EM Studies of Dimeric Molecular Motors

描述（由申请人提供）：在肌球蛋白超家族的马达蛋白中，肌球蛋白II是唯一的成丝类。肌球蛋白II是六聚体，含有2条相同的重链和2对轻链，即必需轻链（ELC）和调节轻链（RLC）以及延伸至LMM丝形成结构域的长卷曲螺旋二聚化结构域S2。被“调节”的肌球蛋白同种型依赖于RLC磷酸化，例如，节肢动物、平滑肌和非肌肉肌球蛋白，或Ca 2+结合，例如，软体动物的横纹肌。基于肌球蛋白的调节共同依赖于两个肌球蛋白头之间的分子内相互作用。这种相互作用是可视化的平滑肌肌球蛋白，最近在节肢动物的粗丝表明，头-头的相互作用和构象变化的一个紧凑的结构是肌球蛋白II调节的一般特征。生物化学和建模研究已经确定了一些构象变化的特征，其中包括依赖于S2长度的关于S2 α-螺旋的扭转运动。这些扭转运动可能会影响其他肌球蛋白的功能，包括双头相互作用与肌动蛋白丝和进行性运动。该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，这些特征对卷曲螺旋结构域的长度有很强的依赖性。肌球蛋白V是一种细胞质肌球蛋白，也形成一种紧凑的抑制构象，与肌球蛋白II的构象有一定的相似性，但肌球蛋白头部和货物结合结构域之间存在相互作用。肌球蛋白II和肌球蛋白V在抑制构象时结合肌动蛋白，但亲和力非常不同。虽然肌球蛋白V被广泛认为是一种进行性运动，需要同时肌动蛋白结合的两个头，同时结合平滑肌肌球蛋白的两个头是有争议的。肌球蛋白II和肌球蛋白V的抑制状态具有共同的基本原理。这个项目寻求答案，这些和其他方面的两个为首的运动功能和调节。最后，在传统的驱动蛋白抑制构象也涉及货物结合结构域和驱动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相似性，也将进行研究。

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.