title: Effects of denaturants on amide proton exchange rates: a test for structure in protein fragments and folding intermediates.
abstract: A method for detecting structure in marginally stable forms of a protein is described. The principle is to measure amide proton exchange rates in the absence and presence of varying concentrations of a denaturant. Unfolding of structure by the denaturant is reflected by an acceleration of amide proton exchange rates, after correction for the effects of the denaturant on the intrinsic rate of exchange. This exchange-rate test for structure makes no assumptions about the rate of exchange in the unfolded state. The effects of 0-8 M urea and 0-6 M guanidinium chloride (GdmCl) on acid- and base-catalyzed exchange from model compounds have been calibrated. GdmCl does not appear to be well-suited for use in the exchange-rate test; model compound studies show that the effects of GdmCl on intrinsic exchange rates are complicated. In contrast, the effects of urea are a more uniform function of denaturant concentration. Urea increases acid-catalyzed, and decreases base-catalyzed, rates in model compounds. The exchange-rate test is used here to study structure formation in the S-protein (residues 21-124 of ribonuclease A). In conditions where an equilibrium folding intermediate of S-protein (I3) is known to be populated (pH 1.7, 0 degree C), the exchange-rate test for structure is positive. At higher temperatures (greater than 32 degrees C) I3 is unfolded, but circular dichroism data suggest that residual structure remains [Labhardt, A. M. (1982) J. Mol. Biol. 157, 357-371].(ABSTRACT TRUNCATED AT 250 WORDS)
mesh_terms: D000577:Amides; D019791:Guanidine; D006146:Guanidines; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010446:Peptide Fragments; D010455:Peptides; D011487:Protein Conformation; D011489:Protein Denaturation; D012260:Ribonucleases
chemical_list: D000577:Amides; D006146:Guanidines; D010446:Peptide Fragments; D010455:Peptides; C019529:polyalanine; D012260:Ribonucleases; D019791:Guanidine