Membrane-bound structure and alignment of the antimicrobial beta-sheet peptide gramicidin S derived from angular and distance constraints by solid state F-19-NMR

The antimicrobial properties of the cyclic beta -sheet peptide gramicidin S are attributed to its destabilizing effect on lipid membranes. Here we pre sent the membrane-bound structure and alignment of a derivative of this pep tide, based on angular and distance constraints. Solid-state F-19-NMR was u sed to study a F-19-labelled gramicidin S analogue in dimyristoylphosphatid ylcholine bilayers at a lipid:peptide ratio of 80:1 and above. Two equivale nt leucine side chains were replaced by the non-natural amino acid 4F-pheny lglycine, which serves as a highly sensitive reporter on the structure and dynamics of the peptide backbone. Using a modified CPMG multipulse sequence , the distance between the two F-19-labels was measured from their homonucl ear dipolar coupling as 6 Angstrom, in good agreement with the known backbo ne structure of natural gramicidin S in solution. By analyzing the anisotro pic chemical shift of the F-19-labels in macroscopically oriented membrane samples, we determined the alignment of the peptide in the bilayer and desc ribed its temperature-dependent mobility. In the gel phase, the F-19-labell ed gramicidin S is aligned symmetrically with respect to the membrane norma l, i.e., with its cyclic beta -sheet backbone lying flat in the plane of th e bilayer, which is fully consistent with its amphiphilic character. Upon r aising the temperature to the liquid crystalline state, a considerable narr owing of the F-19-NMR chemical shift dispersion is observed, which is attri buted the onset of global rotation of the peptide and further wobbling moti ons. This study demonstrates the potential of the F-19 nucleus to describe suitably labelled polypeptides in membranes, requiring only little material and short NMR acquisition times.