F1-ATPase conformational cycle from simultaneous single-molecule FRET and rotation measurements
F1-ATPase conformational cycle from simultaneous single-molecule FRET and rotation measurements
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来自同时单分子 FRET 和旋转测量的 F1-ATPase 构象循环
DOI：
10.1073/pnas.1524720113
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
T.
中科院分区：
文献类型：
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作者：
Sugawa;M. Okazaki;K. I. Kobayashi;M. Matsui;T. Hummer;G. Masaike;T. Nishizaka;T.
尽管进行了广泛的研究，但旋转分子马达F1-ATPase(F1)中机械力化学耦合的结构基础仍然不完整。我们进行了单分子FRET测量，以监测定子环-α3β3的构象变化，同时监测中心轴-γ的旋转。在三磷酸腺苷等待间歇中，三个β亚基中的两个同时采用低FRET非封闭形式。相反，在催化中间体中，两个β-亚基同时处于高FRET闭合状态。这些差异使我们可以直接将晶体结构分配给两种主要的驻留状态，从而解决了一个长期存在的问题，并在F1结构和电机的旋转角之间建立了牢固的联系。值得注意的是，处于ε抑制状态的F1的结构与ATP等待驻留的独特FRET特征一致，而大多数晶体结构捕获了催化驻留的结构。对现有晶体结构的主成分分析进一步阐明了αβ-二聚体在β循环中的五步构象转变，突出了两种主要的模式：αβ的开启/关闭运动和在ATPase-界面的松弛/收紧运动。这些结果为F1-ATPase的化学反应、定子构象和旋转角之间的三方耦合提供了新的观点。
Despite extensive studies, the structural basis for the mechanochemical coupling in the rotary molecular motor F1-ATPase (F1) is still incomplete. We performed single-molecule FRET measurements to monitor conformational changes in the stator ring-α3β3, while simultaneously monitoring rotations of the central shaft-γ. In the ATP waiting dwell, two of three β-subunits simultaneously adopt low FRET nonclosed forms. By contrast, in the catalytic intermediate dwell, two β-subunits are simultaneously in a high FRET closed form. These differences allow us to assign crystal structures directly to both major dwell states, thus resolving a long-standing issue and establishing a firm connection between F1structure and the rotation angle of the motor. Remarkably, a structure of F1in an ε-inhibited state is consistent with the unique FRET signature of the ATP waiting dwell, while most crystal structures capture the structure in the catalytic dwell. Principal component analysis of the available crystal structures further clarifies the five-step conformational transitions of the αβ-dimer in the ATPase cycle, highlighting the two dominant modes: the opening/closing motions of β and the loosening/tightening motions at the αβ-interface. These results provide a new view of tripartite coupling among chemical reactions, stator conformations, and rotary angles in F1-ATPase.