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关联，强调了研究这些肌动蛋白相关的分子马达的重要性。了解这些酶的分子机制最终可以提供对设计合理的治疗方法以避免这些疾病至关重要的信息。I类肌球蛋白是一种小的、单体的机械酶，具有一个运动域，它结合肌动蛋白和核苷酸；一个轻链附着的调节区；以及一个羧基末端尾巴。I类肌球蛋白在哺乳动物细胞中广泛表达，并被预测为介导重要的肌动蛋白依赖的过程，如细胞迁移和细胞内间隔之间的货物运输。我们的长期目标是了解哺乳动物肌球蛋白-I，MYR-1的酶机制，以及它与细胞功能的关系。本实验室所做的关键观察证明了MYR 1独特的机械力化学性质。MYR 1-肌动蛋白与核苷酸呈现缓慢的两相瞬时相互作用，并通过单分子方法观察到两步PowerStroke。这些特性以前在其他肌球蛋白中从未见过，推测反映了MYR 1对特定细胞功能的特殊适应。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。具体目的是：(I)利用突变蛋白研究两个特定的亚区对MYR 1‘S独特的机械力化学性质的贡献。突变的MYR 1与肌动蛋白和核苷酸的相互作用能力将通过稳态和暂态动力学分析、体外动力分析和单分子方法来确定。(Ii)利用来自MYR 1-肌动蛋白冷冻电子显微镜的三维重建来确定MYR 1是否经历了核苷酸依赖的构象变化。这些研究将使MYR 1在肌球蛋白强卒中的结构变化可视化。(Iii)确定MYR 1是否与肌动蛋白细丝的特定亚群相关，以及运动域中的特定结构元件是否调节其与微丝的结合。突变的MYR 1与其他肌动蛋白结合蛋白的复合体与肌动蛋白细丝结合的能力将通过肌动蛋白共沉淀法在体外和在细胞中使用表达的MYR 1来确定。这些研究将为深入了解马达如何靶向细胞中的特定位置提供洞察力，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.