Crystal structure of a high-affinity variant of rat α-parvalbumin

In model peptide systems, Ca2+ affinity is maximized in EF-hand motifs containing four carboxylates positioned on the +x and -x and +z and -z axes; introduction of a fifth carboxylate ligand reduces the affinity. However, in rat β-parvalbumin, replacement of Ser-55 with aspartate heightens divalent ion affinity [Henzl, M. T., et al. (1996) Biochemistry 35, 5856-5869]. The corresponding α-parvalbumin variant (S55D/E59D) likewise exhibits elevated affinity [Henzl, M. T., et al. (2003) Anal. Biochem. 319, 216-233]. To determine whether these mutations produce a variation on the archetypal EF-hand coordination scheme, we have obtained high-resolution X-ray crystallographic data for α S55D/E59D. As anticipated, the aspartyl carboxylate replaces the serine hydroxyl at the +z coordination position. Interestingly, the Asp-59 carboxylate abandons the role it plays as an outer sphere ligand in wild-type rat β, rotating away from the Ca2+ and, instead, forming a hydrogen bond with the amide of Glu-62. Superficially, the coordination sphere in the CD site of α S55D/E59D resembles that in the EF site. However, the orientation of the Asp-59 side chain is predicted to stabilize the D-helix, which may contribute to the heightened divalent ion affinity. DSC data indicate that the α S55D/E59D variant retains the capacity to bind 1 equiv of Na+. Consistent with this finding, when binding measurements are conducted in K+-containing buffer, divalent ion affinity is markedly higher. In 0.15 M KCl and 0.025 M Hepes-KOH (pH 7.4) at 5 °C, the macroscopic Ca2+ binding constants are 1.8 × 1010 and 2.0 × 109 M-1. The corresponding Mg2+ binding constants are 2.7 × 106 and 1.2 × 105 M-1

Conformational stabilities of the rat α- and β-parvalbumins

AbstractIt is widely believed that β-parvalbumin (PV) isoforms are intrinsically less stable than α-parvalbumins, due to greater electrostatic repulsion and an abbreviated C-terminal helix. However, when examined by differential scanning calorimetry, the apo-form of the rat β-PV (i.e. oncomodulin) actually displays greater thermal stability than the α-PV. Whereas the melting temperature of the α isoform is 45.8°C at physiological pH and ionic strength, the Tm for the β isoform is more than 7° higher (53.6°C). This result suggests that factors besides net charge and C-terminal helix length strongly influence parvalbumin conformational stability. Extension of the F helix in the β-PV, by insertion of Ser-109, has a modest stabilizing effect, raising the Tm by 1.1°. Truncation of the α-PV F helix, by removal of Glu-108, has a more profound impact, lowering the Tm by 4.0°