Journal of Biomolecular NMR

, Volume 27, Issue 4, pp 323–339 | Cite as

Solid state NMR sequential resonance assignments and conformational analysis of the 2×10.4 kDa dimeric form of the Bacillus subtilis protein Crh

  • Anja BöckmannEmail author
  • Adam Lange
  • Anne Galinier
  • Sorin Luca
  • Nicolas Giraud
  • Michel Juy
  • Henrike Heise
  • Roland Montserret
  • François Penin
  • Marc Baldus


Solid state NMR sample preparation and resonance assignments of the U-[13C,15N] 2×10.4 kDa dimeric form of the regulatory protein Crh in microcrystalline, PEG precipitated form are presented. Intra– and interresidue correlations using dipolar polarization transfer methods led to nearly complete sequential assignments of the protein, and to 88% of all 15N, 13C chemical shifts. For several residues, the resonance assignments differ significantly from those reported for the monomeric form analyzed by solution state NMR. Dihedral angles obtained from a TALOS-based statistical analysis suggest that the microcrystalline arrangement of Crh must be similar to the domain-swapped dimeric structure of a single crystal form recently solved using X-ray crystallography. For a limited number of protein residues, a remarkable doubling of the observed NMR resonances is observed indicative of local static or dynamic conformational disorder. Our study reports resonance assignments for the largest protein investigated by solid state NMR so far and describes the conformational dimeric variant of Crh with previously unknown chemical shifts.

assignments catabolite repression histidine-containing phosphocarrier protein (Crh) MAS protein dynamics protein structure solid state NMR spectroscopy 


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  1. Andrew, E.R., Bradbury, A. and Eades, R.G. (1958) Nature, 4650, 1659.Google Scholar
  2. Baldus, M. (2002) Prog. Nucl. Magn. Reson. Spectrosc., 41, 1-47.Google Scholar
  3. Baldus, M., Geurts, D.G. and Meier, B.H. (1998a) Solid State Nucl. Magn. Reson., 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. Bennett, A.E., Ok, J.H., Griffin, R.G. and Vega, S. (1992) J. Chem. Phys., 96, 8624-8627.Google Scholar
  6. Bennett, A.E., Rienstra, C.M., Auger, M., Lakshmi, K.V. and Griffin, R.G. (1995) J. Chem. Phys., 103, 6951-6958.Google Scholar
  7. Bloembergen, N. (1949) Physica, 15, 386-426.Google Scholar
  8. Böckmann, A. and McDermott, A.E. (2002) In The Encyclopedia of NMR, Grant, D.M. and Harris, R.K. (Eds.), J. Wiley and Sons, London.Google Scholar
  9. Castellani, F., van Rossum, B., Diehl, A., Schubert, M., Rehbein, K. and Oschkinat, H. (2002) Nature, 420, 98-102.Google Scholar
  10. Cornilescu, G., Delaglio, F. and Bax, A. (1999) J. Biomol. NMR, 13, 289-302.Google Scholar
  11. Creemers, A.F.L., Kiihne, S., Bovee-Geurts, P.H.M., DeGrip, W.J., Lugtenburg, J. and de Groot, H.J.M. (2002) Proc. Natl. Acad. Sci. USA, 99, 9101-9106.Google Scholar
  12. Delaglio, F., Grzesiek, S., Vuister, G.W., Zhu, G., Pfeifer, J. and Bax, A. (1995) J. Biomol. NMR, 6, 277-293.Google Scholar
  13. Detken, A., Hardy, E.H., Ernst, M., Kainosho, M., Kawakami, T., Aimoto, S. and Meier, B.H. (2001) J. Biomol. NMR, 20, 203-221.Google Scholar
  14. Deutscher, J., Galinier, A. and Martin-Verstraete, I. (2001) In Bacillus subtilis and its Closest Relatives: From Genes to Cells, Sonenschein, A.L., Hoch, J.A. and Losick, R. (Eds.), ASM Press, Washington, pp. 129-150.Google Scholar
  15. Deutscher, J., Kuster, E., Bergstedt, U., Charrier, V. and Hillen, W. (1995) Mol. Microbiol., 15, 1049-1053.Google Scholar
  16. Egorova-Zachernyuk, T.A., Hollander, J., Fraser, N., Gast, P., Hoff, A.J., Cogdell, R., de Groot, H.J.M. and Baldus, M. (2001) J. Biomol. NMR, 19, 243-253.Google Scholar
  17. Favier, A., Brutscher, B., Blackledge, M., Galinier, A., Deutscher, J., Penin, F. and Marion, D. (2002) J. Mol. Biol., 317, 131-144.Google Scholar
  18. Fieulaine, S., Morera, S., Poncet, S., Mijakovic, I., Galinier, A., Janin, J., Deutscher, J. and Nessler, S. (2002) Proc. Natl. Acad. Sci. USA, 99, 13437-13441.Google Scholar
  19. Galinier, A., Haiech, J., Kilhoffer, M.C., Jaquinod, M., Stulke, J., Deutscher, J. and Martin-Verstraete, I. (1997) Proc. Natl. Acad. Sci. USA, 94, 8439-8444.Google Scholar
  20. Galinier, A., Kravanja, M., Engelmann, R., Hengstenberg, W., Kilhoffer, M.C., Deutscher, J. and Haiech, J. (1998) Proc. Natl. Acad. Sci. USA, 95, 1823-1828.Google Scholar
  21. Guex, N. and Peitsch, M.C. (1997) Electrophoresis, 18, 2714-2723.Google Scholar
  22. Hediger, S., Meier, B.H. and Ernst, R.R. (1995) Chem. Phys. Lett., 240, 449-456.Google Scholar
  23. Hohwy, M., Rienstra, C.M., Jaroniec, C.P. and Griffin, R.G. (1999) J. Chem. Phys., 110, 7983-7992.Google Scholar
  24. Hong, M. (1999) J. Biomol. NMR, 15, 1-14.Google Scholar
  25. Hong, M. and Griffin, R.G. (1998) J. Am. Chem. Soc., 120, 7113-7114.Google Scholar
  26. Jaroniec, C.P., MacPhee, C.E., Astrof, N.S., Dobson, C.M. and Griffin, R.G. (2002) Proc. Natl. Acad. Sci. USA, 99, 16748-16753.Google Scholar
  27. Johnson, B.A. and Blevins, R.A. (1994) J. Biomol. NMR, 4, 603-614.Google Scholar
  28. Jones, B.E., Dossonnet, V., Kuster, E., Hillen, W., Deutscher, J. and Klevit, R.E. (1997) J. Biol. Chem., 272, 26530-26535.Google Scholar
  29. Juy, M., Penin, F., Favier, A., Galinier, A., Montserret, R., Haser, R., Deutscher, J. and Böckmann, A. (2003), submitted.Google Scholar
  30. Lange, A., Seidel, K., Verdier, L., Luca, S. and Baldus, M. (2003), submitted.Google Scholar
  31. Luca, S., White, J.F., Sohal, A.K., Filipov, D.V., van Boom, J.H., Grisshammer, R. and Baldus, M. (2003) Proc. Natl. Acad. Sci. USA, in press.Google Scholar
  32. Markley, J.L., Bax, A., Arata, Y., Hilbers, C.W., Kaptein, R., Sykes, B.D., Wright, P.E. and Wüthrich, K. (1998) Eur. J. Biochem., 256, 1-15.Google Scholar
  33. McDermott, A., Polenova, T., Böckmann, A., Zilm, K.W., Paulsen, E.K., Martin, R.W. and Montelione, G.T. (2000) J. Biomol. NMR, 16, 209-219.Google Scholar
  34. Metz, G., Wu, X.L. and Smith, S.O. (1994) J. Magn. Reson. Ser. A, 110, 219-227.Google Scholar
  35. Nielsen, N.C., Bildsoe, H., Jakobsen, H.J. and Levitt, M.H. (1994) J. Chem. Phys., 101, 1805-1812.Google Scholar
  36. Nomura, K., Takegoshi, K., Terao, T., Uchida, K. and Kainosho, M. (1999) J. Am. Chem. Soc., 121, 4064-4065.Google Scholar
  37. Pauli, J., Baldus, M., van Rossum, B., de Groot, H. and Oschkinat, H. (2001) Chembiochemistry, 2, 272-281.Google Scholar
  38. Pauli, J., van Rossum, B., Förster, H., de Groot, H.J.M. and Oschkinat, H. (2000) J. Magn. Reson., 143, 411-416.Google Scholar
  39. Penin, F., Favier, A., Montserret, R., Brutscher, B., Deutscher, J., Marion, D. and Galinier, A. (2001) J. Mol. Microbiol. Biotechnol., 3, 429-432.Google Scholar
  40. Petkova, A.T., Baldus, M., Belenky, M., Hong, M., Griffin, R.G. and Herzfeld, J. (2003) J. Magn. Reson., 160, 1-12.Google Scholar
  41. Petkova, A.T., Ishii, Y., Balbach, J.J., Antzutkin, O.N., Leapman, R.D., Delaglio, F. and Tycko, R. (2002) Proc. Natl. Acad. Sci. USA, 99, 16742-16747.Google Scholar
  42. Rienstra, C.M., Hohwy, M., Hong, M. and Griffin, R.G. (2000) J. Am. Chem. Soc., 122, 10979-10900.Google Scholar
  43. Rienstra, C.M., Hohwy, M., Mueller, L.J., Jaroniec, C.P., Reif, B. and Griffin, R.G. (2002a) J. Am. Chem. Soc., 124, 11908-11922.Google Scholar
  44. Rienstra, C.M., Tucker-Kellogg, L., Jaroniec, C.P., Hohwy, M., Reif, B., McMahon, M.T., Tidor, B., Lozano-Perez, T. and Griffin, R.G. (2002b) Proc. Natl. Acad. Sci. USA, 99, 10260-10265.Google Scholar
  45. Seavey, B.R., Farr, E.A., Westler, W.M. and Markley, J.L. (1991) J. Biomol. NMR, 1, 217-236.Google Scholar
  46. Straus, S.K., Bremi, T. and Ernst, R.R. (1996) Chem. Phys. Lett., 262, 709-715.Google Scholar
  47. Straus, S.K., Bremi, T. and Ernst, R.R. (1997) J. Biomol. NMR, 10, 119-128.Google Scholar
  48. Straus, S.K., Bremi, T. and Ernst, R.R. (1998) J. Biomol. NMR, 12, 39-50.Google Scholar
  49. Stulke, J. and Hillen, W. (2000) Annu. Rev. Microbiol., 54, 849-880.Google Scholar
  50. Tan, W.M., Gu, Z., Zeri, A.C. and Opella, S.J. (1999) J. Biomol. NMR, 13, 337-342.Google Scholar
  51. Verel, R., Baldus, M., Ernst, M. and Meier, B.H. (1998) Chem. Phys. Lett., 287, 421-428.Google Scholar
  52. Wüthrich, K. (1986) NMR of Proteins and Nucleic Acids, Wiley, New York, NY.Google Scholar

Copyright information

© Kluwer Academic Publishers 2003

Authors and Affiliations

  • Anja Böckmann
    • 1
    Email author
  • Adam Lange
    • 2
  • Anne Galinier
    • 3
  • Sorin Luca
    • 2
  • Nicolas Giraud
    • 1
  • Michel Juy
    • 1
  • Henrike Heise
    • 2
  • Roland Montserret
    • 1
  • François Penin
    • 1
  • Marc Baldus
    • 2
  1. 1.Institut de Biologie et Chimie des Protéines, C.N.R.S UMR 5086Lyon Cedex 07France
  2. 2.Max-Planck-Institute for Biophysical Chemistry, Solid-state NMRGöttingenGermany
  3. 3.Institut de Biologie Structurale et Microbiologie, C.N.R.S UPR 9043Marseille Cedex 20France

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