EM Studies of Dimeric Molecular Motors

描述（由申请人提供）：在肌凝蛋白超家族的运动蛋白中，肌凝蛋白II是唯一的成丝类。肌球蛋白ii是六聚体，包含2条相同的重链和2对轻链，基本轻链（ELC）和调节轻链（RLC），以及延伸到LMM细丝形成结构域的长螺旋状二聚化结构域S2。肌球蛋白亚型的“调节”依赖于RLC磷酸化，如节肢动物、平滑肌和非肌肉肌球蛋白，或Ca2+结合，如软体动物横纹肌。基于肌凝蛋白的调控共同依赖于两个肌凝蛋白头之间的分子内相互作用。这种相互作用在平滑肌肌球蛋白和节肢动物粗细丝中被观察到，这表明头-头相互作用和向紧凑结构的构象变化是肌球蛋白II调节的一般特征。生物化学和模型研究已经确定了一些构象变化的特征，其中包括依赖于S2长度的S2 α螺旋的扭转运动。这些扭转运动可能影响肌凝蛋白的其他功能，包括与肌动蛋白丝的双头相互作用和进程运动。这项拨款申请旨在研究平滑肌肌球蛋白调节的结构和功能特征，这些特征强烈依赖于线圈结构域的长度。肌凝蛋白V，一种细胞质肌凝蛋白也形成紧凑的抑制构象，与肌凝蛋白II有一些相似之处，但在肌凝蛋白头部和货物结合域之间存在相互作用。肌凝蛋白II和肌凝蛋白V在受抑制的构象中与肌动蛋白结合，但具有非常不同的亲和力。虽然肌凝蛋白V被广泛认为是一种进行性马达，它需要两个头同时结合肌动蛋白，但平滑肌肌凝蛋白同时结合两个头是有争议的。肌凝蛋白II和肌凝蛋白V的抑制状态具有共同的基本原理。这个项目寻求答案的这些和其他方面的双头运动功能和调节。最后，在常规酪蛋白中被抑制的构象也涉及到货物结合域和酪蛋白头之间的相互作用。这种构象可能与肌球蛋白的抑制构象具有结构和功能上的相似性，也将被研究。

DESCRIPTION (provided by applicant): Among the myosin super-family of motor proteins, myosin II is the sole filament forming class. Myosin IIs are hexamers containing 2 identical heavy chains and 2 pairs of light chains, the essential light chain (ELC) and the regulatory light chain (RLC) and a long coiled-coil dimerization domain, S2, that extends into the LMM filament forming domain. Myosin isoforms that are "regulated" depend either on RLC phosphorylation, e.g., arthropod, smooth muscle and nonmuscle myosins, or Ca2+ binding, e.g., molluscan striated muscles. Myosin based regulation has in common a dependency on intramolecular interactions between the two myosin heads. This interaction was visualized in smooth muscle myosin and recently in arthropod thick filaments suggesting that a head-head interaction and a conformational change to a compact structure is a general feature of myosin II regulation. Biochemistry and modeling studies have identified a number of the features of the conformational change among which are torsional motions about the S2 alpha-helices that are dependent on the length of S2. These torsional motions could affect other myosin functions, including double headed interactions with the actin filament and processive motion. This grant application proposes to investigate structural and functional features of regulation in smooth muscle myosin that have a strong dependency on the length of the coiled-coil domain. Myosin V, a cytoplasmic myosin also forms a compact inhibited conformation with some similarity to that of myosin II, but with an interaction between the myosin heads and the cargo binding domain. Both myosin II and myosin V bind actin when in the inhibited conformation, but with very different affinities. While myosin V is widely identified as a processive motor, which requires simultaneous actin binding by both heads, simultaneous binding of smooth muscle myosin by both heads is controversial. The inhibited states of both myosin II and myosin V have common underlying principles. This project seeks answers to these and other aspects of two headed motor function and regulation. Finally, inhibited conformations in conventional kinesin also involve an interaction between the cargo binding domains and the kinesin heads. This conformation may have structural and functional similarities with inhibited conformations of myosin and will also be investigated.