Structure and mechanism of cytoplasmic and axonemal dyneins

 描述(由申请人提供)：马达蛋白在细胞内运输和运动中起关键作用，这是几个基本的细胞过程所必需的，如有丝分裂。动力蛋白是一种大的、复杂的、以微管为基础、负端定向的运动蛋白，属于AAA(与多种细胞活动相关的ATPase)家族。由于细胞质和轴丝动力蛋白在真核细胞中的重要作用，它们的功能缺陷被认为与包括神经退行性疾病和癌症在内的多种病理机制有关。动力蛋白功能障碍如何导致疾病状态的细节仍不清楚，这在很大程度上是因为我们对动力蛋白作为运动蛋白的分子机制了解有限。最近和正在进行的结构生物学和显微技术的进步使得现在是一个令人兴奋的和理想的时间来更详细地探索动力蛋白运动的结构和机械基础。作为一名结构生物学家，我目前的专业领域是X射线结晶学和核磁共振，这两个优秀的工具可以研究结构动力学如何结合在一起，以促进生物大分子的功能。在我博士后的最初阶段，我接受了一些电子显微镜方面的培训，我曾用它来研究酵母细胞质动力蛋白运动的变构机制。现在，我想延长这一培训，成为快速发展的电子冷冻显微镜(Cryo-EM)领域的专家。最近，低温EM结构以极高的分辨率被报道，使其成为研究大型蛋白质复合体如何工作的一个现象工具，这是我的长期兴趣之一。此外，我想在使用单分子光学显微镜研究溶液中的蛋白质动力学方面获得补充专业知识。这次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。该提案的主要目标是剖析细胞质和轴线动力蛋白的结构、动力学和功能。因此，本研究的具体目的是：1)探讨dynein的茎结构域的动力学和功能作用；2)确定全长dyonemal dynein复合体的高分辨结构；3)重组产生dynein在单分子水平上研究突变体；4)dynein-微管复合体的高分辨结构分析本工作将为深入研究dynein的结构-动力学-功能关系提供基础资料，从而为进一步研究疾病相关突变体以及dynein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.