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

我试图观察沿细丝滑动时肌肉交叉桥或肌球蛋白头部的结构，通过快速冷冻电子显微镜获得高分辨率的特殊和时间分辨率，因为伴随滑动而来的任何结构变化都可能是阐明“滑动运动”的分子机制的关键。研究从最简单的系统开始；以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；每个头部的结构变化不可能是均匀的，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。利用改进的云母片技术和快速冷冻深刻蚀相结合的方法，我首次提出了证据，表明在严格的条件下或在ADP存在下，S1分子以倾斜的方式拉长并附着在肌动蛋白上，而在ADP.Vi或ATP的存在下，S1分子是短的和圆形的。随后观察到重的肌球蛋白分子；每个分子有两个头，但不形成细丝；在各种核苷酸存在的情况下，清楚地表明观察到的S1的圆整可能来自肌球蛋白头的弯曲。虽然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，但最近生物化学研究积累的证据与这一观察结果非常吻合。肌球蛋白在超沉淀过程中出现在肌球蛋白标本中，这是与肌肉收缩相对应的假定的体外模型系统；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。关于钙调蛋白的功能域组织和溶液中的构象也获得了一些新的发现，钙调蛋白是一种可能参与平滑肌收缩的调节蛋白。

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.