MOLECULAR CHARACTERIZATION OF A CYTOPLASMIC DYNEIN

细胞质动力蛋白的分子表征

• 批准号: 6285063
• 负责人: MICHAEL P KOONCE
• 金额: $ 24.77万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-01 至 2004-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6285063
• 关键词: Dictyostelium; affinity chromatography; binding proteins; cell motility; cryoelectron microscopy; cytoplasm; density gradient ultracentrifugation; dynein ATPase; gene expression; intracellular transport; laboratory rabbit; microtubules; protein binding; protein sequence; protein structure function

DESCRIPTION (applicant's description) Dynein is one of the fundamental molecules that drive cytoskeletal-based eukaryotic cell movement. Virtually nothing is known regarding the structural details by which this motor utilizes an ATP hydrolysis mechanism to generate movement along a microtubule. This lack of knowledge contrasts sharply with information on kinesin and myosin, and represents a tremendous gap in our understanding of how chemical energy is used to produce movement. This application presents three specific aims that focus on structural determinations within a cytoplasmic dynein motor domain. Specific Aim One will utilize a cryo-electron microscopy approach to produce the first detailed three-dimensional model of a dynein motor domain. Experiments are described that will identify the locations of important functional domains within the motor and will begin to resolve the conformational changes that dynein undergoes as a result of nucleotide hydrolysis. The work will also investigate how dynein fits onto a microtubule and will identify the essential structural changes involved with movement. Specific Aim Two will determine the atomic structure for the dynein microtubule-binding domain. A major mutagenesis effort will also target the function of individual amino acids that bind dynein to a microtubule. This work will demonstrate how dynein interacts with a microtubule and how its affinity is modulated during the nucleotide hydrolysis cycle. Specific Aim Three seeks to determine the atomic structure for the dynein motor domain. Our group will make essential contributions in developing and analyzing the necessary motor domain fragments for a major crystallization effort. Success here will represent a landmark achievement in the field of cell motility. Each one of these aims will provide important and novel information on how the dynein motor works. Taken together, the aims represent a very powerful attack on a fundamental molecule of cell movement, and our laboratory is uniquely positioned to carry out this work. Success of these projects will not only pioneer an understanding of how dynein functions, but will also allow detailed comparisons with myosin and kinesin. All three families of motor proteins are essential for movement, development, and function of eukaryotic organisms. Defects in their activity lie at the root of a number of developmental and neurological diseases, and cancer.

描述（申请人的描述） 动力蛋白是驱动基于细胞骨架的基本分子之一 真核细胞运动。几乎没有关于结构的知之甚少 该电动机利用ATP水解机制生成的细节 沿着微管运动。缺乏知识与 有关运动蛋白和肌球蛋白的信息，并且代表了我们的巨大差距 了解化学能如何用于产生运动。这 应用提出了三个专注于结构的特定目标 细胞质动力蛋白运动结构域内的测定。具体目标 利用冷冻电子显微镜方法生成第一个详细的 动力蛋白运动结构域的三维模型。描述了实验 这将确定重要功能域的位置 电机并将开始解决动力蛋白的构象变化 由于核苷酸水解而进行。工作还将调查 Dynein如何拟合到微管上并确定必需的结构 运动涉及的变化。具体目标两个将确定原子 动力蛋白微管结合结构域的结构。主要的诱变工作 还将靶向将动力蛋白与A结合的单个氨基酸的功能 微管。这项工作将证明Dynein如何与微管相互作用 以及如何在核苷酸水解循环中调节其亲和力。 特定目的三寻求确定动力蛋白电动机的原子结构 领域。我们的小组将在开发和分析方面做出重要贡献 必要的运动域碎片，以进行主要的结晶工作。 这里的成功将代表牢房领域的里程碑成就 运动。这些目标中的每一个都将提供重要而新颖的信息 关于动力蛋白电机的工作原理。综上所述，目的代表了一个非常 对细胞运动的基本分子和我们的实验室的强大攻击 在执行这项工作方面是独特的位置。这些项目的成功将 不仅是对动力蛋白如何发挥的理解，而且还将允许 与肌球蛋白和运动蛋白的详细比较。这三个运动家族 蛋白质对于真核的运动，开发和功能至关重要 有机体。其活动中的缺陷在于许多 发育和神经疾病以及癌症。