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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Sigurskjold, B.W. Exact analysis of competition ligand binding by displacement isothermal titration calorimetry. Anal. Biochem. 277, 260–266 (2000).
Velazquez-Campoy, A. & Freire E. ITC in the post-genomic era...? Priceless. Biophys. Chem. 115, 115–124 (2005).
Velazquez-Campoy, A. & Freire, E. Incorporating target heterogeneity in drug design. J. Cell. Biochem. S37, 82–88 (2001).
Velazquez-Campoy, A., Kiso, Y. & Freire, E. The binding energetics of first- and second-generation HIV-1 protease inhibitors: implications for drug design. Arch. Biochem. Biophys. 390, 169–175 (2001).
Ohtaka, H., Velazquez-Campoy, A., Xie, D. & Freire, E. Overcoming drug resistance in HIV-1 chemotherapy: The binding thermodynamics of amprenavir and TMC-126 to wild-type and drug-resistant mutants of the HIV-1 protease. Protein Sci. 11, 1908–1916 (2002).
Velazquez-Campoy, A., Vega, S. & Freire, E. Amplification of the effects of drug resistance mutations by background polymorphisms in HIV-1 protease from African subtypes. Biochemistry 41, 8613–8619 (2002).
Bradshaw, J.M., Mitaxov, V. & Waksman, G. Investigation of phosphotyrosine recognition by the SH2 domain of the Src kinase. J. Mol. Biol. 293, 971–985 (1999).
Zhang, Y.-L. & Zhang Z.-Y. Low-affinity binding determined by titration calorimetry using a high-affinity coupling ligand: A thermodynamic study of ligand binding to tyrosine phosphatase 1B. Anal. Biochem. 261, 139–148 (1998).
Todd, M.J., Semo, N. & Freire, E. The structural stability of the HIV-1 protease. J. Mol. Biol. 283, 475–488 (1998).
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.
Author information
Authors and Affiliations
Contributions
A.V.-C. and E.F. designed the experiments and wrote the paper; A.V.-C. performed the experiments and analyzed the data.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Rights and permissions
About this article
Cite this article
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
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2006.28
This article is cited by
-
Macromolecular interactions in vitro, comparing classical and novel approaches
European Biophysics Journal (2021)
-
Isothermal titration calorimetry (ITC): a standard operating procedure (SOP)
European Biophysics Journal (2021)
-
A multi-laboratory benchmark study of isothermal titration calorimetry (ITC) using Ca2+ and Mg2+ binding to EDTA
European Biophysics Journal (2021)
-
The Convergence of Cell-Based Surface Plasmon Resonance and Biomaterials: The Future of Quantifying Bio-molecular Interactions—A Review
Annals of Biomedical Engineering (2020)
-
Binding site plasticity in viral PPxY Late domain recognition by the third WW domain of human NEDD4
Scientific Reports (2019)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.