In the native calcium-binding protein calbindin D9k (M(r) 8.700; 75aa; 2 EF-hands), the backbone carbonyl oxygen of Glu60 coordinates the Ca2+ ion in the C-terminal site (site II). The carboxylate group of the same residue forms a hydrogen bond to a water molecule that constitutes a Ca2+ ligand in the N-terminal site (site I). The mutant E60D, with the charge-conserving substitution Glu60-->Asp, has been prepared to study the role of Glu60 in subjoining the two Ca(2+)-binding sites and its role in the cooperative Ca2+ binding. Ca(2+)-binding studies of the mutant show that the overall affinity for calcium has decreased by a factor of 38 in comparison with wild-type calbindin D9k. The largest reduction is seen in the first macroscopic binding step. The Ca2+ affinities for both sites in the protein are reduced to a similar extent. In contrast, the mutation leads to a large increase in the cooperativity of calcium binding. Differential scanning calorimetry has been used to determine the thermal stability which is almost as high as in the wild-type protein. Cadmium binding has been assessed with 1H and 113Cd NMR. X-ray crystallographic studies of the E60D mutant in its calcium-bound form show very small structural changes relative to the wild-type protein. Almost all differences are within the error limits of the method. The largest crystallographic effects are seen in the crystal packing. Two E60D molecules with slightly different structure are found in the asymmetric unit in contrast to the single molecule in the wild-type crystal.(ABSTRACT TRUNCATED AT 250 WORDS)

The EF-hand calcium.binding protein from Saccharopoly~pora erythraea has been shown, using ~3Cd NMR, to possess three Cda*-ion binding sites. This indicates that of the four EF-hand motifs in the molecule, one (probably site 2) is unable to bind Cd2"-ions. Data from the titration of the protein with Ca:', in the presence of Quin2, were fitted to a curve calculated on the assumption that the protein contains three high affinity Ca 2" binding sites, two of which (PKt = $.0, pKa = 9.0) are strongly cooperative, and one single site (pK~ = 7.5). Preliminary ~H NMR experiments indicate marked structural changes upon Caa*-bindins.