Skip to main content
SpringerLink
Log in

NMR Relaxation Dispersion Methods for the Structural and Dynamic Analysis of Quickly Interconverting, Low-Populated Conformational Substates

  • Protocol
  • First Online:
Protein Folding

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2376))

  • 1681 Accesses

Abstract

Most biomolecular processes involve proteins shuttling among different conformational states, particularly from highly populated ground states to the lowly populated excited states that determine the interconversion rates and biological function, and which are invisible to most structural biology techniques. These structural transitions are rare and relatively fast: happen in the millisecond–microsecond timescale (ms–μs). NMR spectroscopy can access these timescales via relaxation dispersion techniques (RD-NMR). The exchange parameters extracted from RD-NMR experiments provide pivotal information on these otherwise invisible states that reports on key properties of the high free energy, reactive regions of the protein’s energy landscape, including the mechanisms of folding/unfolding and of the interconversion between active and inactive states. Here, we describe a simple, step-by-step protocol to carry out RD-NMR experiments on proteins to detect the existence of such conformational substates and characterize their structural properties (chemical shifts).

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 159.99
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. Lipari G, Szabo A (1982) Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules. 1. Theory and range of validity. J Am Chem Soc 104:4546–4559

    Article  CAS  Google Scholar 

  2. Lipari G, Szabo A (1982) Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules. 2. Analysis of experimental results. J Am Chem Soc 104:4559–4570

    Article  CAS  Google Scholar 

  3. Ban D, Sabo TM, Griesinger C et al (2013) Measuring dynamic and kinetic information in the previously inaccessible supra-τ(c) window of nanoseconds to microseconds by solution NMR spectroscopy. Molecules 18:11904–11937

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Kleckner IR, Foster MP (2011) An introduction to NMR-based approaches for measuring protein dynamics. Biochim Biophys Acta 1814:942–968

    Article  CAS  PubMed  Google Scholar 

  5. Tolman JR (2002) A novel approach to the retrieval of structural and dynamic information from residual dipolar couplings using several oriented media in biomolecular NMR spectroscopy. J Am Chem Soc 124:12020–12030

    Article  CAS  PubMed  Google Scholar 

  6. Meiler J, Prompers JJ, Peti W et al (2001) Model-free approach to the dynamic interpretation of residual dipolar couplings in globular proteins. J Am Chem Soc 123:6098–6107

    Article  CAS  PubMed  Google Scholar 

  7. Lange OF, Lakomek N-A, Farès C et al (2008) Recognition dynamics up to microseconds revealed from an RDC-derived ubiquitin ensemble in solution. Science 320:1471–1475

    Article  CAS  PubMed  Google Scholar 

  8. Jeener J, Meier BH, Bachmann P et al (1979) Investigation of exchange processes by two-dimensional NMR spectroscopy. J Chem Phys 71:4546–4553

    Article  CAS  Google Scholar 

  9. Palmer AG 3rd, Kroenke CD, Loria JP (2001) Nuclear magnetic resonance methods for quantifying microsecond-to-millisecond motions in biological macromolecules. Methods Enzymol 339:204–238

    Article  CAS  PubMed  Google Scholar 

  10. Fejzo J, Westler WM, Macura S et al (1990) Elimination of cross-relaxation effects from two-dimensional chemical-exchange spectra of macromolecules. J Am Chem Soc 112:2574–2577

    Article  CAS  Google Scholar 

  11. Fejzo J, Westler WM, Macura S et al (1991) Strategies for eliminating unwanted cross-relaxation and coherence-transfer effects from two-dimensional chemical-exchange spectra. J Magn Reson 92:20–29

    CAS  Google Scholar 

  12. Bouvignies G, Vallurupalli P, Kay LE (2014) Visualizing side chains of invisible protein conformers by solution NMR. J Mol Biol 426:763–774

    Article  CAS  PubMed  Google Scholar 

  13. Forsén S, Hoffman RA (1963) Study of moderately rapid chemical exchange reactions by means of nuclear magnetic double resonance. J Chem Phys 39:2892–2901

    Article  Google Scholar 

  14. Fawzi NL, Ying J, Ghirlando R et al (2011) Atomic-resolution dynamics on the surface of amyloid-β protofibrils probed by solution NMR. Nature 480:268–272

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Rennella E, Huang R, Velyvis A et al (2015) 13CHD2–CEST NMR spectroscopy provides an avenue for studies of conformational exchange in high molecular weight proteins. J Biomol NMR 63:187–199

    Article  CAS  PubMed  Google Scholar 

  16. Vallurupalli P, Bouvignies G, Kay LE (2012) Studying “invisible” excited protein states in slow exchange with a major state conformation. J Am Chem Soc 134:8148–8161

    Article  CAS  PubMed  Google Scholar 

  17. Sanchez-Medina C, Sekhar A, Vallurupalli P et al (2014) Probing the free energy landscape of the fast-folding gpW protein by relaxation dispersion NMR. J Am Chem Soc 136:7444–7451

    Article  CAS  PubMed  Google Scholar 

  18. Ishima R, Torchia DA (2003) Extending the range of amide proton relaxation dispersion experiments in proteins using a constant-time relaxation-compensated CPMG approach. J Biomol NMR 25:243–248

    Article  CAS  PubMed  Google Scholar 

  19. Hansen DF, Vallurupalli P, Kay LE (2008) An improved 15N relaxation dispersion experiment for the measurement of millisecond time-scale dynamics in proteins. J Phys Chem B 112:5898–5904

    Article  CAS  PubMed  Google Scholar 

  20. Lundström P, Vallurupalli P, Religa TL et al (2007) A single-quantum methyl 13C-relaxation dispersion experiment with improved sensitivity. J Biomol NMR 38:79–88

    Article  PubMed  Google Scholar 

  21. Bloom M, Reeves LW, Wells EJ (1965) Spin echoes and chemical exchange. J Chem Phys 42:1615–1624

    Article  CAS  Google Scholar 

  22. Mulder FA, van Tilborg PJ, Kaptein R et al (1999) Microsecond time scale dynamics in the RXR DNA-binding domain from a combination of spin-echo and off-resonance rotating frame relaxation measurements. J Biomol NMR 13:275–288. https://doi.org/10.1023/A:1008354232281

  23. Luz Z, Meiboom S (1963) Nuclear magnetic resonance study of the protolysis of trimethylammonium ion in aqueous solution—order of the reaction with respect to solvent. J Chem Phys 39:366–370

    Article  CAS  Google Scholar 

  24. Farber PJ, Mittermaier A (2015) Relaxation dispersion NMR spectroscopy for the study of protein allostery. Biophys Rev 7:191–200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Yuwen T, Vallurupalli P, Kay LE (2016) Enhancing the sensitivity of CPMG relaxation dispersion to conformational exchange processes by multiple-quantum spectroscopy. Angew Chem Int Ed Engl 55:11490–11494

    Article  CAS  PubMed  Google Scholar 

  26. Davis DG, Perlman ME, London RE (1994) Direct measurements of the dissociation-rate constant for inhibitor-enzyme complexes via the T1 rho and T2 (CPMG) methods. J Magn Reson B 104:266–275

    Article  CAS  PubMed  Google Scholar 

  27. Deverell C, Morgan RE, Strange JH (1970) Studies of chemical exchange by nuclear magnetic relaxation in the rotating frame. Mol Phys 18:553–559

    Article  CAS  Google Scholar 

  28. Ammann C, Meier P, Merbach A (1982) A simple multinuclear NMR thermometer. J Magn Reson 46:319–321

    CAS  Google Scholar 

  29. Ishima R (2013) CPMG relaxation dispersion. In: Methods in molecular biology. Humana, New York, pp 29–49

    Google Scholar 

  30. Lundström P, Hansen DF, Kay LE (2008) Measurement of carbonyl chemical shifts of excited protein states by relaxation dispersion NMR spectroscopy: comparison between uniformly and selectively (13)C labeled samples. J Biomol NMR 42:35–47

    Article  PubMed  Google Scholar 

  31. Delaglio F, Grzesiek S, Vuister GW et al (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293

    Article  CAS  PubMed  Google Scholar 

  32. Lundström P, Teilum K, Carstensen T et al (2007) Fractional 13C enrichment of isolated carbons using [1-13C]- or [2-13C]-glucose facilitates the accurate measurement of dynamics at backbone Cα and side-chain methyl positions in proteins. J Biomol NMR 38:199–212

    Article  PubMed  Google Scholar 

  33. Tugarinov V, Kanelis V, Kay LE (2006) Isotope labeling strategies for the study of high-molecular-weight proteins by solution NMR spectroscopy. Nat Protoc 1:749–754

    Article  CAS  PubMed  Google Scholar 

  34. Lundström P, Ahlner A, Blissing AT (2012) Isotope labeling methods for relaxation measurements. In: Advances in experimental medicine and biology. Springer Nature, Switzerland, pp 63–82

    Google Scholar 

  35. Lundström P, Hansen DF, Vallurupalli P et al (2009) Accurate measurement of alpha proton chemical shifts of excited protein states by relaxation dispersion NMR spectroscopy. J Am Chem Soc 131:1915–1926

    Article  PubMed  Google Scholar 

  36. Goto NK, Gardner KH, Mueller GA et al (1999) A robust and cost-effective method for the production of Val, Leu, Ile (delta 1) methyl-protonated 15N-, 13C-, 2H-labeled proteins. J Biomol NMR 13:369–374

    Article  CAS  PubMed  Google Scholar 

  37. Korzhnev DM, Kloiber K, Kay LE (2004) Multiple-quantum relaxation dispersion NMR spectroscopy probing millisecond time-scale dynamics in proteins: theory and application. J Am Chem Soc 126:7320–7329

    Article  CAS  PubMed  Google Scholar 

  38. Korzhnev DM, Salvatella X, Vendruscolo M et al (2004) Low-populated folding intermediates of Fyn SH3 characterized by relaxation dispersion NMR. Nature 430:586–590

    Article  CAS  PubMed  Google Scholar 

  39. Loria JP, Patrick Loria J, Rance M et al (1999) A relaxation-compensated Carr−Purcell−Meiboom−Gill sequence for characterizing chemical exchange by NMR spectroscopy. J Am Chem Soc 121:2331–2332

    Article  CAS  Google Scholar 

  40. Mulder FAA, Skrynnikov NR, Hon B et al (2001) Measurement of slow (μs−ms) time scale dynamics in protein side chains by15N relaxation dispersion NMR spectroscopy: application to Asn and Gln residues in a cavity mutant of T4 lysozyme. J Am Chem Soc 123:967–975

    Article  CAS  PubMed  Google Scholar 

  41. Millet O, Patrick Loria J, Kroenke CD et al (2000) The static magnetic field dependence of chemical exchange linebroadening defines the NMR chemical shift time scale. J Am Chem Soc 122:2867–2877

    Article  CAS  Google Scholar 

  42. Skrynnikov NR, Dahlquist FW, Kay LE (2002) Reconstructing NMR spectra of “invisible” excited protein states using HSQC and HMQC experiments. J Am Chem Soc 124:12352–12360

    Article  CAS  PubMed  Google Scholar 

  43. 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 56:275–283

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Ishima R, Torchia DA (2005) Error estimation and global fitting in transverse-relaxation dispersion experiments to determine chemical-exchange parameters. J Biomol NMR 32:41–54

    Article  CAS  PubMed  Google Scholar 

  45. Morin S, Linnet TE, Lescanne M et al (2014) relax: the analysis of biomolecular kinetics and thermodynamics using NMR relaxation dispersion data. Bioinformatics 30:2219–2220

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Bieri M, Gooley PR (2011) Automated NMR relaxation dispersion data analysis using NESSY. BMC Bioinformatics 12:421

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Hansen DF, Vallurupalli P, Lundström P et al (2008) Probing chemical shifts of invisible states of proteins with relaxation dispersion NMR spectroscopy: how well can we do? J Am Chem Soc 130:2667–2675

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was funded by Advanced Grant ERC-2012-ADG-323059 from the European Research Council to V.M. V.M. also acknowledges support from the W.M. Keck foundation and the National Science Foundation (grants NSF-MCB-1616759 and NSF-CREST-1547848).

Author information

Authors and Affiliations

  1. IMDEA Nanociencia, Madrid, Spain

    Sivanandam Veeramuthu Natarajan

  2. Unité Génie Enzymatique et Cellulaire, Université Picardie Jules Verne, Amiens, France

    Nicola D’Amelio

  3. Department of Bioengineering and Center for Cellular and Biomolecular Machines, University of California, Merced, Merced, CA, USA

    Victor Muñoz

Authors
  1. Sivanandam Veeramuthu Natarajan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nicola D’Amelio
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Victor Muñoz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Victor Muñoz .

Editor information

Editors and Affiliations

  1. Department of Bioengineering and Center for Cellular and Biomolecular Machines, University of California, Merced, Merced, CA, USA

    Victor Muñoz

Rights and permissions

Reprints and permissions

Copyright information

© 2022 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Veeramuthu Natarajan, S., D’Amelio, N., Muñoz, V. (2022). NMR Relaxation Dispersion Methods for the Structural and Dynamic Analysis of Quickly Interconverting, Low-Populated Conformational Substates. In: Muñoz, V. (eds) Protein Folding. Methods in Molecular Biology, vol 2376. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1716-8_11

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1716-8_11

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1715-1

  • Online ISBN: 978-1-0716-1716-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation