Ca(2+)- and H(+)-dependent conformational changes of calbindin D(28k).
Calbindin D(28k) is a member of a large family of intracellular Ca(2+) binding proteins characterized by EF-hand structural motifs. Some of these proteins are classified as Ca(2+)-sensor proteins, since they are involved in transducing intracellular Ca(2+) signals by exposing a hydrophobic patch on the protein surface in response to Ca(2+) binding. The hydrophobic patch serves as an interaction site for target enzymes. Other members of this group are classified as Ca(2+)-buffering proteins, because they remain closed after Ca(2+) binding and participate in Ca(2+) buffering and transport functions. ANS (8-anilinonaphthalene-1-sulfonic acid) binding and affinity chromatography on a hydrophobic column suggested that both the Ca(2+)-free and Ca(2+)-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 Ca(2+)-induced conformational change was readily detected by several optical spectroscopic methods. Thus, calbindin D(28k) shares some of the properties of Ca(2+)-sensor proteins. However, the Ca(2+)-induced change in exposed hydrophobic surface was considerably less pronounced than that in calmodulin. The data also shows that calbindin D(28k) 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 Ca(2+)- and H(+)-dependent manner.
Berggård
T
T
Physical Chemistry 2 and Department of Biochemistry, Chemical Centre, University of Lund, S-221 00 Lund, Sweden.
Silow
M
M
Thulin
E
E
Linse
S
S
D000817:Anilino Naphthalenesulfonates; D000818:Animals; D064026:Calbindins; D002118:Calcium / Q000494:pharmacology*; D002417:Cattle; D002645:Chickens; D002942:Circular Dichroism; D020832:EF Hand Motifs; D004926:Escherichia coli; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D064030:S100 Calcium Binding Protein G / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D014364:Tryptophan / Q000737:chemistry; D014508:Urea / Q000494:pharmacology
D000817:Anilino Naphthalenesulfonates; D064026:Calbindins; D011994:Recombinant Proteins; D064030:S100 Calcium Binding Protein G; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan; D014508:Urea; D002118:Calcium