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运动蛋白的分子机制。首先，我们开发了电子显微镜新技术。使用我们开发的一项新技术，即定点重原子标记电子显微镜(SHALEM)，我们仅通过电子显微镜就确定了肌球蛋白的第5个氨基酸的位置。此外，我们还重建了等长收缩过程中肌肉的三维结构。其次，我们通过电子显微镜、小角X射线散射和X射线衍射晶体学检测了肌球蛋白在ATP水解过程中的结构变化。这是首次观察到ATP水解过程中肌球蛋白结构变化的检测。第三，我们通过冷冻电子显微镜观察了细丝在添加Ca离子时的结构变化。最后，我们在子结构域上发现了肌球蛋白-肌动蛋白相互作用位点-1的肌动蛋白使用蛋白质工程技术。我们还发现了肌球蛋白的运动域。该部分对于肌动蛋白的滑动运动和力的产生至关重要。此外，我们使用蛋白质工程来生产突变肌动蛋白，它以高度合作的方式激活肌球蛋白 ATP 酶。我们发现仅3个氨基酸残基的替换导致Ca ^ 2+ 诱导的ATP酶激活的高度协同性。该区域也是 Ca 存在下原肌球蛋白的结合位点之一。