No abstract

V1-ATPase (V1), the catalytic domain of an ion-pumping V-ATPase, is a molecular motor that converts ATP hydrolysis–derived chemical energy into rotation. Here, using a gold nanoparticle probe, we directly observed rotation of V1 from the pathogen Enterococcus hirae (EhV1). We found that 120° steps in each ATP hydrolysis event are divided into 40 and 80° substeps. In the main pause before the 40° substep and at low ATP concentration ([ATP]), the time constant was inversely proportional to [ATP], indicating that ATP binds during the main pause with a rate constant of 1.0 × 107 m−1 s−1. At high [ATP], we observed two [ATP]-independent time constants (0.5 and 0.7 ms). One of two time constants was prolonged (144 ms) in a rotation driven by slowly hydrolyzable ATPγS, indicating that ATP is cleaved during the main pause. In another subpause before the 80° substep, we noted an [ATP]-independent time constant (2.5 ms). Furthermore, in an ATP-driven rotation of an arginine-finger mutant in the presence of ADP, −80 and −40° backward steps were observed. The time constants of the pauses before −80° backward and +40° recovery steps were inversely proportional to [ADP] and [ATP], respectively, indicating that ADP- and ATP-binding events trigger these steps. Assuming that backward steps are reverse reactions, we conclude that 40 and 80° substeps are triggered by ATP binding and ADP release, respectively, and that the remaining time constant in the main pause represents phosphate release. We propose a chemo-mechanical coupling scheme of EhV1, including substeps largely different from those of F1-ATPases.

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°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.

We report a general strategy based on digital counting principle that enables an efficient acquisition of enzyme mutants with desired activities from just a few clones within a day. We prepared a high-density femtoliter droplet array, consisting of 1 million uniform droplets per 1 cm2 to carry out high-throughput protein synthesis and screening. Single DNA molecules were randomly distributed into each droplet following a Poisson process to initiate the protein synthesis with coupled cell-free transcription and translation reactions and then recovered by a microcapillary. The protein yield in each droplet was proportional to the number of DNA molecules, meaning that droplets with apparent intensities higher than the Poisson distribution–predicted maximum can be readily identified as the exact hits exhibiting the desired increased activity. We improved the activity of an alkaline phosphatase up to near 20-fold by using less than 10 nl of reagents.

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