STRUCTURE AND ORIENTATION OF THE MAMMALIAN ANTIBACTERIAL PEPTIDE CECROPIN P1 WITHIN PHOSPHOLIPID-MEMBRANES

Cecropins are positively charged antibacterial peptides that act by pe rmeating the membrane of susceptible bacteria. To gain insight into th e mechanism of membrane permeation, the secondary structure and the or ientation 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/phosphati dylglycerol) in a 7:3 (w/w) ratio (a phospholipid composition similar to that of many bacterial membranes), indicated that the peptide is pr edominantly alpha-helical. Polarized ATR-FTIR spectroscopy was used to determine the orientation of the peptide relative to the bilayer norm al 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 s tructure and orientation were found when zwitterionic phosphatidylchol ine phospholipids were used. The incorporation of CecP did not signifi cantly change the order parameters of the acyl chains of the multibila yer, further suggesting that CecP does not penetrate the hydrocarbon c ore of the membranes. Molecular dynamics simulations were used to gain insight into possible effects of transmembrane potential on the orien tation of CecP relative to the membrane. The simulations arrear to con firm that CecP adopts an orientation parallel to the membrane surface and does not insert into the bilayer in response to a cis positive tra nsmembrane voltage difference. Taken together, the results further sup port a ''carpet-like'' mechanism, rather than the formation of transme mbrane pores, as the mode of action of CecP According to this model, f ormation of a layer of peptide monomers on the membrane surface destab lizes the phospholipid packing of the membrane leading to its eventual disintegration. (C) 1996 Academic Press Limited