MOLECULAR CHARACTERIZATION OF A CYTOPLASMIC DYNEIN

描述（申请人的描述） 动力蛋白是一种基本的分子， 真核细胞运动事实上，我们对结构一无所知。 该马达利用ATP水解机制来产生 沿着微管运动。这种知识的缺乏与 关于驱动蛋白和肌球蛋白的信息，代表了我们在 了解化学能如何用于产生运动。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域内的决定。具体目标一将 利用冷冻电子显微镜方法来产生第一个详细的 动力蛋白运动域的三维模型。实验中描述 这将确定重要功能域的位置， 运动和意志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 动力蛋白是如何与微管结合的， 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。一项重大的诱变研究 还将靶向结合动力蛋白的单个氨基酸的功能， 微管这项工作将展示动力蛋白如何与微管相互作用 以及在核苷酸水解循环期间其亲和力是如何调节的。 具体目标三：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 主要结晶化过程中所需的马达区片段 在这里的成功将代表细胞领域的一个里程碑式的成就 能动性这些目标中的每一个都将提供重要而新颖的信息 动力蛋白发动机是如何工作的总的来说，这些目标代表了一个非常 对细胞运动的基本分子的有力攻击，我们的实验室 是执行这项工作的唯一条件这些项目的成功将 不仅开创了对动力蛋白功能的理解， 与肌球蛋白和驱动蛋白的详细比较。三大汽车家族 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问题的根源， 发育和神经系统疾病以及癌症。

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.