Journal of Biomolecular NMR

, Volume 3, Issue 6, pp 627–638 | Cite as

Measurement of amide proton exchange rates and NOEs with water in 13C/15N-enriched calcineurin B

  • Stephan Grzesiek
  • Ad Bax
Research Papers


A rapid and sensitive 2D approach is presented for measuring amide proton exchange rates and the NOE interaction between amide protons and water. The approach is applicable to uniformly 13C/15N-enriched proteins and can measure magnetization exchange rates in the 0.02 to >20s−1 range. The experiments rely on selective excitation of the water resonance, coupled with purging of underlying Hα resonances, followed by NOESY-or ROESY-type transfer to amide protons, which are dispersed by the amide 15N frequencies in an HSQC-type experiment. Two separate but interleaved experiments, with and without selective inversion of the H2O resonance, yield quantitative results. The method is demonstrated for a sample of the calcium-binding protein calcineurin B. Results indicate rapid amide exchange for the five calcineurin B residues that are analogous to the five rapidly exchanging residues in the ‘central helix’ of the homologous protein calmodulin.


Hydrogen exchange NOESY ROESY Water Protein 2D NMR Calcineurin B 


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  1. Anglister J., Grzesiek S., Ren H., Klee C.B. and Bax A. (1993a) J. Biomol. NMR, 3, 121–126.Google Scholar
  2. Anglister, J., Grzesiek, S., Wang, A., Ren, H., Klee, C.B. and Bax, A. (1993b) Biochemistry, in press.Google Scholar
  3. Babu Y., Bugg C.E. and Cook W.J. (1988) J. Mol. Biol., 204, 191–204.Google Scholar
  4. Barbato G., Ikura M., Kay L.E., Pastor R.W. and Bax A. (1992) Biochemistry, 31, 5269–5278.Google Scholar
  5. Bauer C.J., Frenkiel T.A. and Lane A.N. (1990) J. Magn. Reson., 87, 144–152.Google Scholar
  6. Bax A., Sklenar V. and Summers M.F. (1986) J. Magn. Reson., 70, 327–331.Google Scholar
  7. Bax A. and Pochapsky S.S. (1992) J. Magn. Reson., 99, 638–643.Google Scholar
  8. Bodenhausen G. and Ruben D.J. (1980) Chem. Phys. Lett., 69, 185–188.Google Scholar
  9. Clore G.M., Bax A., Wingfield P.T. and Gronenborn A.M. (1990) Biochemistry, 29, 5671–5676.Google Scholar
  10. Englander S.W. and Kallenbach N.R. (1984) Q. Rev. Biophys., 16, 521–655.Google Scholar
  11. Ernst R.R., Bodenhausen G. and Wokaun A. (1987) Principles of Nuclear Magnetic Resonance in One and Two Dimensions, Clarendon Press, Oxford, p. 187.Google Scholar
  12. Grzesiek, S. and Bax, A. (1993) J. Am. Chem. Soc., in press.Google Scholar
  13. Ikura M. and Bax A. (1992) J. Am. Chem. Soc., 114, 2433–2440.Google Scholar
  14. Jeener J., Meier B.H., Bachmann P. and Ernst R.R. (1979) J. Chem. Phys., 71, 4546–4553.Google Scholar
  15. Kay L.E., Nicholson L.K., Delaglio F., Bax A. and Torchia D.A. (1992) J. Magn. Reson., 97, 359–375.Google Scholar
  16. Kogler H., Sørensen O.W. and Ernst R.R. (1983) J. Magn. Reson., 55, 157–163.Google Scholar
  17. Kubinec M.C. and Wemmer D.E. (1992) J. Am. Chem. Soc., 114, 8739–8740.Google Scholar
  18. Liepinsh E., Otting G. and Wüthrich K. (1992) Nucleic Acids Res., 20, 6549–6553.Google Scholar
  19. Liepinsh E., Rink H., Otting G. and Wüthrich K. (1993) J. Biomol. NMR, 3, 253–257.Google Scholar
  20. Marion D., Ikura M., Tschudin R. and Bax A. (1989) J. Magn. Reson., 85, 393–399.Google Scholar
  21. Otting G. and Wüthrich M. (1989) J. Am. Chem. Soc., 111, 1871–1875.Google Scholar
  22. Otting G., Liepinsh E., Farmer B.T. and Wüthrich K. (1991a) J. Biomol. NMR, 1, 209–215.Google Scholar
  23. Otting G., Liepinsh E. and Wüthrich K. (1991b) Science, 254, 974–980.Google Scholar
  24. Otting G., Liepinsh E. and Wüthrich K. (1991c) J. Am. Chem. Soc., 113, 4363–4364.Google Scholar
  25. Peng J.W. and Wagner G. (1992) J. Magn. Reson., 98, 308–332.Google Scholar
  26. Piotto M., Saudek V. and Sklenar V. (1992) J. Biomol. NMR, 2, 661–665.Google Scholar
  27. Qian Y.Q., Otting G. and Wüthrich K. (1993) J. Am. Chem. Soc., 115, 1189–1190.Google Scholar
  28. Spera S., Ikura M. and Bax A. (1991) J. Biomol. NMR, 1, 155–165.Google Scholar
  29. Strynadka N.C.J. and James M.N.G. (1989) Annu. Rev. Biochem., 58, 951–998.Google Scholar
  30. Tegenfeldt J. and Haeberlen U. (1979) J. Magn. Reson., 36, 453–457.Google Scholar
  31. Tüchsen E. and Woodward C. (1985) J. Mol. Biol., 185, 405–419.Google Scholar
  32. Xu R.X., Meadows R.P. and Fesik S.W. (1993) Biochemistry, 32, 2473–2480.Google Scholar

Copyright information

© ESCOM Science Publishers B.V 1993

Authors and Affiliations

  • Stephan Grzesiek
    • 1
  • Ad Bax
    • 1
  1. 1.Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney DiseasesNational Institutes of HealthBethesdaUSA

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