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: 8804578
• 负责人: Gira Bhabha
• 金额: $ 9万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 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 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8804578
• 关键词: ATP phosphohydrolase; Active Sites; Address; Affect; Affinity; Architecture; Area; Baculovirus Expression System; Binding; Biological; Brain; Cell Cycle; Cell physiology; Cilia; Complex; Congenital Heart Defects; Cryoelectron Microscopy; Data; Defect; Development; Disease; Dynein ATPase; Electron Microscopy; Electrons; Engineering; Enzymes; Eukaryotic Cell; Family; Flagella; Functional disorder; Goals; Head; Incubated; Intracellular Transport; Lead; Length; Life; 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; Relative (related person); Reporting; Resolution; Role; Series; Slide; Solutions; Source; Staging; Structural Biologist; Structure; System; Tail; Techniques; Tetrahymena; Time; Training; Vanadates; Work; X-Ray Crystallography; Yeasts; analog; arm; base; biological systems; cell motility; ciliopathy; design; detector; insight; interest; light microscopy; macromolecule; monomer; mutant; novel; protein complex; public health relevance; research study; 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（由适用提供）：运动蛋白在细胞内运输和运动性中起关键作用，这对于几种基本的细胞过程（例如有丝分裂）所必需。动力蛋白是大型，复杂的，基于微管的，负端的定向运动蛋白，属于AAA（与多种细胞活性相关的ATPases）酶家族。由于细胞质和轴突动力蛋白在真核细胞中起着关键作用，其功能缺陷已与包括神经退行性疾病和癌症在内的多种病理相关。动力蛋白功能障碍如何导致疾病状态的细节仍然晦涩难懂，这在很大程度上是由于我们对动力蛋白作为运动蛋白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和持续的进步使其成为更详细地探测动力蛋白运动的结构和机械基础的令人兴奋和理想的时机。我目前作为结构生物学家的专业知识是X射线晶体学和NMR的领域，这是两个出色的工具，用于研究结构动态如何融合在一起以促进生物大分子的功能。在博士后的初始阶段，我已经接受了电子显微镜训练，我用来研究酵母细胞质动力蛋白运动的变构机制。我现在想扩展这种训练，成为电子冷冻显微镜（Cryo-EM）快速前进领域的专家。最近，已经报道了在极高的分辨率下报道了冷冻EM结构，这使其成为研究大蛋白复合物的工作方式的惊人工具，这是我的长期利益之一。此外，我想获得使用单分子光显微镜研究溶液中的蛋白质动力学的完整专业知识。这项培训将为我提供一个独特的工具套件，使我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突动力蛋白的结构，动力学和功能。彼此之间，具体目的是：1）探测动力蛋白的茎域的动态和功能作用2）确定全长轴突动力蛋白复合物的高分辨率结构3）重新组合产生轴突动力蛋白，从而在单分子级别研究突变体，在单分子级别4）高分辨率的结构分析构成构成型的构成型构成型构成型构成型构成蛋白 - 构成型构成型构成型构成型构成型，结构动力学 - 动力学关系在动力蛋白中，因此为疾病相关突变体的进一步分子研究和动力蛋白在细胞功能和疾病中的作用奠定了基础。