title: Measurements of cysteine reactivity during protein unfolding suggest the presence of competing pathways.
abstract: Evidence that proteins may unfold utilizing complex competing pathways comes from a new pulse-labeling protocol in which the change in reactivity of a single cysteine residue in a protein during unfolding is measured, making use of its easily monitored reaction with the Ellman reagent, dithionitrobenzoic acid. The kinetics of unfolding of two single cysteine-containing mutant forms of the small protein barstar, C82A, which contains only Cys40, and C40A, which contains only Cys82, have been studied. The data suggest that unfolding occurs via two parallel pathways, each forming competing intermediates. In one of these early intermediates, Cys40 and Cys82 are already as reactive as they are in the fully unfolded protein, while in the other intermediate, the Cys thiol groups are unreactive. One more long-lived intermediate also needs to be included on the pathway defined by the early intermediate with unreactive Cys thiol groups to account for the difference in the rates of fluorescence change and of change in Cys40 reactivity. The demonstration of multiple intermediates and pathways for unfolding indicates that protein unfolding reactions can be as complex as protein folding reactions.
authors.affiliation: Tata Institute of Fundamental Research, National Centre for Biological Sciences, UAS-GKVK Campus, Bangalore, 560065, India.
authors.forename: S
authors.initials: S
authors.lastname: Ramachandran
authors.forename: B R
authors.initials: BR
authors.lastname: Rami
authors.forename: J B
authors.initials: JB
authors.lastname: Udgaonkar
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001667:Binding, Competitive; D003545:Cysteine / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D005453:Fluorescence; D006859:Hydrogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008433:Mathematics; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; C057438:barstar protein, Bacillus amyloliquefaciens; D006859:Hydrogen; D004228:Dithionitrobenzoic Acid; D003545:Cysteine