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Structural Analysis of Dymamics of Celluar Motors

蜂窝电机动力学结构分析

基本信息

批准号：
09279103
负责人：
SUTOH Kazuo
金额：
$ 136.45万
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 on Priority Areas (A)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 2000
项目状态：
已结题

项目摘要

Based on crystal structures of the motor domain of Dictyostelium myosin II, we carried out of the structure-function analysis of this motor domain. We constructed many types of Dictyostelium myosin II mutants, which were trapped at a specific step of ATP hydrolysis cycle. Biochemical and biophysical studies on these mutants have shown how the three-dimensional structure of the motor domain rearranges at each step of ATP hydrolysis cycle, and how these changes are finally transmitted to the actin-binding site and the converter domain to generate sliding and force generation on an actin filament. Furthermore, we carried out the GFP-based FRET measurements of the fusion protein consisting of GFP, the motor domain and BFP. These FRET measurements have shown that the converter domain really swings at the isomerization step first and then swings back at the Pi release step. The latter step is likely to correspond to the stroke step. We have then constructed a prism-based total reflection fluorescence microscope that can visualize fluorescence spectra of many single molecules in a microscope filed at the same time at the video rate. By using this novel technique, we have detected that the converter of myosin motor domain is trapped in a particular orientation in the presence of various types of nucleotide analogs such as MgADP.Vi, MgADP.BeFx and MgADP.AlFx, consistent with the ensemble measurements mentioned above.

基于Dictyostelium myosin II运动结构域的晶体结构，我们对该运动结构域进行了结构-功能分析。我们构建了多种类型的盘肌桥菌肌球蛋白II突变体，这些突变体被捕获在ATP水解循环的特定步骤中。对这些突变体的生物化学和生物物理研究表明，在ATP水解周期的每一步，马达结构域的三维结构如何重新排列，以及这些变化最终如何传递到肌动蛋白结合位点和转换结构域，从而在肌动蛋白丝上产生滑动和力。此外，我们对由GFP、运动结构域和BFP组成的融合蛋白进行了基于GFP的FRET测量。这些FRET测量表明，转换域确实在异构化阶段首先摆动，然后在Pi释放阶段摆动回来。后一个步骤很可能对应于笔画步骤。然后，我们构建了一个基于棱镜的全反射荧光显微镜，可以在显微镜场中以视频速率同时显示许多单分子的荧光光谱。通过使用这种新技术，我们已经检测到肌凝蛋白运动结构域的转换器在不同类型的核苷酸类似物（如MgADP）存在下被困在一个特定的方向上。第六,MgADP。BeFx和MgADP。AlFx，与上面提到的集合测量一致。