Membrane Topology of a 14-mer Model Amphipathic Peptide: A Solid-State NMR Spectroscopy Study ² Marise Ouellet, Jean-Daniel Doucet, Normand Voyer, and Miche`le Auger* De´ partement de Chimie, Centre de Recherche sur la Fonction, la Structure et l’Inge´ nierie des Prote´ ines, Centre de Recherche en Sciences et Inge´ nierie des Macromole´ cules, UniVersite´ LaVal, Que´ bec, Que´ bec, Canada G1K 7P4 ReceiVed September 28, 2006; ReVised Manuscript ReceiVed April 4, 2007 ABSTRACT: We have investigated the interaction between a synthetic amphipathic 14-mer peptide and model membranes by solid-state NMR. The 14-mer peptide is composed of leucines and phenylalanines modified by the addition of crown ethers and forms a helical amphipathic structure in solution and bound to lipid membranes. To shed light on its membrane topology, 31 P, 2 H, 15 N solid-state NMR experiments have been performed on the 14-mer peptide in interaction with mechanically oriented bilayers of dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), and dipalmitoylphos- phatidylcholine (DPPC). The 31 P, 2 H, and 15 N NMR results indicate that the 14-mer peptide remains at the surface of the DLPC, DMPC, and DPPC bilayers stacked between glass plates and perturbs the lipid orientation relative to the magnetic field direction. Its membrane topology is similar in DLPC and DMPC bilayers, whereas the peptide seems to be more deeply inserted in DPPC bilayers, as revealed by the greater orientational and motional disorder of the DPPC lipid headgroup and acyl chains. 15 N{ 31 P} rotational echo double resonance experiments have also been used to measure the intermolecular dipole-dipole interaction between the 14-mer peptide and the phospholipid headgroup of DMPC multilamellar vesicles, and the results indicate that the 14-mer peptide is in contact with the polar region of the DMPC lipids. On the basis of these studies, the mechanism of membrane perturbation of the 14-mer peptide is associated to the induction of a positive curvature strain induced by the peptide lying on the bilayer surface and seems to be independent of the bilayer hydrophobic thickness. In recent years, there has been a growing interest in studying membrane-active peptides since they represent a potential alternative to ineffective antibiotics (1). Most of the natural occurring antimicrobial peptides are short, diversified in their amino acid composition, cationic, am- phipathic, and can adopt different structures in interaction with membranes (2, 3). Their membrane activity seems to be modulated both by the structural parameters of the peptides, such as helicity, charge, hydrophobicity, and the lipidic composition, and by the physical state of the membranes (4). As reported by Hancock et al., Jenssen et al., and Marr et al., several variants of cationic antimicrobial peptides are currently being investigated and present varied successes in clinical tests (5-7). Even if the development of antimicrobial peptides for clinical applications remains challenging, these agents possess advantages that overcome limitations of conventional antibiotics. The antibacterial activity of natural membrane-active peptides has been extensively studied, and the readers are referred to the paper of Strandberg et al. (8) for a detailed list of synthetic and natural antimicrobial peptides studied by solid-state NMR. 1 Besides their antibacterial activity, some membrane-active peptides like melittin, indolicidin, and cecropin-like human LL-37 show important cytotoxic prop- erties. As reported by Hancock et al., it is very difficult to predict the hemolytic activity of some antibacterial peptides (9), and it is essential for the design of novel synthetic antibacterial agents to better understand the types of interac- tions involved both in the antibacterial and in the hemolytic activities of such agents. However, the ways by which membrane-active peptides perturb lipid bilayers are not well understood, and many researches have relied on synthetic model peptides to shed light on the mode of membrane perturbation and on the structural features that drive these mechanisms (10-12). General modes of action are reported in the literature, namely, the barrel-stave, the carpet-like, the ² This work was supported by the Natural Science and Engineering Research Council (NSERC) of Canada, by the Fonds Que´be´cois de la Recherche sur la Nature et les Technologies (FQRNT), by the Centre de Recherche sur la Structure, la Fonction et l’Inge´nierie des Prote´ines (CREFSIP), and by the Centre de Recherche en Sciences et Inge´nierie des Macromole´cules (CERSIM). M.O. and J.-D.D. also wish to thank NSERC for the award of postgraduate and undergraduate scholarships, respectively. * Address correspondence to this author. Tel: 418-656-3393. Fax: 418-656-7916. E-mail: michele.auger@chm.ulaval.ca. 1 Abbreviations: BOC, tert-butyloxycarbonyl; CP, cross-polarization; CSA, chemical shift anisotropy; DLPC, dilauroylphosphatidylcholine; DMF, N,N-dimethyl sulfoxide; DMPC, dimyristoylphosphatidylcholine; DOTAP, 1,2-dioleoyl-3-(trimethylammonio)propane; DPPC, dipalmi- toylphosphatidylcholine; DVB, divinylbenzene; FWHM, full width at half-maximum; HPLC, high-performance liquid chromatography; LPC, lysophosphatidylcholine; MAS, magic-angle spinning; MLVs, multi- lamellar vesicles; NMR, nuclear magnetic resonance; PC, phosphati- dylcholine; POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; POPE, 1-palmitoyl-2-oleoylphosphatidylethanolamine; POPG, 1-palmitoyl-2- oleoylphosphatidylglycerol; POPS, 1-palmitoyl-2-oleoylphosphati- dylserine; REDOR, rotational echo double resonance; TPPM, two pulse phase modulation. 6597 Biochemistry 2007, 46, 6597-6606 10.1021/bi0620151 CCC: $37.00 © 2007 American Chemical Society Published on Web 05/08/2007