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Intrinsic Thermodynamics of Protein-Ligand Binding by Isothermal Titration Calorimetry as Aid to Drug Design

  • Vaida Paketurytė
  • Asta Zubrienė
  • John E. Ladbury
  • Daumantas MatulisEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1964)

Abstract

Isothermal titration calorimetry (ITC) is one of the main techniques to determine specific interactions between molecules dissolved in aqueous solution. This technique is commonly used in drug development programs when low-molecular-weight molecules are sought that bind tightly and specifically to a protein (disease target) molecule. The method allows a complete thermodynamic characterization of an interaction, i.e., ITC enables direct determination of the model-independent observed interaction change in enthalpy (ΔH) and a model-dependent observed interaction affinity (change in Gibbs free energy, ΔG) in a single experiment. The product of temperature and change in entropy (TΔS) can be obtained by the subtraction of ΔG from ΔH, and the change in heat capacity (ΔCp) can be determined as a slope of the temperature dependence of the binding ΔH.

Despite the apparent value of ITC in characterization of interactions, it is often forgotten that many protein-ligand binding reactions are linked to protonation-deprotonation reactions or various conformational changes. In such cases, it is important to determine the linked-reaction contributions and obtain the intrinsic values of the changes in Gibbs energy (affinity), enthalpy, and entropy. These energy values can then be used in various SAR-type structure-thermodynamics and combined with structure-kinetics correlations in drug design, when searching for small molecules that would bind the protein target molecule. This manuscript provides a detailed protocol on how to determine the intrinsic values of protein-ligand binding thermodynamics by ITC.

Key words

Isothermal titration calorimetry ITC Enthalpy of binding Gibbs energy of binding Drug design Intrinsic thermodynamics of binding 

References

  1. 1.
    Haq I, Ladbury J (2000) Drug-DNA recognition: energetics and implications for design. J Mol Recognit 13:188–197CrossRefGoogle Scholar
  2. 2.
    Ladbury JE (1995) Counting the calories to stay in the groove. Structure 3:635–639CrossRefGoogle Scholar
  3. 3.
    Ladbury JE (2004) Application of isothermal titration calorimetry in the biological sciences: things are heating up! BioTechniques 37:885–887CrossRefGoogle Scholar
  4. 4.
    Ladbury JE, Chowdhry BZ (1996) Sensing the heat: the application of isothermal titration calorimetry to thermodynamic studies of biomolecular interactions. Chem Biol 3:791–801CrossRefGoogle Scholar
  5. 5.
    Ladbury JE, Klebe G, Freire E (2010) Adding calorimetric data to decision making in lead discovery: a hot tip. Nat Rev Drug Discov 9:23–27CrossRefGoogle Scholar
  6. 6.
    Wiseman T, Williston S, Brandts JF, Lin LN (1989) Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal Biochem 179:131–137CrossRefGoogle Scholar
  7. 7.
    Krainer G, Broecker J, Vargas C, Fanghänel J, Keller S (2012) Quantifying high-affinity binding of hydrophobic ligands by isothermal titration calorimetry. Anal Chem 84:10715–10722CrossRefGoogle Scholar
  8. 8.
    Velazquez-Campoy A, Freire E (2006) Isothermal titration calorimetry to determine association constants for high-affinity ligands. Nat Protoc 1:186–191CrossRefGoogle Scholar
  9. 9.
    Zubrienė A et al (2015) Intrinsic thermodynamics of 4-substituted-2,3,5,6-tetrafluorobenzenesulfonamide binding to carbonic anhydrases by isothermal titration calorimetry. Biophys Chem 205:51–65CrossRefGoogle Scholar
  10. 10.
    Baker BM, Murphy KP (1996) Evaluation of linked protonation effects in protein binding reactions using isothermal titration calorimetry. Biophys J 71(4):2049–2055CrossRefGoogle Scholar
  11. 11.
    Baker BM, Murphy KP (1997) Dissecting the energetics of a protein-protein interaction: the binding of ovomucoid third domain to elastase. J Mol Biol 268(2):557–569CrossRefGoogle Scholar
  12. 12.
    Nasief NN, Hangauer D (2014) Influence of neighboring groups on the thermodynamics of hydrophobic binding: an added complex facet to the hydrophobic effect. J Med Chem 57:2315–2333CrossRefGoogle Scholar
  13. 13.
    Brautigam CA, Zhao H, Vargas C, Keller S, Schuck P (2016) Integration and global analysis of isothermal titration calorimetry data for studying macromolecular interactions. Nat Protoc 11:882–894CrossRefGoogle Scholar
  14. 14.
    Goldberg RN, Kishore N, Lennen RM (2002) Thermodynamic quantities for the ionization reactions of buffers. J Phys Chem Ref Data 31:231–370CrossRefGoogle Scholar
  15. 15.
    Sigurskjold BW (2000) Exact analysis of competition ligand binding by displacement isothermal titration calorimetry. Anal Biochem 277:260–266CrossRefGoogle Scholar
  16. 16.
    Baranauskienė L, Matulis D (2012) Intrinsic thermodynamics of ethoxzolamide inhibitor binding to human carbonic anhydrase XIII. BMC Biophys 5:12CrossRefGoogle Scholar
  17. 17.
    Kazlauskas E et al (2012) Thermodynamics of Aryl-Dihydroxyphenyl-Thiadiazole Binding to Human Hsp90. PLoS One 7:e36899CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Vaida Paketurytė
    • 1
  • Asta Zubrienė
    • 1
  • John E. Ladbury
    • 2
  • Daumantas Matulis
    • 1
    Email author
  1. 1.Department of Biothermodynamics and Drug Design, Institute of BiotechnologyVilnius UniversityVilniusLithuania
  2. 2.Department of Molecular and Cell Biology and Astbury Centre for Structural BiologyUniversity of LeedsLeedsUK

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