A set of accurate experimental data is provided for Ca2+ ion binding to calbindin D9k, a protein in the calmodulin superfamily of intracellular regulatory proteins. The study comprises both the role of protein surface charges and the effects of added electrolyte. The two macroscopic Ca2+-binding constants K1 and K2 are determined for the wild-type and eight mutant calbindins in 0, 0.05, 0.10, and 0.15 M KCl from titrations in the presence of Quin 2 or 5,5'-Br2BAPTA. The mutations involve replacement of surface carboxylates (of Glu17, Asp19, Glu26, and Glu60) with the corresponding amides. It is found that K1K2 may decrease by a factor of up to 2.5 X 10(5) (triple mutant in 0.15 M KCl as compared to the wild-type protein in 0 M KCl). Ca2+-binding constants of the individual Ca2+ sites (microscopic binding constants) have also been determined. The positive cooperativity of Ca2+ binding, previously observed at low salt concentration [Linse et al. (1987) Biochemistry 26, 6723-6735], is also present at physiological ionic strength and amounts to 5 kJ.mol-1 at 0.15 M KCl. The electrolyte concentration and some of the mutations are found to affect the cooperativity. K-39 NMR studies show that K+ binds weakly to calbindin. Two-dimensional H-1 NMR studies show, however, that potassium binding does not change the protein conformation, and the large effect of KCl on the Ca2+ affinity is thus of unspecific nature. Two-dimensional H-1 NMR has also been used to assess the structural consequences of the mutations through assignments of the backbone NH and C-alpha-H resonances of six mutants. Comparison with the corresponding chemical shifts in the wild-type protein shows that the mutations E17Q, E26Q, and E60Q do not affect the structure of the protein. The D19N mutation causes a minor rearrangement in the loop region, but the three-dimensional structure appears to be largely similar to the wild type also in this mutant. Assignment of one double (E17Q + E26Q) and the triple (E17Q + D19N + E26Q) mutant shows that the chemical shift effects of the individual substitutions are essentially additive. The presented data may serve as a basis for evaluating different theoretical models of electrostatic interactions in proteins.
