Solid state NMR structure analysis of the antimicrobial peptide gramicidin S in lipid membranes:: concentration-dependent re-alignment and self-assembly as a β-barrel

Antimicrobial peptides can kill bacteria by permeabilizing their cell membrane, as these amphiphilic molecules interact favourably with lipid bilayers. This mechanism of action is attributed either to the formation of a peptide "carpet" on the membrane surface, or to a transmembrane pore. However, the structure of such a pore has not yet been resolved under relevant conditions. Gramicidin S is a symmetrical cyclic beta-sheet decapeptide, which has been previously shown by solid state NMR to lie flat on the membrane surface at low peptide:lipid ratios (<= 1:80). Using highly sensitive F-19-NMR, supported by N-15-labelling, we found that gramicidin S can flip into an upright transmembrane alignment at high peptide;lipid ratios (>= 1:40). Orientational NMR constraints suggest that the peptide may self-assemble as an oligomeric beta-barrel pore, which is stabilized by intermolecular hydrogen bonds. Comparison of different model membranes shows that the observed re-alignment is favoured in thin bilayers with short-chain lipids, especially near the chain melting temperature, whereas long-chain lipids suppress pore formation. Based on the oligomeric structural model and the conditions of pore formation, guidelines may now be derived for rationally designing peptide analogues as antibiotics with improved selectivity and reduced side effects.