Figure 1.
Experimental (a) and simulated (b) 31P powder NMR spectra of lyophilized DMPC.
Experimental (c) and simulated (d) 31P static powder NMR spectra of DMPC vesicles. The peak at 0 ppm in (c) originates from the phosphate buffer.
Figure 2.
Sketches of the geometries (left column) and examples of membrane/AMP systems described by the geometries (right column).
The geometries are referred to a thinned bilayer (a) and toroidal pore (b) [65] and are identical to those proposed by Wi and Kim [28]. See text for further description of the parameters.
Figure 3.
Oriented-sample solid-state 15N and 31P NMR spectra of (a) 15N-Aib8 labeled alamethicin incorporated into oriented DMPC lipids at a peptide:lipid molar ratio of 1∶15 and (b) 15N-Ile14 labeled novicidin in oriented DMPC:DMPG (molar ratio 4∶1) bilayers at 1∶15 peptide:lipid molar ratio.
The resonance in the spectrum of alamethicin is substantially broadened due to mosaic spread [37] and the heterogeneous nature of the peptide-lipid interactions of the peptide [39], [56]. (c,d) 31P spectra of (c) a sample of alamethicin in oriented DMPC lipids with a peptide:lipid ratio of 1∶25 and (d) the novidin sample in (b). (e,f) Models showing the most likely conformations of the peptides and lipids at high peptide:lipid ratio for (e) alamethicin and (f) novicidin in lipid bilayers.
Figure 4.
Oriented-sample solid-state 31P NMR spectra of DMPC lipids with increasing ratios of alamethicin relative to lipid (P:L ratios given to the right in the figure) and numerical simulations of these spectra.
(a,c,e,g) Experimental spectra. (b,d,f,h) Simulated spectra. (a,b) Pure DMPC bilayers. (c,d) P:L = 1∶400. (e,f) P:L = 1∶100. (g,h) P:L = 1∶25. The spectrum at highest P:L ratio (g) is identical to Fig. 3c.
Table 1.
Parameters used for simulation of the 31P spectra for the alamethicin samples (Fig. 4).a
Figure 5.
Oriented-sample solid-state 31P NMR spectra of DMPC:DMPG (ratio 80∶20) bilayers with increasing amounts of novicidin. (a,c,e,g,I,k,m) Experimental spectra. (b,d,f,h,j,l,n) Simulated spectra using the parameters listen in Table 2.
(a,b) Pure lipid bilayers. (c,d) P:L = 1∶400. (e,f) P:L = 1∶200. (g,h) P:L = 1∶100. (i,j) P:L = 1∶50. (k,l) P:L = 1∶25. (m,n) P:L = 1∶15. The spectrum at highest P:L ratio (m) is identical to Fig. 3d.
Table 2.
Parameters used for simulation of the 31P spectra for the novicidin samples (Fig. 5).a
Figure 6.
Model of peptide interactions with the lipid membrane compatible with data from oriented-sample solid-state NMR 31P spectra.
The lipids are illustrated by circles with the radial line illustrating the orientation of the 31P head group. (a) Pure bilayers display a single sharp resonance at around 30 ppm. (b) At low peptide concentrations, weak interactions between peptide and lipids induce a slight disorder causing the 31P resonance to shift to lower values. (c) The peptide insertion involves lipids that change orientation with reduced diffusion as a consequence. This gives rise to a peak at approximately −15 ppm. (d and e) The last step in the method of action of the peptide is the penetration of the lipids and disruption of the bilayer. Alamethicin forms barrel-stave channels without significant perturbation of the lipid (d), while novicidin creates toroidal pores in the lipids (e).