Sergey V. Balandin

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.

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.

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.

Purification and primary structure of novel lipid transfer proteins from germinated lentil (Lens culinaris) seeds

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.

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.

Heterologous expression and solution structure of defensin from lentil Lens culinaris

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.

Lipid-dependent pore formation by antimicrobial peptides arenicin-2 and melittin demonstrated by their proton transfer activity.

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. Copyright

Comparative in vitro study on cytotoxicity of recombinant β-hairpin peptides

Natural antimicrobial peptides (AMPs) are important components of the innate immune system with a wide spectrum of biological activity. In this study, we investigated the cytotoxic effect of three recombinant β‐hairpin cationic AMPs: arenicin‐1 from the polychaeta Arenicola marina, tachyplesin I from the horseshoe crab Tachypleus tridentatus, and gomesin from the spider Acanthoscurria gomesiana. All the three β‐hairpin AMPs were overexpressed in Escherichia coli. Different cell lines were incubated with various concentrations of the investigated AMPs in order to evaluate their cytotoxic activity. Double staining with subsequent flow cytometric analysis was used to determine the predominant way of cell death mediated by each AMP. Hemolytic activity of the peptides was tested against fresh human red blood cells. Our results indicated that all the three AMPs exhibited significant cytotoxic effect against cancer cells that varied depending on the cell line type and, in most cases, on the presence of serum components. Flow cytometric analysis implicitly indicated that tachyplesin I mostly promoted late apoptosis/necrosis, while arenicin‐1 and gomesin induced early apoptosis under the same conditions. Tachyplesin I proved to be the most promising therapeutic candidate as it displayed the highest specific cytotoxicity against cancer cell lines, independent of the serum presence.