Conformation of alamethicin in oriented phospholipid bilayers determined by N-15 solid-state nuclear magnetic resonance

The conformation of the 20-residue antibiotic ionophore alamethicin in macr oscopically oriented phospholipid bilayers has been studied using N-15 soli d-state nuclear magnetic resonance (NMR) spectroscopy in combination with m olecular modeling and molecular dynamics simulations. Differently N-15-labe led variants of alamethicin and an analog with three of the a-amino-isobuty ric acid residues replaced by alanines have been investigated to establish experimental structural constraints and determine the orientation of alamet hicin in hydrated phospholipid (dimyristoylphosphatidylcholine) bilayers; a nd to investigate the potential for a major kink in the region of the centr al Pro(14) residue. From the anisotropic N-15 chemical shifts and H-1-N-15 dipolar couplings determined for alamethicin with N-15-labeling on the Ala( 6), Val(9), and Val(15) residues and incorporated into phospholipid bilayer with a peptide:lipid molar ratio of 1:8, we deduce that alamethicin has a largely linear a-helical structure spanning the membrane with the molecular axis tilted by 10-20 degrees relative to the bilayer normal. In particular , we find compatibility with a straight a-helix tilted by 17 degrees and a slightly kinked molecular dynamics structure tilted by 11 degrees relative to the bilayer normal. In contrast, the structural constraints derived by s olid-state NMR appear not to be compatible with any of several model struct ures crossing the membrane with vanishing tilt angle or the earlier reporte d x-ray diffraction structure (Fox and Richards, Nature. 300:325-330, 1982) . The solid-state NMR-compatible structures may support the formation of a left-handed and parallel multimeric ion channel.