DISULFIDE DETERMINANTS OF CALCIUM-INDUCED PACKING IN ALPHA-LACTALBUMIN

alpha-Lactalbumin (alpha-LA) is a two-domain calcium-binding protein t hat folds through a molten globule intermediate. Calcium binding to th e 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) c ontains only the two disulfide bonds in the alpha-helical domain of al pha-LA, while alpha-LA(beta) contains only the beta-sheet domain and i nterdomain disulfide bonds. We find that only alpha-LA(beta) binds cal cium, leading to the cooperative formation of substantial tertiary int eractions. In addition, the beta-sheet domain acquires a native-like b ackbone topology. Thus, specific interactions within alpha-LA imposed by the beta-sheet domain and interdomain disulfide bonds, as opposed t o the two alpha-helical domain disulfides, are necessary for the calci um-induced progression from the molten globule toward more nativelike structure. Our results suggest that organization of the beta-sheet dom ain, coupled with calcium binding, comprises the locking step in the f olding of alpha-LA from the molten globule to the native state.