Баландин Сергей Владимирович

Избранные публикации

  1. Шамова О.В., Орлов Д.С., Жаркова М.С., Баландин С.В., Ямщикова Е.В., Кнаппе Д., Хоффманн Р., Кокряков В.Н., Овчинникова Т.В. (2016). Минибактенецины ChBac7.5Nα и ChBac7.5Nβ - антимикробные пептиды из лейкоцитов козы Capra hircus. Acta Naturae 8 (3), 147–157 [+]

    Антимикробные пептиды (АМП) нейтрофилов играют важную роль в осуществлении защитных функций организма человека и животных. Из лейкоцитов домашней козы Capra hircus нами выделены два пептида (средние молекулярные массы 2895.5 и 2739.3 Да), обладающие высокой антимикробной активностью и представляющие собой N-концевые фрагменты (1–22 и 1–21) пролин-богатого пептида бактенецина 7.5 козы, структура гена которого представлена в базах данных, но соответствующий белковый продукт до настоящего времени не был выделен. Полученные АМП названы мини-бактенецинами (mini-ChBac7.5Nα и mini-ChBac7.5Nβ) по аналогии с описанным ранее С-концевым фрагментом бактенецина 7.5, выделенным из лейкоцитов овцы [Anderson, Yu, 2003]. Мини-бактенецины козы в концентрации 0.5–4 мкМ проявляют высокую антимикробную активность в отношении грамотрицательных бактерий, включая устойчивые к ряду применяемых в медицине антибиотиков штаммы Pseudomonas aeruginosa, Klebsiella spp., Acinetobacter baumannii, а также некоторых штаммов грамположительных бактерий (Listeria monocytogenes EGD, Micrococcus luteus). Исследуемые пептиды, как и большинство пролин-богатых АМП, оказывают антимикробное действие без существенного повреждения бактериальных мембран, обладают липополисахарид-связывающей активностью. Мини-бактенецины не токсичны по отношению к культивируемым клеткам человека, что дает основание рассматривать их как перспективные прототипы новых антибактериальных терапевтических препаратов. Обнаружение высокоактивных фрагментов антимикробного пептида в нейтрофилах козы свидетельствует в пользу гипотезы о том, что фрагментация АМП кателицидинового семейства важна для образования функционально активных молекул, в ряде случаев более активных, чем полноразмерные пептиды, и может играть значимую роль в антиинфекционной защите.

  2. Sychev S.V., Balandin S.V., Panteleev P.V., Barsukov L.I., Ovchinnikova T.V. (2015). Lipid-dependent pore formation by antimicrobial peptides arenicin-2 and melittin demonstrated by their proton transfer activity. J. Pept. Sci. 21 (2), 71–6 [+]

    This work presents a comparative study of proton transfer activity (PTA) of two cationic (+6) antimicrobial peptides, β-structural arenicin-2 and α-helical melittin. A new approach was proposed for the detection of passive proton transfer by using proteoliposomes containing bacteriorhodopsin, which creates a small light-induced electrochemical proton gradient ∆ΔpH. Addition of several nanomoles of the peptides lowers ∆ΔpH that is proximately indicative of the pore formation. The quantitative analysis of sigmoidal dependences of ∆pH on the peptides concentration was carried out using liposomes prepared from PC, PC/PE, PC/PE/PI and PC/PG. Substitution of PC-containing liposomes with PE-containing ones, having negative spontaneous curvature, reduced the PTA of α-helical melittin and increased that of β-structural arenicin-2. This result indicates an essential difference in the pore formation by these peptides. Further increase of PTA in response to arenicin-2 (in contrast to melittin) was observed in the liposomes prepared from PC/PE/PI. The data analysis leads to the conclusion that PTA is influenced by (i) efficiency of the pore assemblage, which depends on the structure of pore-forming peptides, and the spontaneous curvature of lipids and (ii) the presence of mobile protons in the polar head groups of phospholipids.

  3. Bogdanov I.V., Finkina E.I., Balandin S.V., Melnikova D.N., Stukacheva E.A., Ovchinnikova T.V. (2015). Structural and Functional Characterization of Recombinant Isoforms of the Lentil Lipid Transfer Protein. Acta Naturae 7 (3), 65–73 [+]

    The recombinant isoforms Lc-LTP1 and Lc-LTP3 of the lentil lipid transfer protein were overexpressed in E. coli cells. It was confirmed that both proteins are stabilized by four disulfide bonds and characterized by a high proportion of the α-helical structure. It was found that Lc-LTP1 and Lc-LTP3 possess antimicrobial activity and can bind fatty acids. Both isoforms have the ability to bind specific IgE from sera of patients with food allergies, which recognize similar epitopes of the major peach allergen Pru p 3. Both isoforms were shown to have immunological properties similar to those of other plant allergenic LTPs, but Lc-LTP3 displayed a less pronounced immunoreactivity.

  4. Shenkarev Z.O., Gizatullina A.K., Finkina E.I., Alekseeva E.A., Balandin S.V., Mineev K.S., Arseniev A.S., Ovchinnikova T.V. (2014). Heterologous expression and solution structure of defensin from lentil Lens culinaris. Biochem. Biophys. Res. Commun. 451 (2), 252–7 [+]

    A new defensin Lc-def, isolated from germinated seeds of the lentil Lens culinaris, has molecular mass 5440.4Da and consists of 47 amino acid residues. Lc-def and its (15)N-labeled analog were overexpressed in Escherichia coli. Antimicrobial activity of the recombinant protein was examined, and its spatial structure, dynamics, and interaction with lipid vesicles were studied by NMR spectroscopy. It was shown that Lc-def is active against fungi, but does not inhibit the growth of Gram-positive and Gram-negative bacteria. The peptide is monomeric in aqueous solution and contains one α-helix and triple-stranded β-sheet, which form cysteine-stabilized αβ motif (CSαβ) previously found in other plant defensins. The sterically neighboring loop1 and loop3 protrude from the defensin core and demonstrate significant mobility on the μs-ms timescale. Lc-def does not bind to the zwitterionic lipid (POPC) vesicles but interacts with the partially anionic (POPC/DOPG, 7:3) membranes under low-salt conditions. The Lc-def antifungal activity might be mediated through electrostatic interaction with anionic lipid components of fungal membranes.

  5. Shenkarev Z.O., Panteleev P.V., Balandin S.V., Gizatullina A.K., Altukhov D.A., Finkina E.I., Kokryakov V.N., Arseniev A.S., Ovchinnikova T.V. (2012). Recombinant expression and solution structure of antimicrobial peptide aurelin from jellyfish Aurelia aurita. Biochem. Biophys. Res. Commun. 429 (1-2), 63–9 [+]

    Aurelin is a 40-residue cationic antimicrobial peptide isolated from the mezoglea of a scyphoid jellyfish Aurelia aurita. Aurelin and its (15)N-labeled analogue were overexpressed in Escherichia coli and purified. Antimicrobial activity of the recombinant peptide was examined, and its spatial structure was studied by NMR spectroscopy. Aurelin represents a compact globule, enclosing one 3(10)-helix and two α-helical regions cross-linked by three disulfide bonds. The peptide binds to anionic lipid (POPC/DOPG, 3:1) vesicles even at physiological salt concentration, it does not interact with zwitterionic (POPC) vesicles and interacts with the DPC micelle surface with moderate affinity via two α-helical regions. Although aurelin shows structural homology to the BgK and ShK toxins of sea anemones, its surface does not possess the "functional dyad" required for the high-affinity interaction with the K(+)-channels. The obtained data permit to correlate the modest antibacterial properties and membrane activity of aurelin.

  6. Akkerdaas J., Finkina E.I., Balandin S.V., SantosMagadán S., Knulst A., FernandezRivas M., Asero R., vanRee R., Ovchinnikova T.V. (2011). Lentil (Lens culinaris) Lipid Transfer Protein Len c 3: A Novel Legume Allergen. International archives of allergy and immunology 157 (1), 51–57 [+]

    Background: Lentils are increasingly consumed in many parts of the world.Two allergens, Len c 1 and 2, have been reported previously. Recently, peanut and green bean lipid transfer proteins (LTPs) have been identified as the first two members of an important group of allergens that might be associated with severe food allergies. Objective: To investigate lentil LTP as a potential new allergen. Methods: Efficacy of LTP extraction was monitored at different acidic pH values, using immunoblotting with cross-reactive anti-peach LTP antiserum. Natural LTP was purified from lentil extract and expressed as recombinant allergen in Escherichia coli. Sera from 10 lentil-allergic and/or -sensitized patients (Spain: 6, Italy: 1 and the Netherlands: 3) were used to further characterize lentil LTP. Results: Natural lentil LTP, purified from the homogenized germinated seeds and optimally extracted at pH 3, was identified and designated as allergen Len c 3. By CAP, 9/10 sera showed specific IgE to Len c 3. Recombinant (r) Len c 3 was successfully purified. The natural (n) Len c 3 CAP was completely inhibited by rLen c 3/rPru p 3. IgE binding to lentil pH 3 extract blot was completely inhibited by rLen c 3. Conclusion: The availability of immunochemically active nLen/rLen c 3 as a novel legume allergen facilitates further development and implementation of a third (next to peanut and green bean) legume LTP in component-resolved diagnosis strategies and contributes to evaluate the clinical importance of legume LTPs. Preferential extraction of Len c 3 (pH 3) will affect the production of sensitive extract-based diagnostic tests.

  7. Shenkarev Z.O., Balandin S.V., Trunov K.I., Paramonov A.S., Sukhanov S.V., Barsukov L.I., Arseniev A.S., Ovchinnikova T.V. (2011). Molecular mechanism of action of β-hairpin antimicrobial peptide arenicin: oligomeric structure in dodecylphosphocholine micelles and pore formation in planar lipid bilayers. Biochemistry 50 (28), 6255–65 [+]

    The membrane-active, cationic, β-hairpin peptide, arenicin, isolated from marine polychaeta Arenicola marina exhibits a broad spectrum of antimicrobial activity. The peptide in aqueous solution adopts the significantly twisted β-hairpin conformation without pronounced amphipathicity. To assess the mechanism of arenicin action, the spatial structure and backbone dynamics of the peptide in membrane-mimicking media and its pore-forming activity in planar lipid bilayers were studied. The spatial structure of the asymmetric arenicin dimer stabilized by parallel association of N-terminal strands of two β-hairpins was determined using triple-resonance nuclear magnetic resonance (NMR) spectroscopy in dodecylphosphocholine (DPC) micelles. Interaction of arenicin with micelles and its oligomerization significantly decreased the right-handed twist of the β-hairpin, increased its amphipathicity, and led to stabilization of the peptide backbone on a picosecond to nanosecond time scale. Relaxation enhancement induced by water-soluble (Mn(2+)) and lipid-soluble (16-doxylstearate) paramagnetic probes pointed to the dimer transmembrane arrangement. Qualitative NMR and circular dichroism study of arenicin-2 in mixed DPC/1,2-dioleoyl-sn-glycero-3-phosphoglycerol bicelles, sodium dodecyl sulfate micelles, and lipid vesicles confirmed that a similar dimeric assembly of the peptide was retained in membrane-mimicking systems containing negatively charged lipids and detergents. Arenicin-induced conductance was dependent on the lipid composition of the membrane. Arenicin low-conductivity pores were detected in the phosphatidylethanolamine-containing lipid mixture, whereas the high-conductivity pores were observed in an exclusively anionic lipid system. The measured conductivity levels agreed with the model in which arenicin antimicrobial activity was mediated by the formation of toroidal pores assembled of two, three, or four β-structural peptide dimers and lipid molecules. The structural transitions involved in arenicin membrane-disruptive action are discussed.

  8. Salnikov E.S., Aisenbrey C., Balandin S.V., Zhmak M.N., Ovchinnikova T.V., Bechinger B. (2011). Structure and alignment of the membrane-associated antimicrobial peptide arenicin by oriented solid-state NMR spectroscopy. Biochemistry 50 (18), 3784–95 [+]

    The antimicrobial arenicin peptides are cationic amphipathic sequences that strongly interact with membranes. Through a cystine ring closure a cyclic β-sheet structure is formed in aqueous solution, which persists when interacting with model membranes. In order to investigate the conformation, interactions, dynamics, and topology of their bilayer-associated states, arenicin 1 and 2 were prepared by chemical solid-phase peptide synthesis or by bacterial overexpression, labeled selectively or uniformly with (15)N, reconstituted into oriented membranes, and investigated by proton-decoupled (31)P and (15)N solid-state NMR spectroscopy. Whereas the (31)P NMR spectra indicate that the peptide induces orientational disorder at the level of the phospholipid head groups, the (15)N chemical shift spectra agree well with a regular β-sheet conformation such as the one observed in micellar environments. In contrast, the data do not fit the twisted β-sheet structure found in aqueous buffer. Furthermore, the chemical shift distribution is indicative of considerable conformational and/or topological heterogeneity when at the same time the (15)N NMR spectra exclude alignments of the peptide where the β-sheet lies side ways on the membrane surface. The ensemble of experimental constraints, the amphipathic character of the peptide, and in particular the distribution of the six arginine residues are in agreement with a boatlike dimer structure, similar or related to the one observed in micellar solution, that floats on the membrane surface with the possibility to oligomerize into higher order structures and/or to insert in a transmembrane fashion.

  9. Shenkarev Z.O., Finkina E.I., Nurmukhamedova E.K., Balandin S.V., Mineev K.S., Nadezhdin K.D., Yakimenko Z.A., Tagaev A.A., Temirov Y.V., Arseniev A.S., Ovchinnikova T.V. (2010). Isolation, structure elucidation, and synergistic antibacterial activity of a novel two-component lantibiotic lichenicidin from Bacillus licheniformis VK21. Biochemistry 49 (30), 6462–72 [+]

    A novel synergetic lantibiotic pair, Lchalpha(3249.51 Da) and Lchbeta(3019.36 Da), termed lichenicidin VK21, was isolated from the producer strain Bacillus licheniformis VK21. Chemical and spatial structures of Lchalphaand Lchbeta were determined. Each peptide contains 31 amino acid residues linked by 4 intramolecular thioether bridges and the N-terminal 2-oxobutyryl group. Spatial structures of Lchalpha and Lchbetawere studied by NMR spectroscopy in methanol solution. Lchalpha peptide displays structural homology with mersacidin-like lantibiotics and involves relatively well-structured N- and C-terminal domains connected by a flexible loop stabilized by thioether bridge Ala11-S-Ala21. In contrast, the Lchbetapeptide represents prolonged hydrophobic alpha-helix flanked with more flexible N- and C-terminal domains. A lantibiotic cluster of the Bacillus licheniformis VK21 genome which comprises the structural genes, lchA1 and lchA2, encoding the lantibiotics precursors, as well as the gene of a modifying enzyme lchM1, was amplified and sequenced. The mature peptides, Lchalphaand Lchbetainteract synergistically to possess antibiotic activity against Gram-positive bacteria within a nanomolar concentration range, though the individual peptides were shown to be active at micromolar concentrations. Our results afford molecular insight into mechanism of lichenicidin VK21 action.

  10. Ovchinnikova T.V., Shenkarev Z.O., Balandin S.V., Nadezhdin K.D., Paramonov A.S., Kokryakov V.N., Arseniev A.S. (2008). Molecular insight into mechanism of antimicrobial action of the beta-hairpin peptide arenicin: specific oligomerization in detergent micelles. Biopolymers 89 (5), 455–64 [+]

    Arenicins are 21-residue cationic antimicrobial peptides isolated from marine polychaeta Arenicola marina. The peptides exhibit potent broad-spectrum antimicrobial activity. In water solution arenicin-2 adopts a beta-hairpin conformation, stabilized by one disulfide and nine hydrogen bonds. To determine the propensity for the peptide oligomerization in membrane mimetic systems, the recombinant arenicin-2 was overexpressed as a fused form in Escherichia coli. The arenicin-2 oligomerization and intermolecular packing in membrane mimicking environment were investigated using high-resolution NMR spectroscopy. The present studies show that arenicin-2 preserves a beta-hairpin structure and forms asymmetric dimers upon incorporation into the dodecylphosphocholine micelle. Two monomers of arenicin-2 are aligned parallel to each other by the N-terminal strands of the beta-hairpin (CN upward arrow upward arrowNC type of association). Polyacrylamide gel electrophoresis analysis indicated that in environment of anionic SDS micelles the arenicin-2 might undergo further oligomerization and form tetramers. Our results afford further molecular insight into possible mechanism of antimicrobial action of arenicins.

  11. Ovchinnikova T.V., Shenkarev Z.O., Nadezhdin K.D., Balandin S.V., Zhmak M.N., Kudelina I.A., Finkina E.I., Kokryakov V.N., Arseniev A.S. (2007). Recombinant expression, synthesis, purification, and solution structure of arenicin. Biochem. Biophys. Res. Commun. 360 (1), 156–62 [+]

    Arenicins are 21-residue cationic antimicrobial peptides, isolated from marine polychaeta Arenicola marina. In order to determine a high-resolution three-dimensional structure of arenicin-2, the recombinant peptide was overexpressed as a fused form in Escherichia coli. Both arenicin isoforms were synthesized using the Fmoc-based solid-phase strategy. Recombinant and synthetic arenicins were purified, and their antimicrobial and spectroscopic properties were analyzed. NMR investigation shows that in water solution arenicin-2 displays a prolonged beta-hairpin, formed by two antiparallel beta-strands and stabilized by one disulfide and nine hydrogen bonds. A significant right-handed twist in the beta-sheet is deprived the peptide surface of amphipathicity. CD spectroscopic analysis indicates that arenicin-2 binds to the SDS and DPC micelles, and conformation of the peptide is significantly changed upon binding. Arenicin strongly binds to anionic lipid (POPE/POPG) vesicles in contrast with zwitterionic (POPC) ones. These results suggest that arenicins are membrane active peptides and point to possible mechanism of their selectivity toward bacterial cells.

  12. Finkina E.I., Balandin S.V., Serebryakova M.V., Potapenko N.A., Tagaev A.A., Ovchinnikova T.V. (2007). Purification and primary structure of novel lipid transfer proteins from germinated lentil (Lens culinaris) seeds. Biochemistry Mosc. 72 (4), 430–8 [+]

    A subfamily of eight novel lipid transfer proteins designated as Lc-LTP1-8 was found in the lentil Lens culinaris. Lc-LTP2, Lc-LTP4, Lc-LTP7, and Lc-LTP8 were purified from germinated lentil seeds, and their molecular masses (9268.7, 9282.7, 9121.5, 9135.5 daltons) and complete amino acid sequences were determined. The purified proteins consist of 92-93 amino acid residues, have four disulfide bonds, and inhibit growth of Agrobacterium tumefaciens. Total RNA was isolated from germinated lentil seeds, RT-PCR and cloning were performed, and the cDNAs of six LTPs were sequenced. Precursor 116-118-residue proteins with 24-25-residue signal peptides were found, and two of them are purified proteins Lc-LTP2 and Lc-LTP4.

  13. Ovchinnikova T.V., Balandin S.V., Aleshina G.M., Tagaev A.A., Leonova Y.F., Krasnodembsky E.D., Menshenin A.V., Kokryakov V.N. (2006). Aurelin, a novel antimicrobial peptide from jellyfish Aurelia aurita with structural features of defensins and channel-blocking toxins. Biochem. Biophys. Res. Commun. 348 (2), 514–23 [+]

    A novel 40-residue antimicrobial peptide, aurelin, exhibiting activity against Gram-positive and Gram-negative bacteria, was purified from the mesoglea of a scyphoid jellyfish Aurelia aurita by preparative gel electrophoresis and RP-HPLC. Molecular mass (4296.95 Da) and complete amino acid sequence of aurelin (AACSDRAHGHICESFKSFCKDSGRNGVKLRANCKKTCGLC) were determined. Aurelin has six cysteines forming three disulfide bonds. The total RNA was isolated from the jellyfish mesoglea, RT-PCR and cloning were performed, and cDNA was sequenced. A 84-residue preproaurelin contains a putative signal peptide (22 amino acids) and a propiece of the same size (22 amino acids). Aurelin has no structural homology with any previously identified antimicrobial peptides but reveals partial similarity both with defensins and K+ channel-blocking toxins of sea anemones and belongs to ShKT domain family.

  14. Ovchinnikova T.V., Aleshina G.M., Balandin S.V., Krasnosdembskaya A.D., Markelov M.L., Frolova E.I., Leonova Y.F., Tagaev A.A., Krasnodembsky E.G., Kokryakov V.N. (2004). Purification and primary structure of two isoforms of arenicin, a novel antimicrobial peptide from marine polychaeta Arenicola marina. FEBS Lett. 577 (1-2), 209–14 [+]

    Two novel 21-residue antimicrobial peptides, arenicin-1 and arenicin-2, exhibiting activity against Gram-positive and Gram-negative bacteria and fungi, were purified from coelomocytes of marine polychaeta Arenicola marina (lugworm) by preparative gel electrophoresis and RP-HPLC. Molecular masses (2758.3 and 2772.3 Da) and complete amino acid sequences (RWCVYAYVRVRGVLVRYRRCW and RWCVYAYVRIRGVLVRYRRCW) were determined for each isoform. Each arenicin has one disulfide bond (Cys3-Cys20). The total RNA was isolated from the lugworm coelomocytes, RT-PCR and cloning were performed, and cDNA was sequenced. A 202-residue preproarenicin contains a putative signal peptide (25 amino acids) and a long prodomain. Arenicins have no structure similarity to any previously identified antimicrobial peptides.