Controlling residual dipolar couplings in high-resolution NMR of proteins by strain induced alignment in a gel

Abstract

Water-soluble biological macromolecules can be weakly aligned by dissolution in a strained, hydrated gel such as cross-linked polyacrylamide, an effect termed `strain-induced alignment in a gel' (SAG). SAG induces nonzero nuclear magnetic dipole-dipole couplings that can be measured in high-resolution NMR spectra and used as structural constraints. The dependence of experimental 15N-1H dipolar couplings extracted from two-dimensional heteronuclear single quantum coherence (HSQC) spectra on several properties of compressed polyacrylamide, including the extent of compression, the polyacrylamide concentration, and the cross-link density, is reported for the B1 immunoglobulin binding domain of streptococcal protein G (protein G/B1, 57 residues). It is shown that the magnitude of macromolecular alignment can be widely varied by adjusting these properties, although the orientation and asymmetry of the alignment tensor are not affected significantly. The dependence of the 15N relaxation times T1 and T2 of protein G/B1 on polyacrylamide concentration are also reported. In addition, the results of 15N relaxation and HSQC experiments on the RNA binding domain of prokaryotic protein S4 from Bacillus stearothermophilus (S4 Δ41, residues 43–200) in a compressed polyacrylamide gel are presented. These results demonstrate the applicability of SAG to proteins of higher molecular weight and greater complexity. A modified in-phase/anti-phase (IPAP) HSQC technique is described that suppresses natural-abundance 15N background signals from amide groups in polyacrylamide, resulting in cleaner HSQC spectra in SAG experiments. The mechanism of protein alignment in strained polyacrylamide gels is contrasted with that in liquid crystalline media.

This is a preview of subscription content, access via your institution.

References

  1. Barrientos, L.G., Dolan, C. and Gronenborn, A.M. (2000) J. Biomol. NMR, 16, 329–337.

    Google Scholar 

  2. Bax, A. and Tjandra, N. (1997) J. Biomol. NMR, 10, 289–292.

    Google Scholar 

  3. Blanco, F.J., Angrand, I. and Serrano, L. (1999) J. Mol. Biol., 285, 741–753.

    Google Scholar 

  4. Bothnerby, A.A., Domaille, P.J. and Gayathri, C. (1981) J. Am. Chem. Soc., 103, 5602–5603.

    Google Scholar 

  5. Bothnerby, A.A., Gayathri, C., Vanzijl, P.C.M., Maclean, C., Lai, J.J. and Smith, K.M. (1985) Magn. Reson. Chem., 23, 935–938.

    Google Scholar 

  6. Cavagnero, S., Dyson, H.J. and Wright, P.E. (1999) J. Biomol. NMR, 13, 387–391.

    Google Scholar 

  7. Clore, G.M., Starich, M.R. and Gronenborn, A.M. (1998) J. Am. Chem. Soc., 120, 10571–10572.

    Google Scholar 

  8. Delaglio, F., Grzesiek, S., Vuister, G.W., Zhu, G., Pfeifer, J. and Bax, A. (1995) J. Biomol. NMR, 6, 277–293.

    Google Scholar 

  9. Deloche, B. and Samulski, E.T. (1981) Macromolecules, 14, 575–581.

    Google Scholar 

  10. Emsley, J.W. and Lindon, J.C. (1975) 'NMR Spectroscopy Using Liquid Crystal Solvents', Pergamon Press, New York, NY.

    Google Scholar 

  11. Gallagher, T., Alexander, P., Bryan, P. and Gilliland, G.L. (1994) Biochemistry, 33, 4721–4729.

    Google Scholar 

  12. Garrett, D.S., Gronenborn, A.M. and Clore, G.M. (1995) J. Cell. Biochem. Suppl., 21B, 71–71.

    Google Scholar 

  13. Gayathri, C., Bothnerby, A.A., Vanzijl, P.C.M. and Maclean, C. (1982) Chem. Phys. Lett., 87, 192–196.

    Google Scholar 

  14. Gronenborn, A.M., Filpula, D.R., Essig, N.Z., Achari, A., Whitlow, M., Wingfield, P.T. and Clore, G.M. (1991) Science, 253, 657–661.

    Google Scholar 

  15. Hansen, M.R., Mueller, L. and Pardi, A. (1998) Nat. Struct. Biol., 5, 1065–1074.

    Google Scholar 

  16. Kay, L.E., Nicholson, L.K., Delaglio, F., Bax, A. and Torchia, D.A. (1992) J. Magn. Reson., 97, 359–375.

    Google Scholar 

  17. Koenig, B.W., Hu, J.S., Ottiger, M., Bose, S., Hendler, R.W. and Bax, A. (1999) J. Am. Chem. Soc., 121, 1385–1386.

    Google Scholar 

  18. Lisicki, M.A., Mishra, P.K., Bothnerby, A.A. and Lindsey, J.S. (1988) J. Phys. Chem., 92, 3400–3403.

    Google Scholar 

  19. Lohman, J.A.B. and MacLean, C. (1978) Chem. Phys., 35, 269–274.

    Google Scholar 

  20. Markus, M.A., Gerstner, R.B., Draper, D.E. and Torchia, D.A. (1999) J. Mol. Biol., 292, 375–387.

    Google Scholar 

  21. Markus, M.A., Gerstner, R.B., Draper, R.B. and Torchia, D.A. (1998) EMBO J., 17, 4559–4571.

    Google Scholar 

  22. Ottiger, M. and Bax, A. (1999) J. Biomol. NMR, 13, 187–191.

    Google Scholar 

  23. Ottiger, M., Delaglio, F. and Bax, A. (1998) J. Magn. Reson., 131, 373–378.

    Google Scholar 

  24. Peshkovsky, A. and McDermott, A.E. (1999) J. Phys. Chem., A103, 8604–8611.

    Google Scholar 

  25. Piotto, M., Saudek, V. and Sklenar, V. (1992) J. Biomol. NMR, 2, 661–665.

    Google Scholar 

  26. Plantenga, T.M. and Maclean, C. (1980) Chem. Phys. Lett., 75, 294–297.

    Google Scholar 

  27. Plantenga, T.M., Bulsink, H., Maclean, C. and Lohman, J.A.B. (1981) Chem. Phys., 61, 271–280.

    Google Scholar 

  28. Plantenga, T.M., Dekanter, F.J.J., Bulsink, H. and Maclean, C. (1982) Chem. Phys., 65, 77–81.

    Google Scholar 

  29. Plantenga, T.M., Ruessink, B.H. and Maclean, C. (1980) Chem. Phys., 48, 359–368.

    Google Scholar 

  30. Prosser, R.S., Losonczi, J.A. and Shiyanovskaya, I.V. (1998) J. Am. Chem. Soc., 120, 11010–11011.

    Google Scholar 

  31. Riley, S.A. and Augustine, M.P. (2000) J. Phys. Chem., A104, 3326–3331.

    Google Scholar 

  32. Sanders, C.R. and Landis, G.C. (1995) Biochemistry, 34, 4030–4040.

    Google Scholar 

  33. Sanders, C.R., Hare, B.J., Howard, K.P. and Prestegard, J.H. (1994) Prog. Nucl. Magn. Reson. Spectr., 26, 421–444.

    Google Scholar 

  34. Sass, J., Cordier, F., Hoffmann, A., Cousin, A., Omichinski, J.G., Lowen, H. and Grzesiek, S. (1999) J. Am. Chem. Soc., 121, 2047–2055.

    Google Scholar 

  35. Sass, H.J., Musco, G., Stahl, S.J., Wingfield, P.T. and Grzesiek, S. (2000) J. Biomol. NMR, 18, 303–309.

    Google Scholar 

  36. Saupe, A. and Englert, G. (1963) Phys. Rev. Lett., 11, 462.

    Google Scholar 

  37. Stellwagen, N.C. (1997) Electrophoresis, 18, 34–44.

    Google Scholar 

  38. Tjandra, N. and Bax, A. (1997) Science, 278, 1697–1697.

    Google Scholar 

  39. Tjandra, N., Grzesiek, S. and Bax, A. (1996) J. Am. Chem. Soc., 118, 6264–6272.

    Google Scholar 

  40. Tjandra, N., Omichinski, J.G., Gronenborn, A.M., Clore, G.M. and Bax, A. (1997) Nat. Struct. Biol., 4, 732–738.

    Google Scholar 

  41. Tolman, J.R., Flanagan, J.M., Kennedy, M.A. and Prestegard, J.H. (1995) Proc. Natl. Acad. Sci. USA, 92, 9279–9283.

    Google Scholar 

  42. Tycko, R., Blanco, F.J. and Ishii, Y. (2000) J. Am. Chem. Soc., 122, 9340–9341.

    Google Scholar 

  43. Zweckstetter, M. and Bax, A. (2000) J. Am. Chem. Soc., 122, 3791–3792.

    Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding author

Correspondence to Robert Tycko.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Ishii, Y., Markus, M.A. & Tycko, R. Controlling residual dipolar couplings in high-resolution NMR of proteins by strain induced alignment in a gel. J Biomol NMR 21, 141–151 (2001). https://doi.org/10.1023/A:1012417721455

Download citation

  • dipolar couplings
  • polyacrylamide gel
  • protein NMR
  • structure determination
  • weak alignment