Thermodynamics of denaturation of staphylococcal nuclease mutants: an intermediate state in protein folding.

Abstract

A valuable approach to understanding the forces that maintain protein structure is to analyze the thermodynamic effects of mutations on protein folding. The folding process is most often described using an energetic model that assumes a two-state transition between the native and denatured states. However, some results obtained using this approach for mutants of the protein staphylococcal nuclease have contradicted expectations from our current understanding of protein energetics. The application of differential scanning calorimetry to a set of mutant nuclease proteins allowed us to measure directly the effects of mutations on the enthalpy and heat capacity changes of unfolding, as well as on the cooperativity. We found that most of these effects can be understood with a three-state model of folding including a distinct intermediate, but not with the two-state model. Use of a three-state instead of a two-state model leads to large differences in conclusions about the stability effects of some mutations, suggesting that reevaluation of the effects of mutations on this and other proteins may be necessary to achieve an accurate description of folding energetics. The two-state assumption commonly used in protein stability studies may be an oversimplification in many cases.