Determination of heat capacity of unfolding for marginally stable proteins from a single temperature induced protein unfolding profile

A reliable estimation of heat capacity of denaturation (ΔCp) is necessary to calculate the free energy of unfolding of proteins. For marginally stable proteins, such as mutants of a protein or proteins at low pH or under denaturating conditions, the pre-transition region is not fully populated by the native state. Analysis of differential scanning calorimeter (DSC) data under such conditions may not yield a reliable value of ΔCp and other associated thermodynamic parameters of unfolding. Analysis of denaturation profiles of (a) cytochrome c at pH 2.5, 3 and 8 and (b) myoglobin at pH 4, show that an accurate value of ΔCp can be extracted from a single unfolding profile obtained spectroscopically by including low temperature data.