N. Yu. Kim

Isolation and characterization of a low-molecular-weight immunoglobulin-binding protein from Yersinia pseudotuberculosis.

A low-molecular-weight immunoglobulin-binding protein (IBP) bound with the cell envelope has been isolated from Yersinia pseudotuberculosis cells and partially characterized. This IBP is a hydrophilic protein with a high polarity index of 55.3%. The molecular weight of the protein has been determined by MALDI-TOF mass spectrometry as 14.3 kD. CD spectroscopy showed that the IBP has high contents of the β-structure and random coil structure. The IBP contains glycine as the N-terminal amino acid. The protein can be stored for a long time at acidic pH values but aggregates and loses activity at alkaline and neutral pH. The IBP binds rabbit IgG with optimum at pH of 6.0–7.5. The IBP interacts with IgG molecule in the Fc-fragment region. The protein retains activity after heating at 100°C in the presence of SDS.

[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 for isolation and refolding of recombinant monomer and porin trimer were selected. Their spatial structures were characterized by the intrinsic protein fluorescence and CD spectroscopy. It was shown that recombinant porins are similar in the composition of secondary structure elements to 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.

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 β-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.

OmpC-like porin from Yersinia pseudotuberculosis: Molecular characteristics, physico-chemical and functional properties

Pore-forming protein from the outer membrane of Yersinia pseudotuberculosis cultured at 37°C has been isolated and characterized. Comparative analysis of the primary and three-dimensional structures of this protein and of OmpC porin from E. coli was carried out, functional properties of these proteins have been studied using bilayer lipid membranes (BLM) technique. The degree of homology, molecular mass and pore-forming properties of the isolated porin was found to be closer to those of OmpC porin from E. coli than OmpF porin from Y. pseudotuberculosis. The value of the most probable conductivity of OmpC porin from Y. pseudotuberculosis (0.18 pS) in BLM corresponded to the conductivity of the native trimer of this protein. Using CD spectroscopy, the porins investigated were shown to belong to the β-structured proteins. Data of the primary structure and intrinsic protein fluorescence revealed essential differences in localization and microenvironment of tryptophan residues in the porins investigated. Participation of external loops L2 and L6 in the formation of the antigenic structure of OmpC porin from Y. pseudotuberculosis was demonstrated. On the basis of crystal structure of osmoporin from Klebsiella pneumoniae, three-dimensional models of the monomer and trimer of the Y. pseudotuberculosis porin were obtained. Using Web server AGGRESCAN, the localization of protein structure sites with the increased aggregation capability (hot spots) has been deter-mined. It turned out that some of these zones localize in the region of intramonomeric contacts in the porin trimer; however, a large part of them is located on the external surface of the β-barrel. The process of thermal denaturation has been studied and the melting points of the porins were determined. It was found that significant changes in the microenvironment of the indole fluorophores (especially tryptophan residues of spectral class I) took place in the process of the thermodenaturation of the proteins. These changes preceded the irreversible conformational transition observed for the E. coli porin at 77°C and for the Y. pseudotuberculosis porin at 70°C.

Recombinant phospholipase A1 of the outer membrane of psychrotrophic Yersinia pseudotuberculosis: Expression, purification, and characterization

The pldA gene encoding membrane-bound phospholipase A1 of Yersinia pseudotuberculosis was cloned and expressed in Escherichia coli cells. Recombinant phospholipase A1 (rPldA) was isolated from inclusion bodies dissolved in 8 M urea by two-stage chromatography (ion-exchange and gel-filtration chromatography) as an inactive monomer. The molecular mass of the rPldA determined by MALDI-TOF MS was 31.7 ± 0.4 kDa. The highly purified rPldA was refolded by 10-fold dilution with buffer containing 10 mM Triton X-100 and subsequent incubation at room temperature for 16 h. The refolded rPldA hydrolyzed 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine in the presence of calcium ions. The enzyme exhibited maximal activity at 37°C and nearly 40% of maximal activity at 15°C. The phospholipase A1 was active over a wide range of pH from 4 to 11, exhibiting maximal activity at pH 10. Spatial structure models of the monomer and the dimer of Y. pseudotuberculosis phospholipase A1 were constructed, and functionally important amino acid residues of the enzyme were determined. Structural differences between phospholipases A1 from Y. pseudotuberculosis and E. coli, which can affect the functional activity of the enzyme, were revealed.

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.

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. Oligomers of mutant porins were obtained as result of dialysis and ion-exchange chromatography. Spatial structure of the mutant proteins was found to have special features in comparison with that of the full-structured OmpF porin on the level of both secondary and tertiary structure. As shown using bilayer lipid membrane (BLM) technique the absence of the loops L1, L6 and L8 didn’t affect the conductivity level of Y. pseudotuberculosis porin channel. The absence of the loops mentioned above has a significant influence on the antigenic structure of the mutant porins as demonstrated using immunoblotting technique and ELISA.

Isolation and characterization of OmpF-like porin from Yersinia ruckeri

The polypeptide profile of the porin protein fraction of Yersinia ruckeri, a Gram-negative bacterium causing yersiniosis in fish, has been shown to depend on cultivation temperature. OmpF-like porins are expressed mainly in the outer membrane (OM) of the “cold” variant (4°C) of the microorganism and OmpC-like proteins are expressed in the OM of the “warm” variant (37°C). Both types of porins are present in the OM of Y. ruckeri at room temperature. The OmpF-like porin of the “cold” variant was isolated and characterized. The molecular weight and primary structure of the protein were determined. The methods of optical spectroscopy (circular dichroism and intrinsic protein fluorescence) have shown that the protein has a spatial structure typical of β-structured porins from the OM of Gram-negative bacteria. The functional activity of isolated protein was characterized by the bilayer lipid membrane (BLM) technique. The most probable level of channel conductivity was 320 ± 60 pS, corresponding to the channel conductivity of OmpF porins of the genus Yersinia. The distinctive feature of OmpF porin from Y. ruckeri is high thermostability of its functionally active conformation: the protein forms stable pores in the BLM even after heating to 85°C.

Effects of pH on structural and functional properties of porin from the outer membrane of Yersinia pseudotuberculosis. II. Characterization of pH-induced conformational intermediates of yersinin

AbstractpH-Induced intermediates of Omp F-like porin from the outer membrane of Yersinia pseudotuberculosis (yersinin) were characterized by fluorescence and fluorescent probe spectroscopy and circular dichroism. The most dramatic changes in the intrinsic fluorescence of the protein induced by pH titration correlated with different conformational states of the porin molecule. pH-induced conformational transitions of yersinin can be described in terms of a three-state model: (1) disordering of porin associates and formation of porin trimers structurally similar to the native protein; (2) unfolding of individual porin domains followed by cooperative dissociation of trimers into monomers; (3) formation of two loosely structured forms of monomer intermediates. It is assumed that one of these monomeric forms (at pH 3.0) corresponds to the molten-globule state of porin with native secondary structure, while the other one (at 2.0) represents a partly denatured (misfolded) monomer, which retains no more than 50% of the regular secondary structure. The putative mechanism of low pH-induced β-barrel unfolding is discussed in terms of a theoretical model of yersinin spatial structure.

[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°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 β-structural proteins (they have 59–96% total β structures and 0–17% α 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 β-D-glucoside for SDS: the content of β 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.