Entwicklung und Anwendung von Technologie für die Gewinnung und strukturelle Untersuchung von membranverbindenden Multiproteinkomplexen

RAB/YPT蛋白是RAS小分子GTP酶超家族中最大的一组蛋白，是多步膜转运的关键调节因子。许多调节因子和效应分子与Rab蛋白协同作用，控制囊泡运输中的对接和融合步骤。它们中的几个要么与所有的Rab蛋白相互作用，要么形成相互作用的蛋白质家族。这一建议的目的是研究RAB与两种类型的RAB相互作用的结构基础，这两种类型的RAB在膜和胞浆之间的循环在功能上是大多数的。第二类是含有Gyp结构域的蛋白，属于Rab GTP酶激活蛋白家族(RabGAP)。我们建议解决Rab：GDI复合体和Gyp1的催化结构域(酵母Rab缺口)的结构。提出了一种新的方法，用于在体外产生大量的预烯基化蛋白质，使其能够大量产生Rab：GDI复合体，用于结晶试验。使用类似的方法，我们计划生成荧光标记的Rab：GDI络合物，并分析Rab与膜的相互作用动力学。这种综合的方法将显著地加深我们对Rab蛋白功能的理解。

Rab/Ypt proteins comprise the largest group of the Ras superfamily of small GTPases and are key regulators of multiple steps of membrane transport. Numerous regulatory factors and effector molecules cooperate with Rab proteins in controlling docking an fusion steps in vesicular transport. Several of them either interact with all Rab proteins or form families of interacting proteins. The aim of this proposal is to investigate the structural basis of Rab interactions with two types of the cycling of Rabs between the membrane and the cytosol an is functionally shared among most of them. The second is the Gyp domain containing protein that belongs to the family of Rab GTPase activating proteins (RabGAP). We propose to solve the structure of a Rab: GDI complex and of a catalytic domain of Gyp1 (a yeast rab GAP). A novel methodology is proposed for the generation of preparative amounts of prenylated proteins in vitro that should enable production of Rab:GDI complex in large amounts for crystallization trials. Using similar approach we plan to generate fluorescently labeled Rab:GDI complexes and analyze kinetics of Rab interaction with membranes in vitro and in vivo. This integrated approach should signifigantly further our understanding of Rab protein function.