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

• Published in: Biophysical chemistry Vol. 197; pp. 25 - 39
• Main Authors: Henzl, Michael T., Sirianni, Arthur G., Markus, Lindsey A., Davis, Christine M.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.02.2015
• Subjects: Amino Acid Sequence; Animals; Binding Sites; Ca2 +-binding proteins; Calcium - metabolism; Calcium-Binding Proteins - chemistry; Calcium-Binding Proteins - genetics; Calcium-Binding Proteins - metabolism; DSC; EF-hand proteins; ITC; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Oncomodulin; Parvalbumin; Parvalbumins - chemistry; Parvalbumins - genetics; Parvalbumins - metabolism; Protein Binding; Protein Conformation; Rats; Thermodynamics; Ca2+-binding proteins; ITC; EF-hand proteins; Parvalbumin; DSC; Oncomodulin; Ca(2+)-binding proteins
• ISSN: 0301-4622; 1873-4200
• DOI: 10.1016/j.bpc.2014.12.002

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. [Display omitted] •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.