Structure and mechanism of cytoplasmic and axonemal dyneins

细胞质和轴丝动力蛋白的结构和机制

• 批准号: 9521385
• 负责人: Gira Bhabha
• 金额: $ 24.9万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9521385
• 关键词: ATP phosphohydrolase; Active Biological Transport; Active Sites; Address; Affect; Affinity; Architecture; Area; Baculovirus Expression System; Binding; Biological; Cell Cycle; Cell physiology; Cilia; Complex; Cryoelectron Microscopy; Data; Defect; Development; Disease; Dynein ATPase; Electron Microscopy; Electrons; Engineering; Enzymes; Eukaryotic Cell; Family; Flagella; Functional disorder; Goals; Heart Abnormalities; Incubated; Intracellular Transport; Lead; Length; Link; Malignant Neoplasms; Measures; Mediating; Mentors; Microscopy; Microtubules; Mitosis; Modeling; Molecular; Molecular Conformation; Motion; Motor; Movement; Mutation; Neurodegenerative Disorders; Nucleotides; Organelles; Pathology; Pharmaceutical Preparations; Phase; Play; Postdoctoral Fellow; Process; Protein Dynamics; Protein Isoforms; Proteins; RNA; Recombinants; Reporting; Resolution; Role; Series; Slide; Source; Structural Biologist; Structure; System; Tail; Techniques; Tetrahymena; Time; Training; Vanadates; Work; X-Ray Crystallography; Yeasts; analog; arm; base; biological systems; brain abnormalities; cell motility; ciliopathy; design; detector; experimental study; insight; interest; light microscopy; macromolecule; monomer; mutant; novel; protein complex; public health relevance; single molecule; small molecule; structural biology; tool

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.

描述（由申请人提供）：运动蛋白在细胞内运输和运动中发挥着关键作用，这是多种基本细胞过程（例如有丝分裂）所必需的。动力蛋白是大型、复杂、基于微管、负端定向的运动蛋白，属于 AAA（与多种细胞活动相关的 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关键作用，其功能缺陷与多种病理学相关，包括神经退行性疾病和癌症。动力蛋白功能障碍如何导致疾病状态的细节仍然不清楚，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了解有限。结构生物学和显微镜技术的最新进展使得现在成为更详细地探讨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 射线晶体学和核磁共振领域，这是研究结构动力学如何结合在一起以促进生物大分子功能的两种出色工具。在博士后的初始阶段，我接受了一些电子显微镜方面的培训，我用它来研究酵母细胞质动力蛋白运动的变构机制。我现在想扩展这次培训，成为快速发展的电子冷冻显微镜 (cryo-EM) 领域的专家。最近，冷冻电镜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物如何工作的非凡工具，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丝动力蛋白的结构、动力学和功能。因此，具体目标是： 1) 探究动力蛋白茎结构域的动力学和功能作用 2) 确定全长轴丝动力蛋白复合物的高分辨率结构 3) 重组生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这项工作将为结构-动力学-功能关系提供基本见解动力蛋白，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了基础。