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

• Residues 49, 50, 57–60 were replaced with their counterparts from rat β-parvalbumin.
• E59D, alone or with the other mutations, confers Ca2 +-specificity on the CD site.
• In contrast to the CD site, EF-site affinity is not compromised by the mutations.
• The consequences of replacing L85 with phenylalanine were also examined.
• L85F improves Ca2 + affinity in wild-type rat α but has little impact in the variants.

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 Ca2 +-specificity on the CD site, in the presence or absence of the other mutations. Despite their minimal impact on ΔG, several CD-loop mutations markedly alter ΔH, evidently by perturbing the apo-protein conformation. The L85F mutation was also examined. In wild-type rat α, L85F increases EF-site Ca2 + affinity. In the CD-loop variants, the mutation leaves the ΔG for Ca2 +-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.

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