Tight Chemomechanical Coupling of the F1 Motor Relies on Structural Stability.

The F1 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 F1ßE190D mutant to demonstrate that the stall torque was significantly smaller than the wild-type F1, indicating a loose coupling of this mutant, despite showing similar stepping torque as the wild-type. Experiments on the ATPase activity after heat treatment and gel filtration of the α3ß3-subcomplex revealed the unstable structure of the ßE190D mutant. Our results suggest that the tight chemomechanical coupling of the F1 motor relies on the structural stability of F1. We also discuss the difference between the stepping torque and the stall torque.

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. We show that the separation of CCSs between isomers reflects folding features of N-glycans, which are determined both by chemical compositions and protonation states. Providing a physicochemical basis of CCS for N-glycans helps not only to interpret IM-MS measurements but also to estimate CCSs of complex glycans.

Simultaneous Formation and Spatial Patterning of ZnO on ITO Surfaces by Local Laser-Induced Generation of Microbubbles in Aqueous Solutions of [Zn(NH3)4]2.

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(NH3)4]2+ solution (1.0 × 10-2 M at pH 12.0) under an optical microscope produced ZnO nanocrystals, the presence of which was confirmed by X-ray diffraction analysis and Raman microspectroscopy. Scanning the focused laser beam over the ITO surface generated a spatial ZnO pattern (height: ∼60 nm, width: ∼1 µm) in the absence of a template or mask. The Marangoni convection generated in the vicinity of the microbubbles resulted in a rapid concentration/accumulation of [Zn(NH3)4]2+ around the microbubbles, which led to the formation of ZnO at the solid-bubble-solution three-phase contact line around the bubbles and thus afforded ZnO nanocrystals on the ITO surface upon local heating with a laser.

Crystal structures of the ATP-binding and ADP-release dwells of the V1 rotary motor.

V1-ATPases are highly conserved ATP-driven rotary molecular motors found in various membrane systems. We recently reported the crystal structures for the Enterococcus hirae A3B3DF (V1) 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 V1 crystals in ADP. In the presence of 20 µM ADP, two ADP molecules bind to two of three binding sites and cooperatively induce conformational changes of the third site to an ATP-binding mode, corresponding to the ATP-binding dwell. In the presence of 2 mM ADP, all nucleotide-binding sites are occupied by ADP to induce conformational changes corresponding to the ADP-release dwell. Based on these and previous findings, we propose a V1-ATPase rotational mechanism model.

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. Under these conditions, as the medium pH increased, a blue-shifted photoproduct (defined as Ma), which is different from M, with a pKa of ca. 9.2 was produced. The sequence of the photoinduced proton uptake and release during the photocycle was inverted with the increase in pH. A pKa value of ca. 9.5 was estimated for this inversion and was in good agreement with the pKa value of the formation of Ma (∼ 9.2). In addition, we measured the photoelectric current generated by PRs attached to a thin polymer film at varying pH. Interestingly, increases in the medium pH evoked bidirectional photocurrents, which may imply a possible reversal of the direction of the proton movement at alkaline pH. On the basis of these findings, a putative photocycle and proton transfer scheme in PR under alkaline pH conditions was proposed.

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. Recombinant and native EhV(o)V1 exhibited almost identical dependence of ATP hydrolysis activity on sodium ion and ATP concentrations, indicating their functional equivalence. In a single-molecule rotation assay with a low load probe at high ATP concentration, EhV(o)V1 only showed the "clear" state without apparent backward steps, whereas EhV1 showed two states, "clear" and "unclear." Furthermore, EhV(o)V1 showed slower rotation than EhV1 without the three distinct pauses separated by 120° that were observed in EhV1. When using a large probe, EhV(o)V1 showed faster rotation than EhV1, and the torque of EhV(o)V1 estimated from the continuous rotation was nearly double that of EhV1. On the other hand, stepping torque of EhV1 in the clear state was comparable with that of EhV(o)V1. These results indicate that rotor-stator interactions of the V(o) moiety and/or sodium ion transport limit the rotation driven by the V1 moiety, and the rotor-stator interactions in EhV(o)V1 are stabilized by two peripheral stalks to generate a larger torque than that of isolated EhV1. However, the torque value was substantially lower than that of other rotary ATPases, implying the low energy conversion efficiency of EhV(o)V1.

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. In this study, we derived new force-field parameters for the triphosphate moiety of ATP based on the high-precision quantum calculations of methyl triphosphate. We tested our new parameters on membrane-embedded sarcoplasmic reticulum Ca(2+)-ATPase and four soluble proteins. The ATP-bound structure of Ca(2+)-ATPase remains stable during MD simulations, contrary to the outcome in shorter simulations using original parameters. Similar results were obtained with the four ATP-bound soluble proteins. The new force-field parameters were also tested by investigating the range of conformations sampled during replica-exchange MD simulations of ATP in explicit water. Modified parameters allowed a much wider range of conformational sampling compared with the bias toward extended forms with original parameters. A diverse range of structures agrees with the broad distribution of ATP conformations in proteins deposited in the Protein Data Bank. These simulations suggest that the modified parameters will be useful in studies of ATP in solution and of the many ATP-utilizing proteins.

Thermodynamic analyses of nucleotide binding to an isolated monomeric ß subunit and the α3ß3γ 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. Thus, nucleotide binding is very important for rotation and energy transduction by F1-ATPase. In this study, we introduced a ßY341W mutation as an optical probe for nucleotide binding to catalytic sites, and a ßE190Q mutation that suppresses the hydrolysis of nucleoside triphosphate (NTP). Using a mutant monomeric ßY341W subunit and a mutant α3ß3γ subcomplex containing the ßY341W mutation with or without an additional ßE190Q mutation, we examined the binding of various NTPs (i.e., ATP, GTP, and ITP) and nucleoside diphosphates (NDPs, i.e., ADP, GDP, and IDP). The affinity (1/Kd) of the nucleotides for the isolated ß subunit and third catalytic site in the subcomplex was in the order ATP/ADP > GTP/GDP > ITP/IDP. We performed van't Hoff analyses to obtain the thermodynamic parameters of nucleotide binding. For the isolated ß subunit, NDPs and NTPs with the same base moiety exhibited similar ΔH(0) and ΔG(0) values at 25°C. The binding of nucleotides with different bases to the isolated ß subunit resulted in different entropy changes. Interestingly, NDP binding to the α3ß(Y341W)3γ subcomplex had similar Kd and ΔG(0) values as binding to the isolated ß(Y341W) subunit, but the contributions of the enthalpy term and the entropy term were very different. We discuss these results in terms of the change in the tightness of the subunit packing, which reduces the excluded volume between subunits and increases water entropy.

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. EhV1 rotated in a counterclockwise direction, exhibiting two distinct rotational states, namely clear and unclear, suggesting unstable interactions between the rotor and stator. The clear state was analyzed in detail to obtain kinetic parameters. The rotation rates obeyed Michaelis-Menten kinetics with a maximal rotation rate (Vmax) of 107 revolutions/s and a Michaelis constant (Km) of 154 µM at 26 °C. At all ATP concentrations tested, EhV1 showed only three pauses separated by 120°/turn, and no substeps were resolved, as was the case with Thermus thermophilus V1-ATPase (TtV1). At 10 µM ATP (<>Km), the distribution of the durations of the catalytic pause was reproduced by a consecutive reaction with two time constants of 2.6 and 0.5 ms. These kinetic parameters were similar to those of TtV1. Our results identify the common properties of rotary catalysis of V1-ATPases that are distinct from those of F1-ATPases and will further our understanding of the general mechanisms of rotary molecular motors.

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. To investigate this mechanism in further detail, we performed all-atom molecular dynamics (MD) simulations and replica-exchange MD (REMD) simulations of the Tom20-presequence complex in explicit water. In the REMD simulations, one major distribution and another minor one were observed in the converged free-energy landscape at 300 K. In the major distribution, structures similar to A- and M-poses exist, whereas those similar to Y-pose are located in the minor one, suggesting that A-pose in solution is more stable than Y-pose. A k-means clustering algorithm revealed a new pose not yet obtained by X-ray crystallography. This structure has double salt bridges between Arg14' in the presequence and Glu78 or Glu79 in Tom20 and can explain the binding affinity of the complex in previous pull-down assay experiments. Structural clustering and analyses of contacts between Tom20 and the presequence suggest smooth conformational changes from Y- to A-poses through low activation barriers. M-pose lies between Y- and A-poses as a metastable state. The REMD simulations thus provide insights into the energetics of the multiple-binding forms and help to detail the progressive conformational states in the dynamic-equilibrium model based on the experimental data.

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. We analyzed the photoelectric current by comparison with a simple equivalent electric circuit. Analysis of experimental results obtained at different light intensities suggested that the electromotive force of the bacteriorhodopsin was independent of light intensity. The pH dependence of the photoelectric current suggested that the bacteriorhodopsin could generate a maximum electromotive force at approximately pH 6.

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. We show that the F1-motor achieves 100% thermo dynamic efficiency at the stalled state. Furthermore, the motor reduces the internal irreversible heat inside the motor to almost zero and achieves a highly-efficient free energy transduction close to 100% during rotations far from quasistatic process. We discuss the mechanism of how the F1-motor achieves such a high efficiency, which highlights the remarkable property of the nano-sized engine F1-motor.

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. We pump heat from an isothermal environment by using the information about the thermal fluctuations of a Brownian particle and increase the particle's free energy.

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. Actually, fundamental questions such as how efficiently F(1)-ATPase transduces free energy remain unanswered. Here, we demonstrated reversible rotations of isolated F(1)-ATPase in discrete 120° steps by precisely controlling both the external torque and the chemical potential of ATP hydrolysis as a model system of F(o)F(1)-ATP synthase. We found that the maximum work performed by F(1)-ATPase per 120° step is nearly equal to the thermodynamical maximum work that can be extracted from a single ATP hydrolysis under a broad range of conditions. Our results suggested a 100% free-energy transduction efficiency and a tight mechanochemical coupling of F(1)-ATPase.

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. Because a single micronanobubble was generated by the Nd:YAG laser focusing point, the precise positioning of the ring structure was feasible directly on the gold surface, which makes it possible to fabricate various patterns of rings such as arrays and letters and even a double-ring structure without any photomasks or any templates.

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. Despite large rotational Brownian motion of the detached head about a free joint adjoining the two necks, unidirectional stepping is achieved, in part by the power stroke of the attached head that moves the joint forward. However, the power stroke alone cannot fully account for preferential forward site binding since the orientation and angle stability of the detached head, which is determined by the properties of the recovery stroke, dictate actin binding site accessibility. Here, we directly observe the recovery stroke dynamics and fluctuations of myosin Va using a novel, transient caged ATP-controlling system that maintains constant ATP levels through stepwise UV-pulse sequences of varying intensity. We immobilized the neck of monomeric myosin Va on a surface and observed real time motions of bead(s) attached site-specifically to the head. ATP induces a transient swing of the neck to the post-recovery stroke conformation, where it remains for ∼40 s, until ATP hydrolysis products are released. Angle distributions indicate that the post-recovery stroke conformation is stabilized by ≥ 5 k(B)T of energy. The high kinetic and energetic stability of the post-recovery stroke conformation favors preferential binding of the detached head to a forward site 72 nm away. Thus, the recovery stroke contributes to unidirectional stepping of myosin Va.

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. We show that the sum of the heat flow through the probe's rotational degree of freedom and the work against an external load is almost equal to the free energy change per a single ATP hydrolysis under various conditions. This implies that F1-ATPase works at an efficiency of nearly 100% in a thermally fluctuating environment.

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. This pinning phenomenon was observed even on the alkane thiol modified Au surface as self-assembled monolayers (SAMs) such as hexanethiol, mercaptohexanol, and hexadecanethiol. However, no pinning was observed on the bovine serum albumin (BSA) modified surface. Since optical tweezers can manipulate a DNA modified bead (radius=1.87 µm), the bead was firstly fixed on a solid surface by being compressed with the optical tweezers, and the pulling and pinning of DNA on the bead were achieved. As a consequence, the laser local heating on the Au surface enables us to control the number and position of the one end immobilized DNA strands as DNA nanowires.

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. As a result, a downhill flux in one subsystem with higher chemical-potential difference drives an uphill flux in the other subsystem with lower chemical-potential difference. The direction of the driven flux inverts depending on the direction of the driving flux. The ratio between the fluxes conveyed by the two subsystems is variable and nonstoichiometric. By selecting appropriate parameters, the maximum ratio of the driven flux to driving flux and maximum free-energy transducing efficiency reaches some 90 and 40%, respectively. At a stalled state, the driven flux vanishes while the driving flux remains finite. The allosteric model enables explicit analysis of the timing between binding-unbinding of particles and transitions of potential profile. The behavior of the model is similar to but different from that of the alternate access model, which is a biochemical model for active transport proteins. Our model works also as a regulatory system. We suggest that the correlated transitions of the subsystems (subunits or domains) through allosteric interaction are the origin of the diverse functions of the protein machineries.

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. The site occupancy remains two except for a brief moment after the ATP binding, but the third vacant site can bind a medium nucleotide weakly.