Structural and Functional Studies on Membrane Targeting Peptides

Abstract

PART I A significantly enhanced antibacterial spectrum of D-enantiomeric lipopeptide bactenecin Cationic antimicrobial peptides (CAMPs) are important antibiotics because they possess a broad spectrum of activity against both Gram-positive and Gram-negative bacteria, including those resistant to traditional antibiotics. The cyclic peptide bactenecin is a 12-amino acid CAMP that contains one intramolecular disulfide bond. To improve the antibacterial activity of bactenecin, we designed and synthesized several bactenecin analogs by applying multiple approaches, including amino acid substitution, use of the D-enantiomeric form, and lipidation. Among the synthetic analogs, D-enantiomeric bactenecin conjugated to capric acid, which we named dBacK-(cap), exhibited a significantly enhanced antibacterial spectrum with MIC values ranging from 1 to 8 μM against both Gram-positive and Gram-negative bacteria, including some drug-resistant bacteria. Upon exposure to dBacK-(cap), S. aureus cells were killed within 1 h at the MIC value, but full inactivation of E. coli required over 2 h. These results indicate that covalent addition of a D-amino acid and a fatty acid to bactenecin is the most effective approach for enhancing its antibacterial activity.

PART II Identification of a novel conotoxin pJY15 that interacts genetically Drosophila Ih channel Conotoxins are peptide neurotoxins which are produced in cone snail venom duct. These toxins have been used to development of therapeutic reagent and channelopathy research. Although conotoxins are abundant in the sea snail venome, therapeutic effect and biological function of most of these toxins are yet to be discorverd due to their structural diversity and chemical modifications as post-translational modifications or disulfides. To identify novel bioactive conotoxin in peptide library isolated from Korean cone snail, we established the new screening tool through conotoxin expression in Drosophila neurons. By combining conotoxin mature peptide and fly neuropeptide prepro precursor, the conotoxin transgenes was so designed as to insert into fly. According to the result of sleep behavior screening in conotoxin transgene expressed flies, we identified a novel conotoxin-pJY15 derived from molluscivorous snail Conus Flavidus which reduced sleep duration in Drosophila specific-Ih neurons. The Cys framework and amino acid sequence of conotoxin-pJY15 are similar to the γ-conotoxin-TxVIIA from Conus Textile and γ-conotoxin-PnVIIA from Conus Pennaceus, which is known to affect neuronal pacemaker channels. These findings show that conotoxin-pJY15 may interact with Ih neurons involved dopamine neurons, and conclude that fly expression system may applicable as the basis of screening studies to search bioactive neurotoxins through observation of behavioral defect.