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.
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References
Blake, M. S., and Gotschlich, E. S. (1982).Infect. Immun. 36, 277–283.
Butt, N. J., Lambden, P. R., and Heckels, J. E. (1990).Nucleic Acids Res. 18, 4258.
Carbonetti, N. H., and Sparling, P. F. (1987).Proc. Natl. Acad. Sci. USA 84, 9084–9088.
Carbonetti, N. H., Simnad, V. I., Seifert, H. S., So, M., and Sparling, P. F. (1988).Proc. Natl. Acad. Sci. USA 85, 6841–6845.
Cleary, S., Mulkerrin, M. G., and Kelley, R. F. (1989).Biochemistry 28, 1884–1891.
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.
Douglas, J. T., Lee, M. D., and Nikaido, H. (1981).FEMS Microbiol. Lett. 12, 305–309.
Edelhoch, H. (1967).Biochemistry 6, 1948–1954.
Ferguson, R. N., and Cahnmann, H. J. (1975).Biochemistry 14, 282–289.
Gotschlich, E. C., Seiff, M. E., Blake, M. S., and Koomey, M. (1987).Proc. Natl. Acad. Sci. USA 84, 8135–8139.
Hancock, R. E. W. (1987). InBacterial Outer Membranes as Model Systems, Wiley, New York.
Jaenicke, R. (1987).Prog. Biophys. Mol. Biol. 49, 117–237.
Jaenicke, R., and Rudolph, R. (1986).Meth. Enzymol. 131, 218–250.
Judd, R. C. (1989).Clin. Microbiol. Rev. 2, S41-S48.
Kreusch, A., Neubuser, A., Schiltz, E., Weckesser, J., and Schulz, G. E. (1994).Protein Sci. 3, 58–63.
Lakowicz, J. R. (1983). InPrinciples of Fluorescence Spectroscopy, Plenum Press, New York.
Markovic-Housley, Z., and Garavito, R. M. (1986).Biochim. Biophys. Acta 869, 158–170.
Matsuka, Y. V., Medved, L. V., Brew, S. A., and Ingham, K. C. (1994).J. Biol. Chem. 269, 9539–9546.
Mauro, A., Blake, M., and Labarca, P. (1988).Proc. Natl. Acad. Sci. USA 85, 1071–1075.
McDade, R. L., and Johnston, K. H. (1980).J. Bacteriol. 141, 1183–1191.
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.
Nikaido, H. (1994).J. Biol. Chem. 269, 3905–3908.
Pace, N. C., Vajdos, F., Fee, L., Grimsley, G., and Gray, T. (1995).Protein Sci. 4, 2411–2423.
Puohiniemi, R., Butcher, S., Tarkka, E., and Sarvas, M. (1991).FEMS Microbiol. Lett. 83, 29–33.
Qi, H. L., Tai, J. Y., and Blake, M. S. (1994).Infect. Immun. 62, 2432–2439.
Schirmer, T., Keller, T. A., Wang, Y., and Rosenbusch, J. P. (1995).Science 267, 512–514.
Schmid, B., Kromer, M., and Schulz, G. E. (1996).FEBS Lett. 381, 111–114.
Schulz, G. E. (1996).Curr. Opin. Struct. Biol. 6, 485–490.
Seed, B. (1987).Nature 329, 840–842.
Semisotnov, G. V., Rodionova, N. A., Razgulyaev, O. I., Uversky, V. N., Gripas, A. F., and Gilmanshin, R. I. (1991).Biopolymers 31, 119–128.
Surrey, T., and Jahnig, F. (1995).J. Biol. Chem. 270, 28199–28203.
Weiss, M. F., and Schulz. G. E. (1992).J. Mol. Biol. 227, 493–509.
Wetzler, L. M., Blake, M. S., and Gotschlich, E. C. (1988).J. Exp. Med. 168, 1883–1897.
Young, J. D., Blake, M., Mauro, A., and Cohn, Z. (1983).Proc. Natl. Acad. Sci. USA 80, 3831–3835.
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Matsuka, Y.V., Dilts, D.A., Hoiseth, S. et al. Characterization of a subunit structure and stability of the recombinant porin fromNeisseria gonorrhoeae. J Protein Chem 17, 719–728 (1998). https://doi.org/10.1007/BF02780975
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DOI: https://doi.org/10.1007/BF02780975