The region of alpha-lactalbumin recognized by GroEL

The binding constants between disulfide-intact or various disulfide-reduced bovine alpha-lactalbumins and an Escherichia coli chaperonin, GroEL, were determined by using the equilibrium dialysis method. The disulfide-intact and one-disulfide (Cys6-Cys120)-reduced alpha-lactalbumins were shown not to bind with GroEL both in the presence and absence of Ca2+. The two-disulfide (Cys6-Cys120 and Cys28-Cys111)-reduced alpha-lactalbumin, which has the native-like tertiary structure in its beta-domain region and an unfolded alpha-domain in the presence of Ca2+, showed considerable binding with GroEL. The binding free energy of the two-disulfide-reduced alpha-lactalbumin in the presence of Ca2+ is close to that of the molten globule state of disulfide-intact alpha-lactalbumin. This result suggests that GroEL binds to the unfolded alpha-domain of alpha-lactalbumin regardless of the conformation of the beta-domain. The fully disulfide-reduced and two-disulfide-reduced alpha-lactalbumins were found to bind more strongly with GroEL in the absence of Ca2+ than the two-disulfide-reduced alpha-lactalbumin in the presence of Ca2+, thus indicating that the unfolding of the beta-domain of alpha-lactalbumin leads to stronger interaction with GroEL.