Denaturation versus unfolding: Energetic aspects of residual structure in denatured alpha-lactalbumin

Denaturational changes in alpha-lactalbumin result in different degrees of disordering of the protein molecule. The thermally denatured states have be en studied to elucidate the energetics of residual structure and its contri butions to the stability of the native conformation. The value of the heat capacity increment of alpha-lactalbumin denaturation correlates closely wit h the amount of residual secondary structure in the denatured protein, ther efore reflecting the degree of its disordering and accessibility to solvent . As a result of the observed correlation, the behavior of protein denatura tion functions is influenced by the degree of disordering of protein confor mation in the denatured state. Analysis of the calorimetric data shows that the denaturational transition of alpha-lactalbumin is described by differe nt thermodynamic functions when it proceeds to an ordered compact denatured state and to the disordered unfolded state. This difference is related to unfolding of the compact denatured state known as a molten globule state, w hich is populated differently under different denaturing conditions. The en thalpy and entropy of the transition from the native to the compact denatur ed state are always higher in magnitude than the enthalpy and entropy of th e complete unfolding reaction due to the large negative hydration effect up on molten globule unfolding. Since the hydration effect increases with decr easing temperature, the gap between the partial denaturing and complete unf olding thermodynamic parameters also increases, resulting in a large differ ence at physiological temperatures. The results clearly indicate that a deg ree of residual structure in the denatured state must be taken into account to yield a more accurate description of protein structural energetics.