The origin of pH-dependent changes in m-values for the denaturant-induced unfolding of proteins

Denaturant-induced unfolding is one of the most prevalent means of evaluati ng the structural stability of proteins and of determining the energetic co nsequences of mutations or changes in solution conditions. In spite of the widespread use of this approach, controversies and inconsistencies still pe rsist with regard to the interpretation of the results of such studies. For example, most proteins show either a significant increase or a decrease (a s much as 100%) in the denaturant-dependence of the free energy of unfoldin g (i.e. the m-value) under increasingly acidic conditions. The pH dependenc e of the m-value is given different interpretations depending on whether th e m-values increase or decrease with decreasing pH. In cases where m-values decrease, the decrease is attributed to the presence of an intermediate th at becomes transiently stabilized during the unfolding transition at low pH . Cases where m-values increase as pH is lowered are usually interpreted in terms of an increase in the amount of surface area exposed by the denature d state at low pH. We have developed a general thermodynamic model that acc ounts for both types of behavior in terms of an intermediate that is popula ted throughout the unfolding transition. The model provides a unified frame work for explaining both types of observed behavior, and the validity of th e model was tested through the analysis of the pH dependence of m-values of staphylococcal nuclease. According to the model, the observed increase in ill-values with decreasing pH is consistent with the existence of an interm ediate that is populated during urea and guanidine unfolding. The intermedi ate becomes less populated during the unfolding transition at Lower pH valu es giving rise to the apparent increase in m-values. These results argue th at the prevailing interpretation need not apply to all proteins. (C) 2001 A cademic Press.