Calbindin D(9k): a protein optimized for calcium binding at neutral pH.
The binding of calcium ions by EF-hand proteins depends strongly on the electrostatic interactions between Ca(2+) ions and negatively charged residues of these proteins. We have investigated the pH dependence of the binding of Ca(2+) ions by calbindin D(9k). This protein offers a unique possibility for interpretation of such data since the pK(a) values of all ionizable groups are known. The binding is independent of pH between 7 and 9, where maximum calcium affinity is observed. An abrupt decrease in the binding affinity is observed at pH values below 7. This decrease is due to protonation of acidic groups, leading to modification of protein charges. The pH dependence of the product of the two macroscopic Ca(2+)-binding constants can be formally described by the involvement of two acidic groups with pK(a) = 6.6. Monte Carlo calculations show that the reduction of Ca(2+) binding is strictly determined by variable electrostatic interactions due to pH-dependent changes not only in the binding sites, but also of the overall charge of the protein.
Kesvatera
T
T
Departments of Biophysical Chemistry and Theoretical Chemistry, Lund University, P.O. Box 124, S-221 00 Lund, Sweden.
Jönsson
B
B
Telling
A
A
Tõugu
V
V
Vija
H
H
Thulin
E
E
Linse
S
S
D064026:Calbindins; D002118:Calcium / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D009010:Monte Carlo Method; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D064030:S100 Calcium Binding Protein G / Q000737:chemistry* / Q000378:metabolism*; D055672:Static Electricity
D064026:Calbindins; D011994:Recombinant Proteins; D064030:S100 Calcium Binding Protein G; D002118:Calcium