Energetic basis of structural stability in the molten globule state: alpha-lactalbumin

The denatured states of a-lactalbumin, which have features of a molten glob ule state, have been studied to elucidate the energetics of the molten glob ule state and its contribution to the stability of the native conformation. Analysis of calorimetric and CD data shows that the heat capacity incremen t of alpha-lactalbumin denaturation highly correlates with the degree of di sorder of the residual structure of the state. As a result, the denaturatio nal transition of alpha-lactalbumin from the native to a highly ordered com pact denatured state, and from the native to the disordered unfolded state are described by different thermodynamic functions. The enthalpy and entrop y of the denaturation of alpha-lactalbumin to compact denatured state are a lways greater than the enthalpy and entropy of its unfolding. This differen ce represents the unfolding of the molten globule state. Calorimetric measu rements of the heat effect associated with the unfolding of the molten glob ule state reveal that it is negative in sign over the temperature range of molten globule stability. This observation demonstrates the energetic speci ficity of the molten globule state, which, in contrast to a protein with un ique tertiary structure, is stabilized by the dominance of negative entropy and enthalpy of hydration over the positive conformational entropy and ent halpy of internal interactions. It is concluded that at physiological tempe ratures the entropy of dehydration is the dominant factor providing stabili ty for the compact intermediate state on the folding pathway, while for the stability of the native state, the conformational enthalpy is the dominant factor. (C) 2000 Academic Press.