Skip to main content
SpringerLink
Log in

Comparison of 13CH3, 13CH2D, and 13CHD2 methyl labeling strategies in proteins

  • Article
  • Published:
Journal of Biomolecular NMR Aims and scope Submit manuscript

Abstract

A comparison of three labeling strategies for studies involving side chain methyl groups in high molecular weight proteins, using 13CH3,13CH2D, and 13CHD2 methyl isotopomers, is presented. For each labeling scheme, 1H–13C pulse sequences that give optimal resolution and sensitivity are identified. Three highly deuterated samples of a 723 residue enzyme, malate synthase G, with 13CH3,13CH2D, and 13CHD2 labeling in Ile δ1 positions, are used to test the pulse sequences experimentally, and a rationalization of each sequence’s performance based on a product operator formalism that focuses on individual transitions is presented. The HMQC pulse sequence has previously been identified as a transverse relaxation optimized experiment for 13CH3-labeled methyl groups attached to macromolecules, and a zero-quantum correlation pulse scheme (13CH3 HZQC) has been developed to further improve resolution in the indirectly detected dimension. We present a modified version of the 13CH3 HZQC sequence that provides improved sensitivity by using the steady-state magnetization of both 13C and 1H spins. The HSQC and HMQC spectra of 13CH2D-labeled methyl groups in malate synthase G are very poorly resolved, but we present a new pulse sequence, 13CH2D TROSY, that exploits cross-correlation effects to record 1H–13C correlation maps with dramatically reduced linewidths in both dimensions. Well-resolved spectra of 13CHD2-labeled methyl groups can be recorded with HSQC or HMQC; a new 13CHD2 HZQC sequence is described that provides improved resolution with no loss in sensitivity in the applications considered here. When spectra recorded on samples prepared with the three isotopomers are compared, it is clear that the 13CH3 labeling strategy is the most beneficial from the perspective of sensitivity (gains ≥2.4 relative to either 13CH2D or 13CHD2 labeling), although excellent resolution can be obtained with any of the isotopomers using the pulse sequences presented here.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • A. Bax R.H. Griffey B.L. Hawkins (1983) J. Magn. Reson. 55 301–315

    Google Scholar 

  • G. Bodenhausen D.J. Rubin (1980) Chem. Phys. Lett. 69 185–189 Occurrence Handle10.1016/0009-2614(80)80041-8

    Article  Google Scholar 

  • F. Delaglio S. Grzesiek G.W. Vuister G. Zhu J. Pfeifer A. Bax (1995) J.Biomol. NMR 6 277–293 Occurrence Handle10.1007/BF00197809 Occurrence Handle8520220

    Article  PubMed  Google Scholar 

  • K.H. Gardner L.E. Kay (1997) J. Am. Chem. Soc. 119 7599–7600 Occurrence Handle10.1021/ja9706514

    Article  Google Scholar 

  • K.H. Gardner M.K. Rosen L.E. Kay (1997) Biochemistry 36 1389–1401 Occurrence Handle10.1021/bi9624806 Occurrence Handle9063887

    Article  PubMed  Google Scholar 

  • N.K. Goto K.H. Gardner G.A. Mueller R.C. Willis L.E. Kay (1999) J. Biomol. NMR 13 369–374 Occurrence Handle10.1023/A:1008393201236 Occurrence Handle10383198

    Article  PubMed  Google Scholar 

  • R. Ishima J.M. Louis D.A. Torchia (1999) J. Am. Chem. Soc. 121 11589–11590 Occurrence Handle10.1021/ja992836b

    Article  Google Scholar 

  • R. Ishima A.P. Petkova J.M. Louis D.A. Torchia (2001) J. Am. Chem. Soc. 123 6164–6171 Occurrence Handle10.1021/ja0104711 Occurrence Handle11414851

    Article  PubMed  Google Scholar 

  • J. Janin S. Miller C. Chothia (1988) J. Mol. Biol. 204 155–164 Occurrence Handle10.1016/0022-2836(88)90606-7 Occurrence Handle3216390

    Article  PubMed  Google Scholar 

  • L.E. Kay P. Keifer T. Saarinen (1992) J. Am. Chem. Soc. 114 10663–10665 Occurrence Handle10.1021/ja00052a088

    Article  Google Scholar 

  • L.E. Kay J.H. Prestegard (1987) J.Am. Chem. Soc. 109 3829–3835 Occurrence Handle10.1021/ja00247a002

    Article  Google Scholar 

  • L.E. Kay D.A. Torchia (1991) J. Magn. Reson. 95 536–547

    Google Scholar 

  • D.M. Korzhnev K. Kloiber L.E. Kay (2004) J. Am. Chem. Soc. 126 7320–7329 Occurrence Handle10.1021/ja049968b Occurrence Handle15186169

    Article  PubMed  Google Scholar 

  • Z.L. Mádi B. Brutscher T. Schulte-Herbrüggen R. Brüschweiler R.R. Ernst (1997) Chem. Phys. Lett. 268 300–305 Occurrence Handle10.1016/S0009-2614(97)00194-2

    Article  Google Scholar 

  • W.J. Metzler M. Wittekind V. Goldfarb L. Mueller B.T. Farmer (1996) J. Am. Chem. Soc. 118 6800–6801 Occurrence Handle10.1021/ja9604875

    Article  Google Scholar 

  • E. Miclet D.C. Williams G.M. Clore D.L. Bryce J. Boisbouvier A. Bax (2004) J. Am. Chem. Soc. 126 10560–10570 Occurrence Handle10.1021/ja047904v Occurrence Handle15327312

    Article  PubMed  Google Scholar 

  • O. Millet D.R. Muhandiram N.R. Skrynnikov L.E. Kay (2002) J. Am. Chem. Soc. 124 6439–6448 Occurrence Handle10.1021/ja012497y Occurrence Handle12033875

    Article  PubMed  Google Scholar 

  • D.R. Muhandiram T. Yamazaki B.D. Sykes L.E. Kay (1995) J. Am. Chem. Soc. 117 11536–11544 Occurrence Handle10.1021/ja00151a018

    Article  Google Scholar 

  • L. Müller (1979) J. Am. Chem. Soc. 101 4481–4484 Occurrence Handle10.1021/ja00510a007

    Article  Google Scholar 

  • N. Muller G. Bodenhausen R.R. Ernst (1987) J. Magn. Reson. 75 297–334

    Google Scholar 

  • J.E. Ollerenshaw V. Tugarinov L.E. Kay (2003) Magn. Reson. Chem. 41 843–852 Occurrence Handle10.1002/mrc.1256

    Article  Google Scholar 

  • K. Pervushin R. Riek G. Wider K. Wuthrich (1997) Proc.Natl. Acad. Sci. USA 94 12366–12371 Occurrence Handle10.1073/pnas.94.23.12366 Occurrence Handle9356455

    Article  PubMed  Google Scholar 

  • M.K. Rosen K.H. Gardner R.C. Willis W.E. Parris T. Pawson L.E. Kay (1996) J. Mol. Biol. 263 627–636 Occurrence Handle10.1006/jmbi.1996.0603 Occurrence Handle8947563

    Article  PubMed  Google Scholar 

  • J. Schleucher M. Sattler C. Griesinger (1993) Angew. Chem. Int. Ed. Engl. 32 1489–1491 Occurrence Handle10.1002/anie.199314891

    Article  Google Scholar 

  • A.J. Shaka J. Keeler T. Frenkiel R. Freeman (1983) J. Magn. Reson. 52 335–338

    Google Scholar 

  • B.O. Smith Y. Ito A. Raine S. Teichmann L. Bentovim D. Nietlispach R.W. Broadhurst T. Terada M. Kelly H. Oschkinat T. Shibata S. Yokoyama E.D. Laue (1996) J. Biomol. NMR 8 360–368 Occurrence Handle10.1007/BF00410335

    Article  Google Scholar 

  • O.W. Sørensen (1989) Prog. NMR Spectrosc. 21 503–569 Occurrence Handle10.1016/0079-6565(89)80006-8

    Article  Google Scholar 

  • V. Tugarinov W.Y. Choy V.Y. Orekhov L.E. Kay (2005a) Proc. Natl. Acad. Sci. USA 102 622–627 Occurrence Handle10.1073/pnas.0407792102

    Article  Google Scholar 

  • V. Tugarinov P.M. Hwang L.E. Kay (2004a) Annu. Rev. Biochem. 73 107–146 Occurrence Handle10.1146/annurev.biochem.73.011303.074004

    Article  Google Scholar 

  • V. Tugarinov P.M. Hwang J.E. Ollerenshaw L.E. Kay (2003) J. Am. Chem. Soc. 125 10420–10428 Occurrence Handle10.1021/ja030153x Occurrence Handle12926967

    Article  PubMed  Google Scholar 

  • V. Tugarinov L.E. Kay (2004a) J. Biomol. NMR 29 369–376 Occurrence Handle10.1023/B:JNMR.0000032562.07475.7f

    Article  Google Scholar 

  • V. Tugarinov L.E. Kay (2004b) J. Biomol. NMR, 28 165–172

    Google Scholar 

  • V. Tugarinov J.E. Ollerenshaw L.E. Kay (2005b) J. Am. Chem. Soc. 127 8214–8225 Occurrence Handle10.1021/ja0508830

    Article  Google Scholar 

  • V. Tugarinov R. Sprangers L.E. Kay (2004b) J. Am. Chem. Soc. 126 4921–4925 Occurrence Handle10.1021/ja039732s

    Article  Google Scholar 

  • L.G. Werbelow A.G. Marshall (1973) J. Magn. Reson. 11 299–313

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemistry, University of Toronto, Toronto, Ont., M5S 3H6, Canada

    Jason E. Ollerenshaw, Vitali Tugarinov & Lewis E. Kay

  2. Department of Biochemistry, University of Toronto, Toronto, Ont., M5S 1A8, Canada

    Vitali Tugarinov & Lewis E. Kay

  3. Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ont., M5S 1A8, Canada

    Vitali Tugarinov, Lewis E. Kay & Lewis E. Kay

  4. Department of Chemistry, Purdue University, West Lafayette, IN, 47907-2048, U.S.A.

    Nikolai R. Skrynnikov

Authors
  1. Jason E. Ollerenshaw
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Vitali Tugarinov
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nikolai R. Skrynnikov
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lewis E. Kay
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Lewis E. Kay.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ollerenshaw, J.E., Tugarinov, V., Skrynnikov, N.R. et al. Comparison of 13CH3, 13CH2D, and 13CHD2 methyl labeling strategies in proteins. J Biomol NMR 33, 25–41 (2005). https://doi.org/10.1007/s10858-005-2614-2

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10858-005-2614-2

Keywords

Search

Navigation