Protein Interactions in the Flagellar Export Machinery

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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.