Journal of Biomolecular NMR

, 51:245 | Cite as

Multiplet-filtered and gradient-selected zero-quantum TROSY experiments for 13C1H3 methyl groups in proteins

  • Michelle L. Gill
  • Arthur G. PalmerIIIEmail author


Multiplet-filtered and gradient-selected heteronuclear zero-quantum coherence (gsHZQC) TROSY experiments are described for measuring 1H–13C correlations for 13CH3 methyl groups in proteins. These experiments provide improved suppression of undesirable, broad outer components of the heteronuclear zero-quantum multiplet in medium-sized proteins, or in flexible sites of larger proteins, compared to previously described HZQC sequences (Tugarinov et al. in J Am Chem Soc 126:4921–4925, 2004; Ollerenshaw et al. in J Biomol NMR 33:25–41, 2005). Hahn-echo versions of the gsHZQC experiment also are described for measuring zero- and double-quantum transverse relaxation rate constants for identification of chemical exchange broadening. Application of the proposed pulse sequences to Escherichia coli ribonuclease HI, with a molecular mass of 18 kD, indicates that improved multiplet suppression is obtained without substantial loss of sensitivity.


Methyl TROSY Zero-quantum HZQC HMQC RNase H 



A.G.P. and M.L.G. acknowledge support from National Institute of Health grants GM50291 and GM089047, respectively. A.G.P. is a member of the New York Structural Biology Center (NYSBC). The NYSBC is a STAR center supported by the New York State Office of Science, Technology and Academic Research. Data acquired at 18.8 and 21.1 T were collected at the NYSBC. The 900 MHz (21.1 T) spectrometers were purchased with funds from the National Institute of Health (USA), the Keck Foundation (New York State), and the NYC Economic Development Corporation. A.G.P. and M.L.G. thank Mark Rance (University of Cincinnati) and Daròn Freedberg (CBER/FDA) for helpful scientific discussions.

Supplementary material

10858_2011_9533_MOESM1_ESM.pdf (216 kb)
Supplementary material 1 (PDF 215 kb)


  1. Butterwick JA, Patrick Loria J, Astrof NS, Kroenke CD, Cole R, Rance M, Palmer AG (2004) Multiple time scale backbone dynamics of homologous thermophilic and mesophilic ribonuclease HI enzymes. J Mol Biol 339:855–871CrossRefGoogle Scholar
  2. Cavanagh J, Palmer AG, Wright PE, Rance M (1991) Sensitivity improvement in proton-detected two-dimensional heteronuclear relay spectroscopy. J Magn Reson (1969) 91:429–436CrossRefGoogle Scholar
  3. Davis DG (1989) Elimination of baseline distortions and minimization of artifacts from phased 2D NMR spectra. J Magn Reson (1969) 81:603–607CrossRefGoogle Scholar
  4. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293CrossRefGoogle Scholar
  5. Findeisen M, Brand T, Berger S (2007) A 1H-NMR thermometer suitable for cryoprobes. Magn Reson Chem 45:175–178CrossRefGoogle Scholar
  6. Gardner KH, Kay LE (1997) Production and incorporation of 15N, 13C, 2H (1H-δ1 methyl) isoleucine into proteins for multidimensional NMR studies. J Am Chem Soc 119:7599–7600CrossRefGoogle Scholar
  7. Goto NK, Gardner KH, Mueller GA, Willis RC, Kay LE (1999) A robust and cost-effective method for the production of Val, Leu, Ile (δ1) methyl-protonated 15N-, 13C-, 2H-labeled proteins. J Biomol NMR 13:369–374CrossRefGoogle Scholar
  8. Hollien J, Marqusee S (1999) A thermodynamic comparison of mesophilic and thermophilic ribonucleases H. Biochemistry 38:3831–3836CrossRefGoogle Scholar
  9. Kay L, Keifer P, Saarinen T (1992) Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity. J Am Chem Soc 114:10663–10665CrossRefGoogle Scholar
  10. 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 126:3964–3973CrossRefGoogle Scholar
  11. Kroenke CD, Loria JP, Lee LK, Rance M, Palmer AG (1998) Longitudinal and transverse 1H–15N dipolar 15N chemical shift anisotropy relaxation interference: unambiguous determination of rotational diffusion tensors and chemical exchange effects in biological macromolecules. J Am Chem Soc 120:7905–7915CrossRefGoogle Scholar
  12. Mandel AM, Akke M, Palmer AG III (1995) Backbone dynamics of Escherichia coli ribonuclease HI: correlations with structure and function in an active enzyme. J Mol Biol 246:144–163CrossRefGoogle Scholar
  13. Mandel AM, Akke M, Palmer AG (1996) Dynamics of ribonuclease H: temperature dependence of motions on multiple time scales. Biochemistry 35:16009–16023CrossRefGoogle Scholar
  14. Ollerenshaw JE, Tugarinov V, Kay LE (2003) Methyl TROSY: explanation and experimental verification. Magn Reson Chem 41:843–852CrossRefGoogle Scholar
  15. 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
  16. Palmer AG, Cavanagh J, Wright PE, Rance M (1991) Sensitivity improvement in proton-detected two-dimensional heteronuclear correlation NMR spectroscopy. J Magn Reson (1969) 93:151–170CrossRefGoogle Scholar
  17. Palmer AG, Cavanagh J, Byrd RA, Rance M (1992) Sensitivity improvement in three-dimensional heteronuclear correlation NMR spectroscopy. J Magn Reson (1969) 96:416–424CrossRefGoogle Scholar
  18. Rance M, Byrd RA (1983) Obtaining high-fidelity spin-1/2 powder spectra in anisotropic media: phase-cycled Hahn echo spectroscopy. J Magn Reson (1969) 52:221–240CrossRefGoogle Scholar
  19. Shaka AJ, Keeler J, Frenkiel T, Freeman R (1983) An improved sequence for broadband decoupling: WALTZ-16. J Magn Resn (1969) 52:335–338Google Scholar
  20. Tugarinov V, Kay LE (2004) 1H, 13C–1H, 1H dipolar cross-correlated spin relaxation in methyl groups. J Biomol NMR 29:369–376CrossRefGoogle Scholar
  21. Tugarinov V, Hwang PM, Ollerenshaw JE, 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
  22. Tugarinov V, Sprangers R, Kay LE (2004) Line narrowing in methyl-TROSY using zero-quantum 1H–13C NMR spectroscopy. J Am Chem Soc 126:4921–4925CrossRefGoogle Scholar
  23. Yamazaki T, Yoshida M, Nagayama K (1993) Complete assignments of magnetic resonances of ribonuclease H from Escherichia coli by double- and triple-resonance 2D and 3D NMR spectroscopies. Biochemistry 32:5656–5669CrossRefGoogle Scholar
  24. Yang W, Hendrickson WA, Kalman ET, Crouch RJ (1990) Expression, purification, and crystallization of natural and selenomethionyl recombinant ribonuclease H from Escherichia coli. J Biol Chem 265:13553–13559Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular BiophysicsColumbia UniversityNew YorkUSA

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