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Motility of single-headed kinesin

单头驱动蛋白的运动性

基本信息

批准号：
06454662
负责人：
TOYOSHIMA Yoko
金额：
$ 4.8万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06454662/
关键词：
kinesin microtubule sliding movement single-headed motor molecular motor

项目摘要

Single kinesin molecule can move a microtubule continuously. Whether the two heads of a kinesin molecule are necessary for movement or the single head is enough is yet to be established. To approach this problem, we have prepared single headed kinesin and examined in two different motility assay systems.Kinesin motor domain of 5 different lengths were expressed in E.coli : they consist of N-terminal 340 to 410 amino acids and were supplemented with a cystein residue at the C terminus ends. K340, K351 and K368 were monomers, whereas K386 and K410 existed as dimers.To examine the motility of these kinesin fragments, the C-terminal cystein residue was labeled with maleimide-biotin and tethered to a glass surface via streptavidin and biotinylated BSA. All of 5 expressed kinesin fragments moved microtubules. The velocity of double-headed species was about 0.5 mu m/sec, which is similar to that of intact kinesin. The velocity of single-headed species was about ten times lower than that of double-headed fragments.. These results suggest that the two heads of kinesin stimulate the motility, although they are not essential for the movement.To aim single molecule mechanics, we carried out a bead assay using optical tweezers. In this system, the bead binds very few numbers of kinesin fragments via biotin-avidin, and the velocity and force generated by most likely single molecule was measured from the bead displacement. The maximum velocity (0.5 mu m/sec) and the maximum force (6pN) for K351 (single-headed) was almost the same as those for K410 (double-headed). These results indicate that a single-headed kinesin is sufficient to move microtubules and generate the force at a similar level as double-headed kinesin.

单个动蛋白分子可以连续运动一个微管。一个动蛋白分子的两个头是运动所必需的，还是一个头就足够了，这一点还有待确定。为了解决这个问题，我们制备了单头Kinesin，并在两种不同的运动分析系统中进行了检测。在大肠杆菌中表达了5个不同长度的Kinesin运动域：它们由340到410个氨基酸组成，并在C端添加了一个半胱氨酸残基。K340、K351和K368是单体，而K386和K410则以二聚体的形式存在，为了检测这些运动蛋白片段的运动性，用马来酰亚胺-生物素标记了半胱氨酸残基，并通过链霉亲和素和生物素化的BSA将半胱氨酸残基固定在玻璃表面。5例均表达运动蛋白片段移动的微管。双头虫的运动速度约为0.5微米/秒，与完整的动蛋白的运动速度相似。单头物种的速度比双头碎片的速度慢10倍左右。这些结果表明，两个动蛋白的头部刺激了运动，尽管它们对运动并不是必需的。为了达到单分子力学的目的，我们使用光学镊子进行了珠状分析。在这个系统中，微珠通过生物素-亲和素结合了极少数的动蛋白片段，最有可能由单个分子产生的速度和力是通过微珠的位移来测量的。K351(单头)的最大速度(0.5微米/秒)和最大力(6Pn)与K410(双头)几乎相同。这些结果表明，单头动蛋白足以移动微管，并产生与双头动蛋白类似水平的力。