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NMR based structure-activity relationship analysis of an antimicrobial peptide, thanatin, engineered by site-specific chemical modification: Activity improvement and spectrum alteration |
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| Authors: | Imamura Tomohiro Yamamoto Naoki Tamura Atsuo Murabayashi Shinji Hashimoto Shigeki Shimada Hiroaki Taguchi Seiichi |
| Institution: | a Department of Biological Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda 278-8510, Japan b Department of Chemistry, Graduate School of Science, Kobe University, Rokkodai, Nada, 657-8501 Kobe, Japan c Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, Sapporo 060-0810, Japan d Faculty of Industrial Science & Technology, Tokyo University of Science, Oshamanbe, Hokkaido 049-3514, Japan |
| Abstract: | Activity improvement of an antimicrobial peptide, thanatin, has been achieved up to 4-fold higher than natural original one by site-specific chemical modifications with tert-butyl group at two cysteine residues which form an intramoleular disulfide bridge. The chemically modified thanatin (C11tBu/C18tBu) exhibited improved antimicrobial activity toward Gram-positive bacteria, Micrococcus luteus, whereas lowered activity toward Gram-negative bacteria, Escherichia coli. This finding suggests that disulfide-bridge formation is not only indispensable for exhibition of antimicrobial activity of thanatin but also closely related to the activity specificity towards bacteria. NMR analysis indicates that thanatin acts against E.coli stereospecifically by taking advantage of its C-terminal β-hairpin structure, while the activity against M. luteus does not relate to structures and correlates very well to side-chain hydrophobicity. |
| Keywords: | ACN acetonitrile DIPEA diisopropylethylamine DMF N N-dimethylformamide Fmoc 9-fluorenylmethoxycarbonyl HOBt N-hydroxybenzotriazole HPLC high performance liquid chromatography MALDI matrix-assisted laser desorption/ionization MIC minimal inhibitory concentration PEG polyethylene glycol HATU N-[(dimethylamino)-1H-1 2 3-triazolo-[4 5-b]pyridin-1-yl-methylene]-N-mehylmethanaminium hexafluorophosphate N-oxide tBu tert-butyl group TFA trifluoroacetic acid TOF time of flight NMR nuclear magnetic resonance COSY correlated spectroscopy TOCSY total correlated spectroscopy NOESY nuclear Overhauser enhancement spectroscopy DQF-COSY double quantum filtered-corretated spectroscopy WATERGATE water suppression by gradient-tailored excitation RMSD root mean square deviation Δ delta |
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