A MODEL-INDEPENDENT, NONLINEAR EXTRAPOLATION PROCEDURE FOR THE CHARACTERIZATION OF PROTEIN-FOLDING ENERGETICS FROM SOLVENT-DENATURATION DATA

We have characterized the guanidine-induced denaturation of hen egg wh ite lysozyme within the 30-75 degrees C temperature range on the basis of equilibrium fluorescence measurements, unfolding assays, kinetic f luorescence measurements, and differential scanning calorimetry. Analy sis of the guanidine denaturation profiles according to the linear ext rapolation method yields values for the denaturation Gibbs energy whic h are about 15 kJ/mol lower than those derived from differential scann ing calorimetry. Our results strongly suggest that this discrepancy is not due to deviations from the two-state denaturation mechanism. We p ropose a new method for the determination of denaturation Gibbs energi es from solvent-denaturation data (the constant-Delta G extrapolation procedure). It employs several solvent-denaturation profiles (obtained at different temperatures) to generate the protein stability curve at zero denaturant concentration within the -8 to 8 kJ/mol Delta G range . The method is model-independent and provides a practical, nonlinear alternative to the commonly employed linear extrapolation procedure. T he application of the constant-Delta G method to our data suggests tha t the guanidine-concentration dependence of the denaturation Gibbs ene rgy is approximately linear over an extended concentration range but, also, that strong deviations from linearity may occur at low guanidine concentrations. We tentatively attribute these deviations to the abru pt change of the contribution to protein stability that arises from pa irwise charge-charge electrostatic interactions. This contribution may be positive, negative, or close to zero, depending on the pH value an d the charge distribution on the native protein surface [Yang, A.-S., & Honig, B. (1993) J. Mol. Biol. 231, 459-474], which may help to expl ain why disparate effects have been found when studying protein denatu ration at low guanidine concentrations. Kinetic nz values for lysozyme denaturation depend on temperature, in a manner which appears consist ent with Hammond behavior.