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Heterologous expression and solution structure of defensin from lentil Lens culinaris
Authors:Zakhar O. Shenkarev  Albina K. Gizatullina  Ekaterina I. Finkina  Ekaterina A. Alekseeva  Sergey V. Balandin  Konstantin S. Mineev  Alexander S. Arseniev  Tatiana V. Ovchinnikova
Affiliation:1. Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya Str., 16/10, 117997 Moscow, Russia;2. Moscow Institute of Physics and Technology (State University), Department of Physicochemical Biology and Biotechnology, Institutskii per., 9, 141700 Dolgoprudny, Moscow Region, Russia
Abstract:A new defensin Lc-def, isolated from germinated seeds of the lentil Lens culinaris, has molecular mass 5440.4 Da and consists of 47 amino acid residues. Lc-def and its 15N-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.
Keywords:CSαβ, cysteine-stabilized αβ motif   DOPG, 1,2-dioleoyl-sn-glycero-3-phosphoglycerol   POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine   SUV, small unilamellar vesicles
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