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Isothermal titration calorimetry

Nature Reviews Methods Primers volume 3, Article number: 17 (2023) Cite this article

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Abstract

Isothermal titration calorimetry (ITC) has become the gold standard for studying molecular interactions in solution. Although it is increasingly being used in the soft matter and synthetic chemistry fields, ITC is most widely used for characterizing molecular interactions between ligands and macromolecules. This Primer starts by presenting the technique’s foundations and instrumentation, including a brief description of the standard assay, followed by a review of common applications. Further extensions and modifications of the technique are explored. These adaptations enable key features to be studied, such as cooperative effects associated with complex biological interactions and their regulation, alongside applications to other fields, including partition to membranes, kinetics and soft matter. Advantages and caveats in ITC are discussed, with a focus on best practices, instrument calibration, experimental design, data analysis and data reporting, as well as recent and future developments.

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Fig. 1: Simplified scheme of an ITC instrument.
Fig. 2: Thermogram and binding isotherm.
Fig. 3: Determination of the heat capacity change and number of protons exchanged upon binding.
Fig. 4: Experimental output in current calorimeter software.
Fig. 5: Representations of ITC data.
Fig. 6: Interaction of lipid membranes with a peptide.

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Acknowledgements

The authors acknowledge the key role of ARBRE (Association of Resources for Research in Biophysics in Europe) and COST MOBIEU Action (CA15126, Between Atom and Cell: Integrating Molecular Biophysics Approaches for Biology and Healthcare, supported by COST - European Cooperation in Science and Technology) in fostering collaborations and promoting the exchange of knowledge and experience. The authors also acknowledge M. Brandts (MicroCal/Malvern-Panalytical) and C. Quinn (CSC/TA Instruments) for clarifying some technical details of their respective instruments, as well as L. Hansen (Brigham Young University, Provo, UT, USA) for reading critically the parts of the text dealing with CSC/TA Instruments and for many fruitful discussions with M.B. on calorimeter design and function during the preparation of this Primer. Finally, the authors acknowledge pioneering technical work developing microcalorimeters and early studies showing the application of these instruments to biological systems from J. J. Christensen, R. M. Izatt, L. D. Hansen, S. J. Gill, R. L. Biltonen, E. Freire, J. F. Brandts, V. V. Plotnikov, P. L. Privalov, G. I. Makhatadze, J. M. Sturtevant, I. Wadsö, G. Waksman and R. N. Goldberg — without these key scientists the whole area of biocalorimetry would not be the established and widely used technique that it is today.

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Author notes

  1. These authors contributed equally: Margarida Bastos, Olga Abian.

Authors and Affiliations

  1. CIQUP, Institute of Molecular Sciences (IMS), Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Porto, Portugal

    Margarida Bastos

  2. Institute of Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza, Zaragoza, Spain

    Olga Abian, Sonia Vega, Ana Jimenez-Alesanco, David Ortega-Alarcon & Adrian Velazquez-Campoy

  3. Aragon Institute for Health Research (IIS Aragon), Zaragoza, Spain

    Olga Abian & Adrian Velazquez-Campoy

  4. Biomedical Research Networking Center in Hepatic and Digestive Diseases (CIBERehd), Madrid, Spain

    Olga Abian & Adrian Velazquez-Campoy

  5. Department of Biochemistry and Molecular and Cell Biology, University of Zaragoza, Zaragoza, Spain

    Olga Abian, Sonia Vega, Ana Jimenez-Alesanco, David Ortega-Alarcon & Adrian Velazquez-Campoy

  6. MRC Laboratory of Molecular Biology, Cambridge, UK

    Christopher M. Johnson

  7. i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal

    Frederico Ferreira-da-Silva

  8. IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal

    Frederico Ferreira-da-Silva

Authors
  1. Margarida Bastos
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  2. Olga Abian
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  4. Frederico Ferreira-da-Silva
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Contributions

Introduction (M.B., O.A. and A.V.-C.); Experimentation (M.B., C.M.J. and A.V.-C.); Results (M.B., C.M.J. and A.V.-C.); Applications (O.A., S.V. and A.V.-C.); Reproducibility and data deposition (M.B., F.F.d.S., A.J.-A. and A.V.-C.); Limitations and optimizations (O.A., C.M.J., D.O.-A. and A.V.-C.); Outlook (F.S. and A.V.-C.); Overview of the Primer (A.V.-C.).

Corresponding author

Correspondence to Adrian Velazquez-Campoy.

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Competing interests

The authors declare no competing interests.

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Peer review information

Nature Reviews Methods Primers thanks Carmelo Sgarlata and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

AlphaFold: https://alphafold.ebi.ac.uk/

BindingDB: https://www.bindingdb.org/bind/index.jsp

Expasy: https://www.expasy.org

NIST reference database: https://randr.nist.gov/Default.aspx

Supplementary information

Glossary

Binding isotherm

Calorimetric processed data plotting the heats from peak integration as a function of the reaction progress.

Desolvation

The release of some, or all, of the surface-associated solvent molecules to the bulk solution.

Enthalpy

The sum of the internal energy and the product of pressure and volume. Enthalpy is equal to the heat transferred during a process at constant pressure and zero non-expansion work in a closed system. In a biological interaction, it reflects the net energetic balance due to non-covalent bonds rupture (with solvent) and formation (between binding partners).

Entropy

The contribution to the Gibbs energy that amounts to the dissipated energy that cannot be used to generate work. Entropy is associated with order/disorder and the configurational arrangements for energy distribution over an ensemble of states. In a biological interaction, it reflects the changes in degrees of freedom along intermolecular interactions, for example desolvation, ion/solute exchange, and conformational and vibrational changes.

Gibbs energy

The maximum amount of non-expansion work that can be extracted from a process in a closed system. The Gibbs energy is a quantitative measure of the spontaneity of a chemical reaction. In a biological interaction, it reflects the binding affinity or strength of a given intermolecular interaction, the stability of the complex.

Heat capacity

The amount of heat to be provided to a system to increase its temperature a certain quantity. Thermal inertia to change temperature or capability to store thermal energy in a system.

Isobaric

Describes any process performed under constant pressure.

Isothermal

Describes any process performed under constant temperature.

Microcalorimetry

An experimental technique that uses calorimeters able to detect very small amounts of heat, at microjoule level.

Proton ionization

(Also known as deprotonation). The removal or transfer of a proton from an acid form in an acid–base reaction. Ionization of buffer molecules has an associated ionization enthalpy or proton dissociation enthalpy, which needs to be taken into account if proton ionization occurs upon binding.

Reverse titrations

The study of the same reactions exchanging the position of the reactants between cell and syringe.

Thermogram

Calorimetric raw data plotting thermal power as a function of time.

Titration

The step-wise addition of a reactant to another reactant. Etymologically, quantitative chemical analysis to determine the concentration (titre) of a solution using another reagent solution of known concentration.

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Bastos, M., Abian, O., Johnson, C.M. et al. Isothermal titration calorimetry. Nat Rev Methods Primers 3, 17 (2023). https://doi.org/10.1038/s43586-023-00199-x

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