Ca super(2+)- and H super(+)-Dependent Conformational Changes of Calbindin D sub(28k)

Calbindin D sub(28k) is a member of a large family of intracellular Ca super(2+) binding proteins characterized by EF-hand structural motifs. Some of these proteins are classified as Ca super(2+)-sensor proteins, since they are involved in transducing intracellular Ca super(2+) signals by exposing a hydrophobic patch on the protein surface in response to Ca super(2+) binding. The hydrophobic patch serves as an interaction site for target enzymes. Other members of this group are classified as Ca super(2+)-buffering proteins, because they remain closed after Ca super(2+) binding and participate in Ca super(2+) buffering and transport functions. ANS (8-anilinonaphthalene-1-sulfonic acid) binding and affinity chromatography on a hydrophobic column suggested that both the Ca super(2+)-free and Ca super(2+)-loaded form of calbindin D sub(28k) have exposed hydrophobic surfaces. Since exposure of hydrophobic surface is unfavorable in the aqueous intracellular milieu, calbindin D sub(28k) most likely interacts with other cellular components in vivo. A Ca super(2+)-induced conformational change was readily detected by several optical spectroscopic methods. Thus, calbindin D sub(28k) shares some of the properties of Ca super(2+)-sensor proteins. However, the Ca super(2+)-induced change in exposed hydrophobic surface was considerably less pronounced than that in calmodulin. The data also shows that calbindin D sub(28k) undergoes a rapid and reversible conformational change in response to a H super(+) concentration increase within the physiological pH range. The pH-dependent conformational change was shown to reside mainly in EF-hands 1-3. Urea-induced unfolding of the protein at pH 6, 7, and 8 showed that the stability of calbindin D sub(28k) was increased in response to H super(+) in the range examined. The results suggest that calbindin D sub(28k) may interact with targets in a Ca super(2+)- and H super(+)-dependent manner.