Cleavage Mapping the Topology of Protein Folding Intermediates

In order to identify regions of the polypeptide chain that are brought close together by the protein fold, our laboratory devised a scheme in which, in the presence of reductant, reactive oxygen species are generated at an EDTA-Fe moiety covalently attached to unique cysteine residues in a series of...

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Bibliographic Details
Main Author: Ledman, David W
Format: Report
Language:English
Subjects:
AMINO ACIDS
Biochemistry
CHEMICAL CLEAVAGE
CLEAVAGE
CLEAVAGE MAPPING
CRYSTAL STRUCTURE
CYSTEINE
CYSTEINE MUTANTS
EDTA-FE
EDTA-FE MOIETY
ENZYMES
EXCHANGE REACTIONS
FOLDING
INTERACTIONS
MAPPING
MOLECULAR PROPERTIES
MUTATIONS
NUCLEASE
Organic Chemistry
PEPTIDES
POLYPEPTIDE CHAIN
PROTEIN FOLDING
PROTEINS
STAPHYLOCOCCUS
STAPHYLOCOCCUS NUCLEASE
THIOL EXCHANGE REACTION
THIOLS
TOPOLOGY
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Summary:In order to identify regions of the polypeptide chain that are brought close together by the protein fold, our laboratory devised a scheme in which, in the presence of reductant, reactive oxygen species are generated at an EDTA-Fe moiety covalently attached to unique cysteine residues in a series of single cysteine mutants of the protein under study. Only surface residues that do not participate in side chain interactions are mutated. The mutants undergo a thiol exchange reaction in the presence of EPD-Fe that results in the attachment of the EDTA-Fe moiety. The free radicals generated at the iron center promote intramolecular, conformation dependent cleavage of the peptide backbone.