Abstract
The triggering of Ca^2+^ signaling pathways relies on Ca^2+^/Mg^2+^ specificity of proteins mediating these pathways. Two homologous milk Ca^2+^‐binding proteins, bovine α‐lactalbumin (bLA) and equine lysozyme (EQL), were analyzed using the simplest “four‐state” scheme of metal‐ and temperature‐induced structural changes in a protein. The association of Ca^2+^/Mg^2+^ by native proteins is entropy‐driven. Both proteins exhibit strong temperature dependences of apparent affinities to Ca^2+^ and Mg^2+^, due to low thermal stabilities of their apo‐forms and relatively high unfavorable enthalpies of Mg^2+^ association. The ratios of their apparent affinities to Ca^2+^ and Mg^2+^, being unusually high at low temperatures (5.3–6.5 orders of magnitude), reach the values inherent to classical EF‐hand motifs at physiological temperatures. The comparison of phase diagrams predicted within the model of competitive Ca^2+^ and Mg^2+^ binding with experimental data strongly suggests that the association of Ca^2+^ and Mg^2+^ ions with bLA is a competitive process, whereas the primary Mg^2+^ site of EQL is different from its Ca^2+^‐binding site. The later conclusion is corroborated by qualitatively different molar ellipticity changes in near‐UV region accompanying Mg^2+^ and Ca^2+^ association. The Ca^2+^/Mg^2+^ selectivity of Mg^2+^‐site of EQL is below an order of magnitude. EQL exhibits a distinct Mg^2+^‐specific site, probably arising as an adaptation to the extracellular environment. Proteins 2010. © 2010 Wiley‐Liss, Inc.