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

Nature Protocols volume 3, pages 242–248 (2008)Cite this article

Abstract

A nuclear magnetic resonance (NMR) experiment is described for the direct detection of N–H⋯N hydrogen bonds (H-bonds) in 15N isotope-labeled biomolecules. This quantitative HNN-COSY (correlation spectroscopy) experiment detects and quantifies electron-mediated scalar couplings across the H-bond (H-bond scalar couplings), which connect magnetically active 15N nuclei of the H-bond donor and acceptor. Detectable H-bonds comprise the imino H-bonds in canonical Watson–Crick base pairs, many H-bonds in unusual nucleic acid base pairs and H-bonds between protein backbone or side-chain N–H donor and N acceptor moieties. 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 and as a time average to H-bond dynamics. The time required to detect the H-bonds is typically less than 1 d at millimolar concentrations for samples of molecular weight ≤≈25 kDa. A 15N/13C-labeled potato spindle tuber viroid T1 RNA domain is used as an example to illustrate this procedure.

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Figure 1: Watson–Crick RNA base pairs indicating the N–H⋯N H-bond that can be detected by h2JNN correlations.
Figure 2: Basic HNN-COSY pulse sequence.
Figure 3: 2D HNN-COSY spectrum recorded on the PSTVd T1 RNA domain, mutant U18C/A344G (see ref. 30).

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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.).

Author information

Author notes

  1. Andrew J Dingley and Lydia Nisius: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry and School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland, 1142, New Zealand

    Andrew J Dingley

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

    Lydia Nisius & Stephan Grzesiek

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

    Florence Cordier

Authors
  1. Andrew J Dingley
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Corresponding author

Correspondence to Stephan Grzesiek.

Supplementary information

Supplementary Note 1

Bruker two-dimensional HNN-COSY pulse sequence code (DOC 74 kb)

Supplementary Note 2

Conversion and NMRPipe processing script for 2D HNN-COSY data recorded on a Bruker NMR spectrometer (DOC 29 kb)

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Dingley, A., Nisius, L., Cordier, F. et al. Direct detection of N−H⋯N hydrogen bonds in biomolecules by NMR spectroscopy. Nat Protoc 3, 242–248 (2008). https://doi.org/10.1038/nprot.2007.497

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