Дубовский Пётр Викторович


Период обученияСтрана, городУчебное заведениеДополнительная информация
1978–1984 СССР, Москва МИФИ диплом с отличием
1986–1989 СССР, Москва Институт Тонкой Химической Технологии им. Ломоносова М.В. кандидат химических наук
1994–1995 Япония, Кобэ Университет Кобэ стажировка

Научные интересы

Поли-ионные пептиды, структура и взаимодействие с липидными и био-мембранами.

Гранты и проекты

ПериодДополнительная информация
2001–2001 РФФИ:Структура и топология белка-предшественника ферредоксина в мембранах оболочки хлоропластов
2002–2004 РФФИ:Структурно-функциональное изучение пептида слияния из гемагглютинина вируса гриппа 
2007–2009 РФФИ: Изучение мембранных полипептидов с использованием липидных наночастиц.
2013–2015 РФФИ: Взаимосвязь пространственная структура - химический сдвиг в ЯМР для кардиотоксинов кобр.

Избранные публикации

  1. Dubovskii P.V., Dubinnyi M.A., Volynsky P.E., Pustovalova Y.E., Konshina A.G., Utkin Y.N., Arseniev A.S., Efremov R.G. (2017). Impact of membrane partitioning on the spatial structure of an S-type cobra cytotoxin. J. Biomol. Struct. Dyn. , 1–16 [+]

    Cobra cytotoxins (CTs) belong to the three-fingered protein family. They are classified into S- and P-types, the latter exhibiting higher membrane-perturbing capacity. In this work, we investigated the interaction of CTs with phospholipid bilayers, using coarse-grained (CG) and full-atom (FA) molecular dynamics (MD). The object of this work is a CT of an S-type, cytotoxin I (CT1) from N.oxiana venom. Its spatial structure in aqueous solution and in the micelles of dodecylphosphocholine (DPC) were determined by (1)H-NMR spectroscopy. Then, via CG- and FA MD-computations, we evaluated partitioning of CT1 molecule into palmitoyloleoylphosphatidylcholine (POPC) membrane, using the toxin spatial models, obtained either in aqueous solution, or detergent micelle. The latter model exhibits minimal structural changes upon partitioning into the membrane, while the former deviates from the starting conformation, loosing the tightly bound water molecule in the loop-2. These data show that the structural changes elicited by CT1 molecule upon incorporation into DPC micelle take place likely in the lipid membrane, although the mode of the interaction of this toxin with DPC micelle (with the tips of the all three loops) is different from its mode in POPC membrane (primarily with the tip of the loop-1 and both the tips of the loop-1 and loop-2).

  2. Thien T.V., Anh H.N., Trang N.T.T., Trung P.V., Khoa N.C., Osipov A.V., Dubovskii P.V., Ivanov I.A., Arseniev A.S., Tsetlin V.I., Utkin Y.N. (2017). Low-molecular-weight compounds with anticoagulant activity from the scorpion Heterometrus laoticus venom. Dokl. Biochem. Biophys. 476 (1), 316–319 [+]

    Low-molecular-weight compounds with anticoagulant activity were isolated from the scorpion Heterometrus laoticus venom. The determination of the structure of the isolated compounds by nuclear magnetic resonance and mass spectrometry showed that one of the isolated compounds is adenosine, and the other two are dipeptides leucyl-tryptophan and isoleucyl-tryptophan. The anticoagulant properties of adenosine, which is an inhibitor of platelet aggregation, is well known, but its presence in scorpion venom is shown for the first time. The ability of leucyl-tryptophan and isoleucyl-tryptophan to slow down blood clotting and their presence in scorpion venom are also established for the first time.

  3. Dubovskii P.V., Dubinnyi M.A., Konshina A.G., Kazakova E.D., Sorokoumova G.M., Ilyasova T.M., Shulepko M.A., Chertkova R.V., Lyukmanova E.N., Dolgikh D.A., Arseniev A.S., Efremov R.G. (2017). Structural and Dynamic "Portraits" of Recombinant and Native Cytotoxin I from Naja oxiana: How Close Are They? Biochemistry 56 (34), 4468–4477 [+]

    Today, recombinant proteins are quite widely used in biomedical and biotechnological applications. At the same time, the question about their full equivalence to the native analogues remains unanswered. To gain additional insight into this problem, intimate atomistic details of a relatively simple protein, small and structurally rigid recombinant cardiotoxin I (CTI) from cobra Naja oxiana venom, were characterized using nuclear magnetic resonance (NMR) spectroscopy and atomistic molecular dynamics (MD) simulations in water. Compared to the natural protein, it contains an additional Met residue at the N-terminus. In this work, the NMR-derived spatial structure of uniformly (13)C- and (15)N-labeled CTI and its dynamic behavior were investigated and subjected to comparative analysis with the corresponding data for the native toxin. The differences were found in dihedral angles of only a single residue, adjacent to the N-terminal methionine. Microsecond-long MD traces of the toxins reveal an increased flexibility in the residues spatially close to the N-Met. As the detected structural and dynamic changes of the two CTI models do not result in substantial differences in their cytotoxicities, we assume that the recombinant protein can be used for many purposes as a reasonable surrogate of the native one. In addition, we discuss general features of the spatial organization of cytotoxins, implied by the results of the current combined NMR and MD study.

  4. 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.

  5. Dubovskii P.V., Vassilevski A.A., Kozlov S.A., Feofanov A.V., Grishin E.V., Efremov R.G. (2015). Latarcins: versatile spider venom peptides. Cell. Mol. Life Sci. 72 (23), 4501–22 [+]

    Arthropod venoms feature the presence of cytolytic peptides believed to act synergetically with neurotoxins to paralyze prey or deter aggressors. Many of them are linear, i.e., lack disulfide bonds. When isolated from the venom, or obtained by other means, these peptides exhibit common properties. They are cationic; being mostly disordered in aqueous solution, assume amphiphilic α-helical structure in contact with lipid membranes; and exhibit general cytotoxicity, including antifungal, antimicrobial, hemolytic, and anticancer activities. To suit the pharmacological needs, the activity spectrum of these peptides should be modified by rational engineering. As an example, we provide a detailed review on latarcins (Ltc), linear cytolytic peptides from Lachesana tarabaevi spider venom. Diverse experimental and computational techniques were used to investigate the spatial structure of Ltc in membrane-mimicking environments and their effects on model lipid bilayers. The antibacterial activity of Ltc was studied against a panel of Gram-negative and Gram-positive bacteria. In addition, the action of Ltc on erythrocytes and cancer cells was investigated in detail with confocal laser scanning microscopy. In the present review, we give a critical account of the progress in the research of Ltc. We explore the relationship between Ltc structure and their biological activity and derive molecular characteristics, which can be used for optimization of other linear peptides. Current applications of Ltc and prospective use of similar membrane-active peptides are outlined.

  6. Дубовский П.В., Уткин Ю.Н. (2014). Цитотоксины кобр: структурная организация и антибактериальная активность. Acta Naturae 6 (3), 12–19 ID:1125
  7. Кузнецов А.С., Дубовский П.В., Воронцова О.В., Феофанов А.В., Ефремов Р.Г. (2014). Взаимодействие линейных катионных пептидов с фосфолипидными мембранами и полимерами сиаловой кислоты. Биохимия 79 (5), 583–594583–594 ID:1123
  8. Dubovskii P.V., Utkin Y.N. (2014). Cobra cytotoxins: structural organization and antibacterial activity. Acta Naturae 6 (3), 11–8 [+]

    Cardiotoxins (cytotoxins, CT) are β-structured proteins isolated from the venom of cobra. They consist of 59-61 amino acid residues, whose antiparallel chains form three 'fingers'. In contrast to neurotoxins with an overall similar fold, CTs are amphiphilic. The amphiphilicity is caused by positively charged lysine and arginine residues flanking the tips of the loops that consist primarily of hydrophobic amino acids. A similar distribution of amino acid residues is typical for linear (without disulfide bonds) cationic cytolytic peptides from the venoms of other snakes and insects. Many of them are now considered to be lead compounds in combatting bacterial infections and cancer. In the present review, we summarize the data on the antibacterial activity of CTs and compare it to the activity of linear peptides.

  9. Dubovskii P.V., Konshina A.G., Efremov R.G. (2014). Cobra Cardiotoxins: Membrane Interactions and Pharmacological Potential. Curr. Med. Chem. 21 (3), 270–287 [+]

    Natural polycationic membrane-active peptides typically lack disulfide bonds and exhibit fusion, cell-penetrating, antimicrobial activities. They are mostly unordered in solution, but adopt a helical structure, when bound to phospholipid membranes. Structurally different are cardiotoxins (or cytotoxins, СTs) from cobra venom. They are fully -structured molecules, characterized by the three-finger fold (TFF). Affinity of CTs to lipid bilayer was shown to depend on amino acid sequence in the tips of the three loops. In the present review, CT-membrane interactions are analyzed through the prism of data on binding of the toxins to phospholipid liposomes and detergent micelles, as well as their structural and computational studies in membrane mimicking environments. We assess different hydrophobicity scales to compare membrane partitioning of various CTs and their membrane effects. A comparison of hydrophobic/hydrophilic properties of CTs and linear polycationic peptides provides a key to their biological activity and creates a fundamental basis for rational design of new membrane-interacting compounds, including new promising drugs. For instance, since the viewpoint of the data obtained on model lipid membranes, cytotoxic activity of CTs against cancer cells is discussed.

  10. Dubovskii P.V. (2012). Unusual titration of the membrane-bound artificial hemagglutinin fusion peptide. Eur. Biophys. J. 41 (12), 1077–84 [+]

    E5 is a 20-residue-long analog of the fusion peptide from influenza hemagglutinin (GLFEAIAEFIEGGWEGLIEG). It has been suggested that two of its five glutamates, Glu11and Glu15, are critical in its pH-dependent membrane perturbation. To reveal their specific involvement, a pair of analogs with substitution of either Glu11 or Glu15 for Ala were synthesized. By analysis of the pH-dependence of the chemical shifts of protons of these peptides bound to dodecylphosphocholine micelles we found: (1) the peptides adopt an amphiphilic alpha-helical structure within residues 2-18, similar to the parent peptide; (2) the helix is significantly more disordered at neutral pH than at acidic pH for E5 peptide only; and (3) in E5 and mutant peptides the Glu11 and 15 residues have similar pK (a) values, higher than those of the other glutamates. This excludes their mutual interaction in E5, being a source of the elevated pK (a) values. We attribute this phenomenon to the presence of minor states caused by deepening of the Glu11 and 15 side-chains in the hydrophobic environment of the membrane. As the mid-pH of membrane-perturbation activity of E5 matches the pK (a) value of these glutamates, we conclude their presence contributes to the plasticity of the peptide and determines the pH-dependence of membrane perturbation caused by E5.

  11. Konshina A.G., Dubovskii P.V., Efremov R.G. (2012). Structure and dynamics of cardiotoxins. Curr. Protein Pept. Sci. 13 (6), 570–84 [+]

    Cytotoxins (or cardiotoxins; CTs) are toxins from cobra venom characterized by the three-finger (TF) fold. CTs are on average 60-residue-long peptides, possessing as many as 4 disulfide bonds. The elements of antiparallel β-structure take origin from the hydrophobic core formed by the disulfides. The β-strands adopt the shape of the three loops, giving the name of the fold. While neurotoxins (NTs) - also TF proteins from snake venom - exert their effect through specific interactions with protein receptors, no specific protein target has been found for CTs. Unlike NTs, CTs are amphiphilic and cytotoxic against a variety of cells, including cancer ones. Thus, the hypothesis that the activity of CTs is caused by their interactions with lipid membranes is currently central. To understand molecular basis behind variations in toxicities of CTs highly homologous in their sequences, detailed knowledge of their structure and dynamics is required. The present review summarizes experimental and computational data on the spatial organization of CTs and their dynamics in various environments (aqueous solution, membranous milieus).

  12. Semenova A.A., Chugunov A.O., Dubovskii P.V., Chupin V.V., Volynsky P.E., Boldyrev I.A. (2011). The role of chain rigidity in lipid self-association: Comparative study of dihexanoyl- and disorbyl-phosphatidylcholines. Chem. Phys. Lipids 165, 382–386 [+]

    In the course of structure-function investigations of lipids a phosphatidylcholine molecule with short and rigid tails, di-2,4-hexadienoylphosphatidylcholine (DiSorbPC), was synthesized and studied in comparison with its saturated analog, dihexanoylphosphatidylcholine (DHPC). Conjugated double bonds in the acyl chains in DiSorbPC reduce considerably the number of possible conformers of the lipid within an aggregate. This leads to impaired packing of unsaturated acyl chains and thus, to a surprisingly high (115Å(2)) area per molecule for DiSorbPC at the air-water interface and failure to form micelles of regular size and shape. Details on DiSorbPC aggregation and packing provided by a set of experimental techniques combined with molecular dynamics simulations are presented.

  13. Dubovskii P.V., Vassilevski A.A., Samsonova O.V., Egorova N.S., Kozlov S.A., Feofanov A.V., Arseniev A.S., Grishin E.V. (2011). Novel lynx spider toxin shares common molecular architecture with defense peptides from frog skin. FEBS J. 278 (22), 4382–93 [+]

    A unique 30-residue cationic peptide oxyopinin 4a (Oxt 4a) was identified in the venom of the lynx spider Oxyopes takobius (Oxyopidae). Oxt 4a contains a single N-terminally located disulfide bond, Cys4-Cys10, and is structurally different from any spider toxin studied so far. According to NMR findings, the peptide is disordered in water, but assumes a peculiar torpedo-like structure in detergent micelles. It features a C-terminal amphipathic α-helical segment (body; residues 12-25) and an N-terminal disulfide-stabilized loop (head; residues 1-11), and has an unusually high density of positive charge in the head region. Synthetic Oxt 4a was produced and shown to possess strong and broad-spectrum cytolytic and antimicrobial activity. cDNA cloning showed that the peptide is synthesized in the form of a conventional prepropeptide with an acidic prosequence. Unlike other arachnid toxins, Oxt 4a exhibits striking similarity with defense peptides from the skin of ranid frogs that contain the so-called Rana-box motif (a C-terminal disulfide-enclosed loop). Parallelism or convergence is apparent on several levels: the structure, function and biosynthesis of a lynx spider toxin are mirrored by those of Rana-box peptides from frogs. DATABASE: The protein sequence of oxyopinin 4a (Oxt 4a) has been submitted to the UniProt Knowledgebase (UniProtKB) under the accession number P86350. The coordinates and chemical shifts of Oxt 4a in complex with dodecylphosphocholine micelles have been deposited in the Protein Data Bank and Biological Magnetic Resonance Bank under the accession codes 2L3I and 17194, respectively. The nucleotide sequence encoding Oxt 4a has been submitted to the EMBL Nucleotide Sequence Database under the accession number FN997582.

  14. Dubovskii P.V., Vassilevski A.A., Slavokhotova A.A., Odintsova T.I., Grishin E.V., Egorov T.A., Arseniev A.S. (2011). Solution structure of a defense peptide from wheat with a 10-cysteine motif. Biochem. Biophys. Res. Commun. 411 (1), 14–8 [+]

    Hevein, a well-studied lectin from the rubber tree Hevea brasiliensis, is the title representative of a broad family of chitin-binding polypeptides. WAMP-1a, a peptide isolated from the wheat Triticum kiharae, shares considerable similarity with hevein. The peptide possesses antifungal, antibacterial activity and is thought to play an important role in the defense system of wheat. Importantly, it features a substitution of the conserved serine residue to glycine reducing its carbohydrate-binding capacity. We used NMR spectroscopy to derive the spatial structure of WAMP-1a in aqueous solution. Notably, the mutation was found to strengthen amphiphilicity of the molecule, associated with its mode of action, an indication of the hevein domain multi-functionality. Both primary and tertiary structure of WAMP-1a suggest its evolutionary origin from the hevein domain of plant chitinases.

  15. Dubovskii P.V., Volynsky P.E., Polyansky A.A., Karpunin D.V., Chupin V.V., Efremov R.G., Arseniev A.S. (2008). Three-dimensional structure/hydrophobicity of latarcins specifies their mode of membrane activity. Biochemistry 47 (11), 3525–33 [+]

    Latarcins, linear peptides from the Lachesana tarabaevi spider venom, exhibit a broad-spectrum antimicrobial activity, likely acting on the bacterial cytoplasmic membrane. We study their spatial structures and interaction with model membranes by a combination of experimental and theoretical methods to reveal the structure-activity relationship. In this work, a 26 amino acid peptide, Ltc1, was investigated. Its spatial structure in detergent micelles was determined by (1)H nuclear magnetic resonance (NMR) and refined by Monte Carlo simulations in an implicit water-octanol slab. The Ltc1 molecule was found to form a straight uninterrupted amphiphilic helix comprising 8-23 residues. A dye-leakage fluorescent assay and (31)P NMR spectroscopy established that the peptide does not induce the release of fluorescent marker nor deteriorate the bilayer structure of the membranes. The voltage-clamp technique showed that Ltc1 induces the current fluctuations through planar membranes when the sign of the applied potential coincides with the one across the bacterial inner membrane. This implies that Ltc1 acts on the membranes via a specific mechanism, which is different from the carpet mode demonstrated by another latarcin, Ltc2a, featuring a helix-hinge-helix structure with a hydrophobicity gradient along the peptide chain. In contrast, the hydrophobic surface of the Ltc1 helix is narrow-shaped and extends with no gradient along the axis. We have also disclosed a number of peptides, structurally homologous to Ltc1 and exhibiting similar membrane activity. This indicates that the hydrophobic pattern of the Ltc1 helix and related antimicrobial peptides specifies their activity mechanism. The latter assumes the formation of variable-sized lesions, which depend upon the potential across the membrane.

  16. Dubovskii P.V., Volynsky P.E., Polyansky A.A., Chupin V.V., Efremov R.G., Arseniev A.S. (2006). Spatial structure and activity mechanism of a novel spider antimicrobial peptide. Biochemistry 45 (35), 10759–67 [+]

    Latarcins (Ltc), linear peptides (ca. 25 amino acid long) isolated from the venom of the Lachesana tarabaevi spider, exhibit a broad-spectrum antibacterial activity, most likely acting on the bacterial plasmatic membrane. We study the structure-activity relationships in the series of these compounds. At the first stage, we investigated the spatial structure of one of the peptides, Ltc2a, and its mode of membrane perturbation. This was done by a combination of experimental and theoretical methods. The approach includes (i) structural study of the peptide by CD spectroscopy in phospholipid liposomes and by (1)H NMR in detergent micelles, (ii) determination of the effect on the liposomes by a dye leakage fluorescent assay and (31)P NMR spectroscopy, (iii) refinement of the NMR-derived spatial structure via Monte Carlo simulations in an implicit water-octanol slab, and (iv) calculation of the molecular hydrophobicity potential. The molecule of Ltc2a was found to consist of two helical regions (residues 3-9 and 13-21) connected via a poorly ordered fragment. The effect of the peptide on the liposomes suggests the carpet mechanism of the membrane deterioration. This is also supported by the analysis of hydrophobic/hydrophilic characteristics of Ltc2a and homologous antimicrobial peptides. These peptides exhibiting a helix-hinge-helix structural motif are characterized by a distinct and feebly marked amphiphilicity of their N- and C-terminal helices, respectively, and by a hydrophobicity gradient along the peptide chain. The approach we suggested may be useful in studying not only other latarcins but also a wider class of membrane-active peptides.

  17. Dubinnyi M.A., Lesovoy D.M., Dubovskii P.V., Chupin V.V., Arseniev A.S. (2006). Modeling of 31P-NMR spectra of magnetically oriented phospholipid liposomes: A new analytical solution. Solid State Nucl Magn Reson 29 (4), 305–11 [+]

    31P-NMR spectroscopy is widely used for studies of phospholipid liposomes, a commonly used model of a biological membrane. For the correct analysis of 31P-NMR spectra of the liposomes it is necessary to take into account that they are deformed by the magnetic field of the spectrometer. The liposomes become ellipsoidal and this affects the lineshape of the spectrum. In the present communication we suggest a new analytical formula for modeling of 31P-NMR spectra of the prolate phospholipid liposomes. The formula assumes a Lorentzian broadening function and exactly ellipsoidal shape of the liposomes. Based on the formula a program called P-FIT is designed for the practical analysis of the experimental multicomponent spectra of the prolate liposomes. The versatility of the program developed in a Mathematica environment is demonstrated by simulations of a number of 31P-NMR spectra with different complexity.

  18. Feofanov A.V., Sharonov G.V., Dubinnyi M.A., Astapova M.V., Kudelina I.A., Dubovskii P.V., Rodionov D.I., Utkin Y.N., Arseniev A.S. (2004). Comparative study of structure and activity of cytotoxins from venom of the cobras Naja oxiana, Naja kaouthia, and Naja haje. Biochemistry Mosc. 69 (10), 1148–57 [+]

    Cytotoxins are positively charged polypeptides that constitute about 60% of all proteins in cobra venom; they have a wide spectrum of biological activities. By CD spectroscopy, cytotoxins CT1 and CT2 Naja oxiana, CT3 Naja kaouthia, and CT1 and CT2 Naja haje were shown to have similar secondary structure in an aqueous environment, with dominating beta-sheet structure, and to vary in the twisting angle of the beta-sheet and the conformation of disulfide groups. Using dodecylphosphocholine micelles and liposomes, CT1 and CT2 Naja oxiana were shown to incorporate into lipid structures without changes in the secondary structure of the peptides. The binding of CT1 and CT2 Naja oxiana with liposomes was associated with an increase in the beta-sheet twisting and a sign change of the dihedral angle of one disulfide group. The cytotoxins were considerably different in cytotoxicity and cooperativity of the effect on human promyelocytic leukemia cells HL60, mouse myelomonocytic cells WEHI-3, and human erythroleukemic cells K562. The most toxic CT2 Naja oxiana and CT3 Naja kaouthia possessed low cooperativity of interaction (Hill coefficient h = 0.6-0.8), unlike 10-20-fold less toxic CT1 and CT2 Naja haje (h = 1.2-1.7). CT1 Naja oxiana has an intermediate position on the cytotoxicity scale and is characterized by h = 0.5-0.8. The cytotoxins under study induced necrosis of HL60 cells and failed to activate apoptosis. The differences in cytotoxicity are supposed to be related not with features of the secondary structure of the peptides, but with interactions of side chains of variable amino acid residues with lipids and/or membrane proteins.

  19. Dubovskii P.V., Lesovoy D.M., Dubinnyi M.A., Utkin Y.N., Arseniev A.S. (2003). Interaction of the P-type cardiotoxin with phospholipid membranes. Eur. J. Biochem. 270 (9), 2038–46 [+]

    The cardiotoxin (cytotoxin II, or CTII) isolated from cobra snake (Naja oxiana) venom is a 60-residue basic membrane-active protein featuring three-finger beta sheet fold. To assess possible modes of CTII/membrane interaction 31P- and 1H-NMR spectroscopy was used to study binding of the toxin and its effect onto multilamellar vesicles (MLV) composed of either zwitterionic or anionic phospholipid, dipalmitoylglycerophosphocholine (Pam2Gro-PCho) or dipalmitoylglycerophosphoglycerol (Pam2Gro-PGro), respectively. The analysis of 1H-NMR linewidths of the toxin and 31P-NMR spectral lineshapes of the phospholipid as a function of temperature, lipid-to-protein ratios, and pH values showed that at least three distinct modes of CTII interaction with membranes exist: (a) nonpenetrating mode; in the gel state of the negatively charged MLV the toxin is bound to the surface electrostatically; the binding to Pam2Gro-PCho membranes was not observed; (b) penetrating mode; hydrophobic interactions develop due to penetration of the toxin into Pam2Gro-PGro membranes in the liquid-crystalline state; it is presumed that in this mode CTII is located at the membrane/water interface deepening the side-chains of hydrophobic residues at the tips of the loops 1-3 down to the boundary between the glycerol and acyl regions of the bilayer; (c) the penetrating mode gives way to isotropic phase, stoichiometrically well-defined CTII/phospholipid complexes at CTII/lipid ratio exceeding a threshold value which was found to depend at physiological pH values upon ionization of the imidazole ring of His31. Biological implications of the observed modes of the toxin-membrane interactions are discussed.

  20. Efremov R.G., Volynsky P.E., Nolde D.E., Dubovskii P.V., Arseniev A.S. (2002). Interaction of cardiotoxins with membranes: a molecular modeling study. Biophys. J. 83 (1), 144–53 [+]

    Incorporation of beta-sheet proteins into membrane is studied theoretically for the first time, and the results are validated by the direct experimental data. Using Monte Carlo simulations with implicit membrane, we explore spatial structure, energetics, polarity, and mode of insertion of two cardiotoxins with different membrane-destabilizing activity. Both proteins, classified as P- and S-type cardiotoxins, are found to retain the overall "three-finger" fold interacting with membrane core and lipid/water interface by the tips of the "fingers" (loops). The insertion critically depends upon the structure, hydrophobicity, and electrostatics of certain regions. The simulations reveal apparently distinct binding modes for S- and P-type cardiotoxins via the first loop or through all three loops, respectively. This rationalizes an earlier empirical classification of cardiotoxins into S- and P-type, and provides a basis for the analysis of experimental data on their membrane affinities. Accomplished with our previous simulations of membrane alpha-helices, the computational method may be used to study partitioning of proteins with diverse folds into lipid bilayers.

  21. Iwadate M., Asakura T., Dubovskii P.V., Yamada H., Akasaka K., Williamson M.P. (2001). Pressure-dependent changes in the structure of the melittin alpha-helix determined by NMR. J. Biomol. NMR 19 (2), 115–24 [+]

    A novel method is described, which uses changes in NMR chemical shifts to characterise the structural change in a protein with pressure. Melittin in methanol is a small alpha-helical protein, and its chemical shifts change linearly and reversibly with pressure between 1 and 2000 bar. An improved relationship between structure and HN shift has been calculated, and used to drive a molecular dynamics-based calculation of the change in structure. With pressure, the helix is compressed, with the H-O distance of the NH-O=C hydrogen bonds decreased by 0.021 +/- 0.039 A, leading to an overall compression along the entire helix of about 0.4 A, corresponding to a static compressibility of 6 x 10(-6) bar(-1). The backbone dihedral angles phi and psi are altered by no more than +/- 3 degrees for most residues with a negative correlation coefficient of -0.85 between phi(i) and psi(i - 1), indicating that the local conformation alters to maintain hydrogen bonds in good geometries. The method is shown to be capable of calculating structural change with high precision, and the results agree with structural changes determined using other methodologies.

  22. Dubovskii P.V., Dementieva D.V., Bocharov E.V., Utkin Y.N., Arseniev A.S. (2001). Membrane binding motif of the P-type cardiotoxin. J. Mol. Biol. 305 (1), 137–49 [+]

    Carditoxins (CTXs) from cobra snake venoms, the basic 60-62 residue all-beta sheet polypeptides, are known to bind to and impair the function of cell membranes. To assess the membrane induced conformation and orientation of CTXs, the interaction of the P-type cardiotoxin II from Naja oxiana snake venom (CTII) with perdeuterated dodecylphosphocholine (DPC) was studied using ( 1 )H-NMR spectroscopy and diffusion measurements. Under conditions where the toxin formed a well-defined complex with DPC, the spatial structure of CTII with respect to the presence of tightly bound water molecules in loop II, was calculated using the torsion angle dynamics program DYANA. The structure was found to be similar, except for subtle changes in the tips of all three loops, to the previously described "major" form of CTII in aqueous solution illustrated by the "trans" configuration of the Val7-Pro8 peptide bond. No "minor" form with the "cis" configuration of the above bond was found in the micelle-bound state. The broadening of the CTII backbone proton signals by 5, 16-doxylstearate relaxation probes, together with modeling based on the spatial structure of CTII, indicated a periphery mode of binding of the toxin molecule to the micelle and revealed its micelle interacting domain. The latter includes a hydrophobic region of CTII within the extremities of loops I and III (residues 5-11, 46-50), the basement of loop II (residues 24-29,31-37) and the belt of polar residues encircling these loops (lysines 4,5,12,23,50, serines 11,46, histidine 31, arginine 36). It is suggested that this structural motif and the mode of binding can be realized during interaction of CTXs with lipid and biological membranes.

  23. Orekhov V.Y., Dubovskii P.V., Yamada H., Akasaka K., Arseniev A.S. (2000). Pressure effect on the dynamics of an isolated alpha-helix studied by 15N-1H NMR relaxation. J. Biomol. NMR 17 (3), 257–63 [+]

    Dynamics and structure of (1-36)bacteriorhodopsin solubilized in chloroform/methanol mixture (1:1) were investigated by 1H-15N NMR spectroscopy under a hydrostatic pressure of 2000 bar. It was shown that the peptide retains its spatial structure at high pressure. 15N transverse and longitudinal relaxation times, 15N[1H] nuclear Overhauser effects, chemical shifts and the translation diffusion rate of the peptide at 2000 bar were compared with the respective data at ambient pressure [Orekhov et al. (1999) J. Biomol. NMR, 14, 345-356]. The model free analysis of the relaxation data for the helical 9-31 fragment revealed that the high pressure decreases the overall rotation and translation diffusion, as well as apparent order parameters of fast picosecond internal motions (S2) but has no effect on internal nanosecond motions (S2 and taus) of the peptide. The decrease of translation and overall rotation diffusion was attributed to the increase in solvent viscosity and the decrease of apparent order parameters S2f to a compression of hydrogen bonds. It is suggested that this compression causes an elongation of H-N bonds and a decrease of absolute values of chemical shift anisotropy (CSA). In particular, the observed decrease of S2f at 2000 bar can be explained by 0.001 nm increase of N-H bond lengths and 10 ppm decrease of 15N CSA values.

  24. Dubovskii P.V., Li H., Takahashi S., Arseniev A.S., Akasaka K. (2000). Structure of an analog of fusion peptide from hemagglutinin. Protein Sci. 9 (4), 786–98 [+]

    A 20-residue peptide E5 containing five glutamates, an analog of the fusion peptide of influenza virus hemagglutinin (HA) exhibiting fusion activity at acidic pH lower than 6.0-6.5 was studied by circular dichroism (CD), Fourier transform infrared, and 1H-NMR spectroscopy in water, water/trifluoroethanol (TFE) mixtures, dodecylphosphocholine (DPC) micelles, and phospholipid vesicles. E5 became structurally ordered at pH < or = 6 and the helical content in the peptide increased in the row: water < water/TFE < DPC approximately = phospholipid vesicle while the amount of beta-structure was approximately reverse. 1H-NMR data and line-broadening effect of 5-, 16-doxylstearates on proton resonances of DPC bound peptide showed E5 forms amphiphilic alpha-helix in residues 2-18, which is flexible in 11-18 part. The analysis of the proton chemical shifts of DPC bound and CD intensity at 220 nm of phospholipid bound E5 showed that the pH dependence of helical content is characterized by the same pKa approximately 5.6. Only Glu11 and Glu15 in DPC bound peptide showed such elevated pKas, presumably due to transient hydrogen bond(s) Glu11 (Glu15) deltaCOO- (H+)...HN Glu15 that dispose(s) the side chain of Glu11 (Glu15) residue(s) close to the micelle/water interface. These glutamates are present in the HA-fusion peptide and the experimental half-maximal pH of fusion for HA and E5 peptides is approximately 5.6. Therefore, a specific anchorage of these peptides onto membrane necessary for fusion is likely driven by the protonation of the carboxylate group of Glu11 (Glu15) residue(s) participating in transient hydrogen bond(s).

  25. Kalbitzer H.R., Görler A., Li H., Dubovskii P.V., Hengstenberg W., Kowolik C., Yamada H., Akasaka K. (2000). 15N and 1H NMR study of histidine containing protein (HPr) from Staphylococcus carnosus at high pressure. Protein Sci. 9 (4), 693–703 [+]

    The pressure-induced changes in 15N enriched HPr from Staphylococcus carnosus were investigated by two-dimensional (2D) heteronuclear NMR spectroscopy at pressures ranging from atmospheric pressure up to 200 MPa. The NMR experiments allowed the simultaneous observation of the backbone and side-chain amide protons and nitrogens. Most of the resonances shift downfield with increasing pressure indicating generalized pressure-induced conformational changes. The average pressure-induced shifts for amide protons and nitrogens are 0.285 ppm GPa(-1) at 278 K and 2.20 ppm GPa(-1), respectively. At 298 K the corresponding values are 0.275 and 2.41 ppm GPa(-1). Proton and nitrogen pressure coefficients show a significant but rather small correlation (0.31) if determined for all amide resonances. When restricting the analysis to amide groups in the beta-pleated sheet, the correlation between these coefficients is with 0.59 significantly higher. As already described for other proteins, the amide proton pressure coefficients are strongly correlated to the corresponding hydrogen bond distances, and thus are indicators for the pressure-induced changes of the hydrogen bond lengths. The nitrogen shift changes appear to sense other physical phenomena such as changes of the local backbone conformation as well. Interpretation of the pressure-induced shifts in terms of structural changes in the HPr protein suggests the following picture: the four-stranded beta-pleated sheet of HPr protein is the least compressible part of the structure showing only small pressure effects. The two long helices a and c show intermediary effects that could be explained by a higher compressibility and a concomitant bending of the helices. The largest pressure coefficients are found in the active center region around His15 and in the regulatory helix b which includes the phosphorylation site Ser46 for the HPr kinase. This suggests that this part of the structure occurs in a number of different structural states whose equilibrium populations are shifted by pressure. In contrast to the surrounding residues of the active center loop that show large pressure effects, Ile14 has a very small proton and nitrogen pressure coefficient. It could represent some kind of anchoring point of the active center loop that holds it in the right place in space, whereas other parts of the loop adapt themselves to changing external conditions.

  26. Efremov R.G., Nolde D.E., Volynsky P.E., Chernyavsky A.A., Dubovskii P.V., Arseniev A.S. (1999). Factors important for fusogenic activity of peptides: molecular modeling study of analogs of fusion peptide of influenza virus hemagglutinin. FEBS Lett. 462 (1-2), 205–10 [+]

    Nine analogs of fusion peptide of influenza virus hemagglutinin whose membrane perturbation activity has been thoroughly tested [Murata et al. (1992) Biochemistry 31, 1986-1992; Murata et al. (1993) Biophys. J. 64, 724-734] were characterized by molecular modeling techniques with the aim of delineating any specific structural and/or hydrophobic properties inherent in peptides with fusogenic activity. It was shown that, regardless of characteristics common to all analogs (peripheral disposition at the water-lipid interface, amphiphilic nature, alpha-helical structure, etc.), only fusion active peptides reveal a specific 'tilted oblique-oriented' pattern of hydrophobicity on their surfaces and a certain depth of penetration to the non-polar membrane core. The conclusion was reached that these factors are among the most important for the specific destabilization of a bilayer, which is followed by membrane fusion.

  27. Trakhanova M.N., Zinchenko A.A., Okhanov V.V., Dubovskii P.V., Bairamashvili D.I. (1989). [Structural and functional investigation of polymyxins. Structure and biological properties of polymyxin M analogs]. Antibiot. Khimioter. 34 (1), 20–4 [+]
  28. Okhanov V.V., Dubovskii P.V., Trakhanova M.N., Bairamashvili D.I., Zinchenko A.A. (1987). [Structural and functional research on polymyxins. 1H NMR analysis of the conformation of polymyxin M in water]. Antibiot. Med. Biotekhnol. 32 (10), 738–43 [+]