Tatiana V. Ovchinnikova

Purification and primary structure of two isoforms of arenicin, a novel antimicrobial peptide from marine polychaeta Arenicola marina

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) 1 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.

Lipid-protein nanoscale bilayers: a versatile medium for NMR investigations of membrane proteins and membrane-active peptides.

In the present Communication we demonstrate the possibility to use high-resolution NMR for the investigation of membrane proteins in reconstituted high-density lipoprotein (rHDL) particles. The rHDL particles are nanoscale phospholipid bilayers wrapped around by a dimer of apolipoprotein A-1 (Bayburt, T. H.; Grinkova, Y. V.; Sligar, S. G. Nano Lett. 2002, 2, 853−856). In contrast to the commonly used spherical micelles, the rHDL particles incorporate a lipid bilayer like in biological membranes. These particles still undergo isotropic motion on the NMR time scale, providing the application of high-resolution NMR spectroscopy of the peptides and proteins embedded into their bilayer. As an example, the topology of the membrane-active peptide Antiamoebin-I in the bilayer of the rHDL particles was determined by using the lipid-soluble relaxation probe technique.

Isolation, Structure Elucidation, and Synergistic Antibacterial Activity of a Novel Two-Component Lantibiotic Lichenicidin from Bacillus licheniformis VK21

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 Lchalpha and 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 Lchbeta were studied by NMR spectroscopy in methanol solution. The 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 a thioether bridge Ala11-S-Ala21. In contrast, the Lchbeta peptide represents a 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, Lchalpha and Lchbeta, interact 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 the mechanism of lichenicidin VK21 action.

Molecular Mechanism of Action of β-Hairpin Antimicrobial Peptide Arenicin: Oligomeric Structure in Dodecylphosphocholine Micelles and Pore Formation in Planar Lipid Bilayers

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.

Isolation, purification and de novo sequencing of TBD-1, the first beta-defensin from leukocytes of reptiles

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 β‐defensin 1), represents the first defensin isolated from reptilian leukocytes. It contains three disulfide bonds and shows high structural similarities to β‐defensins isolated from birds and mammals.

A novel defensin from the lentil Lens culinaris seeds.

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.

Molecular dynamics simulation of antimicrobial peptide arenicin‐2: β‐Hairpin stabilization by noncovalent interactions

Arenicin‐2 is a 21 residue antimicrobial cyclic peptide, possessing one disulphide bond between residues Cys3 and Cys20. NMR and CD studies suggested that the structure of arenicin‐2 in water represented a well formed, but highly twisted β‐harpin. 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 β‐hairpin structure during simulations, although the residues close to strand ends were found to escape from the ideal hairpin conformation. The type I′ β‐turn connecting the two strands fluctuated between type IV and II′ β‐turn. Conversely, the right‐handed twist of the β‐hairpin was well conserved with average twist value 203° ± 19° per eight residues. Several nonbonded interactions, like hydrophobic interactions between aliphatic side chains, cation/π‐aromatic interactions, CH…π aromatic bond and water bridges, contributed to the hairpin stabilization.

Structure and Alignment of the Membrane-Associated Antimicrobial Peptide Arenicin by Oriented Solid-State NMR Spectroscopy

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.

Design of antimicrobial peptide arenicin analogs with improved therapeutic indices

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. Copyright

Recombinant production and solution structure of lipid transfer protein from lentil Lens culinaris

Lipid transfer protein, designated as Lc-LTP2, was isolated from seeds of the lentil Lens culinaris. The protein has molecular mass 9282.7Da, consists of 93 amino acid residues including 8 cysteines forming 4 disulfide bonds. Lc-LTP2 and its stable isotope labeled analogues were overexpressed in Escherichia coli and purified. Antimicrobial activity of the recombinant protein was examined, and its spatial structure was studied by NMR spectroscopy. The polypeptide chain of Lc-LTP2 forms four α-helices (Cys4-Leu18, Pro26-Ala37, Thr42-Ala56, Thr64-Lys73) and a long C-terminal tail without regular secondary structure. Side chains of the hydrophobic residues form a relatively large internal tunnel-like lipid-binding cavity (van der Waals volume comes up to ∼600Å(3)). The side-chains of Arg45, Pro79, and Tyr80 are located near an assumed mouth of the cavity. Titration with dimyristoyl phosphatidylglycerol (DMPG) revealed formation of the Lc-LTP2/lipid non-covalent complex accompanied by rearrangements in the protein spatial structure and expansion of the internal cavity. The resultant Lc-LTP2/DMPG complex demonstrates limited lifetime and dissociates within tens of hours.