title: Conformation-dependent cleavage of staphylococcal nuclease with a disulfide-linked iron chelate.
abstract: We report the synthesis and evaluation of (EDTA-2-aminoethyl) 2-pyridyl disulfide. By using this easily prepared cysteine-specific hydrophilic reagent, an ethylenediaminetriacetic acid-Fe3+ complex (EDTA-Fe) was covalently attached to a single genetically engineered cysteine residue in staphylococcal nuclease. Upon addition of the iron reductant ascorbate, the nuclease-EDTA-Fe conjugate underwent a protein self-cleavage reaction mediated by reactive oxygen species. Sequence analysis of the products indicated that cleavage occurs close in tertiary structure to the EDTA-Fe attachment site. In the presence of denaturants, the cleavage pattern changes and the reaction is limited to residues proximal in sequence to the cysteine attachment site. These results indicate that intramolecular protein cleavage reactions mediated by EDTA-Fe can be used to evaluate changes in protein conformation. The reagent described should be a useful tool in the structural mapping of nonnative protein states populated at equilibrium, such as the molten globule, that are frequently refractory to conventional structure analysis.
authors.affiliation: Howard Hughes Medical Institute, Yale University, New Haven, CT 06511.
authors.forename: M R
authors.initials: MR
authors.lastname: Ermácora
authors.forename: J M
authors.initials: JM
authors.lastname: Delfino
authors.forename: B
authors.initials: B
authors.lastname: Cuenoud
authors.forename: A
authors.initials: A
authors.lastname: Schepartz
authors.forename: R O
authors.initials: RO
authors.lastname: Fox
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004220:Disulfides; D007502:Iron Chelating Agents / Q000737:chemistry*; D008836:Micrococcal Nuclease / Q000737:chemistry*; D008958:Models, Molecular; D008970:Molecular Weight; D011487:Protein Conformation; D013329:Structure-Activity Relationship
chemical_list: D004220:Disulfides; D007502:Iron Chelating Agents; D008836:Micrococcal Nuclease