Mechanism of protein motor based on molecular structure using protein-engineering and cryo-electron microscopy

我们的目标是从蛋白质的原子结构出发来理解马达蛋白的分子机制。我们的研究结果如下：(1)我们确定了肌球蛋白N-末端在肌动球蛋白僵硬复合体中的位置。(2)我们确定了肌动蛋白上的原肌球蛋白结合位点(S)，它影响肌动蛋白激活的ATPase在钙调节中的协同作用。(3)甲醇在关闭状态下捕获细丝。(4)应用电子断层扫描技术重建收缩肌交叉桥的三维结构。(5)首次用冷冻电子显微镜观察到溶液中的单个肌球蛋白分子。(6)我们在导通状态下获得了高分辨率的细丝结构。(7)我们在松弛状态下重建了含有粗丝的肌球蛋白的三维结构。(8)我们开发了一个新的环境来开发新的图像分析技术。(9)首次可视化了微管蛋白-动蛋白复合体的三维结构。(10)我们检测了钙的加入引起的细丝的结构变化。(11)我们用细丝的二维晶体观察到了肌钙蛋白。(12)用电子冷冻显微镜确定了天然细丝中星云蛋白的位置。(13)在僵直复合体中检测到肌球蛋白的波动部分。(14)用电子冷冻显微镜观察肌动蛋白波动模式的变化。

Our goal is to understand the molecular mechanism of motor proteins on the basis of atomic structure of proteins. We describe our result from this grant below.(1)We identified the position of myosin N-terminus in actomyosin rigor complexes. (2)We identified the tropomyosin binding site(s) on actin, which affects cooperativity of actin-activated ATPase in regulation of Ca. (3)Methanol traps thin filaments on an off-state. (4)The three-dimensional structure of cross-bridge in contracting muscle was reconstructed by electron tomography. (5)We visualized single myosin molecules in solution by cryo-electron microscopy for the first time. (6)We obtained high resolutional structure of thin filaments on an on-state. (7)We reconstructed three-dimensional structure of myosin containing thick filaments on a relaxd state.(8) We developed a new environment to develop new image analysis techniques. (9)For the first time, we visualized three-dimensional structure of tubulin-kinesin complexes. (10)We detected the structural change of thin filaments, which is induced by the addition of Ca.(11)We observed troponin by two-dimensional crystal of thin filaments. (12)We identified the position of nebulin in native thin filaments by electron cryo-microscopy. (13)We detected fluctuating part of myosin in rigor complexes. (14)We detected the change of actin fluctuation mode by electron cryo-microscopy.