Skip to main content
SpringerLink
Log in

Determining Binding Kinetics of Intrinsically Disordered Proteins by NMR Spectroscopy

  • Protocol
  • First Online:
Intrinsically Disordered Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2141))

Abstract

The unique structural flexibility of intrinsically disordered proteins (IDPs) is central to their diverse functions in cellular processes. Protein–protein interactions involving IDPs are frequently transient and dynamic in nature. Nuclear magnetic resonance (NMR) spectroscopy is an especially powerful tool for characterizing the structural propensities, dynamics, and interactions of IDPs. Here we describe applications of the Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiment in combination with NMR titrations to characterize the kinetics and mechanisms of interactions between intrinsically disordered proteins and their targets. We illustrate the method with reference to interactions between the activation domain of the human T-cell leukemia virus type-I (HTLV-1) basic leucine zipper protein (HBZ) and its cellular binding partner, the KIX domain of the transcriptional coactivator CBP.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 219.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 299.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Wright PE, Dyson HJ (2015) Intrinsically disordered proteins in cellular signalling and regulation. Nat Rev Mol Cell Biol 16(1):18–29. https://doi.org/10.1038/nrm3920

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. 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(3):1220–1229. https://doi.org/10.1021/ja511066q

    Article  CAS  PubMed  Google Scholar 

  3. Sugase K, Dyson HJ, Wright PE (2007) Mechanism of coupled folding and binding of an intrinsically disordered protein. Nature 447(7147):1021–1025. https://doi.org/10.1038/nature05858

    Article  CAS  PubMed  Google Scholar 

  4. Loria JP, Rance M, Palmer AG (1999) A relaxation-compensated Carr-Purcell-Meiboom-Gill sequence for characterizing chemical exchange by NMR spectroscopy. J Am Chem Soc 121(10):2331–2332. https://doi.org/10.1021/ja983961a

    Article  CAS  Google Scholar 

  5. Trott O, Palmer AG 3rd (2002) R1rho relaxation outside of the fast-exchange limit. J Magn Reson 154(1):157–160. https://doi.org/10.1006/jmre.2001.2466

    Article  CAS  PubMed  Google Scholar 

  6. Fawzi NL, Ying J, Ghirlando R et al (2011) Atomic-resolution dynamics on the surface of amyloid-beta protofibrils probed by solution NMR. Nature 480(7376):268–272. https://doi.org/10.1038/nature10577

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Vallurupalli P, Bouvignies G, Kay LE (2012) Studying “invisible” excited protein states in slow exchange with a major state conformation. J Am Chem Soc 134(19):8148–8161. https://doi.org/10.1021/ja3001419

    Article  CAS  PubMed  Google Scholar 

  8. Bouvignies G, Vallurupalli P, Hansen DF et al (2011) Solution structure of a minor and transiently formed state of a T4 lysozyme mutant. Nature 477(7362):111–114. https://doi.org/10.1038/nature10349

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Whittier SK, Hengge AC, Loria JP (2013) Conformational motions regulate phosphoryl transfer in related protein tyrosine phosphatases. Science 341(6148):899–903. https://doi.org/10.1126/science.1241735

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Neudecker P, Zarrine-Afsar A, Davidson AR et al (2007) Phi-Value analysis of a three-state protein folding pathway by NMR relaxation dispersion spectroscopy. Proc Natl Acad Sci U S A 104(40):15717–15722. https://doi.org/10.1073/pnas.0705097104

    Article  PubMed  PubMed Central  Google Scholar 

  11. Meinhold DW, Wright PE (2011) Measurement of protein unfolding/refolding kinetics and structural characterization of hidden intermediates by NMR relaxation dispersion. Proc Natl Acad Sci U S A 108(22):9078–9083. https://doi.org/10.1073/pnas.1105682108

    Article  PubMed  PubMed Central  Google Scholar 

  12. Boehr DD, McElheny D, Dyson HJ et al (2006) The dynamic energy landscape of dihydrofolate reductase catalysis. Science 313(5793):1638–1642. https://doi.org/10.1126/science.1130258

    Article  CAS  PubMed  Google Scholar 

  13. Bhabha G, Lee J, Ekiert DC, Gam J, Wilson IA, Dyson HJ, Benkovic SJ, Wright PE (2011) A dynamic knockout reveals that conformational fluctuations influence the chemical step of enzyme catalysis. Science 332(6026):234–238. https://doi.org/10.1126/science.1198542

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Arai M, Sugase K, Dyson HJ et al (2015) Conformational propensities of intrinsically disordered proteins influence the mechanism of binding and folding. Proc Natl Acad Sci U S A 112(31):9614–9619. https://doi.org/10.1073/pnas.1512799112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Sugase K, Konuma T, Lansing JC et al (2013) Fast and accurate fitting of relaxation dispersion data using the flexible software package GLOVE. J Biomol NMR 56(3):275–283. https://doi.org/10.1007/s10858-013-9747-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Mittermaier AK, Kay LE (2009) Observing biological dynamics at atomic resolution using NMR. Trends Biochem Sci 34(12):601–611. https://doi.org/10.1016/j.tibs.2009.07.004

    Article  CAS  PubMed  Google Scholar 

  17. Carver JP, Richards RE (1972) General 2-site solution for chemical exchange produced dependence of T2 upon Carr-Purcell pulse separation. J Magn Reson 6(1):89. https://doi.org/10.1016/0022-2364(72)90090-X

    Article  CAS  Google Scholar 

  18. Maciejewski MW, Schuyler AD, Gryk MR et al (2017) NMRbox: a resource for biomolecular NMR computation. Biophys J 112(8):1529–1534. https://doi.org/10.1016/j.bpj.2017.03.011

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Korzhnev DM, Salvatella X, Vendruscolo M et al (2004) Low-populated folding intermediates of Fyn SH3 characterized by relaxation dispersion NMR. Nature 430(6999):586–590. https://doi.org/10.1038/nature02655

    Article  CAS  PubMed  Google Scholar 

  20. Yang K, Stanfield RL, Martinez-Yamout MA et al (2018) Structural basis for cooperative regulation of KIX-mediated transcription pathways by the HTLV-1 HBZ activation domain. Proc Natl Acad Sci U S A 115(40):10040–10045. https://doi.org/10.1073/pnas.1810397115

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Delaglio F, Grzesiek S, Vuister GW et al (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6(3):277–293. https://doi.org/10.1007/BF00197809

  22. Vallurupalli P, Hansen DF, Kay LE (2008) Structures of invisible, excited protein states by relaxation dispersion NMR spectroscopy. Proc Natl Acad Sci U S A 105(33):11766–11771. https://doi.org/10.1073/pnas.0804221105

    Article  PubMed  PubMed Central  Google Scholar 

  23. Eisenmesser EZ, Bosco DA, Akke M et al (2002) Enzyme dynamics during catalysis. Science 295(5559):1520–1523. https://doi.org/10.1126/science.1066176

    Article  CAS  PubMed  Google Scholar 

  24. Sugase K, Lansing JC, Dyson HJ et al (2007) Tailoring relaxation dispersion experiments for fast-associating protein complexes. J Am Chem Soc 129(44):13406–13407. https://doi.org/10.1021/ja0762238

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Hansen DF, Yang D, Feng H et al (2007) An exchange-free measure of 15N transverse relaxation: an NMR spectroscopy application to the study of a folding intermediate with pervasive chemical exchange. J Am Chem Soc 129(37):11468–11479. https://doi.org/10.1021/ja072717t

    Article  CAS  PubMed  Google Scholar 

  26. Neudecker P, Korzhnev DM, Kay LE (2006) Assessment of the effects of increased relaxation dispersion data on the extraction of 3-site exchange parameters characterizing the unfolding of an SH3 domain. J Biomol NMR 34(3):129–135. https://doi.org/10.1007/s10858-006-0001-2

    Article  CAS  PubMed  Google Scholar 

  27. Palmer AG 3rd, Massi F (2006) Characterization of the dynamics of biomacromolecules using rotating-frame spin relaxation NMR spectroscopy. Chem Rev 106(5):1700–1719. https://doi.org/10.1021/cr0404287

    Article  CAS  PubMed  Google Scholar 

  28. Bhabha G, Ekiert DC, Jennewein M et al (2013) Divergent evolution of protein conformational dynamics in dihydrofolate reductase. Nat Struct Mol Biol 20(11):1243–1249. https://doi.org/10.1038/nsmb.2676

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Farber PJ, Mittermaier A (2015) Relaxation dispersion NMR spectroscopy for the study of protein allostery. Biophys Rev 7(2):191–200. https://doi.org/10.1007/s12551-015-0166-6

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

We thank Gerard J Kroon and David Oyen for expert assistance with the NMR experiments, and Rebecca Berlow for insightful discussions and proofreading. This work was supported by grants CA096865 and CA214054 from the National Institutes of Health and the Skaggs Institute for Chemical Biology.

Author information

Authors and Affiliations

  1. Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA

    Ke Yang & Peter E. Wright

  2. Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan

    Munehito Arai

Authors
  1. Ke Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Munehito Arai
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Peter E. Wright
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Peter E. Wright .

Editor information

Editors and Affiliations

  1. Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen, København N, Denmark

    Birthe B. Kragelund

  2. Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Copenhagen, Denmark

    Karen Skriver

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Yang, K., Arai, M., Wright, P.E. (2020). Determining Binding Kinetics of Intrinsically Disordered Proteins by NMR Spectroscopy. In: Kragelund, B.B., Skriver, K. (eds) Intrinsically Disordered Proteins. Methods in Molecular Biology, vol 2141. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0524-0_34

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0524-0_34

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0523-3

  • Online ISBN: 978-1-0716-0524-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation