Heat capacity-independent determination of differential free energy of stability between structurally homologous proteins

Under the assumption of equivalent heat capacity values, the differential free energy of stability for a pair of proteins midway between their thermal unfolding transition temperatures is shown to be independent of ΔCp up to its cubic term in ΔTm. For model calculations reflecting the nearly 30 °C difference in Tm for the adenylate kinases from the arctic bacterium Bacillus globisporus and the thermophilic bacterium Geobacillus stearothermophilus, the resultant error in estimating ΔΔG by the formula 0.5 [ΔSTm1(1) + ΔSTm2 (2)] ΔTm is less than 1%. Combined with the analogous thermal unfolding data for the adenylate kinase from Escherichia coli, these three homologous proteins exhibit Tm and ΔSTm values consistent with differential entropy and enthalpy contributions of equal magnitude. When entropy-enthalpy compensation holds for the differential free energy of stability, the incremental changes in Tm values are shown to be proportionate to the changes in free energy.