Yoshihiro Minagawa

Basic Properties of Rotary Dynamics of the Molecular Motor Enterococcus hirae V1-ATPase

Background: The chemomechanical coupling scheme of the rotary motor V1-ATPase is incompletely understood. Results: Enterococcus hirae V1-ATPase (EhV1) showed 120° steps of rotation without substeps, as commonly seen with F1-ATPase. Conclusion: The basic properties of rotary dynamics of EhV1 are similar to those of Thermus thermophilus V1-ATPase. Significance: This study revealed the common properties of V1-ATPases as rotary molecular motors, distinct from those of F1-ATPases. V-ATPases are rotary molecular motors that generally function as proton pumps. We recently solved the crystal structures of the V1 moiety of Enterococcus hirae V-ATPase (EhV1) and proposed a model for its rotation mechanism. Here, we characterized the rotary dynamics of EhV1 using single-molecule analysis employing a load-free probe. EhV1 rotated in a counterclockwise direction, exhibiting two distinct rotational states, namely clear and unclear, suggesting unstable interactions between the rotor and stator. The clear state was analyzed in detail to obtain kinetic parameters. The rotation rates obeyed Michaelis-Menten kinetics with a maximal rotation rate (Vmax) of 107 revolutions/s and a Michaelis constant (Km) of 154 μm at 26 °C. At all ATP concentrations tested, EhV1 showed only three pauses separated by 120°/turn, and no substeps were resolved, as was the case with Thermus thermophilus V1-ATPase (TtV1). At 10 μm ATP (⪡Km), the distribution of the durations of the ATP-waiting pause fit well with a single-exponential decay function. The second-order binding rate constant for ATP was 2.3 × 106 m−1 s−1. At 40 mm ATP (⪢Km), the distribution of the durations of the catalytic pause was reproduced by a consecutive reaction with two time constants of 2.6 and 0.5 ms. These kinetic parameters were similar to those of TtV1. Our results identify the common properties of rotary catalysis of V1-ATPases that are distinct from those of F1-ATPases and will further our understanding of the general mechanisms of rotary molecular motors.

Torque Generation of Enterococcus hirae V-ATPase

Background: Torque generation is important for the energy conversion of rotary ATPases. Results: Enterococcus hirae V-ATPase (EhVoV1) generated larger torque than isolated EhV1. Conclusion: Rotor-stator interactions in EhVoV1 are stabilized by the two peripheral stalks to generate larger torque compared with EhV1. Significance: Torques generated by intact V-ATPase and isolated V1 moiety have been compared quantitatively for the first time. 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.

Rotational mechanism of Enterococcus hirae V1-ATPase by crystal-structure and single-molecule analyses.

In ion-transporting rotary ATPases, the mechanical rotation of inner rotor subunits against other stator subunits in the complex mediates conversion of chemical free energy from ATP hydrolysis into electrochemical potential by pumping ions across the cell membrane. To fully understand the rotational mechanism of energy conversion, it is essential to analyze a target sample by multiple advanced methods that differ in spatiotemporal resolutions and sample environments. Here, we describe such a strategy applied to the water-soluble V1 moiety of Enterococcus hirae V-ATPase; this strategy involves integration of crystal structure studies and single-molecule analysis of rotary dynamics and torque generation. In addition, we describe our current model of the chemo-mechanical coupling scheme obtained by this approach, as well as future prospects.

High-Speed Angle-Resolved Imaging of a Single Gold Nanorod with Microsecond Temporal Resolution and One-Degree Angle Precision

We developed two types of high-speed angle-resolved imaging methods for single gold nanorods (SAuNRs) using objective-type vertical illumination dark-field microscopy and a high-speed CMOS camera to achieve microsecond temporal and one-degree angle resolution. These methods are based on: (i) an intensity analysis of focused images of SAuNR split into two orthogonally polarized components and (ii) the analysis of defocused SAuNR images. We determined the angle precision (statistical error) and accuracy (systematic error) of the resultant SAuNR (80 nm × 40 nm) images projected onto a substrate surface (azimuthal angle) in both methods. Although both methods showed a similar precision of ∼1° for the azimuthal angle at a 10 μs temporal resolution, the defocused image analysis showed a superior angle accuracy of ∼5°. In addition, the polar angle was also determined from the defocused SAuNR images with a precision of ∼1°, by fitting with simulated images. By taking advantage of the defocused image methods full revolution measurement range in the azimuthal angle, the rotation of the rotary molecular motor, F1-ATPase, was measured with 3.3 μs temporal resolution. The time constants of the pauses waiting for the elementary steps of the ATP hydrolysis reaction and the torque generated in the mechanical steps have been successfully estimated. The high-speed angle-resolved SAuNR imaging methods will be applicable to the monitoring of the fast conformational changes of many biological molecular machines.

Thermodynamic analysis of F1-ATPase rotary catalysis using high-speed imaging.

F1‐ATPase (F1) is a rotary motor protein fueled by ATP hydrolysis. Although the mechanism for coupling rotation and catalysis has been well studied, the molecular details of individual reaction steps remain elusive. In this study, we performed high‐speed imaging of F1 rotation at various temperatures using the total internal reflection dark‐field (TIRDF) illumination system, which allows resolution of the F1 catalytic reaction into elementary reaction steps with a high temporal resolution of 72 µs. At a high concentration of ATP, F1 rotation comprised distinct 80° and 40° substeps. The 80° substep, which exhibited significant temperature dependence, is triggered by the temperature‐sensitive reaction, whereas the 40° substep is triggered by ATP hydrolysis and the release of inorganic phosphate (Pi). Then, we conducted Arrhenius analysis of the reaction rates to obtain the thermodynamic parameters for individual reaction steps, that is, ATP binding, ATP hydrolysis, Pi release, and TS reaction. Although all reaction steps exhibited similar activation free energy values, ΔG‡ = 53–56 kJ mol−1, the contributions of the enthalpy (ΔH‡), and entropy (ΔS‡) terms were significantly different; the reaction steps that induce tight subunit packing, for example, ATP binding and TS reaction, showed high positive values of both ΔH‡ and ΔS‡. The results may reflect modulation of the excluded volume as a function of subunit packing tightness at individual reaction steps, leading to a gain or loss in water entropy.

Molecular structure and rotary dynamics of Enterococcus hirae V1‐ATPase

V1‐ATPase is a rotary molecular motor in which the mechanical rotation of the rotor DF subunits against the stator A3B3 ring is driven by the chemical free energy of ATP hydrolysis. Recently, using X‐ray crystallography, we solved the high‐resolution molecular structure of Enterococcus hirae V1‐ATPase (EhV1) and revealed how the three catalytic sites in the stator A3B3 ring change their structure on nucleotide binding and interaction with the rotor DF subunits. Furthermore, recently, we also demonstrated directly the rotary catalysis of EhV1 by using single‐molecule high‐speed imaging and analyzed the properties of the rotary motion in detail. In this critical review, we introduce the molecular structure and rotary dynamics of EhV1 and discuss a possible model of its chemomechanical coupling scheme.