Signal sequences play a central role in the membrane targeting and translocation of nearly all secreted proteins and many integral membrane proteins in both prokaryotes and eukaryotes, yet major questions remain about the molecular details of their involvement in these processes. The present application has as its long-term goal elucidating the conformations and interactions of signal sequences as they participate in the steps that make up the export pathway, namely, targeting of a nascent chain to the appropriate membrane, initiation of interactions with lipids and proteins of the membrane-resident translocation apparatus, and translocation of the polypeptide chain across the membrane. We seek a fundamental physical-chemical understanding of how signal sequences may take part in these steps. We thus propose to: l) determine the conformation of a signal sequence in a lipid bilayer; 2) determine the ability of a signal sequence, in the absence of other components of the secretory apparatus, to mediate topologically specific interactions of its mature passenger with a lipid bilayer; 3) determine the conformation of a signal sequence upon binding to SecA; 4) determine the conformation of a signal sequence upon binding to the signal recognition particle (SRP); 5) map the region of SecA that is involved in signal sequence binding and use this information, in combination with structural and biochemical analyses of SecA, to develop a detailed model for SecA function; 6) map the region of SRP that is involved in signal sequence binding and use this information, in combination with structural and biochemical analyses of SRP, to develop a detailed model for SRP function; 7) as systems become available, use the same methods applied to SecA and SRP to explore the interactions of signal sequences with other proteins of the secretion pathway. In order to accomplish these aims, we will use physical methods, including nuclear magnetic resonance experiments (e.g., measurement of transferred nuclear Overhauser effects) that enable determination of the conformation of a signal peptide bound to a membrane or to a protein of the export pathway. We will also employ biochemical approaches such as cross-linking and limited proteolysis to define the peptide-binding regions of the SecA and SRP54 proteins that recognize the signal sequence in bacteria and mammals, respectively. Since all serum antibodies, digestive enzymes, and peptide hormones are secreted, as are many other physiologically important proteins, this research will shed light on several biomedical problems.
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