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

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.

Simultaneous addition of two ligands: a potential strategy for estimating divalent ion affinities in EF-hand proteins by isothermal titration calorimetry.

Capable of providing a detailed thermodynamic picture of noncovalent association reactions, isothermal titration calorimetry (ITC) has become a popular method for studying protein-ligand interactions. We routinely employ the technique to study divalent ion-binding by two-site EF-hand proteins from the parvalbumin- and polcalcin lineages. The combination of high Ca(2+) affinity and relatively low Mg(2+) affinity, and the attendant complication of parameter correlation, conspire to make the simultaneous extraction of binding constants and -enthalpies for both ions challenging.

Disparate impact of the S33V mutation on conformational stability in rat β-parvalbumin (oncomodulin) and chicken parvalbumin 3.

Rat β-parvalbumin (β-PV) and chicken parvalbumin 3 (CPV3) exhibit diminished Ca(2+) affinity. Their sequences, 70% identical, are unusual in that serine replaces the consensus residue, valine, at position 33. Reasoning that the substitution of a compact, polar hydroxymethyl moiety for a bulky, apolar isopropyl group might contribute to the attenuated Ca(2+) affinities, we have characterized the S33V variants of both proteins.