Шенкарёв Захар Олегович

Доктор физико-математических наук


Ведущий научный сотрудник (Группа структурной биологии ионных каналов)

Эл. почта: zakhar-shenkarev@yandex.ru

Избранные публикации

  1. Paramonov A.S., Lyukmanova E.N., Myshkin M.Y., Shulepko M.A., Kulbatskii D.S., Petrosian N.S., Chugunov A.O., Dolgikh D.A., Kirpichnikov M.P., Arseniev A.S., Shenkarev Z.O. (2017). NMR investigation of the isolated second voltage-sensing domain of human Nav1.4 channel. Biochim. Biophys. Acta 1859 (3), 493–506 [+]

    Voltage-gated Na(+) channels are essential for the functioning of cardiovascular, muscular, and nervous systems. The α-subunit of eukaryotic Na(+) channel consists of ~2000 amino acid residues and encloses 24 transmembrane (TM) helices, which form five membrane domains: four voltage-sensing (VSD) and one pore domain. The structural complexity significantly impedes recombinant production and structural studies of full-sized Na(+) channels. Modular organization of voltage-gated channels gives an idea for studying of the isolated second VSD of human skeletal muscle Nav1.4 channel (VSD-II). Several variants of VSD-II (~150a.a., four TM helices) with different N- and C-termini were produced by cell-free expression. Screening of membrane mimetics revealed low stability of VSD-II samples in media containing phospholipids (bicelles, nanodiscs) associated with the aggregation of electrically neutral domain molecules. The almost complete resonance assignment of (13)C,(15)N-labeled VSD-II was obtained in LPPG micelles. The secondary structure of VSD-II showed similarity with the structures of bacterial Na(+) channels. The fragment of S4 TM helix between the first and second conserved Arg residues probably adopts 310-helical conformation. Water accessibility of S3 helix, observed by the Mn(2+) titration, pointed to the formation of water-filled crevices in the micelle embedded VSD-II. (15)N relaxation data revealed characteristic pattern of μs-ms time scale motions in the VSD-II regions sharing expected interhelical contacts. VSD-II demonstrated enhanced mobility at ps-ns time scale as compared to isolated VSDs of K(+) channels. These results validate structural studies of isolated VSDs of Na(+) channels and show possible pitfalls in application of this 'divide and conquer' approach.

    ID:1921
  2. Panteleev P.V., Myshkin M.Y., Shenkarev Z.O., Ovchinnikova T.V. (2017). Dimerization of the antimicrobial peptide arenicin plays a key role in the cytotoxicity but not in the antibacterial activity. Biochem. Biophys. Res. Commun. 482 (4), 1320–1326 [+]

    The β-hairpin antimicrobial peptides arenicins from marine polychaeta Arenicola marina exhibit a broad spectrum of antimicrobial activity and high cytotoxicity. In this study the biological activities of arenicin-1 and its therapeutically valuable analog Ar-1[V8R] were investigated. The peptide Ar-1[V8R] displays significantly reduced cytotoxicity against mammalian cells relative to the wild-type arenicin-1. At the same time, both peptides exhibit similar antibacterial activities and kinetics of bacterial membrane permeabilization. Comparative NMR analysis of the peptides spatial structures in water and membrane-mimicking environment showed that Ar-1[V8R] in contrast to arenicin has significantly lower dimerization propensity. Thus, dimerization of the antimicrobial peptide arenicin plays a key role in the cytotoxicity but not in the antibacterial activity.

    ID:1792
  3. Парамонов А.С., Кульбацкий Д.С., Локтюшов Е.В., Царев А.В., Долгих Д.А., Шенкарёв З.О., Кирпичников М.П., Люкманова Е.Н. (2017). Рекомбинантная продукция и исследование структуры белков человека Lypd6 и Lypd6b. Биоорг. хим. 43 (6), 620–630 [+]

    Белки человека Lypd6 и Lypd6b экспрессируются во многих тканях и имеют высокую степень гомо-
    логии аминокислотной последовательности (~ 54%). Оба белка в отличие от других белков семейства
    Ly6/uPAR имеют дополнительные протяженные N- и С-концевые аминокислотные последователь-
    ности, примыкающие к трехпетельному LU-домену, роль которых на данный момент не изучена. Из-
    вестно, что Lypd6 увеличивает амплитуду токов кальция, индуцированных никотином в нейронах
    тройничного нерва мыши. Lypd6 рыбки Danio rerio участвует в регуляции Wnt/β-катенин сигнального
    каскада, и блокирование экспрессии гена lypd6 приводит к нарушению эмбрионального развития.
    Экспрессия Lypd6b в ооцитах X. laevis повышает чувствительность никотиновых ацетилхолиновых ре-
    цепторов к ацетилхолину и увеличивает скорость их десенситизации. Молекулярные механизмы дей-
    ствия, равно как и пространственная структура Lypd6 и Lypd6b, до сих пор не изучены. В представ-
    ленной работе получены и экспрессированы гены водорастворимых аналогов трехпетельных белков
    человека Lypd6 и Lypd6b, не содержащих N-концевые последовательности (rLypd6 и rLypd6b), а также
    Lypd6 с N-концевой последовательностью – N-rLypd6. Белки получали в виде цитоплазматических
    телец включения в E. coli с последующей солюбилизацией в денатурирующих условиях и ренатураци-
    ей. С целью оптимизации выхода рекомбинантных белков был проведен поиск условий ренатурации.
    Анализ полученных препаратов N-rLypd6, rLypd6 и rLypd6b методами ЯМР-спектроскопии показал,
    что N-rLypd6, возможно, не структурирован. Получение миллиграммовых количеств изотопно-ме-
    ченных вариантов rLypd6 и rLypd6b позволило охарактеризовать вторичную структуру этих белков
    и исследовать внутримолекулярную подвижность. Установлено, что rLypd6 и rLypd6b обладают струк-
    турными элементами, характерными для трехпетельных белков семейства Ly6/uPAR с некоторыми
    уникальными особенностями, такими как наличие дополнительной дисульфидной связи в третьей
    петле и спиральных участков в первой и третьей петлях.

    ID:1924
  4. Мышкин М.Ю., Парамонов А.С., Кульбацкий Д.С., Люкманова Е.Н., Кирпичников М.П., Шенкарёв З.О. (2017). ПОДХОД “РАЗДЕЛЯЙ И ВЛАСТВУЙ” ДЛЯ СТРУКТУРНЫХ ИССЛЕДОВАНИЙ МУЛЬТИДОМЕННЫХ ИОННЫХ КАНАЛОВ НА ПРИМЕРЕ ИЗОЛИРОВАННЫХ ПОТЕНЦИАЛ-ЧУВСТВИТЕЛЬНЫХ ДОМЕНОВ КАНАЛОВ Kv2.1 И Nav1.4 ЧЕЛОВЕКА1. Биоорг. хим. 43 (6), 608–619 [+]

    Потенциал-зависимые K+- и Na+-ионные каналы вовлечены в широкий спектр физиологических
    процессов, включая возбудимость сердечных, мышечных и нервных клеток, а также секрецию гор-
    монов и нейромедиаторов. Эти каналы имеют модульную структуру и состоят из пяти мембранных
    доменов: четырех потенциал-чувствительных доменов (ПЧД) и одного порового домена. На ПЧД раз-
    личных каналов локализованы уникальные сайты связывания с лигандами, поэтому ПЧД рассматри-
    ваются в качестве перспективных фармакологических мишеней. Модульная организация ионных ка-
    налов позволяет ставить задачи по структурным ЯМР-исследованиям изолированных ПЧД отдельно
    от поры. В настоящей работе рассмотрена возможность таких исследований на примере ПЧД канала
    Kv2.1 человека и первого ПЧД канала Nav1.4 человека. Разработаны сопряженные системы бескле-
    точного синтеза на основе бактериального экстракта S30 из E. coli, позволяющие получать милли-
    граммовые количества препаратов ПЧД, включая меченые стабильными изотопами аналоги. Важным
    этапом ЯМР-исследований является подбор мембраномоделирующей среды, обеспечивающей дол-
    говременную стабильность природной структуры мембранного белка в растворе и высокое качество
    ЯМР-спектров. Скрининг различных сред показал, что домены каналов Kv2.1 и Nav1.4 нестабильны
    в средах, содержащих фосфолипиды: мицеллах короткоцепочечного липида DC7PC и липид-детер-
    гентных бицеллах на основе цвиттер-ионных или анионных насыщенных липидов (DMPC и DMPG).
    Показано, что оптимальной средой для структурных ЯМР-исследований являются смеси цвиттер-
    ионного и слабокатионного детергентов (FOS-12/LDAO). Однако, несмотря на высокое качество
    спектров, образец ПЧД канала Nav1.4 в окружении FOS-12/LDAO необратимо агрегировал в течение
    нескольких дней. Вероятно, ПЧД K+- и Na+-каналов человека не являются полностью автономными
    мембранными доменами и для их стабилизации необходимы контакты с другими доменами канала.

    ID:1925
  5. Васильева Н.А., Локтюшов Е.В., Бычков М.Л., Шенкарёв З.О., Люкманова Е.Н. (2017). Трехпетельные белки семейства Ly6/uPAR: функциональное многообразие в рамках одного структурного мотива. Успехи биологической химии 57, 303–330 ID:1931
  6. Shulepko M.A., Lyukmanova E.N., Shenkarev Z.O., Dubovskii P.V., Astapova M.V., Feofanov A.V., Arseniev A.S., Utkin Y.N., Kirpichnikov M.P., Dolgikh D.A. (2016). Towards universal approach for bacterial production of three-finger Ly6/uPAR proteins: Case study of cytotoxin I from cobra N. oxiana. Protein Expr. Purif. 130, 13–20 [+]

    Cytotoxins or cardiotoxins is a group of polycationic toxins from cobra venom belonging to the 'three-finger' protein superfamily (Ly6/uPAR family) which includes small β-structural proteins (60-90 residues) with high disulfide bond content (4-5 disulfides). Due to a high cytotoxic activity for cancer cells, cytotoxins are considered as potential anticancer agents. Development of the high-throughput production methods is required for the prospective applications of cytotoxins. Here, efficient approach for bacterial production of recombinant analogue of cytotoxin I from N. oxiana containing additional N-terminal Met-residue (rCTX1) was developed. rCTX1 was produced in the form of E. coli inclusion bodies. Refolding in optimized conditions provided ∼6 mg of correctly folded protein from 1 L of bacterial culture. Cytotoxicity of rCTX1 for C6 rat glioma cells was found to be similar to the activity of wild type CTX1. The milligram quantities of (13)C,(15)N-labeled rCTX1 were obtained. NMR study confirmed the similarity of the spatial structures of recombinant and wild-type toxins. Additional Met residue does not perturb the overall structure of the three-finger core. The analysis of available data for different Ly6/uPAR proteins of snake and human origin revealed that efficiency of their folding in vitro is correlated with the number of proline residues in the third loop and the surface area of hydrophobic residues buried within the protein interior. The obtained data indicate that hydrophobic core is important for the folding of proteins with high disulfide bond content. Developed expression method opens new possibilities for structure-function studies of CTX1 and other related three-finger proteins.

    ID:1599
  7. Lyukmanova E.N., Shulepko M.A., Shenkarev Z.O., Kasheverov I.E., Chugunov A.O., Kulbatskii D.S., Myshkin M.Y., Utkin Y.N., Efremov R.G., Tsetlin V.I., Arseniev A.S., Kirpichnikov M.P., Dolgikh D.A. (2016). Central loop of non-conventional toxin WTX from Naja kaouthia is important for interaction with nicotinic acetylcholine receptors. Toxicon 119, 274–9 [+]

    'Three-finger' toxin WTX from Naja kaouthia interacts with nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs). Mutagenesis and competition experiments with (125)I-α-bungarotoxin revealed that Arg31 and Arg32 residues from the WTX loop II are important for binding to Torpedo californica and human α7 nAChRs. Computer modeling suggested that loop II occupies the orthosteric binding site at α7 nAChR. The similar toxin interface was previously described as a major determinant of allosteric interactions with mAChRs.

    ID:1598
  8. Lyukmanova E.N., Shulepko M.A., Shenkarev Z.O., Bychkov M.L., Paramonov A.S., Chugunov A.O., Kulbatskii D.S., Arvaniti M., Dolejsi E., Schaer T., Arseniev A.S., Efremov R.G., Thomsen M.S., Dolezal V., Bertrand D., Dolgikh D.A., Kirpichnikov M.P. (2016). Secreted Isoform of Human Lynx1 (SLURP-2): Spatial Structure and Pharmacology of Interactions with Different Types of Acetylcholine Receptors. Sci Rep 6, 30698 [+]

    Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the α3β2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we describe the structure and pharmacology of a recombinant analogue of SLURP-2. Nuclear magnetic resonance spectroscopy revealed a 'three-finger' fold of SLURP-2 with a conserved β-structural core and three protruding loops. Affinity purification using cortical extracts revealed that SLURP-2 could interact with the α3, α4, α5, α7, β2, and β4 nAChR subunits, revealing its broader pharmacological profile. SLURP-2 inhibits acetylcholine-evoked currents at α4β2 and α3β2-nAChRs (IC50 ~0.17 and >3 μM, respectively) expressed in Xenopus oocytes. In contrast, at α7-nAChRs, SLURP-2 significantly enhances acetylcholine-evoked currents at concentrations <1 μM but induces inhibition at higher concentrations. SLURP-2 allosterically interacts with human M1 and M3 muscarinic acetylcholine receptors (mAChRs) that are overexpressed in CHO cells. SLURP-2 was found to promote the proliferation of human oral keratinocytes via interactions with α3β2-nAChRs, while it inhibited cell growth via α7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the 'classical' orthosteric agonist/antagonist binding sites at α7 and α3β2-nAChRs.

    ID:1597
  9. Lyukmanova E.N., Shulepko M.A., Kudryavtsev D., Bychkov M.L., Kulbatskii D.S., Kasheverov I.E., Astapova M.V., Feofanov A.V., Thomsen M.S., Mikkelsen J.D., Shenkarev Z.O., Tsetlin V.I., Dolgikh D.A., Kirpichnikov M.P. (2016). Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor. PLoS ONE 11 (2), e0149733 [+]

    SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1) differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC50 ~ 4 nM) of human oral keratinocytes (Het-1A cells). Application of mecamylamine and atropine,-non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the hypothesis that the antiproliferative activity of SLURP-1 is related to 'metabotropic' signaling pathway through α7-nAChR, that activates intracellular signaling cascades without opening the receptor channel.

    ID:1420
  10. Lyukmanova E.N., Shenkarev Z.O., Shulepko M.A., Paramonov A.S., Chugunov A.O., Janickova H., Dolejsi E., Dolezal V., Utkin Y.N., Tsetlin V.I., Arseniev A.S., Efremov R.G., Dolgikh D.A., Kirpichnikov M.P. (2015). Structural Insight into Specificity of Interactions between Nonconventional Three-finger Weak Toxin from Naja kaouthia (WTX) and Muscarinic Acetylcholine Receptors. J. Biol. Chem. 290 (39), 23616–30 [+]

    Weak toxin from Naja kaouthia (WTX) belongs to the group of nonconventional "three-finger" snake neurotoxins. It irreversibly inhibits nicotinic acetylcholine receptors and allosterically interacts with muscarinic acetylcholine receptors (mAChRs). Using site-directed mutagenesis, NMR spectroscopy, and computer modeling, we investigated the recombinant mutant WTX analogue (rWTX) which, compared with the native toxin, has an additional N-terminal methionine residue. In comparison with the wild-type toxin, rWTX demonstrated an altered pharmacological profile, decreased binding of orthosteric antagonist N-methylscopolamine to human M1- and M2-mAChRs, and increased antagonist binding to M3-mAChR. Positively charged arginine residues located in the flexible loop II were found to be crucial for rWTX interactions with all types of mAChR. Computer modeling suggested that the rWTX loop II protrudes to the M1-mAChR allosteric ligand-binding site blocking the entrance to the orthosteric site. In contrast, toxin interacts with M3-mAChR by loop II without penetration into the allosteric site. Data obtained provide new structural insight into the target-specific allosteric regulation of mAChRs by "three-finger" snake neurotoxins.

    ID:1394
  11. Berkut A.A., Peigneur S., Myshkin M.Y., Paramonov A.S., Lyukmanova E.N., Arseniev A.S., Grishin E.V., Tytgat J., Shenkarev Z.O., Vassilevski A.A. (2015). Structure of Membrane-active Toxin from Crab Spider Heriaeus melloteei Suggests Parallel Evolution of Sodium Channel Gating Modifiers in Araneomorphae and Mygalomorphae. J. Biol. Chem. 290 (1), 492–504 [+]

    We present a structural and functional study of a sodium channel activation inhibitor from crab spider venom. Hm-3 is an insecticidal peptide toxin consisting of 35 amino acid residues from the spider Heriaeus melloteei (Thomisidae). We produced Hm-3 recombinantly in Escherichia coli and determined its structure by NMR spectroscopy. Typical for spider toxins, Hm-3 was found to adopt the so-called "inhibitor cystine knot" or "knottin" fold stabilized by three disulfide bonds. Its molecule is amphiphilic with a hydrophobic ridge on the surface enriched in aromatic residues and surrounded by positive charges. Correspondingly, Hm-3 binds to both neutral and negatively charged lipid vesicles. Electrophysiological studies showed that at a concentration of 1 μm Hm-3 effectively inhibited a number of mammalian and insect sodium channels. Importantly, Hm-3 shifted the dependence of channel activation to more positive voltages. Moreover, the inhibition was voltage-dependent, and strong depolarizing prepulses attenuated Hm-3 activity. The toxin is therefore concluded to represent the first sodium channel gating modifier from an araneomorph spider and features a "membrane access" mechanism of action. Its amino acid sequence and position of the hydrophobic cluster are notably different from other known gating modifiers from spider venom, all of which are described from mygalomorph species. We hypothesize parallel evolution of inhibitor cystine knot toxins from Araneomorphae and Mygalomorphae suborders.

    ID:1138
  12. 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.

    ID:1105
  13. Shestakova T.S., Shenkarev Z.O., Deev S.L., Chupakhin O.N., Khalymbadzha I.A., Rusinov V.L., Arseniev A.S. (2013). Long-range 1H-15N J couplings providing a method for direct studies of the structure and azide-tetrazole equilibrium in a series of azido-1,2,4-triazines and azidopyrimidines. J. Org. Chem. 78 (14), 6975–82 [+]

    The selectively (15)N labeled azido-1,2,4-triazine 2*A and azidopyrimidine 4*A were synthesized by treating hydrazinoazines with (15)N-labeled nitrous acid. The synthesized compounds were studied by (1)H, (13)C, and (15)N NMR spectroscopy in DMSO, TFA, and DMSO/TFA solutions, where the azide-tetrazole equilibrium could lead to the formation of two tetrazoles (T, T') and one azide (A) isomer for each compound. The incorporation of the (15)N label led to the appearance of long-range (1)H-(15)N coupling constants (J(HN)), which can be measured easily by using amplitude-modulated 1D (1)H spin-echo experiments with selective inversion of the (15)N nuclei. The observed J(HN) patterns enable the unambiguous determination of the mode of fusion between the azole and azine rings in the two groups of tetrazole isomers (2*T', 4*T' and 2*T, 4*T), even for minor isoforms with a low concentration in solution. However, the azide isomers (2*A and 4*A) are characterized by the absence of detectable J(HN) coupling. The analysis of the J(HN) couplings in (15)N-labeled compounds provides a simple and efficient method for direct NMR studies of the azide-tetrazole equilibrium in solution.

    ID:1606
  14. Shenkarev Z.O., Paramonov A.S., Lyukmanova E.N., Gizatullina A.K., Zhuravleva A.V., Tagaev A.A., Yakimenko Z.A., Telezhinskaya I.N., Kirpichnikov M.P., Ovchinnikova T.V., Arseniev A.S. (2013). Peptaibol antiamoebin I: spatial structure, backbone dynamics, interaction with bicelles and lipid-protein nanodiscs, and pore formation in context of barrel-stave model. Chem. Biodivers. 10 (5), 838–63 [+]

    Antiamoebin I (Aam-I) is a membrane-active peptaibol antibiotic isolated from fungal species belonging to the genera Cephalosporium, Emericellopsis, Gliocladium, and Stilbella. In comparison with other 16-amino acid-residue peptaibols, e.g., zervamicin IIB (Zrv-IIB), Aam-I possesses relatively weak biological and channel-forming activities. In MeOH solution, Aam-I demonstrates fast cooperative transitions between right-handed and left-handed helical conformation of the N-terminal (1-8) region. We studied Aam-I spatial structure and backbone dynamics in the membrane-mimicking environment (DMPC/DHPC bicelles)(1) ) by heteronuclear (1) H,(13) C,(15) N-NMR spectroscopy. Interaction with the bicelles stabilizes the Aam-I right-handed helical conformation retaining significant intramolecular mobility on the ms-μs time scale. Extensive ms-μs dynamics were also detected in the DPC and DHPC micelles and DOPG nanodiscs. In contrast, Zrv-IIB in the DPC micelles demonstrates appreciably lesser mobility on the μs-ms time scale. Titration with Mn(2+) and 16-doxylstearate paramagnetic probes revealed Aam-I binding to the bicelle surface with the N-terminus slightly immersed into hydrocarbon region. Fluctuations of the Aam-I helix between surface-bound and transmembrane (TM) state were observed in the nanodisc membranes formed from the short-chain (diC12 : 0) DLPC/DLPG lipids. All the obtained experimental data are in agreement with the barrel-stave model of TM pore formation, similarly to the mechanism proposed for Zrv-IIB and other peptaibols. The observed extensive intramolecular dynamics explains the relatively low activity of Aam-I.

    ID:978
  15. Shenkarev Z.O., Lyukmanova E.N., Butenko I.O., Petrovskaya L.E., Paramonov A.S., Shulepko M.A., Nekrasova O.V., Kirpichnikov M.P., Arseniev A.S. (2013). Lipid-protein nanodiscs promote in vitro folding of transmembrane domains of multi-helical and multimeric membrane proteins. Biochim. Biophys. Acta 1828 (2), 776–84 [+]

    Production of helical integral membrane proteins (IMPs) in a folded state is a necessary prerequisite for their functional and structural studies. In many cases large-scale expression of IMPs in cell-based and cell-free systems results in misfolded proteins, which should be refolded in vitro. Here using examples of the bacteriorhodopsin ESR from Exiguobacterium sibiricum and full-length homotetrameric K(+) channel KcsA from Streptomyces lividans we found that the efficient in vitro folding of the transmembrane domains of the polytopic and multimeric IMPs could be achieved during the protein encapsulation into the reconstructed high-density lipoprotein particles, also known as lipid-protein nanodiscs. In this case the self-assembly of the IMP/nanodisc complexes from a mixture containing apolipoprotein, lipids and the partially denatured protein solubilized in a harsh detergent induces the folding of the transmembrane domains. The obtained folding yields showed significant dependence on the properties of lipids used for nanodisc formation. The largest recovery of the spectroscopically active ESR (~60%) from the sodium dodecyl sulfate (SDS) was achieved in the nanodiscs containing anionic saturated lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPG) and was approximately twice lower in the zwitterionic DMPC lipid. The reassembly of tetrameric KcsA from the acid-dissociated monomer solubilized in SDS was the most efficient (~80%) in the nanodiscs containing zwitterionic unsaturated lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The charged and saturated lipids provided lower tetramer quantities, and the lowest yield (<20%) was observed in DMPC. The overall yield of the ESR and KcsA folding was mainly restricted by the efficiency of the protein encapsulation into the nanodiscs.

    ID:802
  16. 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.

    ID:977
  17. 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.

    ID:535
  18. Deev S.L., Shenkarev Z.O., Shestakova T.S., Chupakhin O.N., Rusinov V.L., Arseniev A.S. (2010). Selective (15)N-labeling and analysis of (13)C-(15)N J couplings as an effective tool for studying the structure and azide-tetrazole equilibrium in a series of tetrazolo[1,5-b][1,2,4]triazines and tetrazolo[1,5-a]pyrimidines. J. Org. Chem. 75 (24), 8487–97 [+]

    Two general methods for the selective incorporation of an (15)N-label in the azole ring of tetrazolo[1,5-b][1,2,4]triazines and tetrazolo[1,5-a]pyrimidines were developed. The first approach included treatment of azinylhydrazides with (15)N-labeled nitrous acid, and the second approach was based on fusion of the azine ring to [2-(15)N]-5-aminotetrazole. The synthesized compounds were studied by (1)H, (13)C, and (15)N NMR spectroscopy in both DMSO and TFA solution, in which the azide-tetrazole equilibrium is shifted to tetrazole and azide forms, respectively. Incorporation of the (15)N-label led to the appearance of (13)C-(15)N J coupling constants (J(CN)), which can be measured easily using either 1D (13)C spectra with selective (15)N decoupling or with amplitude modulated 1D (13)C spin-echo experiments with selective inversion of the (15)N nuclei. The observed J(CN) patterns permit unambiguous determination of the type of fusion between the azole and azine rings in tetrazolo[1,5-b][1,2,4]triazine derivatives. Joint analysis of J(CN) patterns and (15)N chemical shifts was found to be the most efficient way to study the azido-tetrazole equilibrium.

    ID:1607
  19. 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.

    ID:359
  20. Shenkarev Z.O., Lyukmanova E.N., Solozhenkin O.I., Gagnidze I.E., Nekrasova O.V., Chupin V.V., Tagaev A.A., Yakimenko Z.A., Ovchinnikova T.V., Kirpichnikov M.P., Arseniev A.S. (2009). Lipid-protein nanodiscs: possible application in high-resolution NMR investigations of membrane proteins and membrane-active peptides. Biochemistry Mosc. 74 (7), 756–65 [+]

    High-resolution NMR is shown to be applicable for investigation of membrane proteins and membrane-active peptides embedded into lipid-protein nanodiscs (LPNs). (15)N-Labeled K+-channel from Streptomyces lividans (KcsA) and the antibiotic antiamoebin I from Emericellopsis minima (Aam-I) were embedded in LPNs of different lipid composition. Formation of stable complexes undergoing isotropic motion in solution was confirmed by size-exclusion chromatography and (31)P-NMR spectroscopy. The 2D 1H-(15)N-correlation spectra were recorded for KcsA in the complex with LPN containing DMPC and for Aam-I in LPNs based on DOPG, DLPC, DMPC, and POPC. The spectra recorded were compared with those in detergent-containing micelles and small bicelles commonly used in high-resolution NMR spectroscopy of membrane proteins. The spectra recorded in LPN environments demonstrated similar signal dispersion but significantly increased (1)H(N) line width. The spectra of Aam-I embedded in LPNs containing phosphatidylcholine showed significant selective line broadening, thus suggesting exchange process(es) between several membrane-bound states of the peptide. (15)N relaxation rates were measured to obtain the effective rotational correlation time of the Aam-I molecule. The obtained value (approximately 40 nsec at 45 degrees C) is indicative of additional peptide motions within the Aam-I/LPN complex.

    ID:353
  21. Stavrakoudis A., Tsoulos I.G., Shenkarev Z.O., Ovchinnikova T.V. (2009). Molecular dynamics simulation of antimicrobial peptide arenicin-2: beta-hairpin stabilization by noncovalent interactions. Biopolymers 92 (3), 143–55 [+]

    Arenicin-2 is a 21 residue antimicrobial cyclic peptide, possessing one disulphide bond between residues Cys(3) and Cys(20). NMR and CD studies suggested that the structure of arenicin-2 in water represented a well formed, but highly twisted beta-hairpin. To investigate the spatial arrangement of the peptide side chains and to get a clear view of its possible amphipathic properties we performed molecular dynamics in explicit water. Four independent trajectories, 50 ns in length, were produced, starting from various initial conformations or by applying different simulation conditions. Arenicin-2 retained its beta-hairpin structure during simulations, although the residues close to strand ends were found to escape from the ideal hairpin conformation. The type I' beta-turn connecting the two strands fluctuated between type IV and II' beta-turn. Conversely, the right-handed twist of the beta-hairpin was well conserved with average twist value 203 degrees +/- 19 degrees per eight residues. Several nonbonded interactions, like hydrophobic interactions between aliphatic side chains, cation/pi-aromatic interactions, CH...pi aromatic bond and water bridges, contributed to the hairpin stabilization.

    ID:410
  22. 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.

    ID:414
  23. Lyukmanova E.N., Shenkarev Z.O., Paramonov A.S., Sobol A.G., Ovchinnikova T.V., Chupin V.V., Kirpichnikov M.P., Blommers M.J., Arseniev A.S. (2008). Lipid-protein nanoscale bilayers: a versatile medium for NMR investigations of membrane proteins and membrane-active peptides. J. Am. Chem. Soc. 130 (7), 2140–1 ID:356
  24. 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.

    ID:419
  25. Shenkarev Z.O., Paramonov A.S., Nadezhdin K.D., Bocharov E.V., Kudelina I.A., Skladnev D.A., Tagaev A.A., Yakimenko Z.A., Ovchinnikova T.V., Arseniev A.S. (2007). Antiamoebin I in methanol solution: rapid exchange between right-handed and left-handed 3(10)-helical conformations. Chem. Biodivers. 4 (6), 1219–42 [+]

    Antiamoebin I (Aam-I) is a membrane-active peptaibol antibiotic isolated from fungal species belonging to the genera Cephalosporium, Emericellopsis, Gliocladium, and Stilbella. Antiamoebin I has the amino acid sequence: Ac-Phe(1)-Aib-Aib-Aib-Iva-Gly-Leu-Aib(8)-Aib-Hyp-Gln-Iva-Hyp-Aib-Pro-Phl(16). By using the uniformly (13)C,(15)N-labeled sample of Aam-I, the set of conformationally dependent J couplings and (3h)J(NC) couplings through H-bonds were measured. Analysis of these data along with the data on magnetic nonequivalence of the (13)C(beta) nuclei (Deltadelta((13)C(beta))) in Aib and Iva residues allowed us to draw the univocal conclusion that the N-terminal part (Phe(1)-Gly(6)) of Aam-I in MeOH solution is in fast exchange between the right-handed and left-handed 3(10)-helical conformations, with an approximately equal population of both states. An additional conformational exchange process was found at the Aib(8) residue. The (15)N-NMR-relaxation and CD-spectroscopy measurements confirmed these findings. Molecular modeling and Monte Carlo simulations revealed that both exchange processes are correlated and coupled with significant hinge-bending motions around the Aib(8) residue. Our results explain relatively low activity of Aam-I with respect to other 15-amino acid residue peptaibols (for example, zervamicin) in functional and biological tests. The high dynamic 'propensity' possibly prevents both initial binding of the antiamoebin to the membrane and subsequent formation of stable ionic channels according to the barrel-stave mechanism.

    ID:418
  26. Shenkarev Z.O., Paramonov A.S., Balashova T.A., Yakimenko Z.A., Baru M.B., Mustaeva L.G., Raap J., Ovchinnikova T.V., Arseniev A.S. (2004). High stability of the hinge region in the membrane-active peptide helix of zervamicin: paramagnetic relaxation enhancement studies. Biochem. Biophys. Res. Commun. 325 (3), 1099–105 [+]

    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.

    ID:425
  27. Malakhov A.D., Skorobogatyi M.V., Prokhrenko I.A., Gontarev S.V., Kozhich D.T., Stetsenko D.A., Stepanova I.A., Shenkarev Z.O., Berlin Yu.A., Korshun V.A. (2004). 1-Phenylethynylpyrene and 9,10-bis(phenylethynyl)anthracene, useful fluorescent dyes for DNA labeling: excimer formation and energy transfer. Eur.J.Org.Chem  (6), 1298–1307 [+]

    A series of novel modifying reagents, including phosphoramidites and solid supports, have been synthesized, and used for the introduction of 1-(phenylethynyl)pyrene (PEPy) and 9,10-bis(phenylethynyl)anthracene (BPEA) fluorescent dyes into predetermined positions of synthetic oligonucleotides. These two fluorophores have been shown to constitute an energy donor−acceptor pair, and can be used as such in fluorescent oligonucleotide probes, designed for the detection and structural studies of nucleic acids. The sensitivity of the probe to duplex formation is demonstrated. The formation of the PEPy and BPEA excimers is reported for the first time on nucleic acids.

    ID:663