Study of effect of substitution of the penultimate amino acid residue on expression, structure, and functional properties of Yersinia pseudotuberculosis OmpY porin
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The purpose of the study was to compare the expression of two Yersinia pseudotuberculosis proteins, wild-type porin OmpY and the mutant porin OmpY designated as OmpY-Q having the uncharged amino acid residue Gln instead of positively charged Arg at the penultimate position in the same heterologous host. According to the literature, a similar substitution (Lys to Gln) of the penultimate amino acid residue in Neisseria meningitidis porin PorA drastically improved the assembly of the protein in the E. coli outer membrane in vivo. Site-directed mutagenesis was used to replace Arg by Gln (R338Q) in OmpY, and the conditions for optimal expression and maturation of OmpY-Q were selected. It was found that the growth rates of E. coli strains producing OmpY and OmpY-Q and the expression levels of the porins were approximately equal. Comparative analysis of recombinant OmpY and OmpY-Q did not show significant differences in structure, antigenic, and functional properties of the porins, or any noticeable effect of the R338Q substitution in OmpY on its assembly in the E. coli outer membrane in vivo. The probable causes of discrepancies between our results and the previous data on porin PorA are discussed considering the known mechanisms of biogenesis of porins at the periplasmic stage.
Key wordsYersinia pseudotuberculosis outer membrane pore-forming proteins site-directed mutagenesis biogenesis of porins spatial structure computer modeling
bilayer lipid membrane
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- 5.Solov’eva, T. F., Likhatskaya, G. N., Khomenko, V. A., Stenkova, A. M., Kim, N. Yu., Portnyagina, O. Yu., Novikova, O. D., Trifonov, E. V., Nurminski, E. A., and Isaeva, M. P. (2011) A novel OmpY porin from Yersinia pseudotuberculosis: structure, channel-forming activity and trimer thermal stability, J. Biomol. Struct. Dyn., 28, 517–533.PubMedCrossRefGoogle Scholar
- 7.Sambrook, J., and Russel, D. W. (2001) in Molecular Cloning, Laboratory Manual, 3rd Edn., Cold Spring Harbor Laboratory Press, N. Y., pp. 16.33–16.36.Google Scholar
- 15.Likhatskaya, G. N., Novikova, O. D., Solov’eva, T. F., and Ovodov, Yu. S. (1985) Isolation of pore-forming protein from the outer membrane of Yersinia pseudotuberculosis and the study of its effect on conductivity of bilayer lipid membranes, Biol. Membr., 2, 1219–1224.Google Scholar
- 18.Novikova, O. D., Fedoreeva, L. I., Khomenko, V. A., Portnyagina, O. Yu., Ermak, I. M., Likhatskaya, G. N., Moroz, S. I., Solov’eva, T. F., and Ovodov, Yu. S. (1993) The effect of the method of extraction of the pore-forming protein from Yersinia pseudotuberculosis on its macromolecular organization, Bioorg. Khim., 19, 536–547.Google Scholar
- 23.Serdyuk, I., Zaccai, N., and Zaccai, J. (2010) in Methods in Molecular Biophysics: Structure, Dynamics, Function, Vol. 2 (Serdyuk, I. N., ed.) KDU Publishers, Moscow, pp. 503–505.Google Scholar
- 29.Klose, V., Schwarz, H., MacIntyre, S., Freudl, R., Eschbach, M.-L., and Henning, U. (1988) Internal deletions in the gene for an Escherichia coli outer membrane protein define an area possibly important for recognition of the outer membrane by this polypeptide, J. Biol. Chem., 263, 13291–13296.PubMedGoogle Scholar