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.

我试图观察沿细丝的肌肉跨桥或肌球蛋白头的结构，并通过快速冻结电子显微镜获得高的特殊和时间分辨率，因为任何结构性变化都伴随滑动对于阐明“滑动运动的分子机制”至关重要。该研究始于最简单的系统。纯化的肌球蛋白亚纹理1（S1）和F-肌动蛋白作为测试样品的混合物，以避免使用整个肌肉纤维产生的困难；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连接的头部的限制，而且还通过形成厚的丝。使用改进的云母技术，再加上快速冻结深蚀刻方法，我首次提出了证据，表明S1分子在严格的情况下或在ADP的情况下以倾斜的方式延长并连接到肌动蛋白上，而在ADP.VI或ATP ATP中则是短且圆形的。随后观察重毛霉素分子；每个都有两个头，但没有形成细丝。在存在各种核苷酸的情况下，明确表明，观察到的S1的圆形将来自肌球蛋白头的转动。尽管电子显微镜从未观察到肌球蛋白头部如此巨大的构象变化，但是最近生化研究的证据与该观察结果非常兼容。肌球蛋白头在超沉积过程中在肌动蛋白标本中看到；与肌肉收缩相对应的假定体外模型系统；表现出类似的形态特征，表明这种构象变化实际上是在积极收缩肌肉中发生的。推定的调节蛋白参与平滑肌收缩。