The correct transport of proteins must occur across the membranes of all prokaryotic and eukaryotic cells. The targeting and transport of these proteins requires several proteinaceous components that comprise the cellular transport pathway and a signal peptide at the amino-terminus of the secreted protein that directs entry into this pathway. Little is known about how these components function in concert to achieve the transport process. The principal objective of this work is to elucidate the features involved in molecular recognition of the preprotein, including its amino-terminal signal peptide, and components of the transport machinery, with the goal of understanding how these interactions propel Sec-dependent tranport. We will use Escherichia coli as a model system, and a combination of mutagenesis, and biochemical and biophysical strategies to examine associations with two key components, SecA and signal peptidase, and to probe the features which render these components receptive to transfer of the preprotein through the Sec relay system. This will involve a combination of in vitro and in vivo studies with the goal of correlating the molecular features we identify with purified components and their role in protein transport.
The aims of the proposed research are to delineate the requirements for signal peptide interaction with the SecA signal peptide binding groove identified by our laboratory;to characterize the oligomeric state of SecA key for specific stages of the transport process;to elucidate the conformational changes and mechanism by which preprotein interacts with SecA during cycles of membrane insertion and de-insertion;to examine molecular recognition of signal peptides by signal peptidase;and to identify the spatial and temporal relationship of signal peptidase with the translocon and emerging preprotein. These studies will take advantage of the library of synthetic signal peptides and truncated alkaline phosphatase preproteins that we have generated and characterized in vivo and in vitro;strategies that we recently developed for the selective photolabeling and specific proteolysis of transport components to identify sites of preprotein interaction;our experience with fluorescence assays and Cys chemistry to report on protein conformation in solution and in model membranes;and build upon our recent NMR analysis of signal peptidase and signal peptide interaction. Knowledge of how signal peptides enhance correct compartmentalization in bacteria is useful in understanding secretion in normal and diseased cells. The principles that evolve can be applied to the tissue-specific targeting of therapeutic agents and the development of antimicrobials that inhibit interactions of the preprotein and transport machinery as alternatives to classical antibiotics.

Public Health Relevance

Knowledge of how signal peptides interact with the protein transport machinery to enhance correct compartmentalization in bacteria is useful in understanding secretion in normal and diseased cells. The principles that evolve can be applied to the tissue-specific targeting of therapeutic agents and the development of antimicrobials, that inhibit interactions of the preprotein and transport machinery, as alternatives to classical antibiotics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM037639-23
Application #
7781316
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Ainsztein, Alexandra M
Project Start
1989-09-01
Project End
2012-02-29
Budget Start
2010-03-01
Budget End
2011-02-28
Support Year
23
Fiscal Year
2010
Total Cost
$361,204
Indirect Cost
Name
University of Connecticut
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
614209054
City
Storrs-Mansfield
State
CT
Country
United States
Zip Code
06269
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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
Sun, Changqi; Rusch, Sharyn L; Kim, Jinoh et al. (2007) Chloroplast SecA and Escherichia coli SecA have distinct lipid and signal peptide preferences. J Bacteriol 189:1171-5

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