Membrane helix orientation from linear dichroism of infrared attenuated total reflection spectra

Oriented multilamellar systems containing phospholipids and peptides have b een formed on a germanium internal reflection element. Attenuated total ref lection infrared spectra have been recorded and the linear dichroism of pep tide amide I and amide II bands measured. Using peptides for which the orie ntation had been previously studied under similar experimental conditions b y N-15 solid-state nuclear magnetic resonance spectroscopy, important concl usions were drawn on the approach to be used to derive secondary structure orientation in a membrane from dichroic ratios. In particular, it is shown that the influence of the film thickness and refractive index on the orient ation determination can be evaluated from the value of R-ATRiso, i.e., the dichroic ratio of a dipole oriented at the magic angle or with isotropic mo bility. A series of peptides was used to test the validity of our suggestio ns on various helix orientations in the membrane. These include magainin 2 and hydrophobic (h Phi 20) model peptides, the transmembrane segment of gly cophorin (GLY), and LAH(4), a designed peptide antibiotic that changes betw een a transmembrane and an in-plane orientation in a pH-dependent manner.