Single-experiment displacement assay for quantifying high-affinity binding by isothermal titration calorimetry

Area of Science:

• Biochemistry
• Biophysical Chemistry
• Thermodynamics

Background:

• Isothermal titration calorimetry (ITC) is crucial for determining biomolecular binding thermodynamics.
• Standard ITC is limited to dissociation constants (KD) between 1 nM and 100 μM.
• Existing competition assays require multiple experiments, consuming significant time and resources.

Purpose of the Study:

• To develop a fast and efficient ITC displacement assay.
• To enable simultaneous characterization of high- and moderate-affinity ligands.
• To accurately determine KD values and binding enthalpies (ΔH) for both ligands in a single experiment.

Main Methods:

• A novel ITC displacement assay protocol.
• Titration of a high-affinity ligand into a receptor-bound moderate-affinity ligand solution.
• Analysis of the resulting biphasic binding isotherm.

Main Results:

• Simultaneous determination of KD and ΔH for both high- and moderate-affinity ligands.
• Demonstrated application in metal ion chelation studies.
• Validated through simulations and statistical analyses.

Conclusions:

• The developed ITC displacement assay offers a significant improvement over traditional methods.
• It provides accurate and precise thermodynamic data for competing ligands.
• Potential applications include characterizing protein-inhibitor interactions and other high-affinity binding events.