ttermodynamic stability of proteins is often characterized on the basis of chemical denaturation studies, in which unfolding is induced by urea or guanidine. In contrast with the DSC approach, the analysis of chemical denaturation is to a large extent empirical and based upon extrathermodynamic assumptions (for instance, assuming that the denaturant-concentration dependence of the unfolding free energy is linear outside the narrow transition region). It must be recognized, however, that one of the parameters derived from chemical denaturation profiles has found widespread application in protein folding studies: the slope of the plot of folding free energy versus denaturant concentration, referred to as the m value (Greene and Pace 1974), which has been found empirically to be related to the amount of protein surface exposed to the solvent upon denaturation (Myers et al. 1995).
Recent work (Ibarra-Molero et al. 2004; Perez-Jimenez et al. 2004) shows, nevertheless, that denaturant m values can also be calculated from DSC experiments in a simple and, to a large extent, model-independent manner, tte calculation requires knowledge (derived from DSC experiments) of the effect of denaturant concentration on denaturation temperatures and enthalpies, ft does not require, however, values of the denaturation heat capacity change.
One of the main advantages of the calorimetrically determined m values is that they are obtained as a function of denaturant concentration, thus providing detailed information about the denaturant-concentration dependence of the unfolding free energy. In particular, calorimetrically determined m values have been found to change abruptly at low denaturant concentrations when the denaturant is a salt (guanidinium chloride, guanidinium thiocyanate), reflecting very likely the salt-induced screening of interactions between the protein charged groups (Ibarra-Molero et al. 2004; Perez-Jimenez et al. 2004) and providing, therefore, a convenient approach to probe those interactions.
Acknowledgements. Research in the authors' laboratory is supported by Spanish Ministry ofEducation and Science Grant BI02003-02229 and Feder Funds.
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