Systematic Structural Biology of Dynein

动力蛋白的系统结构生物学

• 批准号: 6605782
• 负责人: Stephen M King
• 金额: $ 24.65万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6605782
• 关键词: dynein ATPase; nuclear magnetic resonance spectroscopy; protein structure function; structural biology

Dyneins are microtubule-based molecular motors involved in a wide variety of essential cellular functions including retrograde vesicular trafficking, ciliary/flagellar motility and cell division. These enzymes are highly complex containing up to thirteen different polypeptide components with a total mass of almost 2 MDa. In order to achieve a molecular understanding of mechanism, it will be essential to obtain high resolution structural information for all components of the dynein complex. However, the complexity and size of the dynein particle combined with the inherent flexibility of several domains has made obtaining high resolution structural information for the entire enzyme unfeasible. This application proposes a systematic approach to dynein structural biology by obtaining information for individual proteins or subdomains using multidimensional NMR techniques. These structures will then be used to build ever more complete models of the dynein motor and will enable structure-based hypotheses concerning function to be made. The specific aims for this submission center on analysis of dynein light chains and include: 1) molecular dynamics and refinement of the solution structure of LC1 which is associated with the dynein motor domain; 2) structural analysis of mutant forms of LC1 to allow interpretation of mutant phenotypes at the structural level; 3) completion of the NMR assignments and calculation of the solution structure for the Tctex1 light chain; 4) NMR structure analysis of thioredoxin-related and Ca2+-binding light chains; 5) preparation of other dynein domains for structure analysis following a systematic search for appropriate solution conditions. These studies will provide structure-based insight into the mechanisms of dynein regulation and will provide the necessary background for further structural analysis of this massive molecular motor.

动力蛋白是基于微管的分子电机，参与了各种必需的细胞功能，包括逆行囊泡运输，睫状/鞭毛运动和细胞分裂。这些酶是高度复杂的，含有多达13种不同的多肽成分，总质量将近2 MDa。为了获得对机制的分子理解，必须为动力蛋白复合物的所有组成部分获得高分辨率结构信息。但是，动力蛋白颗粒的复杂性和大小结合了多个结构域的固有柔韧性，使得为整个酶获得高分辨率的结构信息。该应用程序通过使用多维NMR技术获取单个蛋白质或子域的信息，提出了一种系统的动力蛋白结构生物学方法。然后，这些结构将用于构建动力蛋白电动机的更完整的模型，并将实现有关功能的基于结构的假设。该提交中心的特定目的是分析动力蛋白光链的中心，包括：1）与动力蛋白运动结构域相关的LC1溶液结构的分子动力学和完善； 2）LC1突变形式的结构分析，以允许在结构水平上解释突变表型； 3）NMR分配的完成和TCTEX1轻型链的解决方案结构的计算； 4）NMR结构分析与硫氧还蛋白相关的和Ca2+结合的光链； 5）在系统搜索适当的解决方案条件后，准备其他动力蛋白结构域进行结构分析。这些研究将提供基于结构的洞察力，了解动力蛋白调节的机制，并为进一步的结构分析提供必要的背景。