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We have four general objectives: 1) to refine the model of the docking of SecA onto SecB to identify which of the contact sites are responsible for the observed asymmetry in binding, 2) to map docking interfaces on the surface of SecA for its binding partners, 3) to extend our studies to examine events at the membrane translocon and 4) to elucidate the mechanism of transfer of precursor from SecB to SecA.
Non-Technical Summary: Translocation across biological membranes during protein localization is a ubiquitous process. Greater than 30% of proteins in any organism are exported from the site of synthesis into or through a membrane. The subject of the research proposed here is the major route of export of protein from the cytosol of all eubacteria. The pathway through the membrane is provided by a channel that is highly conserved and is found in all three kingdoms, going from single cell organisms to mammals and higher plants. In addition to a pathway through the membrane most translocation processes involve cytosolic chaperones. Molecular chaperones are a family of proteins united by their remarkable ability to recognize and bind polypeptides based on the fact that they are in a nonnative structural state. Members of the chaperone family play roles in many processes in addition to protein localization. They are involved in folding of polypeptides and their assembly into oligomers and in protection of cells against protein aggregation induced by stress. It is likely that underlying fundamental principles are shared among different systems and that knowledge provided by our investigations of one system, protein export in Escherichia coli, will provide insights into the mechanisms of others. <P> Approach: We shall employ approaches that range from an in vitro assay of the translocation process using inverted membrane vesicles to biochemical and biophysical studies of the purified components. The techniques we use to examine complex formation include analytical ultracentrifugation, titration calorimetry, size exclusion chromatography coupled with static light scatter as well as site-directed spin labeling coupled with electron paramagnetic resonance spectroscopy. We believe that what we learn about the functional interactions of proteins during export in E. coli will have relevance not only to secretion but also to chaperone-mediated processes in other systems as well as to the widespread phenomenon of conformational switching between functional states.