title: Folding intermediates of wild-type and mutants of barnase. II. Correlation of changes in equilibrium amide exchange kinetics with the population of the folding intermediate.
abstract: There is an unanswered question from previous studies of 1H/2H-exchange of amide protons of barnase. Under certain conditions, there is a relatively abrupt change from EX2 towards EX1 kinetics as the temperature is slightly increased. The change in kinetics for different mutants is not directly related to their changes in stability. We have measured the stability of the folding intermediate of barnase (I) in 2H2O under a variety of conditions and calculated its population at different temperatures. The change in kinetics correlates with the change in the population of the folding intermediate. At higher temperatures and pH, the free energy of I becomes higher than that of the denatured state, D, and the kinetics becomes EX1. The data fit a simple kinetic scheme. Such changes in kinetics may be used to detect the presence of intermediates in the folding reaction at equilibrium in native conditions, but cannot distinguish whether they are on or off-pathway.
authors.affiliation: Cambridge Centre for Protein Engineering, UK.
authors.forename: P A
authors.initials: PA
authors.lastname: Dalby
authors.forename: J
authors.initials: J
authors.lastname: Clarke
authors.forename: C M
authors.initials: CM
authors.lastname: Johnson
authors.forename: A R
authors.initials: AR
authors.lastname: Fersht
mesh_terms: D000577:Amides; D019943:Amino Acid Substitution; D001426:Bacterial Proteins; D002152:Calorimetry, Differential Scanning; D017666:Deuterium Oxide; D004795:Enzyme Stability; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology; D014867:Water
chemical_list: D000577:Amides; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D014867:Water; D014508:Urea; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease; D017666:Deuterium Oxide