Skip to main content
SpringerLink
Log in

Nitrogen detected TROSY at high field yields high resolution and sensitivity for protein NMR

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

An Erratum to this article was published on 11 December 2017

This article has been updated

Abstract

Detection of 15N in multidimensional NMR experiments of proteins has sparsely been utilized because of the low gyromagnetic ratio (γ) of nitrogen and the presumed low sensitivity of such experiments. Here we show that selecting the TROSY components of proton-attached 15N nuclei (TROSY 15NH) yields high quality spectra in high field magnets (>600 MHz) by taking advantage of the slow 15N transverse relaxation and compensating for the inherently low 15N sensitivity. The 15N TROSY transverse relaxation rates increase modestly with molecular weight but the TROSY gain in peak heights depends strongly on the magnetic field strength. Theoretical simulations predict that the narrowest line width for the TROSY 15NH component can be obtained at 900 MHz, but sensitivity reaches its maximum around 1.2 GHz. Based on these considerations, a 15N-detected 2D 1H–15N TROSY-HSQC (15N-detected TROSY-HSQC) experiment was developed and high-quality 2D spectra were recorded at 800 MHz in 2 h for 1 mM maltose-binding protein at 278 K (τc ~ 40 ns). Unlike for 1H detected TROSY, deuteration is not mandatory to benefit 15N detected TROSY due to reduced dipolar broadening, which facilitates studies of proteins that cannot be deuterated, especially in cases where production requires eukaryotic expression systems. The option of recording 15N TROSY of proteins expressed in H2O media also alleviates the problem of incomplete amide proton back exchange, which often hampers the detection of amide groups in the core of large molecular weight proteins that are expressed in D2O culture media and cannot be refolded for amide back exchange. These results illustrate the potential of 15NH-detected TROSY experiments as a means to exploit the high resolution offered by high field magnets near and above 1 GHz.

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

Similar content being viewed by others

Change history

  • 11 December 2017

    The authors regret a mistake appeared in the supplement of this paper.

References

  • Allegrozzi M, Bertini I, Janik MBL, Lee Y-M, Liu G, Luchinat C (2000) Lanthanide-induced pseudocontact shifts for solution structure refinements of macromolecules in shells up to 40 Å from the metal ion. J Am Chem Soc 122:4154–4161

    Article  Google Scholar 

  • Allen KN, Imperiali B (2010) Lanthanide-tagged proteins—an illuminating partnership. Curr Opin Chem Biol 14:247–254

    Article  Google Scholar 

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

    Article  Google Scholar 

  • Balayssac S, Bertini I, Luchinat C, Parigi G, Piccioli M (2006) 13C direct detected NMR increases the detectability of residual dipolar couplings. J Am Chem Soc 128:15042–15043

    Article  Google Scholar 

  • Banci L, Bertini I, Cavallaro G, Giachetti A, Luchinat C, Parigi G (2004) Paramagnetism-based restraints for Xplor-NIH. J Biomol NMR 28:249–261

    Article  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, 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 

  • Bohlen JM, Bodenhausen G (1993) Experimental aspects of chirp NMR spectroscopy. J Magn Res A 102:293–301

    Article  ADS  Google Scholar 

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

    Article  Google Scholar 

  • Fujiwara T, Nagayama K (1988) Composite inversion pulses with frequency switching and their application to broadband decoupling. J Magn Res (1969) 77:53–63

  • Gal M, Edmonds KA, Milbradt AG, Takeuchi K, Wagner G (2011) Speeding up direct (15)N detection: hCaN 2D NMR experiment. J Biomol NMR 51:497–504

    Article  Google Scholar 

  • Gardner KH, Zhang X, Gehring K, Kay LE (1998) Solution NMR studies of a 42 KDa Escherichia Coli maltose binding protein/β-cyclodextrin complex: chemical shift assignments and analysis. J Am Chem Soc 120:11738–11748

    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 

  • Inagaki F, Miyazawa T (1980) NMR analyses of molecular conformations and conformational equilibria with the lanthanide probe method. Prog Nuc Magn Res Spec 14:67–111

    Article  Google Scholar 

  • Kay L, Keifer P, Saarinen T (1992) Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity. J Am Chem Soc 114:10663–10665

    Article  Google Scholar 

  • Keizers PH, Desreux JF, Overhand M, Ubbink M (2007) Increased paramagnetic effect of a lanthanide protein probe by two-point attachment. J Am Chem Soc 129:9292–9293

    Article  Google Scholar 

  • Kelly AE, Ou HD, Withers R, Dötsch V (2002) Low-conductivity buffers for high-sensitivity NMR measurements. J Am Chem Soc 124:12013–12019

    Article  Google Scholar 

  • Kofuku Y, Ueda T, Okude J, Shiraishi Y, Kondo K, Mizumura T, Suzuki S, Shimada I (2014) Functional dynamics of deuterated β2 -adrenergic receptor in lipid bilayers revealed by NMR spectroscopy. Angew Chem Int Ed 53:13376–13379

    Article  Google Scholar 

  • Kupce E, Nishida T, Freeman R (2003) Hadamard NMR spectroscopy. Magn Reson Chem 42:95–122

    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 

  • Lee D, Hilty C, Wider G, Wüthrich K (2006) Effective rotational correlation times of proteins from NMR relaxation interference. J Magn Reson 178:72–76

    Article  ADS  Google Scholar 

  • Levy G, Richter R (1979) Nitrogen 15 nuclear magnetic resonance spectroscopy. Wiley, Hoboken

    Google Scholar 

  • Lohr F, Katsemi V, Hartleib J, Gunther U, Ruterjans H (2003) A strategy to obtain backbone resonance assignments of deuterated proteins in the presence of incomplete amide 2H/1H back-exchange. J Biomol NMR 25:291–311

    Article  Google Scholar 

  • Miyazawa-Onami M, Takeuchi K, Takano T, Sugiki T, Shimada I, Takahashi H (2013) Perdeuteration and methyl-selective 1H, 13C-labeling by using a Kluyveromyces lactis expression system. J Biomol NMR 57:297–304

    Article  Google Scholar 

  • Morgan WD, Kragt A, Feeney J (2000) Expression of deuterium-isotope-labelled protein in the yeast pichia pastoris for NMR studies. J Biomol NMR 17:337–347

    Article  Google Scholar 

  • Peng J, Thanabal V, Wagner G (1991) Improved accuracy of heteronuclear transverse relaxation time measurements in macromolecules. Elimination of antiphase contributions. J Magn Res 95:421–427

    ADS  Google Scholar 

  • Pervushin K (2000) Impact of transverse relaxation optimized spectroscopy (TROSY) on NMR as a technique in structural biology. Q Rev Biophys 33:161–197

    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 94:12366–12371

    Article  ADS  Google Scholar 

  • Pervushin KV, Wider G, Wüthrich K (1998) Single transition-to-single transition polarization transfer (ST2-PT) in [15N,1H]-TROSY. J Biomol NMR 12:345–348

    Article  Google Scholar 

  • Piotto M, Saudek V, Sklenar V (1992) Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J Biomol NMR 2:661–665

    Article  Google Scholar 

  • Rovnyak D, Hoch JC, Stern AS, Wagner G (2004) Resolution and sensitivity of high field nuclear magnetic resonance spectroscopy. J Biomol NMR 30:1–10

    Article  Google Scholar 

  • Sastry M, Xu L, Georgiev IS, Bewley CA, Nabel GJ, Kwong PD (2011) Mammalian production of an isotopically enriched outer domain of the HIV-1 gp120 glycoprotein for NMR spectroscopy. J Biomol NMR 50:197–207

    Article  Google Scholar 

  • Schanda P, Brutscher B (2005) Very fast two-dimensional NMR spectroscopy for real-time investigation of dynamic events in proteins on the time scale of seconds. J Am Chem Soc 127:8014–8015

    Article  Google Scholar 

  • Schanda P, Brutscher B (2006) Hadamard frequency-encoded SOFAST-HMQC for ultrafast two-dimensional protein NMR. J Magn Reson 178:334–339

    Article  ADS  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

    ADS  Google Scholar 

  • Takeuchi K, Sun ZY, Wagner G (2008) Alternate 13C–12C labeling for complete mainchain resonance assignments using Ca 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 (2010a) High-resolution 3D CANCA NMR experiments for complete mainchain assignments using Ca direct-detection. J Am Chem Soc 132:2945–2951

    Article  Google Scholar 

  • Takeuchi K, Heffron G, Sun ZY, Frueh DP, Wagner G (2010b) Nitrogen-detected CAN and CON experiments as alternative experiments for main chain NMR resonance assignments. J Biomol NMR 47:271–282

    Article  Google Scholar 

  • Takeuchi K, Gal M, Shimada I, Wagner G (2012) Low γ-nuclei detection experiments for bimolecular NMR. In: Clore M, Potts J (eds) Recent Developments in Biomolecular NMR, Royal Society of Chemistry

  • Tjandra N, Bax A (1997) Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. Science 278:1111–1114

    Article  ADS  Google Scholar 

  • Tolman JR, Flanagan JM, Kennedy MA, Prestegard JH (1995) Nuclear magnetic dipole interactions in field-oriented proteins: information for structure determination in solution. Proc Natl Acad Sci 92:9279–9283

    Article  ADS  Google Scholar 

  • Tugarinov V, Kanelis V, Kay LE (2006) Isotope labeling strategies for the study of high-molecular-weight proteins by solution NMR spectroscopy. Nat Protocols 1:749–754

    Article  Google Scholar 

  • Tycko R, Blanco FJ, Ishii Y (2000) Alignment of biopolymers in strained gels: a new way to create detectable dipole–dipole couplings in high-resolution biomolecular NMR. J Am Chem Soc 122:9340–9341

    Article  Google Scholar 

  • Vasos PR, Hall JB, Kummerle R, Fushman D (2006) Measurement of 15N relaxation in deuterated amide groups in proteins using direct nitrogen detection. J Biomol NMR 36:27–36

    Article  Google Scholar 

Download references

Acknowledgments

This work was supported by NIH Grants GM047467 and AI37581 to GW and by METI (Grant Name: development of core technologies for innovative drug development based upon IT) to IS. This work was also partly supported by JST, PRESTO to KT. Maintenance of NMR instruments was in part supported by NIH grant EB002026. We would like thanks Dominique Frueh, Wolfgang Bermel and Arthur Palmer for useful discussions. We would like to thank Ms. Misaki Imai for making the protein samples used in the paper.

Author information

Author notes

    Authors and Affiliations

    1. Molecular Profiling Research Center for Drug Discovery, National Institute for Advanced Industrial Science and Technology, Tokyo, 135-0063, Japan

      Koh Takeuchi & Ichio Shimada

    2. PRESTO, JST, Tokyo, 135-0063, Japan

      Koh Takeuchi

    3. Department of Biochemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA, 02115, USA

      Haribabu Arthanari & Gerhard Wagner

    4. Graduate Schools of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

      Ichio Shimada

    Authors
    1. Koh Takeuchi
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Haribabu Arthanari
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Ichio Shimada
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Gerhard Wagner
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding authors

    Correspondence to Ichio Shimada or Gerhard Wagner.

    Additional information

    Koh Takeuchi and Haribabu Arthanari have contributed equally to this work.

    An erratum to this article is available at https://doi.org/10.1007/s10858-017-0131-8.

    Electronic supplementary material

    Below is the link to the electronic supplementary material.

    Supplementary material 1 (DOCX 1071 kb)

    Rights and permissions

    Reprints and permissions

    About this article

    Check for updates. Verify currency and authenticity via CrossMark

    Cite this article

    Takeuchi, K., Arthanari, H., Shimada, I. et al. Nitrogen detected TROSY at high field yields high resolution and sensitivity for protein NMR. J Biomol NMR 63, 323–331 (2015). https://doi.org/10.1007/s10858-015-9991-y

    Download citation

    • Received:

    • Accepted:

    • Published:

    • Issue Date:

    • DOI: https://doi.org/10.1007/s10858-015-9991-y

    Keywords

    Search

    Navigation