T A Balashova

Russian Academy of Sciences, Moskva, Moscow, Russia

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Publications (46)61.78 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: This study presents purification, activity characterization, and 1H NMR study of the novel antifungal peptide EcAMP1 from kernels of barnyard grass Echinochloa crus-galli. The peptide adopts a disulfide-stabilized α-helical hairpin structure in aqueous solution and thus represents a novel fold among naturally occurring antimicrobial peptides. Micromolar concentrations of EcAMP1 were shown to inhibit growth of several fungal phytopathogens. Confocal microscopy revealed intensive EcAMP1 binding to the surface of fungal conidia followed by internalization and accumulation in the cytoplasm without disturbance of membrane integrity. Close spatial structure similarity between EcAMP1, the trypsin inhibitor VhTI from seeds of Veronica hederifolia, and some scorpion and cone snail toxins suggests natural elaboration of different functions on a common fold.
    Journal of Biological Chemistry 07/2011; 286(28):25145-25153. · 4.65 Impact Factor
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    ABSTRACT: This study presents purification, activity characterization, and (1)H NMR study of the novel antifungal peptide EcAMP1 from kernels of barnyard grass Echinochloa crus-galli. The peptide adopts a disulfide-stabilized α-helical hairpin structure in aqueous solution and thus represents a novel fold among naturally occurring antimicrobial peptides. Micromolar concentrations of EcAMP1 were shown to inhibit growth of several fungal phytopathogens. Confocal microscopy revealed intensive EcAMP1 binding to the surface of fungal conidia followed by internalization and accumulation in the cytoplasm without disturbance of membrane integrity. Close spatial structure similarity between EcAMP1, the trypsin inhibitor VhTI from seeds of Veronica hederifolia, and some scorpion and cone snail toxins suggests natural elaboration of different functions on a common fold.
    Journal of Biological Chemistry 05/2011; 286(28):25145-53. · 4.65 Impact Factor
  • ChemInform 01/2010; 29(17).
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    ABSTRACT: Here we present the study of the chromophore structure of the purple chromoprotein from Condylactis gigantea. Tandem mass spectrometry and 1H and 13C NMR of the chromopeptide reveal that the protein contains a chromophore with a chemical structure identical to that of the red fluorescent protein from Discosoma sp. A single A63G substitution demonstrates that the nature of the first amino acid of the XYG chromophore-forming sequence is dispensable for the chromoprotein red shift development. It has been recently proposed that post-translational reactions at the acylimine, a chemical group that accounts for the red fluorescence, might be an additional source of spectral diversity of proteins homologous to the Aequorea victoria green fluorescent protein (GFP). We have examined the reactivity of the chromophore acylimine group within the C. gigantea purple chromoprotein. Like other proteins with the acylimine-modified chromophore, the purple chromoprotein suffers a hypsochromic spectral shift to the GFP-like absorbance (386 nm) upon mild denaturation. NMR analysis of the chromopeptide suggests this hypsochromic spectral shift is due to H2O addition across the C=N bond of the acylimine. However, unlike the red fluorescent protein from Discosoma sp., denatured under harsh conditions, the wild-type chromoprotein exhibits only slight fragmentation, which is induced by complete hydrolysis of the acylimine. A model suggesting the influence of the amino acid X side chain on protein fragmentation is presented.
    Biochemistry 07/2006; 45(23):7256-64. · 3.38 Impact Factor
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    ABSTRACT: The venom of South American ant Paraponera clavata and its low-molecular-mass fraction were shown to possess insectotoxic and pore-forming activities. A number of glycophospholipid components were isolated from this ant venom by means of gel filtration and reversed-phase chromatography. Some of the compounds cause conductivity fluctuations in lipid bilayer membranes within the ranges 3-25 pS and 200-400 pS at concentrations of 10(-6) to 10(-7) M. N-Acetylglucosamine, a fatty acid, and phosphoric acid residues were found in their structures. A full structure, 3-myristoyl-2-acetamido-2-deoxy-alpha-D-glucopyranosyl phosphate, was elucidated for one of the compounds by the use of 1H, 13C, and 31P NMR spectroscopy and mass spectrometry.
    Bioorganicheskaia khimiia 01/2006; 32(5):530-7.
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    ABSTRACT: A number of protected proline-containing dipeptides Boc-Xaa-Pro-OBu(t) were converted via epimerization-free oxidation with RuO4 to dipeptides with an internal pyroglutamic acid residue, Boc-Xaa-Glp-OBu(t). The latter were subjected to oxidative Hoffman-type rearrangement induced by PhI[OC(O)CF3]2 to give N-(aminoacyl)-pyroglutamates. The behavior of these derivatives under basic conditions was studied, and for two such a derivatives an aminoacyl incorporation reaction was observed, producing otherwise poorly accessible 10-membered-ring dilactams derived from 1,4-diaminobutyric and glutamic acids in practicable yields.
    Journal of Peptide Science 04/2005; 11(3):175-86. · 2.07 Impact Factor
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    ABSTRACT: Deuteroporphyrin IX derivatives containing tryptophan and quinone residues were synthesised and intramolecular interactions between the nitrogen atoms of the porphyrin ring and the NH group of the indole ring of tryptophan were detected in these compounds.
    ChemInform 01/2005; 15(6):234-236.
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    ABSTRACT: Zervamicin IIB is a 16 amino acid peptaibol that forms voltage dependent ion channels with multilevel conductance states in planar lipid bilayers and vesicular systems. Stability of the hinge region and intermolecular interactions were investigated in the N- and C-terminally spin-labelled peptide analogues. Intermolecular and intramolecular paramagnetic enhancement indicates that zervamicin behaves as a rigid helical rod in methanol solution. There are no high amplitude hinge-bending motions, and the peptaibol is monomeric up to concentration 1.5 mM. Stability of the hinge region illustrates the helix stabilising propensity of the Pro residue in membrane mimic environments and implies absence of significant conformational rearrangement due to voltage peptaibol activation.
    Biochemical and Biophysical Research Communications 12/2004; 325(3):1099-105. · 2.41 Impact Factor
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    ABSTRACT: The purple chromoprotein (asFP595) from Anemonia sulcata belongs to the family of green fluorescent protein (GFP). Absorption and emission spectra of asFP595 are similar to those of a number of recently cloned GFP-like red proteins of the DsRed subfamily. The earlier proposed asFP595 chromophore structure [Martynov, V. I.; et al. (2001) J. Biol. Chem. 276, 21012-21016] was postulated to result from an "alternative cyclization" giving rise to a pyrazine-type six-membered heterocycle. Here we report that the asFP595 chromophore is actually very close in chemical structure to that of zFP538, a yellow fluorescent protein [Zagranichny, V. E.; et al. (2004) Biochemistry 43, 4764-4772]. NMR spectroscopic studies of four chromophore-containing peptides (chromopeptides) isolated under mild conditions from enzymatic digests of asFP595 and one chromopeptide obtained from DsRed revealed that all of them contain a p-hydroxybenzylideneimidazolinone moiety formed by Met-65/Gln-66, Tyr-66/67, and Gly-67/68 of asFP595/DsRed, respectively. Two asFP595 chromopeptides are proteolysis products of an isolated full-length polypeptide containing a GFP-type chromophore already formed and arrested at an earlier stage of maturation. The two other asFP595 chromopeptides were isolated as proteolysis products of the purified chromophore-containing C-terminal fragment. One of these has an oxo group at Met-65 C(alpha) and is a hydrolysis product of another one, with the imino group at Met-65 C(alpha). The N-unsubstituted imino moiety of the latter is generated by spontaneous polypeptide chain cleavage at a very unexpected site, the former peptide bond between Cys-64 C' and Met-65 N(alpha). Our data strongly suggest that both zFP538 and asFP595 could be attributed to the DsRed subfamily of GFP-like proteins.
    Biochemistry 11/2004; 43(42):13598-603. · 3.38 Impact Factor
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    ABSTRACT: A green fluorescent protein from the coral Dendronephthya sp. (Dend FP) is characterized by an irreversible light-dependent conversion to a red-emitting form. The molecular basis of this phenomenon was studied in the present work. Upon UV-irradiation at 366 nm, the absorption maximum of the protein shifted from 494 nm (the green form) to 557 nm (the red form). Concurrently, in the fluorescence spectra the emission maximum shifted from 508 to 575 nm. The green form of native Dend FP was shown to be a dimer, and the oligomerization state of the protein did not change during its conversion to the red form. By contrast, UV-irradiation caused significant intramolecular changes. Unlike the green form, which migrates in SDS-polyacrylamide gels as a single band corresponding to a full-length 28-kD protein, the red form of Dend FP migrated as two fragments of 18- and 10-kD. To determine the chemical basis of these events, the denatured red form of Dend FP was subjected to proteolysis with trypsin. From the resulting hydrolyzate, a chromophore-containing peptide was isolated by HPLC. The structure of the chromophore from the Dend FP red form was established by methods of ESI, tandem mass spectrometry (ESI/MS/MS), and NMR-spectroscopy. The findings suggest that the light-dependent conversion of Dend FP is caused by generation of an additional double bond in the side chain of His65 and a resulting extension of the conjugated system of the green form chromophore. Thus, classified by the chromophore structure, Dend FP should be referred to the Kaede subfamily of GFP-like proteins.
    Biochemistry (Moscow) 09/2004; 69(8):901-8. · 1.15 Impact Factor
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    ABSTRACT: Zervamicin IIB (Zrv-IIB) is a channel-forming peptaibol antibiotic of fungal origin. The measured transhydrogen bond (3h)J(NC') couplings in methanol solution heaving average value of -0.41 Hz indicate that the stability of the Zrv-IIB helix in this milieu is comparable to the stability of helices in globular proteins. The N-terminus of the peptide forms an alpha-helix, whereas 3(10)-helical hydrogen bonds stabilize the C-terminus. However, two weak transhydrogen bond peaks are observed in a long-range HNCO spectrum for HN Aib(12). Energy calculations using the Empirical Conformation Energy Program for Peptides (ECEPP)/2 force field and the implicit solvent model show that the middle of the peptide helix accommodates a bifurcated hydrogen bond that is simultaneously formed between HN Aib(12) and CO Leu(8) and CO Aib(9). Several lowered (3h)J(NC') on a polar face of the helix correlate with the conformational exchange process observed earlier and imply dynamic distortions of a hydrogen bond pattern with the predominant population of a properly folded helical structure. The refined structure of Zrv-IIB on the basis of the observed hydrogen bond pattern has a small ( approximately 20 degrees ) angle of helix bending that is virtually identical to the angle of bending in dodecylphosphocholine (DPC) micelles, indicating the stability of a hinge region in different environments. NMR parameters ((1)HN chemical shifts and transpeptide bond (1)J(NC') couplings) sensitive to hydrogen bonding along with the solvent accessible surface area of carbonyl oxygens indicate a large polar patch on the convex side of the helix formed by three exposed backbone carbonyls of Aib(7), Aib(9), and Hyp(10) and polar side chains of Hyp(10), Gln(11), and Hyp(13). The unique structural features, high helix stability and the enhanced polar patch, set apart Zrv-IIB from other peptaibols (for example, alamethicin) and possibly underlie its biological and physiological properties.
    Biophysical Journal 07/2004; 86(6):3687-99. · 3.67 Impact Factor
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    ABSTRACT: The yellow fluorescent protein (zFP538) from coral Zoanthus sp. belongs to a family of green fluorescent protein (GFP). Absorption and emission spectra of zFP538 show an intermediate bathochromic shift as compared with a number of recently cloned GFP-like red fluorescent and nonfluorescent chromoproteins of the DsRed subfamily. Here we report that the zFP538 chromophore is very close, if not identical, in chemical structure to that of DsRed. To gain insight into the mechanism of zFP538 fluorescence and chromophore structure and chemistry, we studied three chromophore-containing peptides isolated from enzymatic digests of zFP538. Like GFP and DsRed chromophores, these contain a p-hydroxybenzylideneimidazolinone moiety formed by Lys-66, Tyr-67, and Gly-68 of zFP538. One of the peptides studied, the hexapeptide FKYGDR derivative, is a proteolysis product of the zFP538 full-length polypeptide containing a GFP-type chromophore already formed and arrested at an earlier stage of maturation. The two other peptides are the derivatives of the pentapeptide KYGDR resulted from the protein in which the chromophore maturation process had been completed. One of these has an oxogroup at Lys-66 C(alpha) and is a hydrolysis product of another one, with the imino group at Lys-66 C(alpha). The N-unsubstituted imino moiety of the latter is generated by spontaneous polypeptide chain fragmentation at a very unexpected site, the former peptide bond between Phe-65 C' and Lys-66 N(alpha). Also observed in the entire protein under mild denaturing conditions, this fragmentation is likely the feature of native zFP538 chromophore that distinguishes it chemically from the DsRed chromophore.
    Biochemistry 05/2004; 43(16):4764-72. · 3.38 Impact Factor
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    ABSTRACT: The goal of this work was to elucidate the structure of the low-molecular components of seeds of the greater plantain ( Plantago major L.) possessing antifungal activity.
    Doklady Biochemistry and Biophysics 01/2004; 396:132-5. · 0.32 Impact Factor
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    ABSTRACT: Thermophilic and thermoresistant strains of bacilli were screened on a medium containing Chrome Azurol S for the producers of siderophores. It was found that the Bacillus licheniformis VK21 strain dramatically increases secretion of the metabolite, a chelator of Fe3+, in response to addition of manganese(II) salts. The growth of the producer on a minimal medium containing MnSO4 under the conditions of iron deficiency is accompanied by the accumulation of a catecholic product, the content of which reaches maximum at the beginning of the stationary growth phase of culture. In the presence of FeCl3, the amount of the catecholic product in the medium considerably decreases. The siderophore, called SVK21, was isolated from the cultural medium and purified by reversed phase HPLC, and its siderophore function was confirmed by the test for the restoration of growth of producer cells in a medium containing EDTA. The UV spectrum of the siderophore has absorption maxima at 248 and 315 nm. According to the amino acid analysis and NMR spectrometry, the metabolite SVK21 is 2,3-dihydroxybenzoyl-glycyl-threonine.
    Russian Journal of Bioorganic Chemistry 10/2003; 29(6):542-549. · 0.52 Impact Factor
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    ABSTRACT: It was previously shown that the catalytic subunit of the plant toxin viscumin induces aggregation of small unilamellar liposomes and this process is inhibited by the mab_TA7 monoclonal antibody produced to the denatured catalytic subunit of viscumin (Agapov, I.I. et al., FEBS Lett., 1999, vol. 464, p. 63). The interaction of the synthetic F101–T105 and A96–T105 fragments of the viscumin catalytic subunit with the mab_TA7 monoclonal antibody was studied by 1H NMR spectroscopy. Results of this study demonstrated that only the A96–T105 fragment is capable of binding to mab_TA7. A nuclear Overhauser effect observed in the antigen–antibody complex and registered on the resonances of the free peptide transferred from the free state to the antibody-bound state was analyzed, the mab_TA7 antigen determinant (H99–T105) was identified, and its conformation and orientation within the complex with the antibody were determined.
    Russian Journal of Bioorganic Chemistry 10/2003; 29(6):534-541. · 0.52 Impact Factor
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    ABSTRACT: A protein corresponding to the extracellular 1-209 domain of the alpha-subunit of the nicotine acetylcholine receptor from the electric organ of Torpedo californica was prepared using the corresponding cDNA domain by culturing Escherichia coli cells on a synthetic medium supplemented with 5-fluoro-L-tryptophan. The presence of a (His)6 fragment preceding the 1-209 sequence allowed purification of the protein isolated from inclusion bodies by affinity chromatography on Ni-NTA Agarose. The incorporation of 5-fluorotryptophan residues was found by 19F NMR to be approximately 50%. The spectrum of the protein reduced under denaturing conditions and subsequently reoxidized in a dilute solution under denaturing conditions in the presence of 0.05% SDS was sufficiently resolved, which allowed partial assignment of 19F resonances using the Trp60Phe mutant protein. The ability of the prepared domains to specifically bind snake alpha-neurotoxins was demonstrated with the use of radioiodinated alpha-bungarotoxin and trifluoroacetylated alpha-cobratoxin.
    Bioorganicheskaia khimiia 07/2003; 29(4):384-90.
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    ABSTRACT: A protein corresponding to the extracellular 1-209 domain of the alpha-subunit of the nicotine acetylcholine receptor from the electric organ of Torpedo californica was prepared using the corresponding cDNA domain by culturing Escherichia coli cells on a synthetic medium supplemented with 5-fluoro-L-tryptophan. The presence of a (His)(6) fragment preceding the 1-209 sequence allowed purification of the protein isolated from inclusion bodies by affinity chromatography on Ni-NTA A-arose. The incorporation of 5-fluorotryptophan residues was found by (FNMR)-F-19 to be similar to50%. The spectrum of the protein reduced in the denaturing conditions and subsequently reoxidized in a dilute solution under denaturing conditions in the presence of 0.05% SDS was sufficiently resolved, which allowed partial assignment of (19)resonances using the Trp60Phe mutant protein. The ability of the prepared domains to specifically bind snake alpha-neurotoxins was demonstrated with the use of radioiodinated alpha-bungarotoxin and trifluoroacetylated a-cobratoxin.
    Russian Journal of Bioorganic Chemistry 07/2003; 29(4):351-357. · 0.52 Impact Factor
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    ABSTRACT: The plant toxin viscumin (60 kD) consists of B- ("binding") and A- ("active") subunits joined by a disulfide bond. The B-subunit is a lectin interacting with galactose-containing glycolipids and glycoproteins of the cell surface. The A-subunit possesses N-glycosidase activity which modifies 28S ribosomal RNA. This results in irreversible inhibition of protein synthesis. After binding and receptor-mediated endocytosis viscumin-containing vesicles are transported to endoplasmic reticulum where the A- (catalytic) subunit is subsequently translocated to cytosol. It is possible that translocation of A-subunit requires its unfolding. For identification of epitopes which might appear during such unfolding, we developed hybridomas producing monoclonal antibodies against denatured viscumin A-chain. Resistance of hybridoma cells to cytotoxic action of viscumin suggests antibody-toxin interaction inside these cells. TA7 hybridoma cells against an epitope which appears only in denatured viscumin are insensitive to the toxin. This suggests that antibody-toxin interaction occurs before transmembrane translocation of the catalytic A-chain into the cytoplasm. Consequently, toxin resistance of TA7 hybridoma cells implies the appearance of a new epitope in viscumin during its intracellular transportation inside of vesicles. Sixty five octapeptides have been synthesized and epitopes have been identified for monoclonal TA7 antibody and immune mouse serum by means of ELISA. Based on the epitopic mapping the peptide A96-ETHLFTGT-T105 was chemically synthesized and binding of this peptide to the monoclonal antibody TA7 and conformation of antigenic determinant (L100-FTGT-T105) was investigated by means of (1)H-NMR spectroscopy.
    Biochemistry (Moscow) 04/2003; 68(3):275-85. · 1.15 Impact Factor
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    ABSTRACT: A protein corresponding to the extracellular 1–209 domain of the α-subunit of the nicotine acetylcholine receptor from the electric organ of Torpedo californica was prepared using the corresponding cDNA domain by culturing Escherichia coli cells on a synthetic medium supplemented with 5-fluoro-L-tryptophan. The presence of a (His)6 fragment preceding the 1–209 sequence allowed purification of the protein isolated from inclusion bodies by affinity chromatography on Ni-NTA Agarose. The incorporation of 5-fluorotryptophan residues was found by 19F NMR to be ∼50%. The spectrum of the protein reduced in the denaturing conditions and subsequently reoxidized in a dilute solution under denaturing conditions in the presence of 0.05% SDS was sufficiently resolved, which allowed partial assignment of 19F resonances using the Trp60Phe mutant protein. The ability of the prepared domains to specifically bind snake α-neurotoxins was demonstrated with the use of radioiodinated α-bungarotoxin and trifluoroacetylated α-cobratoxin.
    Russian Journal of Bioorganic Chemistry 01/2003; 29(4). · 0.52 Impact Factor
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    ABSTRACT: It was previously shown that the catalytic subunit of the plant toxin viscumin induces aggregation of small unilamellar liposomes and this process is inhibited by the mab_TA7 monoclonal antibody produced to the denatured catalytic subunit of viscumin (Agapov, I.I. et al., FEBS Lett., 1999, vol. 464, pp. 63-66). The interaction of the synthetic F101-T105 and A96-T105 fragments of the viscumin catalytic subunit with the mab_TA7 monoclonal antibody was studied by 1H NMR spectroscopy. The results of this study demonstrated that only the A96-T105 fragment is capable of binding to mab_TA7. A nuclear Overhauser effect observed in the antigen-antibody complex and registered on the resonances of the free peptide and exchanging between the free state and the antibody-bound state was analyzed; the mab_TA7 antigen determinant (H99-T105) was identified; and its conformation and orientation within the complex with the antibody were determined. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.
    Bioorganicheskaia khimiia 01/2003; 29(6):589-96.

Publication Stats

271 Citations
61.78 Total Impact Points

Institutions

  • 1996–2005
    • Russian Academy of Sciences
      Moskva, Moscow, Russia
  • 1974–2005
    • Pacific Institute of Bioorganic Chemistry
      Wladiwostok, Primorskiy, Russia