HYDROGEN-EXCHANGE IN CHYMOTRYPSIN INHIBITOR-2 PROBED BY DENATURANTS AND TEMPERATURE

Hydrogen exchange of chymotrypsin inhibitor 2 has been measured in the presence of low concentrations of GdmCl and at different temperatures . The study of exchange at different temperatures allows us to obtain the activation enthalpies for the local exchange processes, and the ch ange in enthalpy between the closed, exchange-incompetent, forms and t he open, exchange-competent, forms. From the GdmCl dependence of excha nge, an m-value, which is a measure of the new surface area exposed to solvent in the equilibrium between open and closed forms, can be dete rmined for individual protons. This parameter therefore provides infor mation about the structural nature of the opening reactions. Ln the ab sence of denaturant, exchange from native and native-like states domin ates. As GdmCl concentration is increased, opening reactions that invo lve global unfolding are selectively promoted for the majority of amid e protons. Three classes of protons emerge: for one set of protons, th ere is a linear and weak dependence on denaturant, indicating that the dominant opening reaction is the same throughout the range of GdmCl c oncentrations and involves local fluctuations with exposure of little new surface. For another set of protons, the most slowly exchanging re sidues, a linear, but much stronger, denaturant dependence is observed . For these protons, global unfolding dominates, and the m-values are similar to that obtained by equilibrium GdmCl denaturation measured by fluorescence under identical conditions. For the remaining protons, t he GdmCl-dependence is weak at low GdmCl concentrations and increases at higher GdmCl concentrations. No segment of sub-global unfolding cou ld be identified. Rather, all protons appear to merge together at high GdmCl concentrations to the global unfolding reaction. (C) 1997 Acade mic Press Limited.