PKa calculations of calbindin D9k: Effects of Ca2 binding, protein dielectric constant, and ionic strength

Abstract

Calbindin is a small (75 residues) helix-loop-helix (“EF-hand”) calcium-binding protein belonging to the calmodulin superfamily. It binds two Ca2+ ions. Continuum electrostatics in combination with the boundary element method was employed for the calculation of the acid-dissociation constants Ka (pKa = −log Ka) values of all titratable residues in the protein. The objectives were to determine quantitatively the effects of divalent ion binding and small ion-induced structural changes on predicted pKa's. Computations were carried out for the apo and holo form of calbindin, for which both X-ray and NMR structures were available. Comparison was made with several sets of experimental pKa values determined by NMR spectroscopy. Different choices of the dielectric constant (ranging from 4 to 78.5) for calbindin and variations in ionic strength (from 0 to 0.3 M) were investigated in a systematic fashion. Removal of the two bound Ca2+ ions increases the pKa values of all residues if no conformational changes were allowed. If conformational differences between the apo and holo were accounted for, shifts in either direction were observed. Titrating groups that are directly involved in Ca2+ binding (Asp and Glu) required a dielectric constant of 78.5 for the holo structure to obtain a reasonable estimate of their pKa's. For the apo structure, passable values for the pKa's of these ligating groups could be determined if the structure was allowed to relax upon ion removal. Proteins 2000;41:554–567. © 2000 Wiley-Liss, Inc.