Structural basis for difference in heat capacity increments for Ca(2+) binding to two alpha-lactalbumins.

Thermodynamic parameters for the unfolding of as well as for the binding of Ca(2+) to goat alpha-lactalbumin (GLA) and bovine alpha-lactalbumin (BLA) are deduced from isothermal titration calorimetry in a buffer containing 10 mM Tris-HCl, pH 7.5 near 25 degrees C. Among the different parameters available, the heat capacity increments (Delta C(p)) offer the most direct information for the associated conformational changes of the protein variants. The Delta C(p) values for the transition from the native to the molten globule state are rather similar for both proteins, indicating that the extent of the corresponding conformational change is nearly identical. However, the respective Delta C(p) values for the binding of Ca(2+) are clearly different. The data suggest that a distinct protein region is more sensitive to a Ca(2+)-dependent conformational change in BLA than is the case in GLA. By analysis of the tertiary structure we observed an extensive accumulation of negatively charged amino acids near the Ca(2+)-binding site of BLA. In GLA, the cluster of negative charges is reduced by the substitution of Glu-11 by Lys. The observed difference in Delta C(p) values for the binding of Ca(2+) is presumably in part related to this difference in charge distribution.