MOLECULAR CHARACTERIZATION OF A CYTOPLASMIC DYNEIN

描述(申请人的描述) 动力蛋白是驱动细胞骨架的基本分子之一 真核细胞运动。关于结构的几乎一无所知 这个马达利用三磷酸腺苷水解机制来产生 沿着微管运动。这种对知识的缺乏与 有关肌动蛋白和肌球蛋白的信息，这代表着我们在 了解化学能如何被用来产生运动。这 应用程序提出了三个具体目标，重点是结构 细胞质动力蛋白运动区的测定。一个人会有明确的目标 利用低温电子显微镜方法制作出第一个详细的 动力系统运动域的三维模型。对实验进行了描述 它将标识重要功能域在 并将开始解决动力作用下的构象变化 作为核苷酸水解的结果而发生。这项工作还将调查 动力蛋白如何装配到微管上，并识别必要的结构 与运动有关的变化。具体目标二将决定原子 动力蛋白微管结合域的结构。重大的诱变努力 也将针对单个氨基酸的功能，这些氨基酸将动力蛋白结合到一个 微管。这项工作将演示动力蛋白如何与微管相互作用。 以及它的亲和力是如何在核苷酸水解循环中被调节的。 具体目标三寻求确定动力蛋白马达的原子结构 域。我们的团队将在开发和分析方面做出重要贡献 主要结晶工作所需的马达结构域片段。 这里的成功将是细胞领域的一项里程碑式的成就 能动性。这些目标中的每一个都将提供重要的和新的信息 动力蛋白马达是如何工作的。总而言之，这些目标代表着一个非常 对细胞运动的基本分子的强大攻击，我们的实验室 是开展这项工作的得天独厚的人选。这些项目的成功将 不仅是了解动力蛋白如何工作的先驱，而且还将使 与肌球蛋白和肌动蛋白进行了详细的比较。所有三个系列的电机 蛋白质是真核生物运动、发育和功能所必需的。 有机体。他们活动中的缺陷是许多 发育和神经疾病，以及癌症。

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.