Journal of Biomolecular NMR

, Volume 3, Issue 2, pp 225–231 | Cite as

A general method for assigning NMR spectra of denatured proteins using 3D HC(CO)NH-TOCSY triple resonance experiments

  • Timothy M. Logan
  • Edward T. Olejniczak
  • Robert X. Xu
  • Stephen W. Fesik
Short Communications

Summary

A general approach for assigning the resonances of uniformly 15N- and 13C-labeled proteins in their unfolded state is presented. The assignment approach takes advantage of the spectral dispersion of the amide nitrogen chemical shifts in denatured proteins by correlating side chain and backbone carbon and proton frequencies with the amide resonances of the same and adiacent residues. The 1H resonances of the individual amino acid spin systems are correlated with their intraresidue amide in a 3D 15N-edited 1H, 1H-TOCSY-HSQC experiment, which allows the spin systems to be assigned to amino acid type. The spin systems are then linked to the adjacent i-1 spin system using the 3D H(C)(CO)NH-TOCSY experiment. Complete 13C assignments are obtained from the 3D (H)C(CO)NH-TOCSY experiment. Unlike other methods for assigning denatured proteins, this approach does not require previous knowledge of the native state assignments or specific interconversion rates between the native and denatured forms. The strategy is demonstrated by assigning the 1H, 13C, and 15N resonances of the FK506 binding protein denatured in 6.3 M urea.

Keywords

Denatured proteins Sequential assignments 3D triple resonance NMR 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Baum, J., Dobson, C.M., Evans, P.A. and Hanley, C. (1989) Biochemistry, 28, 7–13.Google Scholar
  2. Bax, A., Ikura, M., Kay, L.E., Torchia, D.A. and Tschudin, R. (1990) J. Mag. Res., 86, 304–318.Google Scholar
  3. Bax, A., Ikura, M., Kay, L.E. and Zhu, G. (1991) J. Mag. Res., 91, 174–178.Google Scholar
  4. Dobson, C.M. and Evans, P.A. (1984) Biochemistry, 23, 4267–4270.Google Scholar
  5. Dobson, C.M., Evans, P.A. and Williamson, K.L. (1984) FEBS Lett., 168, 331–334.Google Scholar
  6. Dill, K.A. and Shortle, D. (1991) Ann. Rev. Biochemistry, 60, 795–825.Google Scholar
  7. Egan, D.A., Logan, T.M., Liang, H., Matayoshi, E., Fesik, S.W. and Holzman, T.F. (1993) Biochemistry, in press.Google Scholar
  8. Evans, P.A., Kautz, R.A., Fox, R.O. and Dobson, C.M. (1989) Biochemistry, 28, 362–270.Google Scholar
  9. Evans, P.A., Topping, K.D., Woolfson, D.N. and Dobson, C.M. (1991) Proteins, 9, 248–266.Google Scholar
  10. Fesik, S.W. and Zuiderweg, E.R.P. (1990) Quart. Rev. Biophys., 23, 97–131.Google Scholar
  11. Fox, R.O., Evans, P.A. and Dobson, C.M. (1986) Nature, 320, 192–194.Google Scholar
  12. Grzesiek, S. and Bax, A. (1992) J. Am. Chem. Soc., 114, 6291–6293.Google Scholar
  13. Logan, T.M., Olejniczak, E.T., Xu, R.X. and Fesik, S.W. (1992) FEBS Lett., 314, 413–418.Google Scholar
  14. Lyons, B.A. and Montelione, G.T. (1993) J. Mag. Res., in press.Google Scholar
  15. Marion, D., Driscoll, P.C., Kay, L.E., Wingfield, P.T., Bax, A., Gronenborn, A.M. and Clore, G.M. (1989a) Biochemistry, 28, 6150–6156.Google Scholar
  16. Marion, D., Ikura, M., Tschudin, R. and Bax, A. (1989b) J. Mag. Res., 85, 393–399.Google Scholar
  17. Miranker, A., Radford, S., Karplus, M. and Dobson, C.M. (1991) Nature, 349, 633–636.Google Scholar
  18. Mohebbi, A. and Shaka, A.J. (1991) Chem. Phys. Lett., 178, 374–378.Google Scholar
  19. Montelione, G.T., Lyons, B.A., Emerson, S.D. and Tashiro, M. (1992) J. Am. Chem. Soc., 114, 10974–10975.Google Scholar
  20. Neri, D., Wider, G. and Wüthrich, K. (1992) Proc. Natl. Acad. Sci., USA, 89, 4397–4401.Google Scholar
  21. Oh, B.H., Westler, W.M., Darba, P. and Markley, J.L. (1988) Science, 240, 908–911.Google Scholar
  22. Olejniczak, E.T. and Eaton, H.L. (1990) J. Mag. Res., 87, 628–632.Google Scholar
  23. Olejniczak, E.T., Xu, R.X. and Fesik, S.W. (1992) J. Biomol. NMR, 2, 655–659.Google Scholar
  24. Richarz, R. and Wüthrich, K. (1978) Biopolymers, 17, 2133–2141.Google Scholar
  25. Wider, G., Neri, D. and Wüthrich, K. (1991) J. Biomol. NMR, 1, 93–98.Google Scholar
  26. Wüthrich, K. (1986) NMR of Proteins and Nucleic Acids, John Wiley, New York.Google Scholar

Copyright information

© ESCOM Science Publishers B.V. 1993

Authors and Affiliations

  • Timothy M. Logan
    • 1
  • Edward T. Olejniczak
    • 1
  • Robert X. Xu
    • 1
  • Stephen W. Fesik
    • 1
  1. 1.Pharmaceutical Discovery DivisionAbbott LaboratoriesAbbott ParkUSA

Personalised recommendations