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Studies on muscle contraction Mechanism based on molecular structure of mutant motor proteins

基于突变运动蛋白分子结构的肌肉收缩机制研究

基本信息

批准号：
02102009
负责人：
WAKABAYASHI Takeyuki
金额：
$ 129.22万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Specially Promoted Research
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1993
项目状态：
已结题

项目摘要

Our goal is to understand the molecular mechanism of motor proteins on the basis of atomic structure of proteins.First we developed new techniques for electron microscopy. Using one of new techniques we developed, that is, the site-directed heavy atom labelling electron microscopy (SHALEM), we decided the position of the 5^<th> amino acid of myosin only by electron microscopy. Also, we reconstructed three-dimensional structure of muscle during isometric contraction.Second, we detected the structural change of myosin during ATP hyrdrolysis by electron microscopy, small angle X-ray scattering, and crystallography using X-ray diffraction. This is the first observation of detection of myosin structural change during ATP hydrolysis.Third, we observed the structural change of thin filaments in the addition of Ca ions by cryo-electron microscopy.Last, we found the myosin-actin interaction sites on the subdomain-1 of actin using protein engineering techniques. Also we found the motor domain of myosin. This part is essential for sliding movement of actin and production of force. Furthermore, we use protein engineering to produce the mutant actins, which activate myosin ATPase in a highly cooperative manner. We found that the replacement of only 3 amino acid residues cause the high cooperativity in Ca^<2+>-induced activation of ATPase. This region is also one of the binding sites of tropomyosin in the presence of Ca.

我们的目标是在蛋白质原子结构的基础上了解马达蛋白的分子机制。利用我们开发的一种新技术，即重原子定点标记电子显微镜技术（SHALEM），我们<th>仅用电子显微镜就确定了肌球蛋白5^氨基酸的位置。其次，我们利用电子显微镜、小角X射线散射和X射线衍射晶体学技术研究了ATP水解过程中肌球蛋白的结构变化。这是我们首次观察到ATP水解过程中肌球蛋白结构的变化;第三，我们用冷冻电镜观察了Ca ~（2+）对肌球蛋白细丝结构的影响;最后，我们用蛋白质工程技术在肌动蛋白的亚结构域-1上发现了肌球蛋白与肌动蛋白的相互作用位点。我们还发现了肌球蛋白的运动域。这一部分对于肌动蛋白的滑动运动和力的产生是必不可少的。此外，我们使用蛋白质工程产生的突变肌动蛋白，激活肌球蛋白ATP酶在一个高度合作的方式。我们发现，仅3个氨基酸残基的替换就导致了Ca^<2+>诱导的ATP酶激活的高协同性。该区域也是原肌球蛋白在Ca存在下的结合位点之一。