Journal of Biomolecular NMR

, Volume 16, Issue 3, pp 209–219 | Cite as

Partial NMR assignments for uniformly (13C, 15N)-enriched BPTI in the solid state

  • Ann McDermott
  • Tatyana Polenova
  • Anja Bockmann
  • Kurt.W. Zilm
  • Eric K. Paulsen
  • Rachel W. Martin
  • Gaetano T. Montelione


We demonstrate that high-resolution multidimensional solid state NMR methods can be used to correlate many backbone and side chain chemical shifts for hydrated micro-crystalline U-13C,15N Basic Pancreatic Trypsin Inhibitor (BPTI), using a field strength of 800 MHz for protons, magic angle sample spinning rates of 20 kHz and proton decoupling field strengths of 140 kHz. Results from two homonuclear transfer methods, radio frequency driven dipolar recoupling and spin diffusion, were compared. Typical 13C peak line widths are 0.5 ppm, resulting in Cα-Cβ and Cα-CO regions that exhibit many resolved peaks. Two-dimensional carbon–carbon correlation spectra of BPTI have sufficient resolution to identify and correlate many of the spin systems associated with the amino acids. As a result, we have been able to assign a large number of the spin systems in this protein. The agreement between shifts measured in the solid state and those in solution is typically very good, although some shifts near the ion binding sites differ by at least 1.5 ppm. These studies were conducted with approximately 0.2 to 0.4 μmol of enriched material; the sensitivity of this method is apparently adequate for other biological systems as well.

amino acid side chain assignment protocol bovine pancreatic trypsin inhibitor solid state NMR uniform labeling 


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  1. Baldus, M., Geurts, D.G., Hediger, S. and Meier, B.H. (1996) J. Magn. Reson., A 118, 140–144.Google Scholar
  2. Baldus, M. and Meier, B.H. (1996) J. Magn. Reson., A 121, 65–69.Google Scholar
  3. Baldus, M., Geurts, D.G. and Meier, B.H. (1998a) SSNMR, 11, 157–168.Google Scholar
  4. Baldus, M., Petkova, A.T., Herzfeld, J. and Griffin, R.G. (1998b) Mol. Phys., 95, 1197–1207.Google Scholar
  5. Baldus, M., Tomaselli, M., Meier, B.H. and Ernst, R.R. (1994) Chem. Phys. Lett., 230, 329–336.Google Scholar
  6. Bennett, A.E., Ok, J.H., Griffin, R.G. and Vega, S. (1992) J. Chem. Phys., 96, 8624–8627.Google Scholar
  7. Bennett, A.E., Rienstra, C.M., Auger, M., Lakshmi, K.V. and Griffin, R.G. (1995) J. Chem. Phys., 103, 6951–6958.Google Scholar
  8. Bennett, A.E., Rienstra, C.M., Griffiths, J.M., Zhen, W.G., Lansbury, P.T. and Griffin, R.G. (1998) J. Chem. Phys., 108, 9463–9479.Google Scholar
  9. Benzinger, T.L.S., Gregory, D.M., Burkoth, T.S., Miller-Auer, H., Lynn, D.G., Botto, R.E. and Meredith, S.C. (1998) Proc. Natl. Acad. Sci. USA, 95, 13407–13412.Google Scholar
  10. Clore, G.M. and Gronenborn, A.M. (1994) Methods Enzymol., 239, 349–363.Google Scholar
  11. Cole, H.B.R. and Torchia, D.A. (1991) Chem. Phys., 158, 278–281.Google Scholar
  12. de Dios, A.C., Pearson, J.G. and Oldfield, E. (1993) Science, 260, 1491–1495.Google Scholar
  13. Feng, X., Verdegem, P.J.E., Lee, Y.K., Sandström, D., Edén, M., Bovee-Geurts, P., de Grip, W.J., Lugtenburg, J., de Groot, H.J.M. and Levitt, M.H. (1997) J. Am. Chem. Soc., 119, 6853–6857.Google Scholar
  14. Fujiwara, T., Sugase, K., Kainosho, M., Ono, A., Ono, A.M. and Akutsu, H. (1995) J. Am. Chem. Soc., 117, 11351–11352.Google Scholar
  15. Gregory, D.M., Mitchell, D.J., Stringer, J.A., Kiihne, S., Shiels, J.C., Callahan, J., Mehta, M.A. and Drobny, G.P. (1995) Chem. Phys. Lett., 246, 654–663.Google Scholar
  16. Griffin, R.G. (1998) Nat. Struct. Biol., 5, 508–512 S.Google Scholar
  17. Hediger, S., Meier, B.H. and Ernst, R.R. (1995) Chem. Phys. Lett., 240, 449–456.Google Scholar
  18. Hediger, S., Meier, B.H., Kurur, N.D., Bodenhausen, G. and Ernst, R.R. (1994) Chem. Phys. Lett., 223, 283–288.Google Scholar
  19. Hediger, S., Signer, P., Tomaselli, M., Ernst, R.R. and Meier, B.H. (1997) J. Magn. Reson., 125, 291–301.Google Scholar
  20. Hing, A.W., Vega, S. and Schaefer, J. (1992) J. Magn. Reson., 96, 205–209.Google Scholar
  21. Hohwy, M., Jakobsen, H.J., Eden, M., Levitt, M.H. and Nielsen, N.C. (1998) J. Chem. Phys., 108, 2686–2694.Google Scholar
  22. Hohwy, M., Rienstra, C.M., Jaroniec, C.P. and Griffin, R.G. (1999) J. Chem. Phys., 110, 7983–7992.Google Scholar
  23. Hong, M., 1999 ENC Conference Abstract, Orlando, FL, M & T Poster #73.Google Scholar
  24. Hong, M. and Griffin, R.G. (1998) J. Am. Chem. Soc., 120, 7113–7114.Google Scholar
  25. Jansson, M., Li, Y.C., Jendeberg, L., Anderson, S., Montelione, G.L. and Nilsson, B. (1996) J. Biomol. NMR, 7, 131–141.Google Scholar
  26. Kay, L.E., Ikura, M., Tschudin, R. and Bax, A. (1990) J. Magn. Reson., 89, 496–514.Google Scholar
  27. Ketchum, R.R., Hu, W. and Cross, T.A. (1993) Science, 261, 1457–1460.Google Scholar
  28. Lansbury, P.T. Jr., Costa, P.R., Griffiths, J.M., Simon, E.J., Auger, M., Halverson, K.J., Kocisko, D.A., Hendsch, Z.S., Ashburn, T.T., Spencer, R.G.S., Tidor, B. and Griffin, R.G. (1995) Nat. Struct. Biol., 2, 990–998.Google Scholar
  29. Lee, Y.K., Kurur, N.D., Helmle, M., Johannessen, O.G., Nielsen, N.C. and Levitt, M.H. (1995) Chem. Phys. Lett., 242, 304–309.Google Scholar
  30. Long, J.R., Sun, B.Q., Bowen, A. and Griffin, R.G. (1994) J. Am. Chem. Soc., 116, 11950–11956.Google Scholar
  31. Marassi, F.M., Ramamoorthy, A. and Opella, S.J. (1997) Proc. Natl. Acad. Sci. USA, 94, 8551–8556.Google Scholar
  32. Marion, D. and Wüthrich, K. (1983) Biochem. Biophys. Res. Commun., 113, 967–974.Google Scholar
  33. McDowell, L.M., Klug, C.A., Beusen, D.D. and Schaefer, J. (1996) Biochemistry, 35, 5395–5403.Google Scholar
  34. Michal, C.A. and Jelinski, L.W. (1997) J. Am. Chem. Soc., 119, 9059–9060.Google Scholar
  35. Montelione, G.T. and Wagner, G. (1990) J. Magn. Reson., 87, 183–188.Google Scholar
  36. Otting, G., Liepinsh, E. and Wüthrich, K. (1993) Biochemistry, 32, 3571–3582.Google Scholar
  37. Rienstra, C.M., Hatcher, M.E., Mueller, L.J., Sun, B.-Q., Fesik, S.W., Herzfeld, J. and Griffin, R.G. (1998) J. Am. Chem. Soc., 120, 10602–10612.Google Scholar
  38. Rosen, M.K., Gardner, K.H., Willis, R.C., Parris, W.E., Pawson, T. and Kay, L.E. (1996) J. Mol. Biol., 263, 627–636.Google Scholar
  39. Saito, H., Tabeta, R., Shoji, A., Ozaki, T. and Ando, I. (1983) Macromolecules, 16, 1050–1057.Google Scholar
  40. Spera, S. and Bax, A. (1991) J. Am. Chem. Soc., 113, 5490–5492.Google Scholar
  41. States, D.J., Haberkorn, R.A. and Ruben, D.J. (1982) J. Magn. Reson., 48, 286–292.Google Scholar
  42. Straus, S.K., Bremi, T. and Ernst, R.R. (1996) Chem. Phys. Lett., 262, 709–715.Google Scholar
  43. Straus, S.K., Bremi, T. and Ernst, R.R. (1998b) J. Biomol. NMR, 12, 39–50.Google Scholar
  44. Sun, B.Q., Costa, P.R. and Griffin, R.G. (1995a) J. Magn. Reson., A 112, 191–198.Google Scholar
  45. Sun, B.Q., Costa, P.R., Kocisko, D., Lansbury, P.T. and Griffin, R.G. (1995b) J. Chem. Phys., 102, 702–707.Google Scholar
  46. Thompson, L.K., McDermott, A.E., Raap, J., van der Wielen, C.M., Lugtenberg, J., Herzfeld, J. and Griffin, R.G. (1992) Biochemistry, 31, 7931–7938.Google Scholar
  47. Tuchsen, E. and Hansen, P.E. (1988) Biochemistry, 27, 8568–8576.Google Scholar
  48. Tycko, R. (1996) J. Biomol. NMR, 8, 239–251.Google Scholar
  49. Wagner, G. (1983) Quart. Rev. Biophys., 16, 1–57.Google Scholar
  50. Wagner, G. and Bruhwiler, D. (1986) Biochemistry, 25, 5839–5843.Google Scholar
  51. Wagner, G., DeMarco, A. and Wüthrich, K. (1976) Biophys. Struct. Mech., 2, 139–158.Google Scholar
  52. Wishart, D.S., Sykes, B.D. and Richards, F.M. (1991) J. Mol. Biol., 222, 311–333.Google Scholar
  53. Wu, X. and Zilm, K.W. (1993) J. Magn. Reson., A 104, 154–165.Google Scholar
  54. Xu, R., Ayers, B., Cowburn, D. and Muir, T. (1999) Proc. Natl. Acad. Sci. USA, 96, 388–393.Google Scholar

Copyright information

© Kluwer Academic Publishers 2000

Authors and Affiliations

  • Ann McDermott
    • 1
  • Tatyana Polenova
    • 1
  • Anja Bockmann
    • 1
  • Kurt.W. Zilm
    • 2
  • Eric K. Paulsen
    • 2
  • Rachel W. Martin
    • 2
  • Gaetano T. Montelione
    • 3
  1. 1.Department of ChemistryColumbia UniversityNew YorkU.S.A.
  2. 2.Department of ChemistryYale UniversityNew HavenU.S.A.
  3. 3.Center for Advanced Biotechnology and MedicineRutgers UniversityPiscatawayU.S.A.

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