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

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Summary

Two new methods are described for the measurement of three-bond JH NHαcouplings 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 JH NHα, 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 JH NHαvalues 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.

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Authors and Affiliations

  1. Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 20892-0520, Bethesda, MD, U.S.A.

    Hitoshi Kuboniwa, Stephan Grzesiek, Frank Delaglio & Ad Bax

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  1. Hitoshi Kuboniwa
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  2. Stephan Grzesiek
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  3. Frank Delaglio
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  4. Ad Bax
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Kuboniwa, H., Grzesiek, S., Delaglio, F. et al. Measurement of HN-Hα J couplings in calcium-free calmodulin using new 2D and 3D water-flip-back methods. J Biomol NMR 4, 871–878 (1994). https://doi.org/10.1007/BF00398416

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  • DOI: https://doi.org/10.1007/BF00398416

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