Antimicrobial 14-Helical -Peptides: Potent Bilayer Disrupting Agents † Raquel F. Epand, ‡ Tami L. Raguse, § Samuel H. Gellman, § and Richard M. Epand* ,‡ Department of Biochemistry, McMaster UniVersity Health Sciences Centre, Hamilton, ON, L8N 3Z5 Canada, and Department of Chemistry, UniVersity of Wisconsin, Madison, Wisconsin 53706 ReceiVed March 25, 2004; ReVised Manuscript ReceiVed May 17, 2004 ABSTRACT: The interactions of two amphiphilic and cationic, nine-residue -peptides with liposomal membranes were studied. These -peptides are shown to form 14-helices in the presence of bilayers. Membrane binding and membrane permeabilization occur preferentially in the presence of anionic lipids. The -peptides have the ability to cause tranbilayer diffusion of phospholipids, form pores, and promote lipid mixing between liposomes. These -peptides have previously been shown to display antimicrobial activity comparable to that of a longer -peptide, -17, which adopts a different type of helical conformation (12-helix), and to the 23 amino acid (Ala 8,13,18 )-magainin-II-amide, which adopts an R-helical conformation. In addition, these 14-helical -peptides show relatively low hemolytic activity. The biological potency and microbial specificity of the 14-helical -peptides, despite their relatively short length, suggests that 14-helices can be particularly disruptive to microbial membranes. Helix-forming antimicrobial peptides are critical compo- nents of the innate immune system, and these host-defense peptides are potential sources of new antibiotic drugs (1, 2). Recently there has been growing interest in unnatural oligomers that mimic host-defense peptides (3, 4), because unnatural oligomers are not subject to proteolytic degrada- tion, in contrast to peptides composed of proteinogenic residues (5). Several groups have examined oligomers of -amino acids (“-peptides”) in this context (6). These designs have been based on two different types of -pep- tide helix, the 12-helix, containing 12-membered ring CdO(i)- -H-N(i+3) hydrogen bonds, and the 14-helix, containing 14-membered ring CdO(i)- -H-N(i-2) hydrogen bonds. For each of these -peptide secondary structures, sequences that result in formation of an amphiphilic helix, with lipophilic groups aligned on one side and cationic groups on the other, display significant antimicrobial activity against a range of species (7-12). We have recently characterized the interactions of a 12- helical -peptide antibiotic with synthetic lipid vesicles (13), and here we report analogous studies with a pair of 14-helical -peptide antibiotics, 1 and 2. Compound 1 is closely related to a design originally developed by DeGrado et al. (7). The 14-helix has ca. three residues per turn (6); therefore, when -peptides 1 or 2 adopt a 14-helical conformation, the helix has hydrophobic side chains on two-thirds of the helical circumference and cationic side chains on the other third. Most of the residues in -peptides 1 and 2 are derived from -substituted -amino acids (“ 3 -residues”). Previous work has shown that oligomers composed exclusively of  3 - residues adopt 14-helical conformations in organic solvents (e.g., methanol), but usually not in water (6). Three of the hydrophobic residues in -peptide 2 are derived from trans- 2-aminocyclohexanecarboxylic acid (14, 15). This residue is a strong promoter of 14-helicity, even in aqueous solution (16, 17). Circular dichroism (CD) 1 data reported by DeGrado et al. for -peptides related to -peptide 1 indicate that these molecules do not form the 14-helix in water but that 14- helicity can be induced by addition of micelles (7, 9). -Peptide 1 itself also shows no sign of 14-helicity in aqueous solution, but addition of 2,2,2-trifluoroethanol (TFE) induces partial 14-helix formation (12). In contrast, -peptide 2 shows partial 14-helicity even in the absence of TFE (i.e., pure aqueous solution) (12). Both -peptides 1 and 2 display significant bacteriostatic and bactericidal activity against four different microbial † This work was supported by grant MT-7654 from the Canadian Institutes of Health Research and grant GM-56414 from the US National Institutes of Health. T.L.R. was supported in part by a pre-doctoral fellowship from the US National Science Foundation. * To whom correspondence should be addressed. Tel.: (905) 525- 9140. Fax: (905) 521-1397. E-mail: epand@mcmaster.ca. ‡ McMaster University Health Sciences Centre. § University of Wisconsin. 9527 Biochemistry 2004, 43, 9527-9535 10.1021/bi049414l CCC: $27.50 © 2004 American Chemical Society Published on Web 07/03/2004