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Journal of Biomolecular NMR

, Volume 8, Issue 3, pp 351–356 | Cite as

An (H)C(CO)NH-TOCSY pulse scheme for sequential assignment of protonated methyl groups in otherwise deuterated 15N, 13C-labeled proteins

  • Kevin H. Gardner
  • Robert Konrat
  • Michael K. Rosen
  • Lewis E. Kay
Short Communication

Summary

A biosynthetic strategy has recently been developed for the production of 15N, 13C, 2H-labeled proteins using 1H3C-pyruvate as the sole carbon source and D2O as the solvent. The methyl groups of Ala, Val, Leu and Ile (γ2 only) remain highly protonated, while the remaining positions in the molecule are largely deuterated. An (H)C(CO)NH-TOCSY experiment is presented for the sequential assignment of the protonated methyl groups. A high-sensitivity spectrum is recorded on a 15N, 13C, 2H, 1H3C-labeled SH2 domain at 3°C (correlation time 18.8 ns), demonstrating the utility of the method for proteins in the 30–40 kDa molecular weight range.

Keywords

(H)C(CO)NH-TOCSY Multidimensional NMR Deuteration 

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References

  1. Boyd, J. and Soffe, N. (1989) J. Magn. Reson., 85, 406–413.Google Scholar
  2. FarmerII, B.T. and Venters, R.A. (1995) J. Am. Chem. Soc., 117, 4187–4188.Google Scholar
  3. FarmerII, B.T. and Venters, R.A. (1996) J. Biomol. NMR, 7, 59–71.Google Scholar
  4. Farrow, N.A., Muhandiram, D.R., Singer, A.U., Pascal, S.M., Kay, C.M., Gish, G., Shoelson, S.E., Pawson, T., Forman-Kay, J.D. and Kay, L.E. (1994) Biochemistry, 33, 5984–6003.Google Scholar
  5. Fu, R. and Bodenhausen, G. (1995) Chem. Phys. Lett., 245, 415–420.Google Scholar
  6. Gardner, K.H., Rosen, M.K. and Kay, L.E. (1996) Biochemistry, manuscript submitted for publication.Google Scholar
  7. Geen, H. and Freeman, R. (1991) J. Magn. Reson., 93, 93–141.Google Scholar
  8. Grzesiek, S., Anglister, J., Ren, H. and Bax, A. (1993a) J. Am. Chem. Soc., 115, 4369–4370.Google Scholar
  9. Grzesiek, S., Anglister, J., Ren, H. and Bax, A. (1993b) J. Magn. Reson., B101, 114–119.Google Scholar
  10. Grzesiek, S. and Bax, A. (1993a) J. Am. Chem. Soc., 115, 12593–12594.Google Scholar
  11. Grzesiek, S. and Bax, A. (1993b) J. Biomol. NMR, 3, 185–204.Google Scholar
  12. Grzesiek, S., Wingfield, P., Stahl, S., Kaufman, J.D. and Bax, A. (1995) J. Am. Chem. Soc., 117, 9594–9595.Google Scholar
  13. Kay, L.E., Ikura, M., Tschudin, R. and Bax, A. (1990) J. Magn. Reson., 89, 496–514.Google Scholar
  14. Kay, L.E., Keiffer, P. and Saarinen, T. (1992) J. Am. Chem. Soc., 114, 10663–10665.Google Scholar
  15. Kay, L.E. (1993) J. Am. Chem. Soc., 115, 2055–2057.Google Scholar
  16. Kay, L.E., Xu, G.Y. and Yamazaki, T. (1994) J. Magn. Reson., A109, 129–133.Google Scholar
  17. Kupce, E. and Freeman, R. (1995) J. Magn. Reson., A115, 273–276.Google Scholar
  18. Kupce, E. and Wagner, G. (1995) J. Magn. Reson., B109, 329–333.Google Scholar
  19. Li, Y. and Montelione, G.T. (1993) J. Magn. Reson., B101, 315–319.Google Scholar
  20. Logan, T.M., Olejniczak, E.T., Xu, R.X. and Fesik, S.W. (1992) FEBS Lett., 314, 413–418.Google Scholar
  21. Logan, T.M., Olejniczak, E.T., Xu, R.X. and Fesik, S.W. (1993) J. Biomol. NMR, 3, 225–231.Google Scholar
  22. Marion, D., Ikura, M., Tschudin, R. and Bax, A. (1989) J. Magn. Reson., 85, 393–399.Google Scholar
  23. Markus, M.A., Dayie, K.T., Matsudaira, P. and Wagner, G. (1994) J. Magn. Reson., B105, 192–195.Google Scholar
  24. McCoy, M.A. and Mueller, L. (1992) J. Am. Chem. Soc., 114, 2108–2112.Google Scholar
  25. Mohebbi, A. and Shaka, A.J. (1991) J. Chem. Phys., 178, 374–377.Google Scholar
  26. Montelione, G.T., Lyons, B.A., Emerson, D.S. and Tashiro, M. (1992) J. Am. Chem. Soc., 114, 10974–10975.Google Scholar
  27. Muchmore, S.W., Sattler, M., Liang, H., Meadows, R.P., Harlan, J.E., Yoon, H.S., Nettesheim, D., Chang, B.S., Thompson, C.B., Wong, S.-L., Ng, S.-C. and Fesik, S.W. (1996) Nature, 381, 335–341.Google Scholar
  28. Nietlispach, D., Clowes, R.T., Broadhurst, R.W., Ito, Y., Keeler, J., Kelly, M., Ashurst, J., Oschkinat, H., Domaille, P.J. and Laue, E. (1996) J. Am. Chem. Soc., 118, 407–415.Google Scholar
  29. Patt, S.L. (1992) J. Magn. Reson., 96, 94–102.Google Scholar
  30. Rosen, M.K., Gardner, K.H., Willis, R.C., Parris, W.E., Pawson, T. and Kay, L.E. (1996) J. Mol. Biol., in press.Google Scholar
  31. Schleucher, J., Sattler, M. and Griesinger, C. (1993) Angew. Chem. Int. Ed. Engl., 32, 1489–1491.Google Scholar
  32. Shaka, A.J., Keeler, J., Frenkiel, T. and Freeman, R. (1983) J. Magn. Reson., 52, 335–338.Google Scholar
  33. Shan, X., Gardner, K.H., Muhandiram, D.R., Rao, N.S., Arrowsmith, C.H. and Kay, L.E. (1996) J. Am. Chem. Soc., 118, 6570–6579.Google Scholar
  34. Shirakawa, M., Walchli, M., Shimizu, M. and Kyogoku, Y. (1995) J. Biomol. NMR, 5, 323–326.Google Scholar
  35. Stonehouse, J., Shaw, G.L., Keeler, J. and Laue, E. (1994) J. Magn. Reson., A107, 178–184.Google Scholar
  36. Venters, R.A., Metzler, W.J., Spicer, L.D., Mueller, L. and FarmerII, B.T. (1995) J. Am. Chem. Soc., 117, 9592–9593.Google Scholar
  37. Vuister, G.W. and Bax, A. (1992) J. Magn. Reson., 98, 428–435.Google Scholar
  38. Werbelow, L.G. and Grant, D.M. (1977) In Advances in Magnetic Resonance (Ed., Waugh, J.S.), Vol. 9, Academic Press, San Diego, CA, U.S.A., pp. 189–299.Google Scholar
  39. Yamazaki, T., Lee, W., Revington, M., Mattiello, D.L., Dahlquist, F.W., Arrowsmith, C.H. and kay, L.E. (1994a) J. Am. Chem. Soc., 116, 6464–6465.Google Scholar
  40. Yamazaki, T., Lee, W., Arrowsmith, C.H., Muhandiram, D.R. and Kay, L.E. (1994b) J. Am. Chem. Soc., 116, 11655–11666.Google Scholar
  41. Zhou, M.M., Ravichandran, K.S., Olejniczak, E.T., Petros, A.M., Meadows, R.P., Sattler, M., Harlan, J.E., Wades, W.S., Burakoff, S.J. and Fesik, S.W. (1995) Nature, 378, 584–592.Google Scholar

Copyright information

© ESCOM Science Publishers B.V. 1996

Authors and Affiliations

  • Kevin H. Gardner
    • 1
    • 2
    • 3
    • 4
  • Robert Konrat
    • 1
    • 2
    • 3
    • 4
  • Michael K. Rosen
    • 1
    • 2
    • 3
    • 4
  • Lewis E. Kay
    • 1
    • 2
    • 3
    • 4
  1. 1.The Protein Engineering Centers of ExcellenceUniversity of TorontoTorontoCanada
  2. 2.Department of Medical GeneticsUniversity of TorontoTorontoCanada
  3. 3.Department of BiochemistryUniversity of TorontoTorontoCanada
  4. 4.Department of ChemistryUniversity of TorontoTorontoCanada

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