Andrey A. Tagaev

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 nanodiscs: Possible application in high-resolution NMR investigations of membrane proteins and membrane-active peptides

High-resolution NMR is shown to be applicable for investigation of membrane proteins and membrane-active peptides embedded into lipid-protein nanodiscs (LPNs). 15N-Labeled K+-channel from Streptomyces lividans (KcsA) and the antibiotic antiamoebin I from Emericellopsis minima (Aam-I) were embedded in LPNs of different lipid composition. Formation of stable complexes undergoing isotropic motion in solution was confirmed by size-exclusion chromatography and 31P-NMR spectroscopy. The 2D 1H-15N-correlation spectra were recorded for KcsA in the complex with LPN containing DMPC and for Aam-I in LPNs based on DOPG, DLPC, DMPC, and POPC. The spectra recorded were compared with those in detergent-containing micelles and small bicelles commonly used in high-resolution NMR spectroscopy of membrane proteins. The spectra recorded in LPN environments demonstrated similar signal dispersion but significantly increased 1HN line width. The spectra of Aam-I embedded in LPNs containing phosphatidylcholine showed significant selective line broadening, thus suggesting exchange process(es) between several membrane-bound states of the peptide. 15N relaxation rates were measured to obtain the effective rotational correlation time of the Aam-I molecule. The obtained value (∼40 nsec at 45°C) is indicative of additional peptide motions within the Aam-I/LPN complex.

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 Å. 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.

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.

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.

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.

Peptaibol Antiamoebin I: Spatial Structure, Backbone Dynamics, Interaction with Bicelles and Lipid-Protein Nanodiscs, and Pore Formation in Context of Barrel-Stave Model

Antiamoebin I (Aam‐I) is a membrane‐active peptaibol antibiotic isolated from fungal species belonging to the genera Cephalosporium, Emericellopsis, Gliocladium, and Stilbella. In comparison with other 16‐amino acid‐residue peptaibols, e.g., zervamicin IIB (Zrv‐IIB), Aam‐I possesses relatively weak biological and channel‐forming activities. In MeOH solution, Aam‐I demonstrates fast cooperative transitions between right‐handed and left‐handed helical conformation of the N‐terminal (1–8) region. We studied Aam‐I spatial structure and backbone dynamics in the membrane‐mimicking environment (DMPC/DHPC bicelles)1) by heteronuclear 1H,13C,15N‐NMR spectroscopy. Interaction with the bicelles stabilizes the Aam‐I right‐handed helical conformation retaining significant intramolecular mobility on the ms–μs time scale. Extensive ms–μs dynamics were also detected in the DPC and DHPC micelles and DOPG nanodiscs. In contrast, Zrv‐IIB in the DPC micelles demonstrates appreciably lesser mobility on the μs–ms time scale. Titration with Mn2+ and 16‐doxylstearate paramagnetic probes revealed Aam‐I binding to the bicelle surface with the N‐terminus slightly immersed into hydrocarbon region. Fluctuations of the Aam‐I helix between surface‐bound and transmembrane (TM) state were observed in the nanodisc membranes formed from the short‐chain (diC12 : 0) DLPC/DLPG lipids. All the obtained experimental data are in agreement with the barrel‐stave model of TM pore formation, similarly to the mechanism proposed for Zrv‐IIB and other peptaibols. The observed extensive intramolecular dynamics explains the relatively low activity of Aam‐I.