Structure, rotation and regulation of ATP synthase(FoF1)

我们制备了含有一个或两个突变体的F_1-ATP酶。具有改变的催化动力学的β亚基。对它们的不对称步进旋转的分析表明，当观察一个。β亚基在0°结合ATP，~ 200°旋转后切割ATP，~ 320°旋转后进行最终催化反应，推测为产物释放。三.β亚基以120°的差异进行这种“拖弦”式的循环，其循环的域间弯曲运动将诱导γ亚基的旋转，推测分子马达F_1-ATPase中轴γ亚基的旋转与三个催化酶的域运动相关，并可能受其驱动。β亚基。在这里，我们直接观察这些。β运动通过连接的荧光团，伴随着相同单分子中γ的80度和40度子步旋转。我们展示了每个β亚基在三步弯曲中经历的构象序列，一个大约40度的逆时针旋转，然后是两个大约20度的顺时针旋转，与γ的两个子步旋转同步发生。结果表明，大多数以前的晶体结构模拟的构象设置的三个β亚基的催化驻留。此外，一个以前未描述的β构象，开放，封闭和部分封闭，显示在ATP等待驻留。本研究填补了F_1-ATPase中化学和机械步骤之间的差距。

We made F_1-ATPases containing one or two mutant. β subunits that have the altered catalytic kinetics. The analysis of their asymmetric stepwise rotations reveal that, when watching one. β subunit in F_1-ATPase, it binds ATP at 0°, cleaves ATP after～ 200°rotation, and undergoes the final catalytic event, presumably the product release, after～ 320°rotation. Three. β subunits carry out this cycle at 120°difference like "trolling a tune" and their cyclic inter-domain bending motions would induce the rotation of γ subunit.Rotation of the central shaft γ subunit in a molecular motor F_1-ATPase is assumed to correlate with and probably be driven by domain motions of the three catalytic. β subunits. Here we observe directly these. β motions through an attached fluorophore, concomitantly with 80 degrees and 40 degrees substep rotations of γ in the same single molecules. We show the sequence of conformations that each β subunit undergoes in three-step bending, an approximately 40 degrees counterclockwise turn followed by two approximately 20 degrees clockwise turns, occurring in synchronization with two substep rotations of γ. The results indicate that most previous crystal structures mimic the conformational set of three β subunits in the catalytic dwells. Moreover, a previously undescribed set of β conformations, open, closed and partially closed, is revealed in the ATP-waiting dwells. The present study thus bridges the gap between the chemical and mechanical steps in F_1-ATPase.