Engineering of IF -susceptive bacterial F -ATPase.

IF , an inhibitor protein of mitochondrial ATP synthase, suppresses ATP hydrolytic activity of F . One of the unique features of IF is the selective inhibition in mitochondrial F (MF ); it inhibits catalysis of MF but does not affect F with bacterial origin despite high sequence homology between MF and bacterial F . Here, we aimed to engineer thermophilic Bacillus F (TF ) to confer the susceptibility to IF for elucidating the molecular mechanism of selective inhibition of IF .

Digital Cascade Assays for ADP- or ATP-Producing Enzymes Using a Femtoliter Reactor Array Device.

Digital enzyme assays are emerging biosensing methods for highly sensitive quantitative analysis of biomolecules with single-molecule detection sensitivity. However, current digital enzyme assays require a fluorogenic substrate for detection, which limits the applicability of this method to certain enzymes. ATPases and kinases are representative enzymes for which fluorogenic substrates are not available; however, these enzymes form large domains and play a central role in biology.

Rotary properties of hybrid F-ATPases consisting of subunits from different species.

F-ATPase (F) is an ATP-driven rotary motor protein ubiquitously found in many species as the catalytic portion of FF-ATP synthase. Despite the highly conserved amino acid sequence of the catalytic core subunits: α and β, F shows diversity in the maximum catalytic turnover rate and the number of rotary steps per turn. To study the design principle of F, we prepared eight hybrid Fs composed of subunits from two of three genuine Fs: thermophilic PS3 (TF), bovine mitochondria (MF), and (PdF), differing in the and the number of rotary steps.

Correction: Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis.

Correction for 'Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis' by Akihiko Nakamura et al., Phys. Chem.

Rotation of artificial rotor axles in rotary molecular motors.

F- and V-ATPase are rotary molecular motors that convert chemical energy released upon ATP hydrolysis into torque to rotate a central rotor axle against the surrounding catalytic stator cylinder with high efficiency. How conformational change occurring in the stator is coupled to the rotary motion of the axle is the key unknown in the mechanism of rotary motors. Here, we generated chimeric motor proteins by inserting an exogenous rod protein, FliJ, into the stator ring of F or of V and tested the rotation properties of these chimeric motors.

Torque generation of Enterococcus hirae V-ATPase.

V-ATPase (V(o)V1) converts the chemical free energy of ATP into an ion-motive force across the cell membrane via mechanical rotation. This energy conversion requires proper interactions between the rotor and stator in V(o)V1 for tight coupling among chemical reaction, torque generation, and ion transport. We developed an Escherichia coli expression system for Enterococcus hirae V(o)V1 (EhV(o)V1) and established a single-molecule rotation assay to measure the torque generated.

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.

Basic properties of rotary dynamics of the molecular motor Enterococcus hirae V1-ATPase.

V-ATPases are rotary molecular motors that generally function as proton pumps. We recently solved the crystal structures of the V1 moiety of Enterococcus hirae V-ATPase (EhV1) and proposed a model for its rotation mechanism. Here, we characterized the rotary dynamics of EhV1 using single-molecule analysis employing a load-free probe.