Determination of the peptide-binding sites on the bacterial chaperone protein SecB
EMSL Project ID
3997
Abstract
The chaperone protein SecB is required for the efficient export of certain proteins across the periplasmic membrane of E.coli. SecB promotes export by binding to the proteins and delivering them to the translocation apparatus, avoiding the possible fates of degradation and aggregation within the cytoplasm. Previous studies have shown that SecB will bind to short, positively-charged peptides in a manner that mimics physiological binding of proteins destined for export. Therefore, in order to identify the sidechains on SecB that are involved in ligand binding, we have crosslinked short (4-15 amino acids) peptides to SecB using EDC (water-soluble carbodiimide) and N-hydroxysuccinimide (NHS) to form an isopeptide linkage between Asp and Glu sidechains on SecB and the -amino groups of lysines on the peptide. The crosslinking reaction is rapid, highly efficient (up to 50% of SecB monomers become crosslinked to peptides) and irreversible. Despite the efficiency of crosslinking, our efforts to identify the crosslinked residues on SecB by peptide mapping techniques have met with limited success, due to the extensive intermonomer and intramonomer crosslinking that occurs in addition to the peptide crosslinking as well as the large number of acidic residues (8 Asp and 13 Glu) that serve as possible sites of crosslinking. We believe that mass spectrometry will be an excellent technique for analyzing samples of SecB crosslinked to various peptides and will make it possible to identify the residues involved in peptide binding. Specifically, by comparing mass spectra of enzymatic digests of crosslinked and uncrosslinked samples, we would expect to identify modified fragments of SecB and, by application of secondary-ion methods, identify the exact crosslinking sites.
Project Details
Project type
Exploratory Research
Start Date
2003-08-26
End Date
2004-12-09
Status
Closed
Released Data Link
Team
Principal Investigator
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