Molecular mechanism of a mammalian class I myosin motor

哺乳动物 I 类肌球蛋白运动的分子机制

• 批准号: 7116161
• 负责人: LYNNE M COLUCCIO
• 金额: $ 3.25万
• 依托单位: BOSTON BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7116161
• 关键词: actins; bioenergetics; cell migration; chemical kinetics; conformation; cryoelectron microscopy; enzyme activity; enzyme complex; enzyme mechanism; enzyme structure; intermolecular interaction; laboratory rat; microfilaments; microscopy; molecular site; mutant; myosins; nanotechnology; nucleotides; protein structure function; protein transport; tissue /cell culture

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.

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为设计合理的疗法来避免这些疾病提供至关重要的信息。 I 类肌球蛋白是具有运动结构域的小型单体机械酶，可结合肌动蛋白和核苷酸；轻链附着的调控区域；和羧基末端尾。 I 类肌球蛋白在哺乳动物细胞中广泛表达，预计可介导重要的肌动蛋白依赖性过程，例如细胞迁移和细胞内区室之间的货物运输。广泛的长期目标是了解哺乳动物肌球蛋白-I (MYR 1) 的酶促机制及其与细胞功能的关系。该实验室的主要观察结果证明了 MYR 1 独特的机械化学特性。MYR 1-肌动蛋白表现出与核苷酸的缓慢、双相瞬时相互作用，以及用单分子方法观察到的两步动力冲程。这些特性在其他肌球蛋白中是前所未见的，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子结构域对 MYR 1 独特机械化学性质的贡献。突变体 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 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。这些研究将允许可视化肌球蛋白动力冲程期间 MYR 1 的结构变化。 (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。突变体 MYR 1 与肌动蛋白丝与其他肌动蛋白结合蛋白复合物的结合能力将在体外通过肌动蛋白共沉降测定法以及在细胞中使用表达的 MYR 1 进行测定。这些研究将深入了解电机如何针对特定的目标细胞中的位点，是细胞生物学中的一个重要问题。