title: Quantifying high-affinity binding of hydrophobic ligands by isothermal titration calorimetry.
abstract: A fast and reliable quantification of the binding thermodynamics of hydrophobic high-affinity ligands employing a new calorimetric competition experiment is described. Although isothermal titration calorimetry is the method of choice for a quantitative characterization of intermolecular interactions in solution, a reliable determination of a dissociation constant (K(D)) is typically limited to the range 100 μM > K(D) > 1 nM. Interactions displaying higher or lower K(D) values can be assessed indirectly, provided that a suitable competing ligand is available whose K(D) falls within the directly accessible affinity window. This established displacement assay, however, requires the high-affinity ligand to be soluble at high concentrations in aqueous buffer and, consequently, poses serious problems in the study of protein binding involving small-molecule ligands dissolved in organic solvents--a familiar case in many drug-discovery projects relying on compound libraries. The calorimetric competition assay introduced here overcomes this limitation, thus allowing for a detailed thermodynamic description of high-affinity receptor-ligand interactions involving poorly water-soluble compounds. Based on a single titration of receptor into a dilute mixture of the two competing ligands, this competition assay provides accurate and precise values for the dissociation constants and binding enthalpies of both high- and moderate-affinity ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation and high-affinity protein-inhibitor interactions, and explore its potential and limitations with the aid of simulations and statistical analyses.
authors.affiliation: Molecular Biophysics, University of Kaiserslautern, Erwin-Schrödinger-Strasse 13, 67663 Kaiserslautern, Germany.
authors.forename: Georg
authors.initials: G
authors.lastname: Krainer
authors.forename: Jana
authors.initials: J
authors.lastname: Broecker
authors.forename: Carolyn
authors.initials: C
authors.lastname: Vargas
authors.forename: Jörg
authors.initials: J
authors.lastname: Fanghänel
authors.forename: Sandro
authors.initials: S
authors.lastname: Keller
mesh_terms: D002151:Calorimetry / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D008024:Ligands; D011485:Protein Binding / Q000502:physiology; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000378:metabolism
chemical_list: D008024:Ligands; D018160:Receptors, Cytoplasmic and Nuclear