INACTIVATION OF INDIVIDUAL CA2+-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 Ca2+-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 K-Ca 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 K-Mg = 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 K-Mg 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. The fluorescence spectra of the metal-free, Ca2+, and Mg2+ forms of PV-CD are very similar to those of the parent mutant PVF102W In contrast, the spectra of PV-EF indicate the presence of a much less organized hydrophobic core, especially in the metal-free form. The Trp fluorescence of PV-CD/-EF is not sensitive to Ca2+ or Mg2+ and resembles that of metal-free PV-EF. The comparison of ligand-induced UV difference spectra of PV-CD and PV-EF confirms that Trp-102 is located in a hydrophobic core and that inactivation of the EF loop, but not of the CD loop, strongly destabilizes the protein in the absence of Ca2+, thus explaining the impaired cation affinity of PV-EF. Our data indicate that in rat parvalbumin the EF-hand site is the principal structural nucleus of the paired domain.