Ca2+- and H+-dependent conformational changes of calbindin D28k

Calbindin D-28k is a member of a large family of intracellular Ca2+ binding proteins characterized by EF-hand structural motifs. Some of these proteins are classified as Ca2+-sensor proteins, since they are involved in transducing intracellular Ca2+ signals by exposing a hydrophobic patch on the protein surface in response to Ca2+ binding. The hydrophobic patch serves as an interaction site for target enzymes. Other members of this group are classified as Ca2+ buffering proteins, because they remain closed after Ca2+ binding and participate in Ca2+ buffering and transport functions. ANS (8-anilinonaphthalene-1-sulfonic acid) binding and affinity chromatography on a hydrophobic column suggested that both the Ca2+-free and Ca2+-loaded form of calbindin D-28k have exposed hydrophobic surfaces. Since exposure of hydrophobic surface is unfavorable in the aqueous intracellular milieu, calbindin D-28k most likely interacts with other cellular components in vivo. A Ca2+-induced conformational change was readily detected by several optical spectroscopic methods. Thus, calbindin D-28k shares some of the properties of Ca2+-sensor proteins. However, the Ca2+-induced change in exposed hydrophobic surface was considerably less pronounced than that in calmodulin. The data also shows that calbindin D28k undergoes a rapid and reversible conformational change in response to a H+ 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-28k was increased in response to H+ in the range examined. The results suggest that calbindin D-28k may interact with targets in a Ca2+- and H+-dependent manner.