Protein Interactions in the Flagellar Export Machinery

描述(由申请人提供)：运动性在细菌发病中起着关键作用。许多物种利用鞭毛推进来定位宿主并在组织中移动。鞭毛是一种复杂的、可逆的、自组装的纳米机器。它含有自己的III型蛋白输出机制，与毒力因子分泌机制密切相关。输出器官的膜蛋白可能是鞭毛中最鲜为人知的成分。这项拟议研究的总体目标是获得关于鼠伤寒沙门氏菌这些蛋白质之间相互作用的详细知识。此外，了解鞭毛组装的最终目标之一是从组件重新组装鞭毛输出复合体，以确定和分析组成蛋白以及机械本身的作用。为了实现该提案的具体目标并接近长期目标，将开展几项工作：通过亲和印迹、共纯化等生化方法，确定出口器械内膜蛋白(FIhA、FlhB、FliO、FliP、FliQ和FLIR)之间的相互作用，以及膜成分与其他鞭毛蛋白质之间的相互作用；分析与分析超速离心法和荧光共振能量转移研究之间的相互作用的生物物理分析；以及出口器械蛋白质复合体的重组，并分析它们的协调功能。

DESCRIPTION (provided by applicant): Motility plays a critical role in bacterial pathogenesis. Many species utilize flagella propulsion to locate hosts and move through tissues. The flagellum is a complex, reversible, self-assembling nanomachine. It contains its own type III protein export machinery that is closely related to the virulence factor secretion apparatus. The membrane proteins of the export apparatus are probably the least understood components of the flagellum. The overall objective of the proposed research is to obtain detailed knowledge of the interactions among these proteins from Salmonella typhimurium. Additionally, one of the ultimate goals of efforts to understand flagellar assembly is to reassemble the flagellar export complex from components to determine and analyze the roles of constituent proteins as well as the machinery itself. In order to achieve the specific objectives of the proposal and to approach the long term goal, several efforts will be undertaken: identification of interactions among the membrane proteins within the export apparatus (FIhA, FlhB, FliO, FliP, FliQ and FliR) and between the membrane components and other flagellar proteins by biochemical methods such as affinity blotting, copurification, etc.; biophysical analysis of interactions with analytical ultracentrifugation and fluorescence resonance energy transfer studies; and reconstitution of complexes of export apparatus proteins and analysis of their concerted function.