Membrane thinning has been discussed as a fundamental mechanism by which antimicrobial peptides can perturb cellular membranes. To understand which factors play a role in this process, we compared several amphipathic peptides with different structures, sizes and functions in their influence on the lipid bilayer thickness. PGLa and magainin 2 from X. laevis were studied as typical representatives of antimicrobial cationic amphipathic α-helices. A 1:1 mixture of these peptides, which is known to possess synergistically enhanced activity, allowed us to evaluate whether and how this synergistic interaction correlates with changes in membrane thickness. Other systems investigated here include the α-helical stress-response peptide TisB from E. coli (which forms membrane-spanning dimers), as well as gramicidin S from A. migulanus (a natural antibiotic), and BP100 (designer-made antimicrobial and cell penetrating peptide). The latter two are very short, with a circular β-pleated and a compact α-helical structure, respectively. Solid-state 2H-NMR and grazing incidence small angle X-ray scattering (GISAXS) on oriented phospholipid bilayers were used as complementary techniques to access the hydrophobic thickness as well as the bilayer-bilayer repeat distance including the water layer in between. This way, we found that magainin 2, gramicidin S, and BP100 induced membrane thinning, as expected for amphiphilic peptides residing in the polar/apolar interface of the bilayer. PGLa, on the other hand, decreased the hydrophobic thickness only at very high peptide:lipid ratios, and did not change the bilayer-bilayer repeat distance. TisB even caused an increase in the hydrophobic thickness and repeat distance. When reconstituted as a mixture, PGLa and magainin 2 showed a moderate thinning effect which was less than that of magainin 2 alone, hence their synergistically enhanced activity does not seem to correlate with a modulation of membrane thickness. Overall, the absence of a typical thinning response in the case of PGLa, and the increase in the repeat distance and membrane thickening observed for TisB, demonstrate that the concept of peptide-induced membrane thinning cannot be generalized. Instead, these results suggest that different factors contribute to the resulting changes in membrane thickness, such as the peptide orientation in the bilayer, and/or bilayer adaptation to hydrophobic mismatch.

γ-(4S)-Trifluoromethyl proline was synthesised according to a modified literature protocol with improved yield on a multigram scale. Conformational properties of the amide bond formed by the amino acid were characterised using N-acetyl methyl ester model. The amide populations (s-trans vs. s-cis) and thermodynamic parameters of the isomerization were found to be similar to the corresponding values for intact proline. Therefore, the γ-trifluoromethyl proline was suggested as a structurally low-disturbing proline substitution in peptides for their structural studies by (19)F-NMR. Indeed, the exchange of native proline for γ-trifluoromethyl proline in the peptide antibiotic gramicidin S was shown to preserve the overall amphipathic peptide structure. The utility of the amino acid as a selective (19)F-NMR label was demonstrated by observing the re-alignment of the labelled gramicidin S in oriented lipid bilayers.

Peptaibols are promising drug candidates in view of their interference with cellular membranes. Knowledge of their lipid interactions and membrane-bound structure is needed to understand their activity and should be, in principle, accessible by solid-state NMR spectroscopy. However, their unusual amino acid composition and noncanonical conformations make it very challenging to find suitable labels for NMR spectroscopy. Particularly in the case of short sequences, new strategies are required to maximize the structural information that can be obtained from each label. Herein, l-3-(trifluoromethyl)bicyclopent[1.1.1]-1-ylglycine, (R)- and (S)-trifluoromethylalanine, and N-backbone labels, each probing a different direction in the molecule, have been combined to elucidate the conformation and membrane alignment of harzianin HK-VI. For the short sequence of 11 amino acids, 12 orientational constraints have been obtained by using F and N NMR spectroscopy. This strategy revealed a β-bend ribbon structure, which becomes realigned in the membrane from a surface-parallel state towards a membrane-spanning state, with increasing positive spontaneous curvature of the lipids.

The branched chains in diphytanoyl lipids provide membranes with unique properties, such as high chemical/physical stability, low water permeability, and no gel-to-fluid phase transition at ambient temperature. Synthetic diphytanoyl phospholipids are often used as model membranes for electrophysiological experiments. To evaluate whether these sturdy lipids are also suitable for solid-state NMR, we have examined their interactions with a typical amphiphilic peptide in comparison with straight-chain lipids. First, their phase properties were monitored using P NMR, and the structural behaviour of the antimicrobial peptide PGLa was studied byF NMR and circular dichroism in oriented membrane samples. Only lipids with choline headgroups (DPhPC) were found to form stable lipid bilayers in oriented samples, while DPhPG, DPhPE and DPhPS display non-lamellar structures. Hence, the experimental temperature and hydration are crucial factors when using supported diphytanoyl lipids, as both parameters must be maintained in an appropriate range to avoid the formation of non-bilayer structures. For the same reason, a high content of other diphytanoyl lipids besides DPhPC in mixed lipid systems is not favourable. Unlike the situation in straight-chain membranes, we found that the α-helical PGLa was not able to insert into the tightly packed fluid bilayer of DPhPC but remained in a surface-bound state even at very high peptide concentration. This behaviour can be explained by the high cohesivity and the negative spontaneous curvature of the diphytanoyl lipids. These characteristic features must therefore be taken into consideration, both, in electrophysiological studies, and when interpreting the structural behaviour of membrane-active peptides in such lipid environment.