Journal of Biomolecular NMR

, Volume 37, Issue 3, pp 205–224 | Cite as

Improved pulse sequences for sequence specific assignment of aromatic proton resonances in proteins

  • Frank Löhr
  • Robert Hänsel
  • Vladimir V. Rogov
  • Volker Dötsch


Aromatic proton resonances of proteins are notoriously difficult to assign. Through-bond correlation experiments are preferable over experiments that rely on through-space interactions because they permit aromatic chemical shift assignments to be established independently of the structure determination process. Known experimental schemes involving a magnetization transfer across the Cβ–Cγ bond in aromatic side chains either suffer from low efficiency for the relay beyond the Cδ position, use sophisticated 13C mixing schemes, require probe heads suitable for application of high 13C radio-frequency fields or rely on specialized isotopic labelling patterns. Novel methods are proposed that result in sequential assignment of all aromatic protons in uniformly 13C/15N labelled proteins using standard spectrometer hardware. Pulse sequences consist of routinely used building blocks and are therefore reasonably simple to implement. Ring protons may be correlated with β-carbons and, alternatively, with amide protons (and nitrogens) or carbonyls in order to take advantage of the superior dispersion of backbone resonances. It is possible to record spectra in a non-selective manner, yielding signals of all aromatic residues, or as amino-acid type selective versions to further reduce ambiguities. The new experiments are demonstrated with four different proteins with molecular weights ranging from 11 kDa to 23 kDa. Their performance is compared with that of (Hβ)Cβ(CγCδ)Hδ and (Hβ)Cβ(CγCδCɛ)Hɛ pulse sequences [Yamazaki et al. (1993) J Am Chem Soc 115:11054–11055].


Amino-acid type selectivity CC-TOCSY Direct carbon detection Heteronuclear cross-polarization Strong coupling Triple-resonance NMR 



The authors would like to thank Gary Yalloway and Christian Klammt for the preparation of D. vulgaris flavodoxin. The samples of RNase T1 and xylanase were kindly provided by Norman Spitzner and Marco Betz, respectively. Recombinant xylanase was produced in collaboration with Novo-Nordisk A/S (Bagsvaerd, Denmark). Financial support from the Deutsche Forschungsgemeinschaft (BE 1911/4) is gratefully acknowledged. The Centre for Biomolecular Magnetic Resonance is supported by the state of Hesse.

Supplementary material

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Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Frank Löhr
    • 1
  • Robert Hänsel
    • 1
  • Vladimir V. Rogov
    • 1
  • Volker Dötsch
    • 1
  1. 1.Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic ResonanceJohann Wolfgang Goethe-UniversityFrankfurtGermany

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