Отдел «Учебно-научный центр»

Руководитель: Овчинникова Татьяна Владимировна, д. х. н., профессор
+7 (495) 336-44-44 · ovch@ibch.ru

учебный центр, антимикробные пептиды, пептаиболы, антибиотики

Одним из приоритетых направлений деятельности ИБХ РАН является подготовка высококлассных молодых специалистов для физико-химической биологии и биотехнологии. С этой целью в 1982 году академиком Ю.А. Овчинниковым в Институте был организован специализированный Учебный центр, который стал первым учебно-научным центром в нашей стране, объединившем в себе усилия академического института и ряда высших учебных заведений Российской Федерации в деле воспитания молодого поколения исследователей в областибиоорганической химии, физико-химической биологии и биотехнологии. Подробно с образовательной деятельностью УНЦ ИБХ РАН можно ознакомиться на сайте ИБХ РАН в разделе "Обучение". Помимо огромной учебной работы  Отдел ведёт большую научно-исследовательскую работу по изучению функций и свойств нового класса антибиотиков на основе природных антимикробных пептидов.

Направления исследований

Тема НИР:«Конструирование лекарственных средств нового поколения на основе природных пептидных антибиотиков — молекулярных факторов врожденного иммунитета»
Руководитель темы: зав. отделом «Учебно-научный центр» Т. В. Овчинникова

В настоящее время медицина столкнулась с проблемой устойчивости патогенов к используемым в клинике антибиотикам, которые уже не всегда обеспечивают контроль над возбудителями инфекционных заболеваний. Стремительный рост резистентных бактериальных инфекций диктует необходимость поиска принципиально новых лекарств. Исследования Учебно-научного центра ИБХ РАН нацелены на комплексное структурно-фукциональное изучение новых природных пептидных антибиотиков, исследование механизмов их действия как молекулярных факторов врожденного иммунитета, разработку технологий получения их рекомбинантных и синтетических аналогов, предклинические испытания и создание на их основе антибиотиков нового поколения.

Выявление многообразных биологических функций эндогенных антимикробных пептидов (АМП) показывает, что эти природные антибиотики обладают способностью инактивировать широкий спектр микроорганизмов, в том числе бактерии, грибы, простейшие, оболочечные вирусы, и являются перспективными молекулами для создания новых антибиотических средств. АМП как молекулярные факторы системы врожденного иммунитета служат медиаторами фагоцитарных, воспалительных и стрессорных процессов. Учитывая наличие антибиотической и иммуномодулирующей активностей у природных АМП, многие зарубежные фармацевтические компании (в США, Канаде, Франции, Нидерландах и др.) приступили к созданию нового класса антибиотиков на их основе. Успешные клинические испытания прошли пептидные антибиотики для лечения сепсиса, менингита, пневмонии, язвенной болезни желудка, диабетической стопы, кандидоза, мукозита, гингивита, акне, импетиго, а также для заживления ожогов и ран, осложненных вторичной инфекцией.

Исследования Учебно-научного центра ИБХ РАН направлены на поиск, выделение, изучение структуры, биологических свойств и молекулярных механизмов действия природных пептидных антибиотиков, разработку биотехнологических способов их получения и проведение предклинических испытаний. Объектами исследований являются АМП бактериального, грибкового, растительного и животного происхождения, в том числе из бактерий Bаcillus licheniformis, грибов Emericellopsis salmocynnemata и Emericellopsis minima, чечевицы Lens culinaris, морского червя Arenicola marina, медузы Aurelia aurita, осетра Acipencer guldenstadti, черепахи Emys orbicularis, жабы Bufo bufo gargarisans и др. В частности, из целомоцитов морского кольчатого червя Arenicola marina были выделены новые пептиды, обладающие ярко выраженной антимикробной активностью в отношении грамположительных и грамотрицательных бактерий, а также дрожжевых грибков и названные ареницинами. Определенная нами полная первичная структура зрелых ареницинов и генов их предшественников позволяет сделать вывод о выявлении нами пептидов нового семейства, не принадлежащих ни к одной из известных ранее групп АМП. Создана эффективная технология гетерологичной экспрессии, получены штаммы-продуценты, разработаны методики выделения и очистки генно-инженерных ареницинов. На оригинальную структуру ареницинов и биотехнологический способ их получения получены патенты РФ. Параллельно продолжаются фундаментальные исследования механизма действия ареницинов, проводятся детальное исследование их биологической активности и предклинические испытания на животных моделях. Продолжаются поиск и структурно-функциональные исследования других природных пептидных антибиотиков. Исследуемые вещества являются оригинальными и патентоспособными. Конечной целью проекта является создание линейки прототипов новых эффективных лекарственных средств на основе природных пептидных антибиотиков и биотехнологической платформы для их получения.

Ф.И.О.ДолжностьЭл. почта
Алёшина Екатерина Федоровнаст. инж.
Баландин Сергей Владимирович, к. х. н.н.с.
Белогурова-Овчинникова Оксана Юрьевна, к. б. н.н.с.
Богданов Иван Владимировичм.н.с.
Болосов Илья Александровичм.н.с.
Емельянова Анна Андреевнаасп.
Злобина Елизавета Игоревнатех.-лаб.
Калашникова Марьяна Борисовнаасп.
Кузницова Светлана Владимировнаинженер
Кузьмин Денис Владимирович, к. б. н.н.с.
Леонова Юлия Федоровнаст. инж.
Локтюшов Евгений Владимировичасп.
Мельникова Дарья Николаевна, к. х. н.н.с.
Пантелеев Павел Валерьевичм.н.с.
Савельева Юлия Рудольфовнаст. инж.
Симонова Татьяна Николаевнас.н.с.
Снежкова Леона Генриховна, к. х. н.ст. инж.
Стукачёва Елена Анатольевна, к. б. н.с.н.с.
Сурина Анна Михайловнаасп.
Суханов Станислав Владимировичст. инж.
Сычёв Сергей Владимирович, к. х. н.с.н.с.
Тагаев Андрей Азисовичс.н.с.
Тележинская Ирина Никитична, к. х. н.н.с.
Финкина Екатерина Ивановна, к. х. н.н.с.
Фурс Ольга Владимировнам.н.с.
Чичерина Галина Александровнаинженер
Шамборант Ольга Георгиевнас.н.с.
Шереметьева Эльвира Владимировнаст. инж.

Избранные публикации

  1. Bogdanov I.V., Shenkarev Z.O., Finkina E.I., Melnikova D.N., Rumynskiy E.I., Arseniev A.S., Ovchinnikova T.V. (2016). A novel lipid transfer protein from the pea Pisum sativum: isolation, recombinant expression, solution structure, antifungal activity, lipid binding, and allergenic properties. BMC Plant Biol. 16 (1), 107 [+]

    Plant lipid transfer proteins (LTPs) assemble a family of small (7-9 kDa) ubiquitous cationic proteins with an ability to bind and transport lipids as well as participate in various physiological processes including defense against phytopathogens. They also form one of the most clinically relevant classes of plant allergens. Nothing is known to date about correlation between lipid-binding and IgE-binding properties of LTPs. The garden pea Pisum sativum is widely consumed crop and important allergenic specie of the legume family. This work is aimed at isolation of a novel LTP from pea seeds and characterization of its structural, functional, and allergenic properties.

    ID:1507
  2. Sychev S.V., Sukhanov S.V., Telezhinskaya I.N., Ovchinnikova T.V. (2016). Effective lipid-detergent system for study of membrane active peptides in fluid liposomes. J. Pept. Sci. 22 (2), 98–105 [+]

    The structure of peptide antibiotic gramicidin A (gA) was studied in phosphatidylcholin liposomes modified by nonionic detergent Triton X-100. First, the detergent : lipid ratio at which the saturation of lipid membrane by Triton X-100 occurs (Re (sat) ), was determined by light scattering. Measurements of steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene at sublytic concentrations of detergent showed that after saturation of the membrane by Triton X-100 microviscosity of lipid bilayer is reduced by 20%. The equilibrium conformational state of gA in phosphatidylcholine liposomes at Re (sat) was studied by CD spectroscopy. It was found that the conformational state of this channel-forming peptide changed crucially when Triton X-100 induced transition to more fluid membranes. The gA single-channel measurements were made with Triton X-100 containing bilayers. Tentative assignment of the channel type and gA structures was made by correlation of CD data with conductance histograms. Lipid-detergent system with variable viscosity developed in this work can be used to study the structure and folding of other membrane-active peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

    ID:1512
  3. Panteleev P.V., Bolosov I.A., Ovchinnikova T.V. (2016). Bioengineering and functional characterization of arenicin shortened analogs with enhanced antibacterial activity and cell selectivity. J. Pept. Sci. 22 (2), 82–91 [+]

    New bioengineering approaches are required for development of more active and less toxic antimicrobial peptides. In this study we used β-hairpin antimicrobial peptide arenicin-1 as a template for design of more potent antimicrobials. In particular, six shortened 17-residue analogs were obtained by recombinant expression in Escherichia coli. Besides, we have introduced the second disulfide bridge by analogy with the structure of tachyplesins. As a result, a number of analogs with enhanced activity and cell selectivity were developed. In comparison with arenicin-1, which acts on cell membranes with low selectivity, the most potent and promising its analog termed ALP1 possessed two-fold higher antibacterial activity and did not affect viability of mammalian cells at concentration up to 50 μM. The therapeutic index of ALP1 against both Gram-positive and Gram-negative bacteria was significantly increased compared with that of arenicin-1 while the mechanism of action remained the same. Like arenicin-1, the analog rapidly disrupt membranes of both stationary and exponential phase bacterial cells and effectively kills multidrug-resistant Gram-negative bacteria. Furthermore, ALP1 was shown to bind DNA in vitro at a ratio of 1:1 (w/w). The circular dichroism spectra demonstrated that secondary structures of the shortened analogs were similar to that of arenicin-1 in water solution, but significantly differed in membrane-mimicking environments. This work shows that a strand length is one of the key parameters affecting cell selectivity of β-hairpin antimicrobial peptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

    ID:1513
  4. Melnikova D.N., Mineev K.S., Finkina E.I., Arseniev A.S., Ovchinnikova T.V. (2016). A novel lipid transfer protein from the dill Anethum graveolens L.: isolation, structure, heterologous expression, and functional characteristics. J. Pept. Sci. 22 (1), 59–66 [+]

    A novel lipid transfer protein, designated as Ag-LTP, was isolated from aerial parts of the dill Anethum graveolens L. Structural, antimicrobial, and lipid binding properties of the protein were studied. Complete amino acid sequence of Ag-LTP was determined. The protein has molecular mass of 9524.4 Da, consists of 93 amino acid residues including eight cysteines forming four disulfide bonds. The recombinant Ag-LTP was overexpressed in Escherichia coli and purified. NMR investigation shows that the Ag-LTP spatial structure contains four α-helices, forming the internal hydrophobic cavity, and a long C-terminal tail. The measured volume of the Ag-LTP hydrophobic cavity is equal to ~800 A(3) , which is much larger than those of other plant LTP1s. Ag-LTP has weak antifungal activity and unpronounced lipid binding specificity but effectively binds plant hormone jasmonic acid. Our results afford further molecular insight into biological functions of LTP in plants. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

    ID:1370
  5. Алексеева А.С., Третьякова Д.С., Мельникова Д.Н., Молотковский Юл.Г., Болдырев И.А. (2016). Новый флуоресцентный мембранный зонд (2,3;5,6 бисциклогексил)bodipy меченный фосфатидилхолн. Биоорг. хим. 42 (3), 339–344 [+]

    Новый мембранный зонд бисциклогексил BODIPY (BCHB) меченный фосфатидилхолин структурно очень близок в 1,3,5,7 тетраметил BODIPY (TMB) меченному фосфатидилхолину и синтезируется по аналогичной схеме. Системы сопряженных связей BCHB и TMB формально идентичны, однако спектральные характеристики BCHB заметно отличаются, что делают BCHB хорошим акцептором фёрстеровского резонансного переноса (FRET) для TMB. Показано, что FRET пара фосфатидилхолинов на основе BCHB и TMB является перспективным инструментом для исследования мембранных систем, например, межмембранного липидного переноса.
     

    ID:1498
  6. Panteleev P.V., Ovchinnikova T.V. (2015). Improved strategy for recombinant production and purification of antimicrobial peptide tachyplesin I and its analogs with high cell selectivity. Biotechnol. Appl. Biochem. , [+]

    Here we report an efficient procedure for recombinant production and purification of tachyplesin I with a final yield of 17 mg per liter of the culture medium. The peptide was expressed in Escherichia coli as a part of the thioredoxin fusion protein. With the use of soluble expression followed by immobilized metal-ion affinity chromatography, the recombinant protein cleavage and reversed-phase high performance liquid chromatography, a yield of tachyplesin I did not exceed 6.5 mg per liter of the culture medium. Further optimization studies were carried out to improve the protein expression level and simplify purification procedure of the target peptide. To achieve better yield of the peptide, we used high-cell-density bacterial expression. The formed inclusion bodies were highly enriched with the fusion protein, which allowed us to perform direct chemical cleavage of the inclusion bodies solubilized in 6 M guanidine-HCl with subsequent selective precipitation of proteins with trifluoroacetic acid. This enabled us to avoid an extra step of purification by immobilized metal-ion affinity chromatography. The developed procedure has made it possible to obtain biologically active tachyplesin I and was used for screening a number of its mutant analogs. As the result, several selective and non-hemolytic analogs were developed. Significant reduction in hemolytic activity without lose of antimicrobial activity was achieved by substitution of tyrosine or isoleucine residue in the β-turn region of the molecule with hydrophilic serine. The present study affords further insight into molecular mechanism of antimicrobial action of tachyplesin and gains a better understanding of structure-activity relationships in its analogs. This is aimed at searching for novel antibiotics on the basis of antimicrobial peptides with reduced cytotoxicity. This article is protected by copyright. All rights reserved.

    ID:1511
  7. 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.

    ID:1515
  8. Panteleev P.V., Bolosov I.A., Balandin S.V., Ovchinnikova T.V. (2015). Design of antimicrobial peptide arenicin analogs with improved therapeutic indices. J. Pept. Sci. 21 (2), 105–13 [+]

    β-Hairpin antimicrobial peptides are among the most potent peptide antibiotics of animal origin. Arenicins, isolated earlier from marine polychaeta lugworm Arenicola marina, belong to a family of β-hairpin antimicrobial peptides and display a broad spectrum of biological activities. However, despite being potent antimicrobials, arenicins are partially unapplicable as therapeutics as a result of their relatively high cytotoxicity against mammalian cells. In this study, a template-based approach was used to create therapeutically valuable analogs of arenicin-1 and identify amino acid residues important for antibacterial and cytotoxic activities of the peptide. The plasmids encoding recombinant analogs were constructed by mutagenesis technique based on inverse PCR amplification of the whole arenicin-1 expression plasmid. The analogs were produced as a part of the fusion proteins in Escherichia coli. It was shown that an obvious reduction in hemolytic activity without lose of antimicrobial activity can be achieved by a single amino acid substitution in the non-polar face of the molecule with hydrophilic residues such as serine and arginine. As the result, the selective analog with 50-fold improved therapeutic index was developed. The circular dichroism spectra demonstrated that the secondary structure of the analog was similar to the natural arenicin-1 in water solution and sodium dodecyl sulfate micelles but significantly differed in the presence of dodecylphosphocholine micelles mimicking mammalian membranes. Similarly to arenicin-1, the designed analog killed bacteria via induction of the membrane damage, assessed using the fluorescent dye SYTOX Green uptake. Our results afford molecular insight into mechanism of antimicrobial action of the designed arenicin analogs and their possible clinical application.

    ID:1508
  9. 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.

    ID:1315
  10. Panteleev P.V., Bolosov I.A., Balandin S.V., Ovchinnikova T.V. (2015). Structure and Biological Functions of β-Hairpin Antimicrobial Peptides. Acta Naturae 7 (1), 37–47 [+]

    Antimicrobial peptides (AMPs) are evolutionarily ancient factors of the innate immune system that serve as a crucial first line of defense for humans, animals, and plants against infection. This review focuses on the structural organization, biosynthesis, and biological functions of AMPs that possess a β-hairpin spatial structure. Representatives of this class of AMPs are among the most active antibiotic molecules of animal origin. Due to their wide spectrum of activity and resistance to internal environmental factors, natural β-hairpin AMPbased compounds might become the most promising drug candidates.

    ID:1510
  11. Berlov M.N., Umnyakova E.S., Leonova T.S., Milman B.L., Krasnodembskaya A.D., Ovchinnikova T.V., Kokryakov V.N. (2015). [Interaction of Arenicin-1 with C1q Protein]. Bioorg. Khim. 41 (6), 664–8 [+]

    The interaction between arenicin-1, that is an antimicrobial peptide from polychaeta Arenicola marina, and human complement system protein C1q was studied using enzyme-linked receptor sorbent assay and ELISA. We revealed that arenicin-1 and C1q form complex that is stable in high ionic strength condition 0.5 M NaCl. The ability of C1q to interact with arenicin-1 is comparable with the binding activity of C1q towards another antimicrobial peptide, porcine cathelicidin protegrin-1, which has a similar spatial arrangement with arenicin-1. Namely, both arenicin-1 and protegrin-1 form cystine-stabilized antiparallel β-hairpin structure.

    ID:1514
  12. Shamova O.V., Orlov D.S., Balandin S.V., Shramova E.I., Tsvetkova E.V., Panteleev P.V., Leonova Y.F., Tagaev A.A., Kokryakov V.N., Ovchinnikova T.V. (2014). Acipensins - Novel Antimicrobial Peptides from Leukocytes of the Russian Sturgeon Acipenser gueldenstaedtii. Acta Naturae 6 (4), 99–109 [+]

    Antimicrobial peptides (AMPs) play an important role in the innate defense mechanisms in humans and animals. We have isolated and studied a set of antimicrobial peptides from leukocytes of the Russian sturgeon Acipenser gueldenstaedtii belonging to a subclass of chondrosteans, an ancient group of bony fish. Structural analysis of the isolated peptides, designated as acipensins (Ac), revealed in leukocytes of the Russian sturgeon six novel peptides with molecular masses of 5336.2 Da, 3803.0 Da, 5173.0 Da, 4777.5 Da, 5449.4 Da, and 2740.2 Da, designated as Ac1-Ac6, respectively. Complete primary structures of all the isolated peptides were determined, and the biological activities of three major components - Ac1, Ac2, and Ac6 - were examined. The peptides Ac1, Ac2, Ac3, Ac4, and Ac5 were found to be the N-terminal acetylated fragments 1-0, 1-5, 1-9, 1-4, and 1-1 of the histone H2A, respectively, while Ac6 was shown to be the 62-5 fragment of the histone H2A. The peptides Ac1 and Ac2 displayed potent antimicrobial activity towards Gram-negative and Gram-positive bacteria (Escherichia coli ML35p, Listeria monocytogenes EGD, MRSA ATCC 33591) and the fungus Candida albicans 820, while Ac6 proved effective only against Gram-negative bacteria. The efficacy of Ac 1 and Ac2 towards the fungus and MRSA was reduced upon an increase in the ionic strength of the solution. Ac1, Ac2, and Ac6, at concentrations close to their minimum inhibitory concentrations, enhanced the permeability of the E.coli ML35p outer membrane to the chromogenic marker, but they did not affect appreciably the permeability of the bacterial inner membrane in comparison with a potent pore-forming peptide, protegrin 1. Ac1, Ac2, and Ac6 revealed no hemolytic activity against human erythrocytes at concentrations of 1 to 40 μM and had no cytotoxic effect (1 to 20 μM) on K-562 and U-937 cells in vitro. Our findings suggest that histone-derived peptides serve as important anti-infective host defense molecules.

    ID:1509
  13. 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
  14. Shenkarev Z.O., Lyukmanova E.N., Paramonov A.S., Panteleev P.V., Balandin S.V., Shulepko M.A., Mineev K.S., Ovchinnikova T.V., Kirpichnikov M.P., Arseniev A.S. (2014). Lipid-protein nanodiscs offer new perspectives for structural and functional studies of water-soluble membrane-active peptides. Acta Naturae 6 (2), 84–94 [+]

    Lipid-protein nanodiscs (LPNs) are nanoscaled fragments of a lipid bilayer stabilized in solution by the apolipoprotein or a special membrane scaffold protein (MSP). In this work, the applicability of LPN-based membrane mimetics in the investigation of water-soluble membrane-active peptides was studied. It was shown that a pore-forming antimicrobial peptide arenicin-2 from marine lugworm (charge of +6) disintegrates LPNs containing both zwitterionic phosphatidylcholine (PC) and anionic phosphatidylglycerol (PG) lipids. In contrast, the spider toxin VSTx1 (charge of +3), a modifier of Kv channel gating, effectively binds to the LPNs containing anionic lipids (POPC/DOPG, 3 : 1) and does not cause their disruption. VSTx1 has a lower affinity to LPNs containing zwitterionic lipids (POPC), and it weakly interacts with the protein component of nanodiscs, MSP (charge of -6). The neurotoxin II (NTII, charge of +4) from cobra venom, an inhibitor of the nicotinic acetylcholine receptor, shows a comparatively low affinity to LPNs containing anionic lipids (POPC/DOPG, 3 : 1 or POPC/DOPS, 4 : 1), and it does not bind to LPNs/POPC. The obtained data show that NTII interacts with the LPN/POPC/DOPS surface in several orientations, and that the exchange process among complexes with different topologies proceeds fast on the NMR timescale. Only one of the possible NTII orientations allows for the previously proposed specific interaction between the toxin and the polar head group of phosphatidylserine from the receptor environment (Lesovoy et al., Biophys. J. 2009. V. 97. № 7. P. 2089-2097). These results indicate that LPNs can be used in structural and functional studies of water-soluble membrane-active peptides (probably except pore-forming ones) and in studies of the molecular mechanisms of peptide-membrane interaction.

    ID:1106
  15. 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
  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. 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.

    ID:536
  18. 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
  19. Novgorodov S.A., Wu B.X., Gudz T.I., Bielawski J., Ovchinnikova T.V., Hannun Y.A., Obeid L.M. (2011). Novel pathway of ceramide production in mitochondria: thioesterase and neutral ceramidase produce ceramide from sphingosine and acyl-CoA. J. Biol. Chem. 286 (28), 25352–62 [+]

    Reports suggest that excessive ceramide accumulation in mitochondria is required to initiate the intrinsic apoptotic pathway and subsequent cell death, but how ceramide accumulates is unclear. Here we report that liver mitochondria exhibit ceramide formation from sphingosine and palmitoyl-CoA and from sphingosine and palmitate. Importantly, this activity was markedly decreased in liver from neutral ceramidase (NCDase)-deficient mice. Moreover, the levels of ceramide were dissimilar in liver mitochondria of WT and NCDase KO mice. These results suggest that NCDase is a key participant of ceramide formation in liver mitochondria. We also report that highly purified liver mitochondria have ceramidase, reverse ceramidase, and thioesterase activities. Increased accessibility of palmitoyl-CoA to the mitochondrial matrix with the pore-forming peptide zervamicin IIB resulted in 2-fold increases in palmitoyl-CoA hydrolysis by thioesterase. This increased hydrolysis was accompanied by an increase in ceramide formation, demonstrating that both outer membrane and matrix localized thioesterases can regulate ceramide formation. Also, ceramide formation might occur both in the outer mitochondrial membrane and in the mitochondrial matrix, suggesting the existence of distinct ceramide pools. Taken together, these results suggest that the reverse activity of NCDase contributes to sphingolipid homeostasis in this organelle in vivo.

    ID:534
  20. 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.

    ID:533
  21. Macháň R., Hof M., Chernovets T., Zhmak M.N., Ovchinnikova T.V., Sýkora J. (2011). Formation of arenicin-1 microdomains in bilayers and their specific lipid interaction revealed by Z-scan FCS. Analytical and bioanalytical chemistry , [+]

    Z-scan fluorescence correlation spectroscopy (FCS) is employed to characterize the interaction between arenicin-1 and supported lipid bilayers (SLBs) of different compositions. Lipid analogue C8-BODIPY 500/510C5-HPC and ATTO 465 labelled arenicin-1 are used to detect changes in lipid and peptide diffusion upon addition of unlabelled arenicin-1 to SLBs. Arenicin-1 decreases lipid mobility in negatively charged SLBs. According to diffusion law analysis, microdomains of significantly lower lipid mobility are formed. The analysis of peptide FCS data confirms the presence of microdomains for anionic SLBs. No indications of microdomain formation are detected in SLBs composed purely of zwitterionic lipids. Additionally, our FCS results imply that arenicin-1 exists in the form of oligomers and/or aggregates when interacting with membranes of both compositions.

    ID:408
  22. 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
  23. 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
  24. Stegemann C., Kolobov A. Jr, Leonova Y.F., Knappe D., Shamova O., Ovchinnikova T.V., Kokryakov V.N., Hoffmann R. (2009). Isolation, purification and de novo sequencing of TBD-1, the first beta-defensin from leukocytes of reptiles. Proteomics 9 (5), 1364–73 [+]

    A novel peptide with antimicrobial activity was isolated from leukocytes of the European pond turtle Emys orbicularis and purified to homogeneity by preparative gel electrophoresis followed by reversed phase chromatography. It was highly active in vitro against Escherichia coli, Listeria monocytogenes, methicillin-resistant Staphylococcus aureus, and Candida albicans. The isolated peptide was sequenced de novo by tandem mass spectrometry using both collision-induced and electron-transfer dissociation in combination with different chemical derivatization techniques. The 40-residue peptide, called TBD-1 (turtle beta-defensin 1), represents the first defensin isolated from reptilian leukocytes. It contains three disulfide bonds and shows high structural similarities to beta-defensins isolated from birds and mammals.

    ID:409
  25. 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
  26. Ovchinnikova T.V., Murashev A.N. (2009). The peptaibol antibiotic zervamicin displays neurotropic activity. Dokl. Biochem. Biophys. 414, 146–8 ID:415
  27. Finkina E.I., Shramova E.I., Tagaev A.A., Ovchinnikova T.V. (2008). A novel defensin from the lentil Lens culinaris seeds. Biochem. Biophys. Res. Commun. 371 (4), 860–5 [+]

    A novel 47-residue plant defensin was purified from germinated seeds of the lentil Lens culinaris by ammonium sulfate precipitation, gel filtration, chromatography, and RP-HPLC. The molecular mass (5440.41Da) and complete amino acid sequence (KTCENLSDSFKGPCIPDGNCNKHCKEKEHLLSGRCRDDFRCWCTRNC) of defensin, termed Lc-def, were determined. Lc-def has eight cysteines forming four disulfide bonds. The total RNA was isolated from lentil germinated seeds, RT-PCR and subsequent cloning were performed, and cDNA was sequenced. A 74-residue predefensin contains a putative signal peptide (27 amino acid) and a mature protein. Lc-def shows high sequence homology with legumes defensins, exhibits an activity against Aspergillus niger, but does not inhibit proteolytic enzymes.

    ID:412
  28. 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
  29. Ramazanova A.S., Zavada L.L., Starkov V.G., Kovyazina I.V., Subbotina T.F., Kostyukhina E.E., Dementieva I.N., Ovchinnikova T.V., Utkin Y.N. (2008). Heterodimeric neurotoxic phospholipases A2--the first proteins from venom of recently established species Vipera nikolskii: implication of venom composition in viper systematics. Toxicon 51 (4), 524–37 [+]

    For the first time the venom of recently established viper species Vipera nikolskii was fractionated and two heterodimeric phospholipases A(2) (HDP-1 and HDP-2) were isolated. Isolation of HDP-1 and HDP-2 is the first indication of the presence of two heterodimeric phospholipases A(2) in the venom of one viper species. When tested on the frog neuromuscular junction, isolated proteins affected neuromuscular transmission acting presynaptically. Using RP-HPLC, each heterodimer was separated into two monomeric subunits: basic phospholipase A(2) (HDP-1P and HDP-2P) and acidic component without enzymatic activity (HDP-In). The complete primary structures of subunits were deduced from corresponding sequences of cDNAs. The determined amino acid sequences were homologous to those of vipoxin from Vipera ammodytes and vaspin from Vipera aspis. Similar proteins were not found earlier in the well-studied venom of Vipera berus, the species from which V. nikolskii was recently separated. Our finding supports at the biochemical level the correctness of the establishment of V. nikolskii as an independent species. The finding of similar proteins (HDPs and vipoxin) in geographically remote species (V. nikolskii and V. ammodytes) corroborates the hypothesis about the pre-existence of genes encoding these proteins in all true viper species and their expression under certain conditions.

    ID:413
  30. 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
  31. Kordyukova L.V., Serebryakova M.V., Polyakov V.Y., Ovchinnikova T.V., Smirnova Y.A., Fedorova N.V., Baratova L.A. (2008). Influenza A virus M1 protein structure probed by in situ limited proteolysis with bromelain. Protein Pept. Lett. 15 (9), 922–30 [+]

    Influenza A virus matrix M1 protein is membrane associated and plays a crucial role in virus assembly and budding. The N-terminal two thirds of M1 protein was resolved by X-ray crystallography. The overall 3D structure as well as arrangement of the molecule in relation to the viral membrane remains obscure. Now a proteolytic digestion of virions with bromelain was used as an instrument for the in situ assessment of the M1 protein structure. The lipid bilayer around the subviral particles lacking glycoprotein spikes was partially disrupted as was shown by transmission electron microscopy. A phenomenon of M1 protein fragmentation inside the subviral particles was revealed by SDS-PAGE analysis followed by in-gel trypsin hydrolysis and MALDI-TOF mass spectrometry analysis of the additional bands. Putative bromelain-digestion sites appeared to be located at the surface of the M1 protein globule and could be used as landmarks for 3D molecular modeling.

    ID:411
  32. 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
  33. Ovchinnikova T.V., Levitskaya N.G., Voskresenskaya O.G., Yakimenko Z.A., Tagaev A.A., Ovchinnikova A.Y., Murashev A.N., Kamenskii A.A. (2007). Neuroleptic properties of the ion-channel-forming peptaibol zervamicin: locomotor activity and behavioral effects. Chem. Biodivers. 4 (6), 1374–87 [+]

    Zervamicins IIA and IIB are members of the peptaibol family of peptide antibiotics. They are produced by the fungus Emericellopsis salmosynnemata. Peptaibols are known to be of potential usefulness for chemotherapeutic applications, as are other secondary fungal metabolites. Previously, we have found zervamicins to decrease spontaneous locomotor activity in mice, suggesting their neurotropic properties on an equal footing with antimicrobial activity. The current study deals with behavioral effects of zervamicins IIA and IIB in mice. According to our results, both zervamicins induce a reliable decrease in locomotion and exploratory activity measured in the hole-board test. The behavioral effects of zervamicin IIA become apparent at lower dosages (0.05-2.0 mg/kg) as compared with zervamicin IIB (0.5-12.0 mg/kg). The experiments on behavioral effects in the elevated plus maze test showed that both zervamicins caused a reliable decrease in the number of head-dippings, open-arm entries, and rearings. The observed behavioral effects may be rather associated with a decrease in the exploratory activity than with anxiety-related responses in mice. Zervamicins induced depression-like behavior of experimental animals in the forced-swim test. Both peptaibols reduce physical endurance and change motor coordination of experimental animals in the bar-holding test. Taken together, the data obtained clearly indicate that both zervamicins possess neuroleptic activity.

    ID:416
  34. Milov A.D., Tsvetkov Y.D., Gorbunova E.Y., Mustaeva L.G., Ovchinnikova T.V., Handgraaf J.W., Raap J. (2007). Solvent effects on the secondary structure of the membrane-active zervamicin determined by PELDOR spectroscopy. Chem. Biodivers. 4 (6), 1243–55 [+]

    Zervamicin is a voltage-gated ion-channel-forming peptide. Channels are generally considered to be formed by first insertion of amphipathic molecules into the phospholipid bilayer, followed by self-assembly of a variable number of transmembrane helices. We have studied the length of the peptide structure to address the question whether this peptide is long enough to span the phospholipid bilayer. The pulsed electron-electron double resonance (PELDOR) spectroscopic technique was used to determine the length of the helical molecule in membrane-mimicking solvents. This was achieved from the distance-related dipole-dipole interaction between spin labels, which were located at both ends of the linear peptide chain. The data were obtained by using samples of frozen glassy solutions of MeOH, MeOH/toluene, and MeOH/CHCl(3). Contributions of inter- and intramolecular interactions of spin labels were separated to analyze the intramolecular interaction and the distance distribution function between the labels. It is shown that the main maximum of the distribution functions is located at a distance of ca. 3.3 nm, and this distance appears to be only slightly dependent on the solvent composition. The distribution function was observed to narrow after addition of either CHCl(3) or toluene to MeOH. This effect is rationalized in terms of a decreased mobility of the terminal amino acid residues. By molecular-dynamics simulations, it was shown that the conformation, corresponding with the predominant distance found by PELDOR, agrees well with the mixed alpha/3(10)-helical that was previously determined by NMR. However, in the case toluene was added to the MeOH solution to further increase the hydrophobicity of the environment of the membrane-active peptide, the distribution function gives rise to a minor fraction (7-8%) with a distance of 4.2 nm. This distance corresponds most likely to the more extended 2(7)-helix structure.

    ID:417
  35. 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
  36. Berlov M.N., Korableva E.S., Andreeva Y.V., Ovchinnikova T.V., Kokryakov V.N. (2007). Lactoferrin from canine neutrophils: isolation and physicochemical and antimicrobial properties. Biochemistry Mosc. 72 (4), 445–51 [+]

    Lactoferrin has been isolated from canine leukocytes for the first time. Lactoferrin was identified by N-terminal amino acid sequence and by capability to capture ferric cations resulting in a complex with absorbance maximum at 460-470 nm. It is demonstrated that canine lactoferrin resembles the human homolog in some physicochemical properties, i.e. molecular weight, carbohydrate presence, and conditions of protein-iron complex dissociation. Bactericidal activity of dog lactoferrin was demonstrated on the gram-negative bacterium Escherichia coli and gram-positive bacterium Listeria monocytogenes. Bactericidal activity of canine lactoferrin is similar to that of human lactoferrin.

    ID:420
  37. 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.

    ID:421
  38. 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.

    ID:423
  39. Raap J., Hollander J., Ovchinnikova T.V., Swischeva N.V., Skladnev D., Kiihne S. (2006). Trans and surface membrane bound zervamicin IIB: 13C-MAOSS-NMR at high spinning speed. J. Biomol. NMR 35 (4), 285–93 [+]

    Interactions between (15)N-labelled peptides or proteins and lipids can be investigated using membranes aligned on a thin polymer film, which is rolled into a cylinder and inserted into the MAS-NMR rotor. This can be spun at high speed, which is often useful at high field strengths. Unfortuantely, substrate films like commercially available polycarbonate or PEEK produce severe overlap with peptide and protein signals in (13)C-MAOSS NMR spectra. We show that a simple house hold foil support allows clear observation of the carbonyl, aromatic and C(alpha) signals of peptides and proteins as well as the ester carbonyl and choline signals of phosphocholine lipids. The utility of the new substrate is validated in applications to the membrane active peptide zervamicin IIB. The stability and macroscopic ordering of thin PC10 bilayers was compared with that of thicker POPC bilayers, both supported on the household foil. Sidebands in the (31)P-spectra showed a high degree of alignment of both the supported POPC and PC10 lipid molecules. Compared with POPC, the PC10 lipids are slightly more disordered, most likely due to the increased mobilities of the shorter lipid molecules. This mobility prevents PC10 from forming stable vesicles for MAS studies. The (13)C-peptide peaks were selectively detected in a (13)C-detected (1)H-spin diffusion experiment. Qualitative analysis of build-up curves obtained for different mixing times allowed the transmembrane peptide in PC10 to be distinguished from the surface bound topology in POPC. The (13)C-MAOSS results thus independently confirms previous findings from (15)N spectroscopy [Bechinger, B., Skladnev, D.A., Ogrel, A., Li, X., Rogozhkina, E.V., Ovchinnikova, T.V., O'Neil, J.D.J. and Raap, J. (2001) Biochemistry, 40, 9428-9437]. In summary, application of house hold foil opens the possibility of measuring high resolution (13)C-NMR spectra of peptides and proteins in well ordered membranes, which are required to determine the secondary and supramolecular structures of membrane active peptides, proteins and aggregates.

    ID:422
  40. Kropacheva T.N., Salnikov E.S., Nguyen H.H., Reissmann S., Yakimenko Z.A., Tagaev A.A., Ovchinnikova T.V., Raap J. (2005). Membrane association and activity of 15/16-membered peptide antibiotics: zervamicin IIB, ampullosporin A and antiamoebin I. Biochim. Biophys. Acta 1715 (1), 6–18 [+]

    Permeabilization of the phospholipid membrane, induced by the antibiotic peptides zervamicin IIB (ZER), ampullosporin A (AMP) and antiamoebin I (ANT) was investigated in a vesicular model system. Membrane-perturbing properties of these 15/16 residue peptides were examined by measuring the K(+) transport across phosphatidyl choline (PC) membrane and by dissipation of the transmembrane potential. The membrane activities are found to decrease in the order ZER>AMP>>ANT, which correlates with the sequence of their binding affinities. To follow the insertion of the N-terminal Trp residue of ZER and AMP, the environmental sensitivity of its fluorescence was explored as well as the fluorescence quenching by water-soluble (iodide) and membrane-bound (5- and 16-doxyl stearic acids) quenchers. In contrast to AMP, the binding affinity of ZER as well as the depth of its Trp penetration is strongly influenced by the thickness of the membrane (diC(16:1)PC, diC(18:1)PC, C(16:0)/C(18:1)PC, diC(20:1)PC). In thin membranes, ZER shows a higher tendency to transmembrane alignment. In thick membranes, the in-plane surface association of these peptaibols results in a deeper insertion of the Trp residue of AMP which is in agreement with model calculations on the localization of both peptide molecules at the hydrophilic-hydrophobic interface. The observed differences between the membrane affinities/activities of the studied peptaibols are discussed in relation to their hydrophobic and amphipathic properties.

    ID:424
  41. 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
  42. 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.

    ID:426

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