This project has as its overarching goal elucidation of the roles played by signal sequences in various steps of protein export or integration of proteins into the cytoplasmic membrane. Signal sequences serve as cellular 'zipcodes' in that they carry essentially all of the information necessary to target a protein for export from the cell. Despite considerable progress in our understanding of protein secretion and integration into membranes, the means by which signal sequences on nascent or newly synthesized proteins are specifically recognized, stably bound, and then released at the appropriate time for interaction with membrane components is still poorly understood. The proposed work addresses the question of how these processes are accomplished by the two signal sequence-mediated pathways in E. coli, that involving Ffh, which is homologous to the 54 kDa subunit of the mammalian signal recognition particle (SRP), and that involving SecA, a uniquely prokaryotic protein. To address this goal, we will utilize synthetic peptides corresponding to signal sequences of bacterial proteins as probes. The proposed strategy is to identify the signal sequence-binding sites on Ffh and SecA by cross-linking them to signal peptides; to explore how these binding sites are altered in affinity as the signal sequence is bound and released; and to determine the conformation adopted by a signal sequence bound either to Ffh or to SecA. We hypothesize that both of these proteins have intramolecular sequences that bind to the hydrophobic signal sequence-binding site to protect it in the absence of a nascent or precursor protein. Several diseases (e.g., cystic fibrosis, Alzheimer's, Wolman, and some prion-associated diseases) have been shown to be associated with signal peptide polymorphisms that lead to mislocalization of a secreted protein or to hypersecretion. Elucidation of the mode of recognition, binding, and release of signal sequences by SRP will help efforts to develop therapeutic strategies for these diseases. Additionally, SecA is an ideal target for antibiotics, since it is uniquely present in bacteria. Enhanced understanding of its mode of recognition will provide a basis for development of new antibacterial agents.
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