Orientation of Cecropin A Helices in Phospholipid Bilayers Determined by Solid-State NMR Spectroscopy

Abstract

The orientation of the insect antibiotic peptide cecropin A (CecA) in the phospholipid bilayer membrane was determined using 15N solid-state NMR spectroscopy. Two peptide samples, each specifically labeled with 15N at Val 11 or Ala 27, were synthesized by solid phase techniques. The peptides were incorporated into phospholipid bilayers, prepared from a mixture of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol, and oriented on glass slides. The 15N chemical shift solid-state NMR spectra from these uniaxially oriented samples display a single 15N chemical shift frequency for each labeled residue. Both frequencies are near the upfield end of the 15N 15N chemical shift powder pattern, as expected for an α-helix with its long axis in the plane of the membrane and the NH bonds perpendicular to the direction of the magnetic field. These results support a mechanism of action in which CecA binds to and covers the membrane surface, thereby causing a general destabilization and leakiness of the lipid bilayer membrane. The data are discussed in relation to a proposed mechanism of membrane lysis and bacterial killing via an ion channel activity of CecA.