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Biomedical subjects

A S Arseniev

Publications and source records attributed to A S Arseniev.

At least 19 recordsLinked to original sources
PubMed2005

Comparative analysis of proapoptotic activity of cytochrome c mutants in living cells.

A non-traumatic electroporation procedure was developed to load exogenous cytochrome c into the cytoplasm and to study the apoptotic effect of cytochrome c, its K72-substitued mutants and "yeast --> horse" hybrid cytochrome c in living WEHI-3 cells. The minimum apoptosis-activating intracellular concentration of horse heart cytochrome c was estimated to be 2.7 +/- 0.5 microM (47 +/- 9 fg/cell). The equieffective concentrations of the K72A-, K72E- and K72L-substituted mutants of cytochrome c were five-, 15- and 70-fold higher. The "yeast --> horse" hybrid created by introducing S2D, K4E, A7K, T8K, and K11V substitutions (horse protein numbering) and deleting five N-terminal residues in yeast cytochrome c did not evoke apoptotic activity in mammalian cells. The apoptotic function of cytochrome c was abolished by the K72W substitution. The K72W-substituted cytochrome c possesses reduced affinity to the apoptotic protease activating factor-1 (Apaf-1) and forms an inactive complex. This mutant is competent as a respiratory-chain electron carrier and well suited for knock-in studies of cytochrome c-mediated apoptosis.

Animals↗
PubMed2004

Comparative study of structure and activity of cytotoxins from venom of the cobras Naja oxiana, Naja kaouthia, and Naja haje.

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.

Amino Acid Sequence↗
PubMed2004

Peptaibol zervamicin IIb structure and dynamics refinement from transhydrogen bond J couplings.

Zervamicin IIB (Zrv-IIB) is a channel-forming peptaibol antibiotic of fungal origin. The measured transhydrogen bond (3h)J(NC') couplings in methanol solution heaving average value of -0.41 Hz indicate that the stability of the Zrv-IIB helix in this milieu is comparable to the stability of helices in globular proteins. The N-terminus of the peptide forms an alpha-helix, whereas 3(10)-helical hydrogen bonds stabilize the C-terminus. However, two weak transhydrogen bond peaks are observed in a long-range HNCO spectrum for HN Aib(12). Energy calculations using the Empirical Conformation Energy Program for Peptides (ECEPP)/2 force field and the implicit solvent model show that the middle of the peptide helix accommodates a bifurcated hydrogen bond that is simultaneously formed between HN Aib(12) and CO Leu(8) and CO Aib(9). Several lowered (3h)J(NC') on a polar face of the helix correlate with the conformational exchange process observed earlier and imply dynamic distortions of a hydrogen bond pattern with the predominant population of a properly folded helical structure. The refined structure of Zrv-IIB on the basis of the observed hydrogen bond pattern has a small ( approximately 20 degrees ) angle of helix bending that is virtually identical to the angle of bending in dodecylphosphocholine (DPC) micelles, indicating the stability of a hinge region in different environments. NMR parameters ((1)HN chemical shifts and transpeptide bond (1)J(NC') couplings) sensitive to hydrogen bonding along with the solvent accessible surface area of carbonyl oxygens indicate a large polar patch on the convex side of the helix formed by three exposed backbone carbonyls of Aib(7), Aib(9), and Hyp(10) and polar side chains of Hyp(10), Gln(11), and Hyp(13). The unique structural features, high helix stability and the enhanced polar patch, set apart Zrv-IIB from other peptaibols (for example, alamethicin) and possibly underlie its biological and physiological properties.

Hydrogen Bonding↗
PubMed2004

Peptides and proteins in membranes: what can we learn via computer simulations?

Membrane and membrane-active peptides and proteins play a crucial role in numerous cell processes, such as signaling, ion conductance, fusion, and others. Many of them act as highly specific and efficient drugs or drug targets, and, therefore, attract growing interest of medicinal chemists. Because of experimental difficulties with characterization of their spatial structure and mode of membrane binding, essential attention is given now to molecular modeling techniques. During the last years an important progress has been achieved in molecular dynamics (MD) and Monte Carlo (MC) simulations of peptides and proteins with explicit and/or implicit theoretical models of membranes. The first ones allow atomic-resolution studies of peptides behavior on the membrane-water interfaces. Models with implicit consideration of membrane are of a special interest because of their computational efficiency and ability to account for principal trends in protein-lipid interactions. In this approximation, the bilayer is usually treated as continuum whose properties vary along the membrane thickness, and membrane insertion is simulated using either MC or MD methods. This review surveys recent applications of both types of lipid bilayer models in computer simulations of a wide variety of peptides and proteins with different biological activities. Theoretical background of the membrane models is considered with examples of their applications to biologically relevant problems. The emphasis of the review is made on recent MC and MD computations, on structural and/or functional information, which may be obtained via molecular modeling. The approximations and shortcomings of the models, along with their perspectives in design of new membrane active drugs, are discussed.

Animals↗
PubMed2002

Spatial structure of zervamicin IIB bound to DPC micelles: implications for voltage-gating.

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. The spatial structure of zervamicin IIB bound to dodecylphosphocholine micelles was studied by nuclear magnetic resonance spectroscopy. The set of 20 structures obtained has a bent helical conformation with a mean backbone root mean square deviation value of approximately 0.2 A and resembles the structure in isotropic solvents (Balashova et al., 2000. NMR structure of the channel-former zervamicin IIB in isotropic solvents. FEBS Lett 466:333-336). The N-terminus represents an alpha-helix, whereas the C-terminal part has a mixed 3(10)/alpha(R) hydrogen-bond pattern. In the anisotropic micelle environment, the bending angle on Hyp10 (23 degrees) is smaller than that (47 degrees) in isotropic solvents. In the NOESY (Nuclear Overhauser Effect Spectroscopy) spectra, the characteristic attenuation of the peptide signals by 5- and 16-doxylstearate relaxation probes indicates a peripheral mode of the peptaibol binding to the micelle with the N-terminus immersed slightly deeper into micelle interior. Analysis of the surface hydrophobicity reveals that the zervamicin IIB helix is amphiphilic and well suited to formation of a tetrameric transmembrane bundle, according to the barrel-stave mechanism. The results are discussed in a context of voltage-driven peptaibol insertion into membrane.

Anisotropy↗
PubMed2001

Backbone dynamics of the channel-forming antibiotic zervamicin IIB studied by 15N NMR relaxation.

The backbone dynamics of the channel-forming peptide antibiotic zervamicin IIB (Zrv-IIB) in methanol were studied by 15N nuclear magnetic resonance relaxation measurements at 11.7, 14.1 and 18.8 T magnetic fields. The anisotropic overall rotation of the peptide was characterized based on 15N relaxation data and by hydrodynamic calculations. 'Model-free' analysis of the relaxation data showed that the peptide is fairly rigid on a sub-nanosecond time-scale. The residues from the polar side of Zrv-IIB helix are involved in micro-millisecond time-scale conformational exchange. The conformational exchange observed might indicate intramolecular processes or specific intermolecular interactions of potential relevance to Zrv-IIB ion channel formation.

Anisotropy↗
PubMed2001

Membrane binding motif of the P-type cardiotoxin.

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.

Amino Acid Motifs↗
PubMed2000

NMR structure of the channel-former zervamicin IIB in isotropic solvents.

Spatial structure of the membrane channel-forming hexadecapeptide, zervamicin IIB, was studied by NMR spectroscopy in mixed solvents of different polarity ranging from CDCl3/CD3OH (9:1, v/v) to CD3OH/H2O (1:1, v/v). The results show that in all solvents used the peptide has a very similar structure that is a bent amphiphilic helix with a mean backbone root mean square deviation (rmsd) value of ca. 0.3 A. Side chains of Trp1, Ile2, Gln3, Ile5 and Thr6 are mobile. The results are discussed in relation to the validity of the obtained structure to serve as a building block of zervamicin IIB ion channels.

Amino Acid Sequence↗
PubMed2000

Pressure effect on the dynamics of an isolated alpha-helix studied by 15N-1H NMR relaxation.

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.

Anisotropy↗
PubMed2000

Off-resonance effects in 15N T2 CPMG measurements.

The systematic difference between T2 values obtained from CPMG and T1p experiments was observed for backbone 15N nuclei of bacterial ribonuclease barnase. Theoretical consideration suggests that the observed difference is caused by off-resonance effects of 180 degree pulses of the CPMG pulse train. Namely, at off-resonance conditions T1-dependent secondary echo coherence pathways considerably contribute to the signal decay in the CPMG experiment and result in systematic (up to 10%) offset-dependent overestimation of 15N T2 measured by the CPMG technique. Under certain circumstances off-resonance effects result in dependence of 15N T2 on CPMG frequency, which might be erroneously interpreted as conformational exchange on the millisecond time-scale. A procedure for numerical correction of 15N T2 (CPMG) data is proposed.

Artifacts↗
PubMed2000

Stabilization of proteins by enhancement of inter-residue hydrophobic contacts: lessons of T4 lysozyme and barnase.

Although the hydrophobic interactions are considered as the main contributors to the protein stability, not much examples of protein stabilization by rational increasing of this type of interactions still can be found in literature. This is partly due to the lack of proper theoretical "measure" of hydrophobic interactions and their changes upon mutations. In the present paper the molecular hydrophobicity potential approach is used to assess how the changes in type and the strength of inter-residue contacts upon single amino acid mutations are correlated with the changes in thermodynamic stability of T4 lysozyme and barnase mutants, and which factors affect these correlations. Mutations changing unfavorable hydrophilic-to-hydrophobic contacts into favorable hydrophobic were found to enhance the thermodynamic stability in more than 81 % of cases, if these mutations do not create steric bumps and do not involve proline residues and hydrogen-bonded side-chains. Mutations increasing hydrophobic contributions (according to molecular hydrophobicity potential formalism) lead to increase of thermodynamic stability in more than 94% of cases for certain type of mutations (i.e., mutations not involving charged residues, Pro and residues with side-chain hydrogen bonds, when these mutations do not introduce steric bumps and do not involve strongly exposed residues and residues situated at helix N- and C-cap positions). For this type of mutations the correlation was found between the change in hydrophobic contributions of mutated residues deltaCphob and thermodynamic parameters deltaTm (change in melting temperature) and deltadeltaG (change in free energy of unfolding). Although the correlation coefficients were larger if the experimental structures of mutants were used for the calculations (correlation coefficients r(exp) deltaC,deltaT = .85 and r(exp) deltaC,deltadeltaG = 0.87) than if the modeled structures were used instead (r(mod) deltaC,deltaT = 0.74 and r(mod)deltaC,deltadeltaG = 0.76), the modelled structures of mutants in the vast majority of cases can be used for qualitative predictition of the protein stabilization. Basing on the analysis of mutations increasing hydrophobic contributions in T4 lysozyme the substitution matrix was derived, which can be used to decide which new residue should be put instead the old one to increase the stability of protein. The estimation shows that the number of potential mutation sites for enhancement of hydrophobic interactions in T4 lysozyme is quite large, and only approximately 10 per cent of them were studied thus far. Basing on the current analysis of T4 lysozyme and barnase mutations the algorithm for increasing of protein stability via increasing of hydrophobic interactions for the proteins with known spatial structure is proposed.

Algorithms↗
PubMed2000

Structure of an analog of fusion peptide from hemagglutinin.

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).

Circular Dichroism↗
PubMed1999

Factors important for fusogenic activity of peptides: molecular modeling study of analogs of fusion peptide of influenza virus hemagglutinin.

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.

Amino Acid Sequence↗
PubMed1999

Spatial structure of the M2 transmembrane segment of the nicotinic acetylcholine receptor alpha-subunit.

A synthetic peptide corresponding to the transmembrane segment M2 (residues 236-267) of the alpha-subunit of the nicotinic acetylcholine receptor from Torpedo californica has been studied by two dimensional 1H-NMR spectroscopy in a chloroform-methanol (1:1) mixture containing 0.1 M LiClO4. Reconstruction of the spatial structure of M2 from the NMR data resulted in an alpha-helix formed by residues 241-263. Distribution of the molecular hydrophobicity potential on the helix surface is very similar to that in five-helix bundles of proteins with a known three dimensional structure: two hydrophilic bands located on the opposite helix sides separated by strong hydrophobic zones.

Amino Acid Sequence↗
PubMed1999

Structure and stability of recombinant protein depend on the extra N-terminal methionine residue: S6 permutein from direct and fusion expression systems.

Two permuted variants of S6 ribosomal protein were obtained in direct and fusion expression systems, respectively. The product of direct expression contained the extra N-terminal methionine residue. The structural properties and conformational stability of these permuteins were compared using 1-D (1)H-NMR, circular dichroism, intrinsic fluorescence, differential scanning calorimetry and resistance to urea-induced unfolding. A pronounced difference in all the parameters studied has been demonstrated. This means that the structure of recombinant protein can be sensitive to peculiarities of the expression and purification procedures, leading particularly to the presence or absence of the Met at the first position in the target protein sequence.

Calorimetry, Differential Scanning↗
PubMed1999

NMR spatial structure of alpha-conotoxin ImI reveals a common scaffold in snail and snake toxins recognizing neuronal nicotinic acetylcholine receptors.

A 600 MHz NMR study of alpha-conotoxin ImI from Conus imperialis, targeting the alpha7 neuronal nicotinic acetylcholine receptor (nAChR), is presented. ImI backbone spatial structure is well defined basing on the NOEs, spin-spin coupling constants, and amide protons hydrogen-deuterium exchange data: rmsd of the backbone atom coordinates at the 2-12 region is 0.28 A in the 20 best structures. The structure is described as a type I beta-turn (positions 2-5) followed by a distorted helix (positions 5-11). Similar structural patterns can be found in all neuronal-specific alpha-conotoxins. Highly mobile side chains of the Asp-5, Arg-7 and Trp-10 residues form a single site for ImI binding to the alpha7 receptor. When depicted with opposite directions of the polypeptide chains, the ImI helix and the tip of the central loop of long chain snake neurotoxins demonstrate a common scaffold and similar positioning of the functional side chains, both of these structural elements appearing essential for binding to the neuronal nAChRs.

Amino Acid Sequence↗