The denatured states of alpha-lactalbumin, which have features of a molten globule state, have been studied to elucidate the energetics of the molten globule state and its contribution to the stability of the native conformation. Analysis of calorimetric and CD data shows that the heat capacity increment of alpha-lactalbumin denaturation highly correlates with the degree of disorder of the residual structure of the state. As a result, the denaturational transition of alpha-lactalbumin from the native to a highly ordered compact denatured state, and from the native to the disordered unfolded state are described by different thermodynamic functions. The enthalpy and entropy of the denaturation of alpha-lactalbumin to compact denatured state are always greater than the enthalpy and entropy of its unfolding. This difference represents the unfolding of the molten globule state. Calorimetric measurements of the heat effect associated with the unfolding of the molten globule state reveal that it is negative in sign over the temperature range of molten globule stability. This observation demonstrates the energetic specificity of the molten globule state, which, in contrast to a protein with unique tertiary structure, is stabilized by the dominance of negative entropy and enthalpy of hydration over the positive conformational entropy and enthalpy of internal interactions. It is concluded that at physiological temperatures the entropy of dehydration is the dominant factor providing stability for the compact intermediate state on the folding pathway, while for the stability of the native state, the conformational enthalpy is the dominant factor.
Copyright 2000 Academic Press.