Quantitative measurement of exchange dynamics in proteins via 13C relaxation dispersion of 13CHD2-labeled samples

Abstract

Methyl groups have emerged as powerful probes of protein dynamics with timescales from picoseconds to seconds. Typically, studies involving high molecular weight complexes exploit 13CH3- or 13CHD2-labeling in otherwise highly deuterated proteins. The 13CHD2 label offers the unique advantage of providing 13C, 1H and 2H spin probes, however a disadvantage has been the lack of an experiment to record 13C Carr–Purcell–Meiboom–Gill relaxation dispersion that monitors millisecond time-scale dynamics, implicated in a wide range of biological processes. Herein we develop an experiment that eliminates artifacts that would normally result from the scalar coupling between 13C and 2H spins that has limited applications in the past. The utility of the approach is established with a number of applications, including measurement of ms dynamics of a disease mutant of a 320 kDa p97 complex.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  1. Abragam A (1961) Principles of nuclear magnetism. Clarendon Press, Oxford

    Google Scholar 

  2. Baldwin AJ, Religa TL, Hansen DF, Bouvignies G, Kay LE (2010) 13CHD2 methyl group probes of millisecond time scale exchange in proteins by 1H relaxation dispersion: an application to proteasome gating residue dynamics. J Am Chem Soc 132:10992–10995. doi:10.1021/ja104578n

    Article  Google Scholar 

  3. Brath U, Akke M, Yang D, Kay LE, Mulder FA (2006) Functional dynamics of human FKBP12 revealed by methyl 13C rotating frame relaxation dispersion NMR spectroscopy. J Am Chem Soc 128:5718–5727. doi:10.1021/ja0570279

    Article  Google Scholar 

  4. Braun RJ, Zischka H (2008) Mechanisms of Cdc48/VCP-mediated cell death: from yeast apoptosis to human disease. Biochim Biophys Acta 1783:1418–1435. doi:10.1016/j.bbamcr.2008.01.015

    Article  Google Scholar 

  5. Grzesiek S, Anglister J, Ren H, Bax A (1993) 13C line narrowing by 2H decoupling in 2H/13C/15N-enriched proteins. Applications to triple resonance 4D J-connectivity of sequential amides. J Am Chem Soc 115:4369–4370

    Article  Google Scholar 

  6. Ishima R, Louis JM, Torchia DA (1999) Transverse 13C relaxation of CHD2 methyl isotopomers to detect slow conformational changes of protein side chains. J Am Chem Soc 121:11589–11590

    Article  Google Scholar 

  7. Ishima R, Petkova AP, Louis JM, Torchia DA (2001) Comparison of methyl rotation axis order parameters derived from model-free analyses of 2H and 13C longitudinal and transverse relaxation rates measured in the same protein sample. J Am Chem Soc 123:6164–6171

    Article  Google Scholar 

  8. Jiang B, Yu B, Zhang X, Liu M, Yang D (2015) A 15N CPMG relaxation dispersion experiment more resistant to resonance offset and pulse imperfection. J Magn Reson 257:1–7. doi:10.1016/j.jmr.2015.05.003

    ADS  Article  Google Scholar 

  9. Kay LE, Keifer P, Saarinen T (1992) Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity. J Am Chem Soc 114:10663–10665

    Article  Google Scholar 

  10. Kimonis VE, Fulchiero E, Vesa J, Watts G (2008) VCP disease associated with myopathy, Paget disease of bone and frontotemporal dementia: review of a unique disorder. Biochim Biophys Acta 1782:744–748. doi:10.1016/j.bbadis.2008.09.003

    Article  Google Scholar 

  11. Korzhnev DM, Kloiber K, Kanelis V, Tugarinov V, Kay LE (2004) Probing slow dynamics in high molecular weight proteins by methyl-TROSY NMR spectroscopy: application to a 723-residue enzyme. J Am Chem Soc 126:3964–3973

    Article  Google Scholar 

  12. Korzhnev DM, Mittermaier AK, Kay LE (2005) Cross-correlated spin relaxation effects in methyl 1H CPMG-based relaxation dispersion experiments: complications and a simple solution. J Biomol NMR 31:337–342. doi:10.1007/s10858-005-2468-7

    Article  Google Scholar 

  13. Lundstrom P, Vallurupalli P, Religa TL, Dahlquist FW, Kay LE (2007) A single-quantum methyl 13C-relaxation dispersion experiment with improved sensitivity. J Biomol NMR 38:79–88

    Article  Google Scholar 

  14. McConnell HM (1958) Reaction rates by nuclear magnetic resonance. J Chem Phys 28:430–431

    ADS  Article  Google Scholar 

  15. Messerlie BA, Wider W, Otting G, Weber C, Wuthrich K (1989) Solvent suppression using a spin lock in 2D and 3D NMR spectroscopy with H2O solutions. J Magn Reson 85:608–612

    ADS  Google Scholar 

  16. Mittermaier A, Kay LE (2006) New tools provide new insights in NMR studies of protein dynamics. Science 312:224–228

    ADS  Article  Google Scholar 

  17. Mulder FAA, Skrynnikov NR, Hon B, Dahlquist FW, Kay LE (2001) Measurement of slow timescale dynamics in protein sidechains by 15N 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  Google Scholar 

  18. Otten R, Villali J, Kern D, Mulder FA (2010) Probing microsecond time scale dynamics in proteins by methyl 1H Carr–Purcell–Meiboom–Gill relaxation dispersion NMR measurements. Application to activation of the signaling protein NtrC(r). J Am Chem Soc 132:17004–17014. doi:10.1021/ja107410x

    Article  Google Scholar 

  19. Pervushin K, Riek R, Wider G, Wüthrich K (1998) Transverse relaxation-optimized spectroscopy (TROSY) for NMR studies of aromatic spin systems in 13C-labeled proteins. J Am Chem Soc 120:6394–6400

    Article  Google Scholar 

  20. Rennella E, Huang R, Velyvis A, Kay LE (2015) 13CHD2-CEST NMR spectroscopy provides an avenue for studies of conformational exchange in high molecular weight proteins. J Biomol NMR 63:187–199. doi:10.1007/s10858-015-9974-z

    Article  Google Scholar 

  21. Schleucher J, Sattler M, Griesinger C (1993) Coherence selection by gradients without signal attenuation: application to the three-dimensional HNCO experiment. Angew Chem Int Ed Engl 32:1489–1491

    Article  Google Scholar 

  22. Sekhar A, Kay LE (2013) NMR paves the way for atomic level descriptions of sparsely populated, transiently formed biomolecular conformers. Proc Natl Acad Sci USA 110:12867–12874. doi:10.1073/pnas.1305688110

    ADS  Article  Google Scholar 

  23. Shaka AJ, Keeler J, Frenkiel T, Freeman R (1983) An improved sequence for broadband decoupling: WALTZ-16. J Magn Reson 52:335–338

    ADS  Google Scholar 

  24. Shaka AJ, Lee CJ, Pines A (1988) Iterative schemes for bilinear operators—application to spin decoupling. J Magn Reson 77:274–293

    ADS  Google Scholar 

  25. Song C, Wang Q, Li CC (2003) ATPase activity of p97-valosin-containing protein (VCP). D2 mediates the major enzyme activity, and D1 contributes to the heat-induced activity. J Biol Chem 278:3648–3655. doi:10.1074/jbc.M208422200

    Article  Google Scholar 

  26. Sprangers R, Kay LE (2007) Quantitative dynamics and binding studies of the 20S proteasome by NMR. Nature 445:618–622

    Article  Google Scholar 

  27. Tugarinov V, Kay LE (2005) Methyl groups as probes of structure and dynamics in NMR studies of high-molecular-weight proteins. ChemBioChem 6:1567–1577

    Article  Google Scholar 

  28. Tugarinov V, Kay LE (2007) Separating degenerate 1(H) transitions in methyl group probes for single-quantum (1)H-CPMG relaxation dispersion NMR spectroscopy. J Am Chem Soc 129:9514–9521

    Article  Google Scholar 

  29. Tugarinov V, Hwang P, Ollerenshaw J, Kay LE (2003) Cross-correlated relaxation enhanced 1H–13C NMR spectroscopy of methyl groups in very high molecular weight proteins and protein complexes. J Am Chem Soc 125:10420–10428

    Article  Google Scholar 

  30. Tugarinov V, Sprangers R, Kay LE (2007) Probing side-chain dynamics in the proteasome by relaxation violated coherence transfer NMR spectroscopy. J Am Chem Soc 129:1743–1750

    Article  Google Scholar 

  31. Vallurupalli P, Scott L, Williamson JR, Kay LE (2007) Strong coupling effects during X-pulse CPMG experiments recorded on heteronuclear ABX spin systems: artifacts and a simple solution. J Biomol NMR 38:41–46

    Article  Google Scholar 

  32. Werbelow LG, Grant DM (1977) Intramolecular dipolar relaxation in multispin systems. Adv Magn Reson 9:189–299

    Article  Google Scholar 

Download references

Acknowledgments

A.K.S is the recipient of an EMBO Long Term Fellowship and an Early Postdoc Mobility Fellowship from the Swiss National Science Foundation. This work was funded through a Canadian Institute of Health Research grant to L.E.K. L.E.K. holds a Canada Research Chair in Biochemistry.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Lewis E. Kay.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 1930 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Rennella, E., Schuetz, A.K. & Kay, L.E. Quantitative measurement of exchange dynamics in proteins via 13C relaxation dispersion of 13CHD2-labeled samples. J Biomol NMR 65, 59–64 (2016). https://doi.org/10.1007/s10858-016-0038-9

Download citation

Keywords

  • CPMG
  • Conformational dynamics
  • Proteins
  • Methyl labeling
  • Relaxation dispersion
  • 13CHD2