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Working Machinery of Dynein Molecules

动力蛋白分子的工作机制

基本信息

批准号：
18370059
负责人：
TOYOSHIMA Yoko
金额：
$ 7.76万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

Dynein is a huge and complicated molecule and minus-end directed motor of microtubules. To elucidate its machinery as a motor protein, we measured the molecular behavior of single dynein molecules focusing of the interaction with microtubules. Because dynein motor domain is as large as about 15nm in diameter, which is twice of the size of tubulin heterodimer repeat, it is not clear whether dynein uses only a single protofilament or multiple protofilaments. To answer this question, we exploited the property of zinc-induced tubulin sheets that the motor binding sites are only exposed at either edge of the sheet. Previous study has shown that multiple dynein molecules on glass surface can glide zinc-sheets. However, it is possible that multiple dynein molecules interact with the sheets and the gliding doesn't necessarily mean that single dynein molecule can walk on a single protofilament processively. In this study, we investigated the single molecule motility assay on zinc-sheets by TIRF … More M. We tethered the zinc-sheets on the glass using antibody against tubulin or MAPs, so that the dynein binding site is exposed in the solution. To observe single dynein molecules, GFP-tagged yeast Cytoplasmic dynein was prepared. We successfully observed that the GFP-tagged dynein moves processively on zinc-sheet as well as on microtubules. However, the velocity on zinc-sheet was about half of that on microtubules. These observations suggest that two-headed dynein molecule is able to move on single protofilament processively, but it can move more effectively on the multiple protofilaments. We next measured the unbinding force of single-headed dynein by newly developed quick stretch type experiment. The results suggested that the unbinding force was high in no nucleotide or AMPPNP and low in ADP-Vi or ADP, and also polarity dependent; the binding was strong against the pulling force from the plus-end and weak against that from the minus-end. These results showed that rear head within the double-headed structure of dynein is detach faster than the front head, which enable the processive movement of dynein molecules. Less

动力蛋白是一种庞大而复杂的分子，是微管负端定向马达。为了阐明其作为马达蛋白的机制，我们测量了单个动力蛋白分子的分子行为，重点是与微管的相互作用。由于动力蛋白马达结构域的直径约为15 nm，是微管蛋白异源二聚体重复序列的两倍，因此动力蛋白是使用单一的原丝还是多个原丝尚不清楚。为了回答这个问题，我们利用了锌诱导的微管蛋白片的性质，即马达结合位点仅暴露在片的任一边缘。以往的研究表明，玻璃表面的多个动力蛋白分子可以在锌片上滑动。然而，多个动力蛋白分子可能与片层相互作用，并且滑动并不一定意味着单个动力蛋白分子可以在单个原丝原丝上行走。本研究利用TIRF技术对锌膜上的单分子运动进行了研究 ...更多信息 M.我们用微管蛋白或MAPs抗体将锌片固定在玻璃上，使动力蛋白结合位点暴露在溶液中。为了观察单个动力蛋白分子，制备GFP标记的酵母细胞质动力蛋白。我们成功地观察到GFP标记的动力蛋白在锌片层和微管上的运动。而锌片上的速度约为微管上的一半。这些观察结果表明，双头动力蛋白分子能够在单个原丝原丝上运动，但它可以更有效地在多个原丝上运动。接下来，我们通过新开发的快速拉伸型实验测量了单头动力蛋白的解束缚力。结果表明，非核苷酸和AMPPNP的解结合力高，而ADP-Vi和ADP的解结合力低，且具有极性依赖性;正端拉力强，负端拉力弱。这些结果表明，动力蛋白双头结构中的后端比前端更快地脱离，从而使动力蛋白分子进行性运动。少