Vladimir Zav'Yalov - Academia.edu (original) (raw)
Papers by Vladimir Zav'Yalov
The rapid emergence of treatment-resistant bacterial pathogens has become a major threat to publi... more The rapid emergence of treatment-resistant bacterial pathogens has become a major threat to public health. The recent outbreak of new Shiga-toxin–producing Escherichia coli o104H4 infection in Germany illustrates this problem. To colonize host tissues, pathogenic bacteria express surface adhesive organelles. The German strain uses aggregative adherence fimbriae I (aaF/I) to anchor to the intestinal mucosa and induce inflammation. aaF/I belong to the family of chaperone/usher assembled fimbrial polyadhesins. Polyadhesins are functioning as an armory for penetration through the host immune shield. The polyadhesin-binding to the target cells is orchestrated with injectisome (third secretion system), which is extremally important for bacterial virulency, and triggers subversive signals by aggregation of host cell receptors that al-low pathogens to mislead and evade immune defense. Polyadhesins also are involved in biofilm formation making bacteria more resistant to immune re-sponse. Bec...
International Immunopharmacology
Doklady Akademii nauk SSSR
Bioorganicheskaia khimiia
The synthetic peptide SLTCLVKGFY, corresponding to the 364-373 amino acid sequence of the human I... more The synthetic peptide SLTCLVKGFY, corresponding to the 364-373 amino acid sequence of the human IgG heavy chain (Immunorphin), was found to compete with [125I] beta-endorphin for binding by high-affinity receptors on T lymphocytes isolated from the blood of healthy donors (Ki 0.6 nM). The fragments 3-10, 4-10, 5-10, and 6-10 of Immunorphin also inhibited the binding (Ki 2.2, 3.4, 8.0, and 15 nM, respectively). Specificity of these receptors was studied: they turned out to be insensitive to naloxone and, therefore, are not opioid. The Kd values of the specific binding of 125I-labeled Immunorphin and its 6-10 fragment to the receptor were found to be 7.4 and 36.3 nM, respectively.
Journal of chromatography, Jan 10, 1989
The use of pH gradients in borate buffer with a polyhydroxy compound for separation of proteins i... more The use of pH gradients in borate buffer with a polyhydroxy compound for separation of proteins in a free-flow electrophoretic apparatus is outlined. The composition of the pH gradients, the free-flow apparatus design and its operation, and the main causes of failures in the protein separation are considered.
Bioorganicheskaia khimiia
Consensus sequence 30-36 LKDRHDF of human alpha-interferons is inserted into the C- and N-termina... more Consensus sequence 30-36 LKDRHDF of human alpha-interferons is inserted into the C- and N-terminal sequences of the artificial protein albeferon using genetic engineering methods in order to obtain artificial proteins with antiviral activity. Albeferon, obtained by Dolgikh et al. (Protein Eng., 1996, vol. 9, pp. 195-201), already contains the IFN-alpha 2 130-137 fragment and possesses antiproliferative activity comparable with that of IFN-alpha 2. According to CD spectroscopy, both proteins have regular secondary structures similar to that of the precursor protein. They exhibit antiviral activities, and the activity of one of them is comparable with that of IFN-alpha 2. At the same time, their cytotoxic properties are displayed only at relatively high concentrations, which substantially exceed the minimal antiviral concentrations.
Immunology letters, Jan 3, 2002
It was found that the human (hu) myeloma IgG1 Ser, its Fcgamma fragment and the chimeric mouse-hu... more It was found that the human (hu) myeloma IgG1 Ser, its Fcgamma fragment and the chimeric mouse-human monoclonal antibody (chim-mAb), containing the constant part of hu-gamma1-chain, can exist in a long-term metastable conformational state. This state arises as a result of short incubation of IgG molecules and their Fcgamma fragments at pH<2.8 and the consequent rapid neutralisation to pH 7.0-8.0. At pH<2.8 the three-dimensional structure of C(gamma)2 domains is unfolded, but rapidly refolds after neutralisation. At the same time, non-covalent interactions between C(gamma)2 and C(gamma)3 domains are restored very slowly. A metastable state of IgG keeps 70% of complement-binding ability in comparison with the native state.
Peptides, 2001
The synthetic decapeptide H-SLTCLVKGFY-OH (termed immunorphin) corresponding to the sequence 364-... more The synthetic decapeptide H-SLTCLVKGFY-OH (termed immunorphin) corresponding to the sequence 364-373 of the CH3 domain of human immunoglobulin G heavy chain was found to compete with [125I]beta-endorphin for high-affinity receptors on T lymphocytes from the blood of healthy donors (K(i) = 0.6 nM). Besides immunorphin, its synthetic fragments H-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 15 nM), H-Leu-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 8.0 nM), H-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 3.4 nM), H-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 2.2 nM), H-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 1.0 nM) possessed the ability to inhibit specific binding of [125I]beta-endorphin to T lymphocytes. Tests of the specificity of the receptors revealed that they are not sensitive to naloxone and Met-enkephalin, i.e. they are not opioid receptors. K(d) values characterizing the specific binding of 125I- labeled immunorphin and its fragment H-Val-Lys-Gly-Phe-Tyr-OH to the receptors have been determined to be 7.4...
Biofizika
Conformational and dynamic properties of the synthetic peptides, amino acid sequence of which cor... more Conformational and dynamic properties of the synthetic peptides, amino acid sequence of which correspond to biologically active fragments of the following human cytokines: alpha 2, gamma interferons and interleukins 1 beta, 2, were studied in aqueous solution and dimethylsulfoxide by one-dimensional and two-dimensional 1H-NMR spectroscopy (400 MHz). The analysis of nuclear Overhauser effect data, values of vicinal J3(NH-C alpha H) coupling constants of amide protons and their temperature coefficients of chemical shifts indicate an unordered flexible conformation of the peptides in aqueous solutions. The structural and dynamic features of the peptides investigated are discussed together with their biological activity.
Molekuliarnaia biologiia
Using the methods of difference adiabatic scanning microcalorimetry and difference thermal pertur... more Using the methods of difference adiabatic scanning microcalorimetry and difference thermal perturbation spectrophotometry it was established that both the Fc subunits within the intact immunoglobulins G of different subclasses, and the Fc fragments corresponding to them significantly differ in conformational properties. These differences are associated with a different energy of interactions between the CH2 and CH3 domains and also with a different rigidity of structure of the N- and C-terminal parts of the CH2 domains. The analysis of these data allowed to suggest that the "hinge region" interacts with the CH2 domains and the difference in the structure of this region affect the conformation of the Fc subunits. Possible models of the "hinge regions" in immunoglobulins G of the first, second and fourth subclasses, and also their arrangement relative to the CH2 domains were proposed. The analysis of curves of the small-angle scattering of X-rays shows an agreement...
Ukrainskiĭ biokhimicheskiĭ zhurnal
Melting of protein A from Staphylococcus aureus has been studied in neutral medium by the methods... more Melting of protein A from Staphylococcus aureus has been studied in neutral medium by the methods of microcalorimetry and circular dichroism. The melting process of protein A is shown to consist of, at least, 5 independent transitions. The transition with the heat absorption maximum at 38 degrees is ascribed to the melting of the C-terminal domain of protein A. The analysis of amino acid sequences has shown the existence of structural basis for differences in heat stability of the Fc-binding domains of protein A.
Biochimica et biophysica acta, Jan 23, 1977
The conformational changes of antibody structure induced by hapten molecule binding were investig... more The conformational changes of antibody structure induced by hapten molecule binding were investigated by means of thermal perturbation difference spectroscopy. The studies of the free rabit anti-dinitrophenyl antibodies show the conformational transition at temperatures between 25 and 35 degrees C. The changes occurring at the higher temperature are accompanied by the screening of the significant part of exposed tyrosine residues. Binding of the hapten molecules induces a similar transition to that which occurs between the two temperature dependent states of the free antibody. In contrast to our previous results with anti-dansyl rabbit antibodies the dinitrophenyl lysine stabilizes the "low temperature" native state of the protein. The investigation of the MOPC-315 mouse immunoglobulin A myeloma protein possessing anti-dinitrophenyl activity indicates no conformational transition at temperatures between 25 and 35 degrees C and only a small decrease of tyrosine exposure ind...
Biochimica et biophysica acta, Jan 28, 1977
1. The temperature function of the myeloma IgG(K) IVA, Bence-Jones protein (K-type) IVA and its f... more 1. The temperature function of the myeloma IgG(K) IVA, Bence-Jones protein (K-type) IVA and its fragments (Fab(t), Fc'(t), VL and CL) was studied by thermal perturbation difference spectroscopy and circular dichroism. 2. The IgG and Bence-Jones protein studied were found to be capable of a fully reversible structural changes at temperatures between 25 and 35 degrees C. The changes occurring at the higher temperature are accompanied by the screening of the significant part of exposed tyrosine residues. The transition is not accompanied by an appreciable change in the main IgG secondary structure-beta-pleated sheet, according to the CD data. 3. It was found that the temperature-dependent changes of IgG occur in its Fab fragments, the changes of Bence-Jones protein occur in its variable part (VL domains). 4. The temperature changes in the interval 25-35 degrees C are explained by the flexibility of amino acid side chains composed hypervariable loops delineated the "sides"...
Peptides, 2002
The synthetic decapeptide Ser-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr (termed immunorphin) correspond... more The synthetic decapeptide Ser-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr (termed immunorphin) corresponding to the sequence 364-373 of the CH3 domain of human immunoglobulin G heavy chain and its synthetic fragment VKGFY were found to compete with 125 I-labeled -endorphin for high-affinity naloxone-insensitive binding sites on membranes isolated from the rat brain cortex (K i = 1.18 ± 0.09 and 1.58 ± 0.11 nM, respectively). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but to [Met 5 ]enkephalin and [Leu 5 ]enkephalin as well. The K d values characterizing the specific binding of 125 I-labeled immunorphin and its fragment Val-Lys-Gly-Phe-Tyr to these binding sites were determined to be 2.93 ± 0.27 nM and 3.17 ± 0.29 nM, respectively.
Molecular Microbiology, 2002
The F1 antigen of Yersinia pestis belongs to a class of non-pilus adhesins assembled via a classi... more The F1 antigen of Yersinia pestis belongs to a class of non-pilus adhesins assembled via a classical chaperone-usher pathway. Such pathways consist of PapD-like chaperones that bind subunits and pilot them to the outer membrane usher, where they are assembled into surface structures. In a recombinant Escherichia coli model system, chaperone-subunit (Caf1M:Caf1n) complexes accumulate in the periplasm. Three independent methods showed that these complexes are rod- or coil-shaped linear arrays of Caf1 subunits capped at one end by a single copy of Caf1M chaperone. Deletion and point mutagenesis identified an N-terminal donor strand region of Caf1 that was essential for polymerization in vitro, in the periplasm and at the cell surface, but not for chaperone-subunit interaction. Partial protease digestion of periplasmic complexes revealed that this region becomes buried upon formation of Caf1:Caf1 contacts. These results show that, despite the capsule-like appearance of F1 antigen, the basic structure is assembled as a linear array of subunits held together by intersubunit donor strand complementation. This example shows that strikingly different architectures can be achieved by the same general principle of donor strand complementation and suggests that a similar basic polymer organization will be shared by all surface structures assembled by classical chaperone-usher pathways.
Molecular Immunology, 1986
Precipitating and non-precipitating pig anti-2,4-dinitrophenyl group (Dnp) antibodies were invest... more Precipitating and non-precipitating pig anti-2,4-dinitrophenyl group (Dnp) antibodies were investigated by differential adiabatic scanning microcalorimetry in the pH range 3.745. The partial heat capacity functions obtained revealed a notable difference between the two antibody types when the analysis was done at pH 4.5 or 4.0. The transition observed at temps around 50°C in a non-precipitating antibody was absent in a precipitating antibody. Under analogous conditions, pH 4.5, the precipitating antibody was fully resistant to pepsin, while the non-precipitating antibody yielded appropriate F(ab'), and pFc'fragments. At pH 3.7 no substantial difference in the partial heat capacity function could be observed between the two antibody types. Below pH 4.0 the precipitating antibody became susceptible to peptic cleavage and yielded fragments of the same general character as the non-precipitating antibody. This finding lends support to the view that the structural block in immunoglobulin G that melts first, i.e. at temps near 5O"C, is the C,2 domain or a part of it.
Molecular Immunology, 2004
Thermodynamic. conformational and functional properties of the human C1q globular heads (hgC1q) w... more Thermodynamic. conformational and functional properties of the human C1q globular heads (hgC1q) were studied with the experimental approaches, which allow investigating these properties in the intact hC1q molecule in solution. Surprisingly, the scanning calorimetry data reveal a low level of cooperativity of interactions between the hgC1q A, B and C domains even at a neutral pH area. Ionization of His residues due to acidification of the medium at the pH range from 6 to 5 or the chemical modification of His residues completely abolishes the cooperative interactions between the domains without significant effect on their conformation. The thermodynamic data provide evidence that the hgC1q module is composed of three structurally independent A, B and C globular domains characterized by the practically identical thermal stability and very similar enthalpy of melting. The spectroscopic studies and modification with 2-oxy-5-nitrobenzylbromide (ONBB) indicate that Trp residues in the hgC1q A and C domains are accessible to the solvent that has been confirmed by the hgC1q crystal structure solved and refined to 1.9 A. The modification of Trp residues significantly affects the complement-dependent cytotoxicity without noticeable effect on the hC1q conformation. These data provide evidence that Trp residues are the components of immunoglobulin-binding sites both in the hgC1q A and C domains.
International Journal of Biological Macromolecules, 1998
The murine small heat shock protein Hsp25 carries a single cysteine residue in position 141 of it... more The murine small heat shock protein Hsp25 carries a single cysteine residue in position 141 of its amino acid sequence. Interestingly, Hsp25 can exist within the cell as covalently bound dimer which is linked by an intermolecular disulfide bond between two monomers. Oxidative stress caused by treatment of the cells with diamide, arsenite, or hydrogen peroxide leads to an increase in Hsp25-dimerisation which can be blocked by simultaneous treatment with reducing agents. Recombinant Hsp25 was prepared in an oxidized dimeric (oxHsp25) and reduced monomeric (redHsp25) form. The two species were compared with regard to secondary structure, stability, oligomerization properties and their chaperone activity. It is demonstrated by CD measurements in the far UV region that there are no significant differences in the secondary structure and temperature-or pH-stability of oxHsp25 and redHsp25. However, according to CD measurements in the near UV region an increase in the asymmetry of the microenvironment of aromatic residues in oxHsp25 is observed. Furthermore, an increase in stability of the hydrophobic environment of the tryptophan residues mainly located in the N-terminal domain of the protein against urea denaturation is detected in oxHsp25. Both reduced and oxidized Hsp25 form oligomeric complexes of similar size and stability against detergents and both species prevent thermal aggregation of citrate synthase and assist significantly in oxaloacetic acid-induced refolding of the enzyme. Hence, the overall secondary structure, the degree of oligomerization and the chaperone activity of Hsp25 seem independent of the formation of the intermolecular disulfide bond and only the stability of the hydrophobic N-terminal part of the molecule is influenced by formation of this bound. The obtained data do not exclude the possible involvement of dimerization of this protein in other cellular functions, e.g. in intracellular sulfhydryl-buffering or in the protection of actin filaments from fragmentation upon oxidative stress.
Immunology Letters, 1985
platelet-derived growth factors-T-cell growth factors transforming protein p28 sis-interferon p28... more platelet-derived growth factors-T-cell growth factors transforming protein p28 sis-interferon p28 si~ (PDGF), respectively. Thus the homology of hydrophobic cores of the proteins compared is >30%. Probability of chance coincidences of amino acid residues in the hydrophobic cores of p28 ~iS (PDGF) and IFN-/3 is found to be 0.03, and that deduced from comparison of IL-2 and IFN-/3 is 0.05. It was concluded that IL-2, PDGF and p28 sis must have a three-dimensional structure generally similar to that of IFNs.
Immunology Letters, 1997
Introduotlon: It has been shown earlier that short pre-treatment of tumor cells at 3H2"C followed... more Introduotlon: It has been shown earlier that short pre-treatment of tumor cells at 3H2"C followed by the synthesis of heat-inducible proteins renders cells partially resistant to cytotoxicity mediated by different stimuli, such as TNF, metals, cytotoxic cells and other (Jaattela et al., 1990, Stand. J. Immunol. 31: 175). Earlier we have shown that alpha-fetoprotein (AFP) could distinctly increase low dose TNF-cytotoxfcity and practically completely cancel cytotoxicity induced bv hiah TNF in HepG2 cells (Semenkova et al., 1997, Tumor Biol., 18: 58). Here-we-have studied the effect of heating and the treatment with small heat shock protein Hsp 25 and acrystatlln (a-Cry) on the sensttivity of HepGP and MCF7 cells to the-TNF/AFP mediated cytotoxicity. MaterlaIr and Methods: HepG2 cells were pre-treated for 1 h at 42% and after 2-h of adaptation at 37% serially diluted AFP and/or TNF were added to the cells. In the other experiments serially diluted AFP was added to the MCR cells together with recombinant bacterial Hsp25 or bovine a-Cry. Relative cell prolifer&on was assayed by rH]-thymidine tncorporation assay. Results: It was demonstrated that l-h cell heating at 42°C significantly increased (two-fold) cell sensitivity to the AFP mediated cytotoxicity. It was also shown that cell pm-heating also increased HepG2 sensitivity to the low doses of TNF in the presence of 1 mkg/ml ActD and low doses of endogenous AFP. The pm-treatment of MCF7 cells with sHsp25 and a-Cry have been shown to enhance distinctly cell sensitivity to AFP-mediated cytotoxicity. Conclusions: Our data demonstrated, that heat induced synthesis of Hsp's or exogenous Hsp's addition significantly enhanced HepGP cell sensitivity to the AFP-mediated cytotoxicity and also distinctly increased cell cytotoxtc response to low TNF in the presence of low doses of endogenous AFP. These data could explain the effect of heating in the therapy of tumors by the simultaneous action of endogenous AFP. TNF and heat shock proteins.
The rapid emergence of treatment-resistant bacterial pathogens has become a major threat to publi... more The rapid emergence of treatment-resistant bacterial pathogens has become a major threat to public health. The recent outbreak of new Shiga-toxin–producing Escherichia coli o104H4 infection in Germany illustrates this problem. To colonize host tissues, pathogenic bacteria express surface adhesive organelles. The German strain uses aggregative adherence fimbriae I (aaF/I) to anchor to the intestinal mucosa and induce inflammation. aaF/I belong to the family of chaperone/usher assembled fimbrial polyadhesins. Polyadhesins are functioning as an armory for penetration through the host immune shield. The polyadhesin-binding to the target cells is orchestrated with injectisome (third secretion system), which is extremally important for bacterial virulency, and triggers subversive signals by aggregation of host cell receptors that al-low pathogens to mislead and evade immune defense. Polyadhesins also are involved in biofilm formation making bacteria more resistant to immune re-sponse. Bec...
International Immunopharmacology
Doklady Akademii nauk SSSR
Bioorganicheskaia khimiia
The synthetic peptide SLTCLVKGFY, corresponding to the 364-373 amino acid sequence of the human I... more The synthetic peptide SLTCLVKGFY, corresponding to the 364-373 amino acid sequence of the human IgG heavy chain (Immunorphin), was found to compete with [125I] beta-endorphin for binding by high-affinity receptors on T lymphocytes isolated from the blood of healthy donors (Ki 0.6 nM). The fragments 3-10, 4-10, 5-10, and 6-10 of Immunorphin also inhibited the binding (Ki 2.2, 3.4, 8.0, and 15 nM, respectively). Specificity of these receptors was studied: they turned out to be insensitive to naloxone and, therefore, are not opioid. The Kd values of the specific binding of 125I-labeled Immunorphin and its 6-10 fragment to the receptor were found to be 7.4 and 36.3 nM, respectively.
Journal of chromatography, Jan 10, 1989
The use of pH gradients in borate buffer with a polyhydroxy compound for separation of proteins i... more The use of pH gradients in borate buffer with a polyhydroxy compound for separation of proteins in a free-flow electrophoretic apparatus is outlined. The composition of the pH gradients, the free-flow apparatus design and its operation, and the main causes of failures in the protein separation are considered.
Bioorganicheskaia khimiia
Consensus sequence 30-36 LKDRHDF of human alpha-interferons is inserted into the C- and N-termina... more Consensus sequence 30-36 LKDRHDF of human alpha-interferons is inserted into the C- and N-terminal sequences of the artificial protein albeferon using genetic engineering methods in order to obtain artificial proteins with antiviral activity. Albeferon, obtained by Dolgikh et al. (Protein Eng., 1996, vol. 9, pp. 195-201), already contains the IFN-alpha 2 130-137 fragment and possesses antiproliferative activity comparable with that of IFN-alpha 2. According to CD spectroscopy, both proteins have regular secondary structures similar to that of the precursor protein. They exhibit antiviral activities, and the activity of one of them is comparable with that of IFN-alpha 2. At the same time, their cytotoxic properties are displayed only at relatively high concentrations, which substantially exceed the minimal antiviral concentrations.
Immunology letters, Jan 3, 2002
It was found that the human (hu) myeloma IgG1 Ser, its Fcgamma fragment and the chimeric mouse-hu... more It was found that the human (hu) myeloma IgG1 Ser, its Fcgamma fragment and the chimeric mouse-human monoclonal antibody (chim-mAb), containing the constant part of hu-gamma1-chain, can exist in a long-term metastable conformational state. This state arises as a result of short incubation of IgG molecules and their Fcgamma fragments at pH<2.8 and the consequent rapid neutralisation to pH 7.0-8.0. At pH<2.8 the three-dimensional structure of C(gamma)2 domains is unfolded, but rapidly refolds after neutralisation. At the same time, non-covalent interactions between C(gamma)2 and C(gamma)3 domains are restored very slowly. A metastable state of IgG keeps 70% of complement-binding ability in comparison with the native state.
Peptides, 2001
The synthetic decapeptide H-SLTCLVKGFY-OH (termed immunorphin) corresponding to the sequence 364-... more The synthetic decapeptide H-SLTCLVKGFY-OH (termed immunorphin) corresponding to the sequence 364-373 of the CH3 domain of human immunoglobulin G heavy chain was found to compete with [125I]beta-endorphin for high-affinity receptors on T lymphocytes from the blood of healthy donors (K(i) = 0.6 nM). Besides immunorphin, its synthetic fragments H-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 15 nM), H-Leu-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 8.0 nM), H-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 3.4 nM), H-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 2.2 nM), H-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 1.0 nM) possessed the ability to inhibit specific binding of [125I]beta-endorphin to T lymphocytes. Tests of the specificity of the receptors revealed that they are not sensitive to naloxone and Met-enkephalin, i.e. they are not opioid receptors. K(d) values characterizing the specific binding of 125I- labeled immunorphin and its fragment H-Val-Lys-Gly-Phe-Tyr-OH to the receptors have been determined to be 7.4...
Biofizika
Conformational and dynamic properties of the synthetic peptides, amino acid sequence of which cor... more Conformational and dynamic properties of the synthetic peptides, amino acid sequence of which correspond to biologically active fragments of the following human cytokines: alpha 2, gamma interferons and interleukins 1 beta, 2, were studied in aqueous solution and dimethylsulfoxide by one-dimensional and two-dimensional 1H-NMR spectroscopy (400 MHz). The analysis of nuclear Overhauser effect data, values of vicinal J3(NH-C alpha H) coupling constants of amide protons and their temperature coefficients of chemical shifts indicate an unordered flexible conformation of the peptides in aqueous solutions. The structural and dynamic features of the peptides investigated are discussed together with their biological activity.
Molekuliarnaia biologiia
Using the methods of difference adiabatic scanning microcalorimetry and difference thermal pertur... more Using the methods of difference adiabatic scanning microcalorimetry and difference thermal perturbation spectrophotometry it was established that both the Fc subunits within the intact immunoglobulins G of different subclasses, and the Fc fragments corresponding to them significantly differ in conformational properties. These differences are associated with a different energy of interactions between the CH2 and CH3 domains and also with a different rigidity of structure of the N- and C-terminal parts of the CH2 domains. The analysis of these data allowed to suggest that the "hinge region" interacts with the CH2 domains and the difference in the structure of this region affect the conformation of the Fc subunits. Possible models of the "hinge regions" in immunoglobulins G of the first, second and fourth subclasses, and also their arrangement relative to the CH2 domains were proposed. The analysis of curves of the small-angle scattering of X-rays shows an agreement...
Ukrainskiĭ biokhimicheskiĭ zhurnal
Melting of protein A from Staphylococcus aureus has been studied in neutral medium by the methods... more Melting of protein A from Staphylococcus aureus has been studied in neutral medium by the methods of microcalorimetry and circular dichroism. The melting process of protein A is shown to consist of, at least, 5 independent transitions. The transition with the heat absorption maximum at 38 degrees is ascribed to the melting of the C-terminal domain of protein A. The analysis of amino acid sequences has shown the existence of structural basis for differences in heat stability of the Fc-binding domains of protein A.
Biochimica et biophysica acta, Jan 23, 1977
The conformational changes of antibody structure induced by hapten molecule binding were investig... more The conformational changes of antibody structure induced by hapten molecule binding were investigated by means of thermal perturbation difference spectroscopy. The studies of the free rabit anti-dinitrophenyl antibodies show the conformational transition at temperatures between 25 and 35 degrees C. The changes occurring at the higher temperature are accompanied by the screening of the significant part of exposed tyrosine residues. Binding of the hapten molecules induces a similar transition to that which occurs between the two temperature dependent states of the free antibody. In contrast to our previous results with anti-dansyl rabbit antibodies the dinitrophenyl lysine stabilizes the "low temperature" native state of the protein. The investigation of the MOPC-315 mouse immunoglobulin A myeloma protein possessing anti-dinitrophenyl activity indicates no conformational transition at temperatures between 25 and 35 degrees C and only a small decrease of tyrosine exposure ind...
Biochimica et biophysica acta, Jan 28, 1977
1. The temperature function of the myeloma IgG(K) IVA, Bence-Jones protein (K-type) IVA and its f... more 1. The temperature function of the myeloma IgG(K) IVA, Bence-Jones protein (K-type) IVA and its fragments (Fab(t), Fc'(t), VL and CL) was studied by thermal perturbation difference spectroscopy and circular dichroism. 2. The IgG and Bence-Jones protein studied were found to be capable of a fully reversible structural changes at temperatures between 25 and 35 degrees C. The changes occurring at the higher temperature are accompanied by the screening of the significant part of exposed tyrosine residues. The transition is not accompanied by an appreciable change in the main IgG secondary structure-beta-pleated sheet, according to the CD data. 3. It was found that the temperature-dependent changes of IgG occur in its Fab fragments, the changes of Bence-Jones protein occur in its variable part (VL domains). 4. The temperature changes in the interval 25-35 degrees C are explained by the flexibility of amino acid side chains composed hypervariable loops delineated the "sides"...
Peptides, 2002
The synthetic decapeptide Ser-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr (termed immunorphin) correspond... more The synthetic decapeptide Ser-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr (termed immunorphin) corresponding to the sequence 364-373 of the CH3 domain of human immunoglobulin G heavy chain and its synthetic fragment VKGFY were found to compete with 125 I-labeled -endorphin for high-affinity naloxone-insensitive binding sites on membranes isolated from the rat brain cortex (K i = 1.18 ± 0.09 and 1.58 ± 0.11 nM, respectively). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but to [Met 5 ]enkephalin and [Leu 5 ]enkephalin as well. The K d values characterizing the specific binding of 125 I-labeled immunorphin and its fragment Val-Lys-Gly-Phe-Tyr to these binding sites were determined to be 2.93 ± 0.27 nM and 3.17 ± 0.29 nM, respectively.
Molecular Microbiology, 2002
The F1 antigen of Yersinia pestis belongs to a class of non-pilus adhesins assembled via a classi... more The F1 antigen of Yersinia pestis belongs to a class of non-pilus adhesins assembled via a classical chaperone-usher pathway. Such pathways consist of PapD-like chaperones that bind subunits and pilot them to the outer membrane usher, where they are assembled into surface structures. In a recombinant Escherichia coli model system, chaperone-subunit (Caf1M:Caf1n) complexes accumulate in the periplasm. Three independent methods showed that these complexes are rod- or coil-shaped linear arrays of Caf1 subunits capped at one end by a single copy of Caf1M chaperone. Deletion and point mutagenesis identified an N-terminal donor strand region of Caf1 that was essential for polymerization in vitro, in the periplasm and at the cell surface, but not for chaperone-subunit interaction. Partial protease digestion of periplasmic complexes revealed that this region becomes buried upon formation of Caf1:Caf1 contacts. These results show that, despite the capsule-like appearance of F1 antigen, the basic structure is assembled as a linear array of subunits held together by intersubunit donor strand complementation. This example shows that strikingly different architectures can be achieved by the same general principle of donor strand complementation and suggests that a similar basic polymer organization will be shared by all surface structures assembled by classical chaperone-usher pathways.
Molecular Immunology, 1986
Precipitating and non-precipitating pig anti-2,4-dinitrophenyl group (Dnp) antibodies were invest... more Precipitating and non-precipitating pig anti-2,4-dinitrophenyl group (Dnp) antibodies were investigated by differential adiabatic scanning microcalorimetry in the pH range 3.745. The partial heat capacity functions obtained revealed a notable difference between the two antibody types when the analysis was done at pH 4.5 or 4.0. The transition observed at temps around 50°C in a non-precipitating antibody was absent in a precipitating antibody. Under analogous conditions, pH 4.5, the precipitating antibody was fully resistant to pepsin, while the non-precipitating antibody yielded appropriate F(ab'), and pFc'fragments. At pH 3.7 no substantial difference in the partial heat capacity function could be observed between the two antibody types. Below pH 4.0 the precipitating antibody became susceptible to peptic cleavage and yielded fragments of the same general character as the non-precipitating antibody. This finding lends support to the view that the structural block in immunoglobulin G that melts first, i.e. at temps near 5O"C, is the C,2 domain or a part of it.
Molecular Immunology, 2004
Thermodynamic. conformational and functional properties of the human C1q globular heads (hgC1q) w... more Thermodynamic. conformational and functional properties of the human C1q globular heads (hgC1q) were studied with the experimental approaches, which allow investigating these properties in the intact hC1q molecule in solution. Surprisingly, the scanning calorimetry data reveal a low level of cooperativity of interactions between the hgC1q A, B and C domains even at a neutral pH area. Ionization of His residues due to acidification of the medium at the pH range from 6 to 5 or the chemical modification of His residues completely abolishes the cooperative interactions between the domains without significant effect on their conformation. The thermodynamic data provide evidence that the hgC1q module is composed of three structurally independent A, B and C globular domains characterized by the practically identical thermal stability and very similar enthalpy of melting. The spectroscopic studies and modification with 2-oxy-5-nitrobenzylbromide (ONBB) indicate that Trp residues in the hgC1q A and C domains are accessible to the solvent that has been confirmed by the hgC1q crystal structure solved and refined to 1.9 A. The modification of Trp residues significantly affects the complement-dependent cytotoxicity without noticeable effect on the hC1q conformation. These data provide evidence that Trp residues are the components of immunoglobulin-binding sites both in the hgC1q A and C domains.
International Journal of Biological Macromolecules, 1998
The murine small heat shock protein Hsp25 carries a single cysteine residue in position 141 of it... more The murine small heat shock protein Hsp25 carries a single cysteine residue in position 141 of its amino acid sequence. Interestingly, Hsp25 can exist within the cell as covalently bound dimer which is linked by an intermolecular disulfide bond between two monomers. Oxidative stress caused by treatment of the cells with diamide, arsenite, or hydrogen peroxide leads to an increase in Hsp25-dimerisation which can be blocked by simultaneous treatment with reducing agents. Recombinant Hsp25 was prepared in an oxidized dimeric (oxHsp25) and reduced monomeric (redHsp25) form. The two species were compared with regard to secondary structure, stability, oligomerization properties and their chaperone activity. It is demonstrated by CD measurements in the far UV region that there are no significant differences in the secondary structure and temperature-or pH-stability of oxHsp25 and redHsp25. However, according to CD measurements in the near UV region an increase in the asymmetry of the microenvironment of aromatic residues in oxHsp25 is observed. Furthermore, an increase in stability of the hydrophobic environment of the tryptophan residues mainly located in the N-terminal domain of the protein against urea denaturation is detected in oxHsp25. Both reduced and oxidized Hsp25 form oligomeric complexes of similar size and stability against detergents and both species prevent thermal aggregation of citrate synthase and assist significantly in oxaloacetic acid-induced refolding of the enzyme. Hence, the overall secondary structure, the degree of oligomerization and the chaperone activity of Hsp25 seem independent of the formation of the intermolecular disulfide bond and only the stability of the hydrophobic N-terminal part of the molecule is influenced by formation of this bound. The obtained data do not exclude the possible involvement of dimerization of this protein in other cellular functions, e.g. in intracellular sulfhydryl-buffering or in the protection of actin filaments from fragmentation upon oxidative stress.
Immunology Letters, 1985
platelet-derived growth factors-T-cell growth factors transforming protein p28 sis-interferon p28... more platelet-derived growth factors-T-cell growth factors transforming protein p28 sis-interferon p28 si~ (PDGF), respectively. Thus the homology of hydrophobic cores of the proteins compared is >30%. Probability of chance coincidences of amino acid residues in the hydrophobic cores of p28 ~iS (PDGF) and IFN-/3 is found to be 0.03, and that deduced from comparison of IL-2 and IFN-/3 is 0.05. It was concluded that IL-2, PDGF and p28 sis must have a three-dimensional structure generally similar to that of IFNs.
Immunology Letters, 1997
Introduotlon: It has been shown earlier that short pre-treatment of tumor cells at 3H2"C followed... more Introduotlon: It has been shown earlier that short pre-treatment of tumor cells at 3H2"C followed by the synthesis of heat-inducible proteins renders cells partially resistant to cytotoxicity mediated by different stimuli, such as TNF, metals, cytotoxic cells and other (Jaattela et al., 1990, Stand. J. Immunol. 31: 175). Earlier we have shown that alpha-fetoprotein (AFP) could distinctly increase low dose TNF-cytotoxfcity and practically completely cancel cytotoxicity induced bv hiah TNF in HepG2 cells (Semenkova et al., 1997, Tumor Biol., 18: 58). Here-we-have studied the effect of heating and the treatment with small heat shock protein Hsp 25 and acrystatlln (a-Cry) on the sensttivity of HepGP and MCF7 cells to the-TNF/AFP mediated cytotoxicity. MaterlaIr and Methods: HepG2 cells were pre-treated for 1 h at 42% and after 2-h of adaptation at 37% serially diluted AFP and/or TNF were added to the cells. In the other experiments serially diluted AFP was added to the MCR cells together with recombinant bacterial Hsp25 or bovine a-Cry. Relative cell prolifer&on was assayed by rH]-thymidine tncorporation assay. Results: It was demonstrated that l-h cell heating at 42°C significantly increased (two-fold) cell sensitivity to the AFP mediated cytotoxicity. It was also shown that cell pm-heating also increased HepG2 sensitivity to the low doses of TNF in the presence of 1 mkg/ml ActD and low doses of endogenous AFP. The pm-treatment of MCF7 cells with sHsp25 and a-Cry have been shown to enhance distinctly cell sensitivity to AFP-mediated cytotoxicity. Conclusions: Our data demonstrated, that heat induced synthesis of Hsp's or exogenous Hsp's addition significantly enhanced HepGP cell sensitivity to the AFP-mediated cytotoxicity and also distinctly increased cell cytotoxtc response to low TNF in the presence of low doses of endogenous AFP. These data could explain the effect of heating in the therapy of tumors by the simultaneous action of endogenous AFP. TNF and heat shock proteins.