Directional loading of the kinesin motor molecule as it buckles a microtubule
Directional loading of the kinesin motor molecule as it buckles a microtubule
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DOI：
10.1016/s0006-3495(96)79585-1
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发表时间：
1996-01-01
影响因子：
3.4
通讯作者：
Howard, J
中科院分区：
文献类型：
--
作者：
Gittes, F;Meyhofer, E;Howard, J
单个驱动蛋白马达分子被观察到弯曲的微管沿着，他们在一个改进的体外滑行测定。在该测定中，微管的中心部分通过生物素-链霉亲和素键被夹在玻璃基底上，而微管的正端自由地与以低密度吸附到基底上的马达相互作用。对单马达弯曲的微管长度的统计分析表明，平行于细丝的载荷大于4-6 pN时，弯曲的概率降低。这与其他实验中发现的驱动蛋白失速力一致。一些屈曲事件的详细分析，使我们能够估计的大小和方向的加载力，因为它开发了一个垂直的组成部分，往往拉电机远离微管。我们还估计了电机速度作为这个变化的矢量力的函数。当力与马达运动的方向不平行时，驱动蛋白马达总是达到意想不到的高速度。我们的研究结果表明，负载的垂直分量不会阻碍驱动蛋白马达，但相反会导致马达对给定的平行负载移动得更快。因为垂直力分量加速马达但不做净功，所以垂直力充当反应的机械催化剂。一个简单的解释是，有一个空间运动的驱动蛋白分子在其周期是限速下的负载;机械催化的结果，如果这种运动是面向远离表面的微管。
Single kinesin motor molecules were observed to buckle the microtubules along which they moved in a modified in vitro gliding assay. In this assay a central portion of the microtubule was clamped to the glass substrate via biotin-streptavidin bonds, while the plus end of the microtubule was free to interact with motors adsorbed at low density to the substrate. A statistical analysis of the length of microtubules buckled by single motors showed a decreasing probability of buckling for loads greater than 4-6 pN parallel to the filament. This is consistent with kinesin stalling forces found in other experiments. A detailed analysis of some buckling events allowed us to estimate both the magnitude and direction of the loading force as it developed a perpendicular component tending to pull the motor away from the microtubule. We also estimated the motor speed as a function of this changing vector force. The kinesin motors consistently reached unexpectedly high speeds as the force became nonparallel to the direction of motor movement. Our results suggest that a perpendicular component of load does not hinder the kinesin motor, but on the contrary causes the motor to move faster against a given parallel load. Because the perpendicular force component speeds up the motor but does no net work, perpendicular force acts as a mechanical catalyst for the reaction. A simple explanation is that there is a spatial motion of the kinesin molecule during its cycle that is rate-limiting under load; mechanical catalysis results if this motion is oriented away from the surface of the microtubule.