Hierarchical unfolding of the α-lactalbumin molten globule: presence of a compact intermediate without a unique tertiary fold

Abstract

The difference between the framework model and the hydrophobic collapse model of protein folding largely rests on whether a secondary-structure framework can exist independently of native tertiary interactions. Here, we used circular dichroism and disulfide exchange experiments to examine the unfolding mechanism of α-LA(α), a two-disulfide variant of human α-lactalbumin (α-LA) that adopts a molten globule conformation under near physiological conditions. Our results show that as the concentration of denaturant increases, the α-LA molten globule first loses its ability to form a specific, native-like tertiary fold. Subsequently, at a higher denaturant concentration, the protein loses its secondary structure and adopts an extended conformation. A compact, non-native disulfide bond isomer, which does not form significantly under both native and strongly denaturing conditions, was found to be moderately populated in ∼2 M guanidine hydrochloride (GuHCl). Qualitatively the same result was also obtained in urea. These results suggest that formation of secondary structure is a necessary, but not sufficient condition for formation of the native-like tertiary fold and support a hierarchical model of protein folding.