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


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 



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.


  1. 1.
    Ross P et al (1987) Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid. Nature 325(6101):279–281CrossRefPubMedGoogle Scholar
  2. 2.
    Krasteva PV, Sondermann H (2017) Versatile modes of cellular regulation via cyclic dinucleotides. Nat Chem Biol 13:350–359CrossRefPubMedGoogle Scholar
  3. 3.
    Jenal U, Reinders A, Lori C (2017) Cyclic di-GMP: second messenger extraordinaire. Nat Rev Microbiol 15(5):271–284CrossRefPubMedGoogle Scholar
  4. 4.
    Krasteva PV, Giglio KM, Sondermann H (2012) Sensing the messenger: the diverse ways that bacteria signal through c-di-GMP. Protein Sci 21(7):929–948CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Schirmer T, Jenal U (2009) Structural and mechanistic determinants of c-di-GMP signalling. Nat Rev Microbiol 7(10):724–735CrossRefPubMedGoogle Scholar
  6. 6.
    Galperin MY, Nikolskaya AN, Koonin EV (2001) Novel domains of the prokaryotic two-component signal transduction systems. FEMS Microbiol Lett 203(1):11–21CrossRefPubMedGoogle Scholar
  7. 7.
    Amikam D, Galperin MY (2006) PilZ domain is part of the bacterial c-di-GMP binding protein. Bioinformatics 22(1):3–6CrossRefPubMedGoogle Scholar
  8. 8.
    Ryjenkov DA et al (2006) The PilZ domain is a receptor for the second messenger c-di-GMP: the PilZ domain protein YcgR controls motility in enterobacteria. J Biol Chem 281(41):30310–30314CrossRefPubMedGoogle Scholar
  9. 9.
    De N et al (2008) Phosphorylation-independent regulation of the diguanylate cyclase WspR. PLoS Biol 6(3):e67CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Lee VT et al (2007) A cyclic-di-GMP receptor required for bacterial exopolysaccharide production. Mol Microbiol 65(6):1474–1484CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Navarro MV et al (2009) Structural analysis of the GGDEF-EAL domain-containing c-di-GMP receptor FimX. Structure 17(8):1104–1116CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Navarro MV et al (2011) Structural basis for c-di-GMP-mediated inside-out signaling controlling periplasmic proteolysis. PLoS Biol 9(2):e1000588CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Huang YH et al (2012) The structural basis for the sensing and binding of cyclic di-GMP by STING. Nat Struct Mol Biol 19(7):728–730CrossRefPubMedGoogle Scholar
  14. 14.
    Shang G et al (2012) Crystal structures of STING protein reveal basis for recognition of cyclic di-GMP. Nat Struct Mol Biol 19(7):725–727CrossRefPubMedGoogle Scholar
  15. 15.
    Shu C et al (2012) Structure of STING bound to cyclic di-GMP reveals the mechanism of cyclic dinucleotide recognition by the immune system. Nat Struct Mol Biol 19(7):722–724CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Tschowri N et al (2014) Tetrameric c-di-GMP mediates effective transcription factor dimerization to control Streptomyces development. Cell 158(5):1136–1147CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Krasteva PV et al (2010) Vibrio cholerae VpsT regulates matrix production and motility by directly sensing cyclic di-GMP. Science 327(5967):866–868CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Matsuyama BY et al (2016) Mechanistic insights into c-di-GMP-dependent control of the biofilm regulator FleQ from Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 113(2):E209–E218CrossRefPubMedGoogle Scholar
  19. 19.
    Wang YC et al (2016) Nucleotide binding by the widespread high-affinity cyclic di-GMP receptor MshEN domain. Nat Commun 7:12481CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Freyer MW, Lewis EA (2008) Isothermal titration calorimetry: experimental design, data analysis, and probing macromolecule/ligand binding and kinetic interactions. Methods Cell Biol 84:79–113CrossRefPubMedGoogle Scholar
  21. 21.
    Stelitano V et al (2013) Probing the activity of diguanylate cyclases and c-di-GMP phosphodiesterases in real-time by CD spectroscopy. Nucleic Acids Res 41(7):e79CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    De N et al (2010) Biophysical assays for protein interactions in the Wsp sensory system and biofilm formation. Methods Enzymol 471:161–184CrossRefPubMedGoogle Scholar
  23. 23.
    Korovashkina AS et al (2012) Enzymatic synthesis of c-di-GMP using inclusion bodies of Thermotoga maritima full-length diguanylate cyclase. J Biotechnol 164(2):276–280CrossRefPubMedGoogle Scholar
  24. 24.
    Zahringer F, Massa C, Schirmer T (2011) Efficient enzymatic production of the bacterial second messenger c-di-GMP by the diguanylate cyclase YdeH from E. coli. Appl Biochem Biotechnol 163(1):71–79CrossRefPubMedGoogle Scholar
  25. 25.
    De N et al (2009) Determinants for the activation and autoinhibition of the diguanylate cyclase response regulator WspR. J Mol Biol 393(3):619–633CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Wilkins MR et al (1999) Protein identification and analysis tools in the ExPASy server. Methods Mol Biol 112:531–552PubMedGoogle Scholar
  27. 27.
    Turnbull WB, Daranas AH (2003) On the value of c: can low affinity systems be studied by isothermal titration calorimetry? J Am Chem Soc 125(48):14859–14866CrossRefPubMedGoogle Scholar
  28. 28.
    Li Z et al (2012) Structures of the PelD cyclic diguanylate effector involved in pellicle formation in Pseudomonas aeruginosa PAO1. J Biol Chem 287(36):30191–30204CrossRefPubMedPubMedCentralGoogle Scholar

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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