Hydrophobic hydration processes thermal and chemical denaturation of proteins

The hydrophobic hydration processes have been analysed under the light of a mixture model of water that is assumed to be composed by clusters (W₅)_I, clusters (W₄)_II and free water molecules W_III. The hydrophobic hydration processes can be subdivided into two Classes A and B. In the processes of Class A, the transformation A(− ξ_wW_I → ξ_wW_II + ξ_wW_III + cavity) takes place, with expulsion from the bulk of ξ_w water molecules W_III, whereas in the processes of Class B the opposite transformation B(− ξ_wW_III − ξ_wW_II → ξ_wW_I − cavity) takes place, with condensation into the bulk of ξ_w water molecules W_III. The thermal equivalent dilution (TED) principle is exploited to determine the number ξ_w. The denaturation (unfolding) process belongs to Class A whereas folding (or renaturation) belongs to Class B. The enthalpy ΔH_den and entropy ΔS_den functions can be disaggregated in thermal and motive components, ΔH_den = ΔH_therm + ΔH_mot, and ΔS_den = ΔS_therm + ΔS_mot, respectively. The terms ΔH_therm and ΔS_therm are related to phase change of water molecules W_III, and give no contribution to free energy (ΔG_therm = 0). The motive functions refer to the process of cavity formation (Class A) or cavity reduction (Class B), respectively and are the only contributors to free energy ΔG_mot. The folded native protein is thermodynamically favoured (ΔG_fold ≡ ΔG_mot < 0) because of the outstanding contribution of the positive entropy term for cavity reduction, ΔS_red ? 0. The native protein can be brought to a stable denatured state (ΔG_den ≡ ΔG_mot < 0) by coupled reactions. Processes of protonation coupled to denaturation have been identified. In thermal denaturation by calorimetry, however, is the heat gradually supplied to the system that yields a change of phase of water W_III, with creation of cavity and negative entropy production, ΔS_for ? 0. The negative entropy change reduces and at last neutralises the positive entropy of folding. In molecular terms, this means the gradual disruption by cavity formation of the entropy-driven hydrophobic bonds that had been keeping the chains folded in the native protein. The action of the chemical denaturants is similar to that of heat, by modulating the equilibrium between W_I, W_II, and W_III toward cavity formation and negative entropy production. The salting-in effect produced by denaturants has been recognised as a hydrophobic hydration process belonging to Class A with cavity formation, whereas the salting-out effect produced by stabilisers belongs to Class B with cavity reduction.Some algorithms of denaturation thermodynamics are presented in the Appendices.