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ANALYSIS OF THE CONFORMATIONAL CHANGE ASSOCIATED WITH THE SLIDING MOVEMENT OF MYOSIN ALONG ACTIN FILAMENT

肌球蛋白沿肌动蛋白丝滑动的构象变化分析

基本信息

批准号：
63480508
负责人：
KATAYAMA Eisaku
金额：
$ 3.52万
依托单位：
FACULTY OF MEDICINE, THE UNIVERSITY OF TOKYO
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

I attempted to observe the structure of muscle cross-bridges or myosin heads while sliding along thin filaments, with high special and temporal resolution obtained by quick-freezing electron microscopy, since any structural change accompanied with the sliding could be crucial to elucidate the molecular mechanism of "sliding movement". The study was started with the simplest system; a mixture of purified myosin subfragment-1 (S1) and F-actin as test specimen, to avoid the difficulty which arises from using whole muscle fiber; i.e.) the structural change in each head cannot be homogenous because of the restriction not only by two connected heads in a single molecule, but also by forming thick filaments. Using improved mica-flake technique coupled with quick-freeze deep-etch method, I presented the evidences, for the first time, indicating that S1 molecules appear elongated and attached to actin in a tilted manner under rigor condition or in the presence of ADP, whereas they are short and rounded if ADP.Vi or ATP is incorporated. Subsequent observation of heavy meromyosin molecules; each having two heads but does not form filament; in the presence of various nucleotides made it clear that the observed rounding of S1 would have come from the flection in myosin head. Though the presence of such large conformational change in myosin head has never been observed by electron microscopy, accumulating evidences from recent biochemical studies are quite compatible with this observation. Myosin heads seen in actomysin specimen during superprecipitation; the putative in vitro model system corresponding to muscle contraction; showed a similar morphological feature, suggesting the possibility that such conformational change actually occurring in actively contracting muscle.Several new findings were also obtained concerning the functional domain organization and the conformation in solution, of caldesmon; the putative regulatory protein involved in smooth muscle contraction.

我尝试通过速冻电子显微镜获得高特殊分辨率和时间分辨率，观察肌肉横桥或肌球蛋白头沿着细丝滑动时的结构，因为伴随滑动的任何结构变化对于阐明“滑动运动”的分子机制至关重要。这项研究是从最简单的系统开始的；纯化的肌球蛋白亚片段-1（S1）和F-肌动蛋白的混合物作为测试样本，以避免使用全肌纤维带来的困难；即）每个头部的结构变化不能是同质的，因为不仅受到单个分子中两个相连头部的限制，而且还受到形成粗丝的限制。使用改进的云母片技术与快速冷冻深蚀刻方法相结合，我首次提出了证据，表明在严格条件下或存在 ADP 的情况下，S1 分子显得拉长并以倾斜的方式附着在肌动蛋白上，而如果掺入 ADP.Vi 或 ATP，则它们会短而圆形。随后观察重的meromyosin分子；每个都有两个头但不形成细丝；在存在各种核苷酸的情况下，清楚地表明观察到的 S1 的圆整可能来自肌球蛋白头部的弯曲。尽管电子显微镜从未观察到肌球蛋白头部存在如此大的构象变化，但最近生化研究中积累的证据与这一观察结果非常吻合。超沉淀过程中在肌动蛋白标本中看到肌球蛋白头；与肌肉收缩相对应的假定体外模型系统；显示出类似的形态特征，表明这种构象变化实际上发生在主动收缩的肌肉中的可能性。推测的参与平滑肌收缩的调节蛋白。