Characterizing Protein-Protein Interactions Using Solution NMR Spectroscopy

  • Jose Luis Ortega-Roldan
  • Martin Blackledge
  • Malene Ringkjøbing Jensen
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1764)

Abstract

In this chapter, we describe how NMR chemical shift titrations can be used to study the interaction between two proteins with emphasis on mapping the interface of the complex and determining the binding affinity from a quantitative analysis of the experimental data. In particular, we discuss the appearance of NMR spectra in different chemical exchange regimes (fast, intermediate, and slow) and how these regimes affect NMR data analysis.

Key words

Protein-protein interactions Solution NMR spectroscopy Binding affinity Dissociation constant Chemical shift titration Chemical exchange 

References

  1. 1.
    Zuiderweg ERP (2002) Mapping protein-protein interactions in solution by NMR spectroscopy. Biochemistry 41:1–7CrossRefPubMedGoogle Scholar
  2. 2.
    Vaynberg J, Qin J (2006) Weak protein-protein interactions as probed by NMR spectroscopy. Trends Biotechnol 24:22–27CrossRefPubMedGoogle Scholar
  3. 3.
    Takeuchi K, Wagner G (2006) NMR studies of protein interactions. Curr Opin Struct Biol 16:109–117CrossRefPubMedGoogle Scholar
  4. 4.
    Fielding L (2007) NMR methods for the determination of protein-ligand dissociation constants. Prog Nucl Magn Reson Spec 51:219–242CrossRefGoogle Scholar
  5. 5.
    O’Connell MR, Gamsjaeger R, Mackay JP (2009) The structural analysis of protein-protein interactions by NMR spectroscopy. Proteomics 9:5224–5232CrossRefPubMedGoogle Scholar
  6. 6.
    Vinogradova O, Qin J (2012) NMR as a unique tool in assessment and complex determination of weak protein-protein interactions. Top Curr Chem 326:35–45CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Stamenova SD, French ME, He Y et al (2007) Ubiquitin binds to and regulates a subset of SH3 domains. Mol Cell 25:273–284CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Ortega Roldan JL, Casares S, Jensen MR et al (2013) Distinct ubiquitin binding modes exhibited by SH3 domains: molecular determinants and functional implications. PloS One 8:e73018CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Williamson MP (2013) Using chemical shift perturbation to characterise ligand binding. Prog Nucl Magn Reson Spec 73:1–16CrossRefGoogle Scholar
  10. 10.
    Jensen MR, Ortega-Roldan JL, Salmon L et al (2011) Characterizing weak protein-protein complexes by NMR residual dipolar couplings. Eur Biophys J 40:1371–1381CrossRefPubMedGoogle Scholar
  11. 11.
    Waudby CA, Ramos A, Cabrita LD et al (2016) Two-dimensional NMR Lineshape analysis. Sci Rep 6:24826CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Palmer AG, Kroenke CD, Loria JP (2001) Nuclear magnetic resonance methods for quantifying microsecond-to-millisecond motions in biological macromolecules. Methods Enzymol 339:204–238CrossRefPubMedGoogle Scholar
  13. 13.
    Sugase K, Dyson HJ, Wright PE (2007) Mechanism of coupled folding and binding of an intrinsically disordered protein. Nature 447:1021–1025CrossRefPubMedGoogle Scholar
  14. 14.
    Hansen DF, Vallurupalli P, Kay LE (2008) Using relaxation dispersion NMR spectroscopy to determine structures of excited, invisible protein states. J Biomol NMR 41:113–120CrossRefPubMedGoogle Scholar
  15. 15.
    Salmon L, Ortega Roldan JL, Lescop E et al (2011) Structure, dynamics, and kinetics of weak protein-protein complexes from NMR spin relaxation measurements of titrated solutions. Angew Chem 50:3755–3759CrossRefGoogle Scholar
  16. 16.
    Schneider R, Maurin D, Communie G et al (2015) Visualizing the molecular recognition trajectory of an intrinsically disordered protein using multinuclear relaxation dispersion NMR. J Am Chem Soc 137:1220–1229CrossRefPubMedGoogle Scholar
  17. 17.
    Kragelj J, Palencia A, Nanao MH et al (2015) Structure and dynamics of the MKK7-JNK signaling complex. Proc Natl Acad Sci 112:3409–3414CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Delaglio F, Grzesiek S, Vuister GW et al (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293CrossRefPubMedGoogle Scholar
  19. 19.
    Goddard TD, Kneller DG. SPARKY 3, University of California, San FranciscoGoogle Scholar
  20. 20.
    Ortega Roldan JL, Romero Romero ML, Ora A et al (2007) The high resolution NMR structure of the third SH3 domain of CD2AP. J Biomol NMR 39:331–336CrossRefPubMedGoogle Scholar
  21. 21.
    Ortega Roldan JL, Jensen MR, Brutscher B et al (2009) Accurate characterization of weak macromolecular interactions by titration of NMR residual dipolar couplings: application to the CD2AP SH3-C:ubiquitin complex. Nucleic Acids Res 37:e70CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Bodenhausen G, Ruben DJ (1980) Natural abundance nitrogen-15 NMR by enhanced heteronuclear spectroscopy. Chem Phys Lett 69:185–189CrossRefGoogle Scholar
  23. 23.
    Pervushin K, Riek R, Wider G et al (1997) Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc Natl Acad Sci 94:12366–12371CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Ikura M, Kay LE, Bax A (1990) A novel approach for sequential assignment of 1H, 13C, and 15 N spectra of proteins: heteronuclear triple-resonance three-dimensional NMR spectroscopy. Application to calmodulin. Biochemistry 29:4659–4667CrossRefPubMedGoogle Scholar
  25. 25.
    Kay LE, Ikura M, Tschudin R et al (1969) (1990) three-dimensional triple-resonance NMR spectroscopy of isotopically enriched proteins. J Magn Reson 89:496–514Google Scholar
  26. 26.
    Jung YS, Zweckstetter M (2004) Mars – robust automatic backbone assignment of proteins. J Biomol NMR 30:11–23CrossRefPubMedGoogle Scholar
  27. 27.
    Dominguez C, Boelens R, Bonvin AMJJ (2003) HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. J Am Chem Soc 125:1731–1737CrossRefPubMedGoogle Scholar
  28. 28.
    Schumann FH, Riepl H, Maurer T et al (2007) Combined chemical shift changes and amino acid specific chemical shift mapping of protein-protein interactions. J Biomol NMR 39:275–289CrossRefPubMedGoogle Scholar
  29. 29.
    Clore GM, Tang C, Iwahara J (2007) Elucidating transient macromolecular interactions using paramagnetic relaxation enhancement. Curr Opin Struct Biol 17:603–616CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Tolman JR, Flanagan JM, Kennedy MA et al (1995) Nuclear magnetic dipole interactions in field-oriented proteins: information for structure determination in solution. Proc Natl Acad Sci 92:9279–9283CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Tjandra N, Bax A (1997) Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. Science 278:1111–1114CrossRefPubMedGoogle Scholar
  32. 32.
    Blackledge M (2005) Recent progress in the study of biomolecular structure and dynamics in solution from residual dipolar couplings. Prog Nucl Magn Reson Spec 46:23–61CrossRefGoogle Scholar
  33. 33.
    Marley J, Lu M, Bracken C (2001) A method for efficient isotopic labeling of recombinant proteins. J Biomol NMR 20:71–75CrossRefPubMedGoogle Scholar
  34. 34.
    Mulder FAA, Schipper D, Bott R et al (1999) Altered flexibility in the substrate-binding site of related native and engineered high-alkaline Bacillus subtilisins 1. J Mol Biol 292:111–123CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Jose Luis Ortega-Roldan
    • 1
  • Martin Blackledge
    • 2
  • Malene Ringkjøbing Jensen
    • 2
  1. 1.School of BiosciencesUniversity of KentCanterburyUK
  2. 2.Univ. Grenoble Alpes, CEA, CNRS, IBSGrenobleFrance

Personalised recommendations