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

Rab/Ypt蛋白是Ras小gtpase超家族中最大的一组，是膜运输多个步骤的关键调节因子。许多调节因子和效应分子与Rab蛋白合作，控制囊泡运输中的对接和融合步骤。它们中的一些要么与所有Rab蛋白相互作用，要么形成相互作用蛋白家族。本研究的目的是研究两种类型的Rabs在膜和细胞质之间的循环相互作用的结构基础，并且大多数Rabs在功能上是共享的。第二种是含有Gyp结构域的蛋白，属于RabGAP家族的GTPase激活蛋白。我们提出解决Rab: GDI配合物和Gyp1催化结构域（酵母Rab GAP）的结构。提出了一种新的方法，用于在体外产生制备量的戊烯基化蛋白，这应该能够生产大量的Rab:GDI复合物用于结晶试验。使用类似的方法，我们计划生成荧光标记的Rab:GDI复合物，并分析Rab在体内和体外与膜相互作用的动力学。这种综合方法将显著地促进我们对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.