Development of potent anti-infective agents from Silurana tropicalis: Conformational analysis of the amphipathic,alpha-helical antimicrobial peptide XT-7 and its non-haemolytic analogue [G4K]XT-7 |
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Authors: | Anusha P. Subasinghage J. Michael Conlon Chandralal M. Hewage |
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Affiliation: | 1. UCD School of Biomolecular and Biomedical Science, Centre for Synthesis and Chemical Biology, SEC Strategic Research Cluster, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland;2. Department of Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates University, 17666 Al-Ain, United Arab Emirates |
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Abstract: | Peptide XT-7 (GLLGP5LLKIA10AKVGS15NLL.NH2) is a cationic, leucine-rich peptide, first isolated from skin secretions of the frog, Silurana tropicalis (Pipidae). The peptide shows potent, broad-spectrum antimicrobial activity but its therapeutic potential is limited by haemolytic activity (LC50 = 140 µM). The analogue [G4K]XT-7, however, retains potent antimicrobial activity but is non-haemolytic (LC50 > 500 µM). In order to elucidate the molecular basis for this difference in properties, the three dimensional structures of XT-7 and the analogue have been investigated by proton NMR spectroscopy and molecular modelling. In aqueous solution, both peptides lack secondary structure. In a 2,2,2-trifluoroethanol (TFE-d3)-H2O mixed solvent system, XT-7 is characterised by a right handed α-helical conformation between residues Leu3 and Leu17 whereas [G4K]XT-7 adopts a more restricted α-helical conformation between residues Leu6 and Leu17. A similar conformation for XT-7 in 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micellular media was observed with a helical segment between Leu3 and Leu17. However, differences in side chain orientations restricting the hydrophilic residues to a smaller patch resulted in an increased hydrophobic surface relative to the conformation in TFE-H2O. Molecular modelling of the structures obtained in our study demonstrates the amphipathic character of the helical segments. It is proposed that the marked decrease in haemolytic activity produced by the substitution Gly4 → Lys in XT-7 arises from a decrease in both helicity and hydrophobicity. These studies may facilitate the development of potent but non-toxic anti-infective agents based upon the structure of XT-7. |
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Keywords: | CD, circular dichroism DHPC, 1,2-dihexanoyl-sn-glycero-3-phosphocholine MIC, minimum inhibitory concentration NMR, nuclear magnetic resonance NOE, nuclear Overhauser effect NOESY, nuclear Overhauser effect spectroscopy TFE, trifluoroethanol TOCSY, total correlation spectroscopy RMSD, root mean square deviation |
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