Thermodynamic Constraints on the “Hidden” Folding Intermediates

Experimental data on the folding and unfolding of small globular proteins are often well described assuming a two-state equilibrium process. It means that after careful analysis by a combination of experimental techniques, only folded and unfolded states of the protein are found to be populated under various external conditions with no detectable intermediates. One of the consequences of the two-state behavior is that the equilibrium ratio of the folded to unfolded protein states follows a simple thermodynamic relation, and the enthalpy difference between states can be obtained from the temperature dependence of the equilibrium constant. In this paper, we theoretically investigate the criteria for the two-state equilibrium behavior and discuss the thermodynamic constraint on the properties of the “hidden” folding intermediates. The literature data on the folding mechanism of lysozyme in water and glycerol, which follows a two-state equilibrium behavior but includes kinetic intermediates, is analysed in light of this constraint.