Publications

Shoichi Toyabe and Eiro Muneyuki,
Single molecule thermodynamics of ATP synthesis by F1-ATPase,
New Journal of Physics 17, 015008/1-7 (2015).

[Summary] FoF1-ATP synthase is a factory for synthesizing ATP in virtually all cells. Its core machinery is the subcomplex F1-motor (F1-ATPase) and performs the reversible mechanochemical coupling. The isolated F1-motor hydrolyzes ATP, which is accompanied by unidirectional rotation of its central γ−shaft. When a strong opposing torque is imposed, theγ-shaft rotates in the opposite direction and drives the F1 -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 F1 -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.

Hiroshi Ueno, Yoshihiro Minagawa, Mayu Hara, Suhaila Rahman, Ichiro Yamato, Eiro Muneyuki, Hiroyuki Noji, Takeshi Murata, *Ryota Iino,
Torque Generation of Enterococcus hirae V-ATPase,
The Journal of Biology Chemistry 289, 31212-31223 (2014).

[Summary] V-ATPase (VoV1) converts the chemical free energy of ATP into an ion-motive force across the cell membrane via mechanical rotation. This energy conversion requires proper interactions between the rotor and stator in VoV1 for tight coupling among chemical reaction, torque generation, and ion transport. We developed an Escherichia coli expression system for Enterococcus hirae VoV1 (EhVoV1) and established a single-molecule rotation assay to measure the torque generated. Recombinant and native EhVoV1 exhibited almost identical dependence of ATP hydrolysis activity on sodium ion and ATP concentrations, indicating their functional equivalence. In a single-molecule rotation assay with a low load probe at high ATP concentration, EhVoV1 only showed the "clear" state without apparent backward steps, whereas EhV1 showed two states, "clear" and "unclear." Furthermore, EhVoV1 showed slower rotation than EhV1 without the three distinct pauses separated by 120° that were observed in EhV1. When using a large probe, EhVoV1 showed faster rotation than EhV1, and the torque of EhVoV1 estimated from the continuous rotation was nearly double that of EhV1. On the other hand, stepping torque of EhV1 in the clear state was comparable with that of EhVoV1. These results indicate that rotor-stator interactions of the Vo moiety and/or sodium ion transport limit the rotation driven by the V1 moiety, and the rotor-stator interactions in EhVoV1 are stabilized by two peripheral stalks to generate a larger torque than that of isolated EhV1. However, the torque value was substantially lower than that of other rotary ATPases, implying the low energy conversion efficiency of EhVoV1.

Yasuaki Komuro, Suyong Re, Chigusa Kobayashi, Eiro Muneyuki, and *Yuji Sugita,
CHARMM Force-Fields with Modified Polyphosphate Parameters Allow Stable Simulation of the ATP-Bound Structure of Ca²⁺-ATPase,
Journal of Chemical Theory and Computation 10, 4133−4142 (2014).

[Summary] Adenosine triphosphate (ATP) is an indispensableenergy source in cells. In a wide variety of biologicalphenomena like glycolysis, muscle contraction/relaxation, and active ion transport, chemical energy released from ATPhydrolysis is converted to mechanical forces to bring aboutlarge-scale conformational changes in proteins. Investigation of structure−function relationships in these proteins by molecular dynamics (MD) simulations requires modeling of ATP in solution and ATP bound to proteins with accurate force-field parameters. In this study, we derived new force-field parameters for the triphosphate moiety of ATP based on the high-precision quantum calculations of methyl triphosphate. We tested our new parameters on membrane-embedded sarcoplasmic reticulum 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 originalparameters. 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 arameters will be useful in studies of ATP in solution and of the many ATP-utilizing proteins.