Probing the kinesin reaction cycle with a 2D optical force clamp
Probing the kinesin reaction cycle with a 2D optical force clamp
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DOI：
10.1073/pnas.0436709100
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发表时间：
2003-03-04
影响因子：
11.1
通讯作者：
Lang, MJ
中科院分区：
文献类型：
--
作者：
Block, SM;Asbury, CL;Lang, MJ
随着它所采取的每一步，驱动蛋白马达经历机械化学反应循环，包括一个ATP分子的水解，ADP/P-i释放，加上未知数量的额外转换。驱动蛋白的速度取决于所施加载荷的大小和方向。使用专门的仪器，我们使单个驱动蛋白分子受到不同方向的力。侧向和前向载荷高达8 pN时，只产生微弱的影响，而向后方向施加的类似力则会导致失速。这种强烈的方向性偏向表明，主要的工作行程是密切对齐的微管轴。侧向负荷不对称地减慢运动，但仅在较高的ATP水平下，揭示了在循环后期存在额外的、依赖于负荷的转变。波动分析表明，该周期包含至少四个过渡，并确认水解仍然紧密耦合到步进。总之，我们的研究结果对驱动蛋白运动模型提出了挑战。
With every step it takes, the kinesin motor undergoes a mechanochemical reaction cycle that includes the hydrolysis of one ATP molecule, ADP/P-i release, plus an unknown number of additional transitions. Kinesin velocity depends on both the magnitude and the direction of the applied load. Using specialized apparatus, we subjected single kinesin molecules to forces in differing directions. Sideways and forward loads up to 8 pN exert only a weak effect, whereas comparable forces applied in the backward direction lead to stall. This strong directional bias suggests that the primary working stroke is closely aligned with the microtubule axis. Sideways loads slow the motor asymmetrically, but only at higher ATP levels, revealing the presence of additional, load-dependent transitions late in the cycle. Fluctuation analysis shows that the cycle contains at least four transitions, and confirms that hydrolysis remains tightly coupled to stepping. Together, our findings pose challenges for models of kinesin motion.