Kojima H, Muto E, Higuchi H, Yanagida T
Yanagida Biomotron Project, ERATO, JST, Mino, Osaka, Japan.
Biophys J. 1997 Oct;73(4):2012-22. doi: 10.1016/S0006-3495(97)78231-6.
We have analyzed the mechanics of individual kinesin molecules by optical trapping nanometry. A kinesin molecule was adsorbed onto a latex bead, which was captured by an optical trap and brought into contact with an axoneme that was bound to a glass surface. The displacement of kinesin during force generation was determined by measuring the position of the beads with nanometer accuracy. As the displacement of kinesin was attenuated because of the compliance of the kinesin-to-bead and kinesin-to-microtubule linkages, the compliance was monitored during force generation and was used to correct the displacement of kinesin. Thus the velocity and the unitary steps could be obtained accurately over a wide force range. The force-velocity curves were linear from 0 to a maximum force at 10 microM and 1 mM ATP, and the maximum force was approximately 7 pN, which is larger by approximately 30% than values previously reported. Kinesin exhibited forward and occasionally backward stepwise displacements with a size of approximately 8 nm. The histograms of step dwell time show a monotonic decrease with time. Model calculations indicate that each kinesin head steps by 16-nm, whereas kinesin molecule steps by 8-nm.
我们通过光镊纳米技术分析了单个驱动蛋白分子的力学特性。将一个驱动蛋白分子吸附到一个乳胶珠上，该乳胶珠被光镊捕获并与结合在玻璃表面的轴丝接触。在产生力的过程中，驱动蛋白的位移通过以纳米精度测量珠子的位置来确定。由于驱动蛋白与珠子以及驱动蛋白与微管连接的柔韧性，驱动蛋白的位移会衰减，因此在产生力的过程中监测柔韧性，并用于校正驱动蛋白的位移。这样就能在很宽的力范围内准确获得速度和单位步长。在10 microM和1 mM ATP条件下，力-速度曲线在从0到最大力的范围内呈线性，最大力约为7 pN，比之前报道的值大约大30%。驱动蛋白表现出向前以及偶尔向后的约8 nm大小的逐步位移。步长停留时间的直方图显示随时间单调递减。模型计算表明，每个驱动蛋白头部移动16 nm，而驱动蛋白分子移动8 nm。
