title: Study of effect of substitution of the penultimate amino acid residue on expression, structure, and functional properties of Yersinia pseudotuberculosis OmpY porin.
abstract: 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.
authors.affiliation: Elyakov Pacific Institute of Bioorganic Chemistry, Far East Branch of the Russian Academy of Sciences, Vladivostok, 690022, Russia. soltaf@mail.ru.
authors.forename: T F
authors.initials: TF
authors.lastname: Solov'eva
authors.forename: N M
authors.initials: NM
authors.lastname: Tischenko
authors.forename: V A
authors.initials: VA
authors.lastname: Khomenko
authors.forename: O Y
authors.initials: OY
authors.lastname: Portnyagina
authors.forename: N Y
authors.initials: NY
authors.lastname: Kim
authors.forename: G N
authors.initials: GN
authors.lastname: Likhatskaya
authors.forename: O D
authors.initials: OD
authors.lastname: Novikova
authors.forename: M P
authors.initials: MP
authors.lastname: Isaeva
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D005260:Female; D015870:Gene Expression; D057927:Hydrophobic and Hydrophilic Interactions; D007106:Immune Sera / Q000737:chemistry; D008808:Mice, Inbred CBA; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018272:Porins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D017433:Protein Structure, Secondary; D015011:Yersinia pseudotuberculosis
chemical_list: D001426:Bacterial Proteins; D007106:Immune Sera; D018272:Porins