Advertisement

High-Quality Data of Protein/Peptide Interaction by Isothermal Titration Calorimetry

  • Juan Ramirez
  • Yves NominéEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1964)

Abstract

Despite the emergence of high-throughput interaction methods within the last decade, there is still a strong need for careful and accurate measurements of affinities and thermodynamic parameters of single interactions in order to fully dissect the mechanisms of binding. To this end, isothermal titration calorimetry (ITC) is a well-established and convenient label-free technique covering a broad range of affinities.

This review describes the careful use of ITC in the context of protein/peptide interaction in order to measure thermodynamic parameters of the binding with high accuracy and reproducibility. The relative medium-to-low affinities often encountered for protein/peptide binding imply to increase the concentration of the peptide and/or the protein, making the sample quality and data acquisition all the more critical. This chapter emphasizes more specifically the relevance of those points to improve the reproducibility of ITC measurements and to gain high-quality thermodynamic parameters.

Key words

Isothermal titration calorimetry (ITC) Peptide binding Protein/peptide interactions Thermodynamic parameters High-quality sample Reproducibility Sample concentration 

Notes

Acknowledgments

This work was supported by the Association pour la Recherche contre le Cancer (ARC) (no. 3171), and the Agence Nationale de la Recherche (ANR- MIME-2007 EPI-HPV-3D). J.R. was financially supported by ANR. We warmly thank Georges Mer for critical reading of the manuscript.

References

  1. 1.
    Sidhu SS, Fairbrother WJ, Deshayes K (2003) Exploring protein/protein interactions with phage display. Chembiochem 4:15–25Google Scholar
  2. 2.
    Aebersold R, Mann M (2016) Mass-spectrometry exploration of proteome structure and function. Nature 537:347–355CrossRefGoogle Scholar
  3. 3.
    Cassonnet P, Rolloy C, Jacob Y (2011) Benchmarking a luciferase complementation assay for detecting protein complexes. Nat Methods 8:990–992CrossRefGoogle Scholar
  4. 4.
    Vincentelli R, Luck K, Travé …G (2015) Quantifying domain-ligand affinities and specificities by high-throughput holdup assay. Nat Methods 12:787–793CrossRefGoogle Scholar
  5. 5.
    Huibregtse JONM, Scheffner M, Howley PM (1993) Localization of the E6-AP regions that direct human papillomavirus E6 binding, association with p53, and ubiquitination of associated proteins. Mol Cell Biol 13:4918–4927CrossRefGoogle Scholar
  6. 6.
    Zanier K, M’Hamed Ould Sidi AO, Travé G (2012) Solution structure analysis of the HPV16 E6 oncoprotein reveals a self-association mechanism required for E6-mediated degradation of p53. Structure 20:604–617CrossRefGoogle Scholar
  7. 7.
    Zanier K, Nominé Y, Travé …G (2007) Formation of well-defined soluble aggregates upon fusion to MBP is a generic property of E6 proteins from various human papillomavirus species. Protein Expr Purif 51:59–70CrossRefGoogle Scholar
  8. 8.
    Roux S, Zekri E, Fay N (2008) Elimination and exchange of trifluoroacetate counter-ion from cationic peptides: a critical evaluation of different approaches. J Pept Sci 14:354–359CrossRefGoogle Scholar
  9. 9.
    Andruschchenko V, Vogel H, Prenner E (2007) Optimization of the hydrochloric acid concentration used for trifluoroacetate removal from synthetic peptides. J Pept Sci 13:37–43CrossRefGoogle Scholar
  10. 10.
    Wider G, Dreier L (2006) Measuring protein concentrations by NMR spectroscopy. J Am Chem Soc 128:2571–2576CrossRefGoogle Scholar
  11. 11.
    Anthis NJ, Clore GM (2013) Sequence-specific determination of protein and peptide concentrations by absorbance at 205 nm. Protein Sci 22:851–858CrossRefGoogle Scholar
  12. 12.
    Wiseman T, Williston S, Lin …L-N (1989) Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal Biochem 179:131–137CrossRefGoogle Scholar
  13. 13.
    Broecker J, Vargas C, Keller S (2011) Revisiting the optimal c value for isothermal titration calorimetry. Anal Biochem 418:307–309CrossRefGoogle Scholar
  14. 14.
    Schwarz FP, Reinisch T, Surolia …A (2008) Recommendations on measurement and analysis of results obtained on biological substances using isothermal titration calorimetry (IUPAC Technical Report). Pure Appl Chem 80:2025–2040CrossRefGoogle Scholar
  15. 15.
    Myszka DG (1999) Improving biosensor analysis. J Mol Recognit 12:279–284CrossRefGoogle Scholar
  16. 16.
    Han JC, Han GY (1994) A procedure for quantitative determination of Tris(2-carboxyethyl)phosphine, an odorless reducing agent more stable and effective than dithiothreitol. Anal Biochem 220:5–10CrossRefGoogle Scholar
  17. 17.
    Little MJ, Aubry N, Laplante …SR (2007) Quantifying trifluoroacetic acid as a counterion in drug discovery by 19 F NMR and capillary electrophoresis. J Pharm Biomed Anal 43:1324–1330CrossRefGoogle Scholar
  18. 18.
    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

Copyright information

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

Authors and Affiliations

  1. 1.Institute of Nanotechnology (INT), Karlsruhe Institute of Technology (KIT)Eggenstein-LeopoldshafenGermany
  2. 2.Equipe Labelisée Ligue 2015, Department of Integrative Structural BiologyInstitut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U964 UMR 7104 CNRS, Université de StrasbourgIllkirchFrance

Personalised recommendations