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Direct detection of N−H⋯O=C hydrogen bonds in biomolecules by NMR spectroscopy

Nature Protocols volume 3, pages 235–241 (2008)Cite this article

Abstract

A nuclear magnetic resonance (NMR) experiment is described for the direct detection of N-H⋯O=C hydrogen bonds (H-bonds) in 15N and 13C isotope-labeled biomolecules. This quantitative 'long-range' HNCO-COSY (correlation spectroscopy) experiment detects and quantifies electron-mediated scalar couplings across the H-bond (H-bond scalar couplings), which connect the magnetically active 15N and 13C nuclei on both sides of the H-bond. Detectable H-bonds comprise the canonical backbone H-bonds in proteins as well as other H-bonds in proteins and nucleic acids with N–H donors and O=C (carbonylic or carboxylic) acceptors. Unlike other NMR observables, which provide only indirect evidence of the presence of H-bonds, the H-bond scalar couplings identify all partners of the H-bond, the donor, the donor proton and the acceptor, in a single experiment. The size of the scalar couplings can be related to H-bond geometries. The time required to detect the N−H⋯O=C H-bonds in small proteins (≤≈10 kDa) is typically on the order of 1 d at millimolar concentrations, whereas H-bond detection for larger proteins (≤≈30 kDa) may be possible within several days depending on concentration, isotope composition, magnetic field strength and molecular weight. The proteins ubiquitin (8.6 kDa), dimeric RANTES (2 × 8.5 kDa) and MAP30 (30 kDa) are used as examples to illustrate this procedure.

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Figure 1: Backbone H-bond network of ubiquitin detected by h3JNC′ correlations4.
Figure 2: 2D long-range H(N)CO TROSY pulse sequence.
Figure 3: Results of the long-range HNCO.

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Acknowledgements

We gratefully acknowledge our collaborators Professor Barfield, Professor Feigon and Dr. Bax for their continued support and enthusiasm. This work was supported by SNF grant 31-109712 (S.G.) and by a stipend of the Boehringer Ingelheim Fonds (L.N.).

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Author notes

  1. Florence Cordier and Lydia Nisius: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Structural Biology and Chemistry, Institut Pasteur, 28 Rue du Docteur Roux, Paris, 75015, France

    Florence Cordier

  2. Department of Structural Biology, Biozentrum, University of Basel, Basel, CH-4056, Switzerland

    Lydia Nisius & Stephan Grzesiek

  3. Department of Chemistry and School of Biological Sciences, The University of Auckland, Private Bag, Auckland, 92019, New Zealand

    Andrew J Dingley

Authors
  1. Florence Cordier
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  2. Lydia Nisius
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  3. Andrew J Dingley
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  4. Stephan Grzesiek
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Corresponding author

Correspondence to Stephan Grzesiek.

Supplementary information

Supplementary Box 1

Bruker 2D long-range-H(N)CO-TROSY pulse sequence code (DOC 87 kb)

Supplementary Box 2

Conversion and NMRPipe processing script for 2D long-range H(N)CO data recorded on a Bruker NMR spectrometer (DOC 29 kb)

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Cordier, F., Nisius, L., Dingley, A. et al. Direct detection of N−H⋯O=C hydrogen bonds in biomolecules by NMR spectroscopy. Nat Protoc 3, 235–241 (2008). https://doi.org/10.1038/nprot.2007.498

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