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

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的新技术。利用我们开发的一种新技术，即位点定向重原子标记电子显微镜（SHALEM），我们仅用电子显微镜就确定了肌球蛋白5^< >氨基酸的位置。同时，我们重建了肌肉在等距收缩过程中的三维结构。其次，利用电子显微镜、x射线小角散射和x射线衍射检测ATP水解过程中肌球蛋白的结构变化。这是对ATP水解过程中肌球蛋白结构变化的首次观察。第三，用低温电镜观察了钙离子加入后细丝的结构变化。最后，我们利用蛋白工程技术在肌动蛋白亚结构域-1上找到了肌凝蛋白-肌动蛋白相互作用位点。我们还发现了肌凝蛋白的运动结构域。这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，我们利用蛋白质工程产生突变的肌动蛋白，以高度合作的方式激活肌球蛋白atp酶。我们发现，在Ca^<2+>-诱导的atp酶活化中，仅替换3个氨基酸残基就能引起高协同性。当Ca存在时，该区域也是原肌凝蛋白的结合位点之一。

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.