Molecular mechanism of a mammalian class I myosin motor

描述（由申请人提供）：某些形式的心肌病、耳聋和失明与肌球蛋白缺陷的相关性，强调了研究这些肌动蛋白相关分子马达的重要性。对这些酶的分子机制的理解最终可以为设计合理的治疗方法以避免这些疾病提供至关重要的信息。I类肌球蛋白是小的单体机械酶，具有结合肌动蛋白和核苷酸的马达结构域;轻链连接的调节区;和羧基末端尾。I类肌球蛋白在哺乳动物细胞中广泛表达，并被预测介导重要的肌动蛋白依赖性过程，如细胞迁移和细胞内区室之间的货物运输。广泛的，长期的目标是了解哺乳动物肌球蛋白-I，MYR 1的酶机制，以及它如何与细胞功能相关。在该实验室进行的关键观察证明了MYR 1的独特机械化学性质。MYR 1-actin表现出缓慢的，双相的瞬时相互作用与核苷酸和两步powerstroke观察与单分子方法。这些特性在其他肌球蛋白中未见，可能反映了MYR 1对特定细胞功能的专门适应。拟议的实验重点是详细说明MYR 1如何与肌动蛋白和核苷酸相互作用。具体目标是：（i）使用突变蛋白研究两个特定亚结构域对MYR 1独特机械化学性质的贡献。突变MYR 1与肌动蛋白和核苷酸相互作用的能力将通过稳态和瞬态动力学分析、体外运动测定和单分子方法来确定。(ii)使用来自MYR 1-肌动蛋白的冷冻电子显微照片的3D重建来确定MYR 1是否经历核苷酸依赖性构象变化。这些研究将使MYR 1在肌球蛋白动力冲程期间的结构变化可视化。(iii)确定MYR 1是否与肌动蛋白丝的特定亚群相关，以及运动域中的特定结构元件是否调节其与微丝的结合。将在体外用肌动蛋白共沉降测定法和在使用表达的MYR 1的细胞中测定突变MYR 1与肌动蛋白丝结合的能力，所述肌动蛋白丝与其它肌动蛋白结合蛋白复合。这些研究将深入了解马达是如何靶向细胞中的特定位点的，这是细胞生物学中的一个重要问题。

DESCRIPTION (provided by applicant): The association of some forms of cardiomyopathy, deafness, and blindness with defects in myosins, underscores the importance of studying these actin-associated, molecular motors. An understanding of the molecular mechanism of these enzymes can ultimately provide information crucial to the design of rational therapies to avert these diseases. Class I myosins are small, monomeric, mechanoenzymes with a motor domain, which binds actin and nucleotide; a regulatory region to which light chains attach; and a carboxy terminal tail. Class I myosins are widely expressed in mammalian cells and are predicted to mediate important actin-dependent processes such as cell migration and transport of cargo among intracellular compartments. The broad, long-term objective is to understand the enzymatic mechanism of the mammalian myosin-I, MYR 1, and how it relates to cell function. Key observations made in this laboratory have demonstrated the unique mechanochemical properties of MYR 1. MYR 1-actin exhibits a slow, biphasic transient interaction with nucleotide and a two-step powerstroke observed with single-molecule methods. These properties, not seen before for other myosins, presumably reflect the specialized adaptation of MYR 1 for particular cellular functions. The proposed experiments focus on detailing how MYR 1 interacts with actin and nucleotide. The specific aims are: (i) To investigate the contribution of two specific subdomains to MYR 1's unique mechanochemical properties using mutant proteins. The ability of mutant MYR 1 to interact with actin and nucleotide will be determined with steady and transient state kinetic analyses, in vitro motility assays and single molecule methods. (ii) To determine if MYR 1 undergoes nucleotide-dependent conformational changes using 3D reconstructions from cryo-electron micrographs of MYR 1-actin. These studies will allow for visualization of structural changes in MYR 1 during the myosin powerstroke. (iii) To determine if MYR 1 associates with specific subpopulations of actin filaments and whether specific structural elements in the motor domain modulate its binding to microfilaments. The ability of mutant MYR 1 to associate with actin filaments in complex with other actin-binding proteins will be determined in vitro with actin co-sedimentation assays and in cells using expressed MYR 1. These studies will provide insight into how motors are targeted to particular sites in the cell, an important question in cell biology.