Role of the Interaction between Ionizable Groups in the Folding of Bovine α-Lactalbumin1

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Abstract
Previous studies of the reversible unfolding of α-lactalbumin in the acid to neutral pH region have shown that the unfolding transition with guanidine hydrochloride involves a stable intermediate which is similar to a partially unfolded state produced by acid transition. In order to clarify how the interaction of ionizable groups takes part in stabilization of the native structure during the folding of the protein, the transitions were further investigated in the alkaline region in the presence of the denaturant by means of circular dichroism, difference spectra and pH-jump measurements, and the effects of pH on the equilibrium and kinetics of the unfolding in the whole pH region are discussed. The alkaline state is indistinguishable from the acid state in equilibrium and kinetic properties. Thus, as a first approximation, the total unfolding in the whole pH region can be expressed as a three-state mechanism involving the native (N), the intermediate (A), and the fully unfolded (D) states. The strong pH dependence of the N⇄A transition above pH 10 is almost entirely ascribable to the abnormal tyrosines in the N state previously detected by the pH-jump titration method, while the dependence between pH 7 and 10 also suggests the presence of an abnormal α-amino group. The normalization of most of the alkaline and the acidic abnormally ionizable groups in the N state occurs simultaneously in the first step of the unfolding pathway, i.e. , the forward activation of the N⇄A transition, and the final step of the folding may be associated with the interaction of the ionizable groups. Among the abnormally ionizable groups detected, the tyrosyl and carboxyl groups are most important in view of the large changes in their pK values, suggesting the presence of some interactions, even if only indirect, between these groups. Alignment data of amino acid residues also suggest that at least one such abnormal tyrosyl residue (Tyr 50) and its neighbors are conserved throughout in the α-lactalbumin-lysozyme group of proteins. Possible mechanisms of the interaction between the tyrosyl and carboxyl groups are discussed.