Thermodynamics of Molecular Machines Using Incremental ITC

  • Benoît Meyer
  • Cyrielle da Veiga
  • Philippe Dumas
  • Eric EnnifarEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1964)


Molecular biomachines, such as DNA and RNA polymerases or the ribosome, are fascinating biological assemblies able to swiftly perform repeated and highly regulated tasks, with a remarkable accuracy. Significant advances in structural studies during the past 20 years provided a wealth of information regarding their architecture and considerably contributed to a better understanding of their mechanism of action. However, the three-dimensional structure of a biological nanomachine alone does not provide access to its detailed mechanism of action, even when obtained at atomic resolution. When combined with other biophysical approaches, thermodynamic data, together with kinetic data, are essential for a complete description of any binding interaction, revealing forces driving complex formation and providing insights into mechanisms of action. We have developed an incremental ITC approach that is well-suitable for analysis of biomolecular machines. This strategy allows a dissection of molecular biomachine reactions through successive additions in the ITC cell of consecutive substrates.

Key words

HIV reverse transcriptase Incremental ITC Thermodynamics Molecular biomachines Mechanism of action 


  1. 1.
    Klebe G (2015) Applying thermodynamic profiling in lead finding and optimization. Nat Rev Drug Discov 14:95–110CrossRefGoogle Scholar
  2. 2.
    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
  3. 3.
    Bec G, Meyer B, Gerard MA, Steger J, Fauster K, Wolff P, Burnouf D, Micura R, Dumas P, Ennifar E (2013) Thermodynamics of HIV-1 reverse transcriptase in action elucidates the mechanism of action of non-nucleoside inhibitors. J Am Chem Soc 135:9743–9752CrossRefGoogle Scholar
  4. 4.
    Freisz S, Bec G, Radi M, Wolff P, Crespan E, Angeli L, Dumas P, Maga G, Botta M, Ennifar E (2010) Crystal structure of HIV-1 reverse transcriptase bound to a non-nucleoside inhibitor with a novel mechanism of action. Angew Chem 49:1805–1808CrossRefGoogle Scholar
  5. 5.
    Maga G, Radi M, Gerard MA, Botta M, Ennifar E (2010) HIV-1 RT inhibitors with a novel mechanism of action: NNRTIs that compete with the nucleotide substrate. Viruses 2:880–899CrossRefGoogle Scholar
  6. 6.
    Radi M, Maga G, Alongi M, Angeli L, Samuele A, Zanoli S, Bellucci L, Tafi A, Casaluce G, Giorgi G, Armand-Ugon M, Gonzalez E, Este JA, Baltzinger M, Bec G, Dumas P, Ennifar E, Botta M (2009) Discovery of chiral cyclopropyl dihydro-alkylthio-benzyl-oxopyrimidine (S-DABO) derivatives as potent HIV-1 reverse transcriptase inhibitors with high activity against clinically relevant mutants. J Med Chem 52:840–851CrossRefGoogle Scholar
  7. 7.
    Keller S, Vargas C, Zhao H, Piszczek G, Brautigam CA, Schuck P (2012) High-precision isothermal titration calorimetry with automated peak-shape analysis. Anal Chem 84:5066–5073CrossRefGoogle Scholar
  8. 8.
    Dumas P, Ennifar E, Da Veiga C, Bec G, Palau W, Di Primo C, Pineiro A, Sabin J, Munoz E, Rial J (2016) Extending ITC to kinetics with kinITC. Methods Enzymol 567:157–180CrossRefGoogle Scholar
  9. 9.
    Tellinghuisen J (2008) Isothermal titration calorimetry at very low c. Anal Biochem 373:395–397CrossRefGoogle Scholar
  10. 10.
    Tellinghuisen J (2016) Analysis of multitemperature isothermal titration calorimetry data at very low c: global beats van't Hoff. Anal Biochem 513:43–46CrossRefGoogle Scholar
  11. 11.
    Turnbull WB, Daranas AH (2003) On the value of c: can low affinity systems be studied by isothermal titration calorimetry? J Am Chem Soc 125:14859–14866CrossRefGoogle Scholar
  12. 12.
    Burnouf D, Ennifar E, Guedich S, Puffer B, Hoffmann G, Bec G, Disdier F, Baltzinger M, Dumas P (2012) kinITC: a new method for obtaining joint thermodynamic and kinetic data by isothermal titration calorimetry. J Am Chem Soc 134:559–565CrossRefGoogle Scholar
  13. 13.
    Guedich S, Puffer-Enders B, Baltzinger M, Hoffmann G, Da Veiga C, Jossinet F, Thore S, Bec G, Ennifar E, Burnouf D, Dumas P (2016) Quantitative and predictive model of kinetic regulation by E. coli TPP riboswitches. RNA Biol 13:373–390CrossRefGoogle Scholar
  14. 14.
    Spolar RS, Record MT Jr (1994) Coupling of local folding to site-specific binding of proteins to DNA. Science 263:777–784CrossRefGoogle Scholar
  15. 15.
    Ramirez J, Recht R, Charbonnier S, Ennifar E, Atkinson RA, Trave G, Nomine Y, Kieffer B (2015) Disorder-to-order transition of MAGI-1 PDZ1 C-terminal extension upon peptide binding: thermodynamic and dynamic insights. Biochemistry 54:1327–1337CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Benoît Meyer
    • 1
  • Cyrielle da Veiga
    • 1
  • Philippe Dumas
    • 1
  • Eric Ennifar
    • 1
    Email author
  1. 1.Institut de Biologie Moléculaire et CellulaireUniversité de Strasbourg, CNRSStrasbourgFrance

Personalised recommendations