Systematic Structural Biology of Dynein

动力蛋白是一种以微管为基础的分子马达，参与多种重要的细胞功能，包括逆行运输囊泡、纤毛/鞭毛运动和细胞分裂。这些酶高度复杂，含有多达13种不同的多肽成分，总质量几乎为2丙二醛。为了实现对机制的分子理解，获得动力蛋白复合体所有组分的高分辨率结构信息是必不可少的。然而，动力蛋白颗粒的复杂性和大小，加上几个结构域的固有灵活性，使得获得整个酶的高分辨率结构信息是不可能的。这项申请提出了一种系统的方法，通过使用多维核磁共振技术获得单个蛋白质或亚域的信息来研究动力蛋白结构生物学。然后，这些结构将被用来建立动力蛋白马达的更完整的模型，并将使基于结构的关于功能的假设成为可能。本提交文件的具体目标是分析动力蛋白轻链，包括：1)与动力蛋白马达结构域相关的Lc1溶液结构的分子动力学和优化；2)Lc1突变形式的结构分析，以便在结构水平上解释突变表型；3)完成Tctex 1轻链的核磁共振分配和计算；4)硫氧还蛋白相关和钙结合轻链的核磁共振结构分析；5)在系统搜索适当的溶液条件后，制备用于结构分析的其他动力蛋白结构域。这些研究将提供对动力蛋白调控机制的基于结构的洞察，并将为进一步分析这一大规模分子马达的结构提供必要的背景。

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.