Language：English   Publishing type：Research paper (scientific journal)   Publisher：nature  

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

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 M-a), which is different from M, with a pK(a) 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 pK(a) value of ca. 9.5 was estimated for this inversion and was in good agreement with the pK(a) value of the formation of M-a (similar to 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.

DOI： 10.1021/acs.biochem.5b01196

Web of Science

researchmap

Language：Japanese   Publisher：The Physical Society of Japan  

DOI： 10.11316/jpsgaiyo.71.2.0_2645

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：IOP PUBLISHING LTD  

F0F1-ATP synthase is a factory for synthesizing ATP in virtually all cells. Its core machinery is the subcomplex F-1-motor (F-1-ATPase) and performs the reversible mechanochemical coupling. The isolated F-1-motor hydrolyzes ATP, which is accompanied by unidirectional rotation of its central gamma-shaft. When a strong opposing torque is imposed, the gamma-shaft rotates in the opposite direction and drives the F-1-motor to synthesize ATP. This mechanical-to-chemical free-energy transduction is the final and central step of the multistep cellular ATP-synthetic pathway. Here, we determined the amount of mechanical work exploited by the F-1-motor to synthesize an ATP molecule during forced rotations using a methodology combining a nonequilibrium theory and single molecule measurements of responses to external torque. We found that the internal dissipation of the motor is negligible even during rotations far from a quasistatic process.

DOI： 10.1088/1367-2630/17/1/015008

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Biochemistry & Molecular Biology (ASBMB)  

DOI： 10.1074/jbc.m114.598177

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

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 structurefunction 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 Ca2+-ATPase and four soluble proteins. The ATP-bound structure of Ca2+-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.

DOI： 10.1021/ct5004143

Web of Science

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.54.S274_4

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.54.S158_3

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Rotation of the gamma subunit of the F-1-ATPase plays an essential role in energy transduction by F-1-ATPase. Hydrolysis of an ATP molecule induces a 120 degrees step rotation that consists of an 80 degrees substep and 40 degrees substep. ATP binding together with ADP release causes the first 80 degrees step rotation. Thus, nucleotide binding is very important for rotation and energy transduction by F-1-ATPase. In this study, we introduced a beta Y341W mutation as an optical probe for nucleotide binding to catalytic sites, and a beta E190Q mutation that suppresses the hydrolysis of nucleoside triphosphate (NTP). Using a mutant monomeric beta Y341W subunit and a mutant alpha(3)beta(3)gamma subcomplex containing the beta Y341W mutation with or without an additional bE190Q 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/K-d) of the nucleotides for the isolated beta 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 b subunit, NDPs and NTPs with the same base moiety exhibited similar Delta H-0 and Delta G(0) values at 25 degrees C. The binding of nucleotides with different bases to the isolated b subunit resulted in different entropy changes. Interestingly, NDP binding to the alpha(3)beta(Y341W)(3)gamma subcomplex had similar K-d and Delta G(0) values as binding to the isolated beta(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.

DOI： 10.1016/j.bpj.2013.10.018

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Background: The chemomechanical coupling scheme of the rotary motor V-1-ATPase is incompletely understood. Results:Enterococcus hirae V-1-ATPase (EhV(1)) showed 120 degrees steps of rotation without substeps, as commonly seen with F-1-ATPase. Conclusion: The basic properties of rotary dynamics of EhV(1) are similar to those of Thermus thermophilus V-1-ATPase. Significance: This study revealed the common properties of V-1-ATPases as rotary molecular motors, distinct from those of F-1-ATPases.
V-ATPases are rotary molecular motors that generally function as proton pumps. We recently solved the crystal structures of the V-1 moiety of Enterococcus hirae V-ATPase (EhV(1)) and proposed a model for its rotation mechanism. Here, we characterized the rotary dynamics of EhV(1) using single-molecule analysis employing a load-free probe. EhV(1) 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 (V-max) of 107 revolutions/s and a Michaelis constant (K-m) of 154 m at 26 degrees C. At all ATP concentrations tested, EhV(1) showed only three pauses separated by 120 degrees/turn, and no substeps were resolved, as was the case with Thermus thermophilus V-1-ATPase (TtV(1)). At 10 m ATP (?K-m), the distribution of the durations of the ATP-waiting pause fit well with a single-exponential decay function. The second-order binding rate constant for ATP was 2.3 x 10(6) m(-1) s(-1). At 40 mm ATP (?K-m), 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 TtV(1). Our results identify the common properties of rotary catalysis of V-1-ATPases that are distinct from those of F-1-ATPases and will further our understanding of the general mechanisms of rotary molecular motors.

DOI： 10.1074/jbc.M113.506329

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1007/s00249-012-0870-0

PubMed

researchmap

DOI： 10.1016/j.bpj.2012.11.3669

Web of Science

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.53.S264_1

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.53.S230_3

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：EPL ASSOCIATION, EUROPEAN PHYSICAL SOCIETY  

We have developed a novel method to evaluate the potential profile of a molecular motor at each chemical state from only the probe's trajectory and applied it to a rotary molecular motor F1-ATPase. By using this method, we could also obtain the information regarding the mechanochemical coupling and energetics. We demonstrate that the position-dependent transition of the chemical states is the key feature for the highly efficient free-energy transduction by F1-ATPase. Copyright (C) EPLA, 2012

DOI： 10.1209/0295-5075/97/40004

Web of Science

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.52.S77_5

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.52.S128_5

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

F-1-ATPase is a nanosized biological energy transducer working as part of FoF1-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 degrees steps by precisely controlling both the external torque and the chemical potential of ATP hydrolysis as a model system of FoF1-ATP synthase. We found that the maximum work performed by F-1-ATPase per 120 degrees 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.

DOI： 10.1073/pnas.1106787108

Web of Science

researchmap

Language：Japanese   Publisher：一般社団法人 日本生物物理学会  

DOI： 10.2142/biophys.51.159

CiNii Books

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

We have developed a new fabrication method for a ring structure of assembled nanopartides 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.

DOI： 10.1021/la201616s

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

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 similar to 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 similar to 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.

DOI： 10.1371/journal.pbio.1001031

Web of Science

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.51.S131_6

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.51.S103_4

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

In 1929, Leo Szilard invented a feedback protocol(1) in which a hypothetical intelligence-dubbed Maxwell's demon-pumps heat from an isothermal environment and transforms it into work. After a long-lasting and intense controversy it was finally clarified that the demon's role does not contradict the second law of thermodynamics, implying that we can, in principle, convert information to free energy(2-6). An experimental demonstration of this information-to-energy conversion, however, has been elusive. Here we demonstrate that a non-equilibrium feedback manipulation of a Brownian particle on the basis of information about its location achieves a Szilard-type information-to-energy conversion. Using real-time feedback control, the particle is made to climb up a spiral-staircase-like potential exerted by an electric field and gains free energy larger than the amount of work done on it. This enables us to verify the generalized Jarzynski equality(7), and suggests a new fundamental principle of an 'information-to-heat engine' that converts information into energy by feedback control.

DOI： 10.1038/NPHYS1821

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE SA  

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 mu m), 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. (C) 2010 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bioelechem.2010.04.004

Web of Science

researchmap

Publisher：American Physical Society  

DOI： 10.1103/PhysRevLett.104.198103

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

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 similar to 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.

DOI： 10.1016/j.bpj.2009.11.050

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Chemical Society of Japan  

We report a method for manipulating sequential single DNA strands by using a micronanobubble formed by local laser heating on a Au surface. DNA strands near the laser focal point were quickly pulled toward the focal point. This DNA pull-in phenomenon can be explained by Marangoni convection due to a micronanobubble generated by laser heating on the Au surface. The thickness of Au film plays a crucial role for absorbing the IR laser light.

DOI： 10.1246/cl.2010.92

CiNii Books

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER PHYSICAL SOC  

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.

DOI： 10.1103/PhysRevE.81.011137

Web of Science

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.50.S113_2

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.50.S103_6

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.50.S53_1

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Effect of epsilon subunit on the nucleotide binding to the catalytic sites of F-1-ATPase from the thermophilic Bacillus PS3 (TF1) has been tested by using alpha(3)beta(3)gamma and alpha(3)beta(3)gamma epsilon complexes of TF1 containing beta Tyr341 to Trp substitution. The nucleotide binding was assessed with fluorescence quenching of the introduced Trp. The presence of the E subunit weakened ADP binding to each catalytic site, especially to the highest affinity site. This effect was also observed when GDP or IDP was used. The ratio of the affinity of the lowest to the highest nucleotide binding sites had changed two orders of magnitude by the c subunit. The differences may relate to the energy required for the binding change in the ATP synthesis reaction and contribute to the efficient ATP synthesis. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2009.09.092

Web of Science

researchmap

Publisher：2  

DOI： 10.1111/j.1751-1097.2008.00520.x

Web of Science

PubMed

J-GLOBAL

researchmap

Language：English  

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.49.S30_6

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.49.S172_4

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.49.S43_4

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.49.S172_3

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOPHYSICAL SOC  

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 1.2 x 10(6) M-1 s(-1) at 4 degrees C to 4.3 x 10(7) M-1 s(-1) at 40 degrees C, whereas the torque estimated at 2 mM ATP remained around 40 pN.nm over 4-50 degrees C. The rotation was stepwise at 4 degrees C, even at the saturating ATP concentration of 2 mM, indicating the presence of a hitherto unresolved rate-limiting reaction that occurs at ATP-waiting angles. We also measured the ATP hydrolysis activity in bulk solution at 4-65 degrees C. F1-ATPase tends to be inactivated by binding ADP tightly. Both the inactivation and reactivation rates were found to rise sharply with temperature, and above 30 degrees C, equilibrium between the active and inactive forms was reached within 2 s , the majority being inactive. Rapid inactivation at high temperatures is consistent with the physiological role of this enzyme, ATP synthesis, in the thermophile.

DOI： 10.1529/biophysj.107.123307

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

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. The rotation was accelerated by assisting torque and decelerated by hindering torque, but F-1 rarely showed rotations in the ATP synthesis direction. This is consistent with the prediction by models based on the assumption that the rotation is tightly coupled to ATP hydrolysis and synthesis. At low ATP concentrations (2 and 5 mu M), 120 degrees stepwise rotation was observed. Due to the temperature rise during experiment, quantitative interpretation of the data is difficult, but we found that the apparent rate constant of ATP binding clearly decreased by hindering torque and increased by assisting torque. (c) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2007.12.049

Web of Science

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.48.S103_1

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.48.S160_5

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.48.S152_4

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.48.S47_3

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.48.S45_2

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.48.S122_7

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.48.S46_5

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.48.S151_5

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

F-1- ATPase, the catalytic part of FoF1- ATP synthase, rotates the central c subunit within the alpha(3)beta(3) 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 F-1 containing one or two mutant beta subunits with altered catalytic kinetics rotates in an asymmetric stepwise fashion. Analysis of the rotations reveals that for any given beta subunit, the subunit binds ATP at 0 degrees, cleaves ATP at similar to 200 degrees and carries out a third catalytic event at similar to 320 degrees. This demonstrates the concerted nature of the F-1 complex activity, where all three beta subunits participate to drive each 120 degrees rotation of the c subunit with a 120 degrees phase difference, a process we describe as a 'sequential three- site mechanism'.

DOI： 10.1038/nsmb1296

Web of Science

researchmap

Language：Japanese   Publisher：静岡大学  

CiNii Books

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOPHYSICAL SOCIETY  

Motor proteins are essential in life processes because they convert the free energy of ATP hydrolysis to mechanical work. However, the fundamental question on how they work when different amounts of free energy are released after ATP hydrolysis remains unanswered. To answer this question, it is essential to clarify how the stepping motion of a motor protein reflects the concentrations of ATP, ADP, and P-i in its individual actions at a single molecule level. The F, portion of ATP synthase, also called F-1-ATPase, is a rotary molecular motor in which the central gamma-subunit rotates against the alpha(3)beta(3) cylinder. The motor exhibits clear step motion at low ATP concentrations. The rotary action of this motor is processive and generates a high torque. These features are ideal for exploring the relationship between free energy input and mechanical work output, but there is a serious problem in that this motor is severely inhibited by ADP. In this study, we overcame this problem of ADP inhibition by introducing several mutations while retaining high enzymatic activity. Using a probe of attached beads, stepping rotation against viscous load was examined at a wide range of free energy values by changing the ADP concentration. The results showed that the apparent work of each individual step motion was not affected by the free energy of ATP hydrolysis, but the frequency of each individual step motion depended on the free energy. This is the first study that examined the stepping motion of a molecular motor at a single molecule level with simultaneous systematic control of Delta G(ATP). The results imply that microscopically defined work at a single molecule level cannot be directly compared with macroscopically defined free energy input.

DOI： 10.1529/biophysj.106.097170

Web of Science

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.47.S263_2

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.47.S242_2

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.47.S242_1

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Bacteriorhodopsin (BR), which is rich in alpha-helical structure, was spread onto water with single-wall carbon nanotubes (SCNTs). After a Langmuir trough was used to apply compressive surface pressure to maintain the alpha-helices monolayer of denatured BR, the composite films comprising alpha-helices and SCNTs were transferred horizontally onto substrates. Atomic force microscopy (AFM) and fluorescence microscopy observation suggested that alpha-helices in contact with SCNTs changed into beta-sheets. High-resolution transmission electron microscopy (HR-TEM) showed 0.54 nm periodicity characteristic of the turn of alpha-helical structure in the SCNTs-free alpha-helix monolayer region and showed the 0.70 nm periodicity of beta-sheet pleated structure in the region where SCNTs were covered with unfolded BR. Unique features of carbon nanotubes that trigger conformational changes of a protein were revealed.

DOI： 10.1093/jmicro/dfl024

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

Cytochrome bd is a heterodimeric terminal ubiquinol oxidase of Escherichia coli under microaerophilic growth conditions. The oxidase activity shows sigmoidal concentration-dependence with low concentrations of ubiquinols, and a marked substrate inhibition with high concentrations of ubiquinol-2 analogs [Sakamoto, K., Miyoshi, H., Takegami, K., Mogi, T., Anraku, Y., and Iwamura H. (1996) J. Biol. Chem. 271, 29897-29902]. Kinetic analysis of the oxidation of the ubiquinol-2 analogs, where the 2- or 3-methoxy group has been substituted with an azido or ethoxy group, suggested that its peculiar enzyme kinetics can be explained by a modified ping-pong bi-bi mechanism with the formation of inactive binary complex FS in the one-electron reduced oxygenated state and inactive ternary complex (E2S)S-n on the oxidation of the second quinol molecule. Structure-function studies on the ubiquinol-2 analogs suggested that the 6-diprenyl group and the 3-methoxy group on the quinone ring are involved in the substrate inhibition. We also found that oxidized forms of ubiquinone-2 analogs served as weak noncompetitive inhibitors. These results indicate that the mechanism for the substrate oxidation by cytochrome bd is different from that of the heme-copper terminal quinol oxidase and is tightly coupled to dioxygen reduction chemistry.

DOI： 10.1093/jb/mvj087

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOPHYSICAL SOCIETY  

F-1-ATPase, a water-soluble portion of F0F1-ATP synthase, is a rotary motor driven by ATP hydrolysis. The central gamma-subunit rotates in the alpha(3)beta(3) cylinder by repeating four stages of rotation: ATP-binding dwell, rapid 80 degrees substep rotation, catalytic dwell, and rapid 40 degrees substep rotation. In the catalytic dwell, at least two catalytic reactions occur-cleavage of the enzyme-bound ATP and presumably release of the hydrolyzed product(s) from the enzyme - but we found that a slow ATP cleavage mutant of F-1-ATPase from thermophilic Bacillus PS3 rotates at low ATP concentration without substeps and the catalytic dwell. Analysis indicates that in this alternative reaction pathway the two catalytic reactions occur during the preceding long ATP-binding dwell. Thus, F-1-ATPase can operate through (at least) two competing reaction pathways, not necessarily through a simple consecutive reaction.

Web of Science

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.46.S344_1

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.46.S217_4

researchmap

Language：English   Publishing type：Part of collection (book)   Publisher：Wiley Blackwell  

DOI： 10.1002/3527606122.ch10

Scopus

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOPHYSICAL SOCIETY  

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. The rotation is driven by ATP hydrolysis in three catalytic sites on the beta-subunits. How many of the three catalytic sites are filled with a nucleotide during the course of rotation is an important yet unsettled question. Here we inquire whether F-1 rotates at extremely low ATP concentrations where the site occupancy is expected to be low. We observed under an optical microscope rotation of individual F1 molecules that carried a bead duplex on the gamma-subunit. Time-averaged rotation rate was proportional to the ATP concentration down to 200 pM, giving an apparent rate constant for ATP binding of 2 x 10(7) M-1 s(-1). A similar rate constant characterized bulk ATP hydrolysis in solution, which obeyed a simple Michaelis-Menten scheme between 6 mM and 60 nM ATP. F-1 produced the same torque of similar to40 pN . nm at 2 mM, 60 nM, and 2 nM ATP. These results point to one rotary mechanism governing the entire range of nanomolar to millimolar ATP, although a switchover between two mechanisms cannot be dismissed. Below 1 nM ATP, we observed less regular rotations, indicative of the appearance of another reaction scheme.

DOI： 10.1529/biophysj.104.054668

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

In the MF1 crystal structure with the MgADP-fluoroaluminate complex bound to two catalytic sites [Menz, R. I., Walker, J. E., and Leslie, A. G. W. (2001) Cell 106, 331-341], the guanidinium of betaR(337) is within 2.9 Angstrom of the alpha-oxygen of alphaS(370) and 3.7 Angstrom of a methyl group of alphaV(371) at the alphaE-beta(HC) interface. To examine the functional role of this contact, the (alphaV(371)C)(3)(betaR(337)C)(3)gamma subcomplex of the TF1-ATPase was prepared and characterized. Steady state ATPase activity of the reduced double-mutant is 30% of that of the wild type. Inactivation of the double mutant containing empty catalytic sites or MgADP bound to one catalytic site with CuCl2 cross-linked two alpha-beta pairs, whereas a single alpha-beta pair cross-linked when at least two catalytic sites contained MgADP. The reduced double mutant hydrolyzed substoichiometric ATP 100-fold more rapidly than the enzyme containing two cross-linked alpha-beta pairs. Addition of AlCl3 and NaF to the reduced double mutant after incubation with stoichiometric MgADP or 200 muM MgADP irreversibly inactivated the steady state ATPase activity with rate constants of 1.5 x 10(-2) and 4.1 x 10(-2) min(-1), respectively. In contrast, addition of AlCl3 and NaF to the cross-linked enzyme after incubation with stoichiometric or 200 muM MgADP irreversibly inactivated ATPase activity with a common rate constant of similar to10(-4) min(-1). Correlation of these results with crystal structures of MF1 suggests that the catalytic site at the alpha(TP)-beta(TP) interface is loaded first upon addition of nucleotides to nucleoticle-depleted F-1-ATPases and that the catalytic site at the alpha(TP)-beta(TP) interface with bound MgADP in crystal structures represents a catalytic site containing inhibitory MgADP.

DOI： 10.1021/bi047694z

Web of Science

researchmap

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.45.S18_2

researchmap

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.45.S245_2

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

F-1-ATPase is a rotary molecular motor in which unidirectional rotation of the central subunit is powered by ATP hydrolysis in three catalytic sites arranged 120degrees apart around gamma. To study how hydrolysis reactions produce mechanical rotation, we observed rotation under an optical microscope to see which of the three sites bound and released a fluorescent ATP analog. Assuming that the analog mimics authentic ATP, the following scheme emerges: (i) in the ATP-waiting state, one site, dictated by the orientation of gamma, is empty, whereas the other two bind a nucleotide; (ii) ATP binding to the empty site drives an similar to80degrees rotation of gamma; (iii) this triggers a reaction(s), hydrolysis and/or phosphate release, but not ADP release in the site that bound ATP one step earlier; (iv) completion of this reaction induces further 40degrees rotation.

DOI： 10.1038/nsmb721

Web of Science

researchmap

Language：English   Publisher：BIOPHYSICAL SOCIETY  

Web of Science

researchmap

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.44.S226_3

researchmap

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.44.S226_4

researchmap

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.44.S226_1

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

F-1, a water-soluble portion of FoF1-ATP synthase, is an ATIP hydrolysis-driven rotary motor. The central gamma-subunit rotates in the alpha(3)beta(3) cylinder by repeating the following four stages of rotation: ATP-binding dwell, rapid 801 substep rotation, interim dwell, and rapid 40degrees 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 ATIP binding, correspond to these events. To discover which steps, we analyzed rotations of F-1 subcomplex (alpha(3)beta(3)gamma) from thermophilic Bacillus PS3 under conditions where cleavage of ATIP at the catalytic site is decelerated: hydrolysis of ATP by the catalytic-site mutant F, and hydrolysis of a slowly hydrolyzable substrate ATPgammaS (adenosine 5'-[gamma-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 ATPgammaS hydrolysis by the mutant F-1 ensure that cleavage of ATIP 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.

DOI： 10.1073/pnas.2434983100

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

In order to get insight into the origin of apparent negative cooperativity observed for F-1-ATPase, we compared ATPase activity and ATPMg binding of mutant subcomplexes of thermophilic F-1-ATPase, alpha((W463F))(3)beta((Y341W))(3)gamma and alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma. For alpha((W463F)3)beta((Y341W)3)gamma, apparent Km's of ATPase kinetics (4.0 and 233 muM) did not agree with apparent K-m's deduced from fluorescence quenching of the introduced tryptophan residue (on the order of nM, 0.016 and 13 muM). On the other hand, in case of alpha((K175A/T176A/W463F))(3)beta((Y341W)3)gamma, which lacks noncatalytic nucleotide binding sites, the apparent K., of ATPase activity (10 muM) roughly agreed with the highest K. of fluorescence measurements (27 muM). The results indicate that in case of alpha((W463F)3)beta((Y341W))(3)gamma, the activating effect of ATP binding to noncatalytic sites dominates overall ATPase kinetics and the highest apparent K,, of ATPase activity does not represent the ATP binding to a catalytic site. In case of alpha((K175A/T176A/W463F)3)beta((Y341W))(3)gamma, the K-m of ATPase activity reflects the ATP binding to a catalytic site due to the lack of noncatalytic sites. The Eadie-Hofstee plot of ATPase reaction by alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma was rather linear compared with that of alpha((W463F)3)beta((Y341W))(3)gamma, if not perfectly straight, indicating that the apparent negative cooperativity observed for wild-type F-1-ATPase is due to the ATP binding to catalytic sites and noncatalytic sites. Thus, the frequently observed K-m's of 100-300 muM and 1-30 muM range for wild-type F-1-ATPase correspond to ATP binding to a noncatalytic site and catalytic site, respectively. (C) 2003 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbabio.2003.08.006

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

VoV1-ATPase is responsible for acidification of eukaryotic intracellular compartments and ATP synthesis of Archaea and some eubacteria. From the similarity to FoF1-ATP synthase, VoV1-ATPase has been assumed to be a rotary motor, but to date there are no experimental data to support this. Here we visualized the rotation of single molecules of V-1-ATPase, a catalytic subcomplex of VoV1-ATPase. V-1-ATPase from Thermus thermophilus was immobilized onto a glass surface, and a bead was attached to the D or IF subunit through the biotin-streptavidin linkage. In both cases we observed ATP-dependent rotations of beads, the direction of which was always counterclockwise viewed from the membrane side. Given that three ATP molecules are hydrolyzed per one revolution, rates of rotation agree consistently with rates of ATP hydrolysis at saturating ATP concentrations. This study provides experimental evidence that VoV1-ATPase is a rotary motor and that both D and F subunits constitute a rotor shaft.

DOI： 10.1073/pnas.0436796100

Web of Science

researchmap

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.43.S134_2

researchmap

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.43.S134_3

researchmap

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.43.S134_4

researchmap

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.43.S133_3

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOPHYSICAL SOCIETY  

The photovoltage generation by halorhodopsin from Halobacterium salinarum (shR) was examined by adsorbing shR-containing membranes onto a thin polymer film. The photovoltage consisted of two major components: one with a sub-millisecond range time constant and the other with a millisecond range time constant with different amplitudes, as previously reported. These components exhibited different Cl- concentration dependencies (0.1-9 M). We found that the time constant for the fast component was relatively independent of the Cl- concentration, whereas the time constant for the slow component increased sigmoidally at higher Cl- concentrations. The fast and the slow processes were attributed to charge (Cl-) movements within the protein and related to Cl- ejection, respectively. The laser photolysis studies of shR-membrane suspensions revealed that they corresponded to the formation and the decay of the N intermediate. The photovoltage amplitude of the slow component exhibited a distorted bell-shaped Cl- concentration dependence, and the Cl- concentration dependence of its time constant suggested a weak and highly cooperative Cl--binding site(s) on the cytoplasmic side (apparent K-D of similar to5 M and Hill coefficient greater than or equal to5). The Cl- concentration dependence of the photovoltage amplitude and the time constant for the slow process suggested a competition between spontaneous relaxation and ion translocation. The time constant for the relaxation was estimated to be > 100 ms.

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Biochemistry and Molecular Biology  

Motor proteins, myosin, and kinesin have gamma-phosphate sensors in the switch II loop that play key roles in conformational changes that support motility. Here we report that a rotary motor, F1-ATPase, also changes its conformations upon phosphate release. The tryptophan mutation was introduced into Arg-333 in the beta subunit of F1-ATPase from thermophilic Bacillus PS3 as a probe of conformational changes. This residue interacts with the switch II loop (residues 308-315) of the beta subunit in a nucleotide-bound conformation. The addition of ATP to the mutant F1 subcomplex alpha3beta(R333W)3gamma caused transient increase and subsequent decay of the Trp fluorescence. The increase was caused by conformational changes on ATP binding. The rate of decay agreed well with that of phosphate release monitored by phosphate-binding protein assays. This is the first evidence that the beta subunit changes its conformation upon phosphate release, which may share a common mechanism of exerting motility with other motor proteins.

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING LTD  

F-1-ATPase is inactivated by entrapment of MgADP in catalytic sites and reactivated by MgATP or P-i. Here, using a mutant alpha(3)beta(3)gamma complex of thermophilic F-1-ATPase (alphaW463F/betaY341W) and monitoring nucleotide binding by fluorescence quenching of an introduced tryptophan, we found that P-i interfered with the binding of MgATP to F-1-ATPase, but binding of MgADP was interfered with to a lesser extent. Hydrolysis of MgATP by F-1-ATPase during the experiments did not obscure the interpretation because another mutant, which was able to bind nucleotide but not hydrolyse ATP (alphaW463F/betaE190Q/betaY341W), also gave the same results. The half-maximal concentrations of P-i that suppressed the MgADP-inhibited form and interfered with MgATP binding were both approximate to 20 mM. It is likely that the presence of P-i at a catalytic site shifts the equilibrium from the MgADP-inhibited form to the enzyme-MgADP-P-i complex, an active intermediate in the catalytic cycle.

Web of Science

researchmap

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.42.S146_2

researchmap

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.42.S145_1

researchmap

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.42.S146_1

researchmap

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.42.S144_3

researchmap

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.42.S145_4

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

ATP synthase can be thought of as a complex of two motors - the ATP-driven Ft motor and the proton-driven F-o motor - that rotate in opposite directions. The mechanisms by which rotation and catalysis are coupled in the working enzyme are now being unravelled on a molecular scale.

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The effects of alkane derivatives, 1-alcohols (ROH), aliphatic amine hydrochlorides (RNH2. HCl) and sodium aliphatic carboxylates (ROONa), on the proton pumping activity of bacteriorhodopsin (bR) in a purple membrane have been examined. Photocurrents in bR in the purple membrane adsorbed onto polyester thin film were recorded before and after exposure to these test substances. The peak photocurrent in bR was reversibly suppressed by each substance. From the dose-response curve, the concentrations required to reduce the peak capacitive current by 50% were determined for each homolog and then the standard free energies per CH2 for the adsorption of the alkane derivatives to the site of action were estimated: -3.13 kJ mol(-1) for ROH, -3.05 kJ mol(-1) for RNH3+, and -2.95 kJ mol(-1) for ROO-. The proton pumping activity of bR was mainly suppressed by the hydrophobic interaction with the additive. The relative potencies of the functional groups of the alkane derivatives were almost comparable between 1-octanol (C8OH) and octylamine hydrochloride (C8NH3+) and about 10 times less effective for sodium octanoate (C8OO-) than for others. The addition of C8OH or C8OO- changed the absorption spectra of bR with a maximum at 560 nm to the spectra of the intermediate state with a maximum at 480 nm, while the C8NH3+ decreased the intensity of the 560 nm band only with no blue-shift by the 480 nm band. We conclude that the action of the alkane derivatives is nonspecific and directed to all organized purple membrane structures and that the binding sites of the ROH and ROO- are different from that of RNH3+. (C) 2001 Elsevier Science B.V. All rights reserved.

Web of Science

researchmap

Language：Japanese   Publisher：エヌ・ティー・エス  

CiNii Books

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：National Academy of Science(US)  

DOI： 10.1073/pnas.241365698

researchmap

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.41.S62_2

researchmap

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.41.S198_3

researchmap

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.41.S199_4

researchmap

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.41.S198_2

researchmap

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.41.S199_1

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOPHYSICAL SOCIETY  

A model for the primary active transport by an ion pump protein is proposed. The model, the "energization-relaxation channel model," describes an ion pump as a multiion channel that undergoes stochastic transitions between two conformational states by external energy supply. When the potential profile along ion transport pathway is asymmetrical, a net ion flux is induced by the transitions. In this model, the coupling of the conformational change and ion transport is stochastic and loose. The model qualitatively reproduces known properties of active transport such as the effect of ion concentration gradient and membrane potential on the rate of transport and the inhibition of ion transport at high ion concentration. We further examined the effect of various parameters on the ion transport properties of this model. The efficiency of the coupling was almost 100% under some conditions.

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

Rotation of a motor protein, F-1-ATPase, was demonstrated using a unique single-molecule observation system. This paper reviews what has been clarified by this system and then focuses on the role of residues at the hinge region of the beta subunit, We have visualised rotation of a single molecule of F-1-ATPase by attaching a fluorescent actin filament to the top of the gamma subunit in the immobilised F-1-ATPase, thus settling a major controversy regarding the rotary catalysis, The rotation of the gamma subunit was exclusively in one direction, as could be predicted by the crystal structure of bovine heart F-1-ATPase, Rotation at low ATP concentrations revealed that one revolution consists of three 120 degrees steps, each fuelled by the binding of an ATP to the beta subunit, The mean work done by a 120 degrees step was approximately 80 pN nm, a value close to the free energy liberated by hydrolysis of one ATP molecule, implying nearly 100% efficiency of energy conversion. The torque is probably generated by the beta subunit, which undergoes large opening-closing domain motion upon binding of AT(D)P, We identified three hinge residues, beta His179, beta Gly180 and beta Gly181, whose peptide bond dihedral angles are drastically changed during domain motion. Simultaneous substitution of these residues with alanine resulted in nearly complete loss (99%) of ATPase activity. Single or double substitution of the two Gly residues did not abolish the ATPase activity. However, reflecting the shift of the equilibrium between the open and closed forms of the beta subunit, single substitution caused changes in the propensity to generate the kinetically trapped Mg-ADP inhibited form: Gly180Ala enhanced the propensity and Gly181Ala abolished the propensity. In spite of these changes, the mean rotational torque was not changed significantly for any of the mutants.

Web of Science

researchmap

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.40.S208_3

CiNii Books

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PORTLAND PRESS LTD  

Homogeneous preparations of alpha(3)beta(3)gamma complexes with one, two or three non-competent non-catalytic site(s) were performed as described [Amano, Hisabori, Muneyuki, and Yoshida (1996) J. Biol. Chem. 271, 18128-18133] and their properties were compared with those of the wild-type complex. The ATPase activity of the complex with three non-competent non-catalytic sites decayed rapidly to an inactivated state, as reported previously [Matsui, Muneyuki, Honda, Allison, Dou, and Yoshida (1997) J. Biol. Chem. 272, 8215-8221]. In contrast, the complex with one or two non-competent non-catalytic sites displayed a substantial steady-state phase activity depending on the number of non-competent non-catalytic sites in the complex. This result indicates that one competent non-catalytic site can maintain the continuous catalytic turnover of the enzyme and can potentially relieve all three catalytic sites from inhibition by MgADP(-). Furthermore, the results suggest that the interaction between three non-catalytic sites might not be as strong as that between catalytic sites, which are all strictly required for a continuous catalytic turnover.

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE LTD  

We investigated ATP hydrolysis by a mutant (Delta NC) alpha(3)beta(3)gamma subcomplex of F0F1-ATP synthase from the thermophilic Bacillus PS3 that is defective in the noncatalytic nucleotide binding sites. This mutant subcomplex was activated by inorganic phosphate ions (P-i) and did not show continuous ATP hydrolysis activity in the absence of P-i. P-i also activated the wild-type alpha(3)beta(3)gamma subcomplex in a similar manner. Sulphate activated wild-type alpha(3)beta(3)gamma but not Delta NC alpha(3)beta(3)gamma, indicating that P-i activation did not involve noncatalytic sites but that sulphate activation did. P-i also activated ATP hydrolysis and coupled proton translocation by the wild-type and Delta NC F0F1-ATP synthases reconstituted into vesicle membranes.

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The capacitive photoelectric current responses of the halorhodopsins from Halobacterium salinarum (shR) and from Natronobacterium pharaonis (phR) were studied using membrane fragments adsorbed onto a thin polyester film. The electric current of shR was not much affected by ionic strength or cations present in the medium (Na+, K+, Li+, Mg2+, or Ca2+), but was greatly influenced by the Cl- concentration. It increased biphasically as the Cl- concentration increased from 0 to 5 M, then decreased and almost vanished at around 10 or 12 M, Apparent K-d's of about 0.1 and 6 M were deduced for the Kd of Cl- uptake sites. We had to assume a sigmoidal increase of Cl- binding with a Will coefficient of about 8 at the cytoplasmic, Cl- release site(s), The half-maximum Cl- concentration for the sigmoidal binding was about 7.5 M. The electric current of phR had a maximum around 30 mM Cl- and biphasically decreased at higher Cl- concentrations. The apparent K-d for the Cl- uptake site was 5 mM. The biphasic decrease in the transport activity was explained by assuming a sum of simple hyperbolic type binding (K-d = 0.2 M) and sigmoidally increasing binding with a Hill coefficient of 10 on the cytoplasmic side. The half-maximum concentration of the latter cooperative binding was 5.6 M. This great difference between the apparent affinity of the release site of shR and that of phR can explain the previously reported difference between the Cl- dependency of their photocycles, These results also suggest that there may be multiple Cl- binding sites in the Cl- transport pathway. A simple sequence of Cl- transport steps based on a multiion channel model is proposed.

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

In the crystal structure of mitochondrial F-1-ATPase, two beta subunits with a bound Mg-nucleotide are in "closed" conformations, whereas the third beta subunit without bound nucleotide is in an "open" conformation. In this "CCO" (beta-closed beta-closed beta-open) conformational state, Ile-390s of the two closed beta subunits, even though they are separated by an intervening alpha subunit, have a direct contact. We replaced the equivalent Ile of the alpha(3)beta(3)gamma subcomplex of thermophilic F-1-ATPase with Cys and observed the formation of the beta-beta cross-link through a disulfide bond. The analysis of conditions required for the cross-link formation indicates that: (i) F-1-ATPase takes the CCO conformation when two catalytic sites are filled with Mg-nucleotide, (ii) intermediate(s) with the CCO conformation are generated during catalytic cycle, (iii) the Mg-ADP inhibited form is in the CCO conformation, and (iv) F-1-ATPase dwells in conformational state(s) other than CCO when only one (or none) of catalytic sites is filled by Mg-nucleotide or when catalytic sites are filled by Mg2+-free nucleotide, The alpha(3)beta(3)gamma subcomplex containing the beta-beta cross-link retained the activity of uni-site catalysis but lost that of multiple catalytic turnover, suggesting that open-closed transition of beta subunits is required for the rotation of gamma subunit but not for hydrolysis of a single ATP.

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

We constructed a time-resolved photovoltage measurement system and examined the photovoltage kinetics of wild-type bacteriorhodopsin, its D96N mutant, and halorhodopsins from Halobacterium salinarum and Natronobacterium pharaonis. Upon illumination with a laser hash, wild-type bacteriorhodopsin showed photovoltage generation with fast (10-100 mu s range) and slow (ms range) components while D96N lacked the latter, as reported previously [Holz, M., Drachev, L.A., Mogi, T., Otto, H., Kaulen, A.D., Heyn, M.P., Skulachev, V.P., and Khorana, H.G. (1989) Proc. Natl. Acad. Sci. USA 86, 2167-2171]. In contrast, photovoltage generation in halorhodopsins from H. salinarum and N. pharaonis was significant only in the ms time range. On the basis of the photovoltage kinetics and photocycle, we conclude that major charge (chloride) movements within halorhodopsin occur during the formation and decay of the N intermediate in the ms range. These observations are discussed in terms of the "Energization-Relaxation Channel Model" [Muneyuki, E., Ikematsu, M., and Yoshida, M. (1996) J. Phys. Chem. 100, 19687-19691].

Web of Science

researchmap

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.39.S145_4

CiNii Books

researchmap

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.39.S104_4

CiNii Books

researchmap

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.39.S73_1

CiNii Books

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.273.32.20504

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The ATP hydrolysis of the V-1-ATPase of Thermus thermophilus have been investigated with an ATP-regenerating system at 25 degrees C, The ratio of ATPase activity to ATP concentration ranged from 40 to 4000 mu M; from this, an apparent K-m of 240 +/- 24 mu M and a V-max of 5.2 +/-0.5 units/mg were deduced. An apparent negative cooperativity, which is frequently observed in case of F-1-ATPases, was not observed for the V-1-ATPase, Interestingly, the rate of hydrolysis decayed rapidly during ATP hydrolysis, and the ATP hydrolysis finally stopped. Furthermore, the inactivation of the V-1-ATPase was attained by a prior incubation with ADP-Mg. The inactivated V-1-ATPase contained 1.5 mol of ADP/mol of enzyme. Difference absorption spectra generated from addition of ATP-Mg to the isolated subunits revealed that the A subunit can bind ATP-Mg, whereas the B subunit cannot. The inability to bind ATP-Mg is consistent with the absence of Walker motifs in the B subunit, These results indicate that the inactivation of the V-1-ATPase during ATP hydrolysis is caused by entrapping inhibitory ADP-Mg in a catalytic site. Light-driven ATP synthesis by bacteriorhodopsin-VoV1-ATPase proteoliposomes was observed, and the rate of ATP synthesis was approximately constant. ATP synthesis occurred in the presence of an ADP-Mg of which concentration was high enough to induce complete inactivation of ATP hydrolysis of VoV1-ATPase, This result indicates that the ADP-Mg-inhibited form is not produced in ATP synthesis reaction.

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We developed a new assay system for the measurement of capacitive electric currents generated by ion pumps using the thin polymer film 'Lumirror' (Toray Co., Japan), This system enables us to examine the electrogenicity of ion pumps over a wide range of experimental conditions with high reproducibility due to the mechanical and chemical stability, the high electric resistance and the high electric capacitance of the thin polymer film, Using this method, we examined the photoelectric response of wild type bacteriorhodopsin and its D96N mutant over a wide pH range (2.8-10.0), The results were explained in terms of the affinities of the proton binding sites for translocated protons. A possibility that the direction of the proton transfer from the Schiff base was influenced by the protonation/deprotonation state of the surrounding proton binding sites was suggested. We also found that this film can be used as a substrate for atomic force microscopy (AFM) samples and hence the active purple membrane was observed with AFM. (C) 1998 Federation of European Biochemical Societies.

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

ATP hydrolyzing activity of a mutant alpha(3) beta(3) gamma subcomplex of F0F1-ATP synthase (Delta NC) from the thermophilic Bacillus PS3, which lacked noncatalytic nucleotide binding sites, was inactivated completely soon after starting the reaction (Matsui, T., Muneyuki, E., Honda, M., Allison, W. S., Dou, C., and Yoshida, M. (1997) J. Biol. Chem. 272, 8215-8221), This inactivation is caused by rapid accumulation of the "MgADP inhibited form" which, in the case of wild-type enzyme, would be relieved by ATP binding to noncatalytic sites. We reconstituted F0F1-ATP synthase into liposomes together with bacteriorhodopsin and measured illumination-driven ATP synthesis. Remarkably, Delta NC F0F1-ATP synthase catalyzed continuous turnover of ATP synthesis while it could not promote ATP-driven proton translocation. ATP synthesis by Delta NC F0F1-ATP synthase, as well as wild-type enzyme, proceeded even in the presence of azide, an inhibitor of ATP hydrolysis that stabilizes the MgADP inhibited form. The time course of ATP synthesis by Delta NC F0F1-ATP synthase was linear, and gradual acceleration to the maximal rate, which was observed for the wild-type enzyme, was not seen. Thus, ATP synthesis can proceed without nucleotide binding to noncatalytic sites even though the rate is sub-maximal. These results indicate that the MgADP inhibited form is not produced in ATP synthesis reaction, and in this regard, ATP synthesis may not be a simple reversal of ATP hydrolysis.

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Subunit complexes (alpha(3) beta(3) gamma, alpha(3) beta(3) gamma delta, alpha(3) beta(3) gamma epsilon, and alpha(3) beta(3) gamma delta epsilon) of thermophilic F-1-ATPase were prepared, and their catalytic properties were compared to know the role of delta and epsilon subunits in catalysis. The presence of delta subunit in the complexes had slight inhibitory effect on the ATPase activity, The effect of epsilon subunit was more profound, The (-epsilon) complexes, alpha(3) beta(3) gamma and alpha(3) beta(3) gamma delta, initiated ATP hydrolysis without a lag. In contrast, the (+epsilon) complexes, alpha(3) beta(3) gamma epsilon and alpha(3) beta(3) gamma delta epsilon, started hydrolysis of ATP (<700 mu M) with a lag phase that was gradually activated during catalytic turnover. As ATP concentration increased, the lag phase of the (+epsilon) complexes became shorter, and it was not observed above 1 mM ATP, Analysis of binding and hydrolysis of the ATP analog, 2',3'-O-(2,4,6-trinitrophenyl) ATP, suggested that the (+epsilon) complexes bound substrate only slowly. Differing from Escherichia coli F-1-ATPase, the activation of the (+epsilon) complexes from the lag phase was not due to dissociation of epsilon subunit since the re-isolated activated complex retained epsilon subunit. This indicates that there are two alternative forms of the (+epsilon) complex, inhibited form and activated form, and the inhibited one is converted to the activated one during catalytic turnover.

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Previously, we reported the substitution of Tyr(341) of the F-1-ATPase beta subunit from a thermophilic Bacillus strain PS3 with leucine, cysteine,:or alanine (M, Odaka ct al, J. Biochem., 115 (1994) 789-796). These mutations resulted in a great decrease in the affinity of the isolated beta subunit for ATP-Mg and an increase in the apparent K-m of the alpha(3) beta(3) gamma complex in ATP hydrolysis when examined above 0.1 mM ATP. Here, we examined the ATPase activity of the mutant complexes in a wide range of ATP concentration and found that the mutants exhibited apparent positive cooperativity in ATP hydrolysis. This is the first clear demonstration that a single mutation in the catalytic sites converts the kinetics from apparent negative cooperativity in the wild-type alpha(3) beta(3) gamma complex to apparent positive cooperativity, The conversion of apparent cooperativity could be explained in terms of a simple kinetic scheme based on the binding change model proposed by Boyer, (C) 1997 Federation of European Biochemical Societies.

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

A mutant alpha(3) beta(3) gamma complex of F-1-ATPase from thermophilic Bacillus PS3 was generated in which noncatalytic nucleotide binding sites lost their ability to bind nucleotides. It hydrolyzed ATP at an initial rate with cooperative kinetics (K-m(1), 4 mu M; K-m(2), 135 mu M) similar to the wild-type complex. However, the initial rate decayed rapidly to an inactivated form. Since the inactivated mutant complex contained 1.5 mol of ADP/mol of complex, this inactivation seemed to be caused by entrapping inhibitory MgADP in a catalytic site. Indeed, the mutant complex was nearly completely inactivated by a 10 min prior incubation with equimolar MgADP. Analysis of the progress of inactivation after initiation of ATP hydrolysis as a function of ATP concentration indicated that the inactivation was optimal at ATP concentrations in the range of K-m(1). In the presence of ATP, the wildtype complex dissociated the inhibitory [H-3]ADP pre-loaded onto a catalytic site whereas the mutant complex did not. Lauryl dimethylamineoxide promoted release of preloaded inhibitory [H-3]ADP in an ATP-dependent manner and partly restored the activity of the inactivated mutant complex. Addition of ATP promoted single-site hydrolysis of 2',3'-O-(2,4,6-trinitrophenyl)-ATP preloaded at a single catalytic site of the mutant complex. These results indicate that intact noncatalytic sites are essential for continuous catalytic turnover of the F-1-ATPase but are not essential for catalytic cooperativity of F-1-ATPase observed at ATP concentrations below similar to 300 mu M.

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN BIOCHEMICAL SOC  

The interactions of substoichiometric TNP-ATP and F-1-ATPase from Escherichia coli (EF(1)) were examined and compared with those in the case of mitochondrial F-1-ATPase (MF(1)) and F-1-ATPase from thermophilic Bacillus PS3 (TF1). EF(1) hydrolyzed substoichiometric TNP-ATP faster than TF1 or MF(1), although some 20% of the TNP-ATP remained unhydrolyzed even in the presence of excess chase ATP. The affinity of the catalytic site of EF(1) for the product, TNP-ADP, was weaker than that of TF1 or MF(1), and the TNP-ADP was readily released upon addition of excess ATP. The amplitude of the difference absorption spectrum induced by binding of TNP-AT(D)P to EF(1) was smaller than that of MF(1) or TF1 under similar experimental conditions. When an excess amount of TNP-ATP was added to EF(1) and the change of the difference spectrum was measured, the shape of the difference spectrum of the ATP-replaceable fraction was very similar to that in the case of binding of TNP-ATP to the isolated beta subunit of TF1, indicating that the rapidly replaceable fraction of bound TNP-ATP was actually at the catalytic site and most of the non-replaceable portion was bound at noncatalytic sites. Weaker affinity of the catalytic site for TNP-ATP may account for the heterogeneous binding and hydrolysis under the conditions described in this paper.

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：アメリカ化学会  

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

In order to know how many functional catalytic sites are necessary for ATPase activity of F-1-ATPase from a thermophilic Bacillus PS3, a new method of isolating homogeneous preparations of the alpha(3) beta(3) gamma complex with 1, 2, or 3 incompetent catalytic sites was developed. Ten glutamic acids (Glu . Tag) were linked to the C terminus of the catalytically incompetent beta(E190Q) subunit. The Glu Tag itself did not affect ATPase activity of the complexes. Two kinds of alpha(3) beta(3) gamma complexes, one containing beta(wild-type) and the other Glu Tag-linked beta(E190Q), were mixed, urea-denatured, and dialyzed, and alpha(3) beta(3) gamma complexes were reconstituted. Each of the complexes containing a different number of Glu . Tag-linked beta(E190Q) was separated by anion-exchange chromatography and analyzed. The results were as follows. 1) Normal steady state ATPase activity requires three intact catalytic sites. 2) Chase-acceleration, a catalytic cooperativity, requires at least two intact catalytic sites. 3) Single-site catalysis can be mediated by a single intact catalytic site alone. Rescrambling of subunits between complexes could occur when the complex was aged under certain conditions, and this might be one of the reasons for previous contradictory results (Miwa, K., Ohtsubo, M., Denda, K., Hisabori, T., Date, T., and Yoshida, M. (1989) J. Biochem. (Tokyo) 106, 730-734).

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

ATP hydrolyses by the wild-type alpha(3) beta(3) gamma and mutant (alpha D261N)(3) beta(3) gamma subcomplexes of the F-1-ATPase from the thermophilic Bacillus PS3 have been compared. The wild-type complex hydrolyzes 50 mu M ATP in three kinetic phases: a burst decelerates to an intermediate phase, which then gradually accelerates to a final rate, In contrast, the mutant complex hydrolyzes 50 mu M or 2 mM ATP in two kinetic phases, The mutation abolishes acceleration from the intermediate phase to a faster final rate, Both the wild-type and mutant complexes hydrolyze 4TP with a lag after loading a catalytic site with MgADP. The rate of the MgADP-loaded wild-type complex rapidly accelerates and approaches that observed for the wild-type apo-complex. The MgADP-loaded mutant complex hydrolyzes ATP with a more pronounced lag, and the gradually accelerating rate approaches the slow, final rate observed with the mutant ape-complex. Lauryl dimethylamide oxide (LDAO) stimulates hydrolysis of 2 mM ATP catalyzed by wild-type and mutant complexes 4- and 7.5-fold, respectively. The rate of release of [H-3]ADP from the Mg[H-3]ADP-loaded mutant complex during hydrolysis of 40 mu M ATP is slower than observed with the wild-type complex. LDAO increases the rate of release of [H-3]ADP from the preloaded wild-type and mutant complexes during hydrolysis of 40 mu M ATP, Again, release is slower with the mutant complex. When the wild-type and mutant complexes are irradiated in the presence of 2-N-3-[H-3]ADP plus Mg2+ or 2-N-3-[H-3]ATP plus Mg2+ and azide, the same extent of labeling of noncatalytic sites is observed. Whereas ADP and ATP protect noncatalytic sites of the wild-type and mutant complexes about equally from labeling by 2-N-3-[H-3]ADP or 2-N-3-[H-3]ATP, respectively, AMP-PNP provides little protection of noncatalytic sites of the mutant complex. The results suggest that the substitution does not prevent binding of ADP or ATP to noncatalytic sites, but rather that it affects cross-talk between liganded noncatalytic sites and catalytic sites which is necessary to promote dissociation of inhibitory MgADP.

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Except for the case of gradual activation of EF(1) (F-1-ATPase from Escherichia coli) caused by the dissociation of the epsilon subunit [Laget, P.P. and Smith, J.B. (1979) Arch. Biochem. Biophys. 197, 83-89], EF(1) has long been thought not to show a time-dependent change in activity [Senior, A.E. et al. (1992) Arch. Biochem. Biophys. 297, 340-344]. Here, we report the time-dependent inactivation and activation of EF(1), which are apparently similar to those of mitochondrial F-1-ATPases [Vasilyeva, E.A. et al, (1982) Biochem. J. 202, 15-23]. Analysis of these changes as a function of ATP concentrations in relation to negative cooperativity revealed that the initial inactivation phase was attributable to the decrease in the V-max associated with the low K-m (around 10 mu M), and the following activation, probably due to the dissociation of the epsilon subunit, corresponded to the increase in the V-max associated with the high K-m (in the order of 100 mu M). Thus, the time-dependent change in EF(1) activity is closely related to the apparent negative cooperativity (multiple K-m values) of ATP hydrolysis.

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER  

This paper describes how the structure and concentration level of a detergent used for substitution after bacteriorhodopsin (bR) solubilization affect the reconstitution of the bR into phospholipid planar bilayers. A direct insertion method was used for the bR reconstitution into the bilayers. Two detergents representing the two major types were used: sodium deoxycholate with a cholane-ring structure, and octylglucoside with a linear (or chain) structure. We then characterized the reconstitution for the two detergents by considering the detergent separation profiles and the photocurrent variations upon addition of lanthanum chloride and the protonophore FCCP (carbonylcyanide-p-trifluoromethoxyphenylhydrazone). We found that for successful transmembrane reconstitution of bR the detergent with the cholane-ring structure was preferable to that with the linear structure when the detergent concentration was above its critical micellar concentration. This preference was explained by the ease with which the detergent with the cholane-ring structure was removed from protein compared to that with the linear structure. Finally, we proposed a scheme for the reconstitution of the protein. © 1995.

DOI： 10.1016/0301-4622(94)00111-V

Scopus

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The interaction of 2',3'-O-(2,4,6-trinitrophenyl)ATP (TNP-ATP) with bovine mitochondrial F-1-ATPase (MF(1)) was examined under substoichiometric and stoichiometric conditions to investigate the relationship between the amount of bound TNP-AT(D)P and extent of inhibition on steady state ATP hydrolysis. The hydrolysis of bound TNP-ATP under substoichiometric condition proceeded slowly, with a first order rate constant of 0.014 s(-1). However, hydrolysis was greatly accelerated by addition of excess ATP. The hydrolyzed product, TNP-ADP, did not dissociate from the enzyme even after the addition of excess ATP. These properties were the same for both native and nucleotide depleted enzyme. The difference spectrum induced by binding TNP-ATP to MF(1) had a distinct peak at 410 nm and a deep trough at 395 nm, which were similar to those induced when TNP-ATP bound to the isolated beta subunit of the thermophilic F-1-ATPase. The magnitude of difference spectra as a function of TNP-ATP concentration suggested the presence of at least two types of binding sites on the MF(1) molecule. The first site, where substoichiometric TNP-ATP was hydrolyzed, had a very high affinity for TNP-ATP. TNP-AT(D)P bound to this site did not dissociate even in the presence of excess ATP. TNP-AT(D)P bound to the second site dissociated slowly when excess ATP was added. The steady state ATPase activity at 100 mu M ATP was linearly suppressed as pre-loaded TNP-ATP increased. The binding of 2 mol of TNP-ATP per mol of MF(1) was required to abolish ATPase activity. A model which assumes mutually-activating two catalytic sites is presented to explain these results.

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

Residue Tyr-341 of the F-1-ATPase beta subunit from a thermophilic Bacillus strain, PS3, was mutagenized to leucine, cysteine or alanine. Each of the mutated beta subunits was isolated and its affinity for ATP-Mg was examined by means of difference circular dichroism and differential titration calorimetry. The K-d values for ATP-Mg obtained were: beta Y341 (wild type), 0.015mM; beta Y341L, 0.7mM; beta Y341C and beta Y341A, >3mM. All the mutant beta subunits could be reconstituted into the alpha(3) beta(3) gamma complex with alpha and gamma subunits. The alpha(3) beta((mutant)3) gamma complexes hydrolyzed ATP with apparent V-max values larger than that of the alpha(3) beta(wild)(3) gamma complex. The apparent K-m values of the alpha(3) beta((mutant)3) gamma complexes increased in parallel with the K-d values for ATP-Mg of the isolated mutant beta subunits. These results indicate that residue beta Y341 is directly involved in the catalytic ATP-Mg binding and is a major K-m-determining residue of F-1-ATPase.

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

The kinetics of heart and liver mitochondrial ATPase (FoF(1)) were examined using submitochondrial particles (SMPs) purified from the two tissues to obtain information on the role of gamma subunit isoforms. The F-1 portion is mainly composed of the catalytic, common alpha beta subunits and tissue-specific gamma subunits. In contrast to the previous reports on the kinetics and crystallography of various F-1's, the Vmax and Km of the two isoforms of FoF(1) were identical although the SMPs were prepared from different tissues. Moreover sodium azide inhibited the two equally. The ATPase activity of liver SMP showed slightly steeper pH-dependency than that of heart SMP but the pH optima of the two were the same (pH 8). (C) 1994 Academic Press, Inc.

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER  

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The inhibitory effects of NaN3 on the F0F1 ATPase of beef heart submitochondrial particles were investigated. It was shown that NaN3 inhibited the ATPase activity only in the presence of ATP or ADP and the inhibition proceeded slowly. Analysis of the time-course of the inhibition process lead to a conclusion that an ATP binding site which has an apparent K(d) of 14.0 +/- 8.7 muM is responsible for the increase of NaN3 sensitivity. This value agreed well with the low K(m) of ATP hydrolysis characterized before (Muneyuki, E., and Hirata, H. (1988) FEBS Lett. 234, 455-458) and in the range of so-called bi-site catalysis. The same conclusion was derived as for isolated F1 ATPase. From similar analysis, the K(d) of this site for ADP was deduced to be 1.34 +/- 0.45 muM, which also agreed with that reported by Pedersen (Pedersen, P.L. (1975) Biochem. Biophys. Res. Commun. 64, 610-616) and also in the same range as reported for the low K(m) of ATP synthesis by activated submitochondrial particles. These results suggest that hydrolysis through the low K(m) mode of ATPase reaction leads the enzyme NaN3 sensitive form and this reaction cycle corresponds to the low K(m) mode of ATP synthesis.

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Royal Society(UK,London)  

Unlike Escherichia coli chaperonins, a chaperonin (cpn) from a thermophilic bacterium, Thermus thermophilus, consisting of homologues to GroEL (cpn 60) and GroES (cpn 10) is co-purified as a large complex. Thermus chaperonin shows a bullet-like shape in the side view seen by electron microscopy, and antibody against cpn 10 binds only to the round side of the bullet. We conclude that a single cpn 60-heptamer ring with two stripes stacks into two layers and a cpn 10 oligomer binds to one side of the layers. The purified Thermus chaperonin contains endogenously bound ADP, and incubation with ATP causes a partial dissociation of chaperonin into cpn 60 monomers and a cpn 10 heptamer. The effect of Thermus chaperonin on protein refolding upon dilution from guanidine HC1 is different at three temperature ranges. At high temperatures above 55 degrees C, where the native proteins are stable but their spontaneous foldings fail, the chaperonin induces productive folding in an ATP-dependent manner. At middle temperatures (25-55 degrees C) where spontaneous foldings of the enzymes occur, the chaperonin slows down the rate of folding without changing the final yield of productive folding. At lower temperatures below 25 degrees C where spontaneous foldings also occur, the chaperonin arrests the folding even in the presence of ATP. When a solution of relatively heat labile protein is incubated at high temperatures, and then residual activity of the protein is measured at its optimal temperature after incubation with ATP, the temperature that causes irreversible heat denaturation of the protein is elevated about 10 degrees C by inclusion of Thermus chaperonin in the solution.(ABSTRACT TRUNCATED AT 250 WORDS)

DOI： 10.1098/rstb.1993.0029

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The interaction of 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) and TNP-ADP to F1-ATPase from a thermophilic bacterium PS3 (TF1) was investigated. When TNP-ADP or TNP-ATP was added to the isolated alpha or beta-subunit of TF1, characteristic difference spectra were generated for each subunit. Difference spectra generated on addition of these analogs to TF1 resembled those observed for the beta-subunit, indicating TNP analogs bind to the beta-subunits in the molecule of TF1. Results of equilibrium dialysis showed that TNP-ADP binds to a single high affinity site on TF1 in the presence of Mg2+ with a dissociation constant of 2.2 nM. When TNP-ATP was added to TF1 in a substoichiometric molar ratio, it rapidly bound to TF1 and was slowly hydrolyzed. The hydrolysis proceeded nearly to completion without showing stable equilibrium between bound species of TNP-ATP and TNP-ADP. Similar to beef heart mitochondrial F1, this hydrolysis was greatly accelerated by the chase-addition of 100-mu-M ATP. However, the hydrolyzed product, TNP-ADP, remained bound on the beta-subunit even after the chase.

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The hydrolysis of 0.3-mu-M [alpha, gamma-P-32]ATP by 1-mu-M F1-ATPase isolated from the plasma membranes of Escherichia coli has been examined in the presence and absence of inorganic phosphate. The rate of binding of substoichiometric substrate to the ATPase is attenuated by 2 mM phosphate and further attenuated by 50 mM phosphate. Under all conditions examined, only 10-20% of the [alpha, gamma-P-32]ATP that bound to the enzyme was hydrolyzed sufficiently slowly to be examined in cold chase experiments with physiological concentrations of non-radioactive ATP. These features differ from those observed with the mitochondrial F1-ATPase. The amount of bound substrate in equilibrium with bound products observed in the slow phase which was subject to promoted hydrolysis by excess ATP was not affected by the presence of phosphate. Comparison of the fluxes of enzyme-bound species detected experimentally in the presence of 2 mM phosphate with those predicted by computer simulation of published rate constants determined for uni-site catalysis (Al-Shawi, M.D., Parsonage, D. and Senior, A.E. (1989) J. Biol. Chem. 264, 15376-15383) showed that hydrolysis of substoichiometric ATP observed experimentally was clearly biphasic. Less than 20% of the substoichiometric ATP added to the enzyme was hydrolyzed according to the published rate constants which were calculated from the slow phase of product release in the presence of 1 mM phosphate. The majority of the substoichiometric ATP added to the enzyme was hydrolyzed with product release that was too rapid to be detected by the methods employed in this study, indicating again that the F1-ATPase from E. coli and bovine heart mitochondria hydrolyze substoichiometric ATP differently.

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

Web of Science

researchmap

Total pages：430   Language：Japanese   Book type：Scholarly book

researchmap

Responsible for pages：88-100   Language：Japanese   Book type：Scholarly book

researchmap

Responsible for pages：14-21   Language：Japanese   Book type：Scholarly book

researchmap

Responsible for pages：19-40   Language：Japanese   Book type：Scholarly book

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.54.S167_5

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.54.S167_4

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.53.S134_5

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.53.S134_6

researchmap

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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.

DOI： 10.2142/biophysics.9.91

PubMed

researchmap

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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.

DOI： 10.2142/biophysics.9.107

PubMed

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.53.S135_2

researchmap

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

researchmap

Language：Japanese   Publisher：The Physical Society of Japan (JPS)  

CiNii Books

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.52.S78_2

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.52.S17_3

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.52.S79_4

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.51.S60_4

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.51.S107_1

researchmap

Language：English   Publisher：一般社団法人 日本生物物理学会  

DOI： 10.2142/biophys.51.S62_2

researchmap

Language：Japanese   Publisher：一般社団法人 日本物理学会  

CiNii Books

researchmap

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

CiNii Books

researchmap

Other Link： http://dl.ndl.go.jp/info:ndljp/pid/10942999

Language：Japanese   Publisher：The Physical Society of Japan (JPS)  

CiNii Books

researchmap

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：CELL PRESS  

Web of Science

researchmap

J-GLOBAL

researchmap

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.50.S174_5

researchmap

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

CiNii Books

researchmap

Language：Japanese   Publisher：一般社団法人 日本物理学会  

CiNii Books

researchmap

Language：Japanese   Publisher：The Physical Society of Japan (JPS)  

CiNii Books

researchmap

Language：English   Publisher：(一社)日本生物物理学会  

researchmap

Language：English   Publisher：NATURE PUBLISHING GROUP  

Web of Science

researchmap

Language：Japanese   Publisher：The Physical Society of Japan (JPS)  

CiNii Books

researchmap

Language：English   Publisher：一般社団法人 日本生物物理学会  

DOI： 10.2142/biophys.47.S242_3

researchmap

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

DOI： 10.20665/kakyoshi.55.12_610

CiNii Books

researchmap

Language：Japanese   Publisher：The Physical Society of Japan (JPS)  

CiNii Books

researchmap

Language：Japanese   Publisher：一般社団法人 日本生物物理学会  

DOI： 10.2142/biophys.45.S163_4

researchmap

Language：Japanese   Publisher：The Physical Society of Japan (JPS)  

CiNii Books

researchmap

DOI： 10.1038/nsmb721

PubMed

researchmap

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：BIOPHYSICAL SOCIETY  

Web of Science

researchmap

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.42.S144_4

researchmap

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

CiNii Books

researchmap

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

CiNii Books

researchmap

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：日本膜学会  

researchmap

Language：English   Publisher：AMER CHEMICAL SOC  

Web of Science

researchmap

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：共立出版  

researchmap

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：共立出版  

CiNii Books

researchmap

Language：Japanese  

CiNii Books

researchmap

Language：Japanese  

CiNii Books

researchmap

Language：Japanese  

CiNii Books

researchmap

Language：Japanese  

CiNii Books

researchmap

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：秀潤社  

researchmap

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

CiNii Books

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：English   Presentation type：Oral presentation (general)  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

researchmap

Language：Japanese  

<p>ATPの加水分解によって駆動される回転分子モーターF1-ATPaseのエネルギー論を調べた。特に駆動力である化学ポテンシャルの大きさを一分子実験によって決定するために、回転１自由度の有効ポテンシャルによる記述に基づき、回転の角度の時系列に局所詳細釣り合いを当てはめた。その結果、３６０°回転で解放される化学自由エネルギーの大きさがATP3分子分に相当することが明らかになった。</p>

researchmap

Language：Japanese  

<p>従来の古典力場では，r^-12^項とr^-6^項からなる12-6 Lennard-Jones (LJ)モデルが用いられてきた．しかし，このモデルは，電荷-誘起双極子，双極子-誘起双極子間相互作用を考慮していないため，金属蛋白質における金属イオンの配位構造を正確に表現できない．最近，この欠点を克服するため，12-6 LJモデルにr^-4^項を加えた12-6-4 LJモデルが提案された．本研究では，12-6-4 LJモデルと12-6 LJモデルを用いて，金属蛋白質の構造と結合自由エネルギーを計算し，両モデルを比較する．</p>

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

researchmap

Language：English   Presentation type：Poster presentation  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

<p>ATP合成酵素であるF1-ATPaseの反応スキームについて生成物のADPとPiの解離の関係におけるモデルが2つ考えられている。2010年に渡邊らはADPよりもPiが先に解離することを報告したが、下らはADPはPiよりも後に解離することを報告した。そこで、本研究で、私たちは変異体を用いてこの問題を議論することにした。P-loop変異体TF1（βG158A）を用いた一分子回転観察実験を行い、ATP加水分解とPi解離が遅いことを確認した。更に、ハイブリッドF1変異体を作成し、同様に回転実験から得られたdwell timeの解析を行うことで、PiはADPよりも先に解離するモデルを示唆する結果を得た。</p>

researchmap

Language：Japanese  

<p>回転分子モーターF_1-ATPaseは、回転角度の他に化学状態の自由度を持つが、通常の一分子実験では回転角度のみの観察しか行えない隠れた変数を持つ系であり、そのために熱ゆらぎの顕著な系におけるエネルギー収支を評価する枠組みであるゆらぎのエネルギー論は素朴には適用することができない。本講演では、F_1-ATPaseの回転の駆動力であるヌクレオチドの化学ポテンシャル差を回転角度の時系列のみから求める方法について議論する。</p>

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：English   Presentation type：Poster presentation  

researchmap

Language：English   Presentation type：Poster presentation  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：English   Presentation type：Poster presentation  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：Japanese  

researchmap

Language：English   Presentation type：Oral presentation (general)  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：English  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：English   Presentation type：Poster presentation  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

researchmap

Language：English   Presentation type：Poster presentation  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：Japanese  

researchmap

Language：Japanese   Presentation type：Oral presentation (general)  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap