Journal of Biomolecular NMR

, Volume 4, Issue 6, pp 871–878 | Cite as

Measurement of HN-Hα J couplings in calcium-free calmodulin using new 2D and 3D water-flip-back methods

  • Hitoshi Kuboniwa
  • Stephan Grzesiek
  • Frank Delaglio
  • Ad Bax
Short Communication


Two new methods are described for the measurement of three-bond JHNHαcouplings in proteins isotopically enriched with 15N. Both methods leave the water magnetization in an unsaturated state, parallel to the z-axis, and therefore offer significant enhancements in sensitivity for rapidly exchanging backbone amide protons. The J couplings can be measured either from a set of constant-time 2D 1H-15N HMQC spectra, which are modulated in intensity by JHNHα, or from a water-flip-back version of the 3D HNHA experiment. The method is demonstrated for a sample of calcium-free calmodulin. Residues Lys75-Asp80 have JHNHαvalues in the 6–7 Hz range, suggesting that a break in the ‘central helix’ occurs at the same position as previously observed in solution NMR studies of Ca2+-ligated calmodulin.


Three-bond J coupling 3D NMR Quantitative J correlation Calmodulin 


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Copyright information

© ESCOM Science Publishers B.V 1994

Authors and Affiliations

  • Hitoshi Kuboniwa
    • 1
  • Stephan Grzesiek
    • 1
  • Frank Delaglio
    • 1
  • Ad Bax
    • 1
  1. 1.Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney DiseasesNational Institutes of HealthBethesdaU.S.A.

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