title: 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 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 Ca(2+) affinity. In the CD-loop variants, the mutation leaves the ΔG 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.
authors.affiliation: Department of Biochemistry, University of Missouri, Columbia, MO, USA 65211. Electronic address: henzlm@missouri.edu.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Henzl
authors.affiliation: Department of Biochemistry, University of Missouri, Columbia, MO, USA 65211.
authors.forename: Arthur G
authors.initials: AG
authors.lastname: Sirianni
authors.affiliation: Department of Biochemistry, University of Missouri, Columbia, MO, USA 65211.
authors.forename: Lindsey A
authors.initials: LA
authors.lastname: Markus
authors.affiliation: Department of Biochemistry, University of Missouri, Columbia, MO, USA 65211.
authors.forename: Christine M
authors.initials: CM
authors.lastname: Davis
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010320:Parvalbumins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D013816:Thermodynamics
chemical_list: D002135:Calcium-Binding Proteins; D010320:Parvalbumins; D002118:Calcium
keywords: Ca(2+)-binding proteins; DSC; EF-hand proteins; ITC; Oncomodulin; Parvalbumin