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 [1, 2]. 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(ßE190D) 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 Å3 and +88 Å3 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. [1] Nishiyama M. and Y. Sowa. 2012. Microscopic Analysis of Bacterial Motility at High Pressure. Biophys. J.102:1872-1880. [2] Okuno D., M. Nishiyama, and H. Noji. 2013. Single-Molecule Analysis of the Rotation of F1-ATPase under High Hydrostatic Pressure. Biophys. J.105:1635-1642.