Advertisement

Journal of Biomolecular NMR

, Volume 5, Issue 4, pp 376–382 | Cite as

Sequential backbone assignment of isotopically enriched proteins in D2O by deuterium-decoupled HA(CA)N and HA(CACO)N

  • Andy C. Wang
  • Stephan Grzesiek
  • Rolf Tschudin
  • Patricia J. Lodi
  • Ad Bax
Research Papers

Summary

It is demonstrated that sequential resonance assignment of the backbone 1Hα and 15N resonances of proteins can be obtained without recourse to the backbone amide protons, an approach which should be useful for assignment of regions with rapidly exchanging backbone amide protons and for proteins rich in proline residues. The method relies on the combined use of two 2D experiments, HA(CA)N and HA(CACO)N or their 3D analogs, which correlate 1Hα with the intraresidue 15N and with the 15N resonance of the next residue. The experiments are preferably conducted in D2O, where very high resolution in the 15N dimension can be achieved by using 2H decoupling. The approach is demonstrated for a sample of human ubiquitin, uniformly enriched in 13C and 15N. Complete backbone and 13Cβ/1Hβ resonance assignments are presented.

Keywords

Deuterium Triple resonance Isotope labeling Sequential assignment Ubiquitin 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Abragam, A. (1961) Principles of Nuclear Magnetism, Oxford University Press, Oxford, pp. 309, 501.Google Scholar
  2. Bax, A. and Subramanian, S. (1986) J. Magn. Reson., 67, 565–569.Google Scholar
  3. Burum, D.P. and Ernst, R.R. (1980) J. Magn. Reson., 39, 163–168.Google Scholar
  4. Delaglio, F., Torchia, D.A. and Bax, A. (1991) J. Biomol. NMR, 1, 439–446.Google Scholar
  5. DiStefano, D.L. and Wand, A.J. (1987) Biochemistry, 26, 7272–7281.Google Scholar
  6. Freeman, R. (1988) A Handbook of Nuclear Magnetic Resonance, Longman, New York, NY, pp. 14–16.Google Scholar
  7. Garrett, D.S., Powers, R., Gronenborn, A.M. and Clore, G.M. (1991) J. Magn. Reson., 95, 214–220.Google Scholar
  8. Grzesiek, S. and Bax, A. (1992a) J. Am. Chem. Soc., 114, 6291–6293.Google Scholar
  9. Grzesiek, S. and Bax, A. (1992b) J. Magn. Reson., 96, 201–207.Google Scholar
  10. Grzesiek, S. and Bax, A. (1992c) J. Magn. Reson., 96, 432–440.Google Scholar
  11. Grzesiek, S. and Bax, A. (1993) J. Magn. Reson. Ser. B, 102, 103–106.Google Scholar
  12. Ikura, M., Kay, L.E. and Bax, A. (1990) Biochemistry, 29, 4659–4667.Google Scholar
  13. Kay, L.E., Ikura, M., Tschudin, R. and Bax, A. (1990) J. Magn. Reson., 89, 496–514.Google Scholar
  14. Live, D.H., Davis, D.G., Agosta, W.C. and Cowburn, D. (1984) J. Am. Chem. Soc., 106, 1939–1941.Google Scholar
  15. Madsen, J.C., Sørensen, O.W., Sørensen, P. and Poulsen, F.M. (1993) J. Biomol. NMR, 3, 239–244.Google Scholar
  16. Marion, D., Ikura, M., Tschudin, R. and Bax, A. (1989) J. Magn. Reson., 85, 393–399.Google Scholar
  17. McCoy, M.A. and Mueller, L. (1992) J. Magn. Reson., 99, 18–36.Google Scholar
  18. Morris, G.A. and Freeman, R. (1979) J. Am. Chem. Soc., 101, 760–762.Google Scholar
  19. Palmer, A.G., Fairbrother, W.J., Cavanagh, J., Wright, P.E. and Rance, M. (1992) J. Biomol. NMR, 2, 103–108.Google Scholar
  20. Pople, J.A. (1958) Mol. Phys., 1, 168–174.Google Scholar
  21. Powers, R., Gronenborn, A.M., Clore, G.M. and Bax, A. (1991) J. Magn. Reson. 94, 209–213.Google Scholar
  22. Schneider, D.M., Dellwo, M.J. and Wand, A.J. (1992) Biochemistry, 31, 3645–3652.Google Scholar
  23. Shaka, A.J., Barker, P.B. and Freeman, R. (1985) J. Magn. Reson., 53, 313–340.Google Scholar
  24. Shaka, A.J., Barker, P.B. and Freeman, R. (1985) J. Magn. Reson., 64, 547–552.Google Scholar
  25. Vuister, G.W. and Bax, A. (1992) J. Magn. Reson., 98, 428–435.Google Scholar
  26. Wang, A.C., Lodi, P.J., Qin, J., Vuister, G.W., Gronenborn, A.M. and Clore, G.M. (1994) J. Magn. Reson. Ser. B., 105, 196–198.Google Scholar
  27. Weber, P.L., Brown, S.C. and Mueller, L. (1987) Biochemistry, 26, 7282–7290.Google Scholar
  28. Wüthrich, K. (1986) NMR of Proteins and Nucleic Acids, Wiley, New York, NY.Google Scholar

Copyright information

© ESCOM Science Publishers B.V 1995

Authors and Affiliations

  • Andy C. Wang
    • 1
  • Stephan Grzesiek
    • 1
  • Rolf Tschudin
    • 1
  • Patricia J. Lodi
    • 1
  • Ad Bax
    • 1
  1. 1.Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney DiseasesNational Institute of HealthBethesdaUSA

Personalised recommendations