Orientation of the antimicrobial peptide PGLa in lipid membranes determined from 19F-NMR dipolar couplings of 4-CF3-phenylglycine labels

A highly sensitive solid state F-19-NMR strategy is described to determine the orientation and dynamics of membrane-associated peptides from specific fluorine labels. Several analogues of the antimicrobial peptide PGLa were synthesized with the non-natural amino acid 4-trifluoromethyl-phenylglycine (CF3-Phg) at different positions throughout the alpha-helical peptide chain. A simple I-pulse F-19 experiment allows the simultaneous measurement of both the anisotropic chemical shift and the homonuclear dipolar coupling within the rotating CF3-group ill a macroscopically oriented membrane sample. The value and sign of the dipolar splitting determines the tilt of the CF3-rotational axis, which is rigidly attached to the peptide backbone, with respect to the external magnetic field direction. Using four CF3-labeled peptide analogues (With L-CF3-Phg at Ile9, Ala10, Ile13, and Ala14) we confirmed that PGLa is aligned at the surface of lipid membranes with its helix axis perpendicular to the bilayer normal at a peptide:lipid ratio of 1:200. We also determined the azimuthal rotation angle of the helix, which agrees well with the orientation expected from its amphiphilic character. Peptide analogues with a D-CF3-Phg label resulting front racemization of the amino acid during synthesis were separately collected by HPLC. Their spectra provide additional information about the PGLa structure and orientation but allow only to discriminate qualitatively between multiple solutions. The structural and functional characterization of the individual CF3-labeled peptides by circular dichroism and antimicrobial assays showed only small effects for our four substitutions oil the hydrophobic face of the helix. but a significant disturbance was observed in a fifth analogue where Ala8 oil the hydrophilic face had been replaced. Even though the hydrophobic CF3-Phg side chain cannot be utilized in all positions, it allows highly sensitive NMR measurements over a wide range of experimental conditions and dynamic regimes of the peptide. (C) 2004 Elsevier Inc. All rights reserved.