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CACA-TOCSY with alternate 13C–12C labeling: a 13Cα direct detection experiment for mainchain resonance assignment, dihedral angle information, and amino acid type identification

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Abstract

We present a 13C direct detection CACA-TOCSY experiment for samples with alternate 13C–12C labeling. It provides inter-residue correlations between 13Cα resonances of residue i and adjacent Cαs at positions i − 1 and i + 1. Furthermore, longer mixing times yield correlations to Cα nuclei separated by more than one residue. The experiment also provides Cα-to-sidechain correlations, some amino acid type identifications and estimates for ψ dihedral angles. The power of the experiment derives from the alternate 13C–12C labeling with [1,3-13C] glycerol or [2-13C] glycerol, which allows utilizing the small scalar 3JCC couplings that are masked by strong 1JCC couplings in uniformly 13C labeled samples.

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References

  • Arnesano F, Banci L, Piccioli M (2005) NMR structures of paramagnetic metalloproteins. Q Rev Biophys 38:167–219

    Article  Google Scholar 

  • Balayssac S, Jimenez B, Piccioli M (2006) 13C direct detected COCO-TOCSY: a tool for sequence specific assignment and structure determination in protonless NMR experiments. J Magn Reson 182:325–329

    Article  ADS  Google Scholar 

  • Bermel W, Bertini I, Felli IC, Kummerle R, Pierattelli R (2003) 13C Direct detection experiments on the paramagnetic oxidized monomeric copper zinc superoxide dismutase. J Am Chem Soc 125:16423–16429

    Article  Google Scholar 

  • Bermel W, Bertini I, Duma L, Felli IC, Emsley L, Pierattelli R, Vasos PR (2005) Complete assignment of heteronuclear protein resonances by protonless NMR spectroscopy. Angew Chem Int Ed Engl 44:3089–3092

    Article  Google Scholar 

  • Bermel W, Bertini I, Felli IC, Lee YM, Luchinat C, Pierattelli R (2006a) Protonless NMR experiments for sequence-specific assignment of backbone nuclei in unfolded proteins. J Am Chem Soc 128:3918–3919

    Article  Google Scholar 

  • Bermel W, Bertini I, Felli IC, Piccioli M, Pierattelli R (2006b) 13C-detected protonless NMR spectroscopy of proteins in solution. Prog Nucl Magn Res Spec 48:25–45

    Article  Google Scholar 

  • Bermel W, Bertini I, Felli IC, Matzapetakis M, Pierattelli R, Theil EC, Turano P (2007) A method for Cα direct-detection in protonless NMR. J Magn Reson 188:301–310

    Article  ADS  Google Scholar 

  • Bermel W, Bertini I, Csizmok V, Felli IC, Pierattelli R, Tompa P (2009a) H-start for exclusively heteronuclear NMR spectroscopy: the case of intrinsically disordered proteins. J Magn Reson 198:275–281

    Article  ADS  Google Scholar 

  • Bermel W, Bertini I, Felli IC, Pierattelli R (2009b) Speeding up 13C direct detection biomolecular NMR spectroscopy. J Am Chem Soc 131:15339–15345

    Article  Google Scholar 

  • Cai S, Seu C, Kovacs Z, Sherry AD, Chen Y (2006) Sensitivity enhancement of multidimensional NMR experiments by paramagnetic relaxation effects. J Am Chem Soc 128:13474–13478

    Article  Google Scholar 

  • Castellani F, van Rossum B, Diehl A, Schubert M, Rehbein K, Oschkinat H (2002) Structure of a protein determined by solid-state magic-angle-spinning NMR spectroscopy. Nature 420:98–102

    Article  ADS  Google Scholar 

  • Eletsky A, Moreira O, Kovacs H, Pervushin K (2003) A novel strategy for the assignment of side-chain resonances in completely deuterated large proteins using 13C spectroscopy. J Biomol NMR 26:167–179

    Article  Google Scholar 

  • Felli I, Brutscher B (2009) Recent advances in solution NMR: fast methods and heteronuclear direct detection. ChemPhysChem 10:1356–1368

    Article  Google Scholar 

  • Felli I, Pierattelli R, Glaser S, Luy B (2009) Relaxation-optimised Hartmann–Hahn transfer using a specifically Tailored MOCCA-XY16 mixing sequence for carbonyl–carbonyl correlation spectroscopy in 13C direct detection NMR experiments. J Biomol NMR 43:187–196

    Article  Google Scholar 

  • Frueh DP, Arthanari H, Wagner G (2005) Unambiguous assignment of NMR protein backbone signals with a time-shared triple-resonance experiment. J Biomol NMR 33:187–196

    Article  Google Scholar 

  • Goddard TD, Kneller DG (2004) SPARKY 3. University of California, San Francisco

  • Guo C, Geng C, Tugarinov V (2009) Selective backbone labeling of proteins using 1, 2-13C2-pyruvate as carbon source. J Biomol NMR 44:167–173

    Article  Google Scholar 

  • Hennig M, Bermel W, Schwalbe H, Griesinger C (2000) Determination of ψ torsion angle restraints from 3J(Cα, Cα) and 3J(Cα, HN) coupling constants in proteins. J Am Chem Soc 122:6268–6277

    Article  Google Scholar 

  • Hsu S-TD, Bertoncini CW, Dobson CM (2009) Use of protonless NMR spectroscopy to alleviate the loss of information resulting from exchange-broadening. J Am Chem Soc 131:7222–7223

    Article  Google Scholar 

  • Hu J-S, Bax A (1996) Measurement of three-bond 13C–13C J couplings between carbonyl and carbonyl/carboxyl carbons in isotopically enriched proteins. J Am Chem Soc 118:8170–8171

    Article  Google Scholar 

  • Hyberts SG, Takeuchi K, Wagner G (2010) Poisson-gap sampling and forward maximum entropy reconstruction for enhancing the resolution and sensitivity of protein NMR Data. J Am Chem Soc 132:2145–2147

    Article  Google Scholar 

  • Lee D, Vögeli B, Pervushin K (2005) Detection of C′, Cα correlations in proteins using a new time- and sensitivity-optimal experiment. J Biomol NMR 31:273–278

    Article  Google Scholar 

  • LeMaster DM, Kushlan DM (1996) Dynamical mapping of E. coli thioredoxin via 13C NMR relaxation analysis. J Am Chem Soc 118:9255–9264

    Article  Google Scholar 

  • Liu A, Riek R, Wider G, von Schroetter C, Zahn R, Wuthrich K (2000) NMR experiments for resonance assignments of 13C, 15N doubly-labeled flexible polypeptides: application to the human prion protein hPrP(23–230). J Biomol NMR 16:127–138

    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 

  • Peti W, Hennig M, Smith LJ, Schwalbe H (2000) NMR spectroscopic investigation of ψ torsion angle distribution in unfolded ubiquitin from analysis of 3J(Cα, Cα) coupling constants and cross-correlated relaxation rates. J Am Chem Soc 122:12017–12018

    Article  Google Scholar 

  • Serber Z, Richter C, Dotsch V (2001) Carbon-detected NMR experiments to investigate structure and dynamics of biological macromolecules. Chembiochem 2:247–251

    Article  Google Scholar 

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

    Google Scholar 

  • Shaka AJ, Barker PB, Freeman R (1985) Computer-optimized decoupling scheme for wideband applications and low-level operation. J Magn Reson 64:547–552

    Google Scholar 

  • Shimba N, Stern AS, Craik CS, Hoch JC, Dotsch V (2003) Elimination of 13Calpha splitting in protein NMR spectra by deconvolution with maximum entropy reconstruction. J Am Chem Soc 125:2382–2383

    Article  Google Scholar 

  • Takeuchi K, Sun ZY, Wagner G (2008a) Alternate 13C–12C labeling for complete mainchain resonance assignments using C alpha direct-detection with applicability toward fast relaxing protein systems. J Am Chem Soc 130:17210–17211

    Article  Google Scholar 

  • Takeuchi K, Sun ZY, Wagner G (2008b) Alternate 13C–12C labeling for complete mainchain resonance assignments using Cα direct-detection with applicability toward fast relaxing protein systems. J Am Chem Soc 130:17210–17211

    Article  Google Scholar 

  • Takeuchi K, Frueh DP, Hyberts SG, Sun ZJ, Wagner G (2010) High-resolution 3D CANCA NMR experiments for complete mainchain assignments using Cα direct-detection. J Am Chem Soc 132:2945–2951

    Article  Google Scholar 

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Acknowledgments

Dr. Tsyr-Yan (Dharma) Yu, Dr. Arthanari Haribabu, and Dr. Alexander Koglin are acknowledged for stimulating discussions. This work was supported by the NIH (grants AI37581, GM47467 and EB 002026).

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  1. Koh Takeuchi

    Present address: Biomedicinal Information Research Center (BIRC), National Institute of Advanced Industrial Science and Technology (AIST), Aomi 2-4-7, Koto-ku, Tokyo, 135-0064, Japan

Authors and Affiliations

  1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA, 02115, USA

    Koh Takeuchi, Dominique P. Frueh, Zhen-Yu J. Sun, Sebastian Hiller & Gerhard Wagner

  2. Department of Biophysics, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA

    Dominique P. Frueh

  3. Laboratorium für Physikalische Chemie, ETH Zürich, Zurich, 8093, Switzerland

    Sebastian Hiller

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  1. Koh Takeuchi
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  2. Dominique P. Frueh
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Correspondence to Gerhard Wagner.

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Takeuchi, K., Frueh, D.P., Sun, ZY.J. et al. CACA-TOCSY with alternate 13C–12C labeling: a 13Cα direct detection experiment for mainchain resonance assignment, dihedral angle information, and amino acid type identification. J Biomol NMR 47, 55–63 (2010). https://doi.org/10.1007/s10858-010-9410-3

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  • DOI: https://doi.org/10.1007/s10858-010-9410-3

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