title: A method for the reversible trapping of proteins in non-native conformations.
abstract: High-dilution equilibrium macrocyclization is developed as a general approach to trapping proteins in a non-native state with a synthetic cross-linking agent. The approach is illustrated using the N-terminal domain of phosphoglycerate kinase and a synthetic reagent containing two maleimide groups, for selective attachment to cysteines introduced onto the protein surface through mutagenesis, and an aromatic disulfide that can be chemically or photochemically cleaved. Following functionalization of the cysteine residues, thiol-disulfide exchange chemistry under strongly unfolding conditions was used to achieve intramolecular cyclization and a high yield of the cross-linked protein. (1)H NMR, CD, and fluorescence spectroscopies indicate that the conformation of the cross-linked protein is non-native. Chemical cleavage of the aromatic disulfide cross-link by a reducing agent results in the acquisition of a nativelike conformation for the reduced protein. Thus, the cross-link acts as a reversible switch of protein folding.
authors.affiliation: Department of Molecular Biology and Biotechnology, The University of Sheffield, UK.
authors.forename: Lilia
authors.initials: L
authors.lastname: Milanesi
authors.forename: Clare
authors.initials: C
authors.lastname: Jelinska
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Hunter
authors.forename: Andrea M
authors.initials: AM
authors.lastname: Hounslow
authors.forename: Rosemary A
authors.initials: RA
authors.lastname: Staniforth
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Waltho
mesh_terms: D001671:Biochemistry / Q000379:methods*; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D010735:Phosphoglycerate Kinase / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013696:Temperature
chemical_list: D003432:Cross-Linking Reagents; D004220:Disulfides; D011506:Proteins; D010735:Phosphoglycerate Kinase