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。关于结构几乎一无所知 该电机利用 ATP 水解机制产生的详细信息 沿着微管运动。这种知识的匮乏与 关于驱动蛋白和肌球蛋白的信息，代表了我们的巨大差距 了解如何使用化学能来产生运动。这 应用程序提出了三个具体目标，重点关注结构 细胞质动力蛋白运动结构域内的测定。具体目标一将 利用冷冻电子显微镜方法产生第一个详细的 动力蛋白运动域的三维模型。实验描述 这将确定重要功能域的位置 运动并将开始解决动力蛋白的构象变化 由于核苷酸水解而发生。这项工作还将调查 动力蛋白如何适应微管并识别必要的结构 与运动有关的变化。具体目标二将确定原子 动力蛋白微管结合域的结构。重大诱变工作 还将针对将动力蛋白与 微管。这项工作将演示动力蛋白如何与微管相互作用 以及在核苷酸水解循环过程中如何调节其亲和力。 具体目标三旨在确定动力蛋白马达的原子结构 领域。我们的小组将为开发和分析做出重要贡献 主要结晶工作所需的运动域片段。 这里的成功将代表细胞领域的里程碑式成就 动力。这些目标中的每一个都将提供重要且新颖的信息 关于动力蛋白电机的工作原理。总而言之，这些目标代表了一个非常 对细胞运动的基本分子的强大攻击，以及我们的实验室 具有独特的优势来开展这项工作。这些项目的成功将 不仅开创了对动力蛋白如何发挥作用的理解，而且还允许 与肌球蛋白和驱动蛋白的详细比较。所有三个系列的电机 蛋白质对于真核生物的运动、发育和功能至关重要 有机体。他们活动中的缺陷是许多问题的根源 发育和神经系统疾病以及癌症。