Recombinant expression, synthesis, purification, and solution structure of arenicin |
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Authors: | Ovchinnikova Tatiana V Shenkarev Zakhar O Nadezhdin Kirill D Balandin Sergey V Zhmak Maxim N Kudelina Irina A Finkina Ekaterina I Kokryakov Vladimir N Arseniev Alexander S |
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Institution: | Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya Str., 16/10, 117997 Moscow, Russia. ovch@ibch.ru |
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Abstract: | Arenicins are 21-residue cationic antimicrobial peptides, isolated from marine polychaeta Arenicola marina. In order to determine a high-resolution three-dimensional structure of arenicin-2, the recombinant peptide was overexpressed as a fused form in Escherichia coli. Both arenicin isoforms were synthesized using the Fmoc-based solid-phase strategy. Recombinant and synthetic arenicins were purified, and their antimicrobial and spectroscopic properties were analyzed. NMR investigation shows that in water solution arenicin-2 displays a prolonged beta-hairpin, formed by two antiparallel beta-strands and stabilized by one disulfide and nine hydrogen bonds. A significant right-handed twist in the beta-sheet is deprived the peptide surface of amphipathicity. CD spectroscopic analysis indicates that arenicin-2 binds to the SDS and DPC micelles, and conformation of the peptide is significantly changed upon binding. Arenicin strongly binds to anionic lipid (POPE/POPG) vesicles in contrast with zwitterionic (POPC) ones. These results suggest that arenicins are membrane active peptides and point to possible mechanism of their selectivity toward bacterial cells. |
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Keywords: | Antimicrobial peptide Arenicin Lugworm Arenicola marina Marine invertebrate Expression Recombinant Fusion protein Purification Folding Antimicrobial activity NMR CD β-Hairpin |
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