Direct Observation of the Rotary Motion of FOF1-ATP Synthase Driven by Proton Motive Force

Abstract

FoF1-ATP synthase (FoF1) is a rotary motor protein which carries out ATP synthesis by coupling to the rotary motion driven by the proton motive force (pmf) across the membrane. FoF1 consists of two molecular motors; ATP-driven motor (F1) and proton-driven motor (Fo). In 1997, we for the first time observed the ATP-driven rotation of F1, and have thoroughly elucidated its operating principle as a rotary motor protein in this decade. In contrast, the proton-driven rotation of Fo or whole enzyme; FoF1, has not been observed yet. This is mainly due to the defects of pmf generation system and its interference with the detection system for rotary motions. In this year, to resolve these technical issues, we fixed these defects one by one, and eventually developed the experimental system which enabled to monitor the amplitude of pmf and the rotation of FoF1, simultaneously. In this system, we used the pH-sensitive fluorophore; pHrodo, for the measurement of proton gradient across the membrane, which is the main component of pmf. In addition, to directly observe the rotary motion of FoF1, we attached the gold nanoparticle to the rotor part and its rotation was visualized by the total internal reflection dark-field illumination system (TIRFDF). In the presence of pmf, FoF1 showed the clockwise rotation when viewed from Fo to F1, and accelerated its rotational rate depending on the amplitude of pmf, which was essentially consistent with the previous biochemical study. In addition to the rotational rate, we obtained various information related to the “dynamics”, such as the stepwise rotary motion coupled with the proton transport and ATP synthesis, which provide a clue for further understanding of energy conversion mechanism of FoF1 in the physiological condition.