Advertisement

Journal of Protein Chemistry

, Volume 17, Issue 7, pp 719–728 | Cite as

Characterization of a subunit structure and stability of the recombinant porin fromNeisseria gonorrhoeae

  • Yury V. Matsuka
  • Deborah A. Dilts
  • Susan Hoiseth
  • Rasappa Arumugham
Article

Abstract

An outer membrane PIA protein fromNeisseria gonorrhoeae strain FA19 was expressed inEscherichia coli and refoldedin vitro in the presence of zwitterionic detergent. Its proper folding and subunit organization was confirmed by comparison with the native counterpart. The unfolding of PIA has been investigated using fluorescence spectroscopy and analytical size-exclusion chromatography methods. Analysis of the denaturation pathway of the PIA revealed that it forms an unusually labile quaternary structure. In the presence of 1 M guanidinium chloride (GdmCl) or upon heating up to 50°C, dissociation of the PIA oligomer was observed resulting in the formation of folded monomeric intermediates. Unfolding of monomers occurs at 80°C or in the presence of 4.3 M GdmCl, indicating high intrinsic stability toward both GdmCl and elevated temperatures. Both oligomeric and monomeric forms of PIA exhibited affinity to the hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid (ANS) and bind withK d=80 and 130 µM, respectively. Denaturation of the PIA completely abolished affinity to ANS, suggesting that hydrophobicity is a property of the folded state of the porin.

Key words

N. gonorrhoeae porin expression unfolding ANS intermediate state 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Blake, M. S., and Gotschlich, E. S. (1982).Infect. Immun. 36, 277–283.Google Scholar
  2. Butt, N. J., Lambden, P. R., and Heckels, J. E. (1990).Nucleic Acids Res. 18, 4258.CrossRefGoogle Scholar
  3. Carbonetti, N. H., and Sparling, P. F. (1987).Proc. Natl. Acad. Sci. USA 84, 9084–9088.CrossRefGoogle Scholar
  4. Carbonetti, N. H., Simnad, V. I., Seifert, H. S., So, M., and Sparling, P. F. (1988).Proc. Natl. Acad. Sci. USA 85, 6841–6845.CrossRefGoogle Scholar
  5. Cleary, S., Mulkerrin, M. G., and Kelley, R. F. (1989).Biochemistry 28, 1884–1891.CrossRefGoogle Scholar
  6. Cowan, S. W., Schirmer, T., Rummel, G., Steiert, M., Ghosh, R., Pauptit, R. A., Jansonius, J. N., and Rosenbusch, J. P. (1992).Nature 358, 727–733.CrossRefGoogle Scholar
  7. Douglas, J. T., Lee, M. D., and Nikaido, H. (1981).FEMS Microbiol. Lett. 12, 305–309.CrossRefGoogle Scholar
  8. Edelhoch, H. (1967).Biochemistry 6, 1948–1954.CrossRefGoogle Scholar
  9. Ferguson, R. N., and Cahnmann, H. J. (1975).Biochemistry 14, 282–289.CrossRefGoogle Scholar
  10. Gotschlich, E. C., Seiff, M. E., Blake, M. S., and Koomey, M. (1987).Proc. Natl. Acad. Sci. USA 84, 8135–8139.CrossRefGoogle Scholar
  11. Hancock, R. E. W. (1987). InBacterial Outer Membranes as Model Systems, Wiley, New York.Google Scholar
  12. Jaenicke, R. (1987).Prog. Biophys. Mol. Biol. 49, 117–237.CrossRefGoogle Scholar
  13. Jaenicke, R., and Rudolph, R. (1986).Meth. Enzymol. 131, 218–250.CrossRefGoogle Scholar
  14. Judd, R. C. (1989).Clin. Microbiol. Rev. 2, S41-S48.Google Scholar
  15. Kreusch, A., Neubuser, A., Schiltz, E., Weckesser, J., and Schulz, G. E. (1994).Protein Sci. 3, 58–63.CrossRefGoogle Scholar
  16. Lakowicz, J. R. (1983). InPrinciples of Fluorescence Spectroscopy, Plenum Press, New York.Google Scholar
  17. Markovic-Housley, Z., and Garavito, R. M. (1986).Biochim. Biophys. Acta 869, 158–170.Google Scholar
  18. Matsuka, Y. V., Medved, L. V., Brew, S. A., and Ingham, K. C. (1994).J. Biol. Chem. 269, 9539–9546.Google Scholar
  19. Mauro, A., Blake, M., and Labarca, P. (1988).Proc. Natl. Acad. Sci. USA 85, 1071–1075.CrossRefGoogle Scholar
  20. McDade, R. L., and Johnston, K. H. (1980).J. Bacteriol. 141, 1183–1191.Google Scholar
  21. Minetti, C. A. S. A., Tai, J. Y., Blake, M. S., Pullen, J. K., Liang, S., and Remeta, D. P. (1997).J. Biol. Chem. 272, 10710–10720.CrossRefGoogle Scholar
  22. Nikaido, H. (1994).J. Biol. Chem. 269, 3905–3908.Google Scholar
  23. Pace, N. C., Vajdos, F., Fee, L., Grimsley, G., and Gray, T. (1995).Protein Sci. 4, 2411–2423.Google Scholar
  24. Puohiniemi, R., Butcher, S., Tarkka, E., and Sarvas, M. (1991).FEMS Microbiol. Lett. 83, 29–33.CrossRefGoogle Scholar
  25. Qi, H. L., Tai, J. Y., and Blake, M. S. (1994).Infect. Immun. 62, 2432–2439.Google Scholar
  26. Schirmer, T., Keller, T. A., Wang, Y., and Rosenbusch, J. P. (1995).Science 267, 512–514.CrossRefGoogle Scholar
  27. Schmid, B., Kromer, M., and Schulz, G. E. (1996).FEBS Lett. 381, 111–114.CrossRefGoogle Scholar
  28. Schulz, G. E. (1996).Curr. Opin. Struct. Biol. 6, 485–490.CrossRefGoogle Scholar
  29. Seed, B. (1987).Nature 329, 840–842.CrossRefGoogle Scholar
  30. Semisotnov, G. V., Rodionova, N. A., Razgulyaev, O. I., Uversky, V. N., Gripas, A. F., and Gilmanshin, R. I. (1991).Biopolymers 31, 119–128.CrossRefGoogle Scholar
  31. Surrey, T., and Jahnig, F. (1995).J. Biol. Chem. 270, 28199–28203.CrossRefGoogle Scholar
  32. Weiss, M. F., and Schulz. G. E. (1992).J. Mol. Biol. 227, 493–509.CrossRefGoogle Scholar
  33. Wetzler, L. M., Blake, M. S., and Gotschlich, E. C. (1988).J. Exp. Med. 168, 1883–1897.CrossRefGoogle Scholar
  34. Young, J. D., Blake, M., Mauro, A., and Cohn, Z. (1983).Proc. Natl. Acad. Sci. USA 80, 3831–3835.CrossRefGoogle Scholar

Copyright information

© Plenum Publishing Corporation 1998

Authors and Affiliations

  • Yury V. Matsuka
    • 1
  • Deborah A. Dilts
    • 2
  • Susan Hoiseth
    • 2
  • Rasappa Arumugham
    • 1
  1. 1.Department of Protein and Analytical ChemistryWyeth-Lederle Vaccines and PediatricsWest Henrietta
  2. 2.Department of Molecular BiologyWyeth-Lederle Vaccines and PediatricsWest Henrietta

Personalised recommendations