Membrane orientation of the N-terminal segment of alamethicin determined by solid-state N-15 NMR

Alamethicin was synthesized with N-15 incorporated into alanine at position 6 in the peptide sequence. In dispersions of hydrated dimyristoylphosphatidylcholine, solid-state N-15 NMR yields an axially symmetric powder pattern indicating that the peptide is reorienting with a single axis of symmetry when associated with lamellar lipids. When incorporated into bilayers that are uniformly oriented with the bilayer normal parallel to the B-0 field, the position of the observed N-15 chemical shift is 171 ppm. This is coincident with the sigma(parallel to) edge of the axially symmetric powder pattern for non-oriented hydrated samples. Thus the axis of motional averaging lies along the bilayer normal. Two-dimensional separated local field spectra were obtained that provide a measure of the N-H dipolar coupling in one dimension and the N-15 chemical shift in the other. These data yield a dipolar coupling of 17 kHz corresponding to an average angle of 24 degrees for the N-H bond with respect to the B-0 field axis. An analysis of the possible structures and orientations that could produce the observed spectral parameters show that these values are consistent with an alpha-helical conformation inserted along the bilayer normal.