Structure and mechanism of cytoplasmic and axonemal dyneins

 描述（由申请人提供）：马达蛋白在细胞内转运和运动中发挥关键作用，这是几种基本细胞过程（如有丝分裂）所必需的。动力蛋白是一种大的、复杂的、基于微管的、负端定向的马达蛋白，属于AAA（与多种细胞活性相关的ATP酶）酶家族。由于细胞质和轴丝动力蛋白在真核细胞中发挥的关键作用，其功能的缺陷与多种病理学包括神经退行性疾病和癌症有关。动力蛋白功能障碍如何导致疾病状态的细节仍然模糊不清，这在很大程度上是由于我们对动力蛋白作为运动蛋白发挥作用的分子机制的理解有限。结构生物学和显微镜技术的最新进展使其成为更详细地探索动力蛋白运动的结构和机制基础的令人兴奋和理想的时机。 作为一名结构生物学家，我目前的专业知识是X射线晶体学和NMR，这两个优秀的工具可以研究结构和动力学如何结合在一起，以促进生物大分子的功能。在我的博士后的初始阶段，我有一些电子显微镜的培训，我用它来研究酵母细胞质动力蛋白运动的变构机制。现在，我想扩展这项培训，成为快速发展的电子低温显微镜（cryo-EM）领域的专家。最近，cryo-EM结构已被报道具有极高的分辨率，使其成为研究大型蛋白质复合物如何工作的惊人工具，这是我的长期兴趣之一。此外，我想获得使用单分子光学显微镜研究溶液中蛋白质动力学的补充专业知识。这项培训将为我提供一个独特的工具包，使我能够从许多不同的角度研究生物系统的结构-动力学-功能关系。该提案的广泛目标是剖析细胞质和轴丝动力蛋白的结构，动力学和功能。因此，具体目标是：1）动力蛋白柄结构域的动力学和功能研究2）确定全长轴丝动力蛋白复合物的高分辨率结构3）直接合成轴丝动力蛋白以在单分子水平上研究突变体4）动力蛋白-微管复合物的高分辨率结构分析动力蛋白的功能关系，从而为疾病相关突变体的进一步分子研究以及动力蛋白在细胞功能和疾病中的作用奠定基础。

 DESCRIPTION (provided by applicant): Motor proteins play a critical role in intracellular transport and motility, which are required for several basic cellular processes such as mitosis. Dyneins are large, complicated, microtubule-based, minus-end directed motor proteins belonging to the AAA (ATPases Associated with diverse cellular Activities) family of enzymes. Due to the critical roles cytoplasmic and axonemal dyneins play in eukaryotic cells, defects in their function have been linked to a variety of pathologies including neurodegenerative diseases and cancer. The details of how dynein dysfunction leads to disease states remain obscure, in large part due to our limited understanding of the molecular mechanism by which dynein functions as a motor protein. Recent and ongoing advances in structural biology and microscopy techniques make it an exciting and ideal time to probe the structural and mechanistic basis of dynein motility in greater detail. My current expertise as a structural biologist is in the areas of X-ray crystallography and NMR, two excellent tools to study how structure an dynamics come together to facilitate function in biological macromolecules. During the initial phase of my postdoc, I have had some training in electron microscopy, which I have used to study the allosteric mechanism of yeast cytoplasmic dynein motility. I would now like to extend this training to become an expert in the rapidly advancing field of electron cryomicroscopy (cryo-EM). Recently, cryo-EM structures have been reported at extremely high resolutions, making it a phenomenal tool with which to study how large protein complexes work, which is one of my long-term interests. Additionally, I would like to gain complementary expertise in using single-molecule light microscopy to study protein dynamics in solution. This training will provide me with a unique tool kit that equips me to study structure-dynamics-function relationships of biological systems from many different perspectives. The broad goal of the proposal is to dissect the structure, dynamics and function of cytoplasmic and axonemal dyneins. Accordingly, the specific aims are to: 1) Probe the dynamics and functional role of dynein's stalk domain 2) Determine high-resolution structure of the full-length axonemal dynein complex 3) Recombinantly generate axonemal dynein to study mutants at the single molecule level 4) High-resolution structural analyses of dynein-microtubule complexes This work will provide fundamental insights into the structure-dynamics-function relationship in dynein, thus setting the stage for further molecular studies of disease-related mutants and the role of dynein in cellular function and disease.