Characterization of the metal ion-binding domains from rat α- and β-parvalbumins

We have examined the metal ion-binding domains from rat (alpha and beta parvalbumin. We find that the CD-EF fragments differ markedly in their tendency to self-associate. Whereas Ca2+-free alpha CD-EF is monomeric, the Ca2+-free beta peptide dimerizes weakly (K-2 = 2400 +/- 200 M-1). In buffer containing 1.0 mM Ca2+, the apparent dimerization constant for beta CD-EF (191 000 +/- 29 000 M-1) is more than 50 times that of alpha (3400 +/- 200 M-1). alpha CD-EF binds two Ca2+ with positive cooperativity. Titration calorimetry data afford binding constants of 3.7(0.1) x 10(3) M-1 and 8.6(0.2) x 10(4) M-1. beta CD-EF also binds two Ca2+ cooperatively but with lower affinity. Equilibrium dialysis yields Adair constants of 4.2(0.1) x 10(3) and 6.1(0.2) x 10(3) M-1. Significantly, the difference in Ca2+ affinity is substantially smaller than that observed for the full-length proteins-suggesting that the AB domain can modulate divalent ion affinity. Analysis of beta calorimetry data requires explicit consideration of the self-association behavior. Data collected at low CD-EF concentration are consistent with preferential occupation of the EF site, dimerization of singly bound monomers, and cooperative filling of the CD sites. At higher concentrations, apo-protein dimerization can apparently precede cooperative occupation of the EF sites. In the presence of Ca2+, alpha CD-EF exhibits higher thermal stability, consistent with its higher Ca2+ affinity. However, the beta melting temperature shows greater concentration dependence, consistent with its greater tendency to dimerize. Neither fragment exhibits a sigmoidal melting curve in the Ca2+-free state, suggesting that the apo-peptides are disordered.