The correct transport of proteins must occur across the membranes of all prokaryotic and eukaryotic cells. It is well documented that the targeting and transport of these proteins requires several proteinaceous components which comprise the cellular transport pathway and a signal peptide at the amino-terminus of the secreted protein to direct entry into this pathway. Little is known about how these components function in concert to achieve the transport process. The focus of this project is to examine how the signal peptide and the mature region of the preprotein interface with components of the secretion machinery and to probe the structural aspects which render these components receptive to preprotein translocation. Using Escherichia coli as a model system, and a combination of mutagenesis, biochemical, and biophysical strategies, the aims of the proposed research are to: (1) determine the requirements for molecular recognition of SecA, signal peptide and preprotein; (2) examine the structure of the membrane active species of SecA; (3) determine the nature of SecY-signal peptide/preprotein interactions; (4) determine how leader peptidase I interfaces with the translocon and emerging preprotein. These studies will take advantage of the library of synthetic signal peptides, alkaline phosphatase signal peptide mutants, and truncated preproteins that we have generated andcharacterized in vivo and in vitro. Purified SecA, SecYEG, and leader peptidase I will be reconstituted in membrane mimetic systems or examined in inner membrane vesicles to characterize the sites of interaction with preproteins, the relative affinities, and how these critical protein-protein interactions propel secretion overall. Knowledge of how signal peptides enhance correct compartmentalization in bacteria is useful in understanding secretion in normal and diseased cells. The principles which evolve can be applied to the tissue-specific targeting of therapeutic agents and the development of antimicrobials which are secretion inhibitors as alternatives to classical antibiotics.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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Shapiro, Bert I
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University of Connecticut
Schools of Arts and Sciences
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Wowor, Andy J; Yan, Yuetian; Auclair, Sarah M et al. (2014) Analysis of SecA dimerization in solution. Biochemistry 53:3248-60
Bhanu, Meera K; Kendall, Debra A (2014) Fluorescence spectroscopy of soluble E. coli SPase I ?2-75 reveals conformational changes in response to ligand binding. Proteins 82:596-606
Bhanu, Meera K; Zhao, Ping; Kendall, Debra A (2013) Mapping of the SecA signal peptide binding site and dimeric interface by using the substituted cysteine accessibility method. J Bacteriol 195:4709-15
Yu, Dongmei; Wowor, Andy J; Cole, James L et al. (2013) Defining the Escherichia coli SecA dimer interface residues through in vivo site-specific photo-cross-linking. J Bacteriol 195:2817-25
De Bona, Paolo; Deshmukh, Lalit; Gorbatyuk, Vitaliy et al. (2012) Structural studies of a signal peptide in complex with signal peptidase I cytoplasmic domain: the stabilizing effect of membrane-mimetics on the acquired fold. Proteins 80:807-17
Wowor, Andy J; Yu, Dongmei; Kendall, Debra A et al. (2011) Energetics of SecA dimerization. J Mol Biol 408:87-98
Auclair, Sarah M; Moses, Julia P; Musial-Siwek, Monika et al. (2010) Mapping of the signal peptide-binding domain of Escherichia coli SecA using Förster resonance energy transfer. Biochemistry 49:782-92
Musial-Siwek, Monika; Yeagle, Philip L; Kendall, Debra A (2008) A small subset of signal peptidase residues are perturbed by signal peptide binding. Chem Biol Drug Des 72:140-6
Musial-Siwek, Monika; Kendall, Debra A; Yeagle, Philip L (2008) Solution NMR of signal peptidase, a membrane protein. Biochim Biophys Acta 1778:937-44
Rusch, Sharyn L; Kendall, Debra A (2007) Oligomeric states of the SecA and SecYEG core components of the bacterial Sec translocon. Biochim Biophys Acta 1768:5-12

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