In situ-Synthesized cadmium sulfide quantum dots in pore-forming protein and polysaccharide matrices for optical biosensing applications.

The main limitation for practical implementation of quantum dots-based sensors and biosensors is the possible contamination of sensing media with quantum dots (QDs) moved out from the sensor structure, being critical for living systems measurements. Numerous efforts have addressed the challenge of pre-synthesized QDs incorporation into porous matrix provide, on the one hand, proper fixation of quantum dots in its volume and preserving a free analyte transfer from the sensing media to them - on the other hand. Here, we propose an alternative insight into this problem. Instead of using preliminary synthesized particles for doping a matrix, we have in situ synthesized cadmium sulfide QDs in porous biopolymeric matrices, both in an aqueous solution and on a mica substrate. The proposed technique allows obtaining QDs in a matrix acting simultaneously as a ligand passivating surface defects and preventing QDs aggregation. The conjugates were used as a photoluminescence sensor for the metal ions and glutathione detection in an aqueous media. Different kinds of sensor responses have been found depending on the analyte nature. Zinc ions' presence initiates the intraband QDs emission increases due to the reduction of non-radiative processes. The presence of copper ions, in contrast, leads to a gradual photoluminescence decrease due to the formation of the non-luminescent copper-based alloy in the QDs structure. Finally, the presence of glutathione initiates a ligand exchange process followed by some QDs surface treatment enhancing defect-related photoluminescence. As a result, three different kinds of sensor responses for three analytes allow claiming development of a new selective QD-based sensor suitable for biomedical applications.

An Abnormally High Closing Potential of the OMPF Porin Channel from Yersinia Ruckeri: The Role of Charged Residues and Intramolecular Bonds.

Electrophysiological experiments on bilayer lipid membranes showed that the isolated outer membrane major porin of Yersinia ruckeri (YrOmpF) exhibits activity typical of porins from Gram-negative bacteria, forming channels with a mean conductance of 230 pS (in 0.1 M KCl) and slight asymmetry with respect to the applied voltage. Under acidic conditions (up to pH = 3.0), there was no significant decrease in the total conductance of the YrOmpF channel reconstituted into the bilayer. The studied channel significantly differed from the porins of other bacteria by high values of its critical closing potential (Vc): Vc = 232 mV at pH = 7.0 and Vc = 164 mV at pH = 5.0. A theoretical model of the YrOmpF spatial structure was used for the analysis of the charge distribution in the mouth and inside the channel to explain these properties and quantitatively assess the bonds between the amino acid residues in the L3 loop and on the inner wall of the barrel. The parameters of YrOmpF were compared with those of the classical OmpF porin from E. coli. The results of electrophysiological experiments and theoretical analysis are discussed in terms of the mechanism for voltage-dependent closing of porin channels.

Non-Specific Porins of Yersinia pseudotuberculosis as Inductors of Experimental Hyperthyroidism in Mice.

In vivo experiments showed that antibodies to OmpC and OmpF porins of Yersinia pseudotuberculosis increased thyroxine (T4) level in the blood of experimental animals. The mice were immunized with different antigens: recombinant OmpF porin in a soluble monomeric form, trimers of OmpC and OmpF porins isolated from the outer membrane, or antibodies to them. The level of thyroxine in the blood of mice immunized with OmpF and OmpC porins increased by 5.47 and 22.3 times, respectively; after immunization with antibodies to these proteins, blood thyroxine increased by 9.28 and 14.29 times. Immunization with recombinant OmpF porin induced no reliable increase in thyroxine level. Hence, the serum to recombinant OmpF porin contains no antibodies specific to conformational antigenic determinants that are present in the protein trimer and, according to our previous findings from molecular docking studies, determine cross-reactions between OmpF porin of Y. pseudotuberculosis and thyroidstimulating hormone receptor.

Self-Organization of Recombinant Membrane Porin OmpF from Yersinia pseudotuberculosis in Aqueous Environments.

Recombinant porin OmpF (an integral protein of bacterial outer membrane) from Yersinia pseudotuberculosis was synthesized in Escherichia coli cells as inclusion bodies. By combining the methods of anion-exchange and gel filtration chromatographies, recombinant OmpF (rOmpF) was isolated as an individual protein in its denatured state, and its characteristic properties (molecular mass, N-terminal amino acid sequence, and hydrodynamic radius of the protein in 8 M urea solution) were determined. According to the data of gel filtration, dynamic light scattering, optical spectroscopy, and binding of the hydrophobic fluorescent probe 8-anilino-1-naphthalenesulfonic acid, the rOmpF is fully unfolded in 8 M urea and exists in random coil conformation. In aqueous solutions, rOmpF undergoes conformational changes, reversible self-association, and aggregation. When transferred from 8 M urea into water, PBS (containing 0.15 M NaCl, pH 7.4), or buffer containing 0.8 M urea (pH 8.0), fully unfolded rOmpF forms relatively compact monomeric intermediates prone to self-association with formation of multimers. The oligomeric intermediates have high content of native protein-like secondary structure and pronounced tertiary structure. In acidic media (pH 5.0, close to the protein isoelectric point), rOmpF undergoes rapid irreversible aggregation. Therefore, we found that medium composition significantly affects both porin folding and processes of its self-association and aggregation.

[Study of Yersinia pseudotuberculosis surface antigen epitopes using monoclonal antibodies].

A study of the influence of exogenous factors on the immunochemical activity of the bacterium Yersinia pseudotuberculosis and lipopolysaccharide preparations isolated from bacteria was performed using monoclonal antibodies. It was shown that the hybridomas that were obtained in this work produce antibodies against different and, most likely, species-specific epitopes associated with lipopolysaccharide O side chains. The antibody concentrations produced increased with a decrease in the temperature, at which the bacteria were cultivated. An inhibitory effect of proteinase K, pepsin, and trypsin on the immunochemical activity of bacterial cells, determined using a solid-phase enzyme immunoassay, was demonstrated. Treatment with sodium periodate showed no uniform effect on the reactions between monoclonal antibodies and antigens (lipopolysaccharides and microbial cells), as adjudged by an immunoassay, which is most likely a consequence of the different localization of lipopolysaccharide epitopes recognized by the antibodies from four hybridomas.

Study of effect of substitution of the penultimate amino acid residue on expression, structure, and functional properties of Yersinia pseudotuberculosis OmpY porin.

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.

Mutant OmpF porins of Yersinia pseudotuberculosis with deletions of external loops: structure-functional and immunochemical properties.

Recombinant mutant OmpF porins from Yersinia pseudotuberculosis outer membrane were obtained using site-directed mutagenesis. Here we used four OmpF mutants where single extracellular loops L1, L4, L6, and L8 were deleted one at a time. The proteins were expressed in Escherichia coli at levels comparable to full-sized recombinant OmpF porin and isolated from the inclusion bodies. Purified trimers of the mutant porins were obtained after dialysis and consequent ion-exchange chromatography. Changes in molecular and spatial structure of the mutants obtained were studied using SDS-PAGE and optical spectroscopy (circular dichroism and intrinsic protein fluorescence). Secondary and tertiary structure of the mutant proteins was found to have some features in comparison with that of the full-sized recombinant OmpF. As shown by bilayer lipid membrane technique, the pore-forming activity of purified mutant porins was identical to OmpF porin isolated from the bacterial outer membrane. Lacking of the external loops mentioned above influenced significantly upon the antigenic structure of the porin as demonstrated using ELISA.

OmpC-like porin from outer membrane of Yersinia enterocolitica: molecular structure and functional activity.

OmpC-like porin was isolated from the outer membrane (OM) of Yersinia enterocolitica cultured at 37°C (the "warm" variant) and its physicochemical and functional properties were studied. The amino acid sequence of OmpC porin was established, and the primary structure and transmembrane topology of this protein were analyzed in comparison with the OmpF porin isolated from Y. enterocolitica cultured at 6°C (the "cold" variant). Both porins of Y. enterocolitica had a high homology degree (65%) between themselves and with OmpC and OmpF porins from OM of Escherichia coli (58 and 76% homology, respectively). The secondary structure of OmpC and OmpF porins from OM of Y. enterocolitica consists of 16 ß-strands connected by short "periplasmic" and longer "extracellular" loops with disordered structure, according to the topological model developed for porins of E. coli. The molecular structures of OmpC and OmpF porins of Y. enterocolitica have significant differences in the structure of the "extracellular" loops and in the position of one of three tryptophan residues. Using the bilayer lipid membrane (BLM) technique, pores formed by OmpC porin of Y. enterocolitica were shown to differ in electrophysiological characteristics from channels of OmpF protein of this microorganism. The isolated OmpC porin reconstructed into BLM displayed functional plasticity similarly to OmpF protein and nonspecific porins of other enterobacteria. The conductivity level of the channels formed by this protein in the BLM was regulated by value of the applied potential.

Biogenesis of ß-barrel integral proteins of bacterial outer membrane.

Gram-negative bacteria are enveloped by two membranes, the inner (cytoplasmic) (CM) and the outer (OM). The majority of integral outer membrane proteins are arranged in ß-barrels of cylindrical shape composed of amphipathic antiparallel ß-strands. In bacteria, ß-barrel proteins function as water-filled pores, active transporters, enzymes, receptors, and structural proteins. Proteins of bacterial OM are synthesized in the cytoplasm as unfolded polypeptides with an N-terminal sequence that marks them for transport across the CM. Precursors of membrane proteins move through the aqueous medium of the cytosol and periplasm under the protection of chaperones (SecB, Skp, SurA, and DegP), then cross the CM via the Sec system composed of a polypeptide-conducting channel (SecYEG) and ATPase (SecA), the latter providing the energy for the translocation of the pre-protein. Pre-protein folding and incorporation in the OM require the participation of the Bam-complex, probably without the use of energy. This review summarizes current data on the biogenesis of the ß-barrel proteins of bacterial OM. Data on the structure of the proteins included in the multicomponent system for delivery of the OM proteins to their destination in the cell and on their complexes with partners, including pre-proteins, are presented. Molecular models constructed on the basis of structural, genetic, and biochemical studies that describe the mechanisms of ß-barrel protein assembly by this molecular transport machinery are also considered.

[Yersinia pseudotuberculosis mutant OmpF porins with deletions of the external loops: genetic constructions design, expression, isolation and refolding].

Yersinia pseudotuberculosis outer membrane (OM) recombinant mutant OmpF porins with deletions of the external loops L1, L6 and L8 were obtained using site-directed mutagenesis of the recombinant plasmid including ompF gene. Heterologeous expression of the mutant proteins was carried out in strain Rosetta of Escherichia coli (Novagen, USA), porins with the deletions were isolated from the inclusion bodies. Mutant proteins in oligomeric form were obtained as result of dialysis and ion-exchange chromatography. Spatial structure of the mutant proteins was demonstrated to have special features in comparison with that of the full-structured OmpF porin on the level of both secondary and tertiary structure. Lacking of the loops L1, L6 and L8 didn't affect the conductivity level of Y pseudotuberculosis porin channel as shown using bilayer lipid membrane (BLM) technique. Lacking of the loops mentioned above has a significant influence on the antigenic structure of the mutant porins as demonstrated with use of immunoblotting technique and ELISA.

[Immunogenic and protective properties of nanosized constructs based on tubular immunostimulating complexes and pore forming protein of Yersinia pseudotuberculosis].

AIM: Evaluation of immunogenic and protective properties of constructs based on subunit porin antigen from Yersinia pseudotuberculosis, immunostimulating complexes (ISCOM) and tubular immunostimulating (TI) complexes. MATERIALS AND METHODS: Porin antibodies and blood serum cytokines were determined by using EIA. Porin-specific cell immunity was evaluated by DTH reaction inflammation index. Protective activity of porin formulations was determined by measuring specific gravity of animals surviving Yersinia pseudotuberculosis lethal challenge. RESULTS: Porin in TI complexes develops higher immunogenicity when compared with individual protein or protein with complete Freunds adjuvant. Porin in TI complexes develops higher protective activity, inhibits interferon synthesis in mice. Incorporation of porin into TI complexes results in neutralization of porin suppressive activity against DTH mechanisms and interferon system. CONCLUSION: TI complexes may be used as perspective carriers for bacterial antigens. TI complexes have adjuvant properties and can provide protective properties to porin vaccine constructs.

A novel OmpY porin from Yersinia pseudotuberculosis: structure, channel-forming activity and trimer thermal stability.

A novel OmpY porin was predicted based on the Yersinia pseudotuberculosis genome analysis. Whereas it has the different genomic annotation such as "outer membrane protein N" (ABS46310.1) in str. IP 31758 or "outer membrane protein C2, porin" (YP_070481.1) in str. IP32953, it might be warranted to rename the OmpN/OmpC2 to OmpY, "outer membrane protein Y", where letter "Y" pertained to Yersinia. Both phylogenetic analysis and genomic localization clearly support that the OmpY porin belongs to a new group of general bacterial porins. The recombinant OmpY protein with its signal sequence was overexpressed in porin-deficient Escherichia coli strain. The mature rOmpY was shown to insert into outer membrane as a trimer. The OmpY porin, isolated from the outer membrane, was studied employing spectroscopic, electrophoretic and bilayer lipid membranes techniques. The far UV CD spectrum of rOmpY was essentially identical to that of Y. pseudotuberculosis OmpF. The near UV CD spectrum of rOmpY was weaker and smoother than that of OmpF. The rOmpY single-channel conductance was 180 ± 20 pS in 0.1 M NaCl and was lower than that of the OmpF porin. As was shown by electrophoretic and bilayer lipid membrane experiments, the rOmpY trimers were less thermostable than the OmpF trimers. The porins differed in the trimer-monomer transition temperature by about 20°C. The three-dimensional structural models of the Y. pseudotuberculosis OmpY and OmpF trimers were generated and the intra- and intermonomeric interactions stabilizing the porins were investigated. The difference in the thermal stability of OmpY and OmpF trimers was established to correlate with the difference in intermonomeric polar contacts.

[Immunochemical characteristics of synthetic peptides incorporating T- and B-cell epitopes nonspecific porins of pathogenic Yersinia].

Multiple antigenic peptides (MAPs), a sequence which include common antigenic epitopes of outer membrane porins (OM) bacteria of the genus Yersinia (Y. pseudotuberculosis, Y. enterocolitica, Y. pestis), pathogenic for humans have been synthesized. After immunization of BALB/c mice the antiserum to the peptide have been obtained. With the help of ELISA we showed that these sera interact with porins isolated from OM pathogenic Yersinia, and MAP interact with antibodies in sera from rabbits immunized with individual porins, and with antibodies in sera of patients with intestinal yersiniosis and pseudotuberculosis.

Immunogenic characteristics of recombinant OMPF-like porin from Yersinia Pseudotuberculosis outer membrane.

We studied the capacity of outer membrane pore-forming recombinant protein from Yersinia pseudotuberculosis to initiate the development of immune response in CBA mice. Immunization with the recombinant protein induces the production of IgG antibodies with and without adjuvants. High-avidity immune serum was obtained as a result of immunization. Bactericidal activity of peritoneal macrophages from mice immunized with recombinant protein was significantly higher than that of intact mouse macrophages. The use of recombinant porin instead of native porin as the antigen in enzyme immunoassay system for the diagnosis of acute and secondary focal pseudotuberculosis does not reduce the efficiency of detection of specific antibodies in the sera of patients.

[Isolation and characterization of recombinant OmpF-like porin from the Yersinia pseudotuberculosis outer membrane].

The encoding sequence of the pore-forming OmpF-like protein from the Yersinia pseudotuberculosis outer membrane was cloned and expressed in Escherichia coli cells. Conditions were selected for isolation and refolding of recombinant monomer and porin trimer. Their spatial structures were characterized by the intrinsic protein fluorescence and CD spectroscopy. It was shown that the recombinant porins are similar in the composition of secondary structure elements to the isolated porins, but have a considerably less compact tertiary structure. The pore-forming activities of the recombinant proteins are similar to those of Y. pseudotuberculosis native porins. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.

Influence of cultivation conditions on spatial structure and functional activity of OmpF-like porin from outer membrane of Yersinia pseudotuberculosis.

The influence of cultivation conditions of pseudotuberculosis bacteria on the spatial structure and the functional activity of nonspecific OmpF-like porin was studied by means of optical spectroscopy, scanning microcalorimetry, and bilayer lipid membrane technique. With this goal, porin samples isolated from microbial masses grown at different temperatures, nutrient medium densities, and growth phases were characterized. According to CD data, the porin samples under investigation represent beta-sheet proteins. It was found that the protein isolated from the colonial culture of pseudotuberculosis bacteria grown at low temperature has the most compact structure. Using intrinsic protein fluorescence, it was shown that different conditions of pseudotuberculosis bacteria cultivation (temperature, medium, growth phase) led to the changes in spectral properties of porin fluorescence due to the redistribution of the contributions of tyrosine and different classes of tryptophan residues to the total protein emission. Heat inactivation of porin samples was studied using CD spectroscopy, intrinsic protein fluorescence, and scanning microcalorimetry. Spatial features of the porin samples were found to affect their functional activities. Considering all these data, it is possible to correlate the spatial structure and functional activity of porin samples isolated under different cultivation conditions of bacteria and the composition of the outer membrane lipid matrix.

[Immunostimulatory characteristics of a novel carrier on the basis of cucumarioside A2-2 and monogalactosyldiacylgycerol].

A novel antigen carrier has been formulated on the basis of a cucumarioside-A2-2 triterpene glycoside (CD) complex with cholesterol and monogalactosyldiacylglycerol from Ahnfeltia tobuchiensis (MGDG(At)) and Ulva fenestrate (MGDG(Uf)). Morphological and immunostimulative characteristics of the carrier were studied. Electron microscopy experiments demonstrated the formation of homogeneous tubular structures in a mixture of CD, cholesterol, and MGDG in molar ratio of 1:2:3. In animals immunized by the carrier bearing pore forming protein monomer of pseudotuberculosis agent CD and MGDG synergically affected synthesis of specific antibodies, interleukin-2, and gamma-interferon and delayed hypersensitivity reaction when compared to Freund's complete adjuvant or to immunostimulatory complexes between Quillaja saponaria saponins and phosphatidylcholine from egg yolk. The immunostimulatory effect depends upon the composition of polyunsaturated fatty acids of MGDG. The new tubular adjuvant carrier is a competitive adjuvant, as it includes CD obtained from far-eastern sea cucumber commercial species Cucumaria japonica, and MGDG from seaweed.

[Structure and function of pore-forming proteins from bacteria of the genus Yersinia: I. Isolation and a comparison of physicochemical properties and functional activity of Yersinia porins].

The molecular organization and functional activity of porins isolated from the outer membrane (OM) of the Yersinia enterocolitica and three phylogenetically close nonpathogenic Yersinia species (Y. intermedia, Y. kristensenii, and Y. frederiksenii) cultured at 6-8 degrees C were comparatively studied for the first time. The proteins were isolated in two molecular forms (trimeric and monomeric), and their spatial structures were characterized by the methods of optical spectroscopy, CD and intrinsic protein fluorescence. The studied porins were shown to belong to the beta-structural proteins (they have 59-96% total beta structures and 0-17% alpha helices). The spatial structures of the proteins were demonstrated to depend on the nature of the detergent used for solubilization. Unlike the enterobacterial pore-forming proteins, the porin trimers are less stable to sodium dodecyl sulfate (SDS). The spatial structures of the porins become more compact after the substitution of octyl beta-D-glucoside for SDS: the content of beta structures increases and the accessibility of Trp residues to solvent decreases. It was established with the use of the technique of bilayer lipid membranes that the functional properties of the porins are similar to those of the OmpF proteins of Gram-negative bacteria. Trimers are functionally active forms of the porins. Special features of the pore-forming activity of the Yersinia porins were revealed to depend on the microorganism species and the value of the membrane potential.

Molecular characteristics of OmpF-like porins from pathogenic Yersinia.

Nonspecific pore-forming proteins (porins) are the major proteins of the outer membrane of Gram-negative bacteria responsible for diffusion of low-molecular-weight compounds. Nucleotide sequences of the OmpF-like porins from the pathogenic bacteria Yersinia pseudotuberculosis (YPS) and Yersinia enterocolitica (YE) were cloned and determined. Values of molecular weights (MW) and isoelectric points (IEP) calculated for these proteins (for OmpF-YPS: MW 37.7 kD, IEP 4.45; for OmpF-YE: MW 39.5 kD, IEP 4.34) are in good agreement with experimental data. The OmpF-like Yersinia porins are highly homologous to each other (83-92%) and also to the OmpF protein from Serratia marcescens (70%); the homology to the OmpF porin from E. coli is significantly lower (52-58%). Multiple alignment of the amino acid sequences of mature OmpF proteins provided the distribution of conservative amino acid residues typical for porins. Moreover, the OmpF-like porins from Yersinia are characterized by the presence of extended regions with high and low homologies, which coincide with the transmembrane domains and "external" loops, respectively, of the topological model of the OmpF porin from E. coli. By predictive methods, the secondary structure of the OmpF-like porins from Yersinia was obtained. This structure is represented by 16 beta-strands connected by short "periplasmic" and longer "external" loops with unordered structure.