Unfolding of the molten globule state of alpha-lactalbumin studied by 1H NMR.

The urea-induced unfolding of the molten globule state of bovine alpha-lactalbumin was investigated by 1H nuclear magnetic resonance. In the molten globule state, most of the aromatic resonances deviate from their random coil values, indicating that aromatic side chains form some ordered structures in the molten globule state. When the urea concentration increases, the resonances are shifted, and the deviations from the random coil values are diminished. Because the chemical shifts of several random coil peptides are found to be independent of urea concentration, the urea-induced shifts of the resonances in the molten globule state reflect the unfolding transition of some ordered structures. The unfolding transitions measured by individual aromatic resonances do not coincide with each other. The unfolding transition curves obtained from some aromatic resonances are also different from those of the secondary structures measured by circular dichroism spectra. These results clearly show that the unfolding of the molten globule state of alpha-lactalbumin is not a cooperative two-state process.