Torque generation mechanism of F1-ATPase probed by site-specificand reaction-specific single-molecule manipulation

我们通过操纵单个F1分子测定了F1-ATPase上催化反应的速率常数和平衡常数随旋转角的变化。结果表明，ATP结合过程是产生扭矩的主要步骤。建立了一种新颖的单分子成像系统，利用具有高时空分辨率的金纳米棒的偏振散射光来可视化F1扭矩产生单元亚单位的构象变化。结果表明，该亚基发生了强冲型构象变化。利用高速原子力显微镜观察了含有三个亚基的孤立定子环中亚基的构象变化。亚基表现出高度协同的构象转变，与转子旋转一样沿逆时针方向传播构象状态，这一发现意味着结构基础在定子环中编程。

We determined the rate constants and equilibrium constants of catalytic reactions on F1-ATPase as a function of rotary angle by manipulating individual F1 molecules. The results show ATP-binding process is the major torque-generating step. A novel single-molecule imaging system using polarized scattering light from a gold nanorod that has high spatiotemporal resolution was established to visualize the conformational change of the subunit, the torque-generating unit of F1. It was revealed that the subunit makes a power-stroke type conformational change. High-speed AFM was applied to visualize the conformational change of the subunit in the isolated stator ring with three subunits. subunits showed highly cooperative conformational transition, propagating the conformational state in the anticlockwise direction as the same as the rotor rotation.This finding means that the structural basis is programmed in the stator ring.