Protein stability. Analysis of heat and cold denaturation without and with unfolding models

Abstract Protein stability is of utmost importance in many areas of life sciences and life science technology. The gold standard for assessing protein stability is the measurement of the heat capacity Cp(T), showing large peaks at the temperature of cold and heat unfolding. We first demonstrate that important thermodynamic properties, including the free energy, can be obtained from Cp(T) temperature profiles without the necessity of an unfolding model. This is illustrated with experimental examples taken from the literature. We then compare all thermodynamic results with chemical and statistical unfolding models. An often-cited chemical equilibrium two-state model makes partially incorrect predictions. New models are introduced, which are in excellent agreement with the experimental results. We then show that the free energy is not a good criterion to judge protein stability. More useful parameters are discussed, including protein cooperativity. The new parameters are embedded in a well-defined thermodynamic context and are amenable to molecular dynamics calculations.