Journal of Biomolecular NMR

, Volume 47, Issue 3, pp 163–169 | Cite as

Optimal methyl labeling for studies of supra-molecular systems



Selective methyl labeling combined with HMQC spectroscopy that exploits a TROSY effect in 13CH3 spin systems has significantly extended the utility of solution NMR spectroscopy in studies of high molecular weight particles. Herein we compare the utility of 13CH3- versus 13CHD2-labeling of Ile, Leu, Val probes in supra-molecular systems through quantification of relative signal-to-noise ratios in optimized spectra of highly deuterated, 13CH3- and 13CHD2-labeled samples of the half proteasome (α7α7, 360 kDa). It is shown that the sensitivity of spectra recorded on Ile, Leu, Val 13CH3-labeled samples is between 1.5 and 2 fold higher than the corresponding data sets obtained on α7α7 with 13CHD2 probes. Thus, labeling of supra-molecules with 13CH3 isotopomers remains the method of choice, but in applications where 13CHD2 moieties are required, sensitivity will in general not be limiting.


Methyl-TROSY Methyl labeling Sensitivity HSQC HMQC Proteasome 



T.L.R. acknowledges The European Molecular Biology Organization (ALTF 827-2006) and The Canadian Institutes of Health Research (CIHR) for postdoctoral fellowships. L.E.K. holds a Canada Research Chair in Biochemistry. This work was supported by a grant from the CIHR.

Supplementary material

10858_2010_9419_MOESM1_ESM.doc (170 kb)
(DOC 169 kb)


  1. Amero C, Schanda P, Dura MA, Ayala I, Marion D, Franzetti B, Brutscher B, Boisbouvier J (2009) Fast two-dimensional NMR spectroscopy of high molecular weight protein assemblies. J Am Chem Soc 131:3448–3449CrossRefGoogle Scholar
  2. Bax A, Griffey RH, Hawkings BL (1983) Correlation of proton and nitrogen-15 chemical shifts by multiple quantum NMR. J Magn Reson 55:301–315Google Scholar
  3. Bodenhausen G, Rubin DJ (1980) Natural abundance nitrogen-15 NMR by enhanced heteronuclear spectroscopy. Chem Phys Lett 69:185–189CrossRefADSGoogle Scholar
  4. Cavanagh J, Rance M (1993) Sensitivity-enhancement NMR techniques for the study of biomolecules. Ann Reports NMR Spectrosc 27:1–58CrossRefGoogle Scholar
  5. Fischer M, Kloiber K, Hausler J, Ledolter K, Konrat R, Schmid W (2007) Synthesis of a 13C-methyl-group-labeled methionine precursor as a useful tool for simplifying protein structural analysis by NMR spectroscopy. Chembiochem 8:610–612CrossRefGoogle Scholar
  6. Gans P, Hamelin O, Sounier R, Ayala I, Dura MA, Amero C, Noirclerc-Savoye M, Franzetti B, Plevin MJ, Boisbouvier J (2010) Stereospecific isotopic labeling of methyl groups for NMR spectroscopic studies of high molecular weight proteins. Angew Chem Int Ed. doi: 10.1002/anie.200905660 MATHGoogle Scholar
  7. Gelis I, Bonvin AM, Keramisanou D, Koukaki M, Gouridis G, Karamanou S, Economou A, Kalodimos CG (2007) Structural basis for signal-sequence recognition by the translocase motor SecA as determined by NMR. Cell 131:756–769CrossRefGoogle Scholar
  8. Hamel DJ, Dahlquist FW (2005) The contact interface of a 120 kD CheA-CheW complex by methyl TROSY interaction spectroscopy. J Am Chem Soc 127:9676–9677CrossRefGoogle Scholar
  9. Isaacson RL, Simpson PJ, Liu M, Cota E, Zhang X, Freemont P, Matthews S (2007) A new labeling method for methyl transverse relaxation-optimized spectroscopy NMR spectra of alanine residues. J Am Chem Soc 129:15428–15429CrossRefGoogle Scholar
  10. Ishima R, Louis JM, Torchia DA (1999) Transverse C-13 relaxation of CHD2 methyl isotopomers to detect slow conformational changes of protein side chains. J Am Chem Soc 121:11589–11590CrossRefGoogle Scholar
  11. Kreishman-Deitrick M, Goley ED, Burdine L, Denison C, Egile C, Li R, Murali N, Kodadek TJ, Welch MD, Rosen MK (2005) NMR analyses of the activation of the Arp2/3 complex by neuronal Wiskott–Aldrich syndrome protein. Biochemistry 44:15247–15256CrossRefGoogle Scholar
  12. Mueller L (1979) Sensitivity enhanced detection of weak nuclei using heteronuclear multiple quantum coherence. J Am Chem Soc 101:4481–4484CrossRefGoogle Scholar
  13. Ollerenshaw JE, Tugarinov V, Skrynnikov NR, Kay LE (2005) Comparison of 13CH3, 13CH2D, and 13CHD2 methyl labeling strategies in proteins. J Biomol NMR 33:25–41CrossRefGoogle Scholar
  14. Pervushin K, Riek R, Wider G, Wüthrich K (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 USA 94:12366–12371CrossRefADSGoogle Scholar
  15. Pervushin K, Vogeli B, Eletsky A (2002) Longitudinal (1)H relaxation optimization in TROSY NMR spectroscopy. J Am Chem Soc 124:12898–12902CrossRefGoogle Scholar
  16. Religa TL, Sprangers R, Kay LE (2010) Dynamic regulation of archaeal proteasome gate opening as studied by TROSY NMR. Science 328:98–102CrossRefADSGoogle Scholar
  17. Sprangers R, Kay LE (2007) Quantitative dynamics and binding studies of the 20S proteasome by NMR. Nature 445:618–622CrossRefGoogle Scholar
  18. Sprangers R, Gribun A, Hwang PM, Houry WA, Kay LE (2005) Quantitative NMR spectroscopy of supramolecular complexes: dynamic side pores in ClpP are important for product release. Proc Natl Acad Sci U S A 102:16678–16683CrossRefADSGoogle Scholar
  19. Tugarinov V, Kay LE (2004) An isotope labeling strategy for methyl TROSY spectroscopy. J Biomol NMR 28:165–172CrossRefGoogle Scholar
  20. Tugarinov V, Kay LE (2005a) Methyl groups as probes of structure and dynamics in NMR studies of high-molecular-weight proteins. Chembiochem 6:1567–1577CrossRefGoogle Scholar
  21. Tugarinov V, Kay LE (2005b) Quantitative 13C and 2H NMR relaxation studies of the 723-residue enzyme malate synthase G reveal a dynamic binding interface. Biochemistry 44:15970–15977CrossRefGoogle Scholar
  22. 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 125:10420–10428CrossRefGoogle Scholar
  23. Tugarinov V, Hwang PM, Kay LE (2004) Nuclear magnetic resonance spectroscopy of high-molecular-weight proteins. Annu Rev Biochem 73:107–146CrossRefGoogle Scholar
  24. Velyvis A, Yang YR, Schachman HK, Kay LE (2007) A solution NMR study showing that active site ligands and nucleotides directly purturb the allosteric equilibrium in aspartate transcarbamolyase. Proc Natl Acad Sci U S A 104:8815–8820CrossRefADSGoogle Scholar
  25. Velyvis A, Schachman HK, Kay LE (2009) Application of methyl-TROSY NMR to test allosteric models describing effects of nucleotide binding to aspartate transcarbamoylase. J Mol Biol 387:540–547CrossRefGoogle Scholar
  26. Zhu G, Bax A (1992) Two-dimensional linear prediction for signals truncated in both dimensions. J Magn Reson 98:192–199Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  1. 1.Departments of Molecular Genetics, Biochemistry and ChemistryThe University of TorontoTorontoCanada

Personalised recommendations