title: Characterization of the metal ion-binding domains from rat alpha- and beta-parvalbumins.
abstract: 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 Ca(2+)-free alpha CD-EF is monomeric, the Ca(2+)-free beta peptide dimerizes weakly (K(2) = 2400 +/- 200 M(-1)). In buffer containing 1.0 mM Ca(2+), 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+), alpha CD-EF exhibits higher thermal stability, consistent with its higher Ca(2+) 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 Ca(2+)-free state, suggesting that the apo-peptides are disordered.
authors.affiliation: Department of Biochemistry, University of Missouri-Columbia, Columbia, Missouri 65211, USA. henzlm@missouri.edu
authors.forename: Michael T
authors.initials: MT
authors.lastname: Henzl
authors.forename: Sayeh
authors.initials: S
authors.lastname: Agah
authors.forename: John D
authors.initials: JD
authors.lastname: Larson
mesh_terms: D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008954:Models, Biological; D010320:Parvalbumins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013725:Terbium / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D010320:Parvalbumins; D010446:Peptide Fragments; D020033:Protein Isoforms; D011994:Recombinant Proteins; D013725:Terbium; D002118:Calcium