Journal of Biomolecular NMR

, Volume 1, Issue 1, pp 13–22 | Cite as

Stereospecific assignment of β-methylene protons in larger proteins using 3D15N-separated Hartmann-Hahn and13C-separated rotating frame Overhauser spectroscopy

  • G. Marius Clore
  • Ad Bax
  • Angela M. Gronenborn
Research Papers


3Jxβ coupling constants and complementary nuclear Overhauser data on the intraresidue C x H−CβH distances form an essential part of the data needed to obtain stereospecific assignments of β-methylene protons in proteins. In this paper we show that information regarding the magnitude of the3Jxβ coupling constants can be extracted from a semi-quantitative interpretation of relative peak intensities in a 3D15N-separated1H−1H Hartmann-Hahn1H−15N multiple quantum coherence (HOHAHA-HMQC) spectrum. In addition, we demonstrate that reliable information on the intraresidue C x H−CβH distances, free of systematic errors arising from spin diffusion, can be obtained from a 3D13C-separated1H−1H rotating frame Overhauser effect1H−13C multiple quantum coherence (ROESY-HMQC) spectrum. The applicability of these experiments to larger proteins is illustrated with respect to interleukin-1β, a protein of 153 residues and 17.4 kDa molecular weight.


Stereospecific assignment 3D NMR Heteronuclear NMR Hartmann-Hahn ROE 





nuclear Overhauser effect


rotating frame Overhauser effect


homonuclear Hartmann-Hahn spectroscopy


nuclear Overhauser enhancement spectroscopy


rotating frame Overhauser spectroscopy


heteronuclear multiple quantum coherence spectroscopy


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Bauer, C.J., Frenkiel, T.A. and Lane, A.N. (1990)J. Magn. Reson.,87, 144–152.Google Scholar
  2. Bax, A. and Freeman, R. (1981)J. Magn. Reson.,44, 542–561.Google Scholar
  3. Bax, A. and Davis, D.G. (1985)J. Magn. Reson.,63, 207–213.Google Scholar
  4. Bax, A. and Lerner, L.E. (1988)J. Magn. Reson.,79, 429–438.Google Scholar
  5. Bax, A., Griffey, R.H. and Hawkins, B.L. (1983)J. Magn. Reson.,55, 301–315.Google Scholar
  6. Bax, A., Sklenar, V. and Summers, M.F. (1986)J. Magn. Reson.,70, 327–331.Google Scholar
  7. Bax, A., Clore, G.M., Driscoll, P.C., Gronenborn, A.M., Ikura, M. and Kay, L.E. (1990)J. Magn. Reson.,87, 620–627.Google Scholar
  8. Bothner-By, A.A. and Dadok, J. (1987)J. Magn. Reson.,72, 540–543.Google Scholar
  9. Bothner-By, A.A., Stephens, R.L., Lee, J.T., Warren, C.D. and Jeanloz, R.W. (1984)J. Am. Chem. Soc.,106, 811–813.Google Scholar
  10. Bystrov, V.F. (1976)Prog. Nucl. Magn. Reson. Spectrosc.,10, 41–81.Google Scholar
  11. Cavanagh, J., Chazin, W.J. and Rance, M. (1990)J. Magn. Reson.,72, 540–543.Google Scholar
  12. Clore, G.M., Gronenborn, A.M., Carlson, G. and Meyer, E.F. (1986)J. Mol. Biol.,190, 259–267.Google Scholar
  13. Clore, G.M., Appella, E., Yamada, M., Matsushima, K. and Gronenborn, A.M. (1990a)Biochemistry,29, 1689–1696.Google Scholar
  14. Clore, G.M., Bax, A., Driscoll, P.C., Wingfield, P.T. and Gronenborn, A.M. (1990b)Biochemistry,29, 8172–8184.Google Scholar
  15. Clore, G.M., Bax, A., Wingfield, P.T. and Gronenborn, A.M. (1990c)Biochemistry,29, 5671–5676.Google Scholar
  16. Clore, G.M., Wingfield, P.T. and Gronenborn, A.M. (1991)Biochemistry,30, 2315–2323.Google Scholar
  17. Cowburn, D., Live, D.H., Fischman, A.J. and Agosta, W.C. (1983)J. Am. Chem. Soc.,105, 7435–7442.Google Scholar
  18. Davis, D.G. and Bax, A. (1985)J. Am. Chem. Soc.,107, 2820–2821.Google Scholar
  19. Driscoll, P.C., Gronenborn, A.M. and Clore, G.M. (1989a)FEBS Lett.,243, 223–233.Google Scholar
  20. Driscoll, P.C., Gronenborn, A.M., Beress, L. and Clore, G.M. (1989b)Biochemistry,28, 2188–2198.Google Scholar
  21. Driscoll, P.C., Clore, G.M., Marion, D., Wingfield, P.T. and Gronenborn, A.M. (1990a)Biochemistry,29, 3542–3556.Google Scholar
  22. Driscoll, P.C., Gronenborn, A.M., Wingfield, P.T. and Clore, G.M. (1990b)Biochemistry,29, 4668–4682.Google Scholar
  23. Dyson, H.J., Gippert, G.P., Case, D.A., Holmgren, A. and Wright, P.E. (1990)Biochemistry,29, 4129–4136.Google Scholar
  24. Forman-Kay, J.D., Clore, G.M., Wingfield, P.T. and Gronenborn, A.M. (1991)Biochemistry,30, 2685–2698.Google Scholar
  25. Griesinger, C., Sørensen, O.W. and Ernst, R.R. (1982)J. Am. Chem. Soc.,104, 6800–6802.Google Scholar
  26. Gronenborn, A.M., Clore, G.M., Schmeissner, U. and Wingfield, P.T. (1986)Eur. J. Biochem. 161, 37–43.Google Scholar
  27. Güntert, P., Braun, W., Billeter, M. and Wüthrich, K. (1989)J. Am. Chem. Soc.,111, 3997–4004.Google Scholar
  28. Jeener, J., Meier, B.H., Bachmann, P. and Ernst, R.R. (1979)J. Chem. Phys.,71, 4546–4553.Google Scholar
  29. Kay, L.E. and Bax, A. (1989)J. Magn. Reson.,86, 110–126.Google Scholar
  30. Kay, L.E., Marion, D. and Bax, A. (1989)J. Magn. Reson.,84, 72–84.Google Scholar
  31. Kraulis, P.J., Clore, G.M., Nilges, M., Jones, T.A., Pettersson, G., Knowles, J. and Gronenborn, A.M. (1989)Biochemistry,28, 7241–7257.Google Scholar
  32. 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
  33. Marion, D., Ikura, M., Tschudin, R. and Bax, A. (1989b)J. Magn. Reson.,85, 393–399.Google Scholar
  34. McGregor, M.J., Islam, S.A. and Sternberg, M.J.E. (1987)J. Mol. Biol.,198, 295–310.Google Scholar
  35. Montelione, G.T., Winkler, M.E., Rauenbuehler, P. and Wagner, G. (1989)J. Magn. Reson.,82, 198–204.Google Scholar
  36. Mueller, L. (1979)J. Am. Chem. Soc.,101, 4481–4484.Google Scholar
  37. Mueller, L. (1987)J. Magn. Reson.,72, 191–196.Google Scholar
  38. Nilges, M., Clore, G.M. and Gronenborn, A.M. (1990)Biopolymers,29, 813–822.Google Scholar
  39. Omichinski, J.G., Clore, G.M., Appella, E., Sakaguchi, K. and Gronenborn, A.M. (1990)Biochemistry,29, 9324–9334.Google Scholar
  40. Oppenheim, J.J., Kovacs, E.J., Matsushima, K. and Durum, S.K. (1986)Immunol. Today,7, 45–56.Google Scholar
  41. Oschkinat, H., Pastore, A., Pfändler, P. and Bodenhausen, G. (1986)J. Magn. Reson.,69, 559–566.Google Scholar
  42. Ponder, J.W. and Richards, F.M. (1987)J. Mol. Biol.,193, 775–791.Google Scholar
  43. Qian, Y.Q., Billeter, M., Otting, G., Müller, M., Gehring, W.J. and Wüthrich, K. (1989)Cell. 59, 573–580.Google Scholar
  44. Rucker, S.P. and Shaka, A.J. (1988)Mol. Phys.,68, 509–514.Google Scholar
  45. Shaka, A.J., Lee, C.J. and Pines, A. (1988)J. Magn. Reson.,77, 274–293.Google Scholar
  46. Wagner, G., Braun, W., Havel, T.F., Schaumann, T., Go, N. and Wüthrich, K. (1987)J. Mol. Biol.,196, 611–639.Google Scholar
  47. Wider, G., Neri, D., Otting, G. and Wüthrich, K. (1989)J. Magn. Reson.,85, 426–431.Google Scholar
  48. Wingfield, P.T., Payton, M., Tavernier, J., Barnes, M., Shaw, A., Rose, K., Simona, M.G., Demaczuk, S., Williamson, K. and Dayer, J.M. (1986)Eur. J. Biochem.,160, 491–497.Google Scholar

Copyright information

© ESCOM Science Publishers B.V. 1991

Authors and Affiliations

  • G. Marius Clore
    • 1
  • Ad Bax
    • 1
  • Angela M. Gronenborn
    • 1
  1. 1.Laboratory of Chemical Physics, Building 2, National Institute of Diabetes and Digestive and Kidney DiseasesNational Institutes of HealthBethesdaUSA

Personalised recommendations