Journal of Biomolecular NMR

, Volume 31, Issue 3, pp 201–216 | Cite as

Measurement of eight scalar and dipolar couplings for methine–methylene pairs in proteins and nucleic acids

  • Emeric Miclet
  • Jérôme Boisbouvier
  • Ad Bax


A new 3D, spin-state-selective coherence transfer NMR experiment is described that yields accurate measurements for eight scalar or dipolar couplings within a spin system composed of a methylene adjacent to a methine group. Implementations of the experiment have been optimized for proteins and for nucleic acids. The experiments are demonstrated for Cβ–Cα moieties of the third IgG-binding domain from Streptococcal Protein G (GB3) and for C\( ^{5^{\prime}} \)–C\( ^{4^{\prime}} \) groups in a 24-nt RNA oligomer. Chemical shifts of Cα, Cβ and Hβ (respectively C\( ^{4^{\prime}} \), C\( ^{5^{\prime}} \) and H\( ^{5^{\prime}} \)) are dispersed in the three orthogonal dimensions, and the absence of heteronuclear decoupling leads to distinct and well-resolved E.COSY multiplet patterns. In an isotropic sample, the E.COSY displacements correspond to 1JCαHα, 2JCαHβ2+2JCαHβ3, 2JCβHα, 1JCβHβ2+1JCβHβ3, 1JCβHβ22JHβ2Hβ3, 1JCβHβ32JHβ2Hβ3, 3JHαHβ2 and 3JHαHβ3 for proteins, and 1J\( _{{{\rm C}4^{\prime}}{{\rm H}4^{\prime}}} \), 2J\( _{{\rm C}4^{\prime}{{\rm H}5^{\prime}}}+^{2} \)J\( _{{{\rm C}4^{\prime}}{{\rm H}5^{\prime\prime}}} \), 2J\( _{{{\rm C}5^{\prime}}{{\rm H}4^{\prime}}} \), 1J\( _{{{\rm C}5^{\prime}}{{\rm H}5^{\prime}}} \)+1J\( _{{\rm C}5^{\prime}{{\rm H}5^{\prime\prime}}} \), 1J\( _{{\rm C}5^{\prime}{\rm H}5^{\prime}}-^{2} \)J\( _{{\rm H}5^{\prime}{\rm H}5^{\prime\prime}} \), 1J\( _{{\rm C}5^{\prime}{{\rm H}5^{\prime\prime}}}-^{2} \)J\( _{{\rm H}5^{\prime}{\rm H}5^{\prime\prime}} \), 3J\( _{{\rm H}4^{\prime}{\rm H}5^{\prime}} \) and 3J\( _{{\rm H}4^{\prime}{{\rm H}5^{\prime\prime}}} \) in nucleic acids. The experiment, based on relaxation-optimized spectroscopy, yields best results when applied to residues where the methine–methylene group corresponds to a reasonably isolated spin system, as applies for C, F, Y, W, D, N and H residues in proteins, or the C\( ^{5^{\prime}} \)–C\( ^{4^{\prime}} \) groups in nucleic acids. Splittings can be measured under either isotropic or weakly aligned conditions, yielding valuable structural information both through the 3J couplings and the one-, two- and three-bond dipolar interactions. Dipolar couplings for 10 out of 13 sidechains in GB3 are found to be in excellent agreement with its X-ray structure, whereas one residue adopts a different backbone geometry, and two residues are subject to extensive χ1 rotamer averaging. The abundance of dipolar couplings can also yield stereospecific assignments of the non-equivalent methylene protons. For the RNA oligomer, dipolar data yielded stereospecific assignments for six out of the eight C\( ^{5^{\prime}} \)H2 groups in the loop region of the oligomer, in all cases confirmed by 1J\( _{{\rm C}5^{\prime}{{\rm H}5^{\prime}}} > ^{1} \)J\( _{{\rm C}5^{\prime}{\rm H}5^{\prime\prime}} \), and H\( ^{5^{\prime}} \) resonating downfield of H\( ^{5^{\prime\prime}} \).


alignment dipolar coupling E.COSY liquid crystal methylene scalar coupling side chain conformation 


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Supplementary material

Supplementary material


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Copyright information

© Kluwer Academic Publishers 2005

Authors and Affiliations

  • Emeric Miclet
    • 1
    • 2
  • Jérôme Boisbouvier
    • 1
    • 3
  • Ad Bax
    • 1
  1. 1.Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney DiseasesNational Institutes of HealthMarylandUSA
  2. 2.Laboratoire Structure et Fonction de Molécules BioactivesUMR 7613 – Université Pierre et Marie CurieCedex 5France
  3. 3.Laboratoire de RMNInstitut de Biologie Structurale – Jean-Pierre EbelCedex 1France

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