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Isothermal titration calorimetry to determine association constants for high-affinity ligands

Abstract

An important goal in drug development is to engineer inhibitors and ligands that have high binding affinities for their target molecules. In optimizing these interactions, the precise determination of the binding affinity becomes progressively difficult once it approaches and surpasses the nanomolar level. Isothermal titration calorimetry (ITC) can be used to determine the complete binding thermodynamics of a ligand down to the picomolar range by using an experimental mode called displacement titration. In a displacement titration, the association constant of a high-affinity ligand that cannot be measured directly is artificially lowered to a measurable level by premixing the protein with a weaker competitive ligand. To perform this protocol, two titrations must be carried out: a direct titration of the weak ligand to the target macromolecule and a displacement titration of the high-affinity ligand to the weak ligand—target macromolecule complex. This protocol takes approximately 5 h.

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Figure 1: ITC determination of the binding thermodynamics of indinavir to the HIV-1 protease.

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Acknowledgements

This work was supported by US National Institutes of Health grants GM 57144 and GM 56550. We thank S. Vega for protease purification and A. Schön for indinavir purification.

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A.V.-C. and E.F. designed the experiments and wrote the paper; A.V.-C. performed the experiments and analyzed the data.

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Correspondence to Ernesto Freire.

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The authors declare no competing financial interests.

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Velazquez-Campoy, A., Freire, E. Isothermal titration calorimetry to determine association constants for high-affinity ligands. Nat Protoc 1, 186–191 (2006). https://doi.org/10.1038/nprot.2006.28

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