Calbindin D-28k is a member of a large family of intracellular Ca2+ binding
proteins characterized by EF-hand structural motifs. Some of these protein
s are classified as Ca2+-sensor proteins, since they are involved in transd
ucing intracellular Ca2+ signals by exposing a hydrophobic patch on the pro
tein surface in response to Ca2+ binding. The hydrophobic patch serves as a
n interaction site for target enzymes. Other members of this group are clas
sified as Ca2+ buffering proteins, because they remain closed after Ca2+ bi
nding and participate in Ca2+ buffering and transport functions. ANS (8-ani
linonaphthalene-1-sulfonic acid) binding and affinity chromatography on a h
ydrophobic column suggested that both the Ca2+-free and Ca2+-loaded form of
calbindin D-28k have exposed hydrophobic surfaces. Since exposure of hydro
phobic surface is unfavorable in the aqueous intracellular milieu, calbindi
n D-28k most likely interacts with other cellular components in vivo. A Ca2
+-induced conformational change was readily detected by several optical spe
ctroscopic methods. Thus, calbindin D-28k shares some of the properties of
Ca2+-sensor proteins. However, the Ca2+-induced change in exposed hydrophob
ic surface was considerably less pronounced than that in calmodulin. The da
ta also shows that calbindin D28k undergoes a rapid and reversible conforma
tional change in response to a H+ concentration increase within the physiol
ogical 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 respons
e to H+ in the range examined. The results suggest that calbindin D-28k may
interact with targets in a Ca2+- and H+-dependent manner.