Force-dependent stepping kinetics of myosin-V
Force-dependent stepping kinetics of myosin-V
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DOI：
10.1529/biophysj.104.053504
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发表时间：
2005-06-01
影响因子：
3.4
通讯作者：
Rief, M
中科院分区：
文献类型：
--
作者：
Clemen, AEM;Vilfan, M;Rief, M
肌球蛋白V是一种基于肌动蛋白的双头运动蛋白，参与许多细胞内运输过程。了解肌球蛋白V功能及其两个头部之间的通讯的关键问题是其在负荷下的行为。由于体内肌球蛋白V与其他更强的马达如驱动蛋白共定位，因此其在超失速力下的行为特别相关。我们使用具有远程力反馈的光镊来研究肌球蛋白V在其整个运行长度上在受控外部向前和向后载荷下的运动。我们发现，平均步长保持恒定，在类似于36 nm的范围内从5 pN向前到1.5 pN向后负载的力。我们还发现两个力依赖的化学机械循环中的过渡。较慢的ADP释放在低负荷下是速率限制的，并且仅微弱地依赖于力。更快的速率更强烈地依赖于力。较强的力依赖性表明，该速率代表了领先头部对其结合位点的扩散搜索。与驱动蛋白马达相反，肌球蛋白-V的运行长度基本上独立于5 pN的向前负荷到1.5pN的向后负荷之间的力。在5 pN的超失速力下，我们观察到肌球蛋白-V连续向后步进，表明动力冲程的力驱动反转是可能的。
Myosin-V is a processive two-headed actin-based motor protein involved in many intracellular transport processes. A key question for understanding myosin-V function and the communication between its two heads is its behavior under load. Since in vivo myosin-V colocalizes with other much stronger motors like kinesins, its behavior under superstall forces is especially relevant. We used optical tweezers with a long-range force feedback to study myosin-V motion under controlled external forward and backward loads over its full run length. We find the mean step size remains constant at similar to 36 nm over a wide range of forces from 5 pN forward to 1.5 pN backward load. We also find two force-dependent transitions in the chemomechanical cycle. The slower ADP-release is rate limiting at low loads and depends only weakly on force. The faster rate depends more strongly on force. The stronger force dependence suggests this rate represents the diffusive search of the leading head for its binding site. In contrast to kinesin motors, myosin-V's run length is essentially independent of force between 5 pN of forward to 1.5 pN of backward load. At superstall forces of 5 pN, we observe continuous backward stepping of myosin-V, indicating that a force-driven reversal of the power stroke is possible.