Orthogonal ¹⁹F-labeling for solid-state NMR reveals the conformation and orientation of short peptaibols in membranes

Peptaibols are promising drug candidates in view of their interference with cellular membranes. Knowledge of their lipid interactions and membrane-bound structure is needed for understanding their activity and should be in principle accessible by solid-state NMR. However, their unusual amino acid composition and their non-canonical conformations make it very challenging to find suitable NMR labels. Especially in the case of short sequences, new strategies are required to maximize the structural information that can be obtained from each label. Here, we have combined trifluoromethylbicyclopentyl-L-glycine, (R)- and (S)-trifluoromethylalanine, and ¹⁵N-backbone labels, each probing a different direction in the molecule, to elucidate the conformation and membrane alignment of harzianin HK-VI. For the short sequence of 11 amino acids we obtained 12 orientational constraints using ¹⁹F- and ¹⁵N-NMR. This strategy revealed a β-bend-ribbon structure, which becomes re-aligned in the membrane from a surface-parallel state towards a membrane-spanning state with increasing positive spontaneous curvature of the lipids.