Characterization of a subunit structure and stability of the recombinant porin fromNeisseria gonorrhoeae
- 41 Downloads
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 wordsN. gonorrhoeae porin expression unfolding ANS intermediate state
Unable to display preview. Download preview PDF.
- Blake, M. S., and Gotschlich, E. S. (1982).Infect. Immun. 36, 277–283.Google Scholar
- Hancock, R. E. W. (1987). InBacterial Outer Membranes as Model Systems, Wiley, New York.Google Scholar
- Judd, R. C. (1989).Clin. Microbiol. Rev. 2, S41-S48.Google Scholar
- Lakowicz, J. R. (1983). InPrinciples of Fluorescence Spectroscopy, Plenum Press, New York.Google Scholar
- Markovic-Housley, Z., and Garavito, R. M. (1986).Biochim. Biophys. Acta 869, 158–170.Google Scholar
- Matsuka, Y. V., Medved, L. V., Brew, S. A., and Ingham, K. C. (1994).J. Biol. Chem. 269, 9539–9546.Google Scholar
- McDade, R. L., and Johnston, K. H. (1980).J. Bacteriol. 141, 1183–1191.Google Scholar
- Nikaido, H. (1994).J. Biol. Chem. 269, 3905–3908.Google Scholar
- Pace, N. C., Vajdos, F., Fee, L., Grimsley, G., and Gray, T. (1995).Protein Sci. 4, 2411–2423.Google Scholar
- Qi, H. L., Tai, J. Y., and Blake, M. S. (1994).Infect. Immun. 62, 2432–2439.Google Scholar