Advertisement

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

Summary

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.

Keywords

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

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. BarbatoG., IkuraM., KayL.E. and BaxA. (1992) Biochemistry, 31, 5269–5278.Google Scholar
  2. BaxA., KayL.E., SparksS.W. and TorchiaD.A. (1989) J. Am. Chem. Soc., 111, 408–409.Google Scholar
  3. BilleterM., NeriD., OttingG., QianY.Q. and WüthrichK. (1992) J. Biomol. NMR, 2, 257–274.Google Scholar
  4. Campbell-BurkS., DomailleP. and MuellerL. (1991) J. Magn. Reson., 93, 171–177.Google Scholar
  5. GrzesiekS. and BaxA. (1993) J. Am. Chem. Soc., 115, 12593–12594.Google Scholar
  6. HarbisonG. (1993) J. Am. Chem. Soc., 115, 3026–3027.Google Scholar
  7. JohnB.K., PlantD., HealdS.H. and HurdR.E. (1991) J. Magn. Reson., 94, 664–669.Google Scholar
  8. KayL.E., BrooksB., SparksS.W., TorchiaD.A. and BaxA. (1989) J. Am. Chem. Soc., 111, 5488–5490.Google Scholar
  9. KayL.E., KeiferP. and SaarinenT. (1992) J. Am. Chem. Soc., 114, 10663–10665.Google Scholar
  10. KayL.E., XuG.Y. and YamazakiT. (1994) J. Magn. Reson. Ser. A, 109, 129–133.Google Scholar
  11. LudvigsenS., AndersenK.V. and PoulsenF.M. (1991) J. Mol. Biol., 217, 731–736.Google Scholar
  12. MarionD., IkuraM., TschudinR. and BaxA. (1989) J. Magn. Reson., 85, 393–399.Google Scholar
  13. MesserleB.A., WiderG., OttingG., WeberC. and WüthrichK. (1989) J. Magn. Reson., 85, 608–613.Google Scholar
  14. MontelioneG.T. and WagnerG. (1989) J. Am. Chem. Soc., 111, 5474–5475.Google Scholar
  15. NeriD., OttingG. and WüthrichK. (1990) J. Am. Chem. Soc., 112, 3663–3665.Google Scholar
  16. PlateauP. and GueronM. (1982) J. Am. Chem. Soc., 104, 7310–7311.Google Scholar
  17. RoyS., PapastavrosM., SanchezV. and RedfieldA.G. (1984) Biochemistry, 23, 4395–4400.Google Scholar
  18. SeipS., BalbachJ. and KesslerH. (1994) J. Magn. Reson. Ser. B, 104, 172–179.Google Scholar
  19. SmithL.J., SutcliffeM.J., RedfieldC. and DobsonC.M. (1991) Biochemistry, 30, 986–996.Google Scholar
  20. SperaS., IkuraM. and BaxA. (1991) J. Biomol. NMR, 1, 155–165.Google Scholar
  21. StonehouseJ., ShawG.L., KeelerJ. and LaueE.D. (1994) J. Magn. Reson. Ser. A, 107, 178–184.Google Scholar
  22. VuisterG.W. and BaxA. (1993) J. Am. Chem. Soc., 115, 7772–7777.Google Scholar
  23. WagnerG., SchmiederP. and ThanabalV. (1991) J. Magn. Reson., 93, 436–440.Google Scholar
  24. ZhuG. and BaxA. (1990) J. Magn. Reson., 90, 405–410.Google Scholar
  25. ZhuG. and BaxA. (1993) J. Magn. Reson. Ser. A, 104, 353–357.Google Scholar

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.

Personalised recommendations