Systematic Structural Biology of Dynein

动力蛋白是一种基于微管的分子马达，参与多种基本细胞功能，包括逆行囊泡运输、纤毛/鞭毛运动和细胞分裂。这些酶是高度复杂的，含有多达13种不同的多肽组分，总质量几乎为2 MDa。为了实现对机制的分子理解，获得动力蛋白复合物所有组分的高分辨率结构信息至关重要。然而，动力蛋白颗粒的复杂性和大小与几个结构域的固有灵活性相结合，使得获得整个酶的高分辨率结构信息是不可行的。本申请提出了一种系统的方法，动力蛋白结构生物学，通过获得信息的个别蛋白质或子域使用多维NMR技术。然后，这些结构将被用来建立更完整的动力蛋白马达模型，并将使基于结构的假设有关的功能。本申请的具体目标集中在动力蛋白轻链的分析上，包括：1）分子动力学和与动力蛋白马达结构域相关的LC 1的溶液结构的细化; 2）LC 1的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）完成Tctex 1轻链的NMR归属和溶液结构的计算; 4）硫氧还蛋白相关轻链和Ca 2+结合轻链的NMR结构分析; 5）在系统地寻找合适的溶液条件后，制备用于结构分析的其它动力蛋白结构域。这些研究将提供基于结构的动力蛋白调节机制的见解，并将提供必要的背景，为进一步的结构分析，这个巨大的分子马达。

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.