Our overall goal is to elucidate the molecular details of preprotein translocation across biological membranes, utilizing Escherichia coli as a facile genetic and biochemical system. Although essentially all of the components of the Sec machinery have been characterized, most steps in this process remain poorly defined. We will focus on a central component, the SecA ATPase, which interacts with preproteins, the SecB chaperone, anionic phospholipids, the integral membrane proteins SecYEG, and its own mRNA (for autoregulation), and whose translocation-ATPase and membrane-integration activities are at the heart of the energetics and mechanism of protein translocation.
Six specific aims will be investigated. (1) To obtain structural information about SecA and its interactions, the structure of the highly homologous Bacillus subtilis SecA protein will be refined, and structures of SecA bound to ATP analogs, a signal peptide or extended signal peptide, SecY peptide, and an anionic phospholipid analog will be undertaken by crystallographic approaches. (2) To elucidate the structure of the integral-membrane form of SecA and to characterize its interactions, studies of SecA topology will be continued, along with crosslinking studies to identify the nearest neighbors of SecA. (3) To characterize SecA-SecY interactions further, the interacting sites will be mapped, and novel secA and secY mutants will be constructed and characterized. (4) To characterize the biochemistry and mechanism of action of SecA, individual domains of SecA will be produced, purified, and characterized. (5) To elucidate the recognition of preproteins by SecA, a signal-peptide or extended signal-peptide binding assay will be utilized to characterize the features that are recognized for binding, and this site on SecA will be mapped by photocrosslinking or crystallization approaches. (6) To elucidate the regulation of secA and its modulation by protein secretion, the role of GeneX as a secretion sensor will be investigated, the geneX-secA binding site on SecA will be mapped, and secA mutants creating defects in the binding of SecA to the mRNA will be constructed and characterized. These studies should lead to a refined picture of the structure, biochemistry, and mechanism of action of SecA. They should also be of broad significance in understanding these basic processes in other protein-secretion systems, in developing novel anti-bacterial compounds that target protein-secretion pathways, and in engineering protein-secretion pathways for novel substrates.
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