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Molecular mechanism of protein motors revealed by the combination of genetic engineering and single-molecule analysis

基因工程与单分子分析相结合揭示蛋白质马达的分子机制

基本信息

批准号：
06404081
负责人：
SUTOH Kazuo
金额：
$ 18.82万
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 (A)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

项目摘要

We have constructed a myosin motor domain consisted of the heavy chain alone(760 residues), which retained motor functions. By using this construct, we succeeded to crystallize the myosin motor domain, and solve its structure at the atomic resolution (2.7A). By using this structural information, we carried out carried out site-directed mutagenesis of functionally important regions of myosin, especially the switch I and switch II loops. A loop comprising residues 454-459 of Dictyostelium myosin II is structurally and functionally equivalent to the switch II loop of the G-protein family. The consensus sequence of the switch II loop of the myosin family is DIXGFE.In order to determine the functions of each of the conserved residues, alanine scanning mutagenesis was carried out on the dictyostelium myosin II heavy chain gene. Examination of in vivo and in vitro motor functions of the mutant myosins revealed that the I455A and S456A mutants retained those functions while the D454A,G457A,F458A and E459A mutants lost them. Biochemical analysis of the latter myosins showed that the G457A and E459A mutants lost the basal ATPase activity by blocking of the isomerization and hydrolysis speps of the ATPase cycle, respectively. The F458A mutant, however, lost the actin-activated ATPase activity without loss of the basal ATPase activity. These results are discussed in terms of the crystal structure of the Dictyostelium myosin motor domain.

我们构建了仅由重链（760个残基）组成的肌球蛋白运动结构域，其保留了运动功能。通过使用这种结构，我们成功地结晶了肌球蛋白运动域，并以原子分辨率解析了其结构（2.7A）。通过利用这些结构信息，我们对肌球蛋白的功能重要区域，特别是开关 I 和开关 II 环进行了定点诱变。包含盘基网柄菌肌球蛋白II的残基454-459的环在结构和功能上等同于G蛋白家族的开关II环。肌球蛋白家族开关II环的共有序列为DIXGFE。为了确定各保守残基的功能，对盘基网柄菌肌球蛋白II重链基因进行丙氨酸扫描诱变。对突变体肌球蛋白的体内和体外运动功能的检查表明，I455A和S456A突变体保留了这些功能，而D454A、G457A、F458A和E459A突变体则失去了这些功能。后者肌球蛋白的生化分析表明，G457A 和 E459A 突变体分别通过阻断 ATP 酶循环的异构化和水解过程而丧失了基础 ATP 酶活性。然而，F458A 突变体丧失了肌动蛋白激活的 ATP 酶活性，但没有丧失基础 ATP 酶活性。这些结果根据盘基网柄菌肌球蛋白运动域的晶体结构进行了讨论。