Single-Molecule Analysis of the Rotation of F1-ATPase under High Hydrostatic Pressure

Abstract

F1-ATPase is the water-soluble part of ATP synthase and is an ATP-driven rotary molecular motor that rotates the rotary shaft against the surrounding stator ring, hydrolyzing ATP. Although the mechanochemical coupling mechanism of F1-ATPase has been well studied, the molecular details of individual reaction steps remain unclear. In this study, we conducted a single-molecule rotation assay of F1 from thermophilic bacteria under various pressures from 0.1 to 140 MPa. Even at 140 MPa, F1 actively rotated with regular 120° steps in a counterclockwise direction, showing high conformational stability and retention of native properties. Rotational torque was also not affected. However, high hydrostatic pressure induced a distinct intervening pause at the ATP-binding angles during continuous rotation. The pause was observed under both ATP-limiting and ATP-saturating conditions, suggesting that F1 has two pressure-sensitive reactions, one of which is evidently ATP binding. The rotation assay using a mutant F1(βE190D) suggested that the other pressure-sensitive reaction occurs at the same angle at which ATP binding occurs. The activation volumes were determined from the pressure dependence of the rate constants to be +100 Å(3) and +88 Å(3) for ATP binding and the other pressure-sensitive reaction, respectively. These results are discussed in relation to recent single-molecule studies of F1 and pressure-induced protein unfolding.

Reversible Morphological Control of Tubulin-Encapsulating Giant Liposomes by Hydrostatic Pressure

“…To visualize the pressure-induced changes in the structure and function of biomacromolecules, we have developed a high-pressure microscope that enables us to acquire various microscopic images with high-resolution and sensitivity even when hydrostatic pressure is applied. , It revealed that applied pressure dynamically changes the motility of molecular motors such as kinesin, F 1 -ATPase, and bacterial flagellar motors. ,, Thus, in this study, using the high-pressure microscope, we investigated the pressure-induced morphological changes of tubulin-encapsulating giant liposomes. The results obtained demonstrate that the morphology of tubulin-encapsulating giant liposomes is reversibly and repeatedly controllable by regulating the polymerization and depolymerization of MTs inside the giant liposomes by changing the hydrostatic pressure, which means that MTs are an excellent motion device to develop an artificial motile cell model.…”

High Pressure Bioscience

“…The 120 ı step rotation can be divided into 80 ı substep triggered after ATP binding, and 40 ı substep triggered after ATP hydrolysis (Yasuda et al 2001). We conducted a singlemolecule rotation assay of F 1 from thermophilic bacteria under various pressures (Okuno et al 2013). Although the mechanochemical coupling mechanism of F 1 -ATPase has been well studied, the molecular details of individual reaction steps remain unclear.…”

“…We conducted a singlemolecule rotation assay of F 1 from thermophilic bacteria under various pressures (Okuno et al 2013). This is strongly supported by results of a rotation assay using a mutant F 1 ("E190D) (Okuno et al 2013). When 120 MPa pressure was applied to the system, the rotary motion drastically changed.…”

Light‐Addressable Amperometric Sensor with Counter and Working Electrodes of the Same Material

“…Conversely, the second generation is a popular method for commercial purposes. Recently, single‐cell analysis has attracted interest because it helps to reveal unclear mechanisms of cell functions [27–30] and can be successfully performed on small volumes befitting cells. The second generation is highly conducive to small‐volume measurements because it enables a disposable measurement process.…”