title: Substitution of the leucine zipper sequence in melittin with peptoid residues affects self-association, cell selectivity, and mode of action.
abstract: Melittin (ME), a non-cell-selective antimicrobial peptide, contains the leucine zipper motif, wherein every seventh amino acid is leucine or isolucine. Here, we attempted to generate novel cell-selective peptides by substituting amino acids in the leucine zipper sequence of ME with peptoid residues. We generated a series of ME analogues by replacing Leu-6, Lue-13 and Ile-20 with Nala, Nleu, Nphe, or Nlys, and we examined their secondary structure, self-association activity, cell selectivity and mode of action. Circular dichroism spectroscopy indicated that the substitutions disrupt the alpha-helical structure of ME in micelles of sodium dodecyl sulfate and on negatively charged and zwitterionic phospholipid vesicles. Substitution by Nleu, Nphe, or Nlys but not Nala disturbed the self-association in an aqueous environment, interaction with zwitterionic membranes, and toxicity to mammalian cells of ME but did not affect the interaction with negatively charged membranes or antibacterial activity. Notably, peptides with Nphe or Nlys substitution had the highest therapeutic indices, consistent with their lipid selectivity. In addition, all of peptoid residue-containing ME analogues had little or no ability to induce membrane disruption, membrane depolarization and lipid flip-flop. Taken together, our studies indicate that substitution of the leucine zipper motif in ME with peptoid residues increases its selectivity against bacterial cells by impairing self-association activity and changes its mode of antibacterial action from membrane-targeting mechanism to possible intracellular targeting mechanism. Furthermore, our ME analogues especially those with Nleu, Nphe, or Nlys substitutions, may be therapeutically useful antimicrobial peptides.
authors.affiliation: Department of Bio-Materials, Graduate School and Research Center for Proteineous Materials, Chosun University, Gwangju 501-759, Korea.
authors.forename: Wan Long
authors.initials: WL
authors.lastname: Zhu
authors.forename: Yun Mi
authors.initials: YM
authors.lastname: Song
authors.forename: Yoonkyung
authors.initials: Y
authors.lastname: Park
authors.forename: Ka Hyon
authors.initials: KH
authors.lastname: Park
authors.forename: Sung-Tae
authors.initials: ST
authors.lastname: Yang
authors.forename: Jae Il
authors.initials: JI
authors.lastname: Kim
authors.forename: Il-Seon
authors.initials: IS
authors.lastname: Park
authors.forename: Kyung-Soo
authors.initials: KS
authors.lastname: Hahm
authors.forename: Song Yub
authors.initials: SY
authors.lastname: Shin
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics*; D000818:Animals; D001419:Bacteria / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D004912:Erythrocytes / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D016350:Leucine Zippers / Q000235:genetics*; D008555:Melitten / Q000138:chemical synthesis / Q000737:chemistry* / Q000633:toxicity; D008969:Molecular Sequence Data; D034444:Peptoids / Q000235:genetics*; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D013778:Tetrazolium Salts; D013844:Thiazoles
chemical_list: D000596:Amino Acids; D034444:Peptoids; D013778:Tetrazolium Salts; D013844:Thiazoles; D008555:Melitten; C022616:thiazolyl blue