Characterization of the Metal Ion-Binding Domains from Rat α- and β-Parvalbumins

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Abstract
We have examined the metal ion-binding domains from rat α and β parvalbumin. We find that the CD-EF fragments differ markedly in their tendency to self-associate. Whereas Ca2+-free α CD-EF is monomeric, the Ca2+-free β peptide dimerizes weakly (K2 = 2400 ± 200 M-1). In buffer containing 1.0 mM Ca2+, the apparent dimerization constant for β CD-EF (191 000 ± 29 000 M-1) is more than 50 times that of α (3400 ± 200 M-1). α CD-EF binds two Ca2+ with positive cooperativity. Titration calorimetry data afford binding constants of 3.7(0.1) × 103 M-1 and 8.6(0.2) × 104 M-1. β CD-EF also binds two Ca2+ cooperatively but with lower affinity. Equilibrium dialysis yields Adair constants of 4.2(0.1) × 103 and 6.1(0.2) × 103 M-1. Significantly, the difference in Ca2+ affinity is substantially smaller than that observed for the full-length proteinssuggesting that the AB domain can modulate divalent ion affinity. Analysis of β 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+, α CD-EF exhibits higher thermal stability, consistent with its higher Ca2+ affinity. However, the β 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.