Cleavage Mapping the Topology of Protein Folding Intermediates

摘要

In order to identify regions of the polypeptide chain that are brought closetogether 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.