School of Life Sciences, Arizona State University, Tempe, AZ 85287.
Proc Natl Acad Sci U S A. 2014 Mar 11;111(10):3715-20. doi: 10.1073/pnas.1317784111. Epub 2014 Feb 24.
F1-ATPase, the catalytic complex of the ATP synthase, is a molecular motor that can consume ATP to drive rotation of the γ-subunit inside the ring of three αβ-subunit heterodimers in 120° power strokes. To elucidate the mechanism of ATPase-powered rotation, we determined the angular velocity as a function of rotational position from single-molecule data collected at 200,000 frames per second with unprecedented signal-to-noise. Power stroke rotation is more complex than previously understood. This paper reports the unexpected discovery that a series of angular accelerations and decelerations occur during the power stroke. The decreases in angular velocity that occurred with the lower-affinity substrate ITP, which could not be explained by an increase in substrate-binding dwells, provides direct evidence that rotation depends on substrate binding affinity. The presence of elevated ADP concentrations not only increased dwells at 35° from the catalytic dwell consistent with competitive product inhibition but also decreased the angular velocity from 85° to 120°, indicating that ADP can remain bound to the catalytic site where product release occurs for the duration of the power stroke. The angular velocity profile also supports a model in which rotation is powered by Van der Waals repulsive forces during the final 85° of rotation, consistent with a transition from F1 structures 2HLD1 and 1H8E (Protein Data Bank).
F1-ATP 合酶的催化复合物是一种分子马达，它可以消耗 ATP 来驱动环中三个 αβ 亚基异二聚体的γ亚基以 120°的动力冲程进行旋转。为了阐明 ATP 酶驱动旋转的机制，我们从每秒 20 万帧的单分子数据中确定了旋转位置的角速度函数，具有前所未有的信噪比。动力冲程的旋转比以前理解的更为复杂。本文报道了一个意外的发现，即在动力冲程期间会发生一系列角加速度和减速度。与结合停留时间增加不一致的是，与低亲和力底物 ITMP 结合时角速度的降低，提供了直接的证据表明旋转取决于底物结合亲和力。ADP 浓度的升高不仅增加了与催化停留时间一致的 35°停留时间，这与竞争性产物抑制一致，而且还降低了从 85°到 120°的角速度，表明 ADP 可以在整个动力冲程中结合到发生产物释放的催化部位。角速度曲线还支持这样一种模型，即旋转是由范德华排斥力在最后 85°的旋转过程中提供动力的，这与从 2HLD1 和 1H8E（蛋白质数据库）结构的转变一致。
