CA-43 NMR-STUDIES OF CALCIUM-BINDING LYSOZYMES AND ALPHA-LACTALBUMINS

The calcium-binding properties of equine and pigeon lysozyme as well as those of bovine and human alpha-lactalbumin were investigated by Ca-43 NMR spectroscopy. All proteins were found to contain one high-affinity calcium-binding site. The chemical shifts, line widths, relaxation times (T1 and T2), and quadrupole coupling constants for the respective Ca-43 NMR signals were quite similar; this is indicative of a high degree of homology between the strong calcium-binding sites of these four proteins. The measured chemical shifts (delta almost-equal-to -3 to -7 ppm) and quadrupole coupling constants (chi almost-equal-to 0.7-0.8 MHz) are quite distinct from those observed for typical EF-hand calcium-binding proteins, suggesting a different geometry for the calcium-binding loops. The correlation times for bound calcium ions in these proteins were on the order of 4-8 ns, indicating that the flexibilities of these binding sites are limited. The apparent pK(a) values for the high-affinity sites ranged from 3.4 to 4.7, confirming the participation of carboxylate-containing residues in the coordination of the calcium ion. Competition experiments with EDTA showed that the affinities of these proteins for calcium follow the series bovine a-lactalbumin almost-equal-to human alpha-lactalbumin > pigeon lysozyme > equine lysozyme (K(D) almost-equal-to 5 X 10(-8) to 10(-6) M). Evidence for the existence of a second weak calcium-binding site (K(D) = 3 X 10(-3) M) was obtained for bovine a-lactalbumin, but not for the other proteins studied. The involvement of a histidine residue(s) at this second site was inferred from the unusual pH dependence (pK(a)' = 6.3) of the line width of its Ca-43 NMR signal. The Ca-43 NMR findings presented in this paper provide direct spectroscopic evidence in support of an evolutionary link between the functionally distinct lysozymes and alpha-lactalbumins.