Site-directed mutagenesis of rat α-parvalbumin: replacement of canonical CD-site residues with their non-consensus counterparts from rat β-parvalbumin

Abstract Rat β-parvalbumin (β-PV) displays low divalent-ion affinity. Its CD site is distinguished by six non-consensus residues – the “CD-loop residues” – at positions 49, 50, 57–60. Additionally, leucine occupies position 85, rather than phenylalanine, the β-lineage-consensus residue. Replacement of the CD-loop residues in rat β with the canonical residues was previously found to have little effect on divalent-ion affinity, unless L85 is replaced by phenylalanine. Herein, we replace the canonical CD-loop residues in rat α-PV with their rat β-PV counterparts. Although the mutations have a generally modest impact on affinity, E59D confers Ca 2 + -specificity on the CD site, in the presence or absence of the other mutations. Despite their minimal impact on Δ G , several CD-loop mutations markedly alter Δ H , evidently by perturbing the apo-protein conformation. The L85F mutation was also examined. In wild-type rat α, L85F increases EF-site Ca 2 + affinity. In the CD-loop variants, the mutation leaves the Δ G for Ca 2 + -binding largely unaffected. However, several variants display highly exothermic binding enthalpies, indicative of ligation-linked protein-folding. Consistent with that idea, scanning-calorimetry data confirm that L85F has significantly destabilized those proteins.