title: Simultaneous addition of two ligands: a potential strategy for estimating divalent ion affinities in EF-hand proteins by isothermal titration calorimetry.
abstract: 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. Although global analysis of multiple ITC experiments can overcome these hurdles, our current experimental protocol includes upwards of 10 titrations - requiring a substantial investment in labor, machine time, and material. This paper explores the potential for using a smaller suite of experiments that includes simultaneous titrations with Ca(2+) and Mg(2+) at different ratios of the two ions. The results obtained for four proteins, differing substantially in their divalent ion-binding properties, suggest that the approach has merit. The Ca(2+)- and Mg(2+)-binding constants afforded by the streamlined analysis are in reasonable agreement with those obtained from the standard analysis protocol. Likewise, the abbreviated analysis provides comparable values for the Ca(2+)-binding enthalpies. However, the streamlined analysis can yield divergent values for the Mg(2+)-binding enthalpies - particularly those for lower affinity sites. This shortcoming can be remedied, in large measure, by including data from a direct Ca(2+) titration in the presence of a high, fixed Mg(2+) concentration.
authors.affiliation: Department of Biochemistry, 117 Schweitzer Hall, University of Missouri, Columbia, MO 65211, United States. henzlm@missouri.edu
authors.forename: Michael T
authors.initials: MT
authors.lastname: Henzl
authors.forename: Lindsey A
authors.initials: LA
authors.lastname: Markus
authors.forename: Meredith E
authors.initials: ME
authors.lastname: Davis
authors.forename: Andrew T
authors.initials: AT
authors.lastname: McMillan
mesh_terms: D000818:Animals; D001717:Birds; D002118:Calcium / Q000737:chemistry; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002151:Calorimetry / Q000379:methods*; D020832:EF Hand Motifs; D006168:Guinea Pigs; D008024:Ligands; D008274:Magnesium / Q000737:chemistry; D010320:Parvalbumins / Q000737:chemistry; D031784:Phleum; D010940:Plant Proteins / Q000737:chemistry; D051381:Rats; D013816:Thermodynamics
chemical_list: D002135:Calcium-Binding Proteins; D008024:Ligands; D010320:Parvalbumins; D010940:Plant Proteins; C070978:avian thymic hormone; D008274:Magnesium; D002118:Calcium