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 evaluating the structural stability of proteins and of determining the energetic consequences of mutations or changes in solution conditions. In spite of the widespread use of this approach, controversies and inconsistencies still persist with regard to the interpretation of the results of such studies. For example, most proteins show either a significant increase or a decrease (as much as 100 %) in the denaturant-dependence of the free energy of unfolding (i.e. the m-value) under increasingly acidic conditions. The pH dependence of the m-value is given different interpretations depending on whether the m-values increase or decrease with decreasing pH. In cases where m-values decrease, the decrease is attributed to the presence of an intermediate that 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 denatured state at low pH. We have developed a general thermodynamic model that accounts for both types of behavior in terms of an intermediate that is populated throughout the unfolding transition. The model provides a unified framework for explaining both types of observed behavior, and the validity of the model was tested through the analysis of the pH dependence of m-values of staphylococcal nuclease. According to the model, the observed increase in m-values with decreasing pH is consistent with the existence of an intermediate that is populated during urea and guanidine unfolding. The intermediate becomes less populated during the unfolding transition at lower pH values giving rise to the apparent increase in m-values. These results argue that the prevailing interpretation need not apply to all proteins.