Journal of Protein Chemistry

, Volume 9, Issue 5, pp 641–646 | Cite as

Role of a disulfide cross-link in the conformational stability of a thermostable xylanase

  • Utpal Tatu
  • S. K. Murthy
  • P. J. Vithayathil
Article

Abstract

The role of a S-S cross-link in the conformational stability of xylanase fromHumicola lanuginosa has been investigated using CD, UV absorption spectroscopy, and RIA displacement studies. Our studies show that reduction and carboxymethylation of the S-S cross-link in xylanase results in a gross conformational perturbation of the protein. The secondary structure analysis of the CD spectra indicates that the xylanase with an intact S-S contains 66% β-sheet structure and remaining random coil. Cleavage of the S-S bond results in a loss of 25% β-sheet structure. Thermal denaturation studies using CD spectroscopy andpH-dependent tyrosine ionization studies using UV spectroscopy show that the presence of disulfide cross-link offers resistance against unfolding by extremes of temperature andpH. Further, we demonstrate that the heat-induced changes in xylanase with intact S-S bond are almost totally reversible, while those in the S-S cleaved enzyme fail to show any significant reversal. Our studies support the present theory that S-S cross-links exert their stabilizing effect in proteins by destabilizing the unfolded state of the protein and forcing it back to a more folded state.

Key words

CD disulfide bond protein stability RIA secondary structure tyrosine ionization xylanase 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Brown, S. B. (1980).An Introduction to Spectroscopy for Biochemists, Vol. 29, Academic Press, New York.Google Scholar
  2. Creighton, T. E. (1983). InProteins, Structures and Molecular Properties, Freeman, New York, pp. 137–187.Google Scholar
  3. Ellman, G. L. (1959).Arch. Biochem. Biophys. 82, 70.Google Scholar
  4. Fraker, P. J., and Spack Jr. J. C. (1978).Biochem. Biophys. Res. Commun. 80, 849–857.Google Scholar
  5. Frifelder, D. (1976). InPhysical Biochemistry, 394, W. H. Freeman and Co., England.Google Scholar
  6. Imanishi, A., and Isemura, T. (1969).J. Biochem. 65, 309–312.Google Scholar
  7. Johnson, R. E., Adams, P., and Rupley, J. A. (1978).Biochemistry 17, 1479–1484.Google Scholar
  8. Lalitha, A., Murthy, S.K., and Vithayathil, P. J. (1990).Arch. Biochem. Biophys. 276, 546–553.Google Scholar
  9. Levitt, M., and Chothia, C. (1976).Nature 261, 552–558Google Scholar
  10. Lin, S. H., Konishi, Y., Denton, M. E. and Scheraga, H. A. (1984).Biochemistry 23, 5504–5512.Google Scholar
  11. McCubbin, W. D., Edery, I., Altman, M., Sonenberg, N., and Kay, C. M. (1988).J. Biol. Chem. 263, 17,665.Google Scholar
  12. Pace, C. N., Grimsley, E. R., Thomson, J. A., and Barnett, B. J. (1988).J. Biol. Chem. 263, 11,820–11,825.Google Scholar
  13. Provencher, S. W. (1982).Comput. Phys. Commun. 27, 213–227.Google Scholar
  14. Sauer, R. T., Hehir, K., Stearman, R. S., Weiss, M. A., Jeitler-Nilsson, A., Suchanek, E. G., and Pabo, C. O. (1986).Biochemistry 25, 5992–5998.Google Scholar
  15. Wetzel, R. (1987).TIBS 12, 478–482.Google Scholar

Copyright information

© Plenum Publishing Corporation 1990

Authors and Affiliations

  • Utpal Tatu
    • 1
  • S. K. Murthy
    • 1
  • P. J. Vithayathil
    • 1
  1. 1.Department of BiochemistryIndian Institute of ScienceBangaloreIndia

Personalised recommendations