Department of Applied Physics, School of Engineering, The University of Tokyo, Tokyo, Japan.
Okazaki Institute for Integrative Bioscience, Institute for Molecular Science, National Institutes of Natural Sciences, Aichi, Japan.
Nat Chem Biol. 2016 Apr;12(4):290-7. doi: 10.1038/nchembio.2028. Epub 2016 Feb 29.
The dimeric motor protein kinesin-1 walks along microtubules by alternatingly hydrolyzing ATP and moving two motor domains ('heads'). Nanometer-precision single-molecule studies demonstrated that kinesin takes regular 8-nm steps upon hydrolysis of each ATP; however, the intermediate states between steps have not been directly visualized. Here, we employed high-temporal resolution dark-field microscopy to directly visualize the binding and unbinding of kinesin heads to or from microtubules during processive movement. Our observations revealed that upon unbinding from microtubules, the labeled heads were displaced rightward and underwent tethered diffusive movement. Structural and kinetic analyses of wild-type and mutant kinesins with altered neck linker lengths provided evidence that rebinding of the unbound head to the rear-binding site is prohibited by a tension increase in the neck linker and that ATP hydrolysis by the leading head is suppressed when both heads are bound to the microtubule, thereby explaining how the two heads coordinate to move in a hand-over-hand manner.
驱动蛋白-1 是一种二聚体马达蛋白，通过交替水解 ATP 并移动两个马达结构域（“头部”）沿着微管运动。纳米级精度的单分子研究表明，每水解一个 ATP，驱动蛋白会采取规则的 8nm 步长；然而，在这些步长之间的中间状态还没有被直接可视化。在这里，我们采用高时间分辨率暗场显微镜直接观察到在连续运动过程中驱动蛋白头部与微管的结合和解离。我们的观察结果表明，在从微管上解离后，标记的头部向右移动，并经历了束缚扩散运动。对具有改变的颈环长度的野生型和突变型驱动蛋白的结构和动力学分析提供了证据，表明未结合的头部重新结合到后结合位点受到颈环张力增加的阻碍，并且当两个头部都结合到微管上时，领头头部的 ATP 水解受到抑制，从而解释了两个头部如何协调以手拉手的方式移动。
