title: Single-experiment displacement assay for quantifying high-affinity binding by isothermal titration calorimetry.
abstract: Isothermal titration calorimetry (ITC) is the gold standard for dissecting the thermodynamics of a biomolecular binding process within a single experiment. However, reliable determination of the dissociation constant (KD) from a single titration is typically limited to the range 100 μM>KD>1 nM. Interactions characterized by a lower KD can be assessed indirectly by so-called competition or displacement assays, provided that a suitable competitive ligand is available whose KD falls within the directly accessible window. However, this protocol is limited by the fact that it necessitates at least two titrations to characterize one high-affinity inhibitor, resulting in considerable consumption of both sample material and time. Here, we introduce a fast and efficient ITC displacement assay that allows for the simultaneous characterization of both a high-affinity ligand and a moderate-affinity ligand competing for the same binding site on a receptor within a single experiment. The protocol is based on a titration of the high-affinity ligand into a solution containing the moderate-affinity ligand bound to the receptor present in excess. The resulting biphasic binding isotherm enables accurate and precise determination of KD values and binding enthalpies (ΔH) of both ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation, explore its potential and limitations with the aid of simulations and statistical analyses, and elaborate on potential applications to protein-inhibitor interactions.
authors.affiliation: Molecular Biophysics, University of Kaiserslautern, Erwin-Schrödinger-Str. 13, 67663 Kaiserslautern, Germany; B CUBE - Center for Molecular Bioengineering, Technische Universität Dresden, 01307 Dresden, Germany.
authors.forename: Georg
authors.initials: G
authors.lastname: Krainer
authors.affiliation: Molecular Biophysics, University of Kaiserslautern, Erwin-Schrödinger-Str. 13, 67663 Kaiserslautern, Germany. Electronic address: mail@sandrokeller.com.
authors.forename: Sandro
authors.initials: S
authors.lastname: Keller
mesh_terms: D002122:Calcium Chloride / Q000737:chemistry; D002151:Calorimetry / Q000379:methods*; D003198:Computer Simulation; D004492:Edetic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D004533:Egtazic Acid / Q000737:chemistry; D004791:Enzyme Inhibitors; D008024:Ligands; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D004791:Enzyme Inhibitors; D008024:Ligands; D011506:Proteins; D004533:Egtazic Acid; D004492:Edetic Acid; D002122:Calcium Chloride; C026060:N-(hydroxyethyl)ethylenediaminetriacetic acid
keywords: Competition assay; Metal ion chelation; Protein–inhibitor interactions; Protein–ligand interactions