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

  • Bruno Y. Matsuyama
  • Petya V. Krasteva
  • Marcos V. A. S. NavarroEmail author
Protocol
Part of the Methods in Molecular Biology book series (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 (Ka, Kd) 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.

Key words

C-di-GMP C-di-GMP sensor proteins Intermolecular interactions Receptor–ligand interactions Isothermal titration calorimetry (ITC) Dissociation constant (KdBinding stoichiometry 

Notes

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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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Bruno Y. Matsuyama
    • 1
  • Petya V. Krasteva
    • 2
  • Marcos V. A. S. Navarro
    • 1
    Email author
  1. 1.Department of Physics and Interdisciplinary ScienceInstitute of Physics of São Carlos, University of São PauloSão CarlosBrazil
  2. 2.Institute for Integrative Biology of the Cell (I2BC)Université Paris-Saclay, CEA, CNRS, Université Paris SudGif-sur-YvetteFrance

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