Skip to main content
SpringerLink
Log in

Selective backbone labelling of ILV methyl labelled proteins

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

Abstract

Adding the 13C labelled 2-keto-isovalerate and 2-oxobutanoate precursors to a minimal medium composed of 12C labelled glucose instead of the commonly used (2D, 13C) glucose leads not only to the 13C labelling of (I, L, V) methyls but also to the selective 13C labelling of the backbone Cα and CO carbons of the Ile and Val residues. As a result, the backbone (1H, 15N) correlations of the Ile and Val residues and their next neighbours in the (i + 1) position can be selectively identified in HN(CA) and HN(CO) planes. The availability of a selective HSQC spectrum corresponding to the sole amide resonances of the Ile and Val residues allows connecting them to their corresponding methyls by the intra-residue NOE effect, and should therefore be applicable to larger systems.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

TauF1:

Residues 163-441 of the Tau protein

HMQC:

Heteronuclear multiple quantum coherence

HSQC:

Heteronuclear single quantum coherence

NMR:

Nuclear magnetic resonance

NOESY:

Nuclear Overhauser Effect spectroscopy

TOCSY:

Total correlation spectroscopy

References

  • Brutscher B (2002) Intraresidue HNCA and COHNCA experiments for protein backbone resonance assignment. J Magn Reson 156:155–159

    Article  ADS  Google Scholar 

  • Butner KA, Kirschner MW (1991) Tau protein binds to microtubules through a flexible array of distributed weak sites. J Cell Biol 115:717–730

    Article  Google Scholar 

  • Chau MF, Radeke MJ, de Ines C, Barasoain I, Kohlstaedt LA, Feinstein SC (1998) The microtubule-associated protein tau cross-links to two distinct sites on each alpha and beta tubulin monomer via separate domains. Biochemistry 37:17692–17703

    Article  Google Scholar 

  • Cornilescu G, Delaglio F, Bax A (1999) Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J Biomol NMR 13:289–302

    Article  Google Scholar 

  • Diercks T, Coles M, Kessler H (1999) An efficient strategy for assignment of cross-peaks in 3D heteronuclear NOESY experiments. J Biomol NMR 15:177

    Article  Google Scholar 

  • Gardner KH, Kay LE (1998) The use of 2H, 13C, 15N multidimensional NMR to study the structure and dynamics of proteins. Annu Rev Biophys Biomol Struct 27:357–406

    Article  Google Scholar 

  • 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 131:756–769

    Article  Google Scholar 

  • Goode BL, Denis PE, Panda D, Radeke MJ, Miller HP, Wilson L, Feinstein SC (1997) Functional interactions between the proline-rich and repeat regions of tau enhance microtubule binding and assembly. Mol Biol Cell 8:353–365

    Google Scholar 

  • Goto NK, Kay LE (2000) New developments in isotope labeling strategies for protein solution NMR spectroscopy. Curr Opin Struct Biol 10:585–592

    Article  Google Scholar 

  • Goto NK, Gardner KH, Mueller GA, Willis RC, Kay LE (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  Google Scholar 

  • Grzesiek S, Bax A (1992) Improved 3D triple-resonance NMR techniques applied to a 31-Kda protein. J Magn Reson 96:432–440

    Google Scholar 

  • Hanoulle X, Melchior A, Sibille N, Parent B, Denys A, Wieruszeski JM, Horvath D, Allain F, Lippens G, Landrieu I (2007) Structural and functional characterization of the interaction between cyclophilin B and a heparin-derived oligosaccharide. J Biol Chem 282:34148–34158

    Article  Google Scholar 

  • LeMaster DM (1988) Protein NMR resonance assignment by isotropic mixing experiments on random fractionally deuterated samples. FEBS Lett 233:326–330

    Article  Google Scholar 

  • Lippens G, Wieruszeski JM, Leroy A, Smet C, Sillen A, Buee L, Landrieu I (2004) Proline-directed random-coil chemical shift values as a tool for the NMR assignment of the tau phosphorylation sites. Chembiochem 5:73–78

    Article  Google Scholar 

  • Mandelkow EM, Biernat J, Drewes G, Gustke N, Trinczek B, Mandelkow E (1995) Tau domains, phosphorylation, and interactions with microtubules. Neurobiol Aging 16:355–362; discussion 362–353

    Google Scholar 

  • Mikol V, Kallen J, Walkinshaw MD (1994) X-ray structure of a cyclophilin B/cyclosporin complex: comparison with cyclophilin A and delineation of its calcineurin-binding domain. Proc Natl Acad Sci U S A 91:5183–5186

    Article  ADS  Google Scholar 

  • Mueller GA, Kirby TW, DeRose EF, London RE (2003) NMR assignment of protein side chains using residue-correlated labeling and NOE spectra. J Magn Reson 165:237–247

    Article  ADS  Google Scholar 

  • Neidhardt FC, Curtiss III R, Ingraham JL, Lin ECC, Low Jr KB, Magasanik B, Reznikoff WS, Riley M, Schaechter M, Umbarger HE (1996) Escherichia coli and Salmonella. Cellular and Molecular Biology, 2nd edn. American Society for Microbiology, Washington, DC

  • Palmer AG, Cavanagh J, Wright PE, Rance M (1991) Sensitivity improvement in proton-detected two-dimensional heteronuclear correlation NMR spectroscopy. J Magn Reson 93:151–170

    Google Scholar 

  • Pervushin K, Riek R, Wider G, Wuthrich 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 U S A 94:12366–12371

    Article  ADS  Google Scholar 

  • Piotto M, Bourdonneau M, Elbayed K, Wieruszeski JM, Lippens G (2006) New DEFT sequences for the acquisition of one-dimensional carbon NMR spectra of small unlabelled molecules. Magn Reson Chem 44:943–947

    Article  Google Scholar 

  • Rosen MK, Gardner KH, Willis RC, Parris WE, Pawson T, Kay LE (1996) Selective methyl group protonation of perdeuterated proteins. J Mol Biol 263:627–636

    Article  Google Scholar 

  • Sibille N, Bersch B, Coves J, Blackledge M, Brutscher B (2002) Side chain orientation from methyl 1H–1H residual dipolar couplings measured in highly deuterated proteins. J Am Chem Soc 124:14616–14625

    Article  Google Scholar 

  • Sibille N, Sillen A, Leroy A, Wieruszeski JM, Mulloy B, Landrieu I, Lippens G (2006) Structural impact of heparin binding to full-length Tau as studied by NMR spectroscopy. Biochemistry 45:12560–12572

    Article  Google Scholar 

  • Sillen A, Barbier P, Landrieu I, Lefebvre S, Wieruszeski JM, Leroy A, Peyrot V, Lippens G (2007) NMR investigation of the interaction between the neuronal protein tau and the microtubules. Biochemistry 46:3055–3064

    Article  Google Scholar 

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

    Article  Google Scholar 

  • Sprangers R, Velyvis A, Kay LE (2007) Solution NMR of supramolecular complexes: providing new insights into function. Nat Methods 4:697–703

    Article  Google Scholar 

  • Tugarinov V, Kay LE (2003) Ile, Leu, and Val methyl assignments of the 723-residue malate synthase G using a new labeling strategy and novel NMR methods. J Am Chem Soc 125:13868–13878

    Article  Google Scholar 

  • Tugarinov V, Hwang PM, Ollerenshaw JE, Kay LE (2003) Cross-correlated relaxation enhanced 1H[bond]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 

  • Tugarinov V, Choy WY, Orekhov VY, Kay LE (2005) Solution NMR-derived global fold of a monomeric 82-kDa enzyme. Proc Natl Acad Sci U S A 102:622–627

    Article  ADS  Google Scholar 

  • Van Melckebeke H, Simorre JP, Brutscher B (2004) Amino acid-type edited NMR experiments for methyl-methyl distance measurement in 13C-labeled proteins. J Am Chem Soc 126:9584–9591

    Article  Google Scholar 

  • Verdegem D, Dijkstra K, Hanoulle X, Lippens G (2008) Graphical interpretation of Boolean operators for protein NMR assignments. J Biomol NMR 42:11–21

    Article  Google Scholar 

  • Yang D, Zheng Y, Liu D, Wyss DF (2004) Sequence-specific assignments of methyl groups in high-molecular weight proteins. J Am Chem Soc 126:3710–3711

    Article  Google Scholar 

Download references

Acknowledgements

The NMR facilities used in this study were funded by the Région Nord-Pas de Calais (France), FEDER, Ministère de la Recherche, the CNRS, the University of Lille 1 and the Institut Pasteur de Lille. Financial support from the TGIR-TGE RMN for conducting the research is gratefully acknowledged.

Author information

Authors and Affiliations

  1. CNRS UMR 8576 – Unité de Glycobiologie Structurale et Fonctionnelle, Université des Sciences et Technologies de Lille 1, 59655, Villeneuve d’Ascq Cedex, France

    Nathalie Sibille, Xavier Hanoulle, Fanny Bonachera, Dries Verdegem, Isabelle Landrieu, Jean-Michel Wieruszeski & Guy Lippens

Authors
  1. Nathalie Sibille
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xavier Hanoulle
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fanny Bonachera
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dries Verdegem
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Isabelle Landrieu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jean-Michel Wieruszeski
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Guy Lippens
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Guy Lippens.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sibille, N., Hanoulle, X., Bonachera, F. et al. Selective backbone labelling of ILV methyl labelled proteins. J Biomol NMR 43, 219–227 (2009). https://doi.org/10.1007/s10858-009-9307-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10858-009-9307-1

Keywords

Search

Navigation