Disulfide Determinants of Calcium-Induced Packing in α-Lactalbumin

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Abstract
Alpha-Lactalbumin (alpha-LA) is a two-domain calcium-binding protein that folds through a molten globule intermediate. Calcium binding to the wild-type alpha-LA molten globule induces a transition to the native state. Here we assess the calcium-binding properties of the alpha-LA molten globule by studying two variants of alpha-LA. alpha-LA(alpha) contains only the two disulfide bonds in the alpha-helical domain of alpha-LA, while alpha-LA(beta) contains only the beta-sheet domain and interdomain disulfide bonds. We found that only alpha-LA(beta) binds calcium, leading to the cooperative formation of substantial tertiary interactions. In addition, the beta-sheet domain acquires a native-like backbone topology. Thus, specific interactions within alpha-LA imposed by the beta-sheet domain and interdomain disulfide bonds, as opposed to the two alpha-helical domain disulfides, are necessary for the calcium-induced progression from the molten globule toward more native-like structure. Our results suggest that organization of the beta-sheet domain, coupled with calcium binding, comprises the locking step in the folding of alpha-LA from the molten globule to the native state.