SpringerLink
Log in

NMR exchange broadening arising from specific low affinity protein self-association: Analysis of nitrogen-15 nuclear relaxation for rat CD2 domain 1

  • Published:
Journal of Biomolecular NMR Aims and scope Submit manuscript

Abstract

Nuclear spin relaxation monitored by heteronuclear NMR provides a useful method to probe the overall and internal molecular motion for biological macromolecules over a variety of time scales. Nitrogen-15 NMR relaxation parameters have been recorded for the N-terminal domain of the rat T-cell antigen CD2 (CD2d1) in a dilution series from 1.20 mM to 40 μM (pH 6.0, 25 °C). The data have been analysed within the framework of the model- free formalism of Lipari and Szabo to understand the molecular origin of severely enhanced transverse relaxation rates found for certain residues. These data revealed a strong dependence of the derived molecular correlation time τc upon the CD2d1 protein concentration. Moreover, a number of amide NH resonances exhibited exchange broadening and chemical shifts both strongly dependent on protein concentration. These amide groups cluster on the major β-sheet surface of CD2d1 that coincides with a major lattice contact in the X-ray structure of the intact ectodomain of rat CD2. The complete set of relaxation data fit well to an equilibrium monomer–dimer exchange model, yielding estimates of exchange rate constants (kON=5000 M-1 s-1; kOFF=7 s-1) and a dissociation constant (KD ∼ 3–6 mM) that is consistent with the difficulty in detecting the weak interactions for this molecule by alternative biophysical methods. The self-association of CD2d1 is essentially invariant to changes in buffer composition and ionic strength and the associated relaxation phenomena cannot be explained as a result of neglecting anisotropic rotational diffusion in the analysis. These observations highlight the necessity to consider low affinity protein self-association interactions as a source of residue specific exchange phenomena in NMR spectra of macromolecular biomolecules, before the assignment of more elaborate intramolecular conformational mechanisms.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Akke, M. and Palmer, A.G. (1996) J. Am. Chem. Soc., 118, 911–912.

    Google Scholar 

  • Akke, M., Liu, J., Cavanagh, J., Erickson, H.P. and Palmer, A.G. (1998) Nat. Struct. Biol., 5, 55–59.

    Google Scholar 

  • Banci, L., Felli, I. and Koulougliotis, D. (1998) J. Biomol. NMR, 12, 307-318.

    Google Scholar 

  • Barbato, G., Ikura, M., Kay, L. E., Pastor, R. W. and Bax, A. (1992) Biochemistry, 31, 5269–5278.

    Google Scholar 

  • Bartels, C., **a, T.-H., Billeter, M., Güntert, P. and Wüthrich, K. (1995) J. Biomol. NMR, 6, 1–10.

    Google Scholar 

  • Biekofsky, R.R., Martin, S.R., Browne, J.P., Bayley, P.M. and Feeney, J. (1998) Biochemistry, 37, 7617–7629.

    Google Scholar 

  • Bodian, D.L., Jones, E.Y., Harlos, K., Stuart, D.I., and Davis, S.J. (1994) Structure, 2, 755–766.

    Google Scholar 

  • Boucher, W. (1995) AZARA v1.0, Department of Biochemistry, University of Cambridge, U.K.

  • Brunger, A.T. (1993) XPLOR.v.31, Yale University, New Haven, CT.

    Google Scholar 

  • Chen, H.A., Pfuhl, M., Davis, B. and Driscoll, P.C. (1998) J. Biomol. NMR, 12, 457–458.

    Google Scholar 

  • Chen, H.A., Pfuhl, M., McAlister, M. S.B. and Driscoll, P.C. (1999) submitted.

  • Clore, G.M., Driscoll, P.C., Wingfield, P.T. and Gronenborn, A.M. (1990) Biochemistry, 29, 7387.

    Google Scholar 

  • Clore, G.M., Szabo, A., Bax, A., Kay, L.E., Driscoll, P.C. and Gronenborn, A.M. (1990) J. Am. Chem. Soc., 112, 4989–4991.

    Google Scholar 

  • Crawford, D.A. (1994) Structure and Dynamics of a Cell Adhesion Protein, Ph.D. Thesis, Oxford University, Oxford.

    Google Scholar 

  • Davis, S.J., Ikemizu, S., Wild, M.K. and van der Merwe, P.A. (1998) Immunol. Rev., 163, 217–236.

    Google Scholar 

  • Dayie, K.T., Wagner, G. and Lefèvre, J.F., In Dynamics and the Problem of Recognition in Biological Macromolecules, NatoASI Series A, Vol. 288, Plenum Press, New York, NY, pp. 139–162.

  • de la Torre, J.G. and Bloomfield, V.A. (1981) Q. Rev. Biophys., 14, 81–139.

    Google Scholar 

  • de la Torre, J.G., Navarro, S., Martinez, M.C.L., Diaz, F.G. and Cascales, J.J.L. (1994) Biophys. J., 67, 530–531.

    Google Scholar 

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

    Google Scholar 

  • Driscoll, P.C., Cyster, J.G., Campbell, I.D. and Williams, A.F. (1991) Nature, 353, 762–765.

    Google Scholar 

  • Driscoll, P.C., Cyster, J.G., Somoza, C., Crawford, D.A., Howe, P., Harvey, T.S., Kieffer, B., Campbell, I.D. and Williams, A.F. (1993) Biochem. Soc. Trans., 21, 947–952.

    Google Scholar 

  • Duan, Y., Wang, L. and Kollman, P.A. (1998) Proc. Natl. Acad. Sci. USA, 95, 9897–9902.

    Google Scholar 

  • Evans, P.A., Kautz, R.A., Fox, R.O. and Dobson, C.M. (1989) Biochemistry, 28, 362–370.

    Google Scholar 

  • Fushman, D., Cahill, S. and Cowburn, D. (1998) J. Mol. Biol., 266, 173–194.

    Google Scholar 

  • Gryk, M.R., Abseher, R., Simon, B., Nilges, M. and Oschkinat, H. (1998) J. Mol. Biol., 280, 879–896.

    Google Scholar 

  • Grzesiek, S., Bax, A., Hu, J.-S., Kaufman, J., Palmer, I., Stahl, S.J., Tjandra, N. and Wingfield, P. (1997) Protein Sci., 6, 1248–1261.

    Google Scholar 

  • Jardetzky, O. (1996) Prog. Biophys. Mol. Biol., 65, 171–218.

    Google Scholar 

  • Jones, E.Y., Davis, S.J., Williams, A.F., Harlos, K. and Stuart, D.I. (1992) Nature, 360, 232–239.

    Google Scholar 

  • Kay, L.E. (1998) Nat. Struct. Biol., 5, 513–517.

    Google Scholar 

  • Kay, L.E., Torchia, D.A. and Bax, A. (1989) Biochemistry, 28, 8972–8979.

    Google Scholar 

  • Kay, L.E., Keifer, P. and Saarinen, T. (1992) J. Am. Chem. Soc., 114, 10663.

    Google Scholar 

  • Kay, L.E., Nicholson, L.K., Delaglio, F., Bax, A. and Torchia, D.A. (1992) J. Magn. Reson., 97, 359–367.

    Google Scholar 

  • Kieffer, B. and Driscoll, P.C. (1992) unpublished observations.

  • Kraulis, P.J. (1989) J. Magn. Reson., 24, 627–633.

    Google Scholar 

  • Krishnan, V.V. and Cosman, M. (1998) J. Biomol. NMR, 12, 177–182.

    Google Scholar 

  • Lane, A.N. and Lefèvre, J.-F. (1995) Methods Enzymol., 239, 596–619.

    Google Scholar 

  • Le, H. and Oldfield, E. (1994) J. Biomol. NMR, 4, 341–348.

    Google Scholar 

  • Le, H.B. and Oldfield, E. (1996) J. Phys. Chem., 100, 16423–16428

    Google Scholar 

  • Lee, K., Rance, M., Chazin, W.J. and Palmer, A.G. (1997) J. Biomol. NMR, 9, 287–298.

    Google Scholar 

  • Le Master, M. and Kushlan, D.M. (1996) J. Am. Chem. Soc., 118, 9255–9264.

    Google Scholar 

  • Lipari, G. and Szabo, A. (1982a) J. Am. Chem. Soc., 104, 4546–4559.

    Google Scholar 

  • Lipari, G. and Szabo, A. (1982b) J. Am. Chem. Soc., 104, 4559–4570.

    Google Scholar 

  • Luz, Z. and Meiboom, S. (1963) J. Chem. Phys., 39, 366–370.

    Google Scholar 

  • Mandel, A.M., Akke, M. and Palmer, A.G. (1995) J. Mol. Biol., 246, 144–163.

    Google Scholar 

  • McAlister, M.S.B., Mott, H.R., van der Merwe, P.A., Campbell, I.D., Davis, S.J. and Driscoll, P.C. (1996) Biochemistry, 35, 5982–5991.

    Google Scholar 

  • Mulder, F.A.A., de Graaf, R.A., Kaptein, R. and Boelens, R. (1998) J. Magn. Reson., 131, 351-357.

    Google Scholar 

  • Otting, G., Liepinsh, E. and Wüthrich, K. (1993) Biochemistry, 32, 3571–3582.

    Google Scholar 

  • Palmer, A.G., Cavanagh, J., Wright, P.E. and Rance, M. (1991) J. Magn. Reson., 93, 151–170.

    Google Scholar 

  • Palmer, A.G., Williams, J. and McDermott, A. (1996) J. Phys. Chem., 100, 13293–13310.

    Google Scholar 

  • Peng, J.W. and Wagner, G. (1992) J. Magn. Reson., 98, 308–332.

    Google Scholar 

  • Silkowski, H., Davis, S.J., Barcley, A.N., Rowe, A.J., Harding, S.E. and Byron, O. (1997) Eur. Biophys. J., 25, 455–462.

    Google Scholar 

  • States, D.J., Haberkorn, R.A. and Ruben, D.J. (1982) J. Magn. Reson., 48, 286–292.

    Google Scholar 

  • Szyperski, T., Luginbühl, P., Otting, G. and Wüthrich, K. (1993) J. Biomol. NMR, 3, 151–164.

    Google Scholar 

  • Tjandra, N., Feller, S.E., Pastor, R.W. and Bax, A. (1995) J. Am. Chem. Soc., 117, 12562–12566.

    Google Scholar 

  • Tjandra, N., Wingfield, P., Stahl, S. and Bax, A. (1996) J. Biomol. NMR, 8, 273–284.

    Google Scholar 

  • van der Merwe, P.A. (1993) personal communication.

  • Volkman, B.F., Alam, S.L., Satterlee, J.D. and Markley, J.L. (1998) Biochemistry, 37, 10906–10919.

    Google Scholar 

  • Weber, G. (1992) Protein Interactions, Chapman and Hall, New York, NY.

    Google Scholar 

  • Woessner, D.E. (1962) J. Chem. Phys., 37, 647–654.

    Google Scholar 

  • Wolfram, S. (1998) Mathematica-A System for Doing Mathematics by Computer, 2.0 edition, Wolfram Research Inc., Champaign, IL.

    Google Scholar 

  • Wong, K.-B., Fersht, A.R. and Freund, S.M.V. (1997) J. Mol. Biol., 258, 494–511.

    Google Scholar 

  • Wyss, D.F., Dayie, K.T. and Wagner, G. (1997) Protein Sci., 6, 534–542.

    Google Scholar 

  • Yang, D. and Kay, L.E. (1996) J. Mol. Biol., 263, 369–382.

    Google Scholar 

  • Zhang, W.X., Smithgall, T.E. and Gmeiner, W.H. (1998) Biochemistry, 37, 7119–7126.

    Google Scholar 

  • Zinn-Justin, S., Berthault, P., Guenneugues, M. and Desvaux, H. (1997) J. Biomol. NMR, 10, 363–372.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University College London, Gower Street, London, WC1E 6BT, U.K.

    Mark Pfuhl, Ho A. Chen & Paul C. Driscoll

  2. Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100, København Ø, Denmark

    Søren M. Kristensen

Authors
  1. Mark Pfuhl
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ho A. Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Søren M. Kristensen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Paul C. Driscoll
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Pfuhl, M., Chen, H.A., Kristensen, S.M. et al. NMR exchange broadening arising from specific low affinity protein self-association: Analysis of nitrogen-15 nuclear relaxation for rat CD2 domain 1. J Biomol NMR 14, 307–320 (1999). https://doi.org/10.1023/A:1008319917267

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1008319917267

Search

Navigation