In our previous report, we found the reversible pull-in/shrinkage of one end immobilized DNA strands near a Nd:YAG laser focal point on an Au surface. In further experiments, the pinning of DNA strands in the stretched state was observed on the Au surface only when the bubble has touched the free end of DNA.

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.

Allosteric model of an ion pump.

We present a simple model of a free-energy transducer made of allosterically coupled two ratchet subsystems. Each of the subsystems transports particles from one particle reservoir to another. The coupling of the subsystems imposes correlated transitions of the potential profiles of the two subsystems.

Modulation of nucleotide binding to the catalytic sites of thermophilic F(1)-ATPase by the epsilon subunit: implication for the role of the epsilon subunit in ATP synthesis.

Effect of epsilon subunit on the nucleotide binding to the catalytic sites of F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)) has been tested by using alpha(3)beta(3)gamma and alpha(3)beta(3)gammaepsilon complexes of TF(1) containing betaTyr341 to Trp substitution. The nucleotide binding was assessed with fluorescence quenching of the introduced Trp. The presence of the epsilon subunit weakened ADP binding to each catalytic site, especially to the highest affinity site.

A tin oxide transparent electrode provides the means for rapid time-resolved pH measurements: application to photoinduced proton transfer of bacteriorhodopsin and proteorhodopsin.

An electrochemical cell was previously reported in which bacteriorhodopsin (BR, purple membrane) was adsorbed on the surface of a transparent SnO(2) electrode, and illumination resulted in potential or current changes (Koyama et al., Science 265:762-765, 1994; Robertson and Lukashev, Biophys. J.

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.

Effect of external torque on the ATP-driven rotation of F1-ATPase.

F(1)-ATPase is a rotary molecular motor powered by the torque generated by another rotary motor F(0) to synthesize ATP in vivo. Therefore elucidation of the behavior of F(1) under external torque is very important. Here, we applied controlled external torque by electrorotation and investigated the ATP-driven rotation for the first time.

F1-ATPase rotates by an asymmetric, sequential mechanism using all three catalytic subunits.

F1-ATPase, the catalytic part of FoF1-ATP synthase, rotates the central gamma subunit within the alpha3beta3 cylinder in 120 degrees steps, each step consuming a single ATP molecule. However, how the catalytic activity of each beta subunit is coordinated with the other two beta subunits to drive rotation remains unknown. Here we show that hybrid F1 containing one or two mutant beta subunits with altered catalytic kinetics rotates in an asymmetric stepwise fashion.