ELECTRON CRYO-MICROSCOPY OF ACTO-S1 ATPASE INTERMEDIATES

肌肉收缩的分子机制被认为涉及到一些 肌球蛋白与肌动蛋白结合时头部的变化(无论是 头部自身的构象变化或 用肌动蛋白制成的结合角)。有相当多的人知道关于 肌球蛋白的一种附着状态的结构，即所谓的强结合 状态，与ADP结合或不结合核苷酸。然而，几乎没有什么是 已知其他所谓弱束缚态的结构(S) 因为在低蛋白质浓度下，acto-myosin复合体几乎 被三磷酸腺苷完全分解。中描述的工作的初始目标 这一建议是为了分析这些弱束缚的结构 冷冻水合电子显微镜观察肌动球蛋白的状态。一秒钟 这项工作的目的是研究定义的生化的结构 肌动球蛋白ATP水解机制的中间体使用快速- 冷冻淬火电子显微镜。占主导地位的生化 通过改变之前的时间间隔来选择中级 冷冻或实验条件(pH、核苷酸、温度)。在……里面 通过这种方式，我们计划确定肌动球蛋白的结构 随着时间和生化状态的变化而变化。低温电子显微镜是 特别适合于这种类型的研究，因为它允许极快的 固定性好，保存性好。 尽管进行了20年的密集调查，但对 伴随着力量生产的结构性变化仍然是最单一的 了解正常肌肉功能的重要问题。是这样的 知识也可能对理解和 改善骨骼肌和心肌的病理状态。

The molecular mechanism of muscle contraction is thought to involve some change in the myosin head while attached to actin (either a conformational change within the head itself or an alteration in the binding angle made with actin). A considerable amount is known about the structure of one attached state of myosin, the so-called strongly bound state, which occurs with bound ADP or no nucleotide. However, little is known about the structure(s) of other so-called weakly bound states because at low protein concentration the acto-myosin complex is almost completely dissociated by ATP. The initial aim of the work described in this proposal is to analyze the structure of these weakly bound actomyosin states using frozen hydrated electron microscopy. A second aim of this work is to study the structure of defined biochemical intermediates of the actomyosin ATP hydrolysis mechanisms using rapid- freeze quench electron microscopy. The predominant biochemical intermediate will be selected by varying either the time interval before freezing or experimental conditions (pH, nucleotide, temperature). In this manner we plan to determine how the structure of the actomyosin varies with time and biochemical state. Cryo-electron microscopy is especially well suited for this type of study as it allows extremely fast fixation and excellent preservation. Despite two decades of intensive investigations, knowledge of the structural changes accompanying force production remains the single most important problem in understanding normal muscle function. Such knowledge is also likely to be important for understanding and ameliorating pathological conditions in skeletal and cardiac muscle.