Skip to main content
SpringerLink
Log in

Selective editing of Val and Leu methyl groups in high molecular weight protein NMR

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

Abstract

The development of methyl-TROSY approaches and specific 13C–1H labeling of Ile, Leu and Val methyl groups in highly deuterated proteins has made it possible to study high molecular weight proteins, either alone or in complexes, using solution nuclear magnetic resonance (NMR) spectroscopy. Here we present 2-dimensional (2D) and 3-dimensional (3D) NMR experiments designed to achieve complete separation of the methyl resonances of Val and Leu, labeled using the same precursor, α-ketoisovalerate or acetolactate. The 2D experiment can further select the methyl resonances of Val or Leu based on the Cα or Cβ chemical shift values of Val or Leu, respectively. In the 3D spectrum, the methyl cross peaks of Val and Leu residues have opposite signs; thus, not only can the residue types be easily distinguished, but the methyl pairs from the same residue can also be identified. The feasibility of this approach, implemented in both 2D and 3D experiments, has been demonstrated on an 82 kDa protein, malate synthase G. The methods developed in this study will reduce resonance overlaps and also facilitate structure-guided resonance assignments.

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

References

  • 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 6:277–293

    Article  Google Scholar 

  • Gans P, Hamelin O, Sounier R, Ayala I, Dura MA, Amero CD, 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 Engl 49:1958–1962

    Article  Google Scholar 

  • 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 119:7599–7600

    Article  Google Scholar 

  • Geen H, Freeman R (1991) Band-selective radiofrequency pulses. J Magn Reson 93:93–141

    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 

  • 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 

  • Guo C, Tugarinov V (2010) Selective 1H- 13C NMR spectroscopy of methyl groups in residually protonated samples of large proteins. J Biomol NMR 46:127–133

    Article  Google Scholar 

  • Hu W, Zuiderweg ERP (1996) Stereospecific assignments of Val and Leu methyl groups in a selectively 13C-labeled 18 kDa polypeptide using 3D CT-(H)CCH-COSY and 2D 1Jc-c edited heteronuclear correlation experiments. J Magn Reson B 113:70–75

    Article  Google Scholar 

  • Janin J, Miller S, Chothia C (1988) Surface, subunit interfaces and interior of oligomeric proteins. J Mol Biol 204:155–164

    Article  Google Scholar 

  • John M, Schmitz C, Park AY, Dixon NE, Huber T, Otting G (2007) Sequence-specific and stereospecific assignment of methyl groups using paramagnetic lanthanides. J Am Chem Soc 129:13749–13757

    Article  Google Scholar 

  • Johnson BA, Blevins RA (1994) NMR view: a computer program for the visualization and analysis of NMR data. J Biomol NMR 4:603–614

    Article  Google Scholar 

  • Kato H, van Ingen H, Zhou BR, Feng H, Bustin M, Kay LE, Bai Y (2011) Architecture of the high mobility group nucleosomal protein 2-nucleosome complex as revealed by methyl-based NMR. Proc Natl Acad Sci U S A 108:12283–12288

    Article  Google Scholar 

  • Kupce E, Boyd J, Campbell ID (1995) Short selective pulses for biochemical applications. J Magn Reson B 106:300–303

    Article  Google Scholar 

  • Marino JP, Diener JL, Moore PB, Griesinger C (1997) Multiple-quantum coherence dramatically enhances the sensitivity of CH and CH2 correlations in uniformly C-13-labeled RNA. J Am Chem Soc 119:7361–7366

    Article  Google Scholar 

  • Marion D, Ikura M, Tschudin R, Bax A (1989) Rapid recording of 2d Nmr-spectra without phase cycling—application to the study of hydrogen-exchange in proteins. J Magn Reson 85:393–399

    Google Scholar 

  • Marley J, Lu M, Bracken C (2001) A method for efficient isotopic labeling of recombinant proteins. J Biomol NMR 20:71–75

    Article  Google Scholar 

  • McCaldon P, Argos P (1988) Oligopeptide biases in protein sequences and their use in predicting protein coding regions in nucleotide sequences. Proteins 4:99–122

    Article  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 

  • Religa TL, Sprangers R, Kay LE (2010) Dynamic regulation of archaeal proteasome gate opening as studied by TROSY NMR. Science 328:98–102

    Article  ADS  Google Scholar 

  • Ruschak AM, Religa TL, Breuer S, Witt S, Kay LE (2010) The proteasome antechamber maintains substrates in an unfolded state. Nature 467:868–871

    Article  ADS  Google Scholar 

  • Shaka AJ, Keeler J, Frenkiel T, Freeman R (1983) An improved sequence for broad-band decoupling—Waltz-16. J Magn Reson 52:335–338

    Google Scholar 

  • Shen Y, Bax A (2010) SPARTA plus: a modest improvement in empirical NMR chemical shift prediction by means of an artificial neural network. J Biomol NMR 48:13–22

    Article  Google Scholar 

  • Sheppard D, Guo CY, Tugarinov V (2009) 4D (1)H-(13)C NMR spectroscopy for assignments of alanine methyls in large and complex protein structures. J Am Chem Soc 131:1364–1365

    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, Li X, Mao X, Rubinstein JL, Schimmer AD, Kay LE (2008) TROSY-based NMR evidence for a novel class of 20S proteasome inhibitors. Biochemistry 47:6727–6734

    Article  Google Scholar 

  • Tjong H, Qin S, Zhou HX (2007) PI2PE: protein interface/interior prediction engine. Nucleic Acids Res 35:W357–W362

    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, Kay LE (2004a) An isotope labeling strategy for methyl TROSY spectroscopy. J Biomol NMR 28:165–172

    Article  Google Scholar 

  • Tugarinov V, Kay LE (2004b) Stereospecific NMR assignments of prochiral methyls, rotameric states and dynamics of valine residues in malate synthase G. J Am Chem Soc 126:9827–9836

    Article  Google Scholar 

  • Tugarinov V, Muhandiram R, Ayed A, Kay LE (2002) Four-dimensional NMR spectroscopy of a 723-residue protein: chemical shift assignments and secondary structure of malate synthase g. J Am Chem Soc 124:10025–10035

    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 

  • 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 

  • Vuister GW, Bax A (1992) Resolution enhancement and spectral editing of uniformly 13C-enriched proteins by homonuclear broadband 13C decoupling. J Magn Reson 98:428–435

    Google Scholar 

  • Xu XP, Case DA (2002) Probing multiple effects on 15N, 13C alpha, 13C beta, and 13C′ chemical shifts in peptides using density functional theory. Biopolymers 65:408–423

    Article  Google Scholar 

  • Xu Y, Liu M, Simpson PJ, Isaacson R, Cota E, Marchant J, Yang D, Zhang X, Freemont P, Matthews S (2009) Automated assignment in selectively methyl-labeled proteins. J Am Chem Soc 131:9480–9481

    Article  Google Scholar 

  • Zhu G, Bax A (1990) Improved linear prediction for truncated signals of known phase. J Magn Reson 90:405–410

    Google Scholar 

Download references

Acknowledgments

We thank Dr. Vitali Tugarinov (University of Maryland) for the MSG expression plasmid and Mr. Loren Colson for assistance with protein production. The research was supported by NIH grants R01GM074748 and R01GM086171, an AMMI grant, and the NMR core facility of City of Hope.

Author information

Authors and Affiliations

  1. Departments of Immunology and Molecular Medicine, Beckman Research Institute of City of Hope, Duarte, CA, 91010, USA

    Weidong Hu, Andrew T. Namanja, Steven Wong & Yuan Chen

Authors
  1. Weidong Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andrew T. Namanja
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Steven Wong
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yuan Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yuan Chen.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 192 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hu, W., Namanja, A.T., Wong, S. et al. Selective editing of Val and Leu methyl groups in high molecular weight protein NMR. J Biomol NMR 53, 113–124 (2012). https://doi.org/10.1007/s10858-012-9629-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10858-012-9629-2

Keywords

Search

Navigation