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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Special Emphasis Panel (ZRG1-IDM-A (02))
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Ainsztein, Alexandra M
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University of Missouri-Columbia
Schools of Medicine
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Crane, Jennine M; Randall, Linda L (2017) The Sec System: Protein Export in Escherichia coli. EcoSal Plus 7:
Suo, Yuying; Hardy, Simon J S; Randall, Linda L (2015) The basis of asymmetry in the SecA:SecB complex. J Mol Biol 427:887-900
Chada, Nagaraju; Sigdel, Krishna P; Gari, Raghavendar Reddy Sanganna et al. (2015) Glass is a Viable Substrate for Precision Force Microscopy of Membrane Proteins. Sci Rep 5:12550
Sanganna Gari, Raghavendar Reddy; Frey, Nathan C; Mao, Chunfeng et al. (2013) Dynamic structure of the translocon SecYEG in membrane: direct single molecule observations. J Biol Chem 288:16848-54
Mao, Chunfeng; Cheadle, Carl E; Hardy, Simon J S et al. (2013) Stoichiometry of SecYEG in the active translocase of Escherichia coli varies with precursor species. Proc Natl Acad Sci U S A 110:11815-20
Suo, Yuying; Hardy, Simon J S; Randall, Linda L (2011) Orientation of SecA and SecB in complex, derived from disulfide cross-linking. J Bacteriol 193:190-6
Crane, Jennine M; Lilly, Angela A; Randall, Linda L (2010) Characterization of interactions between proteins using site-directed spin labeling and electron paramagnetic resonance spectroscopy. Methods Mol Biol 619:173-90
Randall, Linda L; Henzl, Michael T (2010) Direct identification of the site of binding on the chaperone SecB for the amino terminus of the translocon motor SecA. Protein Sci 19:1173-9
Mao, Chunfeng; Hardy, Simon J S; Randall, Linda L (2009) Maximal efficiency of coupling between ATP hydrolysis and translocation of polypeptides mediated by SecB requires two protomers of SecA. J Bacteriol 191:978-84
Lilly, Angela A; Crane, Jennine M; Randall, Linda L (2009) Export chaperone SecB uses one surface of interaction for diverse unfolded polypeptide ligands. Protein Sci 18:1860-8

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