Pathway of oxidative folding of α-lactalbumin:: A model for illustrating the diversity of disulfide folding pathways

The pathway of oxidative folding of alpha-lactalbumin (alphaLA) (four disulfide bonds) has been characterized by structural and kinetic analysis of the acid-trapped folding intermediates. In the absence of calcium, oxidative folding of alphaLA proceeds through highly heterogeneous species of one-, two-, three-, and four-disulfide (scrambled) intermediates to reach the native structure. In the presence of calcium, the folding intermediates of alphaLA comprise two predominant isomers (alphaLA-IIA and alphaLA-IIIA) adopting exclusively native disulfide bonds, including the two disulfide bonds (Cys(61)-Cys(77) and Cys(73)-Cys(91)) located within the beta-sheet calcium binding domain. alphaLA-IIA is a two-disulfide species consisting of Cys(61)-Cys(77) and Cys(73)-Cys(91) disulfide bonds. alphaLA-IIIA contains Cys(61)-Cys(77), Cys(73)-Cys(91), and Cys(28)-Cys(111) disulfide bonds. The underlying mechanism of the contrasting folding pathways of calcium-bound and calcium-depleted alphaLA is congruent with the cause of diversity of disulfide folding pathways observed among many well-characterized three-disulfide proteins, including bovine pancreatic trypsin inhibitor and hirudin. Our study also reveals novel aspects of the folding, mechanism of alphaLA that have not been described previously.