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c-di-GMP Signaling pp 403–416Cite as

Isothermal Titration Calorimetry to Determine Apparent Dissociation Constants (K d) and Stoichiometry of Interaction (n) of C-di-GMP Binding Proteins

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1657))

Abstract

Isothermal titration calorimetry (ITC) is a commonly used biophysical technique that enables the quantitative characterization of intermolecular interactions in solution. Based on enthalpy changes (ΔH) upon titration of the binding partner (e.g., a small-molecule ligand such as c-di-GMP) to the molecule of interest (e.g., a receptor protein), the resulting binding isotherms provide information on the equilibrium association/dissociation constants (K a, K d) and stoichiometry of binding (n), as well as on changes in the Gibbs free energy (ΔG) and entropy (ΔS) along the interaction. Here we present ITC experiments used for the characterization of c-di-GMP binding proteins and discuss advantages and potential caveats in the interpretation of results.

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Acknowledgments

Work in the Navarro laboratory is supported by Fundação de Amparo à Pesquisa do Estado de São Paulo under Grant 2009/13238-0. The Krasteva laboratory is supported by the Institute for Integrative Biology of the Cell (I2BC) and by a 2016 ATIP-Avenir grant from the Centre National de la Recherche Scientifique.

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Authors and Affiliations

  1. Department of Physics and Interdisciplinary Science, Institute of Physics of São Carlos, University of São Paulo, Av Trabalhador, Saocarlense, 400, São Carlos, SP, 13566-590, Brazil

    Bruno Y. Matsuyama & Marcos V. A. S. Navarro

  2. Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, Université Paris Sud, 1 Avenue de la Terrasse, 91198, Gif-sur-Yvette, France

    Petya V. Krasteva

Authors
  1. Bruno Y. Matsuyama
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  2. Petya V. Krasteva
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  3. Marcos V. A. S. Navarro
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Corresponding author

Correspondence to Marcos V. A. S. Navarro .

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Editors and Affiliations

  1. Department of Biological Sciences, Binghamton University Department of Biological Sciences, Binghamton, New York, USA

    Karin Sauer

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Matsuyama, B.Y., Krasteva, P.V., Navarro, M.V.A.S. (2017). Isothermal Titration Calorimetry to Determine Apparent Dissociation Constants (K d) and Stoichiometry of Interaction (n) of C-di-GMP Binding Proteins. In: Sauer, K. (eds) c-di-GMP Signaling. Methods in Molecular Biology, vol 1657. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7240-1_30

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  • DOI: https://doi.org/10.1007/978-1-4939-7240-1_30

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7239-5

  • Online ISBN: 978-1-4939-7240-1

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