Inactivation of individual Ca(2+)-binding sites in the paired EF-hand sites of parvalbumin reveals asymmetrical metal-binding properties.
Previously a rat parvalbumin mutant protein PVF102W was constructed with a reporter Trp at position 102 in the middle of the hydrophobic center [Pauls, T. L., et al. (1993) J. Biol. Chem. 268, 20897-20903]. In the present study three new parvalbumin mutant proteins, derived from PVF102W and containing alterations at positions essential for Ca2+ binding in either one of the two Ca(2+)-binding sites (PV-CD and PV-EF) or in both (PV-CD/-EF), were expressed and purified. With the flow dialysis method it was established that both PV-CD and PV-EF bind 1 Ca2+ with affinity constants KCa of 1.1 x 10(7) and 3.2 x 10(6) M-1, respectively. Mg2+ binding, monitored by equilibrium gel filtration in the absence of Ca2+, showed that both mutants bind 1 Mg2+ with KMg = 8 10(4) for PV-CD and 3 x 10(3) M-1 for PV-EF. Compared to the parameters of the parent mutant PVF102W (two sites with equal affinities of 2.7 x 10(7) and 3 x 10(4) M-1 for Ca2+ and Mg2+, respectively), these data indicate that inactivation of the EF site, much more than of the CD site, impairs divalent cation binding. The binding of Ca2+ and Mg2+ is mutually exclusive, indicative of a Ca2+/Mg2+ mixed site. However, as for PVF102W, the KMg values obtained from the competition equation are approximately 40-fold lower than the affinities measured by direct binding. PV-CD/-EF binds neither Ca2+ nor Mg2+. Trp fluorimetry revealed that in the three mutant PVs the residue Trp-102 is deeply buried in the hydrophobic core.(ABSTRACT TRUNCATED AT 250 WORDS)
Pauls
T L
TL
Institute of Veterinary Biochemistry, University of Zürich-Irchel, Switzerland.
Durussel
I
I
Berchtold
M W
MW
Cox
J A
JA
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D066298:In Vitro Techniques; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010320:Parvalbumins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D018076:DNA, Complementary; D010320:Parvalbumins; D011994:Recombinant Proteins; D008274:Magnesium; D002118:Calcium