Structure and Orientation of the Mammalian Antibacterial Peptide Cecropin P1 within Phospholipid Membranes

• Published in: Journal of molecular biology 1996-05, Vol.258 (5), p.860-870
• Main Authors: Gazit, Ehud, Miller, Israel R., Biggin, Phil C., Sansom, Mark S.P., Shai, Yechiel
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; amphipathic α-helix; Animals; Anti-Bacterial Agents - chemistry; antibacterial properties; ATR-FTIR; bacteria; cell membranes; Computer Simulation; defense mechanisms; diphosphatidylglycerols; infrared spectroscopy; innate immunity; Lipid Bilayers - chemistry; Magnetic Resonance Spectroscopy; Membrane Lipids - chemistry; membranes; Models, Molecular; molecular conformation; Molecular Sequence Data; orientation; peptide-membrane interaction; Peptides; phosphatidylethanolamines; Phospholipids - chemistry; polypeptides; Protein Binding; protein secondary structure; Protein Structure, Secondary; small intestine; Spectroscopy, Fourier Transform Infrared; structure; Swine; toxic mechanism
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.1996.0293

Cecropins are positively charged antibacterial peptides that act by permeating the membrane of susceptible bacteria. To gain insight into the mechanism of membrane permeation, the secondary structure and the orientation within phospholipid membranes of the mammalian cecropin P1 (CecP) was studied using attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy and molecular dynamics simulations. The shape and frequency of the amide I and II absorption peaks of CecP within acidic PE/PG multibilayers (phosphatidylethanolamine/phosphatidylglycerol) in a 7:3 (w/w) ratio (a phospholipid composition similar to that of many bacterial membranes), indicated that the peptide is predominantly α-helical. Polarized ATR-FTIR spectroscopy was used to determine the orientation of the peptide relative to the bilayer normal of phospholipid multibilayers. The ATR dichroic ratio of the amide I band of CecP peptide reconstituted into oriented PE/PG phospholipid membranes indicated that the peptide is preferentially oriented nearly parallel to the surface of the lipid membranes. A similar secondary structure and orientation were found when zwitterionic phosphatidylcholine phospho lipids were used. The incorporation of CecP did not significantly change the order parameters of the acyl chains of the multibilayer, further suggesting that CecP does not penetrate the hydrocarbon core of the membranes. Molecular dynamics simulations were used to gain insight into possible effects of transmembrane potential on the orientation of CecP relative to the membrane. The simulations appear to confirm that CecP adopts an orientation parallel to the membrane surface and does not insert into the bilayer in response to a cispositive transmembrane voltage difference. Taken together, the results further support a “carpet-like” mechanism, rather than the formation of transmembrane pores, as the mode of action of CecP. According to this model, formation of a layer of peptide monomers on the membrane surface destablizes the phospholipid packing of the membrane leading to its eventual disintegration.