Export of Proteins in Escherichia coli

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DESCRIPTION (provided by applicant): We aim to elucidate the mechanistic details of protein export in Escherichia coli with emphasis on conformational switches between functional states induced by binding interactions among the protein components involved. Protein localization, which requires transfer of polypeptides across biological membranes, is a ubiquitous process essential to all living organisms. The pathway through the membrane, provided by a heterotrimeric complex SecYEG in E. coli, is highly conserved in all three kingdoms of life going from single cell organisms to mammals. In addition to a pathway through the membrane in almost all cases, whether the process occurs in prokaryotes or eukaryotes, chaperones are involved in the early stages. We propose to develop a molecular description of the events that occur in export including the binding of the chaperone SecB carrying a precursor polypeptide to SecA, the ATPase motor of the translocon, the passage of the precursor from SecB to SecA within the complex and tranlsocation of the precursor polypeptide through the SecYEG translocon driven by SecA. We shall delineate the binding interfaces on SecA for each of its binding partners, SecB, precursors and SecYEG, and we shall provide a description of conformational changes that occur during the dynamic transfer of the precursor from SecB to SecA as well as the conformational changes that are involved in gating and opening of the SecYEG channel. To achieve these goals we shall use a combination of approaches ranging from in vitro translocation assays to biophysical techniques such as electron paramagnetic resonance (EPR) spectroscopy that will allow us to proceed from a general description of interactions to a molecular description at the resolution of aminoacyl side chains and polypeptide backbone structure. Protein localization is a process that is essential for all living organisms. The SecYEG complex and its homologs are the ubiquitous component of the machines that provide the channel for protein transport. Therefore, what we learn by studying bacterial export will be applicable to the phenomenon in all cells, from bacteria to humans.